Phần VI. Công nghệ tế bào thực vật

PHN VI
CNG NGH T BO THC VT
Phn VI: CNG NGH T BO THC VT
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S PHT SINH C QUAN T LP MNG T B O CA GING IU (Anacardium occidentale L.) CAO SN BO1 NUI CY in vitro
Hunh Hu c, Nguyn nh S, Nguyn Th Qunh Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U Cy iu (Anacardium occidentale L.) l cy cng nghi p em li hiu qu kinh t cao mt s nc thuc vng nhit i Chu v Chu Phi do nhn h t iu c gi tr cao trong xut khu. Thng th ng cy c nhn ging t ht, nhng phng php ny em l i tnh khng ng nht v di truyn (Philip v Unni, 1994). Cc phng php nhn gi ng v tnh truyn thng nh gim cnh, ghp thng c s dng nhn cc d ng iu c nng sut cao, tuy nhi n h s nhn ging khng p ng nhu cu. Do ph ng php nhn gi ng in vitro c s dng v thnh cng trn nhi u loi cy n tri gn vi cy iu (Barghchi & Alderson, 1983; Litz v cng s, 1984) nn nhn ging in vitro cy iu c tnh kh thi (V Ngc Phng v cs, 2003). Nui cy lp mng t b o l mt phng php cho nhi u u th hn so vi nhng phng php nhn gi ng in vitro truyn thng khc v c ng dng thnh cng trn nhiu loi cy khc nhau (Dng Tn Nht v cs, 2003). Tuy nhin, vi c ng dng phng php nui cy lp mng t bo cy thn g cha c cng b nhiu. Trong bi ny chng ti trnh by m t s kt qu nghi n cu v nui cy lp mng t bo t t thn mm v t t dip ca cy iu. VT LIU V PHNG PHP Mu nui cy l ht trng thnh ca ging iu cao sn BO1 thu c t vn u dng ca Trung tm Hng Lc (Vin Khoa hc K thut Nng nghip Min Nam), ng Nai. Ht iu c 2 lp v, v cng b n ngoi v v la bn trong. Ht c kh trng bng dung dch NaOCl (C s Vn Phng, Qun 11, Tp. H Ch Minh) vi nng 1% (w/v) trong 24 gi, sau ra li bng n c ct v trng 3 ln. Cy mm pht trin t ht nui cy tr n mi trng khong MS khng c ng v vitamin sau 2 tun c dng lm nguyn liu cho th nghim. Lp mng ct
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Hi ngh KHOA HC V CNG NGH 2007
ngang t 2 loi vt liu l t t dip v t thn ca cy mm (c b dy khong 0,3-0,5mm) c cy trn cc a petri ( = 10cm) cha 20ml mi trng. Mi trng nui cy l mi trng MS (Murashige v Skoog, 1962) c b sung nc da 10% (v/v), adenine su lphate (Sigma Chemical Co.,USA) 40 mg/L, saccharose (Cty ng Bin Ho, ng Nai) 20g/L, maltose (Sigma Chemical Co., Missouri, USA) 10 g/L, agar (Cty C phn hp H Long, Qun g Ninh) 9g/L, than hot tnh 3g/L. Mi trng c b sung cc cht iu ho sinh trng thc vt l 6-benzyladenine (BA), kinetin (KN) v naphthalene -1-acetic acid (NAA) cc nng khc nhau. pH ca mi tr ng trc khi kh trng l 5,9. Mi trng c kh trng 121 oC, 1 atm trong 20 pht. Phng nui cy c nhi t 25 2 oC, m 60 5%. a petri ng mu c che ti trong 3 ng y u, sau c t di cng nh sng 40 -50 mol m -2 s -1 vi thi gian chiu sng 12 gi/ngy. Mi a petri c 24 mu cy ca mi loi vt liu (t thn mm hoc t t dip). Th nghim c 3 yu t l 3 cht iu ho tng trng thc vt, mi yu t c hai mc (Bng 1). Th nghim c b tr hon ton ngu nhin v mi nghim thc gm 4 a petri lp li 3 ln. S liu c phn tch thng k bng phn mm MSTATC (i hc Michigan, M ichigan, USA). Th nghi m c theo di trong 28 ngy.
Bng 1: M t th nghim (chung cho 2 loi vt liu)

Nghim thc 1 2 3 4 5 6 7 8 BA (mg/L) 5 5 5 5 10 10 10 10 KN (mg/L) 1 1 0 0 1 1 0 0 NAA (mg/L) 1 0,5 1 0,5 1 0,5 1 0,5
KT QU V THO LUN Kt qu th nghim sau 28 ng y nui cy cho thy cc lt mng v ng t thn ch cho mt chi cn vng t t dip th cho nhiu chi hn. iu ny cho thy l do vng sinh m ch hin din chung quanh t t dip d b kch hot di tc ng ca ch t iu ho sinh trng thc vt (K. Trn Thanh Vn, 2003). (Hnh 1).
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t thn mm
t t dip
Hnh 1. Chi hnh thnh t lp mng t thn mm v t t dip sau 28 ng y.
S chi cao nht (5, 7 chi) t lp mng t t dip th uc nghim thc 6 c b sung 10mg/l BA, 1mg/l KN v 0,5mg/l NAA. Lp mng to chi thp nht (3 chi) khi c nui trn mi trng c b sung 5mg/l BA v 0,5mg/l NAA. nh hng ca BA v KN ln s to chi ca lp mng v ng t t dip thy r nht khi nng BA 10mg/L v KN 1mg/L, trong khi nh hng ca NAA khng thy r lm trong th nghim ny. i vi t thn mm, nng ca cc cht iu ho sinh trng thc vt c s dng trong th nghim n y khng em li s khc bit v s chi hnh thnh c 8 nghim thc (Hnh 2).
6 5 4 3 2 1 0 1 2 3 4 5 6 7 8
So choi/mau
Nghiem thc
o t tha n o t t die p
Hnh 2. S chi to thnh t lt mng t thn mm v t t dip ngy th 28.
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Cc chi hnh thnh t lp mng c tip tc cy chuyn sang mi tr ng MS c b sung 1mg/l BA v 1mg/l KN pht trin thnh cy vi s ln cy chuyn l 2 tun/ln. Chiu cao cy trung bnh t 3-4cm sau 2 thng cy chuyn (Hnh 3).
Hnh 3. Cm chi cy iu pht trin sau 2 thng cy chuyn.
KT LUN Phng php nui cy lp mng t bo c th c s dng to chi trc tip t trc thng dip ca ht iu ny mm. S chi c to trc tip t lp mng t t dip l cao nht khi mi trng c b sung 10mg/l BA, 1mg/l KN v 0,5mg/l NAA. i vi nui cy lp mng ca t thn mm th chi c th pht trin trn mi trng c BA 5mg/L v c hoc khng c kinetin v NAA. Cn tip tc nghin cu tm mi trng thch hp cho s pht trin chi t nui cy lp mng t thn mm. TI LIU THAM KHO 1. 2. Barghchi M, Alderson PG (1983). In vitro production of Pistacia species. Acta Hort. 131, 49-60. Dng Tn Nht, Da Silva JAT, Bi Vn L, Kim Trn Thanh Vn (2003). Thin cell layer (TCL) morphogenesis as a powerful tool in woody plant and fruit crop mircopropagation and biotechnology, floral genetics andgenetic transformation. In: Jain SM & Ishii K (eds.) Micropropagation of woody trees and fruits, pp. 783-814, Kluwer Academic Publishers, Dordretch, The Netherlands. Litz RE, Knight Jr RJ, Gazit S (1984). In vitro somatic embryogenesis from Mangifera indica L. callus. Sci. Hort. 22, 233 -240.
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4.
Philip VJ & Unni PN (1984). In vitro propagation of cashew for crop improvement. In: Bhaskara Rao EVV & Khan HH (eds.) Cashew Research and Development, pp.77-82, CPCRI, Kasargod, India. Trn Thanh Vn K (2003). Thin cell layer concept. In: Duong Tan Nhut, Van Le Bui, Tran Thanh Van K, Thorpe T (eds.) Thin cell layer culture systerm: regeneration and transformation applications. pp.1 -16, Kluwer Academic Publishers, Dordretch, The Netherlan ds.
5.
SUMMARY
The organogenesis via thin cell layers cashew (Anacardium occidentale L.) of the cultivar BO1 cultured in vitro
Huynh Huu Duc, Nguyen Dnh Sy, Nguyen Thi Quynh Institute of Tropical Biology
Cashew (Anacardium occidentale L.) is a profi table cash crop of several tropical countries due to the export value of kernels. A study on the in vitro organogenesis via thin cell layer (TCL) culture of the cultivar BO1 was carried out for an appropriate approach to produce cashew transplants on a lar ge scale. Mature seeds of the cultivar BO1 were surface sterilized with NaOCl 1% (w/v) on the MS sugar -free medium. Epicotyls of seedlings were used as explants for TCL culture and put on a modified MS medium (Murashige and Skoog, 1962) supplemented with B A, KN and NAA at different concentrations, 20g/L sucrose and 10 g/L maltose. The TCL positions on the explants affected the direct shoot induction and number of shoots. Shoot formation from TCLs derived from the cotyledonary nodal position was greater than that from other nodal positions of epicotyls. Concentrations of BA, KN and NAA of 10, 1, and 0.5 mg/L, respectively, were the best for the direct shoot formation of the cultivar BO1.
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NH HNG CA NH SNG T NHI N TRONG QU TRNH NHN GING in vitro LN S TNG TRNG CA CY LAN GING TRONG GIAI ON VN M
Lu Vit Dng, V Ngc Phng, Thi Xun Du Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U Trong nhng nm gn y, nhu cu trng phong lan pht trin TP. H Ch Minh. Tuy nhin, vic cung cp mt s l ng ln cy ging khe mnh, c t l sng khi trng cao trong thi gian qua c n gp nhiu kh khn do khng c n v c kh nng cung cp. Nhu cu trng lan t cy ging bng cch nui cy in vitro ng y cng ln v giai on hin nay. Chnh v th vic nhn nhanh v a ra th trng mt s lng ln nhng cy ging kho mnh l mt nhu cu ca thc t. to c cy con khe mnh, gi th nh thp, bn cnh thnh cng ca nui cy in vitro, giai on v n m l mt vn ht sc quan trng. Lm th no cy con cy m khi trng ra v n m c t l sng cao, pht trin tt khi chuyn t mi trng nhn to n nh trong b nh cy m ra mi trng bin ng gn vi t nhin trong vn m. Ni tip cc kt qu nghi n cu v nui cy m iu kin nh sng t nhi n, vic kho st: nh h ng ca nh sng t nhi n trong qu trnh nhn gi ng in vitro ln s tng trng ca cy lan ging trong giai on v n m l mt bc thu hp khong cch t cc kt qu nghi n cu khoa hc n tay nh vn ng dng vo thc tin, gp phn a khoa hc k thut phc v sn xut nng nghip cng ngh cao. VT LIU V PHNG PHP Cy lan cy m c nhn ging trong iu kin nh sng t nhi n v trong iu kin nh n hunh quang c la chn cc cy cng kch c trng ra vn m.
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Hnh 1. Cy lan Dendrobium c y m trc khi trng ra vn m.
Hnh 2. Cy lan Catleya c y m trc khi trng ra vn om.
Th nghim c tin hnh trn ging Dendrobium, Catleya v Phalaenopsis. S lng cy th nghim: 150/ging/nghim thc x ba ln lp li. Gi th l x da v dn en (l r ca cy dng x). Sau khi ly cy khi b nh cy m cy c ra sch v ngm 10 pht trong dung d ch Dithan M-45 5gr/lt. Cc ch tiu kho st gm: S cy cht tnh theo %. S l ca mt cy tnh theo trung bnh cng. T l s cy ra l mi tnh theo %. Chiu cao cy: o t c r l n ht thn + l cao nht ca cy. Chiu rng l: o chiu rng ca l ln nht, tnh trung bnh cng cc cy. S nhnh trn mt bi tnh theo trung bnh cng. S r hnh thnh mi trn mt cy tnh theo trung bnh cng. S cy cho r mi tnh theo %. Chiu di r tnh theo trung bnh cng. Th nghim c tin hnh vn m ti Th c Tp. HCM, thuc Ph ng Cng ngh T bo, Vin Sinh hc Nhit i, Vin Khoa hc v Cng ngh Vit Nam.
Hnh 3: Cy lan Phalaenopsis c y m trc khi trng ra vn m.
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KT QU V THO LUN Mt trong nhng yu cu u tin khi trng lan l cy phi thch nghi v sng c. Nh c ti luyn trc trong cc iu kin nh sng v nhit gn ging nh trong vn m nn cy cy m trong iu kin nh sng t nhi n t ra thch nghi tt. Cc kt qu trn cy Dendrobium c trnh by bng 1 di y:
Bng 1. So snh tng tr ng cy lan Dendrobium trng trong vn m cy ngun gc nh sng n ngy o s cy cht % s l (trung bnh) s cy ra l mi (%) cao cy (cm) s nhnh ca mt bi s r mi s cy ra r mi (%) Di r (cm) 0 0 3,4 0 5,2 2,0 0 0 8,2 10 4,7 3,4 2 5,2 2,0 1,8 14,8 8,3 30 10,9 3,7 15 6,6 2,8 3,9 42,7 11,4 60 14,4 4,5 52 10,1 3,4 5,4 87,4 16,7 90 14,8 5,3 100 12,9 3,6 12,6 100 19,8 cy ngun gc nh sng t nhi n 0 0 3,6 0 5,0 2,0 0 0 8,4 10 3,0 3,6 6 5,0 2,0 2,4 28,6 8,5 30 5,2 3,8 40 6,4 2,4 4,6 82,1 12,6 60 5,6 4,7 89 10,8 3,1 6,8 100 18,4 90 5,9 5,4 100 14,6 3,8 15,7 100 24,4
A
Hnh 4. Cy dendrobium ngu n gc nh sng t nhin 60 ngy tui
Hnh 5: A- cy gc nh sng t nhin B- cy gc nh sng n
Cy ging sn xut trong iu kin nh sng t nhin c sc sng tt hn, biu hin s cy cht gim, mau ra l mi, ln nhanh n n c chiu cao cy v s nhnh cng nh s r pht sinh mi tnh tr n mt bi cao hn i chng l cy ging bnh thng c sn xut trong phng my lnh v chiu sng bng n. Cc kt qu trn ging lan dendrobium khch l nhng nghin cu tip theo trn cy Catleya. Kt qu nghin cu c trnh by bng 2 sau y:
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Bng 2. So snh tng tr ng cy lan Catleya trng trong v n om

cy ngun gc nh sng n ngy o s cy cht % s cy ra l mi (%) Di l=cao cy cm s nhnh ca mt bi s r mi s cy ra r mi (%) Di r cm 0 0 0 6,5 2,0 0 0 5,2 10 12,2 11,3 6,7 2,0 1,6 5,8 7,4 30 14,6 44,6 7,1 2,2 2,8 28,9 8,7 60 17,5 78,5 8,1 2,8 3,9 76,8 10,5 90 19,4 100 10,8 3,3 5,8 100 13,4 cy ngun gc nh sng t nhi n 0 0 0 6,5 2,0 0 0 5,2 10 3,0 23,4 6,6 2,0 2,3 15,1 8,6 30 4,8 64,8 7,2 2,4 3,4 40,9 9,4 60 5,2 98,1 8,4 2,8 4,8 89,5 12,6 90 6,8 100 11,3 4,4 6,9 100 14,8
S khc bit biu hin ngay t 10 ngy u tin. Trong khi gn mt phn t cy lan Catleya con bung l mi cng thc ngun nh sng t nhi n th ch trn mi phn trm s lan con cng thc i chng c th bung l non. y l vic quan trng v Catleya vn l mt ging lan ln chm. S nhnh mi, chiu cao thn l cng nh s r pht sinh mi cng vt tri so vi i chng.
Hinh 6. Lan Catleya 90 ngay tu i gc nh sng n
Hinh 7. Lan Catleya 90 ngay tu i gc nh sng t nhin
Trn ging lan Phalaenopsis cng ghi nhn c cc kt qu tng t, xem bng 3 di y:
Bng 3. So snh tng tr ng cy lan Phalaenopsis trng trong v n om
cy ngun gc nh sng n ngy o s cy cht % s cy ra l mi (%) rng l cm Di l=cao cy cm s r mi s cy ra r mi (%) Di r cm 0 0 0 1,8 5,1 0 0 4,6 10 2,1 2,8 1,8 5,2 0,6 4,0 4,7 30 5,6 38,4 2,2 5,9 1,8 12,6 5,6 60 10,5 56,8 2,6 6,8 3,1 76,8 6,4 90 16,4 100 3,0 7,9 3,9 100 10,3 cy ngun gc nh sng t nhi n 0 0 0 1,8 5 0 0 4,5 10 2,0 3,0 1,9 5,2 1,1 11,2 4,8 30 3,4 4,1 2,4 6,2 2,4 54,6 6,2 60 5,8 76,2 2,9 7,6 3,8 98,8 8,6 90 6,9 100 3,5 8,4 4,5 100 11,4
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Cc cy ging khi c sn xut trong iu kin nh sng t nhi n c mt mu sc trung gian gia cy i chng c nui trong phng lnh di nh sng n v cy trng trong vn m. Cy nui trong phng lnh thng c mu xanh ti nhng cy nui trong nh sng t nhin thng vng hn v ln sc t . (V ngc Ph ng 2004, 2005). C th do nh sng t nhin mnh gip cy thch nghi mt phn nn khi em trng ra vn m t b cht v sm bt u tng trng v pht trin. KT LUN Cy lan Dendrobium, Catleya v Phalaenopsis nhn ging bng cy m trong nh sng t nhin khi trng ra mau thch nghi vi iu kin v n m hn i chng l cy lan nhn trong phng my lnh nh sng n. Biu hin trc tin nhn thy ngay l t l cht gim, cy mau ra r v bung l mi. Sau 90 ngy trng trong vn m cy c chiu cao, v s mm nhnh cng nh s r pht sinh mi tnh tr n mt bi cao hn i chng. Nh vy khi c nhn ging trong iu kin nh sng v nhit t nhin khng nhng ch l mt bin php gim gi th nh sn xut do khng cn s dng in chiu sng v in my lnh m y cn l mt bin php rn luyn cho cy ging cho thch nghi trc mt phn vi iu kin t nhin.
Hnh 8: Cy Phalaenopsis 60 ngy tu i gc nh sng n
Hnh 9: Cy Phalaenopsis 60 ngy tui gc nh sng t nhi n
TI LIU THAM KHO 1. Nguyn Th Qunh., Phng V. N. , N. . S, H. H c (2006). nh hng ca nng ng v iu kin nh sng ln s tng trng ca lan Dendrobium nui cy in vitro. Tp ch Khoa hc v Cng ngh 44 (3), 100-106. V Ngc Phng. , . T. A. Thuyn, L. V. Dng, T. X. Du & N. V. Uyn (2001). Quy trnh m cy hng (Paulownia fortunei) giai on sau ng nghim. Trong cun: Cng ngh Sinh hc v Nng nghip Sinh thi Bn vng. Vin Sinh hc Nhit i. NXB Nng nghip. 69 -75. V Ngc Phng (2004). S dng cc ngun carbon hydrat khc nhau trong mi trng nui cy Dendrobium iu kin nh sng t nhin. Bo co Nghim thu kt qu nghin cu ti nhnh nm 2004
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V Ngc Phng (2005). S dng nh sng t nhin trong nui cy m cy lan h ip. Bo co Nghim thu kt qu nghi n cu ti c s chn lc nm 2005.
SUMMARY
The impact of natural daylight conditions during micropropagation on growth of greenhouse orchid plantlets
Luu Viet Dung, Vu Ngoc Phuong, Thai Xuan Du Institute of Tropical Biology
Plantlets of Dendrobium, Phalaenopsis and Catleya multiplicated in vitro under naturally light conditions expressed better growth for first days in greenhouse comparing to normal fluorescent lamp illumination. The results suggest that the natural day light is not only a solution for saving the electricity for light and/or air conditio ning but also created a way somehow to harden orchids for better adaptable plantlets to greenhouse conditions.
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NHN in vitro GING TIU GC GHP (Piper columbrium Link.)

ng Gip, on Th i Thuyn. Thi Xun Du Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i M U H tiu (Piper) l mt chi ln, gm khong 1.200 lo i, phn b ch yu cc khu vc c kh hu nhit i in h nh. Trong vng ng Nam c khong 400 loi. nc ta, theo Phm Hong H (1991), chi H tiu c chng 40 loi. Do nghin cu khai thc, pht trin s dng hp l v ton din ngun gen a dng ca chi H ti u nc ta l vn ng c quan tm. Cy H tiu (Piper nigrum L.) rt d b bnh hi do nm gy ra, c bit l nm Phytophthora palmivorra. Ngoi ra cc loi tuyn trng Radopholus spp. cng l mi gy hi i vi cy H tiu nc ta. Do ng thi vi cc bin php ph ng tr thch hp th vic nghin cu, chn lc cc ging H ti u c tnh chng chu kho v cho nng sut cao l hng gii quyt rt tch cc hin ang c quan tm. mt vi nc, ngi ta cng bt u nghin cu vic ghp cc ging H ti u trng ln trn nhng gc ghp l cc loi cng chi c tc sinh trng cao, chng chu bnh kho (nh dng loi Piper columbrium Link.) (L nh Mi, 2002). Trong vi nm gn y nc ta, Vin Khoa hc K thut Nng nghip min Nam ang th nghim ghp ging h ti u trn loi tiu gc ghp (Piper columbrium Link.) v trng trn ng rung. Nhng s sch bnh ca gc ghp v kh nng gii hn trong s nhn nhanh gc ghp l mt vn t ra cn phi gii quyt. ng thi vi nhng thnh cng trong vic nghin cu qui trnh nhn in vitro ging h tiu sch virt (on Th Ai Thuyn v cs, 2003) a ti hng nghin cu vi ghp in vitro trn cy H tiu to ra nhng ging H tiu khng nhng sch virt m cn c kh nng khng bnh. NGUYN LIU V PHNG PHP Ging tiu gc ghp (P. colubrinum L.) do vin Khoa hc K thut min Nam cung cp. Tm hiu s tng thch gia cy H tiu (P. nigum L.) v tiu gc ghp (P. columbrinum Link.) bng phng php gii phu cu trc thn. Ging ti u gc ghp c trng trong bu t ti v n m vin Sinh hc Nhit i. Cy cao 30 -70cm, c t 10-15 t. Ct nhng on thn cha nhng t bnh t kh trng thu nhn mu nui cy in vitro. Nhng mu khng nhim c nui cy trn mi trng Murashige and Skoog, 1962 (MS) c b sung 6 - benzylaminopurine - BA (0,5mg/l) to chi. To cm chi t t trn mi trng MS c b sung BA (0 -5mg/l), indole-3-butyric acid IBA (0,5mg/l). Cc chi di 2-3 cm, c nui cy trn mi trng trn MS to cy
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con hon chnh. m cy nhn ging in vitro ngoi vn m. Cy tiu gc ghp c kim tra virt bng k thut ELISA, tr c, trong v sau khi nui cy in vitro i vi cc loi virt hi chnh trn cy H tiu l: ToMV (tobacco mosaic virus), PVX (potato virus X), PVY (potato virus Y) v CMV (cucumber mosaic virus). KT QU V THO LUN 1. Tm hiu s tng thch cu trc thn gia loi H tiu v tiu gc ghp Vi nm gn y, Vin Khoa hc v K thut Nng nghip min Nam thc hin ghp v trng th nghim ngoi ng rung gia cy H ti u v cy tiu gc ghp. Bng phng php gii phu cu trc thn, chng ti nhn thy c s t ng thch c bn gia cu trc thn ca cy H ti u v cy tiu gc ghp. T c nhng nh hng trong vic nhn ging in vitro cy ti u gc ghp lm ngun nguyn liu gc ghp sch bnh trong thc hin th nghim vi ghp in vitro cy ti u sau ny. 2. V trng v a mu tiu gc ghp vo nui cy in vitro Chn nhng cnh di khong 20-30cm, c t 5-7 t, ct ly nhng t bnh t. Sau ra sch bng s phng pha long v st trng b mt bng cn 70 0C trong 1 pht. Dng dung dch Javel thng mi pha long 1/3 kh trng mu trong khong 30-40 pht. Sau mi bc kh trng, mu c ra li 3 ln bng nc v trng. Tip theo lc mu trong dung dch khng sinh cefotaxin (0,5%) t 12 -24 gi trn my lc (60 vng/pht). Mu v trng c cy trn mi trng MS c b sung BA 0,5mg/l. Sau 21 ngy c t 10-15% cc mu v trng hnh thnh chi mi. Cc chi mi c cy truyn sang m trng MS to ngun gc ghp. 3. Th nghim to cm chi t ct t Cc chi cao 5-7cm, c 3 t c s dng to cm ch i t ct t. Ct mi t c mt l, di khong 1,5-2cm c cy vo mi trng MS c b sung BA (0 -5mg/l), IBA(0,5mg/l). Sau 1 tun nch l xut hin chi mi t gc v t gia. t ngn khng hnh thnh chi mi do u th ngn ca chi nh. Sau 4 tun nui cy, nhng nghim thc c BA cao (2-5mg/l) hnh thnh nhng chi bt nh phn gc ca t cy, ni mu cy tip xc vi mi tr ng. Nghin cu nh hng v tr t cy ln kh nng hnh thnh chi bt nh cho thy c s khc nhau c bn gia t ngn v nhng t cn li. Khng c khc bit r rt gia t gc v t gia. t ngn c th do u th chi ngn pht trin mnh rt sm khi mi bt u cy v o mi trng c ch kh nng hnh thnh chi bt nh, nn t l to chi bt nh thp ch khong t 2-3 chi/mu cy mi trng c BA cao (5mg/l). Trong khi , v tr t gc v t gia s hnh thnh chi bt nh vi t l rt cao sau 4 tun nui cy. Chi ng cng h nh thnh nhng khng pht tri n nhanh nh l t ngn nhng mi trng c BA cao (25mg/l). S hnh thnh chi bt nh cao nht (> 11 chi/ mu cy) t gc vi mi trng MS c b sung BA (5mg/l).
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Nh vy, nhn nhanh in vitro cy ti u gc ghp ta c th dng phng php to cm chi bt nh t ct t. t c ng gn gc chi cng nhiu trn mi trng MS c b sung BA (2-5mg/l). Cc cm chi bt nh sau 6 tun tui c cy truyn sang mi trng MS khng b sung cht iu ho sinh trng thc vt chi pht trin. C c chi mi c th cao t 1-1,5cm s c s dng to cy con in vitro hon chnh. 4. Nhn cy v a cy tiu g c ghp ra trng ngoi vn m Cc chi cao 1-1,5cm c nui cy trong mi trng MS khng b sung cht HSTTV, trc khi a ra vn m vi cc iu kin nui cy sau: S dng nh sng nhn to (ASNT) trong phng sng; S dng nh sng nhn to + nh sng t nhin (ASNT+TN); S dng nh sng t nhin (ASTN); Thi gian nui cy: 4 tun. Kt qu th nghim cho thy, sau 4 tun nui cy cc ch ti u thu c nhng bnh cy nui kt hp trong iu kin nh sng nhn to (2 tun) sau chuyn sang iu kin nh sng t nhin (2 tun) l tt nht. Tuy nhin s chnh lch cc ch tiu ba iu kin nui cy nh trn l khng ng k. Do chng ta c th nui cy trong giai on trc khi ra vn m bng nh sng t nhin tit kim c chi ph trong qu trnh sn xut. 5. a cy ra vn m Cc cy con sau 4 tun nui cy th nghim tr n c a ra trng ngoi vn m vi gi th l hn hp x da + tro tru vi t l 1:1. Sau 3 tun ngoi vn m cho thy t l sng ca cy nui cy m l 100%. Sau 3 thng trng ngoi vn m cy c th cao t 25 - 35cm. Tuy nhin nhng cy c nui cy in vitro trong iu kin c nh sng t nhin thch ng nhanh hn v pht trin tt hn so vi nhng cy nui trong iu kin nhn to. KT LUN Thnh cng trong nhn ging tiu lm gc ghp. S dng phng php ct t trn mi trng MS c b sung BA (2 -5mg/l) to chi bt nh cy ti u gc ghp. Kt hp nui cy m trong iu kin nh sng nhn to v nh sng t nhin giai on nhn cy trc khi ra vn m. TI LIU THAM KHO 1. Bysov A. S, (2001). Virus and viral diseases of black pepper ( Piper nigrum L.) in condition of closed ground and tropical, subtropical agrocenose s. The theasis for obtaining PhD degree. Kyiv National Univercity, Kyiv. Chacko, S. et al, (1996). Roasting studies on black pepper ( Piper nigrum L.). Flavour and Fragrance Journal 11, 305 -310. De Waard, P. W. F & Anunciado, I, S., (1999). Piper nigrum L. Plant Resources of South-East Asia. (13): 189-194.
2. 3.
429
4.
on Th Ai Thuyn, Thi Xun Du, ng Gip, Nguyn Tng Tn (2005). Bc u nghin cu nhn ging in vitro mt s ging H ti u (Piper nigrum L.) sch virt. Tp ch Sinh hc, 27(3): 39 -45. Joseph-B, Joseph-D. (1996). Plant regeneratin from somatic em bryos in black pepper. Plant Cell Tissue Organ Culture., 47(1): 87 -90. Kelkar SM, Krishnamurthy K. (1998). Adventitious shoot regeneration from root, internode, petiole and leaf expl ants of Piper colubrinum Link. Plant Cell Rep. 17(9): 721-725. Hong Quc Tun (2003). Pht trin sn xut ti u hu c - tiu sch. Hi tho H tiu Vit Nam trn ng hi nhp. Hip Hi H ti u Vit Nam. L nh Mi. (2002). Ti nguyn thc vt c tinh du. Nxb Nng nghip, pp158 -173.
5. 6.
7. 8.
SUMMARY
In vitro culture in Piper columbrium Link.

Do Dang Giap, oan Thi Ai Thuyen. Thai Xuan Du Institute of Tropical Biology
Black pepper (Piper nigrum L.) is a major spice used in food and medicine. Phytophthora foot rot is the major disease affecting pepper plant killing the entire vine . P. colubrinum Link, a related species is unaffected by this fungi. Grafting P.nigrum on P. colubrinum is one approach to safeguard the pepper from this disease. Single node P. colubrinum Link was used as material in culture on the basic medium MS (1962) supplemented with BA (0-5mg/l). Within 10 weeks of culture adventitious shoot formation was observed on the best medium MS + BA (5 mg/L). Shoots were rooted on 1/2 MS medium and in vitro culture plant conditions was described.
430
S DNG TINH BT TRONG NHN GING V TNH PHONG LAN BNG NUI CY M IU KIN NH SNG T NHIN
V Ngc Phng, Thi Xun Du Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U Hin nay chi ph in cho my lnh v n chiu sng chim mt t trng cao trong c cu gi thnh cy ging. V nh th khi bnh trong iu kin nhit v nh sng t nhin l mt gii php mang li hiu qu kinh t. Tuy nhin khi bnh nui cy m trong iu kin nh sng t nhi n tit kim in nng th mt vn ln t ra l cy rt d nhim. C mt cch chng nhim l loi b ng hon ton khi mi trng nui cy, nhng thiu ng th cy mc chm li nht l trong giao on u khi cy cn nh cha c kh nng quang hp vi hiu sut cao. Vic b sung tinh bt l mt gii php ang c la chn gip cy mt mt l ln nhanh gi c tc tng trng mt khc gim bt nhim trong iu kin nhit v nh sng t nhin. Bo co ny trnh by h thng cc gii php c th cng nh nhng hiu ng hu ch khi cy c ti luyn trong iu kin trung gian chuyn tip t cy m ra v n m. V kt qu t c l to c cy con khe mnh, gi th nh thp. Vic hon tt quy trnh cn cho php chuyn giao nhng tin b k thut nhn ging cy m phong lan quy m h gia nh nng thn ven th, gp phn a khoa hc k thut phc v sn xut nng nghip cng ngh cao. VT LIU V PHNG PHP Nguyn liu: ging Dendrobium Madam Cherry, Doritanopsis Sarah Jones v Catleya BLC Tainan City . Mi trng: nui cy Dendrobium c khong a lng nh sau (mg/l): KNO 3 1900, NH 4NO3 1050, KH 2PO4 1000, Ca(NO 3)2.2H2O 400, MgSO 4.7H2O 500. Mi trng nui cy Catleya v Doritannopsis c khong a lng Vacin & Went (1949) nh sau nh sau (mg/l): KNO 3 525, ()NH 4)2SO4 500, KH 2PO4 250, Ca3(PO4)2.2H2O 200, MgSO 4.7H2O 122. Vi lng v FeEDTA theo mi trng MS (Murashige &
431
Skoog 1962). Thiamin HCL 1mg/l, m -inositol 100mg/l. Nc da 10%. pH 5,8. ng hoc tinh bt 30gr/l chia u v o tng bnh. Agar 6gr/l.
Hnh 1. Nh plastic nui lan trong i u kin nh sng t nhi n
Th nghim thc hin cho giai on cy cy ln, cy ln cui c ng chun b a ra vn m. Catleyas v Doritanopsis c kch thc trung bnh 3cm. Dendrobium c kch thc trung bnh 5cm. Cy 6 cy mt bnh. Mi bnh cy m cha 65ml mi trng. Mi cng thc gm 30 b nh. Cng thc nui trong phng c chiu sng 2000 lux bng n hunh quang. Thi gian chiu sng 8 gi mi ng y. Nhit 28 oC+3. Cng thc s dng nh sng t nhin ngoi hnh lang hoc nh plastic. nh sng t nhin khc vi nh sng n ch cng chiu sng thay i lin tc trong ngy t 5007000lux. Nhit ngy m cng lin tc thay i v bin dao ng trong iu kin nh sng t nhin l 29oC+8 ln hn nhiu so vi trong phng chy my lnh. KT QU V THO LUN Trn i tng l cy Dendrobium kt qu o c v cm quan cho thy vic s dng carbohydrat dng tinh bt tt hn so vi dng ng trnh by trong bng 1 di y:
Bng 1. So snh tng tr ng cy lan Dendrobium trn mi tr ng ng v tinh bt.
nh sng n
ngy o mi trng ng 30gr/l mi trng tinh bt 30gr/l cao (cm) nng (gr) s r r (cm) cao (cm) nng (gr) s r r (cm) 10 5,8 0,64 6 0,8 5,7 0,57 5 1,2 20 7,1 0,89 8 3,0 6,9 0,86 7 3,2 30 8,0 1,03 11 5,0 7,9 1,14 10 6,2 45 8,8 1,28 11 5,9 9,8 1,48 10 7,2 10 6,2 0,64 5 1,0 6,0 0,60 5 1,5
nh sng t nhin
20 7,5 0,88 8 3,3 7,2 0,79 9 3,5 30 8,2 1,04 11 5,8 8,5 1,21 10 6,5 45 9,0 1,29 11 6,8 10,9 1,61 11 8,0
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Qua bng 1 cho thy trong chu k pht trin b nh thng 10 ngy u ca cy Dendrobium cy m khng ghi nhn s khc bit ng k v trng l ng gia cy trong phng chiu sng bng n v cy bn ngoi s dng nh sng t nhi n.
Hnh 2. Dendrobium cy m 45 ngy tui. (A) cy nh sng t nhin + tinh bt (B) cy nh sng t nhin + ng. (C) cy nh sng n + tinh bt (D) cy nh sng n + ng.
Khi cy n 45 ngy th ghi nhn c nhng khc bit r rt bn ngoi v mu sc. Cy c chiu sng t nhin xanh hn cy trong ph ng. L rng bn hn nhiu so vi cy nui di nh sng n trn cng mt cng thc mi trng nui cy. Khi tinh bt c dng thay cho ng s khc bit c ghi nhn gia cy trong phng dng nh sng n v cy nh sng t nhin. Bt u t ngy 30 tr i cy trn mi trng tinh bt bt u khc bit vi cy sng tr n ng. Nui cy cng ko di th s khc bit cng r rt. S lng cng nh chiu di r cng ghi nhn c s khc bit gia cc cng thc nhng khng r rt nh trng lng thn l. T l nhim gim r rt trn cc cng thc nui cy dng tinh bt lm ngun carbohydrat duy nht. Nh vy vic a cy cy m ra nui di nh sng t nhin khng nhng ch l gii php tit kim in m li tt cho cy. Trng lng sinh khi ca cng thc d ng tinh bt kt hp nh sng t nhi n gip tng c n 30% so vi cng thc cy nui trn ng di nh sng n. Trn i tng l cy lan h ip Doritanopsis cng ghi nhn mt kt qu tt t ng t khi s dng nh sng t nhin thay cho nh sng n tr nh by trong bng 2 di y:
Bng 2. So snh tng tr ng cy lan Doritanopsis tr n mi trng ng v tinh bt
nh sng n
ngy o cao cm mi trng nng gr ng s r 30gr/l r cm mi trng tinh bt 30gr/l cao mm nng gr s r r cm 15 3,1 1,80 3 1,5 3,22 1,82 3 2,1 30 4,3 3,00 3 1,7 5,31 2,50 4 2,3 45 5,7 3,20 3 2,0 6,12 2,96 4 3,2 60 6,1 3,40 4 2,2 7,92 3,26 5 4,5 15 3,3 1,84 3 2,1 3,34 1,91 3 2,1
nh sng t nhin
30 4,2 2,25 4 2,3 4,43 2,82 4 2,3 45 6,0 2,69 4 3,5 6,61 3,50 5 3,5 60 7,9 3,32 4 4,3 8,60 4,12 5 4,0
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Mt c im quan trng c ghi nhn l cy lan h ip ln chm. c th xut cy ra trng cn nui trong b nh n 60 ngy trong khi Dendrobium ch cn 30 ngy l . Khi ko di thi gian nui cy thng lm mi trng b kh. iu kin nh sng t nhin vi bin dao ng nhit ln trao i kh din ra mnh h n lm cy ln nhanh hn nhng cng lm mt nc v mi trng mau kh hn. Vic b sung mi trng lng c cng thnh phn gip gi mi trng khng b cn kit v cy pht trin tt. ng thi khi nui trong ph ng th vic b sung mi trng khng c tc dng r rt nh khi nui iu kin nh sng t nhi n. (V ngc Phng, 2005).
Hnh 3. Doritanopsis c y m 60 ngy tui (A) cy nh sng t nhi n + tinh bt (B) cy nh sng n + ng
Vic s dng mi trng c cha tinh bt l ngun carbohydrat kt hp chiu sng bng nh sng t nhin gip cy vt 50% so vi i chng nu xt ri ng b l. Doritanopsis ln nhanh vi chiu cao cng c r to gn gp i so vi i chng nui trn mi trng ng di nh sng n. Th nghim trn cy lan Catleya cng cho mt kt qu tng t c trnh by trong bng 3 sau y:
Bng 3. So snh tng tr ng cy lan Catleya trn mi trng ng v tinh bt

