-\ lOl NGI I! Kl lOA I IQC CQNC NCI II^SIMH HQC TOAM QU6C 2013 - _
- K ^ T - ^ y ^ N a H t i = M r.i'n i RI V(^C. ^kuA/^jCHi WPN aFNF n n i i i H A N H V A I V A ZmDREB2A VAO MOT S6 D O N G N G 6 ViET NAM
Biii M^nh Cuwng", Nguyin XuSn Thing, Nguyen Vin Truwig, Doan Thj Bich Thao, Nguyin Thj Thu Ho^l, T? Thj Thuj'Dung : , ,. . .
Vifn Nghien ciru Ngb
Vdi myc dinh nghiSn ciiu, hoan thifn quy trinh tfii sinh va c h i ^ n gen cbiu hai v4o m?t s6 dong ngo thuin ciia Viet Nam, chiing toi di tien hSnh danh pk khi nang tii sinh ciia 8 d6ng ngo thuan li bo m? ciia mOt s6 gidng ngo trien vong dang dir^c ip dyng trong sin xuat. Ket qui cho thay b nlrihig nguSn d6ng khac nhau, khi nin^ t?o mfl sfo, tii sinh cay vi tao ciy hoan chinh cho kit qui khac nhau, chliiigt6khinSngtSismhph\ithu§cviokiSugen. 4 trong so 8 dftng cho ty 1^ lii sinh v i t y l ? tao cay hoin chinh cao (21,4- 68,5%) li VI52N, C67, CTN, vi C463 cfl thi sit dung trong cac thi nghiem chuyen gen. Tren co sfl dfl, buo^ dSu cliiing tfli da cbuy£n thinh cflng 2 gen chju h^ui HVAlva ZiiiDR£B2A v ^ nhihig dfln§ ngo thuin cfl khi nan^ tii sinh cao thong qua vi khuin Agrobacterium tum^aciens. Ket qua buflc dau da xac^dinh dtrpc sv hieu hifn ciia cic gen chuyfin tr£n mflt sfl nguSn dflng ngfl a the hf Tl bing phuong phto phin tich PCR Cic dflng chuyin gen cho ty le cSy tai sinh hfln fty mang gen chuyen tmng binh d?t 22,2- 26,6% doi vfli gen cliuyen HVAI vi 2mDREB2A. Cic cSy di dugc chuyin gene ciln tiip tyc dm^c dinh gii bing nhihig cflng n ^ e phin ti^ khic nhau nhim xic d)nh su biiu hi$n ba d|nh ciia cic gen qua cfic the hf sau.
rir khda: chuyin gene, ngfl, HVAI. ZmDREB2A MdDAU
Kp thu^t chuydn gen vSo c§y nga aa du'pc nghien ci>u vi tib thanh cflng cy quan trpng tO- nhOng nSm 90. Hai phu-ong phdp thu'&ng ducfc dip dgng trong chuyin gene a ciy ngfl 1^ chuyin gene tri/c tilp bing sOng ban gene vi chdyin gen giin tilp thdng qua vl khuin Agrobacterium tumefaciens. Uft so tinh trgng du'pc tap taing nghifin cilu chuyin gene vao cSy ngfl tnsng nhOng nam qua nhu- nSng cao tinh chju m|n: BADH-1 (Wu e( al, 2007); chju l?nh: ADH1; ADH2 . NPK1 (Peters and Frenkel. 2004; Shou e( al., 2004); hay tSng tinh chju hgn: ZmPLCl , CspA, CspB (Wang e( al. 2008;
Castiglioni eta/., 2008)
Hgn \i mOt trong nhO'ng ylu t6 bit thuin chlnh Mm giam sinh kh6i thSn. \i vi ning suit ciia ngfl trfin thl gifl'l cOng nhif fl' \fi#t Nam, nhat \i nhO'ng vOng trong ngfl dya vao nirfl'c ty nhifin. De gidi quylt van d l tren thi vifc chon tgo gllng ngfl chju hgn \i mOt trong nh&ng gidi phdp duvc cdc nhd khoa hpc dat Ifin hdng dlu vd co tinh kha thi cao. Chpn tgo giong ngfl chju hgn bing phuong phap truyin thing da 6ugc tiln hdnh tO nhDng nam 1980, tuy nhien vln efln tin tgi nhilu m^t hgn chl. Trong nhO'ng nam qua, nhieu gene chju hgn vd nhan to phlfin ma da duvc phdn l|p va chuyin vdo ngo bing nhilu phircrng phdp khdc nhau d l Idm tdng khd ndng chju hgn cCia ngfl. M$t trong nhOng phuvng phap dang dirpc si> dyng phfll3iln;4ii§n nay Id chuyin gene vdo ngfl thflng qua vi khuin A. tumefaciens Uu dilm cOa phirong phdp ndy id cho tan so biln ngp cao vd cfl tinh I n dinh qua cdc thl h$ sau (Ishida et al., 19S6]. NhO'ng cflng trinh chuyin gen thdnh cflng sir dyng A. tumefaciens du'pc thyc hifin trfin ph6i hOu tinh cOa dfing ngfl A198 (ishida e( al., 1996) vd b trfin t l hpp lai Hi 11 (Zhao et al., 1998). Hi$u qud biln ngp trfin gllng Hi 11 Id 5,8% v6i vector thi> d p , chCing Atume/aciens LBA 4404, ben phdi non.
