31.

Nguyn Thanh Tng, Trng Th Bch Phng. 2009. Nhn nhanh in

vitro cy M dung d lan (Vanda denisoniana Benson). Bo co Khoa hc Hi ngh Cng ngh Sinh hc ton quc. NXB i hc Thi Nguyn. 452-455. QUY TRNH CA TRUNG TM NHIN CU NN CNC TPHCM GII THIU QUY TRNH K THUT NHN GING MT S LOI HOA LAN PH BIN (MOKARA, DENDROBIUM) BNG PHNG PHP NUI CY M. I. CC PHNG PHP NHN GING HOA LAN. 1. Phng php truyn thng Nhn ging bng ht: Ngi ta tnh ra xc sut mc mt cy lan con l 1/5.000, ngha l gieo 5.000 ht mi c mt ht thnh cy v hng nhiu nm mi n hoa thp Nhn ging t hom: y l phng php nhn ging n gin bng cch cy con c ct thng t an trn cng ca cy m, hom ging ti thiu phi bao gm t 2- 3 r m ra t thn cy m. Phng php ny cho h s nhn chi thp. 2. Phng php Nui cy m t bo(nhn ging in vitro) u im ca phng php nhn ging in vitro. Nhn ging bng cch ct ngn: h s nhn

To qun th ln v ng nht v di truyn trong thi gian ngn.

- C kh nng to c nhiu cy con t m v c quan ca cy (lng, thn, phin l, hoa, ht phn, non, chi) m iu ny th ngoi t nhin khng th thc hin c. - Lm sch ngun virus gy bnh cho cy bng cch thc hin nui cy m phn sinh ngn. - Ci tin cc ging cy trng bng ng dng cc thnh tu cng ngh sinh hc v nhn nhanh cc ging chn c. - Khng gian ginh cho nhn ging in vitro khng nhiu bng nhn ging truyn thng (ngoi ng rung).Cc phng php nhn ging in vitro

Nui cy nh sinh trng Nui cy m so Nui cy t bo n Nui cy protoplast Nui cy ht phn dn bi

Cc b phn dng nui cy in vitro

II. QUY TRNH NHN GING HOA LAN BNG PHNG PHP NUI CY M.
Cy u dng (cy mu sch bnh)

nh sinh trng, pht hoa, l non Kh trng Mi trng to chi 2 3 thng 10- 12 thng To c chi 2 3 thng

Nhn chi 3 4 thng Cy con

2 thng Cy hon chnh (cy con cao 5-6 cm, 4-5 l)

Thuyt minh quy trnh. Chn cy u dng Cy m c chn l cy cho hoa c mu sc p, bn, sing bng (cho nhiu pht hoa trong 1 nm), s hoa trn pht hoa nhiu. Sau khi xc nh c cc ch tiu trn tin hnh test virus bng cc Test th nhanh nh kit DAS-ELISA ca

hng DSMZ (D-0187, D-0493, D-0100), cc test th nhanh ca Agdia, cc kit Monoplex, Multiplex RT-PCR, m bo chc chn cc cy m sch bnh. Quy trnh v mu Nguyn liu: Chi non, pht hoa, l non Tin hnh: Thu mu, ra sch b mt bng x phng v di vi nc chy khong 15 pht. a mu vo t cy. Ra mu 3 -4 ln bng nc ct v trng. S dng cc ha cht kh trng mu nh Calcium hypochlorite 5 -10%, sodium hypochlorite 0,5 - 5%, cn 70 kh trng mu ty theo loi mu cy m chn nng cht kh trng v thi gian kh trng thch hp. Sau cc bc kh trng tin hnh tch mu v t mu vo mi trng to chi. To chi Sau khi to c ngun mu sch, tin hnh nui cy trn mi trng c b sung cht iu ha tng trng thc vt Auxin hoc Cytokinin hay kt hp c hai loi. C th s dng mi trng c hay mi trng lng lc nui cy to chi. Sau 2-3 thng trn mi mu cy s xut hin chi hoc protocom. iu kin nui cy 28oC, nh sng 1200 Lux, thi gian chiu sng 8 gi/ngy. Nhn chi sn xut c s lng cy ln cn qua giai on nhn chi, s dng mi trng dinh dng c b sung cht HSTTV. Ty theo k hoch sn xut m tnh ton s ln nhn chi cho hp l trnh hin tng thoi ha ging do phi cy chuyn nhiu ln. i vi lan Mokara thng s dng mi trng MS c b sung cht HSTTV l BA, Kinetin v NAA. i vi lan Dendrobium ngoi Mi trng MS cn s dng mt s mi trng c bn khc nh Knudson C, Vacine Went, White... iu kin nui cy 28 oC, nh sng 2000 Lux, thi gian chiu sng 12 gi/ngy. Cy con