nh sng n ngy o mi trng ng 30gr/l mi trng tinh bt 30gr/l cao cm nng gr s r r cm cao cm nng gr s r r cm 15 3,3 0,64 5 0,8 3,1 0,57 5 1,3 30 4,1 0,89 8 3,0 4,8 0,86 7 3,4 45 5,0 1,03 10 5,0 6,0 1,14 10 6,5 60 6,2 1,28 11 5,9 7,2 1,48 12 7,2 15 3,2 0,64 5 1,0 3,0 0,60 5 1,5 nh sng t nhin 30 4,5 0,88 9 3,4 5,2 0,79 9 3,5 45 5,6 1,04 12 5,9 7,1 1,21 10 6,8 60 7,0 1,29 12 6,8 8,2 1,61 11 7,1
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Nhn chung Catleya c tc tng trng chm hn Dendrobium. Khi nui trong iu kin nh ng t nhin bng mi trng tinh bt s gia tng v chiu cao c khong 32% v trng lng khong 26% so vi i chng l mi trng ng v chiu nh sng n. Sc t ta c trng khi c nh sng mnh xut hin trn cy cy m trong iu kin nh sng t nhin (xem hnh 4). Hin tng ny ghi nhn c tt c cc ging lan nhng c bit r trn Catleya.
Hnh 4: Catleya 60 ngy tu i nui nh sng t nhin trn mi trng tinh bt.
Hnh 5: Catleya 60 ngy tu i nui nh sng n trn mi tr ng ng.
KT LUN C th s dng nh sng t nhi n nh mt gii php tit kim in my lnh v n chiu sng nui cy phong lan Dendrobium, Doritanopsis v Catleya. Dendrobium c tc ln nhanh hn Doritanopsis v Catleya. Khi s dng tinh bt thay cho ng t l nhim gim. Lng agar dng lm ng mi trng gim i. Trong iu kin nh sng t nhin chiu mnh cy c sc t tm . Trn i tng l cy Dendrobium, Doritanopsis v Catleya kt qu o c cho thy mi tr ng nh sng t nhin vic s dng carbohydrat dng tinh bt tt h n so vi dng ng. K t ngy 30 tr i bt u ghi nhn c s khc bit vi cy sng tr n ng. Trong vng 60 ngy, thi gian nui cy cng ko di v sau th s vt tri cng cng r rt. TI LIU THAM KHO 1. 2. Murashige T., & F. Skoog, 1962. A revised medium for rapide growth and bio assays with tobacco tissue cultures. Physiol Plant 15, 473 -497. Vacin E. F. and Went E. W. 1949. Bot.Gaz 110, 605. Trong cu n: DUCHEFA Biochemicals Plant Cell and Tissue Culture. Catalogue 2003 -2005. 78.
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SUMMARY
The use of starch for micropropagation of orchids under natural daylight conditions
Vu Ngoc Phuong, Thai Xuan Du Institute of Tropical Biology
Plantlets cultured under naturally light conditions expressed better growth comparing to normal fluorescent lamp illumination. The results suggest that the natural day light is not only a solution for saving the electricity for light and/or air con ditioning but also created a better biomas growth. The use of starch as lonely source of carbohydrate help the growth plantlet at the late period of cultivation. The same results obtained for Dendrobium, Doritanopsis and Catleya multiplicated in vitro.
436
NHN GING V TNH CY vani BNG NUI CY M
V Ngc Phng, L Hon Ho, Thi Xun Du Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U Vani c ngun gc t vng Mexic, vng Caribbean thu c chu M nhit i. Sn xut vani nm 2001 c khong gn 5000 tn tr n din tch 41.000 ha. Gi cy vani hin nay rt cao bng khong h n 12-15 ln gi c ph. Vit Nam nm trong vng kh hu thch hp cho vic trng cy vani. B n cnh nhng cy c gi tr kinh t khc c a vo nui cy m (V. N. Phng & CTG 2000, 2002, . T. A Thuyn v CTG 2001) vic a cy vani vo sn xut l mt bc a dng ha sn phm nng nghip phc v xut khu. khi u pht trin, nui cy m l mt cng vic hu hiu nhn nhanh trong mt thi gian ngn cc ging u d ng sch bnh. Bi vit trnh by cc kt qu nghin cu t khi u a mu cy vo nui cy, xc nh mi trng v sau l trng trong vn m thnh cy ging hon chnh. VT LIU V PHNG PHP Th nghim nhn ging c tin hnh trn ging Vanilla tahitiensis v Vanilla Indonesia. Th nghim trng c thc hin trn ging Vanilla tahitiensis. Mu c ly t chi bn cy trng thnh. Kh trng bng hypoclorit canxi 3% trong 15 pht. Sau ra sch ba ln bng nc v trng. Mi trng nui cy c khong a l ng Vacin & Went 1949 nh sau (mg/l): KNO3 525, (NH4)2SO4 500, KH 2PO4 250, Ca3(PO4)2. 2H2O 200, MgSO 4. 7H2O 122. Vi lng v Fe-EDTA theo mi trng MS (Murashige & Skoog 1962). Thiamin HCL 1mg/l, m-inositol 100mg/l. Nc da 10%. pH 5,8. ng 30gr/l. Agar 7gr/l. Mi trng to chi c b xung cht sinh tr ng v ph gia hu c l: 6-Benzyn Adenin (BA) 5mg/l. Adenin 15mg/l. Tyrosin 20mg/l. Mi tr ng to cy v ra r c b xung cht sinh trng l: -Naphthalene Acetic Acid (NAA) 0,5mg/l. Bnh cy chi c chiu sng 2000 lux bng n hunh quang. Thi gian chiu sng 8 gi mi ngy. Nhit 28 oC+3. Bnh cy cy s dng nh sng t nhi n ngoi hnh lang. Cng chiu sng thay i li n tc trong ngy t 500-7000lux. Nhit ngy m dao ng trong khong 29 oC+8.
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Cy nui cy 60 ngy t chiu di trn 5cm c m trn dn cng trong 15 ngy. Dn cng l mt loi gi th trng lan c gi n c t, gip cho b r kh ro ph hp vi vic m vani trong 15 ngy u. Trong nhng ngy ny khng s dng phn bn. Khi u r vani bt u nh ln chng t s hi phc cy vani c chuyn sang bu c gi th gm: phn b 60%, tro tru 20%, t 20%. Phn bn trong giai on n y l NPK 30-10-10 v vi lng HP-306. KT QU V THO LUN Khi u t chi nch cy m gim c nh cc t thn c nui trn mi trng to chi. Sau 1 thng nui cy t nch l ny l n chi. Chi c cy chuyn sang mi trng mi li tip tc cho chi kho mnh xem h nh 1: Khi mun c cy chi c tch v cy ln mi trng to cy. Cy hon chnh c th xut ra vn m c trnh by cc hnh 2, hnh 3 v hnh 4. m cy, cy m l mt cng vic i hi k nng thun thc (V.N. Ph ng v CTG 2001). Cy vani r t d cht ng khi trng ra vn m. Cc kt qu th nghim cho thy khi chn dn bng loi c kh nng gi nc cao vn trng lan h ip th t l cy cht rt cao. Khi cy c m trn mt gi th kh ro t gi n c th t l sng c th t gn 100% (L Hon Ho 2005). Khi cy sng v bt u bung r mi cy c chuyn sang bu gm phn b , tro tru v t. Cc kt qu c trnh by hnh 5 sau y. Theo quy trnh trnh by k trn sau 12 thng k t khi bt u tin h nh nui cy sn xut c trn 10 nghn bu cy vani ging cy m. To n b s cy ging k trn do mt thng nhn ngi Php nhp cnh t nc ngoi v v bao tiu ton b s cy ging sn xut ra em trng ti nng tri tnh B nh Thun.
Vanilla tahitiensis
Hnh 1: Chi Vani cy m
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KT LUN Sau mt qu trnh nghin cu xc nh c mi trng ph hp vi nui cy cy vani nh sau: Mi tr ng nui cy c khong a l ng nh sau (mg/l): KNO 3 525, (NH4)2SO4 500, KH 2PO4 250, Ca3(PO4)2.2H2O 200, MgSO 4.7H2O 122. Vi lng v FeEDTA theo mi trng MS (Murashige & Skoog 1962). Vitamin theo Morel (1949). Nc da 10%. pH 5,8. ng 30gr/l. Agar 7gr/l. Mi tr ng to chi c b sung cht sinh trng v ph gia hu c l: 6-Benzyn Adenin (BA) 5mg/l. Adenin 15mg/l. Tyrosin 20mg/l. Mi tr ng to cy v ra r c b sung cht sinh trng l: Naphthalene Acetic Acid (NAA) 0,5mg/l. y m t quy trnh hon chnh cho php nhn ging cy vani bng nui cy m vi quy m sn xut. TI LIU THAM KHO 1. Ho L.H. (2005). nghin cu kh nng thch nghi ca cy Vanilla tahitien sis in vitro trong giai on vn m. Lun vn tt nghip c nhn. Khoa Cng ngh Sinh hc. i hc m - bn cng Tp. HCM. Murashige T., & F. Skoog, (1962). A revised medium for rapide growth and bio assays with tobacco tissue cultures. Physiol Plant 15, 473-497. Phng V. N. , P. . Tr, T. X. Du & N. V. Uyn (2000). Nhn ging v tnh cy xoan n (Azadirachta indica A. Juss.) bng nui cy m. Tp ch Sinh hc 22 (2), 34 -39. Phng V. N. , P. . Tr, . T. A. Thuyn, T. V. Nga, T. X. Du & N. V. Uyn (2002). Nhn ging in vitro cy tre tu (Sinocalamus latiflorus) v tre mnh tng (Dendrocalamus asper) . Tp ch Sinh hc 24 (2), 59 -64. Phng V. N. , . T. A. Thuyn, L. V. Dng, T. X. Du & N. V. Uyn (2001). Quy trnh m cy hng (Paulownia fortune i) giai on sau ng nghim. Trong cun: Cng ngh Sinh hc v Nng nghip Sinh thi bn vng. Vin Sinh hc Nhit i. NXB Nng nghip. 69-75. Thuyn . T. A., V. N. Phng, T. X. Du & N. V. Uyn (2001). Nhn ging v tnh cy hng - Paulownia fortunei (Seem.) Hemsl. bng phng php nui cy m. Tp ch Sinh hc 23 (3), 46 -50. Vacin E. F. and Went E. W. (1949). Bot. Gaz 110, 605. Trong cun: DUCHEFA Biochemicals Plant Cell and Tissue Culture. Catalogue 2003 -2005. 78.
2. 3. 4.
5.
6.
7.
SUMMARY
Micropropagation of Vanilla.
Vu Ngoc Phuong, Le Hoan Hao, Thai Xuan Du Institute of Tropical Biology The authors have establishe a complete in vitro technology to multiply Vanilla tahitiensis and Vanilla Indonesia for mass seed plantlets production. Culture medium contains macro elements (mg.l-1) KNO3 525, (NH4)2SO4 500, KH2PO4 250, Ca3(PO4)2.2H2O 200,
439
MgSO4.7H2O 122. Micro-elements v Fe-EDTA followed MS medium (Murashige & Skoog 1962). Thiamine HCl 1 mg.l -1, m-inositol 100mg.l-1. Coconut milk 10%v/v. pH 5,8. Sucrose 30gr per liter. Agar 7gr per liter. Medium for creation of multiple shoots contains: 6 -Benzyn Adenin (BA) 5mg.l -1. Adenine 15mg.l-1. Tyrosine 20mg.l-1. Medium for root initiation suplemented with -Naphthalne Acetic Acid (NAA) 0.5 mg.l -1.
Hnh 2: Cy vani cy m 60 ngy tui
Hnh 3: Nhn ging i tr cy Vanilla tahitiensis
Hnh 5: Cy
Vanilla tahitiensis hai thng tui trong vn m
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NHIN CU S KH TRNG, TO CHI V TO R in vitro TRN GING IU CAO SN (Anacardium occidentale L.)
Nguyn nh S, Hunh Hu c, Nguyn Th Qunh Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U
Cy iu, Anacardium occidentale L., thuc h Anacardiaceae, l cy cng nghip c tm quan trng v kinh t mt s n c vng nhit i thuc Chu , Chu Phi v Chu M La tinh (Philip, 1984). Vit Nam hin nay l nc sn xut ht iu ln nht ng Nam , ng th ba th gii sau n v Brazil. Tuy nhin, t c din tch trng iu n nm 2010 l 500.000 hecta th phi cn thm mt lng ngun ging khong 25.000.000 cy (VINACAS, 2004). Nhn ging bng ht v ghp cnh khng p ng c yu cu trn do cy sinh trng khng u v t l thnh cng cha cao (50% - 60%). V vy, vic nghin cu phng php nhn ging cy iu bng nui c y m l ht sc cn thit p ng nhu cu sn xut (V Ngc Ph ng v cs, 2003).
Do vic kh trng mu t cy trng thnh to ngun mu ban u c n nhiu kh khn nn cy mm pht trin t ht ca mt s ging iu cao sn c s dng lm ngun vt liu nghin cu phng php ti u trong nhn ging in vitro cy iu. Trong bi ny chng ti trnh by ph ng php kh trng ht, phng php to chi t t thn mm v phng php to r t chi in vitro cy iu. VT LIU V PHNG PHP
1. nh hng ca nng NaOCl khi kh tr ng v la ln t l nhim v ny mm ca ht iu trng thnh
Ht iu trng thnh ca 2 ging cao sn PN1 v BO1 c chn lc t vn u dng ca Trung tm ging Hng Lc, ng Nai, thuc Vin Khoa hc K thut Nn g nghip min Nam. V la c kh trng bng NaOCl (c s nc javel Vn Phng) vi cc nng theo bng 1.
Bng 1. M t th nghim
Nghim thc J1 J2 J3 Nng % (W/V) 0,6 1,0 1,4 Thi gian (pht) 20 20 20
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Sau khi c kh trng, ht c cy trn mi trng khong MS (Murasighe v Skoog, 1962) c b sung agar 9g/L (Cng ty C phn hp H Long, Qung Ninh), than hot tnh 3g/L. Mi nghim thc gm 10 b nh, mi bnh cy 2 nhn ht iu. Mi nghim thc c lp li 3 ln. Cc bnh cha nhn ht iu c t trong iu kin cng nh sng 30mmol m -2 s-1, thi gian chiu sng 8 gi/ngy, nhit 25 2 oC, m tng i 60 5%. Thi gian th nghim l 14 ngy.
2. nh hng ca nng BA v KN ln s to chi ca t thn mm cy iu
t thn mm (t th hai tnh t ngn) pht trin t ht trong iu kin v tr ng c dng lm mu cy. Mi trng nui cy l mi trng khong MS c a lng 1/2 v b sung KH 2PO4 200mg/L, KNO 3 200mg/L, adenine sulphate 40mg/L, than hot tnh 3g/L saccharose 20g/L, maltose 10g/L, agar 8 g/L, 1-naphthaleneacetic acid (NAA) 0,5mg/L. Benzyladenine (BA) v Kinetin (KN) c b sung cc nng khc nhau (Bng 2). Mi nghim thc gm 4 bnh, mi bnh c 5 mu v c lp li 3 ln. Cy c nui trong iu kin cng nh sng 45-50mmol m-2 s-1 , thi gian chiu sng 16 gi/ngy, nhit 25 2oC, m tng i 60 5%. Thi gian th nghim l 30 ngy.
Bng 2. M t th nghim
Nghim thc A1 A2 A3 A4 BA (mg/L) 1 5 1 5 KN (mg/L) 1 1 5 5
3. nh hng ca IBA v NAA ln s to r bt nh ca chi iu in vitro
Chi in vitro pht trin t t thn, chiu cao 2,5 -3,0cm, mang 4 l c dng lm mu th nghim. Gc chi c ngm 1 gi trong dung dch NAA hoc indol -3-butyric acid (IBA) vi nng 100mg/L trong t cy v trng. Sau khi x l auxin, chi c cy trn mi trng khong MS c a lng 1/2 c b sung KH 2PO4 200mg/L, KNO3 200mg/L, saccharose 10mg/L, agar 8g/L, than ho t tnh 1g/L Mi nghim thc gm 10 bnh, mi bnh c 2 mu cy v c lp li 3 ln. Cy c nui trong iu kin cng nh sng 45-50mmol m -2 s-1 , thi gian chiu sng 16 gi/ngy, nhit 25 2oC, m tng i 60 5%. Thi gian th nghim l 30 ngy. Phn mm MSTATC (i hc Michigan, M) c s dng x l cc s li u thu c t cc th nghim trn. KT QU V THO LUN
1. nh hng ca NaOCl khi kh tr ng v la ln t l nhim v ny mm ca ht iu trng thnh
Kt qu thu c khi tin hnh kh trng v la cho thy nng NaOCl 1% , nhn ht iu c t l nhim thp (2,56%), trong khi t l ny mm, % r c chiu d i
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3cm, % chi c chiu cao 3 cm cao hn so vi hai nng 0,6% v 1,4%. Khi phn tch thng k, s khc bit v t l nhim v t l ny mm gia cc nghim thc khng c ngha, trong khi s khc bit v % r c chiu di 3cm mc p 0,05 v % chi c chiu cao 3cm mc p 0,01. Khi ht c tin hnh kh trng nng NaOCl 1%, s tng trng ca cy mm vi % chi c chiu cao 3 cm l cao nht (Bng 3). T kt qu thu c, nng NaOCl 1% vi thi gian kh tr ng 20 pht c chn khi tin hnh kh trng v la to ngun mu v tr ng cho cc th nghim khc sau ny.
Bng 3. nh hng ca NaOCl ln t l nhim, t l ny mm, chiu d i r ca ht iu trng thnh ngy th 7 v chiu cao chi ngy th 14
7 ngy NT T l nhim (%) 12,83 2,56 5,13 NS T l ny mm (%) 56,67 76,67 65,00 NS % r c chiu di 3 cm 53,33b y 76,67a 71,67a * 14 ngy % chi c chiu cao 3 cm 43,33c 73,33a 65,00b **
J1x J2 J3 ANOVAz
z y
NS, *, ** : khng khc bit hoc khc bit c ngha mc p 0,05 v 0,01 Cc tr s c ch ci ging nhau tr n cng mt ct khng khc bit theo trc nghim phn hng LSD test x K hiu nghim thc xem Bng 2 2. nh hng ca nng BA v KN ln s to chi v tng trng chi t t thn mm cy iu
ngy th 30, trn mi trng A4 (BA 5mg/L + KN 5 mg/L) , s chi xut hin l ln nht (1,78 chi/mu), iu n y cho thy khi nui cy to chi cy iu, cn b sung mt lng kh ln cytokinin trong m i trng nui cy. Tuy nhi n s chi to c cng nhiu th s tng trng chiu cao ca chi c ng gim (Hnh 1).
2 1.5 1 S ch 0.5 i/m u A1 S chi
0 A2 A3 A4 Nghi m thc S chi c 4 l S chi c chiu cao 3 cm
Hnh 1. nh hng ca BA v KN ln s to chi v tng trng chi ca t thn mm ngy th 30. K hiu nghim thc xem Bng 2
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S gia tng chiu cao chi ln nht khi mu c nui cy trn mi trng c BA 1mg/L + KN 5mg/L (trung bnh c 0,98 chi/mu c chiu cao 3 cm) (Hnh 2).
Hnh 2. S to chi trn t thn mm. K hiu nghim thc xem Bng 2
Kt qu ny khng ng nht vi kt qu ca Boggetti v cs (1999) thc hin trn mu cy t cy iu nui tr ong nh knh l BA v KN cao (5 mg/L) khng thch hp cho s to chi. Mi trng A3 (BA 1mg/L + KN 5mg/L) hoc A4 (BA 5mg/L + KN 5mg/L) c chn lm mi trng nui cy t thn mm tu thuc vo cc th nghim tip theo c tin hnh.
3. nh hng ca IBA v NAA ln s to r bt nh trn chi iu in vitro
Theo Das v cc cs (1996) khng c s khc bit v s to r khi nui cy chi iu in vitro trn mi trng c b sung IBA hoc NAA. Trong th nghim n y, IBA c nh hng ln s to r tt hn NAA khi s dng cng nng 100mg/L (Bng 4). S r xut hin trn mt chi ln hn gn 2 ln, do cy tng tr ng tt hn, dn n s gia tng trng lng ti cng ln hn (Hnh 3).
Bng 4. nh hng ca IBA v NAA ln t l sng, t l ra r, s r, chiu d i r v gia tng trng lng ti ca chi iu in vitro ng y th 30
Auxin (100 mg/l) IBA NAA ANOVAz
z
T l mu sng (%) 52,5 52,5 NS
T l mu ra r (%) 55 70 NS
S r/chi
Chiu di r (cm) 6,6 8,4 NS
Gia tng trng lng ti (mg) 360 230 **
2,1 1,2 *
NS, *, ** : khng khc bi t hoc khc bit c ngha mc p 0,05 v 0,01
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Hnh 3. S hnh thnh r sau khi x l (a) NAA v (b) IBA
Trong th nghim ny, sau 30 ngy nui cy, c 47,5 % s chi cht sau khi l chuyn vng v rng. S vng l c th lin quan n s hin din ca ethylen nng cao trong bnh nui cy. Bi Trang Vit (2000) cho rng s hin din ca auxin kch thch s sn xut ethylen bng cch kch thch s th nh lp 1-amino-cyclopropane-1carboxylic acid (ACC). Ngoi ra cy iu in vitro c b r gin v d gy, dn n t l sng rt thp ca cy iu in vitro khi chuyn ra v n m. KT LUN T nhng kt qu thu c, chng ti rt ra nhng kt lun nh sau: Nng NaOCl 1% (W/V), thi gian 20 pht thch hp nht cho s kh tr ng v la ht iu. Mi trng nui cy c b sung BA 5mg/L + KN 5mg/L cho s to chi tt nht vi mu cy l t thn mm. Chi cy iu in vitro to r khi c x l vi IBA 100mg/L tt hn so vi NAA cng nng . TI LIU THAM KHO 1. Boggetti B., Jasik J., Mantell S. (1999). In vitro multiplication of cashew (Anacardium occidentale L.) using shoot node explants of glasshouse -raised plants. Plant Cell Rep. (18): 456-461. Bi Trang Vit. (2000). Sinh l thc vt i c ng, phn II: Pht trin. Tr 81-108. Nxb i hc quc gia TPHCM. Das S., Jha TB., Jha S. (1996). In vitro propagation of cashewnut. Plant Cell Rep. (15): 615-619. Murashige T., Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, 473 -479.
2. 3. 4.
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5. 6.
Philip VJ. (1984). In vitro organogenesis and plantlet formation in cashew (Anacardium occidentale L.). Ann. Bot. (54): 149-152. V Ngc Phng, Nguyn Th Qunh, Nguyn Minh Tun, Thi Xun Du. (2003). Bc u nghin cu nhn ging in viro cy iu ( Anacardium occidentale L.). Tuyn tp ca Hi ngh Cng ngh Sinh hc ton quc ln th 2, t chc ti H Ni. Nxb KHKT, pp945-952.
SUMMARY
Study on the sterilization, shoot multiplication and root formation of cashewnut (Anacardium occidentale L.) elite cultivars
Nguyen inh Sy, Huynh Huu uc, Nguyen Thi Quynh Institute of Tropical Biology
Cashew (Anacardium occidentale L.) is an important, economical crop in Vietnam and many tropical areas. Seed and grafting propagation can not meet the demand due to the ununiformity and low successful rate of grafting (50 -60%). Therefore, a rapid method for producing in vitro transplants is n ecessary. Mature seeds of cashews were collected from elite cultivars growing in the Hung Loc Center, Thong Nhat Dist., Dong Nai Province. The seeds were effectively surface sterilized with NaOCl 1 %(W/V) for the soft inner seed coat for 20 minutes. Shoots developed faster on the modified MS medium supplemented with BA and KN from 1 to 5 mg/L after 30 days of culture. Root induction was better when in vitro shoot s were pretreated with IBA (100 mg/L) for 1 h compared with that in the same concentration of NAA.
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NHN GING in vitro MT S GING H TI U (Piper nigrum L.) SCH virus

on Th i Thuyn, ng Gip, Thi Xun Du Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U Trong nhng nm gn y vic tng din tch trng ti u t dn ti tnh trng cy h tiu b bnh, trong c bnh do virut gy ra. Kt qu iu tra t nh hnh bnh virut h tiu mt s tnh min ng Nam B cho thy c 42 - 93% vn h tiu c triu chng nhim virut biu hin tr n l v cy nh m hoa l, nhn phin l, xon mp l, tp ngn, cy ln d dng, [3,6]. Virut c pht hin trong mu l h ti u l cc virut ToMV (tobacco mosaic virus), PVX (potato virus X), PVY (potato virus Y) v CMV (cucumber mosaic virus) [2,3]. Ni dung chnh ca bi bo ny l nghin cu kh nng loi b mm bnh ni sinh trong mu cy h ti u bi s dng mt s cht khng sinh; nh hng ca cc cht iu ha sinh trng thc vt n s hnh thnh chi, ti sinh chi, cy t m so cc t cy cng nh s dng k thut ELISA x c nh ging sch virut trong nhn ging in vitro mt s ging h tiu (Piper nigrum L.) c trng cc tnh min Nam Vit Nam. VT LIU V PHNG PHP Cc ging h tiu c nghin cu l cc ging c trng ph bin ti cc tnh min ng Nam B do Vin Khoa hc K thut Nng nghip min Nam cung cp gm cc ging: Vinh Linh (Vit Nam); Lada Belangtoeng (Indn xia); Karimunda (n ). Cy h tiu c trng trong bu t ti v n m ca Vin Sinh hc Nhit i. Sau 2 3 tun, cy ra chi non. Ct cc chi c chiu di t 2-3 cm v cc cnh c t 5-6 t. Cc chi c ra sch v st trng bng cn 70 0. Mu cy c kh trng b mt bng dung dch ty javel khong 30-40 pht hoc 0,1% HgCl 2-10 pht hoc kt hp vi s dng cht khng sinh 0,5% x fotaxin t 1 - 24 gi. Cc chi non v trng c nui cy trn mi trng Murashige and Skoog, 1962 (MS) c b sung 6 - benzylaminopurine - BA (0-5,0mg/l), indole-3-butyric axit - IBA (0 - 1,0mg/l) hoc thidiazuron - TDZ (0 - 0,22mg/l) to chi. nghin cu s ti sinh chi t m so chng ti b sung BA (0 - 10mg/l), IBA (0,5 mg/l) v kinetin (0,5mg/l) vo mi trng nui cy. i vi mi tr ng to r, chng ti s dng cc
447
chi ln di 3 - 4 cm c t 2 - 3 cp l cy trn mi trng MS c khong a lng gim mt na v b sung 1-naptthalenaceacetic acid - NAA (0,2 - 0,5mg/l) hoc than hot tnh. Tt c cc mu cy c t trong iu kin ph ng nui cy nhit 27 0C 290C, cng chiu sng 2000 lux, thi gian chiu sng: 12 - 14 gi/ngy. kim tra mu sch virut, cc ging h ti u c gim nh bng k thut ELISA tr c v sau khi nui cy in vitro i vi cc loi virus ToMV, PVX, PVY, CMV. KT QU V THO LUN
1. S dng cht khng sinh trong qu tr nh kh trng mu cy
H tiu l mt cy lu nm, c nhiu mm bnh ni sinh nh nm, khun nm bn trong m. Bi vy mt trong nhng vn quan trng u ti n cn gii quyt trong nui cy in vitro h tiu l phi loi b hon ton mm bnh ni sinh . Cc phng php kh trng b mt thng thng u khng thnh cng trong vic kh trng cy h tiu. Nguyn liu ban u l cc chi mi hnh thnh ca cy ging c trng cch ly trong nh li v phun thuc tr nm bnh, qua kim tra virut. Qua thc nghim cho thy, cc mu qu a x l ngay c khi s dng cc cht c kh nng dit mm bnh b mt cao nh 0,1 -1% HgCl2 (t 5 - 10 pht), mu cy vn b nhim tr li sau mt thi gian nui cy. Do nu ch s dng cc cht kh trng b mt nh dng nc ty javel hoc HgCl 2 u khng cho kt qu kh quan; ton b mu s b nhim sau mt thi gian d i nui cy. mu c v trng hon ton, chng ti kt hp s dng mt s cht khng sinh nh: penixillin, streptomyxin v xfotaxin. Trong , xfotaxin v a c tc dng dit khun, nm bn ngoi v va dit c cc mm bnh ni sinh trong mu cy. Cc bc kh trng mu cy ln lt c thc hin nh sau: mu sau khi st trng bng cn 70%, c x l trong nc ty javel (10%) - 15 pht. Sau ngm dung d ch 0,1% HgCl 2 - 10 pht. Bc k tip l lc trong dung dch 0,5% x fotaxin trn my lc t 1 - 24 gi. Sau mi bc mu u c ra 3 ln bng nc ct v trng. Tuy nhin, cc mu qua kh trng sau 20 - 45 ngy vn c th b ti nhim tr li ti nhng v tr tip xc vi mi trng nui cy do mm bnh ni sinh c n trong mu. lm gim s ti nhim khun mu cy, chng ti b sung 0,5% xfotaxin trong mi trng nui cy ban u. Sau , cy truyn mu qua mi tr ng khng c cht khng sinh to chi mi. Nh vy, mu l nhng chi non pht sinh t mu v trng v khng c kh nng ti nhim tr li. Vi ph ng php kh trng mu trn, chng ti c th nhn c 25 - 30% s mu sch hon ton mm bnh ni sinh.
2. nh hng ca v tr t cy v cc cht iu ho sinh trng thc vt (HSTTV) l n kh nng hnh thnh chi h tiu
Sau khi c v trng 20 ngy, cc t H tiu bt u m chi mi. Chi di 5-6 cm c ct thnh cc t di 2cm. Mi t mang mt cp l c cy trn mi trng MS c b xung cc cht HSTTV khc nhau. Sau 3 - 4 tun nui cy, t nch l xut hin chi mi. Qua theo di nh hng ca v tr t cy ln kh nng hnh thnh chi, chng ti nhn thy c s khc bit r rt v kh nng ny cc loi t. t ngn
448
hu nh khng xut hin chi mi, ngay c nng BA cao (5 mg/l). t gia v t gc d dng to chi mi hn, s chi mi hnh thnh c th ti 4 - 6 chi/t. Trong s to chi mi t gc l nhiu hn so vi t ngn v t gia. Khi nghin cu nh hng ca t hp cc cht HSTTV, chng ti nhn thy, cc mi trng ch b sung BA, kh nng to chi thp tt c cc t cy. Th m ch vi nng BA cao (> 5mg/l) hoc TDZ nng 0,02 - 0,22mg/l chi hnh thnh cng t hn so vi cng thc c b sung NAA hoc IBA. Khng phi nng xytokinin v auxin cao quyt nh s to chi mu cy m l t l thch hp gia xytokinin/auxin s l m tng kh nng to chi h tiu. Trong cc th nghim, chng ti nhn thy nng BA = 2mg/l, IBA = 0,5mg/l s chi c hnh thnh t gia v t gc cao nht. iu ny chng t, khi b sung mt lng xytokinin v auxin thch hp, s lm tng r rt s chi hnh thnh t cy. Khi b sung thm NAA hoc IBA trong mi trng to chi vi nng 0,5 - 1,0mg/l lm tng kh nng hnh thnh m so cc gc cy. nng auxin (NAA, IBA) cao ( 1,0mg/l), m so cng nhiu v c ch s hnh thnh chi ca mu cy. 3. nh hng ca cc cht HSTTV l n kh nng bit ho chi t m so Cc m so hnh thnh t nui cy t thn c dng lm nguyn liu cho nghin cu kh nng bit ho chi t m so ca cy h ti u. Qua cc th nghim bit ho chi t m so, chng ti nhn thy tnh cht ca m so c phn ng khc nhau i vi cc cht HSTTV trn cy h tiu. Mi trng MS c b sung BA (5mg/l), Kin (0,5mg/l) v IBA (0,5mg/l) thch hp nht cho kh nng bit ho chi t m so. t hp mi tr ng ny, t l m so to chi v s chi trn mt mu cy ln nht (10,3 chi/mu). Mi tr ng vi nng BA cao (>5mg/l) hoc b sung TDZ u gy c ch kh nng bit ho chi t m so; mt s chi c hnh thnh nhng t l chi b d dng rt ln v chi km pht trin khi cy truyn qua mi trng to cy con hon chnh. 4. To r Trong cc thc nghim to r cho cy h tiu chng ti s dng mi trng 1/2 MS c b sung NAA (0,1 - 1,0mg/l) hoc IBA (0,5 -1,0mg/l). nhng th nghim c b sung NAA, sau 15 ngy nui cy cho thy: trong mi trng c nng NAA thp (0,1 - 0,2mg/l) r hnh thnh khong 2 - 3 r/chi; vi nhng nng NAA cao hn (0,5 - 1,0mg/l) s r hnh thnh rt nhiu, t 10 - 15 r/chi, tuy nhin r thng rt ngn, km pht trin v di gc chi hnh thnh cc khi m so. Nng NAA cng cao, khi m so cng nhiu v gy c ch s pht trin ca r. i vi mi trng c IBA, s to r cng xy ra, tuy s l ng r t hn nhng r pht trin mnh hn so vi mi trng c NAA. Nh vy, s ra r ca cy h tiu in vitro thch hp mi trng 1/2MS c b sung NAA (0,1-0,2mg/l). 5. Kim tra ging h tiu sch virut bng k thut ELISA xc nh cc ging h ti u hon ton sch virut, chng ti tin hnh ly mu kim tra bng k thut ELISA 3 giai on: tr c khi a vo nui cy, giai on nhn chi in vitro v sau khi a cy con ra vn m.
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KT LUN 1. S dng cht khng sinh x fotaxin (0,5%) kt hp vi cc phng php kh trng b mt, khng nhng loi b c cc ngun ly nhim b n ngoi m cn c tc dng dit c cc mm bnh ni sinh b n trong mu h tiu. Mi trng MS c b sung BA = 2,0mg/l, IBA = 0,5mg/l thch hp nht cho qu trnh to chi t t cy h ti u, trong kh nng to chi nhng t c ng gn gc cng cao. S dng k thut ELISA l cn thit xc nh ging sch virut trong quy tr nh nhn ging in vitro h tiu. Kh nng hnh thnh chi t m so tt nht trn mi trng MS c BA = 5,0mg/l, IBA = 0,5mg/l v Kin = 0,5 mg/l. Cy h tiu d dng to r trn mi trng 1/2 MS c b sung NAA nng thp (0,1-0,2mg/l).
2.
3. 4. 5.
TI LIU THAM KHO 1. 2. Bhat S. K, Chandel K. P. S., Malik S. K., (1995). Plant Cell Rep. 14: 398-402. Bysov A. S, (2001). Virus and viral diseases of black pepper ( Piper nigrum L.) in condition of closed ground and tropical, subtropical agrocenoses. The theasis for obtaining PhD degree. Kyiv National Univerc ity, Kyiv. on T. A. T, Thi X. D v cs, (2000). Bc u nghin cu chun on bnh virt cy H tiu (Piper nigrum L.) ti mt s tnh min ng Nam B. Tuyn tp Cng trnh nghin cu KHCN., 189-195. Joseph B., Joseph D., (1996 ). Plant Cell Tiss. Org., 41 (1):87-90. Kellar S. M., Krishnamurthy K., (1998). Plant Cell Rep., 17(9): 721-725. Nguyen T. T., Boico A. L. et al (2001). Virus and viral disease of black Ppepper (Piper nigrum L.) in South East of Viet Nam. III Intenational Coference Bioresoeurse and Viruses , Kyiv, Ucraina.
3.
4. 5. 6.
SUMMARY
In vitro culture in Black pepper ( Piper nigrum L.) free virus

oan Thi Ai Thuyen, o ang Giap, Thai Xuan Du Institute of Tropical Biology The plant regeneration from the single node or the callus culture of black pepper (Piper nigrum L.) was described. Various combinations of media, growth regulators and explant sterization were compared. Problems, probably caused by endogenous pathogens associated with tissue exudated, offen occurred during the establishment of
450
culture. The method to sterilize the black pepper plants using antibiotic agent such as cefotaxin was described. For the shoot initiation and the establishment, the optimal concentration of black pepp er growth was BA 2mg/l, IBA 0.5 mg/l. The high concentrations of BA (5-10mg/l) had no effect on the shoot regeneration from callus and the development of lateral branches. The MS medium containing BA 5.0 mg/l, Kin 0.5mg/l and IBA 0.5mg/l was the best medium for subsequent growth and shoot regeneration from callus under the current condition. Shoots were rooted on 1/2 MS medium containing NAA 0.1 -0.2mg/l. In vitro plants were then transferred to pots in the nursery house. After 8 - 10 weeks, the plants with height of 20 - 25cm were transferred to field for testing.
nh: Nhn ging in vitro cy h ti u (Piper nigrum L.) s ch virt 1. Nui cy nh sinh trng; 2. Bit ho chi t m so; 3. To cm chi t t thn; 4. Cy con hon chnh cy t cm chi; 5. Cy con in vitro sau 3 tun ngo i vn m
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KHO ST S TNG TRNG in vitro CA CY KHOAI LANG Ipomoea batatas L. TRONG IU KIN CHIU SNG T NHIN
Nguyn M Uyn, ng Gip Phng Cng ngh t bo thc vt, Vin Sinh hc Nhit i
M U Vic ct gim chi phi sn xut h gi th nh, trong bao gm c chi ph in nng ang ngy mt tng cao lun l vn rt c quan tm. Gi thnh cy cy m Vit Nam ni chung cn kh cao, trong , chi ph in thp sng v lm lnh phng nui cy chim mt t l ln. Nghi n cu ny kho st kh nng sinh trng v pht trin ca cy khoai lang Ipomoea batatas. L in vitro trong iu kin chiu sng t nhi n nhit mi trng nhm so snh nng sut v cht lng cy ging vi phng php nui cy truyn thng trong phng sng. VT LIU V PHNG PHP Th nghim c tin hnh t thng 4 n thng 8/2006. Mu nui cy l cc t thn th 2 n 4 tnh t ngn cy khoai lang Ipomoea batatas. L in vitro sch virus. Mi t km theo mt l vi trng lung trung b nh 0,07 0,01 g. Cc nghi m thc c b tr nh bng 1 v c theo di trong 28 ngy.
Bng 1: Cc nghim thc nui cy
Cng Nghim thc Ni b tr th nghim nh sng (mol.m-2.s-1) Chiu sng t nhin Chiu sng t nhin c b sung n Phng nui i chng Vn m che li en Vn m c che li en, b sung nh sng n hunh quang, 3 bng/tng k, bt t ng vo 6h00 v 16h00; tt lc 8h00 v 18h00 mi ngy Phng sng 0 - 148.1 Thi gian chiu sng (gi/ ngy) 12 Nhit ngy m (0C) 27 - 37 m (%)
45 - 85
54 - 148.1
12
27 - 37
45 - 85
67.5 - 74.3
12
26 2
40 - 80
Cng nh sng c quy i t lux ra mol.m-2.s-1 theo Warrington v Mitchell (1976).
Cy con in vitro ca 3 nghim thc nui cy trong th nghim tr n c m v theo di s tng trng trong 30 ngy vn m. Cng nh sng trong ngy dao ng t 0 - 148.1 mol.m-2s-1, thi gian chiu sng 12 gi/ng y, nhit ngy m dao ng trong khong 27-370C, m 45-85%. Cc s liu c phn tch thng k theo Anova v trc nghim phn hng LSD.
452
KT QU V THO LUN
1. Bin thin cng nh sng, nhit v m trong ngy cc iu kin nui cy
Cng nh sng, nhit v m c o vo ngy khng ma mt ln/tun trong thi gian tin hnh th nghim. Nhit bn trong bnh nui cy iu kin t nhin ch cao hn nhit mi trng t 0,5 - 10C.
2. S tng trng ca cy khoai lang Ipomoea batatas. L in vitro trong iu kin chiu sng t nhin
Tt c cc nghim thc u cho t l sng t 100%. Khc bit r nht gia cc nghim thc nui cy in vitro iu kin t nhin v iu kin phng nui l chiu cao cy, din tch l, dy l v mu sc thn l. Cy iu kin phng nui c thn ngn, mp, l to, dy, mu xanh m. Ngc li, cy nui iu kin t nhi n c thn di, m, l nh v mng, thn l mu xanh nht. Biu hin ny c thy c hai nghim thc c hay khng c b sun g n, do , chng ti gi thit y khng phi l biu hin ca s thiu sng nh thng thng m l nh hng ca nhit kt hp vi cht lng nh sng. Tia tr ong quang ph nh sng v c bn xc tin s ko di ca cy, to sc t xanh vng, l mng, cn tia xanh c tc dng ngc li, km hm s ko di (Grondzinxki v cs, 1973, Sager v Britz, 1990) Trong khi , quang ph nh sng n hunh quang c nhiu tia xanh hin din hn tia v ngc li nh sng t nhi n (Fuentes v cs, 2005). T l nh sng xanh/ trong quang ph gy ra s bin i hnh thi ca cy con in vitro m khng nht thit ng thi lm thay i cht lng cy con (Warrington v Mitchell, 1976). ng thi, nhit cao (35 0C) lm cho thn cy khoai lang ko di vi tc nhanh hn nhit thp (Kozai v cs, 2003).
Bng 2: S tng trng ca cy khoai lang in vitro cc iu kin chiu sng khc nhau
Cc ch tiu
T l sng ns S r ns Chiu cao cy (cm) ng knh thn (mm) S l dy l (mm) Din tch l (cm 2) T l chlorophyll a/b Hm lng chlo a+b (mg/g l) Gia tng trng lng ti (mg/cy) Gia tng trng lng kh (mg/cy) Phn trm cht kh (%)ns
KIP
100% 5,09 0,25 3,28 0,28 b 2,58 0,08 a 6,62 0,11 29 2 a 4,77 0,36 a 3,05 0,15 51,05 2,51 b 988 46 b 30 6 b 4,2 0,1
KI
100% 5,79 0,27 9,85 0,28 a 1,92 0,08 b 6,65 0,11 24 2 ab 2,88 0,33 b 2,86 0,15 55,05 2,51 ab 1.280 46 a 43 6 a 4,2 0,1
KIT
99% 5,19 0,27 8,92 0,28 a 1,90 0,08 b 6,63 0,12 20 2 b 3,327 0,41 b 2,96 0,15 63,94 2,51 a 1.105 46 ab 34 6 b 3,9 0,1
Anova ns : khc bit khng c ngha v m t thng k. * : khc bit c ngha mc 0,01 < p < 0,05 ** : khc bit c ngha mc p 0,01
453
Do cy c hai nghim thc iu kin t nhi n pht trin nhanh theo hng ko di thn, ch tiu v trng lng ti v kh cao hn nhi u so vi i chng trong phng nui. Ngoi ra, hm lng chlorophyl a v b cao nht nghim thc iu kin t nhin, thp hn iu kin t nhi n c b sung n v thp nht nghim thc phng nui. Kt qu ny cho thy s nh hng phc tp ca cht lng nh sng ch khng ch l cng nh sng l n cy. Cc ch s phn trm cht kh, s l, s r ca ba nghim thc c gi tr t ng ng nhau. Ch s gia tng trng lung kh v phn trm cht kh ca hai nghim thc iu kin t nhi n tng ng so v i i chng loi tr kh nng ngm n c dn n s gia tng trng l ng ti trong thn, cung, l ca cy nui cy nhit mi trng cao. Mt c im khc bit na c ghi nhn l cy khoai lang nui trong i u kin phng nui c gin ca thn l kh cao. Trong khi thn v cu ng l ca cy nui cy u kin phng nui rt gin v d gy, cy nui cy iu kin t nhin kh do dai, vn gi nguyn vn hu ht s l sau khi ly ra khi b nh m khng cn phi thao tc t m. y cng l mt c im ng c xem xt khi nui cy cy khoai lang s l ng ln. Vic y kn cc bnh nui cy bng np giy t ra c hiu qu trong vic trao i nhit trong v ngoi bnh. Hin tng hiu ng nh knh hu nh khng xy ra bn trong bnh, do , nhit bn trong ch cao hn ngoi khong 0,5 0C nu c th m bo vic che chn trnh nh nng chiu trc tip v o bnh nui cy. Trong iu kin th nghim, chng ti ghi nhn t l nhim gia cc ngh im thc nui cy trong v ngoi phng sng khng c s chnh lch ng k.
3. S tng trng ca cy khoai lang Ipomoea batatas. L vn m sau giai an nui cy in vitro trong iu kin chiu sng t nhi n
Tuy s ko di thn, l nh , mng v xanh vng thng c xem l biu hin ca cy yu, km pht tri n, nhng trong iu kin th nghim ca chng ti, nhng cy khoai lang trn v n pht trin ng u so vi cy i chng giai on ex vitro. Sau 30 ngy m ex vitro, cc ch tiu v s l, din tch v dy l, gia tng trng lng ti, trng lng kh, phn trm cht kh, h m lng chlorophyl a v b, v.v u c gi tr tng ng nhau cc nghim thc. B r ca cy m t nghim thc t nhin khng n pht tri n tt hn, c gi tr ln hn hai nghim thc cn li. T l sng ca hai nghim th c iu kin t nhi n cao hn so vi i chng. Tc gi Santamaria v cs (2005) cng cho rng, nhit khng kh v quang ph rng ca nh sng t nhi n c nhng tc ng tch cc l n t l sng v tc tng trng ca cy nhit i giai on ex vitr o mc d trong mt s trng hp c th l m gim hiu sut quang hp ca cy trong giai on in vitro.
454
Bng 3: S tng trung ex vitro ca cy khoai lang nui cy in vitro trong cc iu kin chiu sng khc nhau
Ch tiu
T l sng S r / cy S l / cy dy l (mm) ns Din tch l (cm 2) ns T l chlorophyll a/b ns Hm lng chlorophyll a+b (mg/g l) ns Chiu cao cy (cm) ng knh thn (mm) ns Gia tng trng lng ti (mg/cy) Gia tng trng lng kh (mg/cy) ns Phn trm cht kh (%)
KEP
84% 6,03 0,35b 6,0 0,2 0,24 0,02 32,68 1,55 3,33 0,26 29,85 0,86 19,06 1,19b 2,74 0,12 1.785 180 185 20 8,68 0,35
KE
90% 5,89 0,35 b 6,0 0,2 0,21 0,02 29,56 1,55 3,58 0,27 30,75 0,88 22,79 1,20ab 2,66 0,11 1.791 129 190 20 8,23 0,34
KET
88% 7,19 0,36 a 6,5 0,2 0,26 0,02 34,51 1,55 3,11 0,27 29,54 0,88 23,91 1,21 a 2,72 0,12 2.361 190 201 20 8,23 0,29
Anova ns : khc bit khng c ngha v mt thng k. * : khc bit c ngha mc 0,01 < p < 0,05 ** : khc bit c ngha mc p 0,01
Vic b sung thm n hunh quang vi mc ch tng c ng nh sng khng cho kt qu cao hn giai on ex vitro trong th nghim n y, thm ch c mt s ch tiu cho gi tr thp hn nghim thc iu kin t nhi n khng n. Ko di th i gian chiu sng trong ngy bng cch b sung thm nh sng n vo hai giai an u v cui ngy c v gip cy quang hp tt h n i cht so vi nghim thc khng b sung n giai an in vitro, nhng khng mang li s khc bit giai on hu cy m. Cng c th thy rng cc ch ti u t gi tr cao giai on in vitro khng nht thit s t l thun ex vitro.
Hnh 1. Cy khoai lang in vitro sau 4 tun nui cy
Hnh 2. Cy khoai lang sau 30 ngy trng vn m
KIP/ KEP: cy nui cy iu kin phng nui KI/ KE: cy nui cy iu kin t nhin c b sung n KIT/ KET: cy nui cy iu kin t nhin
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KT LUN S tng ng ca cc ch tiu o m t hai nghim thc nui cy iu kin t nhin cho thy nhit cao, cng nh thay i nhiu trong ngy khng gy ra nhng nh hng tiu cc ln s tng trng ca cy khoai lang in vitro. Cy nui cy iu kin t nhin pht trin hon chnh, khe mnh giai on in vitro v thch ng tt, tng trng mnh khi ra vn m. S dng iu kin t nhin thay th cho phng nui truyn thng nui in vitro cc cy nhit i c th ch u c nhit cao giai on cui trc khi chuyn ra vn m l mt hnh thc c tnh kh thi, gp phn tit kim mt phn ng k chi ph in nng trong gi thnh cy ging. Tuy nhin, vic chn la i tng nui cy v ni b tr k nui cy sao cho thong mt, trnh nh nng trc tip nhng vn m bo ngng nhit v cng nh sng cho php i vi cy trng l nhng yu t quyt nh s thnh cng ca hnh thc nui cy ny. TI LIU THAM KHO 1. 2. Bi Trang Vit (2000), Sinh l thc vt i cng, Phn 1: Dinh Dng, NXB i hc Quc gia Tp. H Ch Minh, tr. 146 -219. Fujiwara M., Kubota C., Kozai T., Sakami K., (2003). Air temperature effect on leaf development in vegetative propagation of sweetpotato single node cutting under artificial lighting, Scientia Horticulture, 99: 249 -256. Grodzinsky A. M., Grodzinsky D. M.,(1973). Concise manual on plant physiology. Naukova Dumka, Kiev. Nguyn Th Qunh. nh hng cc yu t mi trng ln vic sn xut cy con trong iu kin nh sng nhn to. Tuyn tp cc cng tr nh nghin cu khoa hc (1993 - 1998). NXB Nng nghip, tr. 461 - 465. Santamaria, J. M., Talavera, C., Contreras, F., Espadas, F., Fue ntes G., (2005). Cultivating in vitro coconut palms ( Cocos nucifera) under glasshouse conditions with natural light, improves in vitro photosynthesis nursery survival and growth. Plant Cell, Tissue and Organ Culture, 83: 287 -292. Sarijeva G, Knapp M, Licht enthaler H.K., (2006). Differences in photosynthetic activity, chlorophyll and carotenoid levels, and in chlorophyll fluorescence parameters in green sun and shade leaves of Ginkgo and Fagus. Journal of Plant Physiology: 1-6. Thimijan, Richard W. & Royal D . Heins. (1983) Photometric, Radiometric and Quantum Light Units of Measure: A Review of Procedures for Interconversion. HortScience 18(6) V Ngc Phng, 2005. S dng nh sng t nhi n trong nui cy m cy lan h ip. Bo co nghim thu t i, Vin Sinh hc Nhit i. Warrington IJ, Mitchell KJ (1976). The influence of blue - and red-biased light spectra on the growth and development of plants. Agric Meteorol 16: 247 -262
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456
SUMMARY
In vitro study of Ipomoea batatas L. enhanced growth under natural daylight conditions
Nguyen My Uyn, Do Dang Giap Institute of Tropical Biology
For the purpose of saving energy and reducing product cost, an alternative method for cultivating sweet potato in vitro in the last stage prior to transferring to greenhouse was suggested to study seedling in vitro growth in conditions without controlling temperature and lighting. Flasks of sweet potato node cuttings were set in three conditions: traditional culture room with controlled temperature and light intensity; greenhouse with natural temperature and light intensity; green house with natural temperature and natural light intensity and added two periods of lighting by fluorescent lamps in the day beginning and ending. After 4 weeks of cultivation, survival rates were almost 100% in all treatments. The two natural condition treatments gave tall and thin but flexible seedlings, which had small and thin leaves in pale green color while seedlings from culture room had big and thick leaves in dark green color with shorter but fragile stem and petioles. These differences might be the result of light quality. Other parameters like dry percentage, number of leaves, number of roots were similar between these treatments. However, in ex vitro period, seedlings from all three treatments grown equally healthy with high survival rate. Most of parameters from these treatments were similar except heigth and root number of seedlings from natural condition treatment were higher than the two others. These results suggested that the in vitro sweet potato seedling can growth well under natural light and temperature, therefore producers can transfer the invitro sweet potatoes seedlings to natural condition in the last stage of cultivation before acclimatization.
457
NHN GING CY LT Mehico (Cedrela mexicana) PHC V TRNG RNG

Nguyn c Minh Hng, Bi nh Thch PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Do nhu cu s dng g ngy mt gia tng nn dn n vic khai tht rng ba b i, nn ph rng ngy mt gia tng v hu qu ca chng tc ng khng nh n i sng con ngi: ngun ti nguyn thin nhin b cn kit, s xi mn t, l lt, mt cn bng sinh thi, Nn vn c t ra l cn mt phng php p ng kp thi cho ngun cy rng b mt i v mt ging cy thch hp m bo nhu cu tiu th ca con ngui. Lt Mexico l mt cy trng rng c mt s c tnh: tc tng tr ng nhanh, cht lung g tt c th s dng v o mt s lnh vc khc nhau ( mc v trong cng ngh vn dn, vn p thanh,) p ng cc y u cu v m thut, cht lng. Ni dung bi ny, chng ti trnh by mt s kt qu thu c trong phng php nhn ging in vitro v kh nng tng trng ca cy lt Mexico (cedrela mexicana) ngoi t nhin. VT LIU V PHNG PHP
Vt liu:
S dng cy con (2-3 thng tui) c cung cp t Nng trng Hu Lng lm nguyn liu ban u. Mi trng c bn: mi trng MS cha 25g/L ng, 8g/L agar. Cc cht sinh trng: BA, NAA, IBA, KI, TDZ.
Phng php:
nh hng ca nng cht kh tr ng v thi gian x l n t l ti sinh ca mu: Mu ly t cc t thn cy con, c kh trng vi hypochloric Ca vi nng 5- 15%, thi gian thay i 20-40 pht. Mu sau khi kh trng uc nui cy vo mi trng c bn sau 30 ngy, so snh kh nng ti sinh ca mu cy. Ti sinh t lng thn: Mi tr ng nui cy ti sinh l mi trng c bn cng vi t hp cc cht kch thch sinh tr ng nh: BA(0-5ppm), NAA(0-0.1ppm), IBA(0-0.1ppm), KI(0-0.5ppm), TDZ (0-0.5ppm) n s to chi. Mi b nh tam gic cha 4 t thn. Mi ngh im thc thc hin vi trung b nh 40 mu, c lp li 3 ln. Cc mu c cy trong iu kin 9 gi chiu sng/ngy, nhit : 25-28oC. Kt qu c tnh sau 30 ngy k t ngy nui cy.
458
To cy in vitro: Mi trng to cy in vitro l mi trng c bn cng t hp gia than hot tnh (0-1g/L) vi cc cht kch thch sinh tr ng nh: NAA, IBA (0-0.5ppm). Mi bnh tam gic cha 5 chi. Mi nghim thc thc hin vi trung b nh 40 mu, c lp li 3 ln. cc mu c cy trong iu kin 9 gi chiu sng/ngy, nhit : 25-28 oC. Kt qu c tnh sau 30 ngy k t ngy nui cy. Kho st mc tng trng cy trng ngoi t nhin: Cy lt nui cy m c a i trng th nghim ti cc vng kh hu khc nhau v theo di s thch nghi ca cy. Hin nay cy lt cy m c trng th ti tnh Ty Ninh, Bo Lc - Lm ng, Bnh Phc, c Lc, Bnh Thun KT QU V THO LUN Nng kh trng mu cy Lt Mexico bng Hypochloric Canxi cn thit l 10%, trong khong thi gian 30 pht c s dng cht khng s inh cfotaxime nng 150mg/L. Mi trng nhn chi thch hp Lt Mexico tng t l pht sinh nhiu chi, chi cao, c nhiu t l mi trng tng ng vi nghim thc: mi tr ng c bn MS c b sung thm 1ppm BA + 0.1 ppm IBA. H s nhn ging l 3.83312 Mi trng thch hp Lt Mexico ra r v pht trin thnh cy con hon chnh trc khi a cy con ra vn m l mi trng tng ng vi nghim thc: mi trng MS + 1 ppm IBA. Nng Hypochloric Canxi kh tr ng l 10%, thi gian 30 pht, cfotaxime nng 150mg/l. Cy lt in vitro c trng thc nghim ngo i t nhin (2-2,5 tui) cho kt qu tt v tc tng trng.
a. s kt hp nng BA 1 ppm vi: b. NAA(0.1), IBA(0.1), Ki(0.5). c. s kt hp nng BA 2 ppm vi : d. NAA(0.1), IBA(0.1), Ki(0.5). e. s kt hp nng BA 3 ppm vi: f. NAA(0.1), IBA(0.1), Ki(0.5). g. s kt hp nng BA 5 ppm vi : h. NAA(0.1), IBA(0.1), Ki(0.5). i. s kt hp nng KI 0.5 vi j. BA ( 1,2,3,5 ppm) k. nh hng ca TDZ (0.5 ppm) l n s to chi
459
KT LUN: Nng Hypochloric Canxi kh trng l 10%, thi gian 30 pht, cfotaxime nng 150mg/l. Mi trng nhn chi: mi trng c bn MS c b sung 1ppm BA + 0.1 ppm IBA. Mi trng thch hp ra r v pht trin thnh cy con: mi trng MS + 1 ppm IBA. TI LIU THAM KHO 1. 2. 3. 4. 5. 6. 7. Bi Trang Vit, Nguyn Th Ngc Lang, Nguyn Du Sanh, V Th Bch Mai (1997). Gio trnh thc tp sinh l thc vt. H KHTN TPHCM Dng Cng Kin (2002). Nui cy m thc vt T1, T2. NXB HQG TPHCM Mamood, M. (1913). Applications of plant in vitro technology . Proceeding. 16-18 November 1993, malaysia. Nguyn Vn Uyn v cc tc gi - Nui cy m t bo thc vt phc v nhn ging cy trng - NXB Nng Nghip. Trn Vn Minh (1999). Cng ngh sinh hc thc vt (Gio trnh i hc). Vin Sinh hc Nhit i Nguyen Van Uyn (1993). Micropropagation of important economic plants . Workshop, Hochiminh city 1993, Vietnam. V Vn V, V Thanh Tm, Hong Minh Tn (2001). Sinh L hc Thc vt NXB Gio Dc
SUMMARY
Micropropagation and test growing of Mexico plantlets (Cedrela mexicana) at the natural conditions
Nguyn c Minh Hng, Bi nh Thch Institute of Tropical Biology
Micropropagation in Mexico plant using from node stems of plant two month age. Results showed that MS medium supplemented with 1ppm BA + 0.1 ppm IBA induced shoots creating. In vitro plantlets could be elongated and rooted in the MS medium supplemented with 1ppm IBA. The developing of plantlet at natural conditions have good results
460
VI NHN GING CY SUNG M (Ficus carica L.)