Ky thu^t chuyin gene vdo cSy ngfl da dgt du>pc nhOng ^dnh cflng ban dlu b'ong qua trinh ph^t triln gllng ngfl lai chju hgn. Theo bdo do ndm 2008. cflng ty Monsanto, cong ty dypc dlu tir mgnh nhat vfi cflng ngh$ chuyen gene chju hgn trfin thl gidi, da tiln hdnh nghifin ctHu chuyin gen&chju h^n vdo cfiy ngfl. d V i l t Nam, nhO'ng nghifin ciluchuyln gene vdo mOt s l giing cdy tring da thu du'pc nh&ng thdnh cflng ban Siu b mire bf phong thf nghiem. Vln d l chuyin gene chju hgn vdo cdc ding ngfl cung dang du'pc tap trung tiln hdnh nghien c&u. Tuy nhifin, cdc kit quS nghifin c&u efln rat khifim tin vd chya c6 m^t cflng bo chinh thiic nao. Chinh vi vdy chiing tfli da tiln hanh nghifin c&u xdc ^nh khd ndng tdi sinh cOa mOt s6 dflng ngfl vd chuydn gene HVAI va ZmDREB2A vfio cac dflng ngo co kha ndng tdi sinh cao, nhim ndng cao kha ndng tinh chju lign cua mflt s l dong ngo bo mg cua nhOng gllng ngfl dang duvc Vi#n Nghifin c&u Ngfl dp dyng trong sdn xuat. Trong khufln khi bai ndy. chdng toi se trinh bdy mOt s l kit qua nghien cuu bu-oc dau ve chuyin gene chfu hgn tren cdy ngfl tgi Vi|n Nghifin c&u Ngfi.
VAT Ligu VA PHUaNG PHAP %•
Vft llfu
V|t li$u nghifin c&u khS hdng tai sinh thflng qua nufli cly phfii non bao gim 08 dong ngfl thuin Id b l m$ cOa cdc giong ngfl lai cua W?n Nghifin c&u Ngfl {Bdng 1).
Hf thSng mol trubng nubi ciy tai sinh
Mfli tnrbng cdm &ng tgo mfl s^o: N6 + 2 mg/l 2.4D + 10 mgfl AgNOa + 30 g/l sucrose +100 mg/l casein hydrolysate + 25 mM L-prolin + 2i5 gfl phytagel, pH = 5,8;>nhi§l 6f nufli cay 2rC ± 1 trong dilu kifn toi hodn toan Mfli tnffl-ng tgo phfli soma: N6 + 1 mg/l 2,4D + 10 mg/I AgNOa + 20 g/I sucrose +100 mg/I casein hydrolysate + 25 mM L-prolin + 2,5 gfl phytagel, pH = 5,8; nhifit dp nufli cly 27*'c ± 1 trong dilu kifin co dnh sdnQ.
HOI NGH! KHOA HOC C 6 N G N G H $ SINH HQC T O A N Q U O C 2013
Mfli tnr&ng tdi sinh cay. MS + 0.5 mg BAP/1 + 25 mM L-prolin + 10O mg/l casein + 10 mg/l AgNOa + 30 g/1 sucrose + 2.5 gfl phytagel +100 mUt nubc dira, pH = 5,8. Nhifit do phong Id 26°C ± 1, v o i ctrong bf anh sSng la 2000 - 2500 lux, thfl'i gian c h i l u sang 14-16 gio/ngay.