Sau khi to c lng chi cn thit s cy chuyn sang mi trng mi chi bit ha thnh cy con, s dng mi trng to r bng cch b sung than hot tnh hoc auxin gip tng kh nng to r. iu kin nui cy 28 oC, nh sng 2000 Lux, thi gian chiu sng 12 gi/ngy. Cy hon chnh Cy con c cy chuyn sang mi trng dinh dng c b sung cc dch chit t nhin (chui, khoai ty, c rt) gip cy tng trng c v chiu cao, cn nng, s l, s r. iu kin nui cy 28 oC, nh sng 2000 Lux, thi gian chiu sng 12 gi/ngy. Giai on hun luyn cy Mc ch: gip cy lm quen t t vi iu kin bn ngoi khi khng cn c nui trong iu kin phng th nghim trnh cho cy b sc. Khi cy pht trin hon chnh trong cc bnh nui cy s c chuyn ti phng hun luyn thch nghi c thit k n nh sng t nhin v thong gi. Thi gian ca giai on ny khong 10- 15 ngy. Giai on hu cy m PH LC I. MT S MI TRNG DINH DNG TRONG NUI CY M HOA LAN. Bng 1: Mi trng MS (MurashigeSkoog) stt 1 Thnh phn a lng NH4NO3 CaCl2.2H2O MgSO4.7H2O KNO3 KH2PO4 Vi lng Na2EDTA FeSO4.7H2O MnSO4.4H2O Nng (mg/l) 1650 440 370 1900 170 37,3 27,8 22,3

H3BO3 ZnSO4.4H2O KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O VITAMIN Glycine Pirydoxine Meso-inositol Nicotinic acid Thiamin HCl

6,2 8,6 0,83 0,25 0,025 0,025 2 0,5 100 0,5 0,1

Bng 2: Mi trng KNUDSON C stt 1 Thnh phn a lng Ca(NO3)2 KCl MgSO4 NH4NO3 KH2PO4 (NH4)2SO4 Vi lng FeSO4.7H2O MnSO4. H2O Nng (mg/l) 241,3 250 122,15 500 250 500 25 5,68

II. K THUT PHA CC DUNG DCH M MI TRNG MS 1/ Pha dung dch m a lng 2/ Pha dung dch m vi lng 3/ Pha Fe-EDTA 4/ Pha Vitamin 5/ Pha cht iu ha sinh trng TV 1/ Pha dung dch m a lng (x10): s dng cho 10 lit mi trng sn xut STT Thnh phn Nng (mg/l) Dung dch m (x10), mg

1 2 3 4 5

a lng NH4NO3 CaCl2.2H2O MgSO4.7H2O KNO3 KH2PO4

1650 440 370 1900 170

16500 4400 3700 19000 1700

Cn v ha tan ring tng cht sau nh mc thnh 1 lt dung dch m. Khi pha mi trng MS s dng 100ml /l mi trng sn xut
2/ Pha dung dch m vi lng (x100): s dng cho 100 lt mi trng sn xut

STT 1 2 3 4 5 6 7 MnSO4.4H2O H3BO3 ZnSO4.4H2O KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O

Nng (mg/l) 22,3 6,2 8,6 0,83 0,25 0,025 0,025

Dung dch m (x100), mg 2230 620 860 83 (1 ml dd b ngoi) 25 (1 ml dd b ngoi) 2,5 (1 ml dd b ngoi) 2,5 (1 ml dd b ngoi)

Do mt s cht vi lng c khi lng s dng rt nh nn tin hnh pha cc dung dch b ngoi nh sau: - Pha dd b ngoi KI nng 83 mg/ml: cn 8300 mg KI ha tan trong nc ct v ong thm nc cho 100 ml. - Pha dd b ngoi Na2MoO4.2H2O nng 25 mg/ml: cn 2500 mg Na2MoO4.2H2O ha tan trong nc ct v ong thm nc cho 100 ml. - Pha dd b ngoi CuSO4.5H2O nng 2,5 mg/ml: cn 250 mg CuSO4.5H2O ha tan trong nc ct v ong thm nc cho 100 ml.