Nguyn c Minh Hng, Bi nh Thch PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Sung M l mt loi cy n tri ch mi c a vo Vit Nam trong nhng nm gn y, nhng y loi cy gi vai tr quan trng trn th gii, vi sn lng c tnh 1 triu tn qu /nm, trong : Th Nh K 300000 tn/nm, Ai Cp 160000 tn/nm, Algeria 38400 tn/nm, Syria 37000 tn/nm, Tunisis 35000 tn/nm, Libya, Iraq, B Nha,Nhm mc ch p ng nhu cu v s l ng cng nh cht lng cho ging cy trng mi ny, chng ti tin hnh nghin cu ti: Nhn ging cy sung M bng phng php in vitro. VT LIU V PHNG PHP
Nguyn liu:
S dng cy con bu t l m nguyn liu ban u. Mi trng c bn: mi trng MS, 25g/L ng, 8g/L agar. Cc cht sinh tr ng: BA, NAA, IBA.
Phng php: nh hng ca nng cht kh tr ng v thi gian x l n t l ti sinh ca mu:
Mu ly t cc t thn cy con, c kh trng vi hypochloric Ca vi nng 5 15%, thi gian thay i 20-40 pht. Mu sau khi kh trng uc nui cy vo mi trng c bn sau 30 ngy, so snh kh nng ti sinh ca mu cy.
nh hng ca nng cytokinin l n s to chi:
Mi trng nui cy: mi trng c bn cng vi t hp cc cht kch t hch sinh trng nh: BA (0; 0.1; 0.5; 1; 2; 4)ppm, IBA (00.1; 0.3; 0.5) ppm n s to chi. Mi bnh tam gic cha 4 t thn. Mi nghim thc thc hin vi trung b nh 40 mu, c lp li 3 ln. Cc mu c cy trong iu kin 9 gi chiu sng /ngy, nhit : 25-28 o C. Kt qu c tnh sau 30 ngy k t ngy nui cy.
Kho st mi trng ra r v ti sinh cy hon chnh:
Mi trng nui cy: mi trng c bn cng vi cc cht kch thch sinh tr ng nh: NAA (0; 0.1; 0.3; 0.5) ppm , IBA (0; 0.1; 0.3; 0 .5) ppm. Mi bnh tam gic cha 5 chi. Mi nghim thc thc hin vi trung b nh 40 mu, c lp li 3 ln. Cc mu
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c cy trong iu kin 9 gi chiu sng/ngy, nhit : 25-28oC. Kt qu c tnh sau 30 ngy k t ngy nui cy. KT QU V THO LUN Nng kh trng mu sung M bng Hypochloric Canxi cn thit l 10%, trong khong thi gian 30 pht, vi t l 35%. Mi trng ti u cho vic hnh thnh chi l mi trng cn bn c b sung 2 ppm BA v 0.1 ppm IBA (mi tr ng 14), vi h s ti sinh ch i: 5.67 chi / mu sau 3 tun nui cy.
a. 0: 0ppm BA, 0 ppmIBA

1: 0.1ppm BA, 0 ppmIBA 2: 0.1ppm BA, 0.1 ppmIBA 3: 0.1ppm BA, 0.3 ppmIBA 4: 0.1ppm BA, 0.5 ppmIBA 5: 0.5ppm BA, 0 ppmIBA
b. 0: 0ppm BA, 0 ppmIBA 6: 0.5ppm BA, 0.1 ppmIBA 7: 0.5ppm BA, 0.3 ppmIBA 8: 0.5ppm BA, 0.5 ppmIBA 9: 1ppm BA, 0 ppmIBA 10: 1ppm BA, 0.1 ppmIBA
c. 0: 0 ppm BA, 0 ppmIBA 11: 1 ppm BA, 0.3 ppmIBA 12: 1 ppm BA, 0.5 ppmIBA 13: 2 ppm BA, 0 ppmIBA 14: 2 ppm BA, 0.1 ppmIBA
d. 0: 0ppm BA, 0 ppmIBA 16: 2 ppm BA, 0.5 ppmIBA 17: 4 ppm BA, 0 ppmIBA 18: 4 ppm BA, 0.1 ppmIBA 19: 4 ppm BA, 0.3 ppmIBA
15: 2 ppm BA, 0.3 ppmIBA
20: 4 ppm BA, 0.5 ppmIBA
Mi trng ti u cho vic to r v hnh thnh cy hon chnh: mi trng c bn vi: 0.1ppm IBA (I1), kt qu c tnh trn s l trung bnh (10.67 l/ cy) v s r trung bnh ( 2.67 r/ cy) sau 6 tun nui cy.
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a. 0:0 ppm IBA + 0 ppm NAA b. 0: 0 ppm IBA + 0 ppm NAA c. 0: 0 ppm IBA + 0 ppm NAA N1:0 ppm IBA + 0.1ppm NAA I1: 0.1ppm IBA+0ppm NAA IN1:0.1 ppmIBA+0.1ppmNAA N2:0 ppm IBA + 0.3ppm NAA I2: 0.3ppmIBA+0ppmNAA IN2:0.3ppmIBA+0.3ppmNAA N3:0 ppm IBA + 0.5ppm NA A I3: 0.5ppmIBA +0ppmNAA IN3:0.5ppmIBA+0.5 ppmNAA
KT LUN Nng kh trng Hypochloric Canxi l 10%, th i gian 30 pht thch hp cho vic v mu. Mi trng ti u cho ti sinh chi: mi trng MS c b sung 2 ppm BA v 0.1 ppm IBA. Mi trng ti u cho vic to r v hnh thnh cy hon chnh: mi trng MS c b sung 0.1ppm IBA TI LIU THAM KHO 1. 2. 3. 4. Trn Vn Minh (2003). Cng ngh sinh hc thc vt (gio trnh CH&NCS H Nng Lm TPHCM) Dng Cng Kin. Sung M (Ficus Carica) mt ging cy n qu mi Vit Nam. NXB Nng nghip. Joan Tous and Louis Ferger son, Mediterranean Fruits, 1999. M. Grieve, Botanical.Com.fig, common , A Modern Herbalry, 1996. trang web: www.google.com/ ficus carica ; www.tropilab.com/ ficus carica
SUMMARY
Micropropagation of fig plants ( Ficus carica L.)

Nguyn c Minh Hng, Bi nh Thch
Institute of Tropical Biology
Micropropagation in Fig plant using for node stems of plant. Results showed that MS medium supplemented with 2ppm BA + 0.1 ppm IBA induced shoots creating. In vitro plantlets could be elon gated and rooted in the MS medium supplemented with 0.1ppm IBA
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NG DNG CNG NGH T B O THC VT BO TN NGUN GEN LT HOA CN O (Chukrasia tabularis A. Juss)
L Xun i, Bi Thi Tng Thu, Trn Vn Minh
PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Vn Quc gia Cn o thuc h thng rng c dng Vit Nam, l mt trong ba vn quc gia duy nht ca c nc va thc hin chc nng bo tn rng, va thc hin ch c nng bo tn bin. Vi din tch rng t nhi n gn 6.000ha, thm thc vt rng Cn o c v tr v chc nng rt quan trng v mt sinh thi, mi tr ng, cnh quan v kinh t - x hi ca qun o lch s v ni ting ny. Tng kt cc nghin cu khoa hc v thm thc vt rng ti Cn o thng k c 882 loi thuc 562 chi, 161 h trong c nhiu loi thc vt qu him, c hu v Lt hoa (Chukrasia tabularis A. Juss) l mt loi in hnh. S phn b t nhin ca cc loi Lt hoa trong Vn QG Cn o l mt iu l th, do rng t nhin ca cc tnh min ng Nam B v cc hi o pha Nam Vit Nam cha pht hin c s phn b ca loi cy ny. Lt hoa l mt trong nhng loi cy g c gi tr ca Vit Nam. L cy g ln, qu v c vn rt p, th g mn, khng b mi mt, rt c a chung ng cc gia dng cao cp, chnh v vy Lt hoa b sn tm ro rit khai thc g, c thn ln r khng cha li cy ging, nn hin nay Lt hoa trong tnh trng cn kit khng c kh nng t phc hi trn phm vi c nc (Nguyn B Cht, 1998; Nguyn Ho ng Ngha, 1999; Trn nh Hu, 1998). Nm 1997, cn bo Linda (cn bo s 5) b vo Cn o gy thit hi nghim trng, trong c 2.200ha rng thuc V n QG Cn o, lm suy gim v s lng v cht lng cc loi cy g qu bn a v c hu nh Lt hoa, gng nc, qung lng, du Cn Sn Trong bi cnh , thc trng loi cy Lt hoa cn li rt t, ti sinh t nhin km, kh nng gieo ging rt hn ch ng dng CNTBTV trong vic bo tn v phc hi ngun gene Lt hoa Cn o l yu cu cp bch. VT LIU V PHNG PHP
Vt liu: Mu nui cy: Cnh bnh t ca cy Lt hoa c s dng gim cnh trong bu t ti Vn QG Cn o. Cy con gim c nh c a v VSHN. Chi non cnh gim c s dng lm mu nui cy. Mu c ct ta thnh nhng on 3-5cm, c ra sch bng x phng v v trng bng Hypochlorite-Na v HgCl 2. Sau khi v trng, mu c ct thnh nhng on 10-15mm c cha vng nhu m nh sinh trng. Mu nui cy l chi nh hay t thn. iu kin nui cy: Mi trng c v trng 121oC v 1at trong 25 pht. Nhit phng nui cy 28+2oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy.
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Mi trng nui cy: Mi trng dinh dng khong c bn MS (Murashige-Skoog, 1962) v WPM (Lloyd and McCown, 1981), c b sung BA (6-benzyl aminopurine), Kinetin (6-furfurylaminopurine), IAA ( -indol acetic acid), IBA ( -indol butyric acid), NAA (naphthalene acetic acid), Tyrosin, Adenine sulfate v n c da (CW) Phng php:
Th nghim c b tr theo RCBD (1 yu t) v CRD (2 yu t), 4 ln lp li, mi ln lp li nui cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTAT theo M, CV% v LSD(0.05). Ch tiu theo di: s chi/cm, chiu cao chi (mm), s t (no), s l (no), chiu d i l (mm), chiu rng l (mm), to m so (+/ -), s pht trin l (+/-), t l ra r (+/-), s r (no), chiu di r (mm), s l xanh cn li (no), ng knh thn (mm). KT QU V THO LUN
1. Nui cy chi nh sinh trng cy lt hoa in vitro
V trng mu: Do cy lt hoa c mt lp nha bao bc quanh thn v c thu thp trong rng t nhin nn kh nng mu nui cy b tp nhim cao. X l kh tr ng kt hp trc l Hypo-Na sau n HgCl 2 cho hiu qu v trng chp nhn c c t l pht sinh chi cao nh hng ca mi trng khong c bn n nui cy pht sinh chi lt hoa in vitro: Kt qu nghin cu cho thy mi trng c bn MS c b sung BA (0,1mg/l) + CW (10%) thch hp cho pht sinh chi cao (3,5 -6,7 chi/mu) c thn l pht trin nh hng ca BA n kh nng pht sinh cm chi lt hoa in vitro : Nui cy trn mi trng MS cho pht sinh chi (10,5 -17,5 chi/mu) tt hn MS/2. Nng 0,51mg/l BA b sung vo mi trng nui cy MS thch hp cho t o cm chi. Cm chi l n v c bn dng lm nguyn liu cho qu trnh nhn nhanh in vitro v sau.
2. Nhn nhanh cy lt hoa in vitro
nh hng ca BA v IAA n nhn nhanh cy lt hoa in vitro : iu u tin c ghi nhn l hu ht cc nghim thc kt hp BA+IAA u to m so gc v pht sinh thn l km pht trin. Nng 1mg/l BA b sung v o mi trng nui cy cho kt qu pht sinh chi tt h n nh hng ca BA v Kinetin n nhn nhanh cy lt hoa in vitro : Mi trng nui cy MS c b sung BA (1mg/l), + Kinetin (0,1-1mg/l) + CW (10%) cho kt qu pht sinh chi cao (13,2-16,7chi/cm) v c thn l pht trin. BA cn cho pht sinh chi; kinetin v CW cn cho pht trin l. nh hng ca Tyrosin n nhn nhanh cy lt hoa in vitro : T hp BA + Kinetin + Tyrosin b sung vo mi trng nui cy cho pht sinh chi cao v thn l pht trin tt hn hn BA + Kinetin. T hp BA (1mg/l) + Kinetin (1mg/l) + Tyrosisn (3mg/l) cho
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pht sinh chi cao (18,7chi/cm) v c thn l pht trin. Tyrosin ng vai tr h tr pht sinh chi v pht trin thn l trong mi trng nui cy khng c n c da. nh hng ca Adenine sulfate (AS) n nhn nhanh cy lt hoa in vitro : T hp BA + kinetin + AS b sung vo mi trng nui cy cho pht sinh chi v thn l pht trin hn t hp BA + Kinetin. T hp BA (1mg/l) + Kinetin (1mg/l) + AS (3mg/l) cho pht sinh chi cao (18,2chi/cm). Adenine sulfate kch thch pht sinh chi c thn l pht trin trong mi trng nui cy khng b sung n c da nh hng ca nc da (CW) n nhn nhanh cy lt hoa in vitro : Mi trng nui cy c b sung t hp BA+IAA u to m so ngay gc v thn l km pht trin. B sung vo mi trng nui cy t hp BA + Kinetin hoc Tyrosin hoc Adenine sulfate u kch thch pht trin thn l v kh nng pht sinh chi cao h n khi cc t hp ny c b sung nc da. nh hng ca cng chiu sng v trao i kh n nhn nhanh cy lt hoa in vitro: Cng chiu sng 34,2 mol/m2/s kt hp bnh nui cy c y np giy cho kt qu pht sinh chi cao (18,7chi/cm) v c thn l pht trin (chiu di l 34mm, rng l 12,2mm). Cng chiu sng trung b nh kt hp tng cng trao i kh ci thin sinh trng hiu qu. Ti sinh cy lt hoa in vitro : Do cy lt hoa ang nhn nhanh d i dng cm chi, nn thn l cha p ng c yu cu a ra thun ha. Cn c mt giai on trung gian thun ha in vitro trc khi nui cy pht sinh r, v giai on ti sinh cm chi c nghin cu. Kt qu cho thy khi nui cy cm chi tr n mi trng WPM + CW (10%) c b sung 0,1mg/l BA v 0,1mg/l Kinetin cho kh nng ti sinh cao v c thn l pht trin. Sinh trng cy lt hoa in vitro : y l vn nghin cu cn thit xc nh chu k nui cy in vitro. Kt qu qua bng 11 cho thy, mu nui c y l chi n th khng c qu trnh tng sinh chi v mu nui cy l cm chi (3 chi/cm) lun i i vi kh nng tng sinh chi theo thi gian nui cy. Chu k nui cy cho chi n v cm chi l 45 ngy. Tng t kt qu phi hp trong nui cy gi t, trm hng Nui cy pht sinh r cy lt hoa in vitro : So vi t l ra r ca i chng (44%), t l ra r cy lt hoa in vitro t cao nht (100%) tr n mi trng c b sung auxin (0,1-1mg/l). Vi nng NAA cao 1mg/l, gc cy c hin t ng ph xp; ngc li IAA v IBA khng cho phn ng. Nng auxin thp (0,1mg/l) r pht sinh khng phn nhnh; nng 0,5-1mg/l r pht sinh phn nhnh. Cy lt hoa l cy pht sinh r d dng in vitro tng t nh cy gi t.
3. Thun ha cy lt hoa cy m
Cy lt hoa cy m c thun ha trong c cht x da 3 tun l, c t l sng >95%. Cy lt hoa sau c cy chuyn ra bu t vi t l c cht l t: x da : tro tru (1:1:1). Phn chung hoai c b sung c cht: phn chung (3:1). Kt qu cho thy cy lt hoa l loi sinh tr ng nhanh, t kch thc xut vn (50cm) sau 8 thng. Kt qu ghi nhn tng t nh cy trm hng. Cy con cy m t kch thc 1.83m chiu cao vi ng knh c r 7,53cm sau mt nm tui.
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KT LUN Cy Lt hoa Cn o c nhn nhanh v bo tn ngun gene in vitro. Mi tr ng MS l mi trng c bn thch hp cho qu trnh nui cy in vitro. BA, kinetin, tyrosin, adenine sulfate, cng chiu sng trung b nh kt hp tng cng trao i kh kch thch pht sinh chi mnh m. Chu k nui cy in vitro l 45 ngy. Cy lt hoa d dng pht sinh r in vitro. Qu trnh thun ha v t kch thc xut vn sau 8 thng sinh trng trong bu t. Cy con cy m t kch th c 1,8m chiu cao vi ng knh c r 75mm sau mt nm tui. TI LIU THAM KHO 1. Lloyd G. & B. McCown (1981 ). Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot -tip culture. In: Comb. Proc. Intl. Plant Prop. Soc., 30:421-426 Mamood M. (1993). Application of plant in vitro technology. Proc. 16-18 Nov 1993, Univ. of Malaysia, Malaysia. Murashige T. & F. Skoog (1962 ). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15:472 -497 Nguyn B Cht (1998). Nghin cu mt s c im lm hc v bin php k thut gy trng nui dng cy lt hoa (Chukrasia tabularis A. Juss). Lun n Tin s. Nguyn Hong Ngha (1999). Mt s loi cy b e da Vit Nam. NXB Nng nghip. Trn nh Hu (1998). Nghin cu u hp thc vt lt hoa, gng n c, chiu liu nc, trng tri nh ti khu vc t Thm, V n QG Cn o.
2. 3. 4. 5. 6.
SUMMARY
Aplication of plant cell biotechnology in conservation of lat hoa Con ao (Chukrasia tabularis A.Juss)
Le Xuan Ai, Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology Lat Hoa was conserved and restored successfully in in vitro via using micropropagation techniques. MS was used as basic favoured medium for plant culture. BA (1mg/l), kinetin (1mg/l), tyrosin (3mg/l), A denine sulfate (3mg/l), increasing light intensity (34,2 mol/m2/s combined with air exchange stimulates shoots raising healthy. Cultured circle determined by 45 days. Its easily for root induction in vitro. Its acclimatises in nursery bed and reaches the plant height for field evaluation after 8months. Plantlets reach 1.83m in plant height and 7.53cm in diameter after 1 year.
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SINH TRNG V PHT TRIN CY TRM HNG (Aquilaria crassna Pierre ex Lecomte) CY M TRN NG RUNG
inh Trung Chnh, H Nng Lm TPHCM Bi Thi Tng Thu, PTNT pha Nam v CNTBTV Trn Vn Minh, Vin Sinh hc Nhit i
M U
Cy Trm Hng cn c gi l cy K Nam hay cy D Bu (Aquilaria crassna Pierre ex Lecomte), trong g ca n c kh nng hnh thnh loi sn phm c bit gi l trm hng v k nam, c nhiu cng dng c bit n hn 2000 nm qua. T xa n nay, trm hng v k nam l loi c sn rng qu him, c gi tr kinh t cao, c a chung trn th gii. Iu ny lm cho cy trm hng tr thnh mt trong nhng thc vt c hu, c gi tr c bit v khoa hc v kinh t Vit Nam cng nh nhiu nc khc. Tuy nhin, cc nghin cu c h thng v s hnh thnh trm ch mi thc hin t vI thp nin gn y. Trong khi cng vi s mt rng, ngun trm t nhi n hin nay khng cn nhiu. Cc loi thuc chi Aquilaria c kh nng cho trm b khai thc trit . Vit Nam, ng i ta khai thc trm ba bI ngay c nhng cy trm cha thun thc tm trm. Vi cch khai thc nh vy th khng cn cy cho ht gieo ging nn cy trm hng ang ng trc nguy c b tiu dit (Phm Hong H, 1985; L Mng Chn, 1995; Phm nh Tr, 1988). H qu hin nay l c s lng c th v tr lng, nht l nhng cy ln lm nhim v gieo ging cho qu trnh tI sinh ca loI ny cn rt t v ang b e da dit chng (V Vn Cn v V Vn Dng, 1987). Chnh Ph ban hnh Ngh nh s 18-HBT, xp cy D Bu vo nhm 1A, tc l loi cn c bo v nghim ngt. Sau khi thnh cng ng dng CNTBTV trong nhn nhanh cy trm h ng qu him (inh Trung Chnh v cs. 1998; Tr n Vn Minh v cs. 1998). Vn cn t ra l kho st kh nng sinh trng v pht trin cc dng ang cho trm qua cy m, t c c s khoa hc a cc loI cy trm hng ang t trm v nI bn a v pht trin cy trm hng cc vng khai thc truyn thng. VT LIU V PHNG PHP Vt liu Cc nghin cu c tin hnh ti H Nng Lm TPHCM. Ngun nguy n liu s dng trong nui cy m v cy t ht thc sinh c thu thp ti Ph Quc. Cc cy trm hng cy m v cy t ht thc sinh c a vo th nghim c cng chiu cao thn ban u. Cy con c nui cy trong bu t trong giai on 2-8 thng tui. C cht tro tru: phn chung hoai : x da (1:1:1).
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Phng php nghin cu Cc th nghim c b tr theo kiu hon ton ngu nhin RCBD (1 yu t), 6 ln lp li, s cy trn mi ln lp li ph thuc v o tnh cht th nghim. S liu thu thp c phn tch thng k bng phn mm stagraphic plus (v.4) theo M, CV%, LSD(0.05). Cc ch tiu theo di: chiu cao thn (cm), ng knh c r (mm), s nhnh (no), s l (no), chiu di l (mm), chiu rng l (mm) KT QU V THO LUN 1. Sinh trng ca cy trm hng cy m n giai on xut v n (8 thng tui) Kt qu nghin cu cho thy cc ch tiu s nhnh v s l c bin ng khng theo quy lut. Dng ND2 pht sinh nhnh trong v n m (2-6 nhnh/ cy) v s l (17-37 l) tt hn so vi dng BD1, BD2 v ND1. Chi u cao thn, chiu dI l v chiu rng l ca 4 dng trong th nghim khng c s sai khc c ngha 2. Sinh trng v pht trin cy trm hng 12 thng tui Kt qu nghin cu cho thy cc ch tiu sinh trng chiu cao thn, s nhnh, ng knh s r v pht trin chiu dI l v rng l c sai khc gia cc d ng a vo th nghim. Cc ch tiu v chiu cao thn, ng knh c r v chiu dI l gia cc dng BD1, BD2, ND1, ND2 khng sai khc c n gha. Dng ND2 pht sinh s nhnh (6-13 nhnh/cy) v s l (54-65 l) tt hn cc dng BD1, BD2 v ND1. V ch tiu pht trin chiu rng l cho thy cc d ng ND1, ND2 c kch thc nh hn cc dng BD1, BD2. So snh bt cp th khng sai khc gia cc cp ND1-ND2, ND2-BD1, BD1-BD2 (bng 2). Bn dng trm hng c a vo nuI cy m c ngun gc di truyn khc nhau (t ht thc sinh) khng c sai khc nhau v cc ch ti u chiu cao thn, ng knh c r v chiu dI l. Ring cc ch tiu v s nhnh v s l, dng ND2 c ch s sinh trng tt hn BD1, BD2 v ND1. Ch tiu chiu rng l l ch tiu thng c s dng trong dn gian v th n trm dng phn bit hai loI ph (d bu v d me) th khng sai khc cc dng. 3. Sinh trng v pht trin cy trm hng cy m trong Iu kin c v khng che nng Kt qu nghin cu cho thy ngim thc c che nng 50% v khng che nng khng sc chng t c s khc bit gia hai nghim thc v chiu cao thn, ng knh c r, s nhnh, chiu dI l v chiu rng l, s l. Nhng v s trung bnh th hu ht cc ch tiu ca nghim thc cc cy khng che nng u cao h n cc cy c che nng (bng 3). Nh bit, cc loI cy thn g c gi tr rng nhit i th ng c chia thnh hai nhm theo ph n ng vi nh sng: cy chu rp (chu bng) v cy sng (khng chu rp). Thng thng, cy chu rp bao gm cc lo I m trong giai on cy con cn phI c tn che, nhng khi cy ln th cng c nhu cu phI ra nng hon ton; ngc li, cy khng chu rp li c nhu cu nh sng cao ngay c lc c n l cy con. Nhng kt qu ghi nhn th nghim n y ph hp vi nhn nh cy d bu l cy trung tnh, thin v a sng (L Mng Chu v cs. 1995).
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4. Sinh trng ca cy trm hng cy m v cy t ht thc sinh trn ng rung Kt qu nghin cu cho thy: giai on 14 thng tui: cy con cy m v cy t ht thc sinh khng khc nhau nhiu v s nhnh / cy, s l v chiu cao thn cy. Nhng v chiu dI v chiu rng l, th cy cy m c kch th c ln hn v ngc li cy con t ht thc sinh c ng knh c r ln hn cy con cy m. giai on 2 tui: gia cy con cy m v cy t ht thc sinh khng c s khc bit nhau v s nhnh / cy, chiu cao thn cy, ng knh c r v chiu di l. Nhng chiu rng l ca cy cy m c kch th c ln hn cy con t ht. giai on 3 tui: gia cy con cy m v cy t ht khng c s khc bit v chiu cao thn cy v ng knh c r. Qua th nghim so snh s sinh trng ca cc dng cy cy m vi cc cy trng t ht thc sinh th giai on cn nh (14 thng tui) c s khc nhau v ng knh c r v kch thc l; nhng cng v sau (3 nm tui) th s sinh trng v pht trin li ging nhau, chng t cy cy m sinh tr ng v pht trin bnh thng nh cy trng t ht thc sinh. KT LUN Kt qu nghin cu ca tI cho thy sinh trng v pht trin cy nui cy m v cy t ht thc sinh khng sai khc c ngha trong giai on v n m; Iu ny cng c ghi nhn khi trng trn ng rung. Trong qu trnh nui cy nh sinh trng nhng dng c ngun gc di truyn khc nhau, cho cy con c nhng c Im ging nhau nh chiu cao thn, ng knh c r v pht trin chiu di l; ring cc ch tiu v s nhnh, s l th sai khc n hau c ngha. Kt qu nghin cu v phn ng nh sng trn cy con cy m, cho thy d bu l cy trung tnh v thin v a nh sng; do cy cy c th c m m khng cn dn che. Kt qu nghin cu sinh trng v pht trin trong vn m xc nh thi gian xut vn cho cy cy m l 7-8 thng tui; iu ny ph hp vi kt qu trong dn gian khi gieo m t ht thc sinh (gieo vo thng 8, tr ng ra ng rung vo thng 4dl nm sau). Th i gian canh tc trn ng rung cy 3 nm tui cho thy, sinh trng v pht trin ging nhau cy cy m v cy t ht thc sinh th hin qua chiu cao thn v ng knh c r.
TI LIU THAM KHO
1.
inh Trung Chnh, Trn Vn Minh, Bi T Tng Thu & Bi Cch Tuyn (1998). ng dng cng ngh sinh hc thc vt t rong cng tc ging cy d bu (Aquilaria crassna Pierre ex Lecomte). Tp san KHKT Nng Lm nghip 12:50 -55 L Mng Chn (1995). Nghin cu c Im sinh vt hc ca lo i trm hng lm c s bo v, gy trng, nui d ng loi cy qu him nhit i Vit Nam. Kt qu nghin cu KH-CN Lm nghip 1991-1995. NXB Nng nghip, pp89-91. Phm nh Tr,(1988). Tm hiu thm v cy trm hng. Tp ch Lm nghip 4:22-26 Phm Hong H (1985). Thc vt o Ph Quc. NXB TPHCM .
2.
3. 4.
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5.
Trn Vn Minh, Bi T Tng Thu, inh Trung Chnh & Bi Cch Tuyn (1998). Nhn ging cy trm hng qua nui cy nh sinh trng. Tp ch KHCN&KT Lm nghip 11/12:44-45 V Vn Cn & V Vn Dng (1987). Cy trm hng (d bu). Thng tin chuyn Nhng loi thc vt rng qu him cn bo v ca Vit Nam. Vin iu tra Quy hoch Rng, H Ni, pp43-48
6.
SUMMARY
Growth and development of agarwood (Aquilaria crassna Pierre ex Lecomte) plantlets in plantation
inh Trung Chanh(1), Bui Th Tuong Thu(2), Tran Van Minh(2) (1)University of Agriculture and Forestry HCMCity (2)National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Studies the plant growth and development of agarwood plantlets after released from in vitro were set up. In period of 2 -8mos in nursery bed, there are d ifference of plant height, lenghth of leaves and width of leaves of 4 clones tested were not significant. In period of 12mos in nursery bed, clone of ND2 has branches/plant and number of leaves were developed better than clones of BD1, BD2 and ND1; the wid th of leaves of ND1, ND2 were better than BD1, BD2. Plantlet growth and development in nursery bed could be suffered under completed shading or shading 50%. Studies plant growth and development of in plantlets and seedlings in plantation showed that in per iod of 14mos, there were differences in diameter of plant and parameters of leaves; in 2 year -old, the width of leaves of in vitro plantlets were better than in seedlings; in period of 3 year old, there were not difference from in vitro plantlets and see dlings in parameters of plant height and diameter of plant.
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NGHIN CU K THUT NHN GING in vitro TRONG BO TN V PHT TRIN CY Pmu (Fokienia hodginsii)
Phm Th Anh, Bi Thi Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Pmu l loi cy g qu, bn, p, th m, thn thng d ca x, c dng lm m ngh, iu khc, dng lm cu, dng trong xy dng, ct tinh du lm hng liu rt qu v lm dc liu (B NN & PTNT, 2000). Ngo i ra n cn c dng lm cy cnh trang tr v cy trng ng ph (Henry & Thomas, 1991). L loi cy g c gi tr cao nn c xp vo loi g qu Vit Nam. Tnh trng cy pmu l mt loi cy c xp vo danh mc cc loi cy cn c bo v nghim ngt, trong Sch Vit Nam (B KHCN&MT, 1996). Do g qu v r c tinh du gi tr cao nn b khai thc mnh. Hin ch cn ri rc ni xa dn hoc trn nh v ng nh ni him tr. Ti sinh km, sinh trng chm nn s lng gim nhanh chng. c nhiu d n trng rng c thc hin nhng n nay vn cha c nhiu s bin chuyn. Kt qu nhn ging cy cy l kim v cy pmu ni ring bng cng ngh sinh hc c mt s bo co ghi nhn c (Becwar, 1987; Wann, 1989). Trong bo co ny nghin cu quy trnh nui cy m cy pmu in vitro v to c cy con pmu in vitro hon chnh. VT LIU V PHNG PHP
Vt liu
Mu ban u dng nui cy l chi nh, c ly t cy 1-1,5 tui (c ngun gc t Trung tm nghin cu Lm sinh Lm ng) v c gy trng ti VSH, Th c, Thnh ph H Ch Minh. S dng c nh non khong 15-20 ngy tui, ly chi nh ra sch nhiu ln di vi nc my sau ngm trong n c x phng 0,1% trong 15 pht v c ra li bng nc my bnh thng. iu kin nui cy: mi trng v trng 1210C v 1at trong 25 pht. Nhi t phng nui cy 282 0C. Cng chiu sng 34,2mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962), WPM (Lloyd and McCown, 1981), IAA ( -indol acetic acide), NAA ( naphthalene acetic acid), BA (6 -benzylaminopurine), IBA ( -indol butyric acid), Kinetin (6-furfurylaminopurine), Rib (rhizopon), n c da (CW).
472 Phng php
Th nghim c b tr theo RCBD (1 yu t), 3 ln lp li nui cy 5 b nh tam gic 300ml, mi bnh cha 65ml mi trng th nghim v c cy 7 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (p=0,05). Ch ti u theo di: t l nui cy pht sinh chi (%), chiu cao chi (mm), s chi / mu (chi), s chi / b nh (chi). KT QU V THO LUN V trng mu nui cy: Chi nh v chi bn c s dng lm mu nui cy. Chi non c v trng bng hypochlorite-Natri (10%) trong 12 pht. Ch i non v trng c a vo nui in vitro v l nguyn li u cho cc nghim tip theo (bng 1) nh hng ca cc mi trng khong c bn n kh nng ti sinh chi cy pmu: chi in vitro nui cy trn mi trng c bn: WPM, 1/2 WPM, 1/2 MS c b sung BA (0,1mg/l) v Cw (1-5%). Mi trng WPM l khong c bn thch hp nht cho nui cy chi nh nh hng ca BA, KI v Cw n kh nng nhn ging cy p mu in vitro: mi trng dinh dng khong b sung BA (0 v 0,1mg/l) v Kinetin (0,1 v 0,5mg/l). K t qu nghin cu cho thy mi trng WPM c b sung BA (0,1mg/l) c kh nng pht sinh chi nh hng ca BA v IBA n kh nng nhn ging cy p mu in vitro: nhm nng cao kh nng nhn ging, s dng kt hp BA (0,1mg/l) v IBA (0,3mg/l) b sung vo mi trng nui cy WPM. Kt qu cho thy kh nng nhn chi cao nh hng ca cc cht hu c n kh nng nhn ging cy p mu in vitro: cy pmu l mt loi cy rng rt mn cm vi cc cht iu h a sinh trng. V vy t hp BA(0,1 mg/l), yeast extract (0 -1g/l), glutamine (0-0,1g/l) v ng (0-0,1g/l) c b sung vo mi trng dinh dng WPM. Kt qu cho thy mi tr ng WPM b sung BA (0,1mg/l) v yeast extract (1g/l) c kh nng nhn chi cao nh hng ca BA, IBA, yeast extract v than hot tnh n kh nng vn thn cy pmu in vitro: mc ch nng cao kh nng nui cy ti sinh v n thn, s dng phi hp BA (0,3mg/l) + IBA (0,3mg/l), yeast extract (0 -0,1g/l) v than (0-0,1g/l), b sung vo mi trng dinh dng WPM. Kt qu th nghim cho thy, t hp WPM + IBA (0,3mg/l) + yeast extract (1g/l) c kh nng vn thn tt nh hng ca IBA v Rib n qu trnh to r pmu in vitro: cy con pmu in vitro t chiu cao 2,5cm c cy vo mi trng WPM + IBA (0,3-1-3-5mg/l) + Rib (10mg/l). Kt qu cho thy, cy pmu ra r nhiu v di nht trn mi trng WPM + IBA (5mg/l), sau 45 ngy nui c y KT LUN Cy pmu c nghin cu nhn ging in vitro nhm bo tn v pht trin c ngun gen cy g qu him ang b cn kit. Chi non c v trng bng
473
hypochlorite-Natri (10%) trong 12 pht. Mi tr ng WPM l khong c bn thch hp nht cho nui cy chi nh. Mi tr ng WPM c b sung BA (0,1mg/l) c kh nng pht sinh chi cao nht. Kt hp BA (0,1mg/l) v IBA (0,3mg/l) b sung vo vo mi trng nui cy WPM cho kh nng nhn chi cao nht. Mi tr ng WPM b sung BA (0,1mg/l) v yeast extract (1g/l) c i thin kh nng nhn chi. M i trng WPM + IBA (0,3mg/l) + yeast extract (1g/l) thch h p cho vn thn. Chi pmu in vitro ra r nhiu v di nht trn mi trng WPM + IBA (5 mg/l), sau 45 ngy nui c y TI LIU THAM KHO 1. 2. 3. B NN & PTNT (2000). Tn cy rng Vit Nam, quyn 1 trang 113. NXB Nng nghip. 2 Becwar M. R. (1987). Somatic embryo development and plant vegeneration from embryogenic norway spruce callus . Tappi joural 70 (4). Gamborg O. L. (1986). Protolasts anh plant regeneration in culture . In: demain AL, solomon NA (eds) manual of industrial microbiology and biotechnology. American Society for Microbiology, Washiton, DC. Henry &t Thomas (1991). Fokienia hodginsii. Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture . Comb. Proc. Int. Plant Crop Soc. 30:421-427. Wann S. R. (1989). Biochamical difference embryogenic and nonembryogenic callus of confiers. Tree 3:173-178
4. 5.
6.
SUMMARY
Conservation and development of Pomu (Fokienia hodginsii) by micropropagation techniques

Phm The Anh, Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Shoot tips were used as planting materials. The young Shoot tip was sterilized by treated with Hypochlorite-Na (10%) in 12 minutes. Then, young shoot tip was cultured in the WPM medium supplemented with BA (0,1 mg/l), IBA (0,3mg/l), yeast extract (1g/l) for shoots induction. Elongation and micropropagation on the medium WPM + IBA (0,3mg/l) + yeast extract (1g/l). And rooting on the medium WPM + IBA (5mg/l) after 45 days of cultured
474
NGHIN CU GY TO TRM H NG NHN TO TRM TRN CY D BU (Aquilaria crassna Pierre ex. Lecomte) BNG PHNG PHP VI SINH V HO H C
inh Trung Chnh, H Nng Lm TPHCM Bi Thi Tng Thu, PTNT pha Nam v CNTBTV Trn Vn Minh, Vin Sinh hc Nhit i
M U Cy d bu cn c gi l cy trm hng (Aquilaria crassna Pierre ex Lecomte) l loi cy c sn qu, mc t nhin rng Vit Nam, c xp vo nhm cy g qu him (nhm 1A) cn c bo tn v cm khai thc. Trn nhiu cy d bu c th tm thy cc dng nha tch t gi l trm k hay k nam c gi tr l m thuc v xut khu cao. Tuy nhin, s hiu bit cc c tnh ca loi cy ny cn qu t. M c d c phm vi phn b rng Vit Nam, t Qung Bnh n Thun Hi v huyn o Ph Quc, loi cy ny ang b e da tuyt chng v c xp vo danh mc nhng loi c nguy c b tuyt dit (hng EN). Nhn dn trng cy d bu theo truyn thng t ht, tuy nhin khng phi cy t ht no cng cho trm. Pht hin, nghi n cu v pht trin cc dng d bu ang tng trm, kt hp x l to trm l phng thc nng cao hiu qu cy d bu. Vic phn tch trm v tinh du trm c Erhartdt, L Cng Thun v Hopwood (1997) thc hin bng phng php sc k kh (GC) v sc k kh kt hp vi khi ph (GC/MS), ghi nhn c hai sesquiterpen tn ti ph bin trong g cng nh trong tinh du, l (+)-aromadendrene v (+)-selinene. Theo Phm Hong H (1985) d bu c trm l cy d bu b bnh, ngun gc ca bnh ch gn y mi c bit. Kho st ca Julaluddin (1977) cho thy rng v ng tc cha mt loi nm c xc nh l Cryptosphaeria mangifera . ng xc nhn iu ny bng cch cho nhng cy d bu l nh mnh nhim nm. Sau mt thi gian, vng b xm nhim tr nn xm mu v tr thnh tc r rt v khi t cng ta ra mi trm. Tuy nhin, mc du c mt s du hiu b c u, vic nghin cu ny cn qu ngn i n giai on trm k (K nam). Phi cn 1 0-20 nm na mi c kt qu chc chn. VT LIU V PHNG PHP Nghin cu ny c mc ch gp phn lm sng t c ch v thm d mt s phng php kch thch s to trm. Sau khi iu tra tr n din rng v hin tng to trm v tp on vi sinh vt hin din trong phn g c hin tng to trm, mt th nghim c thc hin bng cch to ra mt vt th ng nhn to trong phn g (xylem) ca nhng cy Aquilaria crassna Pierre ex Lecomte b ng khoan Pressler v a vo vt thng mt s tc nhn kch thch c ngun gc vi sinh hay ha hc. S bin i ca m g trong v ng
475
x l c quan st; s hnh thnh trm c xc nh bng cch i chi u cc kt qu sc k lng cao p trn dch chit ca mu g c s bin i c trng. iu tra thu thp mu: cc cy d bu c du hiu tng trm Kin Giang (rng trng VQG Ph Quc (Bc o) v rng t nhin ( Nam o), Qung Nam (d bu t rng trng 8-10 nm, ng knh 18-25cm ang khai thc cy cn ti), Khnh Ha (d bu t rng trng, mt s t t t nhin, cy c mt kin (trong giai on c tc). Phn lp cc loi vi sinh: S dng phng php thng thng, vi mi trng bt bp nui cy v phn lp vi sinh vt hin din trong cc m g c hin t ng to thnh trm: Bt bp (50g/l) + Agar (20g/l). Mu vt chia l m hai loi, (a) nguyn v (b) kh trng b mt bng cn 70%, dng dao kh trng tch phn g trung gian gia phn cn sng v phn bin tnh cy vo mi trng nui cy. Phn lp bng cch cy chuyn v nh danh nm da vo cc c im hnh thi di knh hin vi v khun lc trong mi trng nui cy. Phn tch ha hc: Phng php sc k kh cao p (GC/MS) c p dng trn dch chit ca mu g c hin tng hnh thnh trm: (1) Chit bng aceton (2) Chit bng CH2Cl2 (3) Chit bng ethanol trong mi trng kim Phng php ly nhim v kch thch: Th nghim c thc hin ba a im: i Lnh (Khnh Ha), Tho Cm Vin (TPHCM), Vn cy (Vnh Long). Mi a im chn 3-4 cy. Dng khoan tng trng Pressler ng knh 10-12mm khoan vo thn cy hay cnh cy n su 5-8cm, cc l khoan cch nhau 20cm v khng nm trn cng ng thng dc th ca mch g. Cy sau khi khoan l c x l vi 7 nghim thc bao gm: - i chng: Khoan, nhng khng x l tc nhn vi sinh hay ha hc - Vi sinh: Hai nghim thc, l hai tc nhn vi sinh phn lp c - Ha hc: Bn ch phm ha hc c thnh phn chnh l (1) du, (2) Cl, (3) SO4, (4) NO3. Cc nghim thc c b tr ngu nhin. Do cc iu kin thc t cc a im nghi n cu, c vi bin i nh trong cch b tr th nghim, tuy nhin, mt s chung ca b tr th nghim hon ton ngu nhin c p dng nh trong Bng 2. Chi tit ng knh, chiu cao v tui cy c ghi nhn trc khi th nghim. KT QU V THO LUN 1. S hin din ca nm tro ng cc m g bin i: Kt qu phn lp v nh danh nm t cc mu vt ly t Qung Nam, Khnh H a v Tho Cm Vin TPHCM c trnh by trong (bng 1). Chng ti cng trnh by trong ct cui cng ca bng ny s i chiu vi kt qu ca mt s cng tr nh gn y v tp on nm trn cc m c bin i tng t ca cc tc gi: Siripatanadilok (1991), Blanchette etal. (2002), v Parman (2002). T kt qu ny, c th kt lun rng s loi nm hin din nhiu nht tr n cc mu vt c xut x Khnh Ha (10 loi), k l Qung Nam (4 loi) v Tho Cm Vin (2 loi). Cc loi c Qung Nam v Tho Cm Vin cng c Khnh Ha.
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Bng 1: Nm phn lp v nh danh c v i chiu vi mt s tc gi khc

Tn nm Khnh Ha Tho Cm Vin Tc nhn c th
Diplodia sp. 1 1 3 Fusarium sp. 1 1 Fusarium lateritium I Erysiphe cichoracearum 1 1 3 Botryodiplodia sp. 1 2 xT Botryodiplodia theobromae Pennicillium sp. 2 x Aspergillus phoenicis 2 x Fusarium sp. 2 Pythium sp. 2 Rhizoctonia sp. 2 Scytalium lignicola 2 x Phialophora parasitica x Torula sp. x Cladosporium sp. x Epicoecum granulatum x Cylindrocladium sp. x Sphaeropsis sp. x Trichoderma sp. x Phomopsis sp. x Cunninghamella echinulata T Phellinus rimosus T Phellinus ferreus T Lentinus edides Ghi ch: 1. Tin Phc; 2. i lnh; 3. Tho Cm Vin; x. Theo Blanchette et al., (2002). T. Theo Siripatanadilok (1991). I. Theo Parman, (2002).
2. Kt qu th nghim ly nhim v kch thch
+ Thnh phn vi sinh vt: Kt qu phn tch vi sinh vt tr n cy th nghim a im i Lnh - Khnh Ha, Tho Cm Vin TPHCM v Vn cy Vnh Long uc trnh by ln lt trong bng 2. Thnh phn nm trong tng a im c v t ng t nhau. Rhizoctonia sp. chim u th c ba a im, cng cc loi nm bt ton khc nhau v c bit l s hin din ca tuyn trng. Cc loi nm bt ton nh Pennicellium sp. ch xut hin nghim thc i chng gy ra s bin m u g c trng, nh hng ln s quan st cc du hiu h nh thnh trm trn phn g x l. Cng c mt s khc bit v thnh phn nm gia cc nghim thc.
Bng 2: Cc loi nm v tuyn trng hin din trn cc nghim thc a im
Nghi m thc
Cy A Cy B Cy C
1
i chng
2
Nm trng
3
Nm en
4
Cl
5
Du
6
SO4
7
NO3
i lnh Khnh Ho Penicillium sp. Rhizoctonia sp. Botryo-diolodia Rhizoctonia sp Scytalium lignicola Diplodia sp. Rhizoctonia sp. Rhizoctonia sp. Tuyn trng Rhizoctonia sp. Fusarium sp. Pythium sp. Botryo-diplodia Rhizoctonia Botryo-diplodia
477
Tho Cm Vin TPHCM