Mfli tnrong ra rS t^o cdy hoan chtnh: MS + lmg/1 tt-NAA+ 100 mg/l myo-tnostoi + 100 mg/l c a s e i n l * 30 g/1 sucrose + 2.5 g/1 than hogt tinh + 2,5 g/1 phytagel. pH = 5.8. Nhi#t dp phflng la 26°C ± 1 . v d i c u d n g dp dnh sdng Id 2000 - 2500 lux. CSy hoan thi^n d i r p c ra n§di trfin nen trau hun vd h r o i bdng dung djcn 1/10 MS.
Bang 1. Danh s i c h c<ic dflng ngo thuan si> dyng trong nufli c i y phdi non TT
1 2 3 4 5 fi
/
U Ten dflng V64 V152N CTN C502N C88N M67 C433 C436
Tham gia ^flng/THL LVN154 LVN154 LVN146 LVN092 LVN885 CN12-1 G26S 6466
Vai trfl cua dflng Dflng m?
Dflng bfl Dflng b l Dflng b l Dflng m?
Dflng me Dflng me Dflng m?
Nguin Vi|n Nghien ciru Ngfl Vrfin Nghifin c&u Ngfl Vifin Nghifln c&u Ngfl Vifln Nghien c&u NgO Vlfln Nghien c&u Ngfl Vlfln Nghifin c&u Ngfl Vien Nghi§n c&u Ngfl Vi|n Nghifin cOnj Ngfl
Chuyen gene vdo 3 dflng ngo cb khi ndng tai sinh cao 1^ V152N, 0 4 3 6 . C7N thong qua chung vi k h u i n Agrobacterium LBA4404 mang vector c h u y i n gene.
Vector c h u y i n gene pBY520 mang gene HAV1 v d vector c h u y i n gene pCAMBIA1300 mang gene 2 m D R E B 2 A d y p c V i f n Nghifin ciJu H$ gen p h ^ \fp vi t h i l t k l .
Hf thong m6i tnrbng si> dyng trong chuyin gene
Mfli tnrdng gdy n h i l m : N6 + 1.5 mg/l 2.4D + 6S.4 g/I sucrose + 36 g/1 glucose + 2 5 mM L-proIin + 10 pM A S . pH = 5,8 , . _ •
Mflt tnrdng nufli u: N6 + 1.5 mg/l 2,4D + 10 mg/l /\gN03 + 30 g/1 sucrose + 25 m M L-prolin + 10uM A S + 2.5 g/1 phytagel, pH = 5,8; nhifit dp nufii cay 27^0 ± 1 trong <Seu k i f n t i l hodn toan.
Mfli tardng phyc h i i : N6 + 1,5 mg/l 2,4D + 10 mg/I AgNOg + 30 g/1 sucrose + 25 m M L-prolin + 0,5 g/1 MES + 100 mg/l cefotaxime + 100 mg/l vancomycin + 2.5 g/1 phytagel. pH = 5,8; nhi?t dp nufli d y 28''C ± 1 trong d i l u kifin t i i hc^n todn. )
• Mfli btPdng chpn Ipc- N6 + 1,5 mgfl 2.4D + 10 mg/l Agl^JOj + 30 g/1 sucrose + 25 m M L-prolin + 0,5 g/1 MES + 100 mg/l cefotaxime + 100 mg/l vancomycin + 30 mg/I hygromycin + 2,5g/l phytagel, pH = 5,8; nhifit 6b nufli cay 2 a ' ' c ± 1 trong d i l u ki$n t i i hodn todn.
Mfli b v d n g tdi sinh: MS + 100 mg/l mycninositol + 30 g/l sucrose + 100 mg/I casein + 2,5 g/1 than hoat tinh + 2.5 g/1 phytagel, pH = 5,8; nhifit do nufli c4y 28''c ± 1 trong d i l u l d | n c h i l u sdng 16 gld/ngdy.
