M U Vn su hi l mt kh khn ln trong sn xut nng nghip.

. Tn tht do su gy ra rt nghim trng, hng nm gy thit hi 29,7 t USD, khong 13,8% ma mng - cao nht so vi cc loi dch hi khc. phng tr su hi, ngoi cc bin php truyn thng nh canh tc, ha hc, c gii, vt l ..., s dng ging khng su l mt bin php mi v hiu qu. Trc y, mun to ra ging c c tnh mi ny, ngi ta phi lai to trong thi gian di. Tuy nhin, cng ngh gen ra i gii quyt c nhc im . Nhiu ging cy trng chuyn gen c kh nng khng su nh: bng, ng, la, u tng... c mt trn th trng nh phng php chuyn gen v khng nh u th vt tri. Trn th gii, cc nc u - M nghin cu thnh cng nhng thnh tu ny v ng dng i tr phc v cho sn xut nng nghip, nhm mang li hiu qu kinh t, gim chi ph cho ngi sn xut v to ra sn phm an ton cho ngi tiu dng v bo v mi trng. u tin, B nghin cu thnh cng trn cy thuc l chuyn gen khng su vo nm 1985. T nm 1995 n nm 1996, M cng nghin cu v ng dng i tr cc loi cy trng chuyn gen khng su nh: bp, khoai ty, bng vi. Cn i vi chu , cng c nhiu quc gia t c nhng thnh tu ng k nh Trung Quc, n , Nht Bn, Thi Lan c bit, Philippine l mt trong nhng nc i u nghin cu v s dng cy trng chuyn i gen khng su trn bp, u tng, bng vi, ci du..., trong cy bp ng th hai sau cy la. Ring i vi Vit Nam, cc vin, trng, cc Trung tm Cng ngh sinh hc cng bc u nghin cu v kho nghim s dng cc ging cy trng c gen khng mt s loi su hi trn rau mu nh vic chuyn gen khng su xanh da lng, su c tri trn cy u tng. Hin nay, trn th gii c hn 150 triu ha trng cc loi cy chuyn gen, y l du hiu ng mng cho cc nh sn xut nng nghip trn ton th gii

CHNG 1. I CNG V K THUT CHUYN GEN THC VT 1.1. nh ngha cc khi nim chnh

Chuyn gen (transgenesis) l a mt on DNA ngoi lai vo genome ca mt c th a bo, sau on DNA ngoi lai ny s c mt hu ht cc t bo v c truyn li cho th h sau. Khi nim ny ch s dng cho thc vt v ng vt. Nm men, vi khun v t bo nui cy mang mt on DNA ngoi lai c gi l cc t bo ti t hp (recombinant cell) hoc t bo bin np (transformed cell). Qu trnh bin np (transformation) l qu trnh a mt DNA ngoi lai vo genome (h gen) ca mt sinh vt. Gen chuyn (transgene) l gen ngoi lai c chuyn t mt c th sang mt c th mi bng k thut di truyn. Gen chuyn c th c phn lp t nhng loi thc vt c quan h h hng hoc t nhng loi khc bit hon ton. Cy trng chuyn gen (transgenic crops) hay cn gi l cy trng bin i gen (genetically modified crops) l mt thc vt mang mt hoc nhiu gen c a vo nhn to thay v thng qua lai to. Ni cch khc, thc vt chuyn gen l thc vt c gen ngoi lai (gen chuyn) xen vo trong DNA genome ca n. Gen ngoi lai ny phi c truyn li cho tt c mi t bo, k c cc t bo sinh sn mm.

1.2. Mc ch chuyn gen

Hnh 1.1. T l phn b cc mc tiu cng ngh gen thc hin trn cy trng M (thng k nm 2004). Ni chung, mc ch ca chuyn gen l thm mt thng tin di truyn ngoi lai vo genome, cng nh c ch mt gen ni sinh. Trong mt s trng hp, s thay th mt gen hot ng chc nng bng mt gen hot ng chc nng khc l cn thit. Gen ngoi lai c th l mt th t bin ca gen ni sinh hoc mt gen hon ton khc. S thm gen c th c thc hin cung cp sinh vt mang protein mi. S thm gen cng c th c s dng nghin cu c ch hot ng ca mt promoter trong ton c th. S kt hp ca gen reporter vi promoter l nguyn tc chung ca phng php ny. S thay th gen c s dng ch yu lm bt hot mt gen bit. Trn thc t, n bao gm s thay th gen ni sinh bng mt th t bin bt hot. Phng php ny c dng cho thng tin v chc nng sinh hc ca gen nh trng hp thm gen. Thc vy c thm gen v bt hot gen c th gy ra cc bin i sinh vt chuyn gen, m cc bin i ny c th c quan st hoc nh gi. Cc th t bin ca gen thay th c th cung cp thng tin bng mt cch thc tinh vi hn. S thay th mt gen bng mt gen c chc nng khc nhau hon ton l kh hn nhng c th thc hin a mt marker hoc mt gen chn lc vo genome. V tr hp nht vo genome c th c chn lc i vi kh nng cha ca n biu hin gen ngoi lai bng mt cch chc chn [4]. 1.3. Nguyn tc c bn ca vic chuyn gen 1.3.1. Mt s nguyn tc sinh hc Khi t ra mc ch v thc hin th nghim chuyn gen cn ch mt s vn sinh hc nh hng n qu trnh chuyn gen nh sau: - Khng phi ton b t bo u th hin tnh ton nng (totipotency).
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- Cc cy khc nhau c phn ng khng ging nhau vi s xm nhp ca mt gen ngoi lai. - Cy bin np ch c th ti sinh t cc t bo c kh nng ti sinh v kh nng thu nhn gen bin np vo genome. - M thc vt l hn hp cc qun th t bo c kh nng khc nhau. Cn xem xt mt s vn nh: ch c mt s t t bo c kh nng bin np v ti sinh cy. cc t bo khc c hai trng hp c th xy ra: mt s t bo nu c to iu kin ph hp th tr nn c kh nng, mt s khc hon ton khng c kh nng bin np v ti sinh cy. - Thnh phn ca cc qun th t bo c xc nh bi loi, kiu gen, tng c quan, tng giai on pht trin ca m v c quan. - Thnh t bo ngn cn s xm nhp ca DNA ngoi lai. V th, cho n nay ch c th chuyn gen vo t bo c thnh cellulose thng qua Agrobacterium, virus v bn gen hoc phi ph b thnh t bo chuyn gen bng phng php xung in, siu m v vi tim. - Kh nng xm nhp n nh ca gen vo genome khng t l vi s biu hin tm thi ca gen. - Cc DNA (tr virus) khi xm nhp vo genome ca t bo vt ch cha m bo l lin kt n nh vi genome. - Cc DNA (tr virus) khng chuyn t t bo ny sang t bo kia, n ch