Cy A Cy B Cy C Rhizoctonia sp. Rhizoctonia sp. Pythium sp. Rhizoctonia sp. Tuyn trng Botryo-diplodia sp. Rhizoctonia sp. Rhizoctonia sp. Tuyn trng Rhizoctonia sp. Pythium sp. Rhizoctonia sp. Rhizoctonia sp. Tuyn trng Diplodia sp. Tuyn trng Diplodia sp. Diplodiasp. Tuyn trng Diplodia sp. Thermomyces Tuyn trng Rhizoctonia sp. Diplodia Rhizoctonia sp. Botryo-diplodia Tuyn trng Pythium
Vn cy Vnh Long
Cy A Cy B Cy C Cy D B bt kn Rhizoctonia sp. B bt kn Rhizoctonia sp. Rhizoctonia sp. Macrophoma sp. Botryo-diplodia sp. Trichoderma sp. Botryo-diplodia sp. Rhizoctonia sp. Rhizoctonia sp. Botryo-diplodia sp. Diplodia sp. Rhizoctonia sp. Diplodia sp. Rhizoctonia sp. Diplodia sp.
S bin i ca m g: Cc du hiu ban u ca s to trm xut hin mt thi gian ngn (1 tun) sau khi x l vt th ng trong vng g, c trng bi s bin mu t trng xm sang nu nht, tuy nhi n, s tch ly nha i hi thi gian di hn (bng 3). Kt qu cho thy x l bng Cl mang li kt qu cao hai trong ba a im v cng c xp hng chung cao nht. X l bng nm en v NO3 cho kt qu th 2 i Lnh v Tho Cm Vin; th nht Vnh Long
Bng 3: S bin m u g v s xut hin ca mi c trng khi t
Nghim thc 1 i chng 2 Nm trng 3 Nm en 4 Cl 5 Du 6 SO4 7 NO3
i Lnh - Khnh Ha
CyA CyB CyC Mu Mi trm Mu Mi trm Mu Mi trm Vng + Nu rt nht + Trng 0 Nu rt nht + Nu en + Nu nht + Nu rt nht + Nu nht + Nu nht ++ Nu en +++ Nu +++ Nu + Nu nht 0 Nu nht 0 Nu nht ++ Nu + Nu + Nu ++ Nu nht + Nu + Nu ++
Tho Cm Vin
CyA CyB CyC Mu Mi trm Mu Mi trm Mu Mi trm en nht ++ Nu m ++ Vng nht 0 Nu rt nht + Nu nht + Nu nht + Nu rt nht + Nu m ++ Nu m ++ Nu en +++ Nu en +++ Nu nht + Nu nht 0 Nu nht + Nu nht + Nu + Nu nht + Nu nht + Nu nht + Nu nht + Nu m +++
Vnh Long
Cy A CyB Mu Mi trm Mu Mi trm CyC Cy D Mu Mi trm Mu M trm Trng 0 Nu nht 0 Khng mu 0 Nu nht + Nu nht + Nu nht (sm) + Nu nht + Nu en +++ Nu hi en ++ Nu sm (nht) ++ Nu m + Nu m ++ Nu hi m + en +++ Nu nht + Nu nht + Nu nht ++ Nu en ++ Nu en ++ Nu en ++ Nu nht ++ Nu nht + Nu nht + Nu rt m +++ Nu nht ++ Nu m en +++ Nu nht +++ Nu nht +++
Ghi ch: Mi trm c ng gi bng cch t mu g bin m u vi n cn v kt qu qua 3 ngi nh gi theo thang im: 0 : Khng r ; +: Thong nh; ++: Trung bnh, +++: m mi
478
3. Thnh phn ha hc ca du trm do th nghim ly nhim

Bng 4: Thnh phn cu t (tnh bng %) ghi nhn qua sc k ca 4 mu c hin tng c trm t th nghim kch thch ly nhim t cy A im Vnh Long.
S hiu mu
Tn cu t Benzaldehyde Cyclopentasiloxane, decamethyl 2-Butanone, 4-phenylCyclopentasiloxane, dodecamethyl Cyclohexane, 3,5-dimethyl1,3,6-Octatriene, 3,7-dimethyl2-Butanone, 4-(4-merthoxyphenyl-) C th Alpha-FarnesenceneAcetic acid, (m-(trimethylsiloxy) phenyl -) trimethylsilyl ester Alpha-Eudesmol Acetophenone, 4-methoxyBeta-Eudesmene Elemol X2 Myrtenol Pyrethrolone X3 Cinnamaldehyde Benzaldehyde, 3-(phenylmethoxy)X4 3 Nm en 1.42 1.37 1.47 0.51 4 Cl 1.59 6.25 2.41 3.83 6 SO4 5.16 5.40 2.33 1.59 7 NO3 20.98 30.45 0.73 2.4 1.55 1.55 1.93 2.22 2.45 0.85 5.80 5.09 2.20 0.56 0.20 5.52 12.46
1.83 6.97 1.62 6.05 2.50 45.7 15.19 7.56 3.43 0.32 2.45 1.24 0.33
2.13 3.84 3.88 3.40 1.54 31.13 13.02 8.83 2.20 0.43 8.47 6.59 0.45
4.43 6.17 4.63 4.03 3.48 19.33 16.27 0.32 6.11 16.22
4.53
Ghi ch: X1 v X2: cu t khng nh danh c
KT LUN Kt qu nghin cu gp phn lm sng t mt s c s ca s hnh thnh trm trn cy : S phn lp v nh danh cho thy tn ti mt tp o n nm phong ph, nhng cc bin php x l khc nhau c n h hng ln tp on ny. Phn tch cm quan kt qu cho thy x l bng Cl mang li kt qu cao hai trong ba a im v cng c xp hng chung cao nht. X l bng nm en v NO3 cho kt qu th 2 i Lnh v Tho Cm Vin v th nht Vnh Long. S hnh thnh g trm ch xy ra trn g sng. C th s dng mt s ho cht kch thch s h nh thnh nha cho cc t bo chung quanh vt thng c bit l Clorua. S tch ly nha trm c th pht hin bng cm quan sm nht l mt thng sau khi x l. Nha cha cc tinh cht v c s hin din ca cc terpen v ng thm. Kt qu phn tch gip pht hin mt lot cc hp cht a dng c gc keton, alcol v aldehyde. TI LIU THAM KHO 1. 2. Blanchette R. A. (2002). Cultivated Agarwood. US Patent Application 20020194780 December 26, 2002. Blanchette R. A. (2003). Agarwood Formation in Aquilaria Trees: Resin Production in Nature and How It Can be Induced in Plantation Grown Trees. First International Agarwood Conference Nov.10-15, 2003 HCMC and An Giang, Vietnam, sponsored by EC.
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3.
4.
5. 6. 7.
8.
9.
Ito M. (2003). Sesquiterpenoid Production in Cell Culture of Aquilaria sinensis . In: The First International Agarwood Conference Nov.10-15, 2003 HCMC and An Giang, Vietnam, sponsored by EC. L Cng Kit (2003). History and Ecology of Agarwood in Vietnam. In: the First International Agarwood Conference Nov.10-15, 2003 An Giang, Vietnam, sponsored by EC. L Cng Kit (2005). A New Species of Aquilaria in Vietnam . Seminar, University of Natural Sciences HCMCity. Nguyn Hng Lam. (2003). Nghin cu K thut gy to trm hng trn cy d trm (Aquilaria crassna Pierre ex Lecomte). http://www.mekonginfo.org/mrc/article.nsf/ Nobuchi T. & S. Siripatanadilok (1991). Preliminary observation of Aquilaria crassna-wood associated with the formation of Aloeswood. Bull. Kyoto Univ. Forests. (63) 226-236. Parman & T. Mulyaningsih (2002). Cultivation of Gaharu Tree Toward Sustainable Production System of Gubal Gaharu. Seminar on World Sustainable Development Expo (WSDE 2002), 4-7 Hune 2002, Bali, Indonesia. Siripatanadilok, S. (1991). Utilization and Propagation of Agarwood Trees (Aquilaria spp.). Final Report. IFS Research Grant Agreement D/0731 .
SUMMARY
Studies of resin induction in agawood (Aquilaria crassna Pierre ex. Lecomte) by microbial and chemical techniques
Dinh Trung Chanh(1), Bui Thi Tuong Thu(2), Tran Van Minh(2) (1)University of Agriculture and Forestry HCMC (2)National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Research results give more clear about fundamental of resin induction: Isolation and determination of the microbes show that there was a remaining of a commnity of microbes correlated to the effects of different treatments. If it does not treatment, the tumors was appeared to inhibit the resin formation. The plant was wounded by making a hole through the xylem tissue and treatmented by chemicals and microbes. Analysis by sensivity of the results showed t hat treatments with Cl gave the high effects in 3 sites and graded in the highest. It could be used chemicals especially with Clor to stimulates the resin induction in the surround of the wounds. Agarwood resin is a composed of pured chemical compounds wer e isolated by buffer and to show the presence of terpen compounds. The results of analysis found that there was a complex compound having keton, alcol and aldehyde in structure.
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NUI CY PHT SINH PHI GI (protocorm like body) TRN GING DA Cayenne (Ananas comosus cv. Cayenne L.)
Trn Th Ho, Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Cy da c trng ch yu cc vng nhit i, c gi tr dinh dng rt cao v l mt trong 6 loi cy n tri ch lc ti Vit Nam (Dng Tn Li, 2002). Hin nay, nc ta ang pht trin ngnh trng da trn quy m cng nghip. Trong ging da Cayenne c chn l ging ch lc v c cht lng, nng sut cao, ph hp vi cng ngh ch bin. Tuy nhin, da Cayenne c rt t con ging (1-2 chi/ nm), v vy cng tc ging da Cayenne tr nn cp thit (inh Trng Sn, Nguyn Quang Thch, Nguyn Th Nhn, 2003). Trong cng tc ging da, vic ng dng c ng ngh sinh hc thc vt l rt cn thit (Dng Cng King, 2002). Quy trnh nhn nhanh gi ng da cy m c nhiu tc gi nghin cu v cng b nhng cn nhiu mt hn ch cn c nghin cu v b sung (Nguyn Vn Uyn v cs. 1993; Lm Ngc Phng, ng Phng Trm, 2003). Hin nay, cng ngh phi soma ang c ng dng rng ri vi li th: din tch tc nghip nh, gim thp nht chi ph lao ng, t b o nhn nhanh trong thi gian ngn (Trn Vn Minh, 1997, 2004). Trong bo co ny chng ti tin hnh nghin cu pht sinh phi gi (PLB) trn ging da Cayenne (Ananas comosus cv. Cayenne ) gp phn hon thin quy trnh nhn nhanh da Cayenne phc v cng tc pht trin ging da . VT LIU V PHNG PHP
Vt liu
Vt liu: ging da Cayenne. c nhp t Thi Lan v ang trng kho nghim ti nng trng Th Vc, tnh ng Nai. Mi trng nui cy: mi trng khong MS (Murashige-Skoog, 1962) b sung BA, NAA, Kinetin, Vitamin B 1, Glycin, Tyrosin, Pacloputrazol (PAC), n c da (CW). Mu da Cayenne in vitro (4 chi/cm) c ph nh sinh trng lm vt liu nui cy cho cc th nghim to cm chi, to PLB. To ht nhn to: s dng mi trng MS b sung cht kch thch sinh tr ng v Na-alginate, dung dch nh hnh ht nhn to l CaCl2 bc v bo qun protocorm ca da Cayenne in vitro. iu kin nui cy: mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 282 oC, cng chiu sng 34,2mol/m2/s. Thi gian chiu sng 8gi/ngy.
481
Phng php
Cc nghim thc c b tr ngu nhin vi ba ln lp li, mi ln lp li c cy 5 mu trong bnh tam gic cha 65ml mi trng, 9 mu trong hp nha cha 100ml mi trng. Sau 30-45 ngy nui cy s thu thp s liu, xc nh s chi, chiu cao chi, lng PLB c to, s cy ti sinh, theo d i tnh trng protocorm khi c bc trong ht nhn to. S liu s c x l thng k bng phn mm MSTATC. KT QU V THO LUN
1. To cm chi.
nh hng ca Kinetine n kh nng to chi: khi b sung Ki (1-3-5mg/l) vo mi trng MS, kt qu cho s lng chi cao (trung bnh 61-89 chi/ cm) nhng chiu cao chi cao (2,4-3cm). Vi nng Kin cao (10mg/l) gy pht sinh chi t (trung bnh 53 chi/ cm) nhng chiu cao chi thp (1,5cm) thch hp cho vic to PLB. nh hng ca BA n kh nng to chi: trong mi trng MS c b sung Kin (10mg/l), NAA (0,2mg/l), Tyr (5mg/l), B 1 (5mg/l), Ade (5mg/l), Gly (5mg/l), CW (10%), v BA (0,1-0,5-1-3-5mg/l). Kt qu th nghim cho thy: BA (0,5mg/l) cho s chi t nht (54 chi/cm) v c s khc bit rt c ngha vi cc nghim thc c n li theo thng k, vi BA (1mg/l) cho s lng chi cao nht (t 100 chi/cm). Chiu cao chi khng c s khc bit theo thng k gia cc nghim thc nhng theo quan st, BA nng cng thp th kch thc chi cng ngn thch hp cho vic to PLB. nh hng ca NAA n kh nng to chi: trong nui cy m t bo thc vt, pht sinh chi cn c t l Cytokinin/ Auxin thch hp. Do , th nghim c b tr cho NAA (0,1-0,3-0,5-0,7-1-3-5mg/l) kt hp vi BA (5mg/l) hoc Kinetin (5mg/l) b sung vo mi trng MS + CW (10%). Kt qu th nghim cho thy t hp NAA (3mg/l) + BA (5mg/l) cho s chi nhiu nht (87 chi/cm) v khng c s khc bit vi nghim thc: NAA (5mg/l) + BA (5mg /l) nhng c s khc bit rt c ngha vi cc nghim thc cn li theo thng k, ng thi chiu cao chi t ng i thp. nh hng ca t l BA/ Ki n kh nng to chi: th nghim c b tr cho BA (1-3-5mg/l) kt hp vi Kinetin (1-3-5mg/l) b sung vo mi trng MS + NAA (0,5mg/l), CW (10%). K t qu: t hp BA (1mg/l) + Kin ( 5mg/l) cho s chi nhiu nht (36 chi/cm), chiu cao chi (3,20cm) t ng i thp hn so vi cc nghim thc khc nhng khng ph hp vi mc ch to PLB. nh hng ca t l BA/ Kinetine/ NAA n ph t sinh chi: th nghim c b tr NAA (0,5-1-3-5mg/l) kt hp vi BA (3-5mg/l) v Kinetin (3 -5mg/l) b sung vo mi trng MS + CW (10%). Kt qu: t hp BA (5mg/l) + Kin (3mg/l) + NAA (5mg/l), v t hp BA (3mg/l) + Kin (3mg/l) + NAA (3mg/l) to s chi cao (91 -89 chi/cm), chiu cao chi l 3,4 cm. Trong khi , t hp BA (3 mg/l) + Kin (5 mg/l) + NAA (3 mg/l) c s chi thp hn (83 chi/cm) nhng kch thc chi thp thch hp cho vic to PLB h n.
2. To protocorm like body (PLB)
nh hng ca Pacloputrazol (PAC) n kh nng to PLB: th nghim c b tr
482
PAC (1-3-5mg/l) kt hp vi BA (0,1-0,5-1mg/l) b sung vo mi trng MS + NAA (0,2mg/l) + Tyr (5mg/l) + B 1 (5mg/l) + Ade (5mg/l) + Gly (5mg/l) + CW (10%). K t qu: s pht sinh PLB da Cayenne in vitro c to thnh cng vi nng PAC (3mg/l) kt hp vi: BA (3mg/l) + Kin (5mg/l) + NAA (3mg/l) + CW (10%) cho s PLB cao nht: 86 PLB/ cm.
3. Ti sinh PLB ca cy da Cayenne in vitro
Vi nng BA thp (0,1-0,5-1mg/l) b sung vo mi trng MS + NAA (0,2mg/l) + Tyr (5mg/l) + B 1 (5mg/l) + Ade (5mg/l) + Gly (5mg/l) + CW (10%) th PLB da Cayenne in vitro ti sinh rt tt, gn 100%. ng thi, cy da Cayenne in vitro d cho ra r, c th ra r ngay trn mi trng ti sinh v vic thun ha cy con vn m cng c thc hin d dng.
4. To ht nhn to
S dng Na-alginate (30g/l) b sung vo mi trng MS + BA (1,5mg/l) + NAA (0,1mg/l) to dung dch dinh dng. Dung dch dinh dng + PLB tch ri c nh git vo dung dch nh hnh CaCl 2 (2%) to ht. Ht da nhn to c kh nng bo tn 6 thng trong iu kin nhit 15 oC v ti sinh thnh cy hon ch nh trn mi trng MS khng c b sung cht iu h a sinh trng. Cch thc hin: cho Na-alginate (30g/l) vo mi tr ng MS c BA (1,5mg/l), NAA (0,1mg/l), va un va khuy u cho n khi d ung dch chuyn sang dng do nh tng. Sau em hp v trng 1210C, 1atm trong 25 pht. Ti n hnh bc PLB da Cayenne in vitro c tch ri bng dung dch bao ht trong t cy v tr ng, v ngm vo dung dch CaCl 2 (2%) trong 30 pht nh hnh ht nhn to. Kt qu: Sau su thng, PLB trong h t to vn gi c trng thi ban u. Nh vy, ht nhn to c ng dng thnh cng trong bo qun protocorm da Cayenne in vitro. KT LUN Mu nui cy l chi da ang pht trin in vitro. Chi da c ct loi b l v nui cy trn mi trng khong MS + BA (3mg/l) + NAA (3mg/l) + Kin (5mg/l) + CW (10%) thch hp cho nui cy pht sinh cm chi. Chi da c nui cy to PLB trn mi trng khong MS + PAC (3mg/l) + BA (3mg/l) + NAA (3mg/l) + Kin (5mg/l) + Tyr (5mg/l) + B1 (5mg/l) + Ade (5mg/l) + Gly (5mg/l) + CW (10%). PLB c nui cy ti sinh trn mi trng MS + BA (0,1-0,5mg/l) + CW (10%). PLB c bo qun di dng ht nhn to qua 6 thng tr n mi trng MS + Na-alginate (30g/l) + BA (1,5mg/l) + NAA (0,1mg/l). H t nhn to c nh hnh bng dung dch CaCl 2 (2%). TI LIU THAM KHO 1. Dng Cng King (2002). Nui cy m thc vt (tp 1, 2). NXB HQG.TPHCM
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2. 3.
Dng Tn Li (2002). K thut trng cy n qu - khm (Da). NXB Nng nghip inh Trng Sn, Nguyn Quang Thch, Nguyn Th Nhn (2003). Nghin cu ci tin v xy dng quy trnh sn xut cy ging da Cayenne bt ngun t nui cy m. NXB Khoa hc v K Thut. Lm Ngc Phng, ng Phng Trm (2003). Nhn ging v tnh khm i Nng (Ananas comosus. Merr). NXB Khoa hc v K thut Nguyn Vn Uyn v cs. (1993). Nui cy m thc vt phc v cng tc ging cy trng. NXB Nng nghip. Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cu ltures. Physiol. Plant. 15:431-497 Trn Vn Minh (1997). Cng ngh sinh hc cy n tri. NXB Tr 1997. Trn Vn Minh (2004). Cng ngh t bo thc vt. H Nng Lm TPHCM.
4. 5. 6. 7. 8.
SUMMARY
Studies of tissue culture techniques in protocorm like body induction on cayenne (Ananas comosus cv. Cayenne L.) pineapple
Tran Thi Hao, Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
In vitro pineapple single shoots were cultured for multiple -shoots formation on the basic medium MS (1962) supplemented with BA, NAA, Kinetin, Coconut water. Medium was favoured for multiple -shoots induction was MS + BA (3mg/l) + NAA (3mg/l) + Kin (5mg/l) + CW (10%). The single shoots were cultured to induce PLB on the medium of MS + PAC (3mg/l) + BA (3mg/l) + NAA (3mg/l) + Kin (5mg/l) + Tyr (5mg/l) + B1 (5mg/l) + Ade (5mg/l) + Gly (5mg/l) + CW (10%). PLB was regenerated on the medium MS + BA (0,1 -0,5mg/l) + CW (10%). PLB was conserved through 6 months in the medium MS + Na -alginate (30g/l) + BA (1,5mg/l) + NAA (0,1mg/l). Synthetic seeds were immobilized in the solution of CaCl2 (2%).
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VI NHN GING CY TRAI NAM B (Fagarea cochinchinensis A.chev.) NHM MC TIU BO TN

Khu Hong Minh, Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Cy Trai Nam B (Fagarea cochinchinensis A. chev.) l loi cy g qu him, g Trai thuc g nhm I (Trn Hp, 2002). G c m i chua, mu vng, rt cng, g nng c t trng d=0,85, chu n c v chn lu di t, dng lm ct nh, ng g ni tht cao cp, g xy dng, g lt s n nh, khung tu (Nguyn Thng Hin, 1998; Phm Hong H, 1972). Cy Trai Nam B c xp vo cc loi cy ang b e da v mc e da theo phn hng ca UICN (2001) l CRC (rt nguy cp) v nguy c tuyt chng trong t nhi n cao trong mt tng lai gn (Nguyn Hong Ngha, 1997, 2005). Nui cy nh sinh trng hay chi nh l phng php thng c s dng trong bo tn cy lm nghip (Gladfelter & Phillips, 1987; Trn Vn nh, 2005). Vi nhn ging cy Trai Nam B nhm mc tiu bo tn ngun gen qu ang b tuyt dit v cung cp ngun cy ging sch bnh phc v cng tc ti sinh rng. VT LIU V PHNG PHP
Vt liu
Mu nui cy: Cy trai Ph Quc c s dng lm nguyn liu nghin cu. Cnh cy non khong 30-45 ngy ca cy mt nm tui ngo i vn m c kh trng trong dung dch Hypochlorite-Ca v dung dch HgCl 2 (0,05%). iu kin nui cy: Nhit phng 28+2oC, m 60-70%, thi gian chiu sng 8 gi/ ngy; cng chiu sng 34,2 mol/m2/s. Mi trng nui cy: MS (Murashige-Skoog, 1962), WPM (Lloy-McCown, 1980) c b sung BA (6-benzylaminopurine), IBA ( -indolbutyric acid), CW (n c da), ng sucrose (30g/l), agar (7,5g/l).
Phng php
Th nghim c b tr theo RCB. C 4 ln lp li, Mi ln cy 5 b nh tam gic 300ml, mi bnh tam gic cy 5 mu. Kt qu th nghim c x l bng phn mm MSTATC theo phn tch ANOVA. Sau ki m tra trc nghim phn hng bng LSD vi mc xc sut c ngha P = 0,05.
485
KT QU V THO LUN V trng mu nui cy: Khi s dng ring r Hypochlorite-Ca v trng mu Trai thc sinh th hiu qu kh trng cha cao. C th khi chng ta kh trng nng 25% trong thi gian 30 pht cho t l mu sng v tr ng cao nht l 4,4% so vi cc nghim thc cn li. Cn khi chng ta s dng nng Hypochlorite -Ca (25%) trong 20-30 pht kt hp vi HgCl 2 (0,05%) trong 15 pht th t l mu sng v trng tng ln 6-6,2 % cao nht trong cc nghim thc c n li. T l mu sng v trng tng ln l do dung dch Hypocholorite-Ca c kh nng dit nm tt ng thi dung dch HgCl 2 dit khun kh tt. Tuy nhin, HgCl 2 gy c cho ngi s dng v mi trng. Do , chng ta nn hn ch vic s dng HgCl 2 v ch s dng khi tht cn thit. Nh vy, phng php v trng m u Trai thc sinh tt nht l s dng kt hp Hypochlorite -Ca (25%) trong thi gian 20-30 pht kt vi HgCl 2 (0,05%) trong 15 pht. Nui cy pht sinh chi trn cc mi trng khong c bn: Th nghim gm hai nghim thc s dng mi trng khong c bn l MS v WPM c b sung BA (0,1mg/l). Kt qu thc nghim cho thy: Mi tr ng MS b sung BA (0,1mg/l) kch thch pht sinh chi/cm l 8,6 chi. Trong khi WPM + BA (0,1mg/l) cho pht sinh chi/cm l 15,4 chi. C hai nghim thc c du hiu ph gc chng t cy Trai nhy vi BA (0,1mg/l). Nh vy, mi trng khong c bn c la chn cho cc th nghim tip theo l WPM To cm chi: Th nghim c tin hnh trn mi trng WPM c b sung BA (0-1 mg/l). Kt qu th nghim l nng BA cng tng th kh nng to cm chi cy Trai cng tng theo. Khi nng BA (1mg/l) th cc chi mi hnh thnh c dng li ti rt nhiu, rt nh v chiu cao thp. C th nng BA (1mg/l) th t c s chi/cm l 30,2 chi v chiu cao chi l 10,2mm. Nh vy, nng BA (1mg/l) thch hp cho to cm chi cy Trai in vitro Tc ng ca BA n nhn cm chi cy Trai in vitro: Th nghim c tin hnh trn mi trng WPM + BA (0-0,5mg/l). Kt qu thu c l nng BA cng cao th s chi/cm cng tng v chiu cao thn chi gim dn cc nghim thc. Trong nghim thc c nng BA (0,5mg/l) th s chi/cm l 20,5 chi v chiu cao thn chi l 10,9 mm so vi cc nghim thc cn li. Nh vy, nng BA s dng nhn cm chi l 0,5mg/l Ti sinh cm chi trn cc mi trng khong c bn: Th nghim c tin hnh trn hai mi trng khong c bn l MS v WPM. Kt qu th nghim nhn chi tr n cho thy cm chi cy Trai in vitro pht sinh mnh nhng chiu cao thn chi thp nn cn c giai on trung gian ti sinh cm chi. T thc nghim, cm chi nui cy tr n mi trng WPM ti sinh t c chiu cao l 17,5mm; s chi /cm l 6,5 chi v l pht trin tt (+). Trong khi , nui cy tr n mi trng MS th chiu cao t c l 15,3mm; s chi/cm l 4,3 chi v l km pht trin hn (-). Nh vy, mi trng WPM thch hp cho ti sinh cm chi cy Trai in vitro Vn thn cy Trai in vitro: Th nghim c tin hnh trn mi trng WPM c b sung BA (0,1mg/l) v nc da (0-10 %). Kt qu cho thy cc nghim thc th
486
nghim u c kh nng pht sinh chi. Tuy nhi n, s chi/cm, chiu cao chi, s l sai bit rt c ngha v mt thng k hc vi P = 0,05. Nghim thc khng b sung nc da pht sinh chi km h n so vi hai nghim thc cn li. Khi b sung nc da hm lng t 5- 10% vo mi trng nui cy th s chi/cm, chiu cao ch i, s l tng ln r rt. Hm lng nc da (10%) th pht sinh chi cao nht (15,5 chi/cm), kch thch vn thn chi (32,4mm) v pht trin l (8,4 l). Nh vy, vn thn cy Trai chng ta nn b sung thm nc da vo mi trng nui cy vi hm lng 10% To r cy Trai in vitro: Th nghim c tin hnh trn mi trng WPM c b sung IBA (0-0,5mg/l). Kt qu sau 15 ngy nui cy, cy Trai ra r d dng trn mi trng c IBA nng 0,3mg/l so vi cc nghim thc c n li. Nh vy, mi trng ra r l WPM + IBA (0,3mg/l) KT LUN Phng php v trng m u Trai thc sinh tt nht l s dng kt hp Hypochlorite Ca nng 25% trong thi gian 20 -30 pht vi HgCl 2 0,05% trong 15 pht. Mi tr ng khong c bn c la chn cho cc th nghim l WPM. Nng BA (1mg/l) thch hp cho to cm chi cy Trai in vitro. Nng BA thch hp nhn cm chi l 0,5mg/l. Mi trng WPM thch hp cho ti sinh cm chi cy Trai in vitro. v n thn cy Trai cn b sung nc da vo mi trng nui cy vi hm lng 10%. Mi trng ra r l WPM + IBA (0,3mg/l). TI LIU THAM KHO 1. Gladfelter H. J. & G. C. Phillips (1987). De novo organogenesis of Pinus eldarica in vitro. I. Reproducible regeneration from long -term callus cultures. Plant Cell Rep. 6:163-166 Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture . Comb. Proc. Int. Plant Crop Soc. 30:421-427 Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 431-497 Nguyn Thng Hin (1998). Thc vt v c sn rng. H Nng lm TPHCM Nguyn Hong Ngha (1997). Bo tn ti nguyn di truyn thc vt rng. Nh xut bn Nng nghip Nguyn Hong Ngha (2005). Kt qu nghin cu bo tn ngun gen cy rng. Thng tin chuyn Lm nghip s 1-2005. Vin khoa hc Lm nghip Vit Nam. Phm Hong H (2004). Cy c Min Nam Vit Nam, tp 2. NXB Tr Trn Hp (2002). Ti nguyn cy g Vit Nam. Nh xut bn Nng nghip
2.
3. 4. 5. 6. 7. 8.
487
9.
Trn Vn nh, 2005. Bo tn ngun gen cy thng (Taxus bacata var Wallichiana Zucc.) bng k thut nui cy ti sinh nh sinh tr ng in vitro. Lun vn thc s Khoa hc Nng nghip. H Nng lm TPHCM
SUMMARY
Conservation of Fagarea cochinchinensis A.chev by micropropagation techniques

Khuu Hoang Kinh, Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Sample was sterilized by combination of Hypochlorite -Ca (25%) in 20-30 minutes and HgCl2 (0,05%) in 15 minutes gave the best results. WPM was the b asic medium for cultivation. WPM was supplemented with BA (1mg/l) for multiple -shoots induction, with BA (0,5mg/l) multiple -shoots micropropagation. WPM medium was supplemented with CW (10%) for elongation and with IBA (0,3mg/l) for rooting.
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NH HNG CA CC CHT IU HO SINH TRNG TRONG NHN NHANH CY blueberry (Vaccinium sp.) in vitro
Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Cy du tm blueberry xut thn t mt loi du tm hoang di, c trng trt phc v cho vic trng cy du ly l nui con tm. Sau nhiu nm nghin cu, lai to ging v chn lc ging, cc nh khoa hc Hoa K chn c nhiu ging du tm s dng l cho nui tm v tri l mt loi c sn ca vng n i c gi tr cao trong xut khu (nh km). Hin nay cng nghip blueberry pht trin nhanh cc nc Bc M, Nam M, Chu u, Nam Phi, c, New Zealand, Trung QucV ISHS (International Society of Horticulture Science - www.ishs.org ) t chc 4 cuc hi ngh quc t v nghin cu v pht trin loi cy ny. Hin nay gi xut khu 100g/18USD (Singapore). Cy du tm blueberry l mt loi cy thn bi, c chiu cao thn 1.5-2m; l l loi l n mc cch, dng trong chn nui tm; thch hp cho nhng vng cao 800-1500m. asl, c nhit thp 15-20oC ko di 1500-2000 gi trong nm ra hoa v u tri. Tri l mt loi c sn ca vng n i xut khu phc v cho gii th ng lu v giu c do trong tri c cha nhiu vitamin C v cc dn xut khc c hot tnh sinh hc mang tnh chng oxy ha, hn ch v phng nhng bnh lo suy v ngn chn bnh ung th y loi tri cy khng ht, kh nhn ging truyn thng nh gim cnh, ghp cnh... (Gajdosova etal, 2006). Lt, vng cao nguyn lm vin ca Vit Nam, thch hp cho trng loi cy ny. Vi 100 cy ban u c nhp t c, nay ra hoa kt tri, mang li hiu qu cao. M rng din tch nhm khai thc tt nht iu kin sinh thi ca Lt, th nhu cu v ging trng trt l ro cn u tin. ng dng k thut vi nhn ging l con ng hiu qu ch ng ging (Tsao etal, 2000; OHerlihy etal, 2003) VT LIU V PHNG PHP
Vt liu
Theo cc nh khoa hc c, trong c ging Jubilee v ONeal thch hp cho vng Lt Vit Nam. Ging Jubilee: C chiu cao trung bnh 1.5m, nhit thch hp 15-25oC, thn bi, c l pht trin mnh, tri c mu xanh thm, tri mc ni u nhnh. Tri mc thnh tng chm, kch thc tri trung bnh, cy ra sai tri, thi gian tri chn ko di 2 tun.
489
L vo ma thu chuyn qua mu vng. Ging ny c B Nng nghip Hoa K khuyn co trng nhng vng cao pha Nam ni c nhi t thp 15-20oC ko di 500 gi thch hp cho ra hoa u tri (nh k m) Ging ONeal: C chiu cao trung bnh 1.5-2m. nhit thch hp 15-25oC, thn bi, c l pht trin mnh, tri c m u xanh da tri, tri mc ni u nhnh. Thn cy khe, thn l pht trin mc nhanh tr n cc loi t c kt cu khc nhau v c chn lc trng bao bc bn ngoi v bn trong trng vi ging Jubilee, tng hiu sut u tri. Tri mc thnh tng chm, kch thc tri ln, cy ra sai tri, thi gian tri chn ko di 2 tun. Ging ny c B Nng nghip Hoa K khuyn co trng nhng vng cao pha Nam ni c nhi t thp 15-20oC ko di 500 gi thch hp cho ra hoa u tri (nh km) Mu nui cy: chi non c kch thc 5cm iu kin nui cy: nhit phng 26+2oC, RH=65%, cng chiu sng 33mol/m2/s, thi gian chiu sng 8 gi/ng y Mi trng nui cy: WPM, MS, LV c b sung cc cht iu ha sinh trng BA (6-benzyladenine), 2iP (6-[,-dimethylallylamino] purine [2 -isopentenyladenine]), TDZ (N-phenyl-N-1,2,3-thidiazol-5-yl-ure, thidiazol), NAA (-nathalene acetic acid), IBA (indol-3-butyric acid), IAA (indol-3-acetic acid), nc da (CW)
Phng php
Thc nghim c b tr theo khi y (RBD), c 3 ln l p li, mi ln cy 5 bnh. S liu thu thp c x l thng k bng phn mm MSTATC (p=0.05). Ch ti u theo di: S chi, s pht trin l KT QU V THO LUN Nui cy chi nh: chi non sau khi v trng, c a vo nui cy trn 3 mi trng khong c bn WPM, MS, LV c b sung BA (1mg/l) v TDZ (1mg/l). Kt qu cho thy, mi trng WPM v MS t ra khng thch hp, khng ti sinh c chi. Mi trng LV c b sung BA v TDZ cho kt qu BA khng thch hp, chi xut hin sau thi gian 7-10 ngy th vng l v ch t, TDZ b sung thch hp h n, cho chi pht trin. Nhn chi in vitro: y l giai on quan trng quyt nh tc nhn ging. Chi non ti sinh in vitro c a vo nui cy trn mi trng LV c b sung BA (0.1 -0.51mg/l), TDZ (0.1-0.5-1mg/l) v 2iP (0.1-0.5-1mg/l). Kt qu cho thy: BA khng thch hp cho nhn ging, chi pht trin c hai cp l th vng v cht; TDZ cho kt qu kh quan hn, chi pht trin c 4 cp l v ng li khng tip tc pht trin; 2iP (0.5mg/l) cho pht trin chi mnh m, pht sinh 4 -6 chi cho mt t a vo nui cy ban u, chiu cao chi t 12cm sau 45 ng y nui cy, h s nhn ging l 10-14 ln tnh cho mt ln cy truyn. Nui cy to r: Do l loi cy trng khng c kh nng nhn ging truyn thng, qu trnh ra r cnh gim thp 2-5%. Mi trng LV c s dng cho nui cy, b sung cc loi auxin khc nhau NAA (0.1 -0.5-1mg/l), IBA (0.1-0.5-1mg/l) v IAA (0.1-
490
0.5-1mg/l). Kt qu ghi nhn IAA (0.5mg/l) thch hp cho qu trnh nui cy ra r in vitro, vi tn sut ra r > 90% sau 45 ngy nui cy Thun ha: Thun ha cy blueberry hai iu kin khc nhau: TPHCM, c nhit khng kh trong nh m l 30oC v Lt, l 26oC. Kt qu cho thy t l cy sng sau 10 ngy l 75% (TPHCM) v 95% ( Lt). cy sinh trng v pht trin tt giai on vn m vi chiu cao cy sau 2 thng l 30cm ( Lt) v 12cm (TPHCM) ng rung: Cy cy m ra hoa kt tr i sau 2 nm trng trt, vi nng sut v bi 200g/cy (mt 5000 cy/ha), chiu cao tn t 75cm, chi pht sinh t nhi n 8-12 chi/cy Hin trng: Hin nay nhu cu cn khong 0.5 triu cy pht trin h ng nm. KT LUN Mi trng thch hp cho nhn nha nh in vitro cy blueberry c xc nh: LV + 2iP (0.5mg/l) vi h s nhn ging bnh qun 10-15 ln sau mi ln cy truyn. Mi trng thch hp cho ra r l LV + IAA (0.5mg/l). H thng nhn ging in vitro t ra hiu qu i vi cy trng kh nhn gi ng, nht l cc loi cy n tri TI LIU THAM KHO 1. Gajdosova A, MG Ostrolucka, G Libiakova, E Ondruskova, D Simala. Microclonal propagation of Vaccinium sp. and Rubus sp. And detection of genetic variability in culture in vitro. J. Fruit and Ornamental P lant Res. Vol14, pp103-119, 2006 Tsao CWV, JD Postman, BM Reed. Virus infection reduce in vitro multiplication of Malling Landmark raspberry. In vitro cell dev. Biol. - Plant, Vol36, pp65-68, 2000 OHerlihy EA, JT Crock, AC Cassel. Influence of in vitro fa ctorson titre and elemination of model fruit tree virus. Plant cell, tissue, and organ culture, Vol72, pp33-42, 2003
2. 3.
SUMMARY
Effects of plant growth regulators on micropropagation of blueberry (Vaccinium sp.)

Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
LV was a basic medium supplemented with 2iP (0.5mg/l) favoured for micropropagation and IAA (0.5mg/l) favoured for rooting. Acclimatization of in vitro plantlets were carried out in Da lat with mild climate to enhance the survivals. The trees was primarily flower and fruit after 2 years with yield of 200g/plant. Micropropagation is an effective tools for propagation of difficult fruit trees
491
Effect of Improper Storage of Shrimp Shell Materia ls on the Quality of Chitin and Chitosan
Nguyen Van Toan(1), Tran Van Minh(1,2) (1)School of Biotechnology, International University, Ho Chi Minh, Vietnam. (2)Institute of Tropical Biology Research, Ho Chi Minh, Vietnam
INTRODUCTION
Nowadays, earth-environmental pollution or disruption is becoming a serious problem in the world. The polymer industry has numerous problems regarding recycling or disposal of polymeric waste at the present. Therefore, material development without an accompanying disruptio n of the earth - environment are getting much more important even in the polymer industry. Among them, the natural polymers have undergone a re-evaluation regarding their ability to biodegrade. The international shrimp industries produce a huge pile of was te that consists mainly of heads and shells. Crude shrimp heads and skin materials have only a small economical value and are treated as biowaste or sold to the animal feed manufacturers1. The shrimp biowaste in the tropical region contains 10 -20 % calcium, 30-65% protein content and 8 10% chitin as a dry basis2 (Rao et al., 1996). Chitosan, which is obtained by N deacetylation of chitin, has attracted some attention since chitin is the most abundant polysaccharide. Typically, chitosan is mainly produced from waste generated from the crustacean processing (e.g. shrimp and crab). Chitin and its derivative chitosan are of commercial interest due to their excellent biocompatibility, biodegradability, non -toxicity, chelating, and adsorption power. With these s pecial characteristics, chitin and chitosan have many attractive applications in biotechnology, food and pharmaceutical industry, in cosmetics, environmental engineering, in agriculture and aquaculture. Chitin and its deacetylated derivative chitosan are p roduced from shrimp waste, conventionally using a combination of acid and alkali extraction. Though several standard chemical procedures are widely used to produce chitin and chitosan utilizing shrimp waste as raw material3, there is not much information a bout how to improve the production process so as to produce chitosan with consistent quality, especially from stored material source. The effect of the decaying process that starts as soon as the shrimp waste is discarded or stored at ambient temperature on the quality of chitin and chitosan is unknown or has not been scientifically documented. This paper describes experiments to determine the suitable process for chitin/chitosan production from the decayed shrimp shell, and that suggests a way to utilize a source of low quality shrimp shells, of which the shells have already been soften and degraded at some degrees.
492 MATERIALS AND METHOD S
Shrimp shells from black tiger shrimp (Penaeus monodon) were obtained from a local shrimp processing factory. The shrimp shells were peeled and collected in big plastic bags with addition of ice to preserve the shrimp shell from decomposition. During transportation to the ambient lab (the taken time was one and haft hour), the shrimp waste in the plastic bags was kept in the dark and surrounded with ice outside to avoid the direct sunlight so as to maintain its freshness during transportation. After arrival, the shrimp waste was directly used for making chitosan, following the proposed process. The shrimp shell that used in t his way was named fresh shrimp shell. The collected shrimp shell that wa s being stored in the freezer (at minus 180C) for several months before being used as initial material for making chitosan was named as frozen shrimp shell. In these materials, the chi tin is bound to proteins, inorganic material (mainly CaCO 3) and pigments and lipids. Various procedures have been adopted to remove the non -chitin components. Demineralization is most frequently carried out by treatment with HCl and deproteination by treatment with NaOH but other methods have been used. The sequence of these processes may be varied with different workers, although in most instances deproteination has been carried out prior to demineralization4 . .A difference in the above scheme is that in the revised method, shrimp shell was allowed to decay before it was being used to produce chitin and chitosan. The selected decaying times were 1, 2, 3 and 4 days. A non -decay shrimp shell used as the control for this experiment is marked as 0 day (Figure 1). The decayed shrimp shell was demineralized with 0.68 M HCl solution (1:5 w/v) at ambient temperature (28 -320C) for 12 hrs. The residue was washed and soaked in tap water for 6 -8 hrs. It was then dewatered and deproteinized with 0.62 M NaOH solution (1: 5 w/v) at ambient temperature for 20 hrs. The chitin obtained from above processes was deacetylated in 12.5 M NaOH (1:5 w/v) solution at 65 0C for 20 hrs. After deacetylation, the chitosan was washed and dried in sunlight and assayed for moisture content, a sh content, protein content, degree of deacetylation, viscosity, solubility, turbidity and molecular weight5. The protein content in the chitosan sample was determined using the micro -biuret method6. The degree of deacetylation was analyzed by first deriva tive ultraviolet (UV) spectrophotometry 7. Weight average molecular weight was determined by gel permeation chromatography (Waters GPC) with a differential refractometer detector. Dextrans of various molecular weights ranging from 9.9 x 103 to 2 x 106 were used as standards. Ash content was determined by the standard AOAC - method8. Various physico-chemical criteria were investigated for chitosan in a 1% solution in 0.35 M acetic acid. Turbidity was assessed using a turbidimeter (Model 2100P portable turbidimeter, HACH Company, USA) and viscosity by a Brookfield Model DV - VII + Viscometer. Solubility was measured using the transglucosidase method9: the pH of 50 ml 1% (w/v) chitosan solution was adjusted to 4.8 with 30% (w/v) sodium acetate and mixed with Transglucosidase L-500 (Genencor International, 500 l). After incubation at 600C for 24 hrs, the insoluble material was collected by filtration using a pre -weighed Whatmann GF/C filter paper (1.2 m). The filter paper was dried and weighed and the amount of insolubles was calculated from its weight gain.
493
RESULTS AND DISCUSSI ONS 1. Characterization of chitosan samples
The demineralized shrimp shell, chitin, was determined the ash and protein content to see if the two critical values are below or higher 1%. Th is step is necessary because the quality of the final chitosan product will be affected very much by some characteristics such as the degree of deacetylation, solubility is low and turbidity is high. Table 1 shows the ash and protein content in the chitin samples. The purpose of this study is to adapt the existing protocol to compensate for less quality due to decay of the starting materials and to improve the quality of chitosan product. Hence, chitosan samples obtained from the two different production sc hemes were characterized and collated with each other and with the chitosan samples produced using common method. All chitosan samples had a uniform quality with less than 1% protein content (by Microbiuret assay), 1% ash and 8 -10% moisture content. Most samples had a degree of deacetylation close to 90%, solubility near 100%, and turbidity 20.38 ~ 22.13 NTU. Data on solubility, turbidity, degree of deacetylation and molecular indicate that there is no significant difference among all treatments for the chi tosan produced from fresh and frozen shrimp shell. There is, however, a difference in the ash content, protein content and in particular, the viscosity of chitosan solutions (Table 2 and Table 3). A possible explanation for the highness of ash content in t he chitosan obtained from the fresh shrimp shell compared to the chitosan obtained from frozen shrimp shell is that the matrix of proteins, inorganic material (mainly CaCO 3). Pigments and lipids in the fresh shell are tougher. The mentioned components in t he matrix are closely and strongly linked together, making the naturally existed shell firm enough to tolerate the harsh environmental conditions. Therefore, it is rather difficult for the 4% HCl solution to have proper contact with the shrimp shell and in teract with inorganic material in the shell. However, the shrimp shell that has been subjected to long - term freezing could have been weaken in its calcified structure, making it ease for HCl to contact and react with. The protein content of chitosan sam ples in all treatments are well below 1% (Table 2 and Table 3). The protein content ranges from 0.53% to 0.94% and 0.38% to 0.66% for the fresh and frozen shrimp shells, respectively. It can be seen that the protein content of chitosan produced from fresh shrimp shells higher than those from frozen shrimp shells in every treatment. This may be because of the matrix of protein and another component in the fresh shrimp shell is tougher than that in the frozen shrimp shell. Being stored for several months, the protein in the shrimp shell would have been degraded. Numerous studies on the chitin -protein complexes have been carried out and its biological role was thought to be that of a defense mechanism against the action of chitinases and mild alkaline condition s (Robert 1992)10. For the frozen shell, it might have the loose structure in which the matrixes of proteins, inorganic materials, and lipids have already been weaken during the storage time. Hence, it allows NaOH solution to easily breakdown the protein a nd removes it from the shell. The same behavior has been seen when using HCl solution to demineralize inorganic materials from shrimp shells.
494
Normally, the decaying proc ess accelerates the degradation of biological compositions in the system. It is general ly thought that chitosan produced from this process will have a lower viscosity since chain degradation seems to be unavoidable consequences. However, there are a lot of surprises as shown in the Table 2 and Table 3. The chitosan produced from fresh shrimp shell has the highest viscosity after one day decay. But the chitosan produced from the frozen shrimp shell has the highest viscosity after 2-day decay. The viscosity of chitosan in these two cases was nearly identical. Viscosities of chitosan obtained f rom fresh and frozen shell are 3575 cps and 3577cps, respectively. All these viscosities were found significantly higher (t<5%) than the none-decay treatment. This phenomenon is also supported by the data on molecular weight (Table 4 and Table 5). The reco rded values of Mp, Mn, Mw and polydispersity index had shown that there is no significant difference between the starting materials. The main difference among treatments is Mn values. The 2 -day decays treatment has the highest Mn value of 1.4 M Dalton comp ared to the control at 0 day of 0.9 M Dalton. All the polydispersity index values are less than 2.This shows that no major chain degradation has been occurred during the process. All these information indicates that the 2 - day decay chitosan has the minimum amount of chain degradation during the process.
2. The phenomenon of high viscosity of chitosan solution
The high viscosity of chitosan in the 2 day - decay (chitosan production started with frozen shrimp shell) and 1 day decay (started with fresh materi al) could be a consequence of many parameters. Looking into the processing procedure, there are variables that might bring about the facts of high viscosity (Figure 2). The changes in the protein-chitin complex brought about by the decaying process during 1-4 days may be the result of the action of enzyme in the shrimp tissue, activated during decay or due to the appearance of new metabolites. In case of an enzyme mediated process, the enzyme might act on protein -chitin complex. 1- By an direct action on the CaCO3 2- By an action on the protein scaffold, resulting in an improved exposure of the CaCO3 3- By an indirect action on tissue structures in general. In all cases, it might be assumed that the changes brought about result in a better access of the CaCO 3 for the demineralized agent HCl. Shorter time and lower concentration of HCL may suffice and damage due to hydrolytic action of HCl on the polymer backbone structure of chitin is reduced. In case of new metabolite mediated effect, some particular compoun d might synthesized during decay that helps the demineralization (and deproteination) to occur. An alternative is that the metabolite is activated by preceding the chitin chain to HCl hydrolysis, or to influence its physiochemical properties. Since HCl is much more effective in the destruction of the chitin polymer, the change brought about by the decay are firstly attributed to a more efficient and less damaging action of HCl, more than to an effect on increased deproteination. Deproteination is only damag ing chitosan at high temperature during longer treatment time.
495
a. Indirect examination of the calcified and proteineous structure
Normally, the demineralization process was conducted in 4% HCl for a period of 12hrs. As shown in Table 6, HCl with 2% can red uce the ash content to less than 1% within 6hrs of treatment and attained a result that similar to 4% HCl treatment. Not only the demineralization can be conducted in a much easier way, the deproteination process has also similar observation (Table 7 and F igure 3). In which the protein content treated by 2% NaOH similar to the treatment carried in 4% NaOH. More surprisingly, it took not more than 6hrs to obtain a protein residue of 0.31%. Therefore, accompanying with the result of demineralization, it can be realized that the chemical structure of the shell has been changed during the decaying process. This change has allowed HCl and NaOH to conduct their job in a much easier way, the damage on chitin chain reduced greatly. The statement mentioned above has been strengthened by the viscosity performance of these treatments, in which viscosity of chitosan solution reached higher values (Table 8 and Figure 4)
b. Effect of ash content on viscosity of chitosan sample
As shown in the Table 2 and Table 3, chitosan produced from fresh shell gave the highest viscosity after 1 day-decay and the chitosan produced from frozen shrimp shell gave the highest viscosity after 2 day - decay. The first one was 3591 cps and the later was 3577 cps and they were quite high compared with the other chitosan preparations, especially compared with the chitosan obtained from the shrimp shell taken without decay. The ash content of the chitosan was also relatively high in these treatments. Therefore, it was checked whether a certain amoun t of ash (mainly CaCO 3) present in chitosan would affect the viscosity of a particular chitosan sample. So, an extra amount of as (CaCO 3) was added to the chitosan sample to make the total amount of ash content the same with that of the highest viscosity c hitosan sample (Table 9 and Table 10; Figure 5 and Figure 6). The result indicated that the viscosity of chitosan solution was even decreased after adding CaCO 3. Based on this evidence, it can be said that the amount of CaCO 3 present in chitosan sample wil l not be sufficient to increase the viscosity of that chitosan. The viscosity of studied chitosan may be influenced and impacted by other parameters. Therefore, it may be concluded that the high viscosity of chitosan is not a consequence of higher ash cont ent.
CONCLUSIONS
The viscosity of chitosan sample is a crucial parameter that indicates the quality of the chitosan sample. From the results obtained through this study, it can be concluded that chitosan sample with an enhanced viscosity can be obtained wh en the shrimp biowaste is allowed to a limited decay. In particular, the viscosity of chitosan samples extracted after one day decaying process of fresh shells and two day decaying process of frozen shells has high values. Normally, viscosity, solubility a nd molecular weight of chitosan are closely related. But the result of this study shows that in spite of the increase of the viscosity, other characteristics of chitosan such as the degree of deacetylation, molecular weight, solubility and turbidity are no t changed compared to the chitosan from fresh material.
496
It has been suggested that the amount of ash present in the chitosan sample might affect the viscosity of chitosan solution. But in this study, it has been proven that the increase in the ash content in the chitosan samples does not result in an increase of the viscosity of the chitosan.
REFERENCES
1. 2.
Suchiva K, Chandrkrachang S, Methacanon P and Peter MG, (2002). Advances in Chitin Science.Vol. V. ISBN 974 - 229 - 412 - 7, pp3 Rao MS, Yu P, Stevens WF, Chandrkrachang S, Kungsuwan A., and Hall GM (1996). Drum reactor for extraction of chitin from shrimp waste using lactic acid bacteria. Environmental Friendly and Versatile Biomaterials. (Eds. Stevens WF, Rao MS and Chandrkrachang, S) Bangkok, Thailand, pp 63-67 Roberts GAF (1997). Chitosan production routes and their role in determining the structure and properties of the product, in Advances in Chitin Science Vol.II, Ed. Domard A, Roberts GAF and Vrum KM, Jacquers Andre Publisher, Lyon, France, pp22-30 Roberts GAF (1992a). Preparation of chitin and chitosan: In Chitin Chemistry. First Edition, The Macmillan Press. London.U.K., pp54 -84 Trung TS, Thein-Han WW, Qui NT, Ng CH and Stevens WF (2006). Functional characteristics of shrimp chitosan and its membran es as affected by the degree of deacetylation. Bioresource Technol., pp659 -663 Hein S, Ng CH, Chandrkrachang S and Stevens WF (2002). A systematic approach to a quality assessment system of chitosan, in Chitin and Chitosan - Chitin and Chitosan in LifeScience, ed by Uragami T, Kurita K and Fukamizo T. Kodansha Scientific Ltd. Tokyo, pp332 - 335 Muzzarelli RAA, Rochetti R (1985). Determination of the degree of deacetylation of chitosan by first derivative ultraviolet spectrophotometry. Carbohydrate Polym. 5: 461 - 472 AOAC (1984). Official methods of analysis 14 th, edition. Association of Official Analytical Chemists, Washington, DC Hein S, Ng CH, and Stevens WF (2003). Quantification and characterization of insoluble chitinous materials in viscous chitosan s olutions. Biotechnology Letters 25: 863 - 868
3.
4. 5.
6.
7. 8. 9.
10. Roberts GAF (1992b). Analysis of chitin and chitosan: In chitin Chemistry, First Edition, The Macmillan Press. London.U.K.pp.85 -115
497
SUMMARY
Effect of Improper Storage of Shrimp Shell Materials on the Quality of Chitin and Chitosan
Nguyen Van Toan(1), Tran Van Minh(1,2) (1)School of Biotechnology, International University HCM City (2)Institute of Tropical Biology Research HCM City A special focus on the effect of storage of material sources and the decay ing process on chitin/chitosan quality has been investigated. The shrimp waste materials sources were stored for this purpose was in a 30 oC incubator for different period of time (0 day, 1day, 2 days, 3 days and 4 days). It was found that the chitosan prod uced from shrimp shell brought in fresh form giving the highest viscosity after 1 -day decay and the chitosan produced from shrimp shell in frozen form giving the highest viscosity after 2 -day decay. The viscosities of chitosan obtained from these two treatments were rather high, 3591 cps and 3577 cps for fresh and frozen shell as starting materials, respectively. The viscosities of chitosan solutions obtained from fresh and frozen shell without decay were 2200 cps and 2700 cps, respectively. Normally, viscosity, solubility and molecular weight of chitosan are closely related. But the result of this study shows that in spite of the increase of the viscosity, other characteristics of chitosan such as degree of deacetylation, molecular weight, solubility and tu rbidity are not changed, compared to the chitosan from fresh material.
Apendices (a) Common scheme for chitin/chitosan producti on
Shrimp shells
(b) Scheme under this study