Phtra>ng p h d p nghifin cChi
Danh gli khi ning til sinh ciy ngo ft>nudr cay p / i d / n o n : Phoi non cOa cdc n g u i n vgt li$u sau khi th\i p h i n 12 ngdy dirpc thu mSu, x& IJ Ignh. k h d trung hgt va d y trfin bfi mgt 6\a petri c6 ch&a mfli trudng dm ung tao mfl s§o v & i m | t 6b 20 phfli/dTa. Moi n ^ n dflng d u ^ c cay lap lai 3 l l n . moi l l n l | p Igi 300 phoi hrong i>ng vdi 15 dTa petn. Mfl s e o hlnh thdnh dirpc d y chuyen sang mfli tardng nufli d y tgo phfli soma, mfli frydng tao c h l l vd re. N h d n g c§y hodn chinh d i r o c tien hdnh ra ngoi.
Bien nap nhb Agrobacterium: 1) GSy nhlSm vi nuol 0: Mfl s f o 7 ngdy sau c l y c h u y i n d y p c sO dyng d l gdy nhiSm bdng each cho vdo I n g falcon ch&a dung djch Agrobacterium khd b i l n {ODgoo = 0.7), ISc nh? frong khodng 10 phiit, sau dfl v f l i mfl seo vd t h i m kho dung djch vi k h u i n b i n g g i l y t h i m k h d tning. Mfl s ^ da gay n h i l m d i r p c t J y lai tren mfii birdng nufii ii (15-20 mfl sgo I6ia pehi) vd nufli trong d i l u kifin t i i d 27''C trong 3 ngdy. 2) Phuc ha va chon loc m6 seo:
Sau 3 ngdy nufli 0. d y c h u y i n toan bo mo seo sang mfli t a r d n g phyc hoi, nufli frong loi 7 ngdy d 27*'C Sau khi nufli phyc hoi, mfl seo du'pc d y chuyen sang rnfli frydng chpn ipc co c h d a hygromycin hogc bialaphos, vd d y c h u y i n 2 hjdn mbt Idn. 3) Tii sinh ciy-. Cac mfl seo mdu sdng d y p c c l y c h u y i n sang mfii fru'dng tai sinh vd nufii trong d i l u ki#n chieu i d n g 16h/ngay. Cac cay hodn chinh d i r p c tien hanh ra ngfli vd trfing frong nha Ii«7i.
^^r^-rS^.-^X^*^ " ^ ^ "^"^ ^ ^ " ® * ' ^ ' ' ' ^ ^ " 3 * * *^"^' ''^f^- Pfian d n g PCR s d dung cgp m l i ZmDREB2A-F:
^^JTUtl'i^^^^^'^'^^^^^^ ^ f^°s T-R: TAAATAAAAAGAGTGCTTGC cho gene ZmDREB2A vd c g p m i l HVAI-F: TGGCCTCCAACCAGAACCAG va H V A l - R : GACTCCTTGGTGTACTGCGC cho gene HVAl.Thdnh p h i n phan u-ng bao gflm Buffer Dream Taq (IX). dNTPs (1 mM). m i l xufli (0,6 pm), m i l ngiroc (0,6 pm). Dream Taq (2.5 U), DNA Wiuen 10 ng; Chu chlnh nhifit cua phdn d n g PCR n h y sau: 94''c frong 5 phOt; (g^^C b o n g 4 5 giay. SS^C frong 30 gidy.
72"C frong 1 phut) Igp lgi-30 chu ky; 72''C trong 8 phut vd g i y d 4''C.
1 ^ QUA VA TH A o LUAN
Xic djnh k h i nang tdi s i n h cua cac d o n g n g f i t h f i n g qua n u f i i d y p h f l i n o n
K i t q u i phdn boh khd nang tdi sinh cua 8 dfing ngfl t h u i n dang d y p c s d dung ldm b l m ? cOa mW s l l l hop lai friiwng mgi bong nam 2012 dirpc t h l h i # n d Bang 2. . v v - r K i t qud ddnh gia kha nang tai sinh cay ngfl t d phfli non trfin 8 dflng thuan t d Bang 2 cho I h l y :
' MOI NGM! KHOA HQC C 6 N G NGHE SINH HOC T O A N Q U 6 C 2013
Cr nhiwig n g u i n dfing khic nhau. khd n i n g tgo mo sgo. tai sinh cay va tao cdy hodn chinh cho ket qua khdc nhau.
chdng & klid ii3ng~tdj iiiiili phu Uiupc vuu kieu gener ' — • • — Dflng CTN ra khd nSng Igo mfl sgo cao nhat (73.5%), t i l p 6b Id dong V152N (63.2%). Dong C88N co khd nang tgo m f i sgo thap nhat (3.2%). C^c dong con Igi co ty I f Igo m o s?o I d 13.3 - 29.8%.