- Pha dd b ngoi CoCl2.6H2O nng 2,5 mg/ml: cn 250 mg CoCl2.6H2O ha tan trong nc ct v ong thm nc cho 100 ml. 3/ Dung dch m Fe-EDTA (x100): s dng cho 100 lt mi trng sn xut stt Thnh phn Fe- EDTA Na2EDTA FeSO4.7H2O Nng (mg/l) Dung dch m (x100), mg 1 2 37,3 27,8 3730 2780

Ha tan tng cht, c th gia nhit 80 0C, sau nh mc thnh 500ml. Dung dch m Fe- EDTA: 5ml/ lt.
4/ Pha dung dch m Vitamin MS (x100): s dng cho 100 lt mi trng sn xut

VITAMIN 1 2 3 4 5 Glycine Pirydoxine Meso-inositol Nicotinic acid Thiamin HCl

Nng (mg/l) 2 0,5 100 0,5 0,1

Dung dch m (x100), mg 200 50 10000 50 10

Cn v ha tan ring tng cht sau nh mc thnh 500 ml dung dch m. Dung dch m Vitamin MS: 5ml/lt. Nh vy sau khi pha song cc dung dch m, pha 1 lt mi trng MS sn xut cn phi c y cc thnh phn sau: 1/ Dung dch m a lng: 100ml 2/ Dung dch m Fe-EDTA: 5ml 3/ Dung dch m vi lng: 5 ml 4/ Dung dch m Vitamin: 5 ml Ghi nhn tn dung dch, nng s dng, ngy pha, ngi pha. 5/ Cch pha mt s cht iu ha sinh trng thc vt

Pha BA c nng 1 mg/ml: cn 100 mg BA, ha tan BA trong NaOH 1N v thm nc ct cho 100 ml. Pha NAA c nng 1 mg/ml: cn 100 mg NAA, ha tan NAA trong NaOH 1N v thm nc ct cho 100 ml.

III. Thc hnh k thut pha mi trng - Pha mi trng sn xut - o pH - Ch mi trng vo chai - Hp kh trng mi trng 1/ Pha mi trng cy chuyn lan Dendrobium- mi trng MS stt 1 2 3 4 5 6 7 8 Thnh phn Dung dch m a lng Nng (1 lt mi trng) 100ml 5 ml 5 ml 5 ml 25 g 150 ml 1g 8g 5,6-5,8

Dung dch m Fe-EDTA Dung dch m vi lng Dung dch m Vitamin ng Nc da Than hot tnh Agar pH 2/ o pH ca mi trng

Sau khi chun b y cc thnh phn trn theo s lng (lt) mi trng cn s dng phi tin hnh o pH ca mi trng. i vi lan dendrobium gi tr pH dao ng trong khong 5,6 n 5,8. C th iu chnh pH mi trng tng hoc gim nh dung dch NaOH 1N (tng pH) hoc HCl 1N (gim pH). 3/ Rt mi trng vo chai Sau khi o pH, mi trng c phn phi vo cc bnh cy (loi bnh nc bin 500 ml). Th tch mi trng khong 75 ml/ bnh. 4/ Hp v trng mi trng Mi trng c hp v trng trong iu kin 121 C trong 20 pht. IV. Thc hnh k thut cy chuyn

1/ Quan st cc thao tc ca ngi hng dn 2/ Chun b t cy, mu cy v mi trng nui cy 3/ Thao tc v trng t cy v cc dng c 4/ Cch ly mu, tch mu v cy mu V. TRANG B PHNG NUI CY M T BO Phng cy v trng T cy v trng Phng chun b mi trng + Cn phn tch 4 s l 0,0001-210 g + Cn k thut 0,01-3100 g + My o pH bn + My Khuy t gia nhit + T lnh + L viba + My ct nc 1 ln + Ni hp v trng Phng nui cy + K nui cy 5 tng v n chiu sng + My o nhit , m + My o cng nh sng Phng hun luyn cy + My o nhit , m + My o cng nh sng Vn m hu cy m + My o nhit , m + My o cng nh sng

+ My xt thuc + H thng ti phun sng