Shrimp shells
Deproteination
Demineralization
Decay (1, 2, 3 and 4 days)
Demineralization
Deproteination
Demineralization: (4% HCl, 30 0C, 12 hrs)
Deproteination (4% NaOH, 30 0C, 20 hrs)
Chitin
Chitin
Deacetylation
Deacetylation (50% NaOH, 65 0C, 20hrs)
Chitosan
Chitosan
498
Figure 1: A comparison between (a) the common process and (b) rev ised process under this study for the production of chitin and chitosan
Shrimp shells (Fresh and frozen) Enzymatic process: - To destabilize tissue - To facilitate Ca ++ removal - To facilitate Ca ++ removal by enhanced deproteination Decay 1 and 2 days 30oC
Demineralization
Action of particular metabolites: - To protect chitin backbone - To combine with chitosan to improve its physio-chemical behavior deproteination
Deproteination
Chitin
Chitosan
499
Figure 2: Possible variables that influence the apparent viscosity of chitosan solution
Figure 3: Change of protein content with different treatment conditions

1 0.8 0.6 0.4 0.2 0 1day 2days 2%/6h 0.31 0.52 2%/12h 0.34 0.55 4%/6h 0.37 0.73
Figure 4: the increased viscosity of chitosan sample
CaCO3
500
Figure 5. Viscosity of chitosan (from frozen shrimp shell) decreased after adding extra CaCO 3
CaCO3
Figure 6.Viscosity of chitosan (from fresh shrimp shell) decreased after adding extra CaCO 3 Table1. Ash and protein content of chitin
Sample Chitin 0 Chitin 1 Chitin 2 Chitin 3 Chitin 4 Ash content (%) Fresh Frozen 0.67 0.02 0.40 0.05 0.61 0.16 0.51 0.04 0.67 0.11 0.41 0.04 0.71 0.06 0.53 0.04 0.77 0.02 0.97 0.04 Protein content (%) Fresh Frozen 0.83 0.16 0.65 0.08 0.78 0.06 0.58 0.05 0.84 0.12 0.48 0.04 0.55 0.06 0.45 0.04 0.61 0.01 0.41 0.06
* The ash content of the original shells was 24.5% * The protein content of the original shell was 23.5%
Table2. Properties of chitosan produced from fresh shrimp shells

Characteristics of chitosan produced from fresh shrimp shells Sample Chitosan 0 Chitosan 1 Chitosan 2 Chitosan 3 Chitosan 4
Ash content (%) 0.68 0.07 0.78 0.04 0.51 0.03 0.27 0.06 0.61 0.02 Protein content (%) 0.86 0.06 0.73 0.05 0.94 0.01 0.53 0.06 0.57 0.01 Solubility (%) 98.80 0.22 98.50 0.42 98.60 0.24 98.70 0.13 97.70 0.06 Degree of deacetylation (%) 88.10 0.12 88.63 0.18 86.20 0.18 87.95 0.20 86.72 0.14 Turbidity (NTU) 21.50 0.58 20.63 0.75 20.38 0.48 21.25 0.50 21.50 0.41 Viscosity (cps) 2195 66 3575 66 2538 80 1230 23 1115 65 Molecular weight (Dalton x10 -6) 1.71 0.03 1.72 0.02 1.74 0.02 1.70 0.02 1.72 0.03
Table3. Properties of chitosan produced from frozen shrimp shells

Characteristics of chitosan produced from frozen shrimp shells Sample
Ash content (%) Chitosan 0 Chitosan 1 Chitosan 2 0.37 0.05 0.55 0.04 0.60 0.04 Protein content (%) 0.66 0.04 0.56 0.04 0.48 0.04 Solubility (%) 98.60 0.06 97.90 0.06 98.70 0.07 Degree of deacetylatio n (%) 87.12 0.82 88.32 0.33 89.82 0.19 Turbidity (NTU) 22.13 0.25 20.46 0.42 20.21 0.25 Viscosity (cps) 2724 323 2060 63 3577 471 Molecular weight (Daltonx10 -6) 1.70 0.05 1.72 0.04 1.75 0.04
501
98.80 0.13 98.80 0.15 89.35 0.92 88.90 0.30 21.44 0.52 21.38 0.48 1953 152 2092 280 1.71 0.05 1.71 0.02
Chitosan 3 Chitosan 4
0.30 0.04 0.27 0.04
0.38 0.04 0.41 0.05
* Chitin/chitosan 0: 0 days of shrimp shell decaying * Chitin/chitosan 1: 1 day of shrimp shell decaying * Chitin/chitosan 2:2 days of shrimp shell decaying * Chitin/chitosan 3: 3 days of shrimp shell decaying * Chitin/chitosan 4:4 days of shrimp shell decaying
Table4. General molecular weight of chitosan obtained from fresh shrimp shell
Sample Chitosan 0 Chitosan 1 Chitosan 2 Chitosan 3 Chitosan 4 Mp ( M Dalton) 1.94 0.003 1.94 0.002 1.95 0.001 1.94 0.002 1.94 0.002 Mn ( M Dalton) 0.90 1.850 1.11 1.100 1.38 1.060 1.10 1.480 0.87 1.650 Mw ( M Dalton) 1.71 0.03 1.72 0.02 1.74 0.02 1.70 0.02 1.72 0.03 Polydispersity 1.82 0.384 1.44 0.125 1.25 0.075 1.48 0.153 1.89 0.434
Table5. General molecular weight of chitosan obtained from frozen shrimp shell
Sample Chitosan 0 Chitosan 1 Chitosan 2 Chitosan 3 Chitosan 4 Mp ( M Dalton) 1.94 0.003 1.94 0.002 1.95 0.001 1.94 0.002 1.94 0.002 Mn ( M Dalton) 0.90 1.850 1.11 1.100 1.40 1.060 1.10 1.480 0.94 1.650 Mw ( M Dalton) 1.70 0.05 1.72 0.04 1.75 0.04 1.71 0.05 1.71 0.02 Polydispersity 1.82 0.224 1.44 0.125 1.25 0.075 1.48 0.153 1.89 0.524
Table6. The Ash content of chitosan sample

Description of demineralization 2% HCl for 6hrs 2% HCl for 12hrs 4% HCl for 6hrs Fresh/1day decay 0.79 0.02 0.72 0.03 0.76 0.02 Ash content (%) Frozen/2 days decay 0.71 0.01 0.67 0.01 0.68 0.02
Table7. The protein of chitosan sample

Description of demineralization 2% HCl for 6hrs 2% HCl for 12hrs 4% HCl for 6hrs Fresh/1day decay 0.31 0.06 0.34 0.07 0.37 0.02 Protein content (%) Frozen/2 days decay 0.52 0.01 0.55 0.05 0.73 0.02
Table8. The viscosity of chitosan sample

Description of demineralization 2% HCl for 6hrs Fresh/1day decay 4790 27 Protein content (%) Frozen/2 days decay 3380 32
502
2% HCl for 12hrs 4% HCl for 6hrs 4812 46 4534 68
3292 24 3188 47
Table9. Adding extra CaCO 3 to chitosan sample (from frozen shrimp shell) to examine its effect on the viscosity performance of each treatment
Sample Initial amount of ash [as CaCO 3] (g) 0.0037 0.0055 0.0060 0.0030 0.0027 Added CaCO 3 (g) After adding CaCO3 ( g) 0.0060 0.0060 0.0060 0.0060 0.0060 Original viscosity (cP) 2724 2060 3777 1953 2092 Viscosity after adding CaCO 3 2090 1780 3577 1650 850
Chitosan 0 Chitosan 1 Chitosan 2 Chitosan 3 Chitosan 4
0.0023 0.0005 0.0000 0.0030 0.0033
Table10. Adding extra CaCO 3 to chitosan sample (from fresh shrimp shell) to examine its effect on the viscosity performance of each treatment
Sample Initial amount of ash [as CaCO 3] (g) 0.0068 0.0078 0.0051 0.0027 0.0061 Added CaCO 3 (g) After adding CaCO3 ( g) 0.0078 0.0078 0.0078 0.0078 0.0078 Original viscosity (cP) 2195 3591 2538 1318 1115 Viscosity after adding CaCO 3 1820 3591 2178 1025 980
Chitosan 0 Chitosan 1 Chitosan 2 Chitosan 3 Chitosan 4
0.0010 0.0000 0.0027 0.0051 0.0017
503
NH HNG CA CC CHT IU HO SINH TRNG N QU TRNH NUI CY PHT SINH T BO soma V PHI V TNH CY HOA LAN (Dendrobium, Phalaenopsis, Cymbidium )
Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV. Vin Sinh hc Nhit i
M U Nhng k thut ca cng ngh t b o pht trin nhanh trong nhng nm gn y. Pht sinh phi soma v pht sinh hnh thi l k thut nui cy c ng dng nhiu trong nhn nhanh cc loi cy tr ng. Do vy, nghin cu pht sinh v ti sinh in vitro l nhng nghin cu c bn ban u cho cc ng dng v sau. H n na, k thut nui cy dng ha t bo phi ha in vitro t ngun nguyn liu di truyn ban u xc nh cho php sn xut mt qun th ng u c c im di truyn ging nhau, kh nng ti sinh in vitro cao, sinh trng nhanh v ng dng (Wang etal. 2002). Dendrobium c nui cy ti sinh thnh cng t t bo soma (Meesawat v Kanchanapoom, 2002). Sagawa (1990ab) nui cy to t bo soma thnh cng. Ichihashi v Hiraiwa (1996) nghin cu s sinh trng v pht trin ca t bo soma. V Kobayashi etal (1993) ti sinh thnh cng t bo soma. Phi gi (PLB) ca Cymbidium cng c nui cy thnh cng ca Begum etal (1994b), Huan v Tanaka (2004ab), Nhng nghin cu hin i v cng ngh t bo thc vt ang tp trung nghin cu kh nng nui cy pht sinh t bo soma c kh nng phi ha. Trong qu tr nh nui cy, mi trng dinh dng khong v cc cht iu ha sinh trng tc ng mt cch tch cc qua cc giai on pht trin in vitro. Trong cng nghip hoa lan, ging l tin pht trin, v cng ngh vi nhn ging chim vai tr pht trin cc ging cy con cht l ng cao c gi thnh h. Dendrobium, Phalaenopsis v Cymbidium c th trng tiu th ni a v xut khu vng chc; tuy nhin, cng tc ging vn l ro cn u tin pht trin. Ging nhp hin nay ch yu t Trung Quc, i Loan, Thi Lan vi gi thnh cy con cao. Gii quyt k thut nhn ging nhanh l yu cu bc thit ch ng pht trin hoa lan thnh ngnh cng nghip. VT LIU V PHNG PHP Nguyn liu Ging: Dendrobium (D. Sonia 17) Phalaenopsis (P Sweet Candy) Cymbidium (Cym Enzan Myth) Mu nui cy: L chi non in vitro, phi gi in vitro (PLB)
504
Mi trng nui cy: VW (Vacin and Went), c b sung BA (6-benzyladenine), NAA (-nathalene acetic acid), 2.4D (dichlorophenoxy acetic acid), TDZ (N -phenyl-N1,2,3-thidiazol-5-yl-ure, thidiazol), nc da (CW), kinetin (6 -fufurylaminopurine), peptone, than hot tnh (AC) iu kin nui cy: nhit phng 26+2 oC, RH: 65%, thi gian chiu sng 10 gi/ngy, cng chiu sng 33.3mol/m 2/s Phng php Thc nghim c b tr theo khi y (RBD), c 3 ln lp li, mi ln cy 5 bnh. S liu thu thp c x l thng k bng phn mm MSTATC (p=0.05). Ch ti u theo di: kh nng pht sinh t bo soma (%), kh nng pht sinh phi gi PLB (protocorm like body), kh nng ti sinh chi (%) KT QU V THO LUN Phong lan (Dendrobium) Nui cy pht sinh t bo soma: Mu nui cy l chi non, c ct thnh lt mng, nui cy trn mi trng VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) + peptone (1g/l) + sucrose (20g/l) + AC (1g/l). Sau th i gian 30 ngy nui cy, t bo soma pht sinh trn vt ct. T l to t bo soma l 100% trn cc m u nui cy. T bo soma, c mu trng sng, d dng nhn sinh khi Nui cy tng sinh t bo soma: T bo soma c nui cy tng sinh trn 6 mi trng thc nghim. Kt qu cho thy, t b o soma tng sinh mnh m trn mi trng VW + CW (10%) v VW + BA (1mg/l) + NAA (0.3mg/l) + peptone (1g/l) Nui cy pht sinh v tng sinh phi gi (PLB): T bo soma tng sinh c nui cy trn mi trng thc nghim. Kt qu cho thy, phi gi pht sinh v tng sinh nhanh trn mi trng VW + CW (10%) v VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%). Nui cy ti sinh PLB: Phi gi c kh nng ti sinh trn cc mi trng thc nghim. Hiu qu trn mi trng VW + peptone (1g/l) v VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) Nhn xt: trn mi trng c bn VW, cc cht iu ha sinh trng b sung BA, NAA kch thch pht sin h t bo soma v phi gi (bng 1) H ip (Phalaenopsis) Nui cy pht sinh t bo soma: Mu nui cy l phi gi (PLB) in vitro c ct lp mng. Lp mng c nui cy trn mi trng VW c b sung 2.4D (0 -0.11mg/l), BA (0-0.01-0.1-1mg/l), sucrose (40g/l), CW (20%). Kt qu cho thy sau 4 tun: mi trng VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + sucrose (40g/l) thch hp cho nui cy pht sinh t b o soma v PLB (bng 2)
505
Nui cy tng sinh t b o soma: T bo soma c nui cy tng sinh mnh m trn mi trng VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + sucrose (40g/l) + CW (20%) sau 4 tu n. Nc da kch thch qu tr nh phn chia v bi t ha t bo. Nui cy pht sinh phi gi (PLB): T bo soma c nui cy pht sinh phi gi trn mi trng c ng v khng c ng sucrose. Kt qu cho thy sau 4 tun: t bo soma tip tc tng sinh khi tr n mi trng VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + sucrose (40g/l) + CW (20%) v hnh thnh phi gi trn mi trng khng ng VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + CW (20%) (bng 3) Nui cy ti sinh phi gi (PLB): phi gi c nui cy ti sinh trn mi trng VW + NAA (0.1mg/l) + BA (0.1mg/l) + CW (20%) + sucrose (20g/l) + AC (1g/l) Nhn xt: T bo soma pht sinh v tng sinh kh i mnh m trn mi trng VW c b sung 2.4D+BA+Sucrose v s hnh thnh phi gi khi khng c b sung ng vo mi trng. Phi gi c ti sinh trn mi trng VW+BA+NAA+sucrose a lan (Cymbidium) Nui cy pht sinh v tng sinh t bo soma: Phi gi in vitro c ct thnh lt mng v c nui cy trn mi trng VW + 2.4D (0.1mg/l) + Kinetin (0.1mg/l) + peptone (1g/l) + sucrose (30g/l). T bo soma pht sinh sau 30 ngy nui cy, t l pht sinh t 50 -60% trn s mu nui cy. T b o soma c tch ra v nui cy tng sinh mnh m trn cng mi trng, thi gian gia hai ln cy truyn 30 ng y. Nui cy pht sinh phi gi (PLB): T bo soma c nui cy trn hai mi trng pht sinh phi: VW + NAA (0.1mg/l) + Kinetin (0.1mg/l) + peptone (1g/l) + sucrose (20g/l) v VW + NAA (0.1mg/ l) + TDZ (0.1mg/l) + peptone (1g/l) + sucrose (20g/l). K t qu cho thy: phi gi h nh thnh sau 50 ngy nui cy trn c hai mi trng, cho t l hnh thnh phi gi cao >90% (bng 4) Nhn xt: Mi trng VW c b sung NAA+Kinetin(+TDZ)+ Kinetin+CW quyt nh n qu trnh pht sinh v tng sinh t bo soma v PLB KT LUN Mi trng VW b sung BA, NAA, kinetin, TDZ, n c da, ng sucrose thch hp cho nui cy pht sinh v tng sinh t bo soma, pht sinh v ti sinh phi gi (PLB). Cng ngh t bo soma l cng ngh nn cho nhn ging cng nghip trong bioreactor
506
Bng 1: Kh nng nui cy pht sinh v tng sinh t bo soma, pht sinh phi gi (PLB) v ti sinh trn loi phong lan dendrobium
Cc giai on nui cy Nui cy pht sinh t bo soma Mi trng nui cy Sau 4 tun VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) + peptone (1g/l) + sucrose (20g/l) Sau 4 tun VW VW + CW (10%) VW + peptone (1g/l) VW + BA (1mg/l) + NAA (0.3mg/l) VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) VW + BA (1mg/l) + NAA (0.3mg/l) + peptone (1g/l) Sau 8 tun VW VW + CW (10%) VW + peptone (1g/l) VW + BA (1mg/l) + NAA (0.3mg/l) VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) VW + BA (1mg/l) + NAA (0.3mg/l) + peptone (1g/l) Sau 12 tun VW VW + CW (10%) VW + peptone (1g/l) VW + BA (1mg/l) + NAA (0.3mg/l) VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) VW + BA (1mg/l) + NAA (0.3mg/l) + peptone (1g/l) Kt qu T bo soma hnh thnh trn mt ct. T l hnh thnh t bo soma 100% Tng sinh t bo soma / 100mg nui cy ban u 55 a 82 ab 95 ab 61 a 102 ab 115 b Tng sinh phi gi / 100mg nui cy ban u 45 + 15 ab 124 + 48 d 51 + 20 ab 46 + 12 a 215 + 51 cd 122 + 43 bc S chi / 1mg PLB 7.2 ns 3.2 ns 16.1 ns 6.8 ns 10.6 ns 0.0
Nui cy tng sinh t bo soma
Nui cy pht sinh v tng sinh phi gi (PLB)
Nui cy ti sinh chi
Bng 2: S hnh thnh t bo soma v phi gi (PLB) trn hoa lan h ip

Suc 40 40 40 40 40 40 40 0 40 PGR (mg/l) 2.4D BA 0 0 0.1 0 0.1 0.01 0.1 0.1 1 0 1 0.1 1 1 0 0 0 0 CW (%) Tng s mu 25 25 25 25 25 25 25 25 25 Cal(+++) Mu nui cy Cal(++) Cal(+) 4.5 10 9 4.5 9 8.5 2.5 11 5 3 6.5 7.5 5 7 0 0 5.5 2.5 Cal+PLB 6.5 7 2 1 2.5 1.5 2 0 3.5 PLB 2 1.2 2.2 1 0 2.2 1.5 24 0
20 20
2 0.2 1 0 0 0 13
Cal(+++): t bo soma mu vng ln, Cal(++): t bo soma mu vng nh, Cal(+): t bo soma mu vng rt nh, PLB: phi gi
Bng 3: S hnh thnh phi gi (PLB)
Sucrose (g/l) 0 20 Tng sinh khi t bo soma (s lng/ 100mg.cm) Tng s mu Tng sinh t bo soma Phi gi (PLB) 25 0 25 25 25
507
Bng 4: S hnh thnh phi gi

Mu nui cy PLB % PLB 92.2a 86.4b S PLB/mu 7.8a 4.2b % T bo soma 1.8b 5.9a
TI LIU THAM KHO 1. Begum AA, M Takami, M Tahara, S Sako. Somatic embryogenesis in Cymbidium through in vitro culture of inner tissue of protocorm -like body. J. Japan Soc. Hort. Sci. Vol63, pp419-427, 1994a Begum AA, M Takami, S Kato. Formation of protocorm -like body and shoot development through in vitro culture of outer tissue of Cymbidium. . Japan Soc. Hort. Sci. Vol63, pp663-673, 1994b Meesawat U, K Kanchanapoom. In vitro plant regeneration through embryogenesis and organogenesis from callus culture of pigeon orchid ( D. crumenatum Sw.). Thamasat Intl.J. Sc.Tech. Vol7, No2, pp9 -17, 2002 Huan LVT, M Tanaka. Effec ts of red and blue light emitting diodes on callus induction, callus proliferation and protocorm -like body formation from callus of Cymbidium orchid. Emviron. Controll Biol. Vol42, pp57 -64, 2004a Huan LVT, M Tanaka. Callus induction from protocorm -like body segments and plant regeneration in Cymbidium. Emviron. Controll Biol. Vol79, pp406 -410, 2004b Ichihashi S, H Hiraiwa. Effects of solidifiers, coconut water, carbohydrate source on growth of embryogenic callus in Phalaenopsis and allied genera. J. Orchid Soc. India, Vol10, pp81-88, 1996 Ishii Y, T Takamura, M Goi, M Tanaka. Callus induction and somatic embryogenesis of phalaenopsis. Plant cell report, Vol17, pp446 -450, 1998 Jaime A, T da Sylva, N Singh, M Tanaka. Priming biotic factors for optimal protocorm like body (PLB) and callus induction in hybrid Cymbidium and assessment of cytogenetic stability in regenerated plantlet. Plant cell, tissue, and organ culture, Vol84, pp135 -144, 2006 Kobayashi S, T Kameya, S Ichihashi. Plant regeneration from protoplast s derived from callus of Phalaenopsis. Plant Tissue Cult. Letters, Vol10, pp267 -270, 1993
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7. 8.
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10. Sagawa Y. Biotechnology in orchid. In: KimuraT, S Ichihashi, H Nagata (eds) Proc. Nagaya Intl. Orchid Show 1990. NIOS, Kariya, pp46 -48, 1990a 11. Sagawa Y. Orchid, other considerations. In: Ammirato PV, DA Evans, WR Sharp, YSP Bajaj (eds) Handbook of plant cell culture, Vol5. McGraw -Hill, New York, pp638-653, 1990b 12. Wang Z, D Lehmann, J Bell, A Hopkins. Development of an efficient plant regeneration system for Russian wildr ye. Plant cell report, Vol20, pp797 -801, 2002
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SUMMARY
Effects of plant growth regulators on somatic cell and protocorm-like body (PLB) induction, proliferation and regeneration of Dendrobium, Phalaenopsis and Cymbidium
Young shoots of Dendrobium were used as samples. Samples were cut into small pieces and was cultured for somatic cell induction on the medium VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%) + peptone (1g/l) + sucrose (20g/l) + AC (1g/l). After 30 days, somatic cells were induced on cut surface. Somatic cell has white color and proliferated strongly on the medium VW + CW (10%) v VW + BA (1mg/l) + NAA (0.3mg/l) + peptone (1g/l). Somatic cell was induced on the medium cultured VW + CW (10%) v VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%). And regeneration on the medium VW + peptone (1g/l) v VW + BA (1mg/l) + NAA (0.3mg/l) + CW (10%). On the basic medium VW, plant growth regulator supplemen ted as BA, NAA were favoured for induction of somatic cell and embryogenesis Young PLB of Phalaenopsis were used as samples and were cultured on the medium VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + sucrose (40g/l) for somatic cell induction after 4 weeks. Soma tic cells were proliferated on the medium VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + sucrose (40g/l) + CW (20%) after 4 weeks. Coconut water stimulates cell division and differentiation. Somatic cells were cultured for embryogenesis after 4 weeks to form PLB on the medium VW + 2.4D (0.1mg/l) + BA (0.01mg/l) + sucrose (40g/l) + CW (20%). The medium supplemented with sucrose for somatic cells induction continuously and free -sucrose for PLB induction. PLB s were regenerated to plantlets after 4 weeks on the medium VW + NAA (0.1mg/l) + BA (0.1mg/l) + CW (20%) + sucrose (20g/l) + AC (1g/l). Somatic cells proliferated strongly on the medium VW+2.4D+BA+sucrose and PLB formation on the free -sucrose medium. PLBs were regenerated on VW +BA+NAA+AC+sucrose Cymbidium PLBs were cut to the segments and were cultured for somatic cell induction and proliferation after 30 days on the medium VW + 2.4D (0.1mg/l) + Kinetin (0.1mg/l) + peptone (1g/l) + sucrose (30g/l).. Somatic cells were cultured for PLB induction after 50 days with t e rate of over 90% performance on the medium VW + NAA (0.1mg/l) + Kinetin (0.1mg/l) + peptone (1g/l) + sucrose (20g/l) or VW + NAA (0.1mg/l) + TDZ (0.1mg/l) + peptone (1g/l) + sucrose (20g/l).
509
NG DNG CNG NGH T B O THC VT NHN NHANH LOI G QU GI T (Tectona grandia Linn.F)
M U Cy gi t t nhin phn b v tuyn 9 o-26oN v kinh tuyn 73o-104oN trong rng nhit i trung v nam n , Myanmar, bc Thi Lan v Lo (White, 1991). Cy gi t cn c pht trin nhiu n cc quc gia khc trong khu vc ng Nam (Indonesia, Sri Lanka, Vit nam, Malaysia, Solomon), Chu Phi (Ivory Coast, Nigeria, Togo), trung v nam Chu M (Costa Rica, Brasil). y l loi cy g qu, do cht lng g bn vng, c s dng nhiu trong xy dng v cng nghip, c xp hng u trn th gii. Tuy nhin, sn xut khng tha mn c nhu cu (Dupuy, 1990). iu ny dn n vic pht trin trng gi t cht l ng cao vi cy con ging thun chng (Wellendorf etal, 1987; Kaosa -ard, 1986; Mascarenhas & Muralidharan, 1993). Trong dn gian, cy gi t c nhn ging bng ht v stump. Cy gi t c nhn ging bng ht c nhiu hn ch v cht l ng (Mascarenhas etal, 1987; Kaosa -ard, 1986) nh: Kh nng sn xut ht ca cy thp (Wellendorf & Kaosa-ard, 1988; White, 1991). Khi cy ra hoa, thn phn nhnh ph t trin mnh, thn thng v di thn quyt nh gi bn bun nn ph thuc nhiu vo kh nng duy tr chi nh trong thi gian di (White, 1991). T l ny mm thp (Kaosa ard, 1986; Mascarenhas etal, 1987; White, 1991). a d ng v di truyn lm gim cht lng g (Dupuy & Verhaegen, 1993; Mascarenhas & Muralidharan, 1993). Cho n nay, con ngi vn cn gii hn kin thc v di truyn cc c tnh (Wellendorf & Kaosa-ard, 1988) Nhn ging bng hom, hay cn c gi l stump cy gi t l nhn ging v tnh vi s lng ln mt hn hp cc kiu di truyn khc nhau m khng duy tr bt k mt tnh cht c th no. Phng php ny cho php tng s lng cc kiu di truyn c tr ha, ng thi cng gia tng tnh khng ng u trong qun th g sn xut v iu ny dn n tng tnh bin d cy gi t. Nh ng duy tr c c tnh tng kiu di truyn v ph thuc vo tc nhn ging... sn xut ra nhng chi gi t thch hp c tim nng tt ra r. Nhn ging bng phng php CNTBTV, th c tnh di truyn c duy tr qua chu k nhn v tnh v bo lu hng th k. m bo c c tnh di truyn cy m v to ra qun th cy rng ng u (Zobel & Talbert, 1984; Timmis, 1985; Ahuja & Libby, 1993a,b). Hn na, kiu di truyn c chn lc vi nhn ging th hin hin tng thun thc v sinh l nh gim hay mt hn tim nng ra r nhnh (Timmis, 1985; Wareing, 1987). Mt c tnh sinh l tr, l trng thi cn thit cho hom ra r.
510
Kh nng ra r bt nh (Monteuuis etal, 1995) trong vi nhn ging cy gi t dn n cy con ging cy m v ng u v hnh dng. Nhng c tnh qu hin nay ca cy gi t u dng cn c nhn rng pht trin: sinh tr ng nhanh, ra hoa chm, thn thng v cc tnh cht g qu khc (Zobel & Talbert, 1984; Wellendorf & Kaosa -ard, 1988). Tnh ng u khng c c khi nhn ging bng ht hay stump. VT LIU V PHNG PHP
Vt liu:
Mu nui cy: Mu nui cy l chi non t stump cy gi t ging Golden Teak ca Myanmar, c kch th c 10-15mm. c ra sch bng x phng v v trng bng HgCl2 trong 7 pht. Nui cy v trng: Sau khi c v trng, mu nui cy c ct thnh tng mnh nh c cha chi non, l vng nhu m nh sinh trng, c kch thc 3-5mmm v c cy vo nui cy trn mi trng MS. Sau 20 ngy nui cy, chi non pht sinh in vitro, chi non c s dng lm nguyn liu trong cc nghin cu in vitro. iu kin nui cy: Mi trng v dng c nui cy c v trng 121oC v 1at trong 25 pht. Bnh nui cy l bnh tam gic 300ml cha 65ml mi trng nui cy v c y kn bng np cao su hay np giy. Hay c cy vo hp nha 500ml c cha 100ml mi trng nui cy v c y kn bng np nha. Nhit ph ng nui cy l 28+1oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng l 8gi/ngy. Mi trng nui cy: l mi trng MS (Murashige-Skoog, 1962) hay WPM (Lloyd & McCown, 1981), c b sung nc da (CW), Tyrosin, Adenine sulfate v cc cht iu ha sinh trng nh BA (6-benzyl amino acid), IAA (b -indol acetic acid), IBA (b-indol butyric acid), NAA (a-naphthalene acetic acid).
Phng php:
Cc th nghim c b tr theo phng php RCBD (1 yu t) v RCD (2-3 yu t), c 4 ln lp li, mi ln nui cy 5 b nh (bnh tam gic hay hp nha), mi bnh cy 5 mu. S liu c o c vo ngy th 60 sau nui cy cho tt c cc th nghim. S liu thu c, c x l bng phn mm thng k MSTAT theo M, CV%, LSD(0.05). Cc ch tiu theo di nh: chiu cao thn (mm), s t (no), s l (no), chiu d i l (mm), s pht trin l (+/-), chiu rng l (mm), t l ra r (%), s r (no), chiu di r (mm), thi gian xut hin r (ngy) KT QU V THO LUN
1. Nui cy chi nh sinh trng cy gi t in vitro
Mi trng c bn MS v WPM u thch hp cho nui cy chi nh sinh tr ng gi t. Trn hai mi trng c b sung BA (0,1 v 0,5mg/l) u tc ng n kh nng pht sinh chi. Tng tc gia mi trng nui cy v BA khng c ngha v thng
511
k. V tr tuyt i, mi trng WPM t ra thch hp cho qu tr nh pht sinh chi so vi MS qua thi gian ti sinh chi ngn h n, c hai nng BA
2. Nhn nhanh cy gi t in vitro
nh hng ca mi trng dinh dng n nhn nhanh cy gi t in vitro: Mi trng nui cy (A) khng tc ng c ngha n cc ch ti u sinh trng: chiu cao thn, s t, s l, chiu di l. Tuy nhin rt c ngha n ch tiu pht trin chiu rng l. Vi mi trng nui cy MS, mu nu i cy l chi ngn (N) c s pht trin hn mu nui cy l t thn (); iu ny khng cho thy s khc bit gia hai mu khi nui cy trn mi trng WPM. Trn mi trng MS, mu nui cy N cho pht trin rng l hn mu nui cy ; trn mi trng WPM, khng sai khc gia hai mu nui cy. Mu nui cy pht trin tt tr n mi trng WPM so vi MS. M t thn () l n v nhn ging v mi trng WPM c chn s dng cho cc th nghim vi nhn ging v sau. nh hng ca khong a lng WPM v BA n nhn nhanh cy gi t in vitro : Yu t hm lng khong a lng mi trng WPM (A) nh hng c ngha n cc ch tiu s t, s pht trin chiu d i l v rng l. Trn mi trng khong chun WPM nh hng tt ln cc ch tiu ni trn so vi WPM/2. Trong mi tng tc (AxB) WPM nh hng c ngha trn ch tiu chiu cao thn so vi WPM/2 cc nng BA (0-0,1mg/l). Trong mi tng tc (AxBxC), WPM nh hng c ngha n ch tiu chiu cao thn v chiu rng l so vi WPM/2 cc nng BA. Vi hm lng khong chun WPM v nng 0,1mg/l BA cho kt qu c ngha cc ch ti u chiu cao thn, s t, chiu di l v chiu rng l trn c hai loi mu nui cy N v . nh hng ca Tyrosin n nhn nhanh cy gi t in vitro: Trn nn mi trng WPM, Tyrosin (10mg/l) c nh hng r rt n cc ch tiu sinh trng chiu cao, s l, s pht trin di l v rng l. Trong tng tc (AxB) nh hng n ch tiu s l. Tr tuyt i ca ch s s l hai loi mu nui cy c ngha nng 1 0mg/l Tyrosin so vi 0 v 30mg/l. Tyrosin l mt loi acid amin c s dng nhiu trong vi nhn ging kch thch pht sinh chi v tng cng pht trin thn l. nh hng ca Adenine sulfate (AS) n nhn nhanh cy gi t in vitro: Adenine sulfate c ngha n s pht trin di l. nng 10mg/l c ngha n s pht trin di l trn hai loi mu nui cy, nht l vi mu nui cy l t thn. Tng tc (AxB) c ngha trong s pht trin s t. S c mt ca Adenine sulfate dn n hiu qu tng t nh b sung BA vo mi trng nui cy (khi nng Adenine sulfate l 0mg/l). BA hay Adenine sulfate ng vai tr kch thch pht sinh chi v pht trin l bnh thng loi cy l rng. nh hng ca BA v nc da (CW) n nhn nhanh cy gi t in vitro: BA (A) tc ng su sc n cc ch ti u sinh trng chiu cao thn, s t, s l v s pht trin chiu di l; kt qu ghi nhn nh trong th nghim 3. Nng 0,1mg/l BA cho cc ch s sinh trng v pht trin tt hn nng 0,5mg/l BA. Tng tc (AxB) tc ng c ngha n cc ch ti u sinh trng chiu cao thn, s pht trin d i l v rng
512
l qua t hp [BA (0,1mg/l) + CW (5%)]. T ng tc (AxBxC) tc ng c ngha n cc ch tiu s t, s l, v chiu di l. Vi hai loi mu nui cy, chi ngn (N) v t thn (), cho kt qu c ngha trong t hp [BA (0,1mg/l) + CW (5%)] khi so snh gia hai loi mu nui cy. Vi nng thch hp ca BA, n c da khng c vai tr r rt, ngc li hiu qu tc ng ca n c da cao khi nng BA s dng thp nh hng ca th tch bnh nui cy v s thng gi n nhn nhanh cy gi t in vitro: Th tch bnh nui cy (A) tc ng n cc ch ti u s t, s l, s pht trin di l v rng l. Bnh nui cy c thng kh tt hn, th hin qua y np giy hay hp nha (th tch ln) cho cc ch s sinh tr ng tt hn h kn (y bng np cao su). Tng tc (AxB) c tc ng su sc n cc ch ti u sinh trng chiu cao thn, s t, s l v s pht trin rng l. Th tch b nh nui cy v tng cng trao i kh c nh hng su sc n sinh trng cy con in vitro. nh hng ca cng chiu sng n nhn nhanh cy gi t in vitro: Cng chiu sng (A) tc ng su sc n cc ch ti u sinh trng. Vi cng chiu sng 22,8 mol/m 2/s cho cc ch s sinh trng v pht trin tt hn so vi hai cng chiu sng c n li. Cng chiu sng thp (11,4 mol/m 2/s) v cao (34,2-45,6 mol/m 2/s) khng ci thin sinh trng. Tng tc gia (AxB) tc ng c ngha n cc ch ti u sinh trng nh chiu cao thn, s t, s l, s pht trin di l v rng l. Cng chiu sng 11,4 -22,8 mol/m 2/s thch hp cho chi gi t sinh trng v pht trin so vi s cn thit c ng chiu sng cao >34,2 mol/m 2/s a s cc loi cy trng khc. iu ny cho thy hiu qu s dng nh sng cy gi t. nh hng ca v tr t nui cy n nhn nhanh cy gi t in vitro: Hu ht cc mu nui cy u pht sinh chi v c cc ch tiu sinh trng v pht trin tt. Mu nui cy l chi ngn, t 1, t 2 cho cc ch s sinh tr ng v pht trin khng sai khc c ngha. Tng t kt qu c ghi nhn t 3 v t 4 (bng 9). Chi pht sinh bt c v tr no trn thn chng minh s thun nht v sinh l cc v tr mu nui cy. Sinh trng v pht trin cy gi t in vitro: Vi mu nui cy l chi ngn cho thy cc ch s sinh trng tng n tun th 6 v chm dn gn nh khng c ngha v thng k sau tun th 6. Tng t vi mu nui cy l t thn. Tun th 6 l thi im thch hp cho cy truyn, l chu k nhanh cy thn g, kt qu t ng t ghi nhn cy trm hng, hng, xoan, keo lai, b ch n Chu k nui cy quyt nh h s nhn ging v k hoch sn xut cy con. Nui cy pht sinh r cy gi t in vitro : Hu ht cc nghim thc c b sung auxin u kch thch pht sinh r (100%) v c t l ra r cao hn so vi i chng (62,5%). Vi nng auxin thp cng kch thch ra r (100%).
3. Thun ha cy gi t cy m
Ch trong thun ha l phun sng duy tr m v gim nhit khng kh. Bn cnh c cht chim vai tr quan trng; c cht cn phi ti xp, thot nc
513
nhanh, duy tr m v khng ng c s dng. Kt qu cho thy [phun s ng + x da] thch hp cho thun ha cy gi t cy m. KT LUN Chi nh cy gi t nhp ni c s dng nh nguyn liu nui cy ban u. Mi trng nui cy chi nh l MS v WPM. Ch i non c s dng lm nguyn liu cho cc th nghim v sau. Mi tr ng c bn thch hp cho vi nhn ging cy gi t l WPM c b sung BA (0,1mg/l). Tyrosin, Adenine sulfate, n c da, v tr mu nui cy, th tch b nh nui cy, trao i kh, c ng chiu sng nh hng su sc n qu tr nh nhn ging in vitro. Chu k nui cy in vitro l 6 tun. Cy gi t in vitro pht sinh r vi n ng auxin thp (0,01mg/l). Thun ha hiu qu trn mi trng ti xp v gi m [phun sng + x da]. Cng ngh nhn nhanh in vitro cy gi t c xc nh. TI LIU THAM KHO 1. Ahuja M. R. & W. J. Libby (1993a). Clonal forestry I. Genetics and Biotec hnology. Springer-Verlag, Berlin, Heidelberg, NewYork, London, Paris, Tokyo, Barcelona, Budabest. Ahuja M. R. & W. J. Libby (1993b). Clonal forestry II. Conservation and Application. Springer-Verlage, Berlin, Heidelberg, New York, Paris, Tokyo, Barcelona, Budabest. Dupuy B. (1990). Notes de voyage en Chie tropicale lors dun seminaire regional sur le teck. Bois et foret des tropiques, 226:69 -76 Dupuy B. & D. Verhaegen (1993). Le teck de plantation Tectona grandis en Cote dIvoir. Bois et Foret des Tropiqus 225:9-24 Lloyd G. & B. MacCown (1981). Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot -tip culture. In: Comb. Proc. Intl. Plant Prop. Soc., 30:421-426 Kaosar-ard A (1986). Teak, Tectona grandis Linn f.: Nursery techniques with special reference to Thailand. Seed leaflet No.4, 11/1986. Danida Forest Seed Centre, Denmark, Mascarenhas A. F., S. V. Kendurkar, P. K. Gupta, S. S. Khuspe & D. C. Agrawal (1987). Teak. In: Cell and Tissue Culture in Forestry. Vol. 3. Eds: JM Bonga and DJ Durzan, The Netherland, pp310 -315 Mascarenhas A. F. & E. M. Muralidharan (1993). Clonal forestry with tropical hardwoods. In: Clonal forestry II. Conservation and Application. Eds. MR Ahuja and WJ Libby. Springer-Verlag. Berlin, Heidelberg, New York, London, Paris, Tokyo, Barcelona, Budabest. Pp169 -187
2.
3. 4. 5.
6.
7.
8.
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9.
Monteuuis O., D. Vallauri, C. Poupard, L. Hazard, Y. Yusof, L. A. Wahap, C. Garcia & M. Chauviere (1995). Propagation clonale de tecks matures par bouturage horticole. Bois et Forets des Tr opiques 243:25-39
10. Murashige T. & F. Skoog (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 472-497 11. Timmis R. (1985). Factors influencing the use of clonal material in commercial forestry. In: Proc. An Intl. Conf. on managing fortestry trees as cultivated plants, Helsinki, Finland, 23-28 Jul 1998. pp259-272 12. Wareing P. F. (1987). Phase change and vegetative prpagation. In: Improveing vegetatively propagated crops. Academic Press, London. Pp263 -270 13. Wellendorf H. & A. Kaosar-ard (1988). Teak improvement strategy in Thailand. Technical Note No.21. Danida Forest Seed Centre, Denmark. 14. White K. J. (1991). Teak: some aspects of research and development. Publication 1991/17. FAO regional office for Asia and the P acific (RAPA), Bangkok. 15. Zobel B. & J. Talbert (1984). Applied forest tree improvement. John Willey & Son, New York, Chichester, Brisbane, Toronto, Singapore.
SUMMARY
Application of plant cell biotechnology in propagation of valuable teak tree ( Tectona grandis Linn.F)
An improved micropropagation system has been developed for teak. Shoot tips placed on MS or WPM medium supplemented with 0,1 -,05mg/l BA for shoot induction. Shoots were used as planting materials for far micropropagation. WPM is absic media culture for this purpose. The admenments of BA (0.1mg/l), Tyrosin (10mg/l), Adenine sulfate (10mg/l), coconut water (10%), position of noda ls, volume of cultivated glass jar (500ml), lighting intensity (34,2 mol/m2/s) and incresing air exchange leading high efficiency for in vitro raised micro shoots. Its reaches 100% for rooting and over 95% for hardiness acclimatization.
515
NGHIN CU KH NNG TNG SINH M SO THANH HAO VNG (Artemisia annua L.) TRONG NUI CY LNG in vitro
Bi Thi Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Thc vt l ngun dc phm quan trng t hng ngn nm nay. WHO c tnh khong 80% dn s vn da ch yu vo cc phng thuc truyn thng nh tho dc trong cha tr bnh. Cc loi tn dc ngy nay cng c ngun gc t thc vt. Khong mt phn t cc loi thuc c k toa cha cc chit xut t thc vt. Thuc cha st rt hu hiu nht hin nay l artemisinin c chit xut t cy thanh hao hoa vng. Thanh hao (Artemisisa annua L.) bt ngun t chu (Trung Quc, Vit Nam), hin nay l mt loi cy rt ph thng c mt nhiu ni trn th gii, mc tt kh hu n i hay cao nguyn. Vit Nam, pht hin thanh hao hoa vng mc t nhin 4 tnh, thuc 20 huyn - th x v hn 70 x (Vin Dc liu, 1986-1990) Phng php sn xut artemisinin t cy thanh hao ngoi t nhin ph thuc vo sn lng thu hoch cy thanh hao, vng sinh thi, nhi t khng kh, ma v... Cng ngh sinh hc ng vai tr quan trng trong vic la chn, nhn ging v bo tn cc ngun gen ca cy thuc qu (Hara etal, 1998). K thut ti sinh invitro to ra sinh khi t bo soma hay r c tim nng to ln trong vic to ra cc ngun thc vt lm thuc c cht lng cao (Nalammai v Chan, 2004). C c hp cht th cp c thu nhn thng qua nui cy t bo soma, t bo dch huyn ph, hay r c nghin cu t nhiu loi cy thuc (Chan v Nalammai, 2001). Trong bi bo ny chng ti nghin cu vn nui cy to t bo soma v r trn cy thanh hao chn dng in vitro (Nguyen T Kim Uyen v Trn Vn Minh, 2007), qua lm ngun nguyn liu cho vic nui cy t bo v r in vitro sn xut artemisinin phng v cha bnh st rt. VT LIU V PHNG PHP
Vt liu
Ging thanh hao: dng thanh hao c chn lc qua v trng trt 2007 tr n Lt. Ging nhp t Trung Quc. Chia hai loi da v o chiu cao thn: dng thp v dng cao. Dng thp c hm lng artemisinin tch ly cao, c s dng lm nguyn liu nui cy.
516
Mi trng nui cy: mi trng LV c b sung cc cht iu h a sinh trng nh BA (6-benzyladenine), kinetin (6-fufurylaminopurine), 2.4D (dichlorophenoxy acetic acid), NAA (-nathalene acetic acid), IBA (indol -3-butyric acid), zeatin, peptone iu kin nui cy: nhit phng 26+2oC, RH=65%, cng chiu sng 33.1mol/m 2/s, thi gian chiu sng 8 gi/ng y Phng php Thc nghim c b tr theo khi y (RBD), c 3 ln lp li, mi ln cy 5 bnh. S liu thu thp c x l thng k bng phn mm MSTATC (p=0.05). Ch ti u theo di: kh nng pht sinh t bo soma, kh nng tng sinh t bo soma, pht sinh chi, pht sinh r KT QU V THO LUN
1 nh hng ca cht iu ha sinh trng n kh nng pht sinh m so, chi v r
Cy thanh hao in vitro c nui cy trn mi trng LV (sucrose 30g/l). Nhit nui cy 26+2 oC. Thi gian chiu sng 10 gi/ngy. Thn l r c s dng nui cy m so. M so c cy chuyn cch khong 3 tun/ln. Kt qu nghin cu: m so hnh thnh trn mu nui cy trn mi trng c b sung NAA (1mg/l) hay vi NAA-BA (1-0.05, 1.2-0.2mg/l) v 2.4D (1mg/l). M s o nui cy trong ti, pht trin chm, d chuyn qua mu nu nhanh chng. M s o hnh thnh sau 2-3 tun nui cy. M so nui cy trn mi trng c NAA, c mu xanh v cu trc cht; trong khi m so nui cy trn mi trng c 2.4D th c mu trng hay vng nht hay nu. ng thi sinh trng rt khc nhau cc nghim thc: vi NAA (m=0.06/ng y) v vi 2.4D (m=0.22/ngy) (Bng 1) ng thi sinh trng ca dch huyn ph t cao nht sau 2 tun nui cy; tr n mi trng c NAA (1) m so kt cm c m u vng-xanh, trn mi trng c 2.4D (1) dch huyn ph ng nht hn. Kh nng tng sinh trong iu kin c chiu sng cao hn trong ti. ng thi sinh trng cng khc nhau khi so snh trong ti (T) v ngoi sng (AS) ( th 1) kt qu ghi nhn khi nui cy tr n mi trng c NAA v 2.4D. Vi 2.4D, t bo sinh trng chm, tng sinh km, trong ti v ngoi sng. Kh nng pht sinh c quan qua nui cy m so khi c s kt hp BA (3mg/l) + NAA (0.2mg/l). Tn sut hnh thnh chi 50%. Mi trng nui cy c BA (3mg/l) + NAA (0.2mg/l) hay BA (3mg/l) ring r pht sinh m so v chi. Nng zeatin cao hnh thnh m so xanh c kh nng quang hp v km pht sinh c quan chi hay r. Cm chi c t m so di gc c s dng lm vt liu vi nhn ging. Chi c nhn nhanh trn mi trng BA (3) bn rn hay lng (tr n my lc). Artemisinin hnh thnh trong thn l r cy in vitro (Woerdenbang etal, 1991 ), vic s dng hormone gip ci thin pht trin thn l r (Shukla etal, 1992) Hn na theo Kudakasseril etal (1987) trong thn l r c cha nhng hp phn enzyme cn thit cho qu trnh tng hp artemisinin. Ngo i ra Woerdenbang etal (1991)
517
ghi nhn bn thnh r cn c cha cc hp phn artemisinin v cc tin cht v Jha etal (1988) artemisinin c trong ch i v r cy in vitro Nhn xt: mi trng LV c b sung BA+NAA vi nng NAA thp nui cy pht sinh m so xanh di cng chiu sng ca ph ng th nghim. LV b sung NAA hay 2.4D thch hp cho nui cy pht sinh r. M so xanh c tch ri nui cy tng sinh (bng 1)
mg (DW)
Ngy
th 2: ng thi sinh trng ca dch huyn ph, t trn xung
(1) NAA+T (2) NAA+AS (3) 2.4D+AS (4) 2.4D+T
2 nh hng ca r i vi kh nng tng sinh chi
Ht thanh hao c gieo v trng in vitro trn mi trng LV (sucrose 30g/l). Nhit nui cy 25+2 oC. Thi gian chiu sng 16 gi/ngy. Cy cy m c s dng trong nghin cu tng quan gia r v chi trong qu trnh sn xut artemisinin trong in vitro. Kt qu nghin cu: trn mi trng bn rn, chi c r v l pht trin, cho kh nng tng hp artemisini n nhiu. Trn mi trng lng, chi c r v l, c bit r pht trin mnh m trn mi trng lng, artemisinin tng hp h n hn nghim thc c chi v khng r (Bng 2)
Kh nng nui cy tng sinh m so xanh trong mi tr ng lng
C 3 mi trng khong c bn c s dng B5 (Gamborg), LV (Litvey), MS (Murashige-Skoog). Nhit nui cy 26+2oC. Thi gian chiu s ng 10gi/ngy. Mu m so xanh c s dng trong nui cy. M so c a vo nui cy c sinh khi 100mg/mnh giai on cy truyn 3. Song song tin hnh nui cy trn cng mi trng trong bnh tam gic 300ml, cha 50ml mi trng lng, t trn my lc c tc 90rpm. Kho st kh nng sinh tr ng ca t bo m so v dch huyn ph t bo sau 30 ngy nui cy. Kt qu s hnh thnh t bo m so v huyn ph ha t bo c nh du theo ma trn. Mi trng B5 v LV thch hp cho s hnh thnh m so xp xanh khi c b sung hp phn auxin+cytokinin, mi tr ng MS t ra khng thch hp. Vi mi tr ng LV,
518
cho php kt hp ph auxin+cytokinin rng hn. S hnh thnh m so v dch huyn ph t bo thch hp vi 2.4D+BA hn l NAA+Kin. Khng th y c s hnh thnh m so xp v dch huyn ph khi nui cy ring r cc hormone. Nng 2.4D (0.5 0.2mg/l) + BA (0.025-0.1mg/l) v NAA (0.5-2mg/l) + Kin (0.5-1mg/l) cho thch hp cho nui cy dch huyn ph t bo xanh. Tuy nhin, khi b sung peptone vo mi trng LV, th t hp BA (0.1-0.5mg/l) + NAA (0.1-0.5mg/l) t ra hiu qu tng sinh nhanh mnh m hn t hp 2.4D+BA v NAA+Kin
Bng 1: nh hng ca cht iu h a sinh trng n kh nng pht sinh m so, chi, r
Cht iu ha sinh trng thc vt (mg/l)
2.4D 0 0 0 0.01 0.02 0.03 0 0 0 0 0 0 0 0 0 0 0 0 NAA 0.1 0.2 0.2 0 0 0 0.05 0.1 0.2 0.5 0 0 0 0 0 0.2 0.5 1 Kin 0 0 0 0 0 0 1 2 5 1 2 5 0 0 0 0 0 0 BA 2 3 5 2 3 5 0 0 0 0 0 0 2 3 5 0 0 0 Zeatin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 5 10 IBA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0.5 5 5 0 0 m so xanh, chi chi r, chi r, chi 0 m so xanh, chi chi chi 0
Hiu qu
M so Chi m so xanh, chi chi chi
Bng 2: nh hng ca r i vi kh nng tng sinh chi