Khd nang tai srnh vd tgo cdy hodn chlnh: Dflng V152N cho ty 1$ tdi sinh vd tao cSy hodn chinh cao n h i t (tirong d n g ii 29.5 vd 68,5%). Hai dong M67 vd C433 biy co l y 1$ p h i n fram dy lai sinh cao ( h j v n g dng Id 31,3% va 21%). song Tgf c 6 t j 1$ tgo cdy hodn chinh thap, chl d^il 13 vd 5.3%. Cac dflng khSc cung d i o ft I f tai sinh cao nhy CTN (22,4%) vd C436 (21,4%), vd cho t j \f tgo cdy hodn chinh b j o n g flyong Id 33.3% vd 15,4%. D a v d i dflng V64. m | c dil cfi t# I f tdi sinh cSy t h i p (12.4%) nhyng Igi cfi ty I f igo dy hodn chlnh cao (21.5%). Dgc b i f t dflng C88N khflng cfi kha ndng tgo mfl s?o trong d c Iogi mfli t n r o n g vd d i l u k i ^ thf nghigm nSy. Thfing qua k i t qua ddnh gid kha nang tgo mfl sgo, tai sinh vd tgo cdy hodn chinh, chOng tfii da xic djnh d\«rc 4 nguon v | t lieu cfl kha ndng tai sinh cao la V152N. V64. C7N vd C436.
N g u i n vdt l i f u ndy cfi fril d u v c s d dung bt)ng cdc thi nghifm c h u y i n gene.
Bing 2. K h i nang t^o mfl s$o, tai sinh vd tgo cay h o i n chlnh c6a 8 dflng ngfl thuan TT Tfln dflng Tham gia THL S l phfli Ty 1$ tgo T9 lfi cdy t£
d y callus (%) sJrA (%)
Ty If tgo cty hoflnchhh(%) 1
2 3 4 b 6 7 8
V64 V152N CTN C502N C88N M67 C433 0436
LVN154 LVN1M LVN146 LVN092 LVN885 CN12-1 G269 G466
1800 2550 3700 1800 2500 1300 690 1000
23.1 63.2 73,5 23.4 3,2 24,4 29,8 13,3
12.4 29,5 -22.4 12.1 31.3 21.0 21.4
21,5 6B.5 33,3 13,3 13.2 5.3 15.4
Hlnh 1. Tai smh c&y ngo tir nufli cay phoi non A, Phfli non 12 ngay tuiidLFs v^o nuoi cay. B Mo s ^ d u ^ c taora t d nufli c l y phfli non, C' T^i sinh cdy: D. Cdy cfl dO thdn la dirpc c^y chuyen de t^o r^. E Cay tren m6i trvong t$o r l , ' P: CSy ra ngoi trfin gifl the ti^u .huii,GjCay'iromlrong.nhaiinSi;H:Cayyablp thehflTo. . . . .^ . . , _ . _ , , . _ ,_ ,
Ket qua b i r f i v dau ve c h u y i n gene ZmDREB2A va H V A I
Bing 3. K i t qua chuyin gene Z(nDREB2A vd HVAI vio mflt sfl dflng ngo thuan fl- the h# TO Gene ZmDREB2A Gene HVAI
. ^ Nguin Tflng so Sfl cdy S l % cay tdi % cdy mang Tong s l S l cSy Cfiy % cdy tSi % cSy mang vdt nfiu phfli tdi^nh cdy sinh sau gen^caytdi phfit tdi sinh mang sinh sau gene/cdytii
dupc gdy hOu mang chon IQC sinh hl>u thy dirpc Idy turu - gene chon Ipc sinh Mru thy nhilm thu gens hihi thy nhilm tfiy '^hOti thy 1 V152N
2 C436 3 C7N Ting/TB
1140 890 1416 3446
IS 22 30 67
9 8 17
1.32 2.47
zn
1.97 60,00 26.67 28.89
1037 619 1186 2842
19 37 IS . 74
2 7 4 13
1 ^ 5,S8 1,52 3.11
10.53 18.92
17.22 K i t c h u y i n gene dirpc tflm tat trong Bang 3 i^io thay. buoc d l u da c h u y i n thanh cfing hai gene chju hgn ZmDREB2A vd H V A I vdo ba dflng ngfl cfi khd nSng tdi sinh cao. D 6 vfii gene ZmDREB2A ty I f cay tdi sinh hOu thg frung blnh dat 1,97% vd 19 I f d y mang gene frung binh dgt 28.89%; d l i von gene H V A 1 . ty I f cdy tdi sinh hOu thu tmng blnh dgt 3 . 1 1 % , tuy nhifin t j I f cdy mang gene tnjng blnh I h i p hem (17.22%) so v o l gene ZmDREB2A. Dflng V152N co ty I f cay t n a n g g e n e d i u y l n Z m D R E B 2 A c a o n h l t ( 6 0 % ) n h [ m g l g j c h o t ^ l f c S y mang gene c h u y i n HAV1 thdp n h i t (10,53%).