Mi trng Agar Lng R C r C r Khng r Sinh trng (mgTLkh) R 8.2b 68.4a Chi 29.6b 98.4a 105.2a 63a 52b 33c L / chi (%) Artemisinin (%) R 0.00 0.00 Chi 0.170a 0.078b 0.031b
519
Bng 3: S ma trn nghin cu kh nng sinh tr ng dch huyn ph t bo xanh

NT 1: nh hng ca B5+BA+2.4D n sinh tr ng t bo DHP
BA(mg/l)\2.4D(mg/l) 0.1 0.5 0.05 X X 0.10 X X 0.50 X 0.01 X NT 2: nh hng ca B5+Kin+NAA n sinh tr ng t bo DHP Kin(mg/l)\NAA(mg/l) 0.1 0.5 0.05 X 0.10 X 0.50 1.00 X NT 3: nh hng ca LV+BA+2.4D n sinh tr ng t bo DHP BA(mg/l)\2.4D(mg/l) 0.1 0.5 0.05 X 0.10 X X 0.50 X 1.00 X NT 4: nh hng ca LV+Kin+NAA n sinh tr ng t bo DHP Kin(mg/l)\NAA(mg/l) 0.1 0.5 0.05 0.10 X 0.50 X 1.00 NT 5: nh hng ca MS+BA+2.4D n sinh trng t bo DHP BA(mg/l)\2.4D(mg/l) 0.1 0.5 0.05 0.10 X 0.50 1.00 NT 6: nh hng ca MS+Kin+NAA n sinh tr ng t bo DHP Kin(mg/l)\NAA(mg/l) 0.1 0.5 0.05 X 0.10 X X 0.50 X 1.00 X NT 7: nh hng ca B5+BA+2.4D n sinh tr ng t bo DHP BA(mg/l)\2.4D(mg/l) 0.1 0.3 0.05 X 0.10 X 0.50 1.00 NT 8: nh hng ca B5+Kin+NAA n sinh tr ng t bo DHP Kin(mg/l)\NAA(mg/l) 0.1 0.5 0.02 X 0.03 X 0.04 X X 0.05 X X NT 9: nh hng ca LV+BA+2.4D n sinh tr ng t bo DHP BA(mg/l)\2.4D(mg/l) 0.1 0.5 1.0 2.0 X X
1.0 X X X
2.0 X X X
1.0 X
2.0
1.0
2.0
X X 1.0
2.0
1.0 X X 0.4 X
2.0 X X
0.5 X X
1.0
2.0
X X 1.0 2.0
520
0.025 X X 0.050 X 0.075 X 0.100 X X NT 10: nh hng ca LV+Kin+NAA n sinh tr ng t bo DHP Kin(mg/l)\NAA(mg/l) 0.1 0.5 0.50 X X 0.75 1.00 X 1.25 NT 11: nh hng ca LV+BA+2.4D n sinh tr ng t bo DHP BA(mg/l)\2.4D(mg/l) 0.1 0.5 0.025 X X 0.050 X 0.075 X 0.100 X X NT 12: nh hng ca LV+Kin+NAA n sinh trng t bo DHP Kin(mg/l)\NAA(mg/l) 0.1 0.5 0.50 X 0.75 X 1.00 1.25 X X NT 13: nh hng ca LV+BA+2.4D n sinh tr ng t bo DHP BA(mg/l)\2.4D(mg/l) 0.1 0.5 0.025 X X 0.050 X 0.075 X X 0.100 NT 14: nh hng ca LV+Kin+NAA n sinh tr ng t bo DHP Kin(mg/l)\NAA(mg/l) 1.5 2.0 0.50 X X 0.75 X X 1.00 X 1.25 X NT 15: nh hng ca LV+BA+NAA+peptone n sinh tr ng t bo DHP BA(mg/l)\NAA(mg/l) 0.1 0.3 0.5 0.05 0.1 0.5 X 1.0
X X X X 1.0 X
X X
2.0 X X
1.0 X X X X 1.0 X X X X 1.0 X X X
2.0 X X X
2.0 X X X 2.0 X X X
1.0 X
ng thi sinh trng dch huyn ph m so xanh T bo dch huyn ph, c c khi lc khi t bo m so trn my lc. V mt l thuyt, khi nhng t bo mi hnh thnh, chng tri ni trong dch huyn ph, kt cm v lin kt nhau li. T bo dch huyn ph c tc phn chia cao hn t bo m so. Qu trnh tng sinh m so trong mi trng lng c th hin qua bng 4.7 v th 4.5, chng ti nhn thy trong c ng thi gian nui cy l 3 tun th khi lng t bo m so pht sinh (17,20g) nhiu h n so vi nui cy trn mi trng agar (12,59g).
521
Theo Helgeson (1979), khi m s o c cy vo dch lng ta c ngay giai on lag phase, y l giai on u tin cho n khi c du hiu phn chia t b o u tin, sau l giai on exponential-phase l linear-phase, l giai on t bo phn chia, t bo tng s lng v tng qun th nhanh, sau cng t bo i vo giai on stationary-phase, l giai on t bo khng phn chia, lng t bo sinh ra v cht cn bng nhau, v t bo c cy truyn khi vo gn cui giai on linear-phase (Trn Vn Minh, 2003). Vn duy tr t bo m so trong cng iu kin nui cy, chng ti nhn thy t thi im 21 ngy tr i th khi lng t bo m so thanh hao khng tng na. Qua n ngy th 24 th khi lng t bo m so vn l 17,20g. C th ni t 21 ngy tr i th t bo dch huyn ph ang i vo stationary-phase, lc ny khi lng t bo khng tng na Vy nu ly iu kin l khi lng t bo m so tng sinh lm yu t xc nh thi im cy truyn th chng ti ly mc thi gian l 3 tun cy truyn t bo dch huyn ph.
ng cong sinh tr ng cu a mo se o 20 18 16 Kho i l ng mo se o (g) 14 12 10 8 6 4 2 0 0 3 6 9 18 20 24 Th i gian theo do i (nga y)
2.856 1.26 6.12 13.14 17.2 17.2
Hnh 2: ng thi tng sinh dch huyn ph m so xanh
KT LUN Thn, l, r cy thanh hao c th c s dng trong nui cy to m so. D i iu kin nui cy c nh sng v ti, cho hai dng t bo c mu sc khc nhau: m so xanh v m so trng. M so xanh c cu trc phi c a vo nui cy to dch huyn ph trn mi trng LV + BA (0.5mg/l) + NAA (0.5mg/l) + peptone (1g/l). D ch huyn ph m so xanh tng sinh mnh m, v c s dng lm nguyn liu cho nui cy sn xut artemisinin trong bioreactor
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TI LIU THAM KHO 1. Chan LK & S Nalammai (2001). Preparation of cell suspension of Artemisia annua. Proceedings of the seminar on medicinal plants - Towards of modernization of rsearch and technology in herbal industries. Pp277 -281. Kepong Selangor, Malaysia Hara Y, Y Yukimune, J Hiratsuka, T Nakano, KHT Chris & LK Chan (1998). Studies in tissue and cell culture of medicinal plants. In : JBA & NEDO (eds) Proceedings of the Tokyo international forum on conservation and sustainable use of tropical bioresources. Pp236 -238. Tokyo, Japan Nalammai S & Chan LK (2004). Effect of type of callus, MS -sugar and pH on cell suspension of Artemisia annua, an antimalarial plant. In: Chang YS, M Mastura & MY Nurhanan (eds) Proceedings of the seminar on Tongkat ali, Kacip fatima & Pagaga new dimension in complimentary health care. Pp109 -113. FRIM, Kuala Lumpur, Malaysia Nguyn T Kim Uyn, Trn Vn Minh. Dng ha cy thanh hao in vitro. H i ngh NCCB2007, pp872-875, 2007 Trn Vn Minh. Gio trnh Cng ngh sinh hc thc vt, Cao hc, H Nng l m TPHCM, 2003
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4. 5.
SUMMARY
Study the capacities of proliferation of somatic cells of of Artemisia annua L. in liquid culture in vitro
Bui Thi Tuong Thu, Tran van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Shoots, stems, roots of in vitro plants could be used for cultivation of som atic cell induced. Somatic cells were induced favoured on the medium of LV supplemented with NAA (0.1-1mg/l) or with NAA-BA (1-0.05, 1.2-0.5mg/l) and 2.4D (1mg/l). Somatic cells has white color in darkness and dark -green under lighting. Green -cells were cultured in liquid on shaker. The favoured medium for green -cells proliferated was LV + BA (0.5mg/l) + NAA (0.5mg/l) + peptone (1g/l). Green -cells has structure of proembryo and was used for far study artemisin production in bioreactor
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NG DNG CNG NGH T BO THC VT PHT TRIN CY NGUYN LIU GIY THNG caribaea (Pinus caribaea)
H Th Loan, Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i, M U Pinus caribaea gm 3 loi ph l Pinus caribaea var. Hondurensis ph n b t nhin cc nc Mexico v Trung M, P. C. var. caribaea Cuba v P. C. var. bahamensis qun o Bahamas. Pinus caribaea c trng khp cc chu lc nhit i v nhit i (Birkes & Barnes, 1990; Dvorak & Donahue, 1992) trn 100 n c v c nhiu ni thnh cng rc r nh Queensland (c) ni m P. C. var. hondurensis v cy lai (P. caribaea x cc lo i thng khc) c trng trn din tch ln v c nng sut cao. Vit Nam, Pinus caribaea c trng Lm ng t nm 1965. Sau c trng nhiu ni trn ton quc v t ra l loi sinh trng nhanh. V vy trong thi gian qua c nhiu cng tr nh trng th nghim cy thng caribaea nhiu v ng sinh thi khc nhau v chn ra c loi v xut x Pinus caribaea thch hp (Ph Quang in , 1996). Trong cc loi thng, thng nh a (Pinus merkusii) c gi tr va cho g v va cho nha, nhng li l loi thng b su xm n l theo chu k, cng v sau chu k cng ngn. Thng Pinus caribaea hn thng nha v kh nng khng bnh v sng c nhng vng t ngho kit nh thng nha. T c nhng cng trnh nghin cu di thc cy thng caribaea v tin n trng thng caribaea xen hay thay th dn thng nha nhng vng sn xut cy nguyn liu giy, v xc nh c vng Trung Trung B l vng thch hp cho sn xut ht chc nh Qung Bnh, Hu v Nng (L Vn Ngoan, 1996). c nhng nghin cu trc y v thng caribaea nh kh nng duy tr tnh trng chiu cao thn (Joseph etal, 1999), lai to ging mi c gi tr kinh t Quee nsland (Dieters & Nikles, 1997), tng quan gia cc c tnh ht v cy thc sinh (Toon etal, 1991), s dng k thut m hom l ch yu trong nhn ging v rt t bo co v k thut nui cy m thng caribaea c cng b. ng dng CNTBTV trong nhn nhan h cc loi cy trng kinh t (Mamood, 1993) ang c vn dng c hiu qu nhiu n c trn th gii v nc ta nh cc loi bch n (Eucalyptus sp.) v bch n lai nh Brasil, Congo v Trung Quc; cy vn sam (Picea sp.) Chu u; thng radiata (P inus radiata) New Zealand; thng P. taeda v P. elliottii Chu M; Tre trc Thi Lan v n ; phi lao Thi Lan v Trung Quc v c trn 64 loi l rng v 32 loi l kim nui cy thnh cng in vitro (Nguyn Hong Ngha, 2001; Trn Vn Minh, 2003). Trong bi bo co ny chng ti nghin cu kh nng nhn nhanh in vitro lo i thng caribaea u dng thch hp cho trng rng nguyn liu vng cao nguyn v ty nguyn nc ta, l bc u to ngun gene nguyn liu u dng sn xut dng v tnh.
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VT LIU V PHNG PHP

Vt liu:
Mu nui cy: Cy bu t l ging thng caribaea (Pinus caribaea var. hondurensis) c Cng ty Giy ng Nai chn d ng v a vo trng rng nguyn liu giy i tr trn cc vng cao nguyn v ty nguyn. Ch i non cy thng caribaea trong bu t 1 nm tui c s dng lm vt liu nui cy. Mu c ct ta thnh nhng on 3-5cm, c ra sch bng x phng v v trng bng Hypochlorite-Na v HgCl2. Sau khi v trng, mu c ct thnh nhng on 10-15mm c cha vng nhu m nh sinh trng. Mu nui cy l chi nh. iu kin nui cy: Mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+1oC. Cng chiu sng 22,8 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: Mi trng dinh dng khong c bn MS (Murashige-Skoog, 1962) v WPM (Lloyd and McCown, 1981), c b sung BA (6-benzyl aminopurine), Kinetin (6-furfurylaminopurine), IAA ( -indol acetic acid), IBA ( -indol butyric acid), NAA ( -Naphthalene acetic acid), Tyrosin, Adenine sulfate v nc da (CW)
Phng php:
Th nghim c b tr theo RCBD (1 yu t), 4 ln lp li, mi ln lp li nu i cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTAT theo M, CV% v LSD(0.05). Ch tiu theo di: s chi/cm, chiu cao chi (mm), to m so (+/ -), t l ra r (+/-), s r (no), chiu di r (mm), mu l (X-xanh, XL-xanh lt, V-vng) KT QU V THO LUN 1. Nui cy chi nh sinh trng cy thng Caribaea in vitro V trng chi nh sinh trng cy thng Caribaea in vitro : Cy thng l loi cy c nha bao bc v khi chun b mu nui cy c lp nha tit ra ni vt ct nn kh nng v trng thp. S dng Hypochlorite -Na hay HgCl2 u cho kt qu v trng thp. Kt hp v trng bng Hypochlorite-Na trc v sau v trng bng HgCl 2 cho kt qu t l v trng chp nhn (65,4%) nh hng ca mi trng khong c bn n kh nng pht sinh chi thng Caribaea in vitro: C bn loi mi trng khong c bn c s dng nh MS, WPM, SH v SKB. Kt qu cho thy mi trng khong c bn WPM t ra thch hp, cho cc ch s tuyt i v sinh trng hn hn nhng mi trng cn li nh hng ca sinh l mu nui cy n kh nng ph t sinh chi thng Caribaea in vitro: Sinh l mu nui cy c quy nh theo ngy tui ca chi non. Kt qu nghin cu cho thy, tui chi 24 -26 ngy tui a vo nui cy cho pht sinh cm chi (89-75%), s chi (7,3-6,3 chi) v chiu cao chi (24,3-18,7mm) cao nht
525
nh hng ca BA v NAA n kh nng pht sinh chi thng Caribaea in vitro : Kt qu nghin cu cho thy vi nng 5mg/l BA kt hp vi CW (10%) b sung v o mi trng nui cy WPM thch hp cho nu i cy pht sinh chi vi t l to cm ch i cao (58%), s chi nhiu (7chi/cm), c chiu cao chi thp (13mm). T hp BA (3mg/l) + Kinetin (0,1mg/l) pht sinh ch i km nhng pht trin c chiu cao thn chi tt hn (21mm). Vn thn l m t trong nhng c tnh kh kim sot trong nui cy cy thng. nh hng ca BA v Kinetin n kh nng pht sinh chi thng Caribaea in vitro: B sung vo mi trng nui cy c bn WPM t hp BA (5mg/l) + Kinetin (0,1-0,5mg/l) thch hp cho nui cy pht sinh chi cho ch s t l to cm chi cao (60,7-91,9%), s chi (8,3-10,0 chi) v chiu cao cm chi c ci thin so vi i chng. BA + Kinetin + CW ci thin kh nng pht sinh chi v sinh trng chiu cao thn chi 2. Nhn nhanh cy thng Caribaea in vitro nh hng ca BA v Kinetin n nhn nhanh cy thng Caribaea in vitro : Trn mi trng nui cy c bn WPM + CW (10%) c b sung BA (0,5 -3mg/l) + Kinetin (0,1-0,5mg/l) cho kt qu nng thch hp l 1mg/l BA + Kinetin (0,1-0,5mg/l) pht sinh chi mnh m (9,3-5,0 chi), sinh trng mnh (27,3-19,3mm). BA+Kinetin ng vai tr quan trng pht sinh chi trong nhn nhanh in vitro. nh hng ca nc da (CW) n nhn nhanh cy thng Caribaea in vitro : Trn nn mi trng nui cy nhn chi c bn WPM + BA (1mg/l) + Kinetin (0,1mg/l) c b sung nc da CW (5-20%) cho kt qu nng 10% CW vn thch hp. Tuy nhi n vn cha ci thin c chiu cao cm chi qua bng 6 (27,3mm) v (25,7mm). nh hng ca s ln cy truyn (F) n nhn nhanh cy thng Caribaea in vitro : S ln cy truyn in vitro biu hin kh nng tr ha in vitro. S ln cy truyn c ng nhiu cho thy tnh thun thc v sinh l in vitro c ng cao. S ln cy truyn cng tng (F4) ci thin s chi pht sinh (7,1 chi) v chiu cao chi (31,2mm) trn mi trng nhn chi c bn WPM + BA (1mg/l) + Kinetin (0,1mg/l) + CW (10%). nh hng ca cng chiu sng v trao i kh n nhn nhanh cy thng Caribaea in vtro: Nhn ging cy thng Caribaea c nhng kt qu ng c li vi nhn ging cc cy trng khc th hin qua s thch hp ca cng chiu sng thp trong nui cy (22,8 mol/m2/s) v dng bnh nui cy c y np cao su (tng c ng trao i kh lm cho l thng b chy) cho pht sinh chi cao (10,7 chi) v sinh trng chi ci thin (31,6mm). Hn ch chy l thng th cn a nhit xung thp 25 oC, khng ci thin sinh trng v dn n tng chi ph nng l ng trong vi nhn ging. ng thi sinh trng cy thng Caribaea in vitro : Chu k nui cy quyt nh n kh nng duy tr tc nhn ging cao, sinh tr ng v pht trin chi in vitro duy tr c trng thi tr v d dng ln k hoch nhn ging. Mc d y bnh bng np cao su hay np giy, chu k nui cy in vitro c xc nh l 6 tun l hiu qu nht v s chi pht sinh v chiu cao chi.
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Nui cy vn thn cy thng caribaea in vitro: C mt vn trong vi nhn ging cy thng l chiu cao chi khng pht trin. Trong cc thc nghim, chiu cao thn chi t cao nht l 32mm (bng 10). Ci thin chiu cao thn chi quyt nh kh nng ra r v thun ha sau ny. Kt qu cho thy Tyrosin hay Adenine sulfate tc ng ring r khng ci thin c s pht trin chiu cao thn. Tuy nhi n khi kt hp Tyrosin v Adenin sulfate cho pht sinh chi (14,3 chi) v sinh trng chiu cao thn chi cao (51,7mm) 3. Thun ha cy thng Caribaea cy m Cy thng Caribaea c nui cy to r trn mi trng WPM c b sung IAA v IBA. Kt qu bng 14 cho thy tc ng ring r ca IAA hay IBA dn n pht sinh r trong nui cy thp; kt hp IAA+IBA ci thin c kh nng pht sinh r cng cc ch s v chiu di r v chiu cao thn chi. S lng r pht sinh t (1 r). Kh nng thun ha trn bu t t 80-90% trong iu kin phun sng gi m 100% v nhit khng kh thp 28+2oC. KT LUN xy dng c cng ngh vi nhn ging cy thng Caribaea in vitro. Mi trng WPM l mi trng khong c bn thch hp cho qu trnh nui cy. B sung BA (1mg/l), Kinetin (0,5mg/l), CW (10%), Tyrosin (5mg/l), Adenine sulfate (5mg/l) nh hng su sc n qu trnh to cm chi, nhn nhanh v vn thn ch i in vitro. Kt hp IAA+IBA kch thch pht sinh r cao trong nui cy. Thun ha cn c mt iu kin mi trng vi kh hu c nhit khng kh thp v m cao. TI LIU THAM KHO 1. Birkes J. S. & R. D. Barnes, (1990). Provenance variation in Pinus caribaea, P. oocarpa and P. patula Tecunumanii. Tropical Forestry No21. Oxford Forestry Institute. Univ. Oxford, UK. 40p Dvorak W.S. & J.K. Donahue (1992). CAMCORE Cooperation research review 1980-1992. CAMCORE North Carolina State Univ., Raleigh, USA, 93p Dieters M. J. & D. G. Nikles (1997). The genetic improvement of Caribbean pine (Pinus caribaea Morlet) - building on a firm foundation. In: Proc. 24 th Southern Forest Tree Improvement Conf. Orlando, Florida, June 10 -12 1997, pp33-52 Joseph A. A., M. . Dieters, P. G. Toon & H. S. Dungey (1999). Trends in heritability of height upto 10 years of age in Pinus caribaea var. hondurensis. In: Proc. Intl. Worshop BIO-REFOR 1999, Kathmandu, Nepal, 28Nov -2Dec 1999, pp299-301 L Vn Ngoan, NT Son, LT Son v LH Thng (1996). Di thc cy thng caribaea vo thanh ha. Kt qu nghin cu KH-CN Lm nghip 1991-1995, pp169-173 Lloyd G. & B. McCown (1981 ). Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot -tip culture. In: Comb. Proc. Intl. Plant Prop. Soc., 30: 421-426
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7. 8. 9. 10.
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Mamood M. (1993). Application of plant in vitro technology. Proc. , 16 -18 Nov 1993, Univ. of malaysia, Malaysia. Murashige T. & F. Skoog (1962 ). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 472-497 Nguyn Hong Ngha (2001). Nhn ging v tnh v trng rng dng v tnh. NXB Nng nghip, pp70-71. Ph Quang in (1996). Nghin cu ging thng caribaea ( Pinus caribaea) Vit Nam. Di thc cy thng caribaea vo Thanh Ha. Kt qu nghin cu KH-CN Lm nghip 1991-1995, pp165-169. Powell M. B. & D. G. Nikles (1996). Genetic parameter estimates and predicted breeding values for diameter, height and stem straightness of Pinus elliotti, P. caribaea var hondurensis and their F1 hybrid. In: Dieters Mj etal (eds), Proc. Tree improvement for sustainable tropical forestry 1996, Caloundra, Queensland, Australia, 27Oct - 1Nov 1996, Vol1, pp169 -172 Toon P. G., R. J. Haines & M. J. Dieters (1991). Relatioship between seed weight, germination time and seedling height growth in Pinus caribaea Morlet var. hodurensis Barrett and Golfari. Seed science and technology, 19: 397-402. Trn Vn Minh (2003). Gio trnh Cng ngh Sinh hc Thc vt. Cao hc v NCS. i hc Nng lm TPHCM.
SUMMARY
Application of plant cell biotechnology in development of paper material caribaea pine ( Pinus caribaea var. Honduraensis)
Application of plant cell biotechnology for establishement of caribbean pine micropropagation was achieved. WPM was used as basic meadia in cultures. The admenments of BA (1mg/l), Kinetin (0,5mg/l), Tyrosin (5mg/l), Adenine sulfate (5mg/l), CW (10%) play an important role in enhancements of shoots induction and proliferation, and shoot stem elongation. Low light intensity (22,4 mol/m2/s) was favour for shoot growth and proliferation. IAA+IBA stimulates rooting. Acclimatization in low temperature (28+2oC) and high humidity (100%) in micro climate enviroment improved plant survivals.
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NG DNG CNG NGH PHI soma TRONG CNG TC GING CY TRM HNG (Aquilaria crassna Pierre ex Lecomte)
inh Trung Chnh, H Nng Lm TPHCM Bi Th Tng Thu, PTNT pha Nam v CNTBTV Trn Vn Minh, Vin Sinh hc Nhit i M U Cy trm hng l loi cy c sn vng nhit i. c im sinh tinh du trm hng l sn phm khai thc ch yu. Qu tr nh hnh thnh v c ch to trm hng n nay vn l iu b n. Vi gi tr kinh t cao, cy trm hng hin nay ang ng trc nguy c tuyt dit (B KH&CN, 2000). Vi nhng cy trm h ng u dng hin nay cn rt t trong cc cnh rng nguy n sinh. Phc trng ging v dng ha dng v tnh bng k thut nui cy phi soma l rt cn thit (Pierik, 1987). Vi k thut nui cy phi soma, cy trm h ng c phc trng v tr v trng thi tr nhanh chng (Thorpe, 1980). Vi trng thi tr, cy trm h ng u dng l ngun cung cp nguyn liu cho qu trnh pht trin vn ging u ngun cung cp nguyn liu cho pht trin cc vng chuyn canh trm hng xut khu. VT LIU V PHNG PHP Vt liu Mu nui cy: cy trm hng in vitro, dng ND (c thu thp nam o Ph Quc). Mu cy con in vitro c chn ang sinh trng khe v c thn l r y . Thn l r cn non c s dng trong nui cy. Thn v r c ct tng n 1cm, l c ct thnh tng mnh 1cm 2 iu kin nui cy: Mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+1oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy Mi trng nui cy: Mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962) v WPM (Lloyd and McCown, 1981), c b sung BA (6-benzyl aminopurine), IAA ( -indol acetic acid), IBA ( -indol butyric acid), NAA (naphthalene acetic acid), glycin, vitamin B1 v n c da (CW) Phng php Th nghim c b tr theo RCBD (1 yu t), 4 ln lp li, mi ln lp li nui cy 5 bnh tam gic 300ml, m i bnh tam gic cha 65ml mi trng th nghim v c
529
cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (P=0.05). T l pht sinh t bo soma (%), sinh khi t bo sau 15, 30 ngy nui cy (g), tng sinh khi (g), mt t b o (tbx10 4/ml) KT QU, THO LUN Nui cy pht sinh t bo soma: thn l r l b phn c s dng lm mu nui cy. Mu nui cy c t trn b mt thch, c vt ct tip xc vi mi tr ng nui cy. Mi trng c bn MS c s dng, c b sung BA, kinetin, NAA, IBA, 2.4D, vitamin B5. Kt qu nghin cu cho thy t l pht sinh t b o soma cao vi mu nui cy l r (dng ND, nam o Ph Quc). Trn mi trng nui cy c b sung 2.4D, t bo soma pht sinh nhanh, c mu tr ng trong; mi trng c b sung BA + NAA + Kin + vitB5 cho t bo soma trc c sa, nhn di knh hin vi thy t b o c dng hnh cu chuyn qua dng t bo phi soma. Vi mu nui cy r ND, nghim thc A1 cho pht sinh t bo soma thp hn nghim thc A2; nhng kh nng t bo soma chuyn trc tip qua phi soma cao. Phi soma pht sinh trn mi tr ng nghim thc A1 c s dng trong cc nghi n cu v sau Nhn sinh khi dch huyn ph phi soma trn mi trng agar: Phi soma pht sinh t r c a nui cy trn mi trng MS c b sung BA, kinetin, NAA, I BA, 2.4D, vitB5. Kt qu nghin cu cho thy t bo tng sinh nhanh trn nghi m thc B1. Di knh hin vi, cc t b o tin phi c t bo cht m c, khng bo nh, phn chia pht sinh phi th cp nhanh Nhn sinh khi dch huyn ph phi soma trn mi trng lng: T bo phi soma pht sinh t nui cy r, c a vo nui cy trn mi trng lng, vi sinh khi ban u nui cy l 1g/65ml. Kt qu nghin cu cho thy mt tng sinh cao nht l mi trng c bn MS c b sung 2.4D hay b sung BA + Kin + IBA. Tuy nhin, trn mi trng c b sung 2.4D, t b o tng sinh nhanh v c khuynh h ng phn bit ha thnh t bo soma; ngc li, trn mi trng c b sung BA + Kin + IBA t bo phi soma i vo qu trnh phn chia v bit ha Tri dch huyn ph phi soma trn mi trng agar: Dch huyn ph t bo phi soma trn nghim thc C5 c ht 10ml tri trn mi trng nui cy c agar. Mi trng nui cy l MS c b sung BA, 2.4D, NAA, IBA, Kin, vitB5. Kt qu nghi n cu cho thy, sau 15 v 30 ngy nui cy, nghim thc D1 v D2 c tng sinh khi cao so vi nghim thc D3. So snh gia nghim thc D1 v D2, cho thy trn nghim thc D1, phi soma bit ha mnh m chuyn t m u trng sa sang mu vng nht, bc chuyn qua ti sinh. Phi soma trn nghim thc D1 c s dng trong nghin cu ti sinh Ti sinh phi soma: T bo phi soma t nghim thc D1 c s dng trong nghin cu ti sinh. Vi mi trng nghim thc D1, t bo phi soma tri qua qu trnh pht trin hnh cu (sau 45 ngy), hnh tim (sau 60 ngy), hnh thy li (sau 90 ngy) v ti sinh xut hin chi n c l tin sinh (sau 120 ngy) Nhn nhanh cy d bu in vitro: Chi n c a vo nhn nhanh trn mi trng WPM + BA (0,1mg/l) pht sinh cm chi. Cm chi nh n nhanh sau vi ln nui cy truyn. Cm chi c s dng lm protocol trong nhn nhanh in vitro.
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KT LUN R cy d bu cy m c s dng trong nui cy. T b o phi soma c nui cy pht sinh trn mi tr ng MS + BA (7mg/l) + Ki (5mg/l) + IBA (0,5mg/l) + vitaminB5 (10mg/l); nhn sinh khi t bo phi trn mi trng bn rn MS + BA (1mg/l) + Ki (1mg/l) + NAA (0,5mg/l) + vitaminB5 (5mg/l); nhn sinh kh i trn mi trng lng MS + BA (7mg/l) + Ki (5mg/l) + IBA (0,5mg/l); v c tri trn mi trng bn rn MS + BA (1mg/l) + Ki (5mg/l) + NAA (0,5mg/l) + vitaminB5 (5mg/l). T bo phi c nui cy ti sinh sau 120 ng y trn mi trng WPM + BA (0,1mg/l). Chi non t phi c tip tc vi nhn ging tr n mi trng WPM + BA (0,5mg/l). Ln u tin ti sinh thnh cng t bo phi soma cy d bu TI LIU THAM KHO 1. 2. B Khoa hc v Cng ngh (2000). Sch Vit Nam, phn Thc vt. NXB KH&KT, H Ni. Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc. 30: 421-427 Murashige T. & R.Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:431-497 Pierik R. L. M. (1987). In vitro culture of higher plants . Martinus Nijhoff, Dordrecht, pp183-230 Thorpe T. A. (1980). Organogenesis in vitro: structural, physiological and biochemical aspects. Int. Rev. Cytol. Suppl. 11A: 71-112
3. 4. 5.
SUMMARY
Application of somatic embryogenesis culture techniques in agarwood (Aquilaria crassna Pierre ex Lecomte) improvement
Dinh Trung Chanh(1), Bui Thi Tuong Thu(2), Tran Van Minh(2) (1)Nong Lam University HCMCity (2)National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology Root explants were the well samples for somatic embryogen esis cultivation. Embryogenic cell was initiated on MS medium supplemented with BA (7mg/l) + K in (5mg/l) + IBA (0,5mg/l) + vitB5 (10mg/l); mass propagation on solid MS medium supplemented with BA (1mg/l) + K in (1mg/l) + NAA (0,5mg/l) + vitB5 (5mg/l); mass propagation on liquid MS medium supplemented with BA (7mg/l) + K in (5mg/l) IBA (0,5mg/l); and layer on the solid MS medium supplemented with BA (1mg/l) + K in (5mg/l) + NAA (0,5mg/l) + vitB5 (5mg/l). Embryogenic cell was regenerated on WPM medium supplemented with BA (0,1mg/l) after 120 days after culture. Single shoot was used as for far micropropagation or other studies in agarwood improvement. This is the first report on agarwood regeneration
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NGHIN CU K THUT NUI CY PHI GI TRONG NHN NHANH CY TR M HNG (Aquilaria crassna Pierre ex Lecomte)
inh Trung Chnh H Nng Lm TPHCM Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Trong qu trnh ti sinh, c nh ng loi thc vt ti sinh tng i d dng v trong thi gian ngn; tuy nhin, c nhng loi thc vt ti sinh chm v cn thi gian di, i khi phi nui cy ti sinh qua nhiu giai on (Zimmerman etal., 1986). nng cao hiu qu ti sinh, chi ti sinh xut hin c a vo nui cy vi nhn ging hay nui cy tng sinh phi soma hay phi gi, nhm mc ti u nng cao qu trnh ti sinh v nng cao h s nhn ging (Mantell etal., 1985). Tng sinh phi soma hay phi gi th nhanh hn nui cy tng sinh chi. Ring i vi cy trm hng, qu trnh ti sinh phi soma thng xut hin chi n, v phi qua nhiu giai on nui cy mi pht sinh cm chi, tc nhn nhanh cm chi gim dn theo thi gian, v kh nng pht sinh r cng gim dn (Minh & Thu, 2003). N n, cn phi c h thng nui cy th ch hp sau khi ti sinh tng sinh nhanh in vitro, th h thng nui cy pht sinh phi gi t ra hiu qu VT LIU V PHNG PHP Vt liu Mu nui cy: Chi ti sinh t phi soma, c hai l tin sinh, d ng ND (c thu thp nam o Ph Quc). Chi con c tch ri v dng trong nghin cu iu kin nui cy: Mi trng c v trng 121oC v 1at trong 25 pht. Nhit phng nui cy 28+1oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: Mi trng dinh dng khong c bn WPM (Lloyd and McCown, 1981), c b sung BA (6-benzyl aminopurine), Kinetin (6 furfurylaminopurine), IBA ( -indol butyric acid), NAA ( - naphthalene acetic acid), glycin, vitamin B1 Phng php Th nghim c b tr theo RCBD (1 yu t), 4 ln lp li, mi ln lp li nui cy 5 bnh tam gic 300ml, m i bnh tam gic cha 65ml mi trng th nghim v c
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cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (P=005). S phi PLB nui cy pht sinh / n v (n v l mt chi c l tin sinh ti sinh t phi soma) KT QU V THO LUN Mu nui cy: Trong qu trnh ti sinh phi soma tr m hng in vitro, chi ti sinh thng xut hin n l, khng to thnh cm chi, nn hiu sut nhn ging s khng cao. Chi n xut hin c 1-2 l tin sinh hay c 2 l thnh thc. Nhng chi n ny c tch ri, c s dng nh protocol trong nghin cu nuI cy pht sinh phi gi nh hng ca BA v kinetin (K) n kh nng to phi gi : Chi n c nui cy trn mI trng WPM c b sung BA (1 -10mg/l) v kinetin (1-10mg/l). Kt qu nghin cu cho thy phi gi pht sinh nhiu tr n mi trng nui cy c b sung BA (5mg/l) hay kinetin (5mg/l) . Phi gi xut hin c dng hnh cu nh, c mu trng, c 1-2 l tin sinh c mu xanh l cy nht. Kh nng pht sinh phi gi tr n m6i trng c b sung BA hay kinetin u thch hp nh hng ca BA/NAA v K/NAA n kh nng to phi gi : nng cao hiu sut nui cy pht sinh phi gi, mi tr ng nui cy WPM c b sung cc t hp BA/NAA hay K/NAA. Kt qu nghin cu cho thy, phi gi pht sinh hiu qu tr n mi trng c b sung BA (5mg/l) + NAA (0,2mg/l) hay K (3mg/l) + NAA (0,2mg/l) so vi BA hay kinetin tc ng ri ng l. Phi gi xut hin c h nh cu trng, c 1-2 l tin sinh. nui cy ko d i sau hai thng th phi gi i vo qu trnh ti sinh c xut hin l tht. Kh nng pht sinh phi gi cho thy c kh nng nng cao tr n mi trng c b sung BA + NAA hay K + NAA nh hng ca BA/K/NAA n kh nn g to phi gi: Nhm mc ch nghin cu n s hon ho ca mi trng nui cy. Trn mI trng WPM c b sung s kt hp BA, kinetin v NAA. Kt qu nghin cu cho thy, kt hp BA (5mg/l) + K (3mg/l) + NAA (0,2mg/l) cho tn sut pht sinh phi gi tng ln r rt (8,2 phi gi / n v). Kt qu cho thy BA, kinetin v NAA ng vai tr quan trng trong qu trnh nui cy pht sinh phi gi Nhn nhanh phi gi: Duy tr nui cy pht sinh phi gi in vitro nhn nhanh l rt cn thit. Mi trng nui cy WPM c b sung BA (5mg/l), kinetin (3mg/l) v NAA (0,2mg/l) ring l hay kt hp. Kt qu nghi n cu cho thy phi gi nui cy pht sinh lin tc in vitro. Kt hp BA + K + NAA cho hiu qu nhn nhanh phi gi r rt (10,6 phI gi / n v) so vi BA hay BA + K Ti sinh phi gi: Phi gi c nui cy ti sinh trn mi trng WPM c b sung IBA (0,3-0,7mg/l). Kt qu nghin cu cho thy IBA (0,7mg/l) thch hp cho qu tr nh ti sinh c chiu cao chi 65mm v pht sinh r sau 45 ngy nui cy KT LUN Chi ti sinh t phi c s dng lm nguyn liu cho cc nghin cu nui cy pht sinh phi gi. Mi trng thch hp cho nui cy pht sinh phi gi: WPM + BA
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(5mg/l) + Ki (3mg/l) + NAA (0,2mg/l). Phi gi c ti sinh trn mi trng WPM + IBA (0,7mg/l), t chiu cao chi 65mm sau 45 ng y nui cy. Mt h thng nhn nhanh cy d bu hiu qu bng k thut nui cy phi gi. TI LIU THAM KHO 1. Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc. 30:421-427 Mantell S.H., Matthews J.A. & McKee R.A. (1985). Principles of plant biotechnology. Blackwell Scientific, Boston, pp130 -157 Zimmerman R.H., Griesbach R.J., Hmmerschlag F.A. & Lawson R.H. (ed s) (1986). Tissue culture as a production system for horticulture crops . Martinus Nijhoff, Dordrecht. ISBN:90-247-3378-2 Minh TV & BTT Thu (2003). ng dng cng ngh t bo thc vt phc hi loI cy c sn trm hng (Aquilaria crassna Pierre ex Lecomte). K yu Hi ngh NCCB 2003, Hu, pp358-360
2. 3.
4.
SUMMARY
Study of PLB initiation culture tecniques in micropropagation of agarwood (Aquilaria crassna Pierre ex Lecomte)
Dinh Trung Chanh(1), Bui Thi Tuong Thu(2), Tran Van Minh(2) (1)Nong Lam University HC MCity (2)National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Shoot regeneration from somatic embryogenesis was used as protocol for far micropropagation through protocorm like body (PLB) initiation culture. The favoured medium for protocorm like body initiated in vitro was WPM supplemented with BA (5mg/l) + K (3mg/l) + NAA (0,2mg/l) and for micropropagation was the same medium. protocorm like body was regenerated on medium of WPM + IBA (0,7mg/l) with the shoots reached 65mm after 4 5 days in culure and having roots developed. An effective rapid micropropagation system was established
534
NG DNG CNG NGH PHI soma TRONG DNG HO CY KEO LAI (Acacia sp.) in vitro
Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh h c Nhit i M U Cy keo lai l dng lai gia cy keo l trm v keo tai tng. Do l dng lai nn rt d b phn ly. Duy tr dng lai ch bng con ng gim hom truyn thng. Cy keo lai l loi cy c u im sinh trng v pht trin nhanh, l nguyn liu chnh cho ngnh giy. Dng ha cy keo lai bng k thut nui cy phi soma u im a cy keo lai v trng thi tr v dng ha dng v tnh bng k thut vi nhn ging (Christianson & Warnick, 1987). Cy keo lai cy m c s dng l cy m u dng trong cc vn ging u ngun, sn xut nguyn liu ging c cht lng ng u v sinh trng v cht lng g. C nhng loi cy thn g d ti sinh, nhng vi cy keo lai, kh nng ti sinh rt hn ch, hin nay ch c bo co ti sinh c y keo l trm (Deyu etal., 2001) khi b sung TDZ (Huetteman & Preece 1973) v o mi trng nui cy, bo co ti sinh trn cy keo lai t n bn VT LIU V PHNG PHP Vt liu Mu nui cy: cy keo lai in vitro, dng KL84 chn lc. Cy con in vitro ang sinh trng khe v c thn l r y . Thn l r cn non c s dng trong nui cy. Thn v r c ct tng on 1cm, l c ct thnh tng mnh 1cm 2. iu kin nui cy: Mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+2oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: Mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962) v WPM (Lloyd & McCown, 1981), c b sung BA (6-benzyl aminopurine), TDZ (thidiazur on), Kinetin (6-furfurylaminopurine), glycin, vitamin B1, Calci panthothenate (Ca-Pan), v nc da (CW) Phng php Th nghim c b tr theo CBD n yu t, 4 ln lp li, mi ln lp li nu i cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (P=0.05). T l pht sinh t bo soma (%), sinh khi t bo sau 15 ngy nui cy (g), t
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l tng sinh khi t bo (%), mt t bo (tbx104/ml), s chi / cm (chi), chiu cao chi (mm), ra r (+/-) KT QU V THO LUN Nui cy pht sinh t bo phi soma: Mu nui cy l thn, l, r cy keo lai in vitro. Mu nui cy c t trn mi trng WPM c b sung BA, TDZ, v nc da (CW). Kt qu nghin cu cho thy nghim thc A2, A3 v A4 thch hp cho nui cy pht sinh t bo soma c ba loi mu a vo nui cy. Trong nghim thc A3: WPM + TDZ (0,5mg/l) + CW (10%), t bo soma bit ha thnh phi soma c kho st qua knh hin vi Nhn sinh khi t bo phi soma trn mi tr ng agar: Sau 15 ngy nui cy: Trn mi trng agar, t bo phi soma tng sinh kh i nhanh trn nghim thc C2: WPM + TDZ (0,5mg/l) + CW (10%); v m t t bo nui cy trong mi trng lng l 1650 x 104/ml (vi sinh khi a vo nui cy ban u l 1g/65ml). T bo phi phn chia v bit ha nhanh trn mi trng c b sung TDZ v CW Tri dch huyn ph phi soma trn mi trng agar: Sinh khi ban u c a vo nui cy to dch huyn ph l 1g (1650x10 4/ml) v 2g (3324x104/ml). 10ml dch huyn ph c ht ra, v tri trn mi trng agar (trong bnh tam gic 300ml), cho thy sinh khi tng sau 15 ngy nui cy l 1,8g (nghim thc C2) v 2,1g (nghim thc C5). Mi trng WPM c b sung TDZ v CW thch hp cho tri dch huyn ph t bo phi Ti sinh phi soma: T bo phi soma nui cy trn mi trng WPM + TDZ (0,5mg/l) + CW (10%) c cy chuyn sang mi tr ng ti sinh MS c b sung TDZ v CW, kt qu nghin cu cho thy, t bo phi soma ti sinh hiu qu trn mi trng nghim thc D2 v D3. Trong nghim thc D3: MS + TDZ (0,2mg/l) + CW (10%) cho ti sinh chi cao hn nghim thc D2 Nhn nhanh cy keo lai in vitro: T bo phi soma keo lai ti sinh in vitro d i dng cm chi c a vo mi trng nhn nhanh in vitro MS + BA (0,5mg/l) + K in (0,5mg/l) (nghim thc E3). Cy keo lai c duy tr v nhn v hn in vitro. Kh nng ra r d dng trn mi trng vi nhn ging in vitro KT LUN Cy keo lai in vitro, dng KL84 chn lc. Cy con in vitro ang sinh tr ng khe v c thn l r y . Thn l r cn non c s dng trong nui cy. Thn v r c ct tng on 1cm, l c ct thnh tng mnh 1cm 2. Kt qu nghin cu cho thy mi trng WPM + TDZ (0.2-0.5mg/l) + BA (1mg/l) + CW (10%) thch h p cho nui cy pht sinh t bo soma c ba loi mu a vo nui cy. Trong mi trng WPM + TDZ (0,5mg/l) + CW (10%), t ra thch hp t bo soma bit ha thnh phi soma c kho st qua knh hin vi. Sau 15 ng y nui cy, t bo phi soma tng sinh khi nhanh 1,8g v 2,1g vi sinh khi nui cy ban u l 1g v 2g/50ml trn mi trng agar WPM + TDZ (0,5mg/l) + CW (10%); v mt t bo nui cy trong mi trng lng l 1650 x 104/ml (vi sinh khi a vo nui cy ban u l 1g/65ml). Dch
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huyn ph t bo phi soma c nui cy tri v ti sinh trc tip trn mi trng WPM + TDZ (0,2mg/l) + CW (10%). T bo phi soma c ti sinh di dng cm chi v c nhn nhanh lin tc trn mi trng MS + BA (0,5mg/l) + K in (0,5mg/l). Cy keo lai c duy tr v nhn v hn in vitro. TI LIU THAM KHO 1. Christianson M. L. & D. A. Warnick (1987). Physiological genetics of organogenesis in vitro. In: Hanover JW, Keathly DE (eds). Genetic manipulation of woody plants. Plenum Press, New York, pp101 -115 Deyu X., H. Yan, D. Y. Xie & Y. Hong (2001). Regeneration of Acacia mangium through somatic embryogenesis . Plant Cell Reports (20) 34-40 Huetteman CA & J. E. Preece (1973). Thiadiazuron: a potential cytokinin for woody pant tissue culture. Plant Cell Tissue Organ Cutu re (33) 105-119 Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc . (30) 421-427 Murashige T. & R. Skoog (1962). A revised medium for rapid growth an d bioassays with tobacco tissue cultures. Physiol. Plant. (15) 431-497
2. 3. 4.
5.
SUMMARY
Application of somatic embryogenesis culture techniques for in vitro hybrid acasia cloning
Le Huu Thuong(1), Bui Thi Tuong Thu(2), Tran Van Minh(2) (1)University of Agriculture and Forestry HCMCity (2)National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology
Stem, leaves, and roots could be used as explants in cultivation. Somatic cell was initiated on WPM medium supplemented with TDZ (0,5mg/l) + CW (10 %) having somatic embryogenesis cell differentiation. Mass propagation and cell layer were established in the solid and liquid medium supplemented with TDZ (0,5mg/l) + CW (10%). Embryogenic cell was regenerated on MS medium supplemented with TDZ (0,2mg/l) + CW (10%). Plant regeneration appearance in multiple -shoot, it was used as protocols for far micropropagation. A system juvenization and cloning was established
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NG DNG CNG NGH PHI soma TRONG CNG TC GING CY LT HOA (Chukrasia tabularis A. Juss)
Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i M U Cy lt hoa l loi cy cy g ln, qu him, qu v c vn rt p, th g mn, khng b mi mt, rt c a chung ng cc gia dng cao cp, chnh v vy lt hoa b sn tm ro rit khai thc g, c thn ln r khng cha li cy ging, n n hin nay lt hoa trong tnh trng cn kit khng c kh nng t phc hi tr n phm vi c nc (Nguyn B Cht, 1998; Nguyn Ho ng Ngha, 1999; Trn nh Hu, 1998). c lit k trong Sch Vit Nam (B KH&CN, 2000). Kh nng ti sinh bng ht trong t nhin km. Nm 1997, cn b o Linda (cn bo s 5) b vo Cn o gy thit hi nghim trng, trong c 2.200ha rng thuc V n QG Cn o, lm suy gim v s lng v cht lng cc loi cy g qu bn a v c hu nh lt hoa, gng no, qung lng, d u Cn Sn Trong bi cnh , thc trng lo i cy lt hoa cn li rt t, ti sinh t nhin km, kh nng gieo ging rt hn ch. Cy m u d ng c i sng di nhiu nm, rt kh tr ha (Mamood, 1993). Nhm mc ti u bo tn, tr ha v dng ha, k thut nui cy phi soma t ra thch hp (Thorpe, 1980; Phillips, 1988), m hin nay cha c bo co v vic ng dng k thut n y. ng dng cng ngh phi soma trong cng tc ging cy lt hoa l cn thit VT LIU V PHNG PHP Vt liu Mu nui cy: cy lt hoa in vitro. Mu cy con in vitro c chn ang sinh trng khe v c thn l r y . Thn l r cn non c s dng trong nui cy. Thn v r c ct tng an 1cm, l c ct thnh tng mnh 1cm 2, iu kin nui cy: Mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+1oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: Mi trng dinh dng khong c bn MS (Murashige-Skoog, 1962) v WPM (Lloyd and McCown, 1981), c b sung BA (6-benzyl aminopurine), Kinetin (6-furfurylaminopurine), IAA ( -indol acetic acid), NAA ( -naphthalene acetic acid), glycin, vitamin B1 v nc da (CW) Phng php Th nghim c b tr theo RCBD (1 yu t), 4 ln lp li, mi ln lp li nui cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (P=005). T l pht
538
sinh t bo soma (%), sinh khi t bo sau 15 ngy nui cy (g), tng sinh khi t bo (g), mt t bo (tbx104/ml), s chi / cm (chi), chiu cao chi (mm), r (+/ -) KT QU V THO LUN Nui cy pht sinh t bo soma: Mu thn l r c nui cy trn mi trng MS c b sung BA, kinetin, NAA. Kt qu nghi n cu cho thy t bo soma pht sinh trc tip trn vt ct sau 10 ngy nui cy. Thn v l cho pht sinh t bo soma cao. Nghim thc A1 c b sung BA (4mg/l) + Kin (2mg/l) + NAA (0,5mg/l) thch h p cho nui cy pht sinh phi soma trc tip tr n mu thn v l nui cy (bng 1), t bo phi soma c dng hnh trn v c mu trng sa. T bo phi soma t nghim thc A1 vi mu nui cy t l c s dng cho nhng nghin cu v sau Nhn sinh khi dch huyn ph phi soma trn mi tr ng agar: T bo phi so ma c nui cy tng sinh trn mi trng agar MS c b sung BA, kinetin, NAA. Kt qu nghin cu cho thy, t bo phi soma phn chia v bi t ha tt trn mi trng nghim thc B1: BA (4mg/l) + K in (2mg/l) + NAA (0,5mg/l) (b ng 2). Trn mi trng nghim thc B1, t bo phi soma bit ha su sc, t bo c mu xanh nht v c vi chi c ti sinh trong giai on n y Nhn sinh khi dch huyn ph phi soma trn mi tr ng lng: T bo phi soma pht sinh trn mi trng nghim thc A1 c a vo nui cy trn lng. Mi trng nui cy lng l MS c b sung BA, kinetin, NAA. Kt qu nghi n cu cho thy, t bo phn chia v bit ha nhanh trn mi trng nghim thc C1: BA (4mg/l) + K in (2mg/l) + NAA (0,5mg/l) (bng 3). Trn mi trng ny, t bo phi phn chia v bit ha t bo phi thnh hnh cu, hnh tim v hnh thy li; chun b y iu kin cho qu trnh ti sinh Tri dch huyn ph phi soma trn mi tr ng agar: T bo dch huyn ph c nui cy trong mi trng lng trn nghim thc C1 c s dng tri t bo. Mi trng tri t bo l MS c b sung BA (4mg/l) + Kin (2mg/l) + NAA (0,5mg/l). Sau 3 tun nui cy, lp t bo ph y mt agar, t bo phi mn v hu ht c dng hnh cu. T bo phi trong giai on ny thch hp cho nui cy ti sinh Ti sinh phi soma: T bo phi soma c nui cy ti sinh trn mi trng MS c b sung BA, kinetin v CW. Kt qu nghin cu cho thy, t bo phi soma bit ha nhanh chng pht sinh hnh thnh ch i, chi xut hin va c chi n v va c cm chi. Chi n pht sinh d dng pht sinh thnh cm chi. Nghim thc B1: MS + BA (0,1mg/l) + CW (10%) thch h p cho qu trnh ti sinh pht sinh cm chi Nhn nhanh cy lt hoa in vitro : cm ci hay chi n ti sinh c nui cy trn mi trng vi nhn ging MS + K in (1mg/l) + CW (10%). Chi pht sinh lin tc trong qu trnh vi nhn ging trn mi trng nghim thc E4 KT LUN Thn, l, r cy lt hoa nui cy m c s dng lm vt liu nui cy. Thn v l thch hp cho cc nghin cu nui cy pht sinh t bo soma v phi soma. L c s dng trong nghin cu nui cy pht sinh t bo soma, nhn sinh khi t bo trn mi trng bn rn v
539
lng, v c tri nui cy pht sinh phi soma tr n mi trng MS + BA (4mg/l) + Ki (2mg/l) + NAA (0,5mg/l). Phi soma c nui cy ti sinh sau 30 ngy trn mi trng MS + BA (0,1mg/l) + CW (10%) hnh thnh ch i n hay cm chi. Cm chi c s dng trong vi nhn ging trn mi trng MS + Ki (1mg/l) + CW (10%). Mt h thng nhn nhanh cy lt hoa bng k thut nui cy phi soma c xc nh TI LIU THAM KHO 1. 2. 3. 4. 5. B Khoa hc v Cng ngh (2000). Sch Vit Nam, phn Thc vt. NXB KH&KT, H Ni Lloyd G, McCown B (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc. 30:421-427 Mamood M. (1993). Application of plant in vitro technology. Proc., 16-18 Nov 1993, Univ. of Malaysia, Malaysia. Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 431-497 Nguyn B Cht (1998). Nghin cu mt s c im lm hc v bin php k thut gy trng nuI dng cy lt hoa (Chukrasia tabularis A. Juss). Lun n Tin s Lm nghip. Nguyn Hong Ngha (1999). Mt s loi cy b e da Vit Nam. NXB Nng nghip Phillips GC (1988). Developmental models f or the expression of totipotency and plant regeneration in vitro. South Assoc. Agri. Sci. Bull. Biochem. Biotech. 1:12-16 Trn nh Hu (1998). Nghin cu u hp thc vt lt hoa + gng no + chi u liu nc + trng tri nh ti khu vc t Thm, V n QG Cn o. Bo co Cn o 2001 Thorpe T. A. (1980). Organogenesis in vitro: structural, physiological and biochemical aspects. Int. Rev. Cytol. Suppl. 11A: 71-112
6. 7. 8. 9.
SUMMARY
Application of somatic embryogenesis culture techniques in lat hoa (Chukrasia tabularis A. Juss) improvement
Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology Stem, leaves and roots were used explants in cultivation. Stem and leaves were favoured for far studies. Leaves was initiated somatic cell, mass propagation on semi solid and liquid, and layer on medium MS + BA (4mg/l) + K in (2mg/l) + NAA (0,5mg/l). Embryogenic cell was regenerated on medium MS + BA (0,1mg/l) + CW (10%) to produce single or multi -shoot. Thease shoots were used for far micropropagation on medium MS + K in (1mg/l) + CW (10%). A system for cloning of lat hoa was established through somatic culture techniques
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VI NHN GING CY LIM XANH (Erythrophloeum fordii Oliv)

Bi Th Tng Thu, Trn Vn Minh PTNT v pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Lim xanh l loi cy g qu him c trong Sch Vit Nam v c xp vo trong danh mc Thc vt rng qu him quc gia (B NN&PTNT, 2000). Lim xanh l loi cy d trng, nhanh ln, thn cng chc n n thng c s dng trng rng ph ng h, phc hi rng (Nguyn Thng Hin, 2001). Vi gi tr kinh t cao nh vy, lim xanh ang l tm ngm ca nhng nh kinh doanh g. Loi cy ny ang b khai thc ro rit v ang trn b tiu dit (Nguyn Hong Ngha, 2005). Do vy cn c cc bin php k thut gy tr ng, phc hi v pht trin loi cy g c gi tr ny ca Vit Nam. Lim xanh cn c nghin cu bng phng php nhn ging nhanh in vitro (Gladfelter HJ, GC Phillips, 1987), nh m cung cp mt s lng ln cy ging phc v cng tc ti sinh rng (Nguyn Thng Hin, 2001) VT LIU V PHNG PHP
Vt liu
Mu nui cy l chi non cy lim xanh khong 20 ngy tui. iu kin nui cy: mi trng v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 282 oC. cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962), WPM (Lloyd and McCown, 1981) c b sung IAA ( -indolacetic acide), NAA ( -naphthaleneacetic acid), BA (6 -benzylaminopurine), IBA ( -indolbutyric acid), Kinetin (6-furfurylaminopurine), nc da.
Phng php
Th nghim c b tr theo RCBD (1 yu t), 3 ln lp li nui cy 5 b nh tam gic 300ml, mi bnh cha 65ml mi trng th nghim v c cy 7 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (p=0,05). T l nui cy pht sinh chi (%), chiu cao chi (mm), s chi / mu ((chi), s chi / b nh (chi). S liu c x l bng phn mn excel, mstatc. KT QU V THO LUN Nui cy nh sinh trng lim xanh in vitro: Mu nui cy l chi nh ca cy ngoi rng c v trng vi Hypochlorite-Ca (10%) trong 12 pht. duy tr cc
541
dng v tnh trong t nhin th nui cy in vitro chi nh l mt trong nhng phng php tch cc c kh nng lm tr ha m nui cy. Trong giai o n ny, cytokinin gi vai tr quan trng trong vic pht sinh chi b n v to th chi. WPM + BA (0,1mg/l) l mi trng thch hp nht cho nui cy nh sinh tr ng To cm chi lim xanh: Sau giai on nui cy nh sinh trng in vitro, th giai on to cm chi rt quan trng v cn thit v y l vt liu u tin c s dng nhn nhanh in vitro. Mu c nui cy trn mi trng khong WPM c b sung BA (0-0.05mg/l) v Kinetin (0-0,05mg/l). Kt qu th nghim cho thy WPM + BA (0,05mg/l) l mi trng thch hp nht cho to cm chi nh hng ca BA n nhn ging lim xanh in vitro : Nhm nhn nhanh, quyt nh hiu qu nui cy in vitro. Mi trng nui cy khong c bn WPM c b sung BA (0,05-0,1-0,3-0,5mg/l). Kt qu cho thy mi trng WPM + BA (0,05mg/l) l mi trng thch hp nht cho nhn ging lim xanh in vitro nh hng ca Kinetin n nhn ging cy lim xanh in vitro: Mi trng nui cy l khong WPM c b sung Kinetin (0,05-0,1-0,3-0,5mg/l). Tt c cc nghim thc th nghim u pht sinh chi mi. Khi nng Kin (0,05mg/l) cho s chi/mu cao nht, chiu cao chi cao, l c m u xanh m, thn chi vn khe. Khi tng nng Ki ln (0,3mg/l) hoc Kin (0,5mg/l) th c ch pht sinh chi, l ng vng sau 20 ngy nui cy, thn chi yu, mc ph gc trung bnh, chi thp. Mi trng khong c bn WPM c b sung Kin (0,05mg/l) thch hp cho nhn ging cy lim xanh in vitro nh hng ca BA, Ki n nhn ging cy lim xanh in vitro: Mi trng nui cy l khong c bn WPM. Khi b sung glut amin (1g/l) vo mi trng c BA (0,05mg/l) + Kin (0,05mg/l) th s chi mi t v chi vn cao, khng cn hin tng ph gc, l ln, c mu xanh m, thn chi vn khe nh hng ca BA, Ki, NAA n nhn chi cy lim xanh in vitro : nhm tng nhanh h s nhn chi ng thi kch thch chi v n cao l ngun nguyn liu trc khi nui cy pht sinh r. Mi tr ng nui cy c s dng l WPM b sung BA (0,05mg/l), kinetin (0,05mg/l), NAA (0 -0,1mg/l) v glutamin (1g/l). K t qu cho thy khi s dng WPM + BA (0,05mg/l) + Kin (0,05 mg/l) + glutamin (1g/l) l mi tr ng thch hp nht nhn ging lim xanh in vitro Nui cy pht sinh r cy lim xanh in vitro: Mi trng kch thch ra r c chn l mi trng khong c bn WPM sau b sung th m cc cht iu ha sinh trng nh: Kinetin nng 0,05mg/l, NAA nng 0,1mg/l. Cy lim xanh in vitro thch hp nht l s dng mi trng khong c bn WPM v b sung NAA vi nng 0,1mg/l cho nui cy to r KT LUN xy dng hon chnh c quy trnh nhn ging cy lim xanh (Erythrophloeum fordii Oliv), nhm bo tn ngun gen cy qu v pht trin din tch rng Vit Nam: nui cy nh sinh trng [WPM + BA (0,1mg/l)], to cm chi [WPM + BA
542
(0,05mg/l)], nhn nhanh ch i [WPM + BA (0,05mg/l) + Ki (0,05 mg/ l) + glutamin (1g/l)] v nui cy to r in vitro [WPM + NAA (0,1mg/l)]. TI LIU THAM KHO 1. 2. B NN&PTNT (2000). Tn cy rng Vit Nam, quyn 1 trang 113. Nh xut bn Nng nghip. Gladfelter H. J. & G. C. Phillips (1987). De novo organogenesis of Pinus eld arica in vitro. I. Reproducible regeneration from long -term callus cultures. Plant Cell Rep. 6: 163-166 Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel , Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Cro p Soc. 30:421-427. Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures . Physiol. Plant. 15:431-497 Nguyn Hong Ngha (2005). Kt qu nghin cu bo tn ngun gen cy rng. Thng tin chuyn Lm nghip s 1-2005. Vin khoa hc Lm nghip Vit Nam. Nguyn Thng Hin (2001). Thc vt v c sn rng. NXB i hc Nng lm.
3.
4. 5. 6.
SUMMARY
Micropropagation of lim xanh ( Erythrophloeum fordii Oliv)

Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plan t Cell Biotechnology, Institute of Tropical Biology
WPM medium was favoured for lim xanh (Erythrophloeum fordii Oliv) cultivation. A system cultivation in in vitro was establishsed for young shoot tip cultivation was WPM + BA (0,1mg/l); for multiple -shoots induction was WPM + BA (0,05mg/l); for multiple-shoots micropropagation was WPM + BA (0,05mg/l) + Kin (0,05mg/l) + glutamine (1g/l); and for rooting cultivation was WPM + NAA (0,1mg/l)
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NG DNG CNG NGH PHI soma TRONG BO TN V PHT TRIN CY QU (Cinnamomum cassia Nees et Eberth)
Bi Th Tng Thu, Trn Vn Minh PTNT v pha Nam v CNTBTV, Vin Sinh hc Nhit i M U Cy qu l mt trong nhng loi cy c sn Vit Nam, c gi tr kinh t cao. Qu c trng nhiu vng trong nc t Bc vo Nam. Tuy nhin, hin nay nhn dn chia ra lm hai loi qu: qu v mng v qu v dy. trong qu v dy cho nhiu tinh du hn. Cy qu c du nhp v trng trt theo tp qun tng a ph ng, ch yu t cy thc sinh bng ht, c nhiu ging khc nhau, cy qu thoi ha dn, dn n h m lng tinh du khai thc thp. Cng tc ging qu trong thi gian qua c gng chn cy u dng bo tn v pht trin, nhng nhng k thut dng ha cn hn ch nh gim hom v ghp cnh (Gamborg, 1993). Nhng r o cn u tin vn l cng tc ging. Ging thun chng sinh tr ng nhanh v c nng sut cao c chn lc. Cy qu c cha nhiu tinh du, rt kh ti sinh in vitro (Mamood, 199 3). ng dng cng ngh phi soma trong bo tn v dng ha cy qu m u dng l cn thit trong pht trin cc vng chuyn canh cy qu. VT LIU V PHNG PHP Vt liu Mu nui cy: cy qu u dng c thu thp ti Tin Phc, Qung Nam. Mu c v trng bng hypochlorite calci (10%) trong 10 pht, sau c v trng bng HgCl2 (0,1%) trong 5 pht. Chi non on 1-1,5cm c s dng trong nui cy. iu kin nui cy: mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+2oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962) v WPM (Lloyd and McCown, 1981), c b sung BA (6-benzyl aminopurine), Kinetin (6-furfurylaminopurine), NAA ( -naphthalene acetic acid), glutamine, glycin, vitamin B1 v nc da (CW) Phng php Th nghim c b tr theo CBD n yu t, 4 ln lp li, mi ln lp li nu i cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC
544
(P=0.05). T l mu nui cy pht sinh t bo soma (%), sinh khi t bo sau 60 ngy nui cy (g), tng sinh khi (g), t l pht sinh phi (%). KT QU V THO LUN Nui cy pht sinh t bo soma: Chi non cha ha g ca cy qu sau khi v tr ng c nui cy trn mi trng pht sinh t bo soma. Mi trng nui cy MS, WPM v B5. Kt qu nghin cu cho thy hu ht cc mi trung khong c bn nui cy u cho pht sinh t bo soma. Trong mi tr ng nghim thc A2: WPM + BA (1mg/l) + Kin (1mg/l) nui cy cho pht sinh phi. Phi soma qu c m u trng sa v t bo cht m c nh hng ca mu nui cy n pht sinh t b o soma: Thn l r cy qu in vitro c s dng trong nui cy pht sinh phi tr n mi trng nghim thc A2. Kt qu nghin cu cho thy c 3 loi mu nui cy u cho pht sinh t b o soma. Trong mu nui cy thn cho pht sinh t b o soma cao (nghim thc B1) Nui cy tng sinh t bo soma trn mi trng agar: T bo phi soma (nghim thc B2) c nui cy tng sinh trn mi trng WPM v MS c b sung BA, kinetin, 2.4D, glutamine v CW. K t qu nghin cu cho thy mi trng nghim thc C2: WPM + BA (1mg/l) + Kin (1mg/l) cho tng sinh t bo soma mnh m (14,333g) sau 15 ngy nui cy. T bo soma c mu nu ca nha cy qu sau 45 ngy nui cy Nui cy pht sinh t bo phi soma: T bo soma nui cy trn mi trng nghim thc C2 c nui cy chuyn sang mi tr ng nui cy pht sinh phi. Mi trng nui cy pht sinh phi l WPM c b sung BA, kinetin, CW. Kt qu nghi n cu cho thy, t bo soma phn chia v bit ha mnh m sang t b o phi soma sau 45 ngy nui cy trn mi trng nghim thc D5: WPM + BA (1mg/l) + K in (1mg/l). T bo phi c dng hnh cu, tch ri rc v khng thnh cm, c mu trng nu ng nh sng ca tinh du qu nh hng ca s ln cy truyn n pht sinh phi soma : T bo soma c nui cy truyn nhiu ln trn mi trng nghim thc D5, thi gian gia hai ln cy truyn l 45 ngy, cng v sau t bo phi soma xut hin cng nhiu t th h cy truyn F5 tr i, n th h F8 ton b t bo phi soma bit ha thnh phi hnh ng c l tin sinh. Ti sinh phi soma: T bo phi soma nui cy truyn trn mi trng nghim thc D5, c nui cy ti sinh trn mi trng WPM c b sung BA, kinetin, CW. Kt qu nghin cu cho thy, mi trng nghim thc E3 v E4 c t l ti sinh cao (8 v 7 chi / bnh). Trn mi trng nghim thc E4: WPM + BA (0,5mg/l) + Ki n (1mg/l) + CW (10%) cho pht sinh chi c l pht trin KT LUN Chi non cy qu thu t rng phng h c v trng v nui cy pht sinh t bo soma in vitro trn mi tr ng WPM + BA (1mg/l) + K in (1mg/l). Thn l r u c kh nng nui cy pht sinh t bo soma. T bo soma tng sinh mnh m (14,333g) sau 15
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ngy nui cy trn mi trng WPM + BA (1mg/l) + K in (1mg/l). T bo soma c mu nu ca nha cy qu sau 45 ngy nui cy. T bo soma bit ha thnh t bo phi soma trn mi trng WPM + BA (1mg/l) + K in (1mg/l). T bo phi c dng hnh cu, tch ri rc v khng thnh cm, c mu trng nu ng nh sng ca tinh du qu. T ln cy truyn th 5 tr i, kh nng pht sinh phi nhiu sau nui cy. Phi soma c ti sinh trn mi trng WPM + BA (0,5mg/l) + Ki n (1mg/l) + CW (10%) cho pht sinh chi c l pht trin. ng dng cng ngh phi soma trong bo tn v pht trin cy qu chng minh c hiu qu TI LIU THAM KHO 1. Gamborg O. L. (1986). Protoplasts and plant regeneration in culture. In: Demain AL, Solomon NA (eds) Manual of industrial micro biology and biotechnology. American Society for Microbiology, Washington, DC Gamborg O. L. (1993). Potential of biotechnology in micropropagation and genetic improvement of woody species. In: Proc Application of plant in vitro technology. 16-18 Nov 1993, University of Malaysia, Malaysia Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc. (30) 421-427 Mamood M. (1993). The total phenols and growth of co coa (Theobroma cacao L.) tissue culture after supplemented with phenylalanine in vitro. In: NA Shamaan etal. (eds) Proc. Application of plant in vitro technology. 16-18 Nov 1993, University of Malaysia, Malaysia Murashige T. & R. Skoog (1962). A revised me dium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. (15) 431-497
2.
3. 4.
5.
SUMMARY
Application of somatic embryogenesis culture techniques in conservation and development of Que (Cinnamomum cassia Nees et Eberth)
Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnology, Institute of Tropical Biology Stems were the well explants in cultivation. The explants were culture for somatic cell initiation well, for somatic cell mass propagation, for somatic embryogenesi s on medium WPM + BA (1mg/l) + K in (1mg/l). Regeneration of somatic embryogenesis was cultured on medium WPM + BA (0,5mg/l) + K in (1mg/l) + CW (10%). A system of initiation, mass propagation, and regeneration culture for conservation and cloning to develop the orchard and export planting area were established
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NGHIN CU K THUT NUI CY NH SINH TRNG TRONG BO TN V PHT TRIN CY THNG (Taxus wallichiana Zucc)
Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i M U Cy thng l loi cy qu him c s dng trong dc liu. Trn nh Liang Biang hin nay s lng cn li rt t, ang ng trc nguy c b tuyt dit. Cy thng nm trong Sch Vit Nam (B KH&CN, 2000). Phc hi li rng thng hin nay ch yu da vo phng php gim cnh truy n thng, chi ngn mang tnh u th ngn mnh m (Pierik, 1987) v cy con bu t thng c ng nghing cao. Cy thng li sinh trng chm, nn kh nng khai thc thng v s dng cho cng nghip dc i hi thi gian lu. Phc hi v pht trin cy thng l mt thch thc ln i vi khoa hc (Coke, 1996). Nghi n cu k thut nui cy nh sinh tr ng nhm mc tiu bo tn (Mantell etal., 1985) v pht trin cy thng l hng nghin cu kh thi. VT LIU V PHNG PHP Vt liu Mu nui cy: cy thng bu t c gim cnh (c thu thp vng ni Liang Biang, Lt) c 10 thng tui. iu kin nui cy: mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+1oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy Mi trng nui cy: mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962) v WPM (Lloyd and McCown, 1981), B5 (Gamborg, 1986), WV3 (Coke, 1996), c b sung BA (6-benzyl aminopurine), Kinetin (6 -furfurylaminopurine), IBA ( -indol butyric acid), NAA ( - naphthalene acetic acid), glyc in (5mg/l), vitamin B1 (5mg/l) v nc da (CW). Cht chng ha nu: AC (than hot tnh), PVP (polyvinylpyrolidon) v AgNO 3 Phng php Th nghim c b tr theo RCBD (1 yu t), 4 ln lp li, mi ln lp li nui cy 5 bnh tam gic 300ml, m i bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC
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(P=0.05). ng knh mi trng di gc thn b ha nu (mm), t l mu nu i cy pht sinh chi (%), s chi / mu (chi), s chi / b nh (chi), chiu cao chi (mm) KT QU V THO LUN V trng mu nui cy: Mu nui cy c ly t cy m bu t 18 thn g tui. Chi nh v chi bn c s dng lm mu nui cy. Chi non c v trng hypochlorite-Na (10%) trong 15 pht. Chi v trng c a vo nui cy in vitro nh hng ca mi trng khong c bn n nui cy pht sinh chi thng in vitro: Chi non c nui cy trn mi trng khong c bn MS, WPM, B5, WV3 c b sung BA (5mg/l). Kt qu nghi n cu cho thy, mi trng khong c bn MS (nghim thc A1) t ra thch h p nh hng ca cht chng ha nu n nui cy pht sinh chi thng in vitr o: Cy thng thng c nha khi b to vt th ng. Chi non a v cy sau 2 ngy th thy xut hin nha tit ra mi tr ng nui cy lm hn ch kh nng pht sinh chi. Trn mi trng nui cy MS + BA (5mg/l) c b sung than hot tnh, PVP v AgNO3. Kt qu nghin cu cho thy khi b sung than hot tnh (1000mg/l), PVP (150mg/l) v AgNO 3 (150mg/l) hn ch r rt s tit nha loang vo mi trng nui cy. Trong AgNO 3 t ra hiu qu nh hng ca sinh l mu nui cy n pht sinh chi thn g in vitro: Sinh l mu nui cy c nh hng rt ln n kh nng pht sinh chi b n v chi bt nh. Sinh l mu nui cy c thc nghim vi nhiu loi mu nui cy: (1) ngn chnh + t 1 (2) thn chnh + t 2 (3) thn chnh + t 3 (4) ngn c nh + t 1 (5) thn cnh + t 2 (6) thn cnh + t 3. Mu c nui cy trn mi trng pht sinh chi MS + BA (5mg/l) + AgNO 3 (150mg/l). Kt qu nghin cu cho thy chi pht sinh cao nht mu (1) v (2), c s chi / mu nui cy: 1,5 v 3 chi nh hng ca tui mu nui cy n pht sinh chi thng in vitro : Tui mu nui cy chim vai tr quan trng, mu phi non v trng thnh cn thit, s cho pht sinh chi cao. Mu nui cy c tui sinh l 12-15-18 thng, c nui cy trn mi trng pht sinh chi MS + BA (5mg/l) + AgNO 3 (150mg/l). Kt qu nghin cu cho thy, t l chi pht sinh cao nht vi tui mu 18 thng, v cho s chi nhiu / mu nui cy (3,1 chi) nh hng ca BA n pht sinh chi thng in vitro : Vi mi trng nui cy c bn MS c b sung nng BA (0,5 -2,5-5,-10,-15mg/l) + AgNO 3 (150mg/l). Kt qu nghin cu cho thy BA (5mg/l) thch hp cho nui cy pht sinh chi nh hng ca BA v kinetin n pht sinh chi thng in vitro : Nhm nng cao kh nng nui cy pht sinh chi, t hp BA (5mg/l) + K in (0,1-0,5-1,-2,5-5mg/l) + AgNO3 (150mg/l) c b sung vo mi trng nui cy MS. Kt qu nghi n cu cho thy t hp BA (5mg/l) + K in (1mg/l) + AgNO 3 (150mg/l) thch hp cho nui cy pht sinh chi, c s chi / mu nui cy nhiu (3,1 chi)
548
nh hng ca mu nui cy n t l ng nghing chi thng in vitro: Cy thng c c tnh khi gim c nh bn ngoi vn m thng c t l cy ng nghin cao. Mi trng nui cy pht sinh chi l MS + BA (5mg/l) + Kin (1mg/l) + AgNO 3 (150mg/l). Mu nui cy (1) nh thng ng (2) thn thng ng (3) nh c nh (4) thn cnh. Kt qu nghin cu cho thy, t l ng nghing cao khi mu nui cy l (3) v (4) so vi thng ng ca trc tung (0 o) l >45 o, mu (1) v (2) c t l ng nghing <45o. Vy nui cy thch hp in vitro l (1) nh thng ng (2) thn thng ng nh hng ca tnh bo lu cc b n sinh trng chi thng in vitro : Trong gim cnh, vi mu l cnh (nhnh) bn, thng cho cy pht trin ng nghing. Nghin cu tnh bo lu cc b in vitro cho thy: tr n mi trng nui cy c bn MS + BA (5mg/l) + Kin (1mg/l) + AgNO 3 (150mg/l), vi mu nui cy l (1) nh cnh v (2) thn cnh, cho kt qu: gc nghing so vi trc tung (0 o), mu nui cy nh cnh (1) c t l ng nghing thp nh hng ca iu kin nui cy thng kh v cng chiu sng n sinh trng chi thng in vitro: Trn mi trng nui cy pht sinh chi c bn MS + BA (5mg/l) + Kin (1mg/l) + AgNO 3 (150mg/l), mu l thn chnh + t 2 c s dng trong nui cy, s dng np giy v np cao su, trong iu kin chiu sng 11,4 -22,834,2 mol/m2/s, kt qu nghin cu cho thy trong iu kin chiu sng 11,4 22,8 mol/m2/s v y bng np giy cho pht sinh chi cao (2,8 chi) Nhn nhanh cy thng in vitro: Chi non pht sinh trong nui cy c s dng lm protocol cho qu trnh vi nhn gi ng. Trn mi trng c bn MS c b sung BA, K. Kt qu nghin cu cho thy mi trng nghim thc E2: MS + BA (5mg/l) + Kin (1mg/l) cho pht sinh chi cao (3,4 chi / cm ). Cy thng in vitro, c tnh u th ngn mnh m, nui cy t thn cho pht sinh chi, nui cy chi nh ch cho v n thn Nui cy pht sinh r chi thng in vitro : Cy con thng pht sinh r in vitro trn mi trng nui cy WPM + NAA (1mg/l) Rhizopon (50mg/l) sau 75 ng y nui cy KT LUN Chi nh v t thn cy thng (1-2 nm tui) c s dng trong nui cy in vitro. Chi non pht sinh sau 45 ng y nui cy trn mi trng MS + BA (5mg/l). Chi non ti sinh c s dng lm propagules trong vi nhn gi ng trn mi trng MS + BA (5mg/l) + Ki (1mg/l). Ch i non c nui cy pht sinh r tr n mi trng WPM + NAA (1mg/l) + Rhizopon (50mg/l) sau 75 ngy nui c y. AgNO 3 thch hp cho nui cy c ch s ha nu mu. Chi nh chim nhiu u th trong qu trnh pht sinh v sinh trng ca chi bn. Nui cy chi nh c xem l k thut hiu qu trong bo tn loi thng Vit Nam. TI LIU THAM KHO 1. 2. B KH&CN (2000). Sch Vit Nam, phn Thc vt. NXB KH&KT, H Ni Coke J. E. (1996). Basal medium for in vitro cultures of loblolly pine. US Patent No. 5,534,434. July 9, 1996
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3.
Gamborg O. L. (1986). Protoplasts and plant regeneration in culture. In: Demain AL, Solomon NA (eds) Manual of industrial microbiology and biotechno logy. American Society for Microbiology, Washington, DC Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc . 30:421-427 Mantell S. H., Matthews J. A. & McKee R. A. (1985). Principles of plant biotechnology. Blackwell Scietific, Boston, pp130 -157 Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:431-497 Philips G. C. (1988). Development models for the expression of totipotency and plant regeneration in vitro. South Assoc. Sci. Bull. Biochem. Biotech . 1: 12-16 Pierik R. L. M. (1987). In vitro culture of higher plants. Martinus Nijhoff, Dordrecht, pp183-230
4.
5. 6. 7. 8.
SUMMARY
Study of shoot-tip culture techniques in conservation and development of taxus trees ( Taxus wallichiana Zucc)
Young shoot-tips of taxus plant were used as explants in cultivation. The explants initiated shoots well on medium MS + BA (5mg/l), micropropagated on medium MS + BA(5mg/l) + K (1mg/l), rooted well on medium MS + WPM + NAA (1mg/l) Rhizopon (50mg/l) after 75 days. AgNO 3 was favoured for inhibition of browning the explants cultured. Shoots had the rule in inhibition the adventitous shoots initiated. Stem noding was used as protocol for far micropropagation. A system to permit producing planlets of taxus around the year in vitro. This is the first rep ort by using micropropagation techniques for conservation and development of taxus trees in Vietnam
550
NUI CY TI SINH PHI SOMA CY TEAK (TECTONA GRANDIS LINN.F)

M U Teak (Tectona grnadis Linn. f.) l mt loi g qu quan trng v gi tr g ca n (Rijuta & Sharon, 1996). Tuy nhin, cy teak sinh tr ng chm vi chu k khai thc 15-20 nm, ngoi ra t l ny mm ca ht thp. Nui cy m l mt phng php nhn nhanh cy teak c s dng nhiu n i trong khu vc Chu nhit i nh Myanmar, Thi Lan, Indonesia, Malaysia (Kaosar -ard & Apavatjrut, 1988). C nhiu cng trnh nghin cu v vi nhn ging bng ph ng php nui cy t thn hay chi nh (Bonga & Aderka s, 1992; Goh & Monteuuis, 1995), nui c y pht sinh c quan qua nui c y l non v l gi (Graudal etal. 1999), nui c y pht sinh phi soma trc tip v gin tip (Rijuta & Sharon, 1996). Ci thin ging teak bng con ng cng ngh sinh hc nhm mc ti u to ra ging mi c u im nh thi gian sinh trng ngn, thn thng, t phn nhnh, ra hoa chm, m u sc g, kh nng chng bnh th ro cn u tin l k thut nui cy ti sinh phi soma. Bo co ny nghin cu qu trnh pht sinh v ti sinh phi soma cy teak m hi n nay cn cha cng b VT LIU V PHNG PHP Vt liu: Mu nui cy l cy teak in vitro, ging Myanmar nhp ni. Mi tr ng nui cy c bn l MS (1962) v WPM (1981). Cc ch t c b sung vo mi trng nui cy nh: BA (6-Benzylaminopurine), Kinetin (6-Furfurylaminopurine), NAA ( Naphthalene acetic acid), IBA ( -Indol butyric acid), TDZ (Thidiazurone), vitamin B5, nc da (CW). iu kin nui cy: nhit ph ng 28+2 oC, m 65%, thi gian chiu sng 8gi/ngy, cng chiu sng 3000lux. Phng php: Nui cy to t bo soma trong bnh tam gic (300ml). Nui c y lng trong cc bnh tam gic (300ml), c ch a 50ml mi trng nui cy, c t trn my lc, c tc 90 vng/pht. T bo dch huyn ph c tri trn a petri (cha 15ml mi trng); v bnh tam gic (300ml) ch a 50ml mi trng. Th nghim c b tr 3 ln lp li, mi ln lp li c 3 b nh tam gic hay 5 a petri, mi bnh tam gic hay mi a petri c cy 5 mu. S liu thu thp c x l bng phn mm thng k MSTATC.
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KT QU V THO LUN
1. Nui cy to t bo soma
Hu ht cc mu nui cy u to t b o soma. Mu nui cy thn l r trn mi trng A4, A8, A12 u cho pht sinh t bo soma cao. Vitamin B5 v Ca-panthothenate c b sung vo mi trng nui cy cho thy pht sinh t bo tin phi soma
2. Nui cy pht sinh t bo tin phi soma trn mi trng agar
Vi kh nng pht sinh t b o soma mnh m, giai on hai l giai on nghin cu kh nng nui cy t bo soma bit ha hnh thnh t bo tin phi soma. Kt qu nghin cu cho thy, cc t bo tin phi c cu trc cht pht sinh tr n khi m c mu trng sa trn mi trng B2. Nghim thc B1 v B3 cho pht sinh t bo tin phi soma thp.
3. Nui cy tch ri t bo tin phi soma v nhn nhanh t bo tin phi soma
Trong qu trnh nui cy pht sinh t bo tin phi soma, t bo pht sinh bao quanh khi m, cn nghin cu tch ri t bo tin phi soma ra khi khi m. Kt qu nghin cho thy, cc cm t bo mn bt u tch ra sau 3 ngy nui cy. Cc cm t bo tin phi soma tch hn ra khi khi m sau 5 ngy nui cy to dch huyn ph trn mi trng nui cy C4. Mi trng C4 c s dng l mi trng nhn nhanh sinh khi t bo tin phi soma v t bo tng nhanh sinh khi sau 5 ngy nui cy
4. Nui cy pht sinh t bo phi soma v mi tr ng iu kin
Vi mu nui cy l nhng cm t bo tin phi soma sau giai on nhn nhanh tng sinh khi trong mi trng lng c nui cy mi trng pht sinh bit ha th nh phi soma. Kt qu nghin cu cho thy, t bo tin phi soma bit ha th nh phi soma sau 10 ngy nui cy trn mi trng lng D4. ti sinh phi soma teak, sau nhiu nghin cu ti sinh khng thnh cng, cn thit phi nghin cu mi trng iu kin tri t bo phi soma sau giai on nui cy trong lng. Kt qu nghin cu cho thy, phi soma pht sinh phi mm tr n mi trng D7
5. Ti sinh phi soma
Cc cm t bo phi mm c a qua nui cy ti sinh. Kt qu nghi n cu cho thy chi non ti sinh trn mi trng sau 30 ngy nui cy KT LUN Thn, l, non cy teak cy m c s dng lm nguyn liu nui cy. T bo soma c nui cy pht sinh trn mi trng NS + BA (1mg/l) + Ki (1mg/l) + NAA (0,5mg/l) + B5 (5mg/l) + Ca -pan (4mg/l) sau 21 ngy nui c y. T bo soma bit ha thnh t bo tin phi soma sau 14 ngy nui cy trn mi trng bn rn MS + 2.4D
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(1mg/l). T bo tin phi c nui cy tch ri trn my lc sau 5 ngy nui cy trn mi trng MS + BA (0,5mg/l) + TDZ (0,2mg/l) + Ca -pan (4mg/l). Sau t bo tin phi c nui cy tng sinh trn mi trng lng c lc trn mi trng MS + BA (0,5mg/l) + TDZ (0,2mg/l) + Ca -pan (4mg/l) sau 5 ngy. T bo tin phi c nui cy bit ha thnh t bo phi trn mi trng lng MS + BA (0,5mg/l) + TDZ (0,2m g/l) + Ca-pan (4mg/l) sau 10 ngy. Tr c khi a qua mi trng ti sinh, t bo phi c nui cy trn mi trng iu kin 21 ngy MS + BA (0,1mg/l) + Ki (0,1mg/l). T bo phi soma c nui cy ti sinh sau 90 ng y nui cy trn mi trng WPM + TDZ (0,2mg/l) + IBA (0,2mg/l) + CW (10%). M t h thng nui cy ti sinh ho n chnh cy teak t phi soma c xy dng TI LIU THAM KHO 1. 2. 3. Bonga J. M. & V. P. Aderkas (1992). In vitro culture of trees. Kluwer Academic Press Publishers, Dordrecht, The Netherl ands Goh D. & O. Monteuuis (1995). Vegetative propagation of teak. Tropical Forest Update 7(2): 12-13 Graudal L., E. D. Kjaer, V. Suangtho, P. Saardavut & A. Kaosa -ard, 1999. Conservation of genetic resources of teak (Tectona grandis L. f). Technical Note No52. Danida Forest Seed Centre, Denmark. Kaosar-ard A. P. Apavatjrut (1988). Teak ( Tectona grandis L. F) tissue culture: rooting and transplanting techniques. PSTC Conference on Biotechnology for Health and Agiculture, Washington DC, USA 6 -9 June 1988 Lloyd G. & L. McCown (1981). Commercial feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot -tip culture. In: Comb. Proc. Intl. Plant Prop. Soc 30: 421-42 Murashige T. & F. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-479 Kushalkar R. & M. Sharon (1996). Direct and indirect somatic embryogenesis in teak (Tectona grandis Linn.f). Current Science, 1996 (71)9: 712-715
4.
5.
6. 7.
553
SUMMARY
Regeneration of teak somatic embryos