Trcng khi dfi dflng C7N cho t f I f cdy mang gene H V A I vd ZmDREB2A t y o n g d n g la 22.22% vd 26,67%. Bgc bift doi v d i dfing C436 khflng thu d i r p c cdy c h u y i n gerie ZmDREB2A d u o n g b'nh. nhimg doi vdi gene hlAV1 thl ty I f dy d i r o n g
HOI NGH! KHOA HQC C O N G N G H $ SINH HQC T O A N Q U 6 C 2013
tinh Id 18.92%. K i t qua cho t h i y doi v d i cdc dong ngfi khac nhau thl t? I f cSy mang gene c h u y i n dircmg tinh frfin cay tdi sinh hiru Uiu khac rrtiau. Ty I f nSy d i n g phy tfiufc vdo cdc vecteir c h u y i n gene khac nhau.
K i t q u d Wem tra s i r co m#t cOa gene c h u y i n ZmDREB2A va H V A I d c d y n g f i c h u y i n g e n e t h e h f T l K i t qud phdn b'ch s y cfi mat cua cac gene c h u y i n d cdy ngfl c h u y i n gene fril h f T l bang PCR d i r p c t h l h i f n trong Hlnh 2 (ZmDREB2A) va H i i ^ 3 (hlVAl).
Hinh 2. Ket qua PCR phan agarose 1%. M- Thang DNA ch&ng dm (cay khflng chu
gene t h i h f T l trdn anh d i f n dl nuyen gene 6 I h l h f T l ; (-) Boi
Hinh 3. K i t qud PCR phdn b'ch s y c6 mat cua gene chuyen HVA1 b m f t sfl cdy c h u y i n gene the h f T l t r i n dnh d i f n di agarose 1%. M: Thang DNA chuin 100 bp, Dirong chay tCr 33-140. ti/cmg dng vfli cdc dflng cdy chuyin gene d t h l h f T l ; (-) D l i chdng am (cdy khflng chuyin gene); (+)€}6i(^i>ng duong (plasmid mang gene HVA1)
Xac djnh khd nang tdi sinh cua m f l k i l u gene d y p c xem ta b u d c quan trpng dau tifin quyet dinh s y thdnh cflng trang qud frinh chuyin gene. Cdc k i l u gene khdc nhau dirpc phdn togi b d i t i n s l tgo mfl sgo va kha nang ldi sinh cSy (Shan et al., 2009). Phfii non tir ki&j gene Hill d u ^ c bdo cao cfl kha nang tdi sinh cao. tuy nhifin do s y phdn fy nfin chung thydng dirpc s d dyng n h y n g u i n thy frong dc phdng thl nghiSm c h u y i n gene (Frame ef al., 2002).
K i t qud nghifin c d u ndy v l khd ndng chuyen gene Id phO hpp v d i nh&ng k i t qud nghifin c&u da d i r p c cfing b l t a r d c ddy (Frame at al., 2006; Ishida e( at., 1996; Takavar e( al.. 2010). Khi nghifin c d u dnh h y d n g cOa k i l u gene d i n tan s f l chuyfin gene, n h i l u tac gia cho r i n g tfin s6 chuyen gene b ngfi thfing qua Agrobactenum phy t h u f c vdo k i l u gene trong cung m f t lodi. H i f u qua chuyfin gene dgt 6,45% doi v d i dfing S61 (Takavar ef a/.. 2010) vd t d 5-30% d l i v o l dflng n g f i A188 (Ishida et al. 1996) Frame ef al (2006) cung cho rang gida cac dong ngfl khac nhau cho h i f u qud c h u y i n gene k h d c n h a u . C g t t i l Id dflng 8104 dai 6.4%. dflng B l 14 dat 2,8%, dflng Ky21 dat 8%, frong khi t i n so d i u y l n gene c u a dfing H 9 v d W 1 7 7 la 0%.
S d dung h f t h i n g sifiu vector nht t h l (super binary vector), Ishida ef al (1996) da thdnh cfing bong v i f c c h u y i n gene v d o dfing ngfi A188 v d i t i n s6 c h u y i n gene dgt 5-30%. Cflng b l cua Frame ef a/ (2002) cho r i n g h i f u qija c h u y i n gene cOa ngufin Hill dgt 5.5% khi s d dyng vector nhj I h l (standard binary vector) vd cho h i f u qua cao h o n khi s d dyng h f t h i n g vector lifin hpp (co-lntergrated vector).
Dgng T-plasmid cua Agrobacterium cung cfi vai frfi frong qud frinh c h u y i n T-DNA va dnh hirdng d i n h i f u qud c h u y i n gene. Cac nghifin cdu cho I h l y . Tl-friasmid dang nopalin co h i f u qud hon frong v i f c Idy n h i l m Agrobacteium vdo ngfl s o vfii Ti-plasmkl dgng octopin (Shen et ai, 1993).
Ba xdy dyng dirpc quy trinh tdi sinh vd xdc dinh d i r p c 4 dflng ngfl t h u i n Id V152N, V64, C7N, 0 4 3 6 cfi k h i nSng H i sinh cdy cao phyc vy cho cdc Ihi nghifm c h u y i n gene. Day la nhdng dfing bo, mg dang tham gia v d o m f t s l g l l n g ngfl t r i l n vpng.
Birdc d l u da c h u y i n tfidnh cfing hai gene chiu hgn id HVA1 vd ZmDREB2A vdo cay ngfl s d dyng phirong phdp c h u y i n gene frifing qua vi khuan Agrobacfe/fum.
Oa phdn bch s y cfl m f t cua cdc gene c h u y i n trong cac dflng ngfi c h u y i n gene d t h l he T l b i n g PCR.
HQI NGHI KHOA HQC CdNG NGHg ^lNH-HO&TQAN-QU6C-2ei3 - -
T A I LIEU THAM K H A O
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Takavar S. Rahnama H, R&himlan H and Kazemltabar K (2010). Agrobacterium mediated bansfbrmalifln of maize {Zea mays L.).
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THE PRELIMINARY RESULTS OF TRANSFORMING TWO DROUGHT TOLERANT GENES HVA1 AND 2mDREB2A INTO SOME INBRED MAIZ£ LINES OF VIETNAM
Bul Manh Cuong', Nguyen Xuan Thang, Nguyen Van Truong, Doan Thi Bich Thao, Nguyen Thl Thu Hoai, T a Thi Thuy Dung
National Maize Research Institute
SUMMARY
With die purposes of studying and improving regeneration and transfomiation of some maize inbred Unes of Vietnam, w e evaluated the regeneration ability of eight parental lines from some promising hybrid maize varieties. The results indicated that the inbred lines , have different ability of callus formation induction and plant regeneration, suggestmg fiiat the plant regeneration ability depends on - flie genotype. Four oul of eight mbied lines with high plant regeneration rate (21,4 to 68,5%) were V152N, C67. CTN, and C463, Next, two drought tolerant genes HVAIand ZmDREB2A were successfiiliy transformed into three inbred lines with high regeneration rate V152N, C436, and CTN using Agrobacterium tumefaciens. The preliminary transformation results showed that the average rate of transgenic plants positive wifli PCR at flie T I generation is from 23.2% fen" HVAI to 26.6% for ZmDREBSA.
Key words: Transformation, maize, H V A I , ZmDREB2A
"Author far correspondence, [email protected]