Young stems, leaves, and roots were used to initiated cultures. Somatic cells were initiated on MS medium supplented with BA (1mg/l) + K in (1mg/l) + NAA (0,5mg/l) + B5 (5mg/l) + Ca-pan (4mg/l) after 21 days. Somatic cells were differentiated to proembryogenic cells after cultured on semi -solid medium MS supplemented with 2.4D (1mg/l) after 14 days. Proembryonic cells were seperated on liqu id medium cultures of MS supplemented with BA (0,5mg/l) + TDZ (0,2mg/l) + Ca -pan (4mg/l) after 5 days. Proembryogenic cells were multipled on liquid medium of MS supplemented with BA (0,5mg/l) + TDZ (0,2mg/l) + Ca -pan (4mg/l) after 5 days. Proembryogenic c ells were differentiated to embryogenic cells on liquid medium of MS supplemented with BA (0,5mg/l) + TDZ (0,2mg/l) + Ca -pan (4mg/l) after 10 days. The conditioned medium MS + BA (0,1mg/l) + Kinetin (0,1mg/l) was required before transfering embryogenic cells to regeneration. Shoots were regenerated on medium WPM + TDZ (0,2mg/l) + IBA (0,2mg/l) + CW(10%). A system for regeneration of teak somatic embryogenesis culture was established
554
DNG HO CY THANH HAO (Artemisia annua L.) in vitro

Phm Th Anh, L Thi Hin, Bi Thi Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i
M U Thc vt l ngun dc phm quan trng t h ng ngn nm nay. WHO c tnh khong 80% dn s vn da ch yu v o cc phng thuc truyn thng nh tho dc trong cha tr bnh. Cc loi tn d c ngy nay cng c ngun gc t thc vt. Khong mt phn t cc loi thuc c k toa cha cc chit xut t thc vt. Thuc cha st rt hu hiu nht hin nay l artemisinin c chit xut t cy thanh hao hoa v ng. Thanh hao (Artemisisa annua L.) c ngun gc Chu (Trung Quc, Vit Nam), hin nay l mt loi cy rt ph thng c mt nhiu n i trn th gii, mc tt kh hu n i hay cao nguyn. Vit Nam, theo kt qu iu tra c th ca Vin D c liu t nm 1986-1990, pht hin thanh cao hoa vng mc t nhin 4 tnh, thuc 20 huyn - th x v hn 70 x Phng php sn xut artemisinin t cy thanh hao ngo i t nhin ph thuc vo sn lng thu hoch cy thanh hao hay thi tit. Cng ngh sinh hc ng vai tr quan trng trong vic la chn, nhn ging v bo tn cc ngun gen ca cy thuc qu (Hara etal, 1998). K thut ti sinh invitro nm gi tim nng to ln trong vic to ra cc ngun thc vt lm thuc c cht lng cao (Nalammai & Chan, 2004) . Cc hp cht th cp c thu nhn thng qua nui cy t b o soma, t bo dch huyn ph c nghin cu t nhiu loi cy thuc (Chan & Nalammai, 2001). Trong b i bo ny chng ti nghin cu vn chn dng cy thanh hao invitro, qua lm ngun nguyn liu cho vic nui cy t b o soma t thn, l, r nhm mc ti u sn xut artemisinin phng chng bnh st rt. VT LIU V PHNG PHP
Vt liu
Mu nui cy: Thanh hao (Artemisia annua L.) thc sinh chn lc c ly t Trung tm Dc liu Lt (Lm ng). Mu l nhng mnh l ct thnh on 2-3cm c v trng bng Na-hypochlorite (1-5%) trong 10 pht v c nui cy trong ng nghim. Sau nui cy 10 ngy, nhng mu khng nhim c a vo nui cy ti sinh trc tip trn mi trng MS + BA (0,5mg/l) + Kinetin (0,5mg/l). Sau 30 ngy nui cy, cm chi non invitro pht sinh. Nhng chi non ny c s dng lm nguyn liu cho cc nghin cu trong ti.
555
iu kin nui cy: mi trng v trng 121oC v 1at trong 25 pht. Nhit phng nui cy 282oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy. Mi trng nui cy: Mi trng khong c bn MS (Murashige-Skoog, 1962), LV (Phillips-Collins, 1979) c b sung ng sucrose, v cc cht iu ho sinh trng nh: BA (6-benzyladenine), Kinetin (6-furfurylaminopurine), NAA ( -naphthalene acetic acid ), 2.4D (2.4-dichlorophenoxy acetic acid). Phng php Th nghim c b tr theo CBD (1 yu t), 4 ln lp li, mi ln lp li nu i cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (P=0.05) sinh khi t bo sau nui cy (g), chiu cao chi (mm) KT QU V THO LUN Chn la v kho st sinh trng v pht trin trn mi trng nui cy MS v LV: xc nh mi trng khong thch hp nui cy cy thanh hao invitro. Cc chi non cy thc sinh bu t c v trng bng hypochlorite-Na (5%) trong 10 pht. Ch i non v trng c a vo nui cy trn hai mi trng khong MS v LV. Theo di v nh gi kh nng to chi sau 30 ngy nui cy. Kt qu nghin cu cho thy: Mi trng khong c bn MS v LV khng c b sung cht iu ha sinh trng u c kh nng pht sinh chi cy thanh hao. Tuy nhi n mi trng LV l thch hp hn vi l cy mc to hn, vn thng, xanh tt Chn dng cy thanh hao: Trong th nghim trn quan st thy c hai dng cy thanh hao khc nhau. Mt dng cao, thn chc, l vn thng; mt dng thn thp. Theo nhiu ti liu ghi nhn dng thn cao s cho hm lng artemisinin cao hn, do chng ti chn nhng cy ny lm vt liu cho nhng th nghim k tip. D ng thanh hao thn cao c nui cy nhn ging in vitro tr n mi trng LV + BA (0-0,1-0,3-0,5-0,7mg/l). Chng ti nhn thy vi nng BA thp (0,3mg/l) chi pht trin c v chiu cao v s lng. Chi xanh tt, thn chi vn thng, l to. Trong khi , mi tr ng c BA (0.5mg/l) v BA (0,7mg/l), chi pht trin khng bnh thng, l xon, thn mng nc. Do mi trng c b sung BA (0,3mg/l) thch hp cho s nhn chi nh hng ca BA, NAA v 2,4 D n kh nng nui cy pht sinh t b o soma: Mi trng nui cy LV c b sung BA, NAA, v 2.4D ring r vi nng s dng cho mi loi cht l 0-0,1-0,5-0,7-1mg/l. Kt qu nghin cu cho thy, BA, NAA v 2.4D ring r khng kch thch pht sinh t b o soma v b sung NAA v 2.4D ring r cho thy kch thch to r (bng 3) nh hng phi hp ca BA v NAA n kh nng nui cy pht sinh t b o soma: Mi trng khong LV c b sung BA (0,1-0,5-0,7-1,1,5-2,-2,5mg/l) v NAA (0,1-0,30,5-0,7-1-1,5-2mg/l) c s dng trong nghi n cu nui cy pht sinh t b o soma trn thn l r cy in vitro a vo nui cy. Trong nghim thc LV + BA (0,5mg/l) + NAA (0,5mg/l) cho pht sinh t bo soma ng u. Vi mu nui cy t mnh l, t bo soma c mu trng xanh, mn, xp. Vi mu nui cy t thn, t bo soma c mu trng xanh. Vi mu nui cy t r, t b o soma c mu vng trng, xp
556
nh hng ca BA, NAA v 2.4D n kh nng nui cy tng sinh t bo soma: Mi trng khong LV c b sung ri ng r BA (0,1-2,5mg/l), NAA (0,1-2,5mg/l) v 2.4D (0,1-2,5mg/l) trong nghin cu nui cy tng sinh t b o soma. Kt qu nghin cu cho thy, mi trng khong LV c b sung BA (1mg/l) hay NAA (1mg/l ) hay 2.4D (1mg/l) cho kh nng tng sinh t bo ln lt 10.670mg/cm, 10.500mg/cm, 10.830mg/cm. iu ny cho php nhn xt rng BA, NAA v 2.4D c tc ng kch thch tng sinh t bo soma nh hng phi hp BA v NAA n kh nng nui cy tng sin h t bo soma: Mi trng khong nui cy LV c b sung BA (0,1 -2,5mg/l) kt hp vi NAA (0,12,5mg/l) c s dng trong nghin cu nui cy tng sinh t b o soma. Kt qu nghin cu cho thy mi trng thch hp cho nui cy tng sinh l LV + BA (0,5mg/l) + NAA (0,5mg/l). Trn mi trng nui cy ny, t bo soma c ngun gc t l hay thn, sau khi tip tc cy truyn s mt i m u xanh dip lc, v chuyn sang mu vng ti xp nh hng ca iu kin chiu sng n qu trnh nui cy t bo soma: Cc mu nui cy t bo soma c t trong 2 iu kin mi tr ng: (a) mi trng c che ti (b) mi trng c t trong iu kin 8 gi chiu sng/ngy, nhit 26-28oC. Xc nh iu kin nui cy thch hp nht cho s nui cy t b o soma. Kt qu nghin cu cho thy: Trong iu kin chiu sng, cm t b o soma chuyn dn sang mu vng m, t bo hi rn li, vn cc, v chuyn sang mu nu sau 2 tun nui cy. Ngc li, khi c t trong iu kin ti, cm t b o soma c mu vng trng, ti, xp. Nhng nu khng c cy truyn sau 1 thng th cm t bo soma ny cng chuyn sang mu vng m ri ha nu. Kh nng to artemisinin: Trong qu trnh nui cy pht sinh t bo soma t l cy thanh hao in vitro trn mi tr ng MS + BA (0,5mg/l) + NAA (0,5m g/l) xut hin 3 dng t bo soma sau 3 tun nui cy: (a) t bo soma c mu trng vng nht v xp (b) t bo soma c mu xanh dip lc v cng (c) t bo soma c mu nu nht. Kim tra artemisinin bng k thut chy in di lp mng (TLC - thin layer chromatography) u cho thy c s hin din. Kho st s tng sinh t b o v phn tch artemisinin b ng k thut HPLC cho thy h m lng artemisinin t bo soma mu xanh dip lc > mu trng vng nht > mu nu. Kt qu ny cho thy s cn thit ca nui c y t bo soma trong iu kin c chiu sng trong iu kin bn rn v lng. Qua nghin cu ng cong sinh trng ca t bo soma trong nui cy lng cho thy t tun th 1 -3 t bo tng sinh mnh m, v t tun th 3 tr i s tng sinh khi bt u i v o giai on n nh (stationary phase) l giai on sinh tng hp artemisinin. Phn tch h m lng artemisinin trong giai o n nui cy tng sinh mnh m cho thy h m lng tng theo thi gian t 3-6-9-18 ngy sau nui cy. iu ny cho thy hm lng artemisinin tng hp c xut hin trong giai on nui cy tng sinh. KT LUN Vi vic chn dng cy thanh hao invitro, cng v i vic nui cy t bo soma t cc b phn ca cy (l, thn, r), hy vng s m ra mt h ng mi trong vic nghi n cu sn xut artemisinin bng k thut nui cy t bo nhm gp phn phng chng bnh st rt.
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TI LIU THAM KHO 1. Hara Y., Y. Yukimune, J. Hiratsuka, T. Nakano, K. H. T. Chris & L. K. Chan (1998). Studies in tissue and cell culture of medicinal plants. In: JBA & NEDO (eds) Proceedings of the Tokyo international forum on conservation and sustainable use of tropical bioresources . Pp236-238. Tokyo, Japan Chan L. K. & S. Nalammai (2001). Preparation of cell suspension of Artemisia annua. In: Proceedings of the seminar on me dicinal plants - Towards of modernization of rsearch and technology in herbal industries . Pp277-281. Kepong Selangor, Malaysia Nalammai S. & L. K. Chan (2004). Effect of type of callus, MS -sugar and pH on cell suspension of Artemisia annua, an antimalarial plant. In: Chang YS, M Mastura & MY Nurhanan (eds) Proceedings of the seminar on Tongkat ali, Kacip fatima and Pagaga new dimension in complimentary health care. Pp109-113. FRIM, Kuala Lumpur, Malaysia Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 431-497
2.
3.
4.
SUMMARY
In vitro cloning of Artemisia annua for bioactive compounds production

Pham The Anh, Le Thi Hien, Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Ce ll Biotechnology, Institute of Tropical Biology
Artemisinin is effective against both chloroquine resistant and sensitive strains of P. falciparum. Yield of artemisinin in plant is low, while demand is increasing and extremely difficult synthesis in labo ratory. So that we select invitro Artemisia annua L. plant, make somatic cell from parts of artemisia annua (roots, stems, leaves) to provide material for producing artemisinin. LV basic medium supplemented with BA (0.3mg/l) was chosen for using micropropa gation with good healthy plant. Using BA, NAA and 2.4D separately was not stimulated somatic cell induction. The combination of plant hormone LV + BA (0.5mg/l) + NAA (0.5mg/l) was favoured for somatic cell mass proliferation. Somatic cells were cultured in the conditions of in darkness and under fluorescent light, the somatic cells was to change green color under light effected with high artemisinin content analysed by TLC and HPLC
558
NGHIN CU K THUT NUI CY NH SINH TRNG TRONG PHC TRNG V DNG HO CY THNG BA L (Pinus kesiya Royle ex Gordon)
Bi Th Tng Thu, Trn Vn Minh PTNT pha Nam v CNTBTV, Vin Sinh hc Nhit i M U Cy thng ba l l loi thng c trng trn din rng Lt. c du nhp v trng t thi Php thuc. L loi cy uc s dng nhiu trong cng nghip v xy dng. Hin nay, cy thng ba l c hin t ng thoi ha ging nh sinh trng chm, b nhiu loi nm bnh gy hi. Phc trng v dng ha l nhu c u cp thit pht trin rng thng tr n quy m cng nghip. Hin nay trong cng tc ging thng ba l, cy con tr ng rng t ht thc sinh c s dng ph bin. Ch a c mt h thng ging hiu qu cho ng nh trng thng (Minh & Loan, 2003). C nhiu bo co trn th gii nghin cu v dng ha cy thng, ch yu qua nui cy pht sinh phi soma (Gladfelter & Phillips, 1987), bo co v nui cy nh sinh trng cn t t liu ni n. Nhm mc ti u nghin cu p dng k thut nui cy nh sinh trng phc trng v dng ha cy thng ba l xy d ng cc vn ging u ngun sn xut cy con ging pht trin cc v ng trng thng chuyn canh, v cng l nhng nghin cu c bn u tin p dng vo bo tn cy thng nm l v hai l d t VT LIU V PHNG PHP Vt liu Mu nui cy: cy thng ba l gim cnh tro ng bu t (c thu thp t nhng cy thng ba l chn dng). Mu nui cy l chi nh non iu kin nui cy: mi trng c v trng 121oC v 1at trong 25 pht. Nhi t phng nui cy 28+2oC. Cng chiu sng 34,2 mol/m2/s. Thi gian chiu sng 8gi/ngy Mi trng nui cy: mi trng dinh dng khong c bn MS (MurashigeSkoog, 1962), WPM (Lloyd and McCown, 1981), LV (Litvay etal., 1985), c b sung BA (6-benzyl aminopurine), IAA ( -indol acetic acid), IBA ( -indol butyric acid), NAA ( -naphthalene acetic acid), Rib (rhizopon), tyrosin, adenin, glycin, vitamin B1 v nc da (CW)
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Phng php Th nghim c b tr theo CBD n yu t, 4 ln lp li, mi ln lp li nui cy 5 bnh tam gic 300ml, mi bnh tam gic cha 65ml mi trng th nghim v c cy 5 mu. S liu thu thp c phn tch thng k bng phn mm MSTATC (P=0.05). T l mu nui cy pht sinh chi (%), s chi / mu (chi), chiu cao chi (mm) KT QU V THO LUN V trng mu nui cy: Mu nui cy l chi nh cy con bu t gim cnh 12 thng tui. Cy m thng ba l u d ng c chn ly mu gim hom. Mi tr ng nui cy WPM + BA (1mg/l). Mu chi nh c v trng bng hypochlorite natrium (10%) trong 10 pht t mc v trng 82,2% nh hng ca mi trng khong c bn n nui cy pht sinh chi in vitro : Chi nh sau khi v trng c nui cy trn mi trng pht sinh chi. Mi tr ng c bn c s dng trong nui cy l MS, WPM v LV. Kt qu nghin cu cho thy mi trng khong c bn WPM thch hp cho nui cy cy thng ba l in vitro c t l pht sinh chi cao (100%), s chi / mu nhiu (4.2 chi / mu) v chiu cao tng i (6,1mm). nh hng ca BA n nui cy pht sinh chi in vitro : Chi in vitro c nui cy trn mi trng pht sinh cm chi WPM c b sung BA. Kt qu nghi n cu cho thy mi trng nghim thc B1: WPM + BA (0,5mg/ l) + NAA (0,1mg/l) cho t l pht sinh chi cao, s chi / mu nhiu (5,2 chi / mu) nh hng ca BA n nhn nhanh cy thng ba l in vitro : Chi non pht sinh c nui cy trn mi trng nui cy c nng BA (0,5 -1-2 mg/l) thay i, nhm xc nh nng BA thch hp cho vi nhn ging. Kt qu nghi n cu cho thy WPM + BA (1mg/l) thch hp cho vi nhn ging, c t l pht sinh cao (88,9%) v cho nhiu chi / mu nui cy (6,4 chi ) nh hng ca BA-IAA, BA-IBA n nhn nhanh cy thng ba l in vitro : Trn mi trng nui cy c bn, c b sung kt hp BA -IAA, BA-IBA v BA-NAA. Kt qu nghin cu cho thy mi trng nghim thc C4 v C5: WPM + BA (1mg/l) + IBA (0,1mg/l) thch hp cho vi nhn ging, so vi nghim thc B1 nh hng ca tyrosine, adenine, tyrosin/adenine n nhn nhanh cy thng ba l in vitro: Trn mi trng nui cy vi nhn ging WPM + BA (1mg/l) + IBA (0,1mg/l) b sung tyrosin (10mg/l), adenine (10mg/l) v tyrosin (10mg/l) + adenine (10mg/l). K t qu nghin cu cho thy s chi pht sinh / cm khng sai khc c ngha so vi nghim thc C5 (bng 3) nh hng ca thng kh v cng chiu sng n n nhn nhanh cy thng ba l in vitro: Trn mi trng nui cy vi nhn ging WPM + BA (1mg/l) + IBA (0,1mg/l) c x l c thng kh (np giy) v kn (np cao su) c t di cng chiu sng 11,4-22,8-34,2 mol/m2/s. Kt qu nghin cu cho thy, vi cng chiu sng 22,8 mol/m2/s trong iu kin nui cy kn (np cao su) s chi pht sinh
560
t cao nht (6,2 chi). Trong khi nui cy c thng kh (np giy), cy thng ba l in vitro d b ho do mt nc Nui cy pht sinh r in vitro: Cy thng ba l c nui cy trn mi trng pht sinh r WPM c b sung NAA v Rhizopon. Kt qu nghin cu cho thy, r pht sinh sau 45 ngy nui cy trn mi trng nghim thc E2. S pht sinh r cao (88,9%), nhiu r (8 r) v r di (11,1mm) KT LUN Mu chi nh c v trng bng hypochlorite natrium (10%) trong 10 pht t mc v rng 82,2%. Mi trng khong c bn WPM thch hp cho nui cy cy thng ba l in vitro c t l pht sinh chi cao (100%), s chi / mu nhiu (4.2 chi / mu) v chiu cao tng i (6,1mm). To cm chi dng cho vi nhn ging thch hp vi mi trng WPM + BA (0,5mg/l) cho t l pht sinh chi cao, s chi / mu nhiu (5,2 chi / mu). Th chi c vi nhn ging hiu qu tr n mi trng WPM + BA (1mg/l) c t l pht sinh cao (88,9%) v cho nhiu chi / mu nui cy (6,4 chi) v mi trng nhn chi hiu qu WPM + BA (1mg/l) + IBA (0,1mg/l). Vi c ng chiu sng 22,8 mol/m2/s trong iu kin nui cy kn (np cao su) cho s chi pht sinh t cao (6,2 chi) so vi nui cy c thng kh, do cy thng ba l in vitro d b ho do mt nc. Nui cy pht sinh r sau 45 ng y nui cy trn mi trng WPM + rhizopon (50mg/l) cho pht sinh r cao (88,9%), nhiu r (8 r) v r di (11,1 mm) TI LIU THAM KHO 1. Gladfelter H. J. & G. C. Phillips (1987). De novo organogenesis of Pinus eldarica in vitro. I. Reproducible regeneration from long -term callus cultures. Plant Cell Rep. (6) 163-166 Litvay J. D., D. C. Verma & M. A. Johnson (1985). Effect of abscisic acid, osmoticum, and desiccation on synthesis of storag e proteins during the development of white spruce somatic embryos. Ann. Bot. (71) 11-22 Lloyd G. & B. McCown (1980). Commercially feasible micropropagation of laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. Int. Plant Crop Soc . (30) 421-427 Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. (15) 431-497 Trn Vn Minh, H Th Loan (2003). ng dng cng ngh t bo thc vt pht trin cy nguyn liu giy thng Caribeae. K yu Hi ngh NCCB 2003 , Hu, pp372-376
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4. 5.
561
SUMMARY
Application of shoot-tip culture techniques in degeneration and cloning of thong ba la ( Pinus kesiya Royle ex Gordon)
Bui Thi Tuong Thu, Tran Van Minh National Key Lab of Plant Cell Biotechnol ogy, Institute of Tropical Biology
Young shoots from cutting pot were used as explants in cultivation. Young shoots were cultured for initiation, mass propgation on medium WPM + BA (1mg/l) + IBA (0,1mg/l). The planlets were cultured for root initiation o n medium WPM + Rhizopon (50mg/l). Plantlets were acclamitization in nursery bed. A system of pine tree degeneration and cloning was established.
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NGHIN CU K THUT TI SINH PHI soma CY TIU (Piper nigrum L.)

M U Tiu en (Piper nigrum L.) l vua ca cc loi gia v l mt trong nhng loi gia v c xa nht v ph bin nht trn ton cu. Mt s loi gia v v dc phm c sn xut t ht ca n c bit n v s dng cho n ngy hm nay. Cy tiu ch yu c nhn ging bng mt s cc ph ng php truyn thng nh: gieo ht, gim cnh, chit cnh. Chi nh c s dng trong nui cy c y tiu (Philip etal, 1992; Sarma & Kalloo, 2004). Cng ngh phi soma c nghin cu nhiu trn cc i tng cy lm nghip, cy cng nghip, cc lo i cy thn tho v c bit trn cy tiu (Josep etal, 1996; Ramakrishnan & Gupta, 2006). Nng cao nng su t ng u trn cc vn tiu trng thm canh thng qua k thut ti sinh phi soma l hng nghin cu kh thi. VT LIU V PHNG PHP
Vt liu
Mu nui cy: cy tiu gim cnh trong b u t c 12 thng tui. iu kin nui cy: Mi trng c v trng 121oC, trong 18 pht, nhit phng nui cy l 282 oC, cng chiu sng l 34,2 mol/m2/s, thi gian chiu sng l 8 gi/ngy. Mi trng nui cy: mi trng dinh dng khong SH (Schenk Hildebrandt, 1972), MS (Murashige & Skoog, 1962) c b sung 2.4D (2.4-dichlorophenoxy acetic acid), BA (6-benzyl aminopurine), NAA ( -naphthalene acetic acid), Kinetin (6 furfuryl-aminopurine), nc da, ng sucrose.
Phng php
Th nghim c b tr theo CBD (n yu t) 3 ln lp li, mi ln 3 b nh tam gic 250ml, mi bnh c cha 50ml mi trng. S liu thu thp c phn tch theo phn mm MSTATC (P=0.05) kh nng to t b o soma, kh nng tng sinh t bo soma, s chi to thnh/mu. KT QUV THO LUN V trng mu nui cy: Mu nui cy c ly t cy m bu t 12 thng tui, ging n . Chi nh c s dng lm mu nui cy. Chi non c v trng bng Hypochorite-Na (10%) trong thi gian 10 pht v HgCl2 (0,05%) trong thi gian 3 pht
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cho thy kh nng nhim l thp, chp nhn c. Chi non v trng c a vo nui cy to th chi invitro nh hng ca nng ng v 2.4D n kh nng pht sinh t b o soma: t thn (di 1cm) ca cy tiu in vitro c ch dc theo chiu di thn v c nui cy trn mi trng khong c bn SH c b sung ng sucrose (15-30-45g/l) v 2.4D (0,5-1-2 mg/l). Kt qu nghin cu cho thy nghim thc SH + sucrose (30g/l) + 2.4D (1mg/l) cho kt qu pht sinh t bo soma cao nht trn mi mu nui cy nh hng ca khi lng t bo a vo nui cy ban u n kh nng tng sinh t bo soma: Mi trng thch hp cho nui cy pht sinh t bo soma SH + sucrose (30g/l) + 2.4D (1mg/l) c dng nui cy kho st s tng sinh ca t b o; V nui cy trong hai iu kin: dng bn rn v dng lng. Khi lng ban u ca t bo soma c a vo nui cy ba mc l 300mg, 500mg, 1000mg. Kt qu sau 8 tun nui cy cho thy vi khi l ng nui cy ban u 500mg t b o soma tng sinh trn mi trng bn rn cho kt qu cao nht l 8811,75mg; V sau 4 tu n nui cy trong mi trng lng, trn my lc (110rpm), nhit 24 oC, vi khi lng ban u nui cy 300mg cho t bo soma tng sinh cao nh t l 29,37 ln. Dch huyn ph t bo soma c nui cy tri trn mi trng bn rn SH + BA (1mg/l) + Sucrose (45g/l) cho pht sinh phi soma ng nht vi hiu sut cao sau 60 ng y nui cy nh hng ca Kinetin, NAA, BA n kh nng ti sinh phi soma : Mi trng c bn MS c b sung Kin (3mg/l), NAA (0,5mg/l), BA (0 -1-3-5-7mg/l) cho nghin cu ti sinh phi soma. Kt qu cho thy nghim thc MS + BA ( 5mg/l) + Kin (3mg/l) + NAA (0,5mg/l) cho kt qu ti sinh cao Vn thn v nhn ging cy phi invitro: Cy tiu in vitro ti sinh t t bo phi soma c cy chuyn vo mi trng vn thn v nhn ging bng phng thc cm chi MS + BA (0,5mg/l) + CW (10%) Nui cy pht sinh r cy tiu in vitro: Cy phi sau khi ln c cy chuyn qua mi trng kch thch pht sinh r sau 34 ng y nui cy MS + NAA (0,1mg/l) KT LUN Trn con ng tm kim m hnh nhn ging thch hp i vi nhng cy cng nghip, c mt c im ging nhau l kh nng to cc hp phn phenol ln chit ra mi trng nui cy lm hn ch kh nng nhn ging. Cy ti u qua nui cy pht sinh t bo soma [SH + sucrose (30g/l) + 2.4D (1mg/l)], pht sinh phi soma [SH + BA (1mg/l) + Sucrose (45g/l)], ti sinh phi soma [MS + B A (5mg/l) + Kin (3mg/l) + NAA (0,5 mg/l)], nhn nhanh th chi [MS + BA (0,5mg/l) + CW (10%)], nui cy pht sinh r [MS + NAA (0,1mg/l)] cho thy l mt h thng nhn nhanh cng nghip hiu qu TI LIU THAM KHO 1. Joshep B., D. Joseph & V. J. Philip (1996). Plant regeneration from somatic embryos in Black pepper . Plant cell, tissues and organ culture (47) 87-90.
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2. 3.
Murashige T. & R. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. (15) 431-497 Nair R. R. & S. D. Gupta (2003). Somatic embryogenesis a nd plant regeneration in black pepper (Peper nigrum L.): I Direct somatic embryogenesis from tissues fo germinating seeds and ontogeny of somatic embryos . The journal of Horticulture Science and Biotechnology (3) 416- 421. Nair R. R. & S. D. Gupta (2006). High-frenquency plant regeneration through cyclic secondary somatic embryogenesis in black pepper ( Piper nigrum L.). Plant cell Rep (24) 699-707 Philip V. J., D. Joseph, G. S. Triggs & N. M. Dickinson (1992). Micropropagation of black piper (piper nigrum L) through shoot tip cultures. Plant cell reports (12) 41-44 Sarma Y. R. & G. Kalloo (2004). Status of current research towards increased production and productivity in black piper in India. Focus on Piper (1) 69-86 Schenk R. U. & A. C. Hildebrandt (1972). Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot (50) 199-204
4.
5. 6. 7.
SUMMARY
Study of regeneration culture techniques of black pepper (Piper Nigrum L.) somatic embryos
Black pepper, the king of spices, is one of the oldest and the most popular spice in the world. Some hotly pungent sp ice and medical products which made from its berries are some of the earliest products to be known and are probably the most widely used in the world today. Black pepper is maily cultured by seedlings, stem cuttings, marcottings. In order to increase the y ield and resistant capability to some of popular deseases. Study of somatic embryogenesis and manipulation is realizable direction for those purposes: somatic cell induction [SH + sucrose (30g/l) + 2.4D (1mg/l)], embryogenesis [SH + BA (1mg/l) + Sucrose (4 5g/l)], embryogenic cell regeneration [MS + BA (5mg/l) + Kin (3mg/l) + NAA (0,5 mg/l)], elongation and micropropagation [MS + BA (0,5mg/l) + CW (10%)], and rooting culture [MS + NAA (0,1mg/l)] proved that there was an efficient system for black piper in vit ro propagation through embryogenesis culture established
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NGHIN CU NUI CY PHT SINH V TI SINH PHI soma THNG (Taxus wallichiana Zucc) in vitro
M U Cy thng dng Taxus sp. var wallichiana (Zucc.) Hook l loi cy b n a ca Vit Nam, cn st li khng qu 16 cy ti Cao nguyn lm vin Liang Biang, c ng knh thn trn 75cm, l loi cy sinh tr ng chm. Bo tn v pht trin cy thng Lt l mt yu cu cp thit i vi loi cy qu him c gi tr chit xut taxol phng chng bnh ung th. Phng thc nhn ging truyn thng hin nay l gieo ht hoc gim cnh hoc ghp cnh (Ho, 1998). Tuy nhin, h t mau mt sc ny mm v s lng ht cho mt ma tri rt thp; hn na cy gim cnh thng c hin tng sinh trng chm v ng nghing (plagiotropically) v khng thng ng (Chang etal, 2001). Nui cy vi nhn ging cy thng dng Taxus media v Taxus mairei c ghi nhn trong nhng nm gn y (Cerdeira, 1994; Chang etal, 1998), tuy nhin cy con in vitro sinh tr ng chm v bo lu cc b tnh ng nghing. Nui cy phi soma l mt k thut nui cy in vitro vt qua kh nng ny mm km ca ht ging v hn ch vn sinh trng chm cy con in vitro nht l i vi cy thn g l kim (Gupta & Durzan, 1987). Kt hp k thut nui cy pht sinh, ti sinh phi soma v dng ha in vitro s hn ch mc thp nht tnh bo l u ng nghing cc b bng k thut vi nhn ging nng cao hiu sut nhn nhanh cy thng in vitro (Amos & McCown, 1981; Chee, 1995) VT LIU V PHNG PHP
Vt liu
Vt liu c a vo nui cy pht sinh t bo soma l thn v l cy thng c dng ha in vitro. T bo soma thu nhn c qua nui cy t thn c a vo nghin cu nui cy tng sinh trn mi trng agar v lng. Dch huyn ph thu nhn c sau 6 tun nui cy c xc nh mt v c s dng trong nui cy pht sinh v ti sinh phi soma Mt t bo: c m bng bung m hng c u (c cu to khung m Thoma, bao gm 25 ln v mi ln c 16 nh, din tch mi nh l 1/400mm 2, chiu cao mi l 0,1mm) trong mt git dung dch, sau c tnh ra trong 1ml dung dch vi nng pha lo ng l 10 -1 vi cng thc tnh: S t bo / ml mu = [ a x 4000 x 1000 ] / H
566
Vi: a: s t bo trung bnh c trong mt din tch vi trng ( nh). 4000: s quy i 1/400mm2 thnh 1mm 3. 1000: s quy i t 1mm 3 thnh 1ml. H: h s pha long. Mi trng nui cy c bn trong nghin cu pht sinh v tng sinh t bo soma trn agar v lng, pht sinh v ti sinh phi: WPM + Cw (10%) + vitaminB 1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l). iu kin nui cy: mi trng c v trng 121oC, 1at, trong 25 pht. Nhi t phng 28+2oC, cng chiu sng 33,4mol/m 2/s, thi gian chiu sng 8 gi/ng y. Ht nhn to: Phi soma c trn vo hn hp: dung dch alginate(3 o/oo) + mi trng nui cy c bn WPM, hn hp c nh git vo dung dch CaCl 2(2%). Ht nhn to cha phi c tn tr trong phytotron nhit 15 oC, thi gian chiu sng 8gi/ngy, v cng chiu sng 11,1mol/m 2/s.
Phng php:
Th nghim c b tr theo CBD mt yu t, mi nghim thc c 5 ln lp li. Mi ln lp li c nui cy trong 3 bnh tam gic 300ml. Mi bnh tam gic nui cy 7 mu. S liu c x l bng phn mm thng k MSTATC KT QU V THO LUN
1. Nui cy pht sinh t bo soma
Mc tiu ca th nghim l tm mi trng thch hp cho nui cy pht sinh t bo soma v kh nng tng sinh khi. Mu nui cy ban u l thn v l cy in vitro, vi trng lng lng mu ban u a vo nui cy l 0,5g. Trng lng ti tng ln c tnh bng cch ly trng lng ti 8 tun sau nui cy tr i cho trng lng ban u. Nui cy pht sinh t bo soma trong iu kin che ti hon ton: mi trng nui cy c bn: WPM + Cw (10%) + vitaminB 1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) + BA (0,1mg/l) + NAA (0-1-3-5-7mg/l). T l pht sinh phi soma cao nht 93.33% (vi mu nui cy l thn) v 96.67% (vi mu nui cy l l) nng NAA (3mg/l), kt qu tng t 90% (mu thn) v 93.33% (mu l) c ghi nhn vi nng NAA (5mg/l). Trng lng ti t bo soma tng ln cao nh t 2.10g (vi mu nui cy l thn) v 2.23g (vi mu nui cy l l) nng NAA (3mg/l), kt qu c ghi nhn thp hn 1.57g (mu thn) v 1.67g (mu l) nng NAA (5mg/l). Vy vi mi trng c bn ban u, c b sung nng NAA bin thi n khc nhau (0-7mg/l), cho thy mu thn hay l u c kh nng pht sinh t b o soma cao (90-96.67%) nng NAA (3-5mg/l); v trng lng ti tng ln sau 8 tun nui cy c ghi nhn mc 2.10-1.57g (mu thn) v 2.23-1.67g (mu l) vi nng NAA (3 -5mg/l). Nui cy pht sinh t bo soma trong iu kin c chiu sng (22,2mol/m2/s): mi trng nui cy c bn: WPM + Cw (10%) + vitaminB 1 (5mg/l) + Glycin (5mg/l) + sucrose
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(30g/l) + BA (0,1mg/l) + NAA (0-1-3-5-7mg/l). T l pht sinh phi soma cao nht 86.67% (vi mu nui cy l thn) v 90% (vi mu nui cy l l ) nng NAA (3mg/l), kt qu tng t 73.33% (mu thn) v 76.67% (mu l) c ghi nhn vi nng NAA (5mg/l). Trng lng ti t bo soma tng ln cao nht 1.53g (vi mu nui cy l thn) v 1.90g (vi mu nui cy l l) nng NAA (3mg/l), kt qu c ghi nhn thp hn 1.27g (mu thn) v 1.60g (mu l) nng NAA (5mg/l). Vy vi mi tr ng c bn ban u, c b sung nng NAA bin thi n khc nhau (0-7mg/l), cho thy mu thn hay l u c kh nng pht sinh t bo soma cao (73.33-90%) nng NAA (3-5mg/l); v trng lng ti tng ln sau 8 tun nui cy c ghi nhn mc 1.53-1.27g (mu thn) v 1.90-1.60g (mu l) vi nng NAA (3-5mg/l). Kt lun: vi mi trng nui cy c bn ban u c thay i nng NAA (0 7mg/l) b sung, kt qu cho thy mu thn v l c a vo nui cy u c kh nng pht sinh v tng sinh kh i t bo soma; vi mu nui cy l l cho t l pht sinh t bo soma v kh nng tng sinh khi cao nng NAA (3 -5mg/l) c hai iu kin nui cy trong ti hon ton v c chiu sng 2000lux. V y l mi trng c bn ban u c s dng cho cc nghi n cu tip sau.
2. Nhn sinh khi t bo soma trn mi trng Agar
Mu nui cy ban u l t bo soma sau nui cy 8 tun t th nghim 1 loi b cc b phn thn v l cha pht sinh, vi trng lng ti nui cy ban u l 1g v nui cy trong 6 tun. Trng lng ti tng sinh c tnh bng cch ly trng l ng ti sau 6 tun nui cy tr i cho trng l ng ti ban u. Mi trng nui cy c bn: WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) + TDZ (0-0.1-0.5-1-3mg/l) hay + BA (0.5-1-2-4mg/l). Kt qu nghin cu cho thy: khi b sung TDZ (0 -3mg/l), kh nng tng sinh khi t bo soma cao nht (5.07g) nng TDZ (0.5mg/l) v gim dn khi tng dn nng TDZ. T bo soma tng sinh c dng xp. Mu sc thay i t vng (TDZ=00.1mg/l), vng m (TDZ=1mg/l), vng nu (TDZ=3mg/l) v n vng xanh (TDZ=0.5mg/l). Khi b sung BA (0-4mg/l), kh nng tng sinh khi t b o soma cao nht (3.67g) nng BA (2mg/l) v gim khi tng dn nng BA. T b o soma tng sinh c dng m cng. Mu sc thay i t vng trng (BA=0-0.5mg/l), vng (BA=1mg/l), v n vng xanh (BA=2-4mg/l) Kt lun: khi nui cy t bo soma trn mi tr ng c bn c b sung TDZ cho thy kh nng tng sinh khi cao (5.07g), t b o soma xut hin c dng xp, l loi t bo cho nui cy tng sinh nhanh trong cy truyn; ng c li khi b sung BA, t bo xut hin c dng t b o cht, l loi t bo cho kh nng tng sinh chm khi cy truyn. TDZ thch hp cho nui cy tng sinh khi v BA thch hp cho nui cy pht sinh phi.
3. Nhn sinh khi t bo soma trn mi trng lng
Mu nui cy ban u l t bo soma t th nghim 1, trng lng ban u l 1g v c nui cy trn my lc vi nhit n nh l 14oC trong thi gian 30 ngy. Mi
568
trng nui cy c bn: WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) + TDZ (0.1-0.5-1mg/l) hay + BA (1-2mg/l) Kt qu nghin cu cho thy: mi trng nui cy c bn c b sung TDZ (1mg/l) cho kh nng tng sinh cao nht vi mt t b o l 1410x10 3 t bo /ml; tng t vi BA (2mg/l) cho kh nng tng sinh vi mt 1023x10 3 t bo /ml. Kt qu nui cy trn mi trng lng cho thy TDZ vn l cht iu ha sinh trng thch hp cho nui cy tng sinh t bo so vi BA
4. Ti sinh t bo phi soma
Mu nui cy l t bo soma thu nhn c qua nui cy lng tr n mi trng nui cy c bn c b sung TDZ (1mg/l). Dch huyn ph t bo c tri trn mi trng pht sinh v ti sinh phi soma trc tip. Mi trng nui cy c bn: WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) v b sung t hp cc cht iu ha sinh trng TDZ (0-0.1-0.5-1-3mg/l) + ABA (0.5mg/l) hay TDZ (0.1 -0.51-3mg/l) + BA (2mg/l) hay TDZ (0.1-0.5-1-3mg/l) + kinetin (2mg/l). Kt qu nghin cu cho thy, dch huyn ph t bo soma bit ha phi soma sau 8 tun (2 thng) nui cy v chuyn qua qu trnh ti sinh sau 6 thng nui cy. Mi trng nui cy c bn c b sung cc t hp TDZ+ABA, TDZ+BA hay TDZ+kinetin u cho pht sinh phi sau 2 thng nui cy. Khi b sung Ki v BA vo mi trng nui cy th t bo soma c dng xanh v cht; ng thi khi ko di thi gian nui cy th mt phn m bn di tip xc vi mt thch bi en, m t trn to nn mt lp m trng v ri rc. Trn mi trng khi c b sung ABA m c dng m u xanh vng v xp, nh phi xut hin nhiu hn. Khi nui cy ko di m cng tr nn xanh hn, mi trng b ha nu t hn, v t bo b cht cng t hn. Ring mi trng nui cy c b sung t hp cht iu h a sinh trng TDZ (1mg/l) + ABA (0.5mg/l) xut hin chi ti sinh sau 8 thng nui cy. Cy con t phi c nui cy trn mi trng pht sinh r WPM + NAA (3mg/l) + Rhizopon (50mg/l). Ring mi trng nui cy c b sung t hp cht iu h a sinh trng TDZ (1mg/l) + ABA (0.5mg/l) xut hin chi ti sinh sau 8 thng nui cy
5. Bo tn t bo phi soma
Bo tn t bo phi thng in vitro: Phi soma chuyn qua giai on pht sinh chi (sau 6 thng nui cy trn mi trng pht sinh phi), phi c nh chi nhn, c tch tng cm 3-5 phi, trn vo hn hp alginate v nh git vo dung dch CaCl2 (2%) to ht. Ht c tn tr trong phytotron. Ht phi nhn to c a vo nui cy ti sinh pht sinh chi sau 2 thng nui cy trn mi trng c bn c b sung TDZ (1mg/l) + ABA (0.5mg/l) KT LUN Mi trng nui cy c bn c b sung TDZ thch hp cho nui cy tng sinh t bo soma trn mi tr ng agar v mi trng lng; t hp cht iu h a sinh trng TDZ+ABA b sung vo mi trng nui cy thch hp cho pht sinh v ti sinh phi soma.
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TI LIU THAM KHO 1. 2. 3. 4. 5. 6. 7. Amos R. R. & B. McCown (1981). Micropropagation of members of the Coniferae. Hortic. Sci. (16): 453 Cerdeira R. M., J. D. McChesney & C. Jr. Burandt (1994). Microcuttings of Taxus media cv. Hicksii. In Vitro Cell Dev. Biol. Anim (30A): 59 Chang S. H., C. K. Ho, Z. Z. Chen & J. Y. Tsay (2001). Micropropagation of Taxus mairei from mature trees. Plant Cell Report (20): 496-502 Chee P. P. (1995). Organogenesis in Taxus brevifolia tissue cultures. Plant Cell Report (14):753-757 Gupta P. K. & D. J. Durzan (1987). Micropropagation and phase specificity in mature elite Douglas fir. J. Am. Soc. Hortic. Sci. (112):969-971 Ho C. K., S. H. Chang & J. Y. Tsai (1998). Selection breeding, propagation and cultivation of Taxus mairei in Taiwan. Halb Taiwan For. Res. Inst . (88): 65-82 Lloyd G. & B. McCown, 1981. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot -tip culture. In: Comb. Proc. Intl. Plant Prop. Soc., (30): 421-426 Murashige T. & F. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-479
8.
SUMMARY
Study of somatic cell induction and regeneration of embryogenic cell of taxus trees (Taxus wallichiana Zucc) in vitro
The basic medium WPM was used for somatic cells induction. WPM medium was supplemented with BA+NAA favoured for somatic cells induction via in vitro stems or leaves cultured in the conditions of darkness or fluorescence. The composition of medium was determined WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucros (30g/l) + BA (0,1mg/l) + NAA (0-1-3-5-7mg/l). The basic medium WPM supplemented with TDZ was favoured to enhance the somatic cell biomass on semi -solid cultivation in the composition of WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) + TDZ (0 -0.1-0.51-3mg/l) /or + BA (0.5-1-2-4mg/l) and liquid cultivation in the composition of WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) + TDZ (0.1 -0.5-1mg/l) /or + BA (1-2mg/l). The composition of plant growth regulators supplemented TDZ+ABA to m edium was favoured for embryogenic induction and regeneration cultured such as WPM + Cw (10%) + vitaminB1 (5mg/l) + Glycin (5mg/l) + sucrose (30g/l) + TDZ (1mg/l) + ABA (0.5mg/l). Embryogenic cells was immobilized by alginate-Na solution (2o/oo) for conservation under temperature of 15oC and light intensity of 11.1 mol/m2/s in phytotron conditions.

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PHN VI

CNG NGH T BO THC VT

Phn VI: CNG NGH T BO THC VT

Hi ngh KHOA HC V CNG NGH 2007

Bng 1: M t th nghim (chung cho 2 loi vt liu)

Phn VI: CNG NGH T BO THC VT

Hnh 2. S chi to thnh t lt mng t thn mm v t t dip ngy th 28.

Hi ngh KHOA HC V CNG NGH 2007

Hnh 3. Cm chi cy iu pht trin sau 2 thng cy chuyn.

Phn VI: CNG NGH T BO THC VT

Hi ngh KHOA HC V CNG NGH 2007

Phn VI: CNG NGH T BO THC VT

Hnh 1. Cy lan Dendrobium c y m trc khi trng ra vn m.

Hnh 2. Cy lan Catleya c y m trc khi trng ra vn om.

Hnh 3: Cy lan Phalaenopsis c y m trc khi trng ra vn m.

Hi ngh KHOA HC V CNG NGH 2007

Hnh 5: A- cy gc nh sng t nhin B- cy gc nh sng n

Phn VI: CNG NGH T BO THC VT

Bng 2. So snh tng tr ng cy lan Catleya trng trong v n om

Hinh 6. Lan Catleya 90 ngay tu i gc nh sng n

Hinh 7. Lan Catleya 90 ngay tu i gc nh sng t nhin

Hi ngh KHOA HC V CNG NGH 2007

Hnh 8: Cy Phalaenopsis 60 ngy tu i gc nh sng n

Hnh 9: Cy Phalaenopsis 60 ngy tui gc nh sng t nhi n

Phn VI: CNG NGH T BO THC VT

Hi ngh KHOA HC V CNG NGH 2007

NHN in vitro GING TIU GC GHP (Piper columbrium Link.)

Phn VI: CNG NGH T BO THC VT

Hi ngh KHOA HC V CNG NGH 2007

Phn VI: CNG NGH T BO THC VT

In vitro culture in Piper columbrium Link.

Hi ngh KHOA HC V CNG NGH 2007

Phn VI: CNG NGH T BO THC VT

Hnh 1. Nh plastic nui lan trong i u kin nh sng t nhi n

Hi ngh KHOA HC V CNG NGH 2007

Phn VI: CNG NGH T BO THC VT

Bng 3. So snh tng tr ng cy lan Catleya trn mi trng ng v tinh bt

Hi ngh KHOA HC V CNG NGH 2007

Hnh 5: Catleya 60 ngy tu i nui nh sng n trn mi tr ng ng.

Phn VI: CNG NGH T BO THC VT

Vu Ngoc Phuong, Thai Xuan Du Institute of Tropical Biology

Hi ngh KHOA HC V CNG NGH 2007

NHN GING V TNH CY vani BNG NUI CY M

Phn VI: CNG NGH T BO THC VT

Hnh 1: Chi Vani cy m

Hi ngh KHOA HC V CNG NGH 2007

Phn VI: CNG NGH T BO THC VT

Hnh 2: Cy vani cy m 60 ngy tui

Hnh 3: Nhn ging i tr cy Vanilla tahitiensis

Vanilla tahitiensis hai thng tui trong vn m

Hi ngh KHOA HC V CNG NGH 2007

Phn VI: CNG NGH T BO THC VT

3. nh hng ca IBA v NAA ln s to r bt nh ca chi iu in vitro

Hi ngh KHOA HC V CNG NGH 2007

0 A2 A3 A4 Nghi m thc S chi c 4 l S chi c chiu cao 3 cm

Phn VI: CNG NGH T BO THC VT

T l mu sng (%) 52,5 52,5 NS

Chiu di r (cm) 6,6 8,4 NS

Gia tng trng lng ti (mg) 360 230 **

NS, *, ** : khng khc bi t hoc khc bit c ngha mc p 0,05 v 0,01

Hi ngh KHOA HC V CNG NGH 2007

Hnh 3. S hnh thnh r sau khi x l (a) NAA v (b) IBA

Phn VI: CNG NGH T BO THC VT

Hi ngh KHOA HC V CNG NGH 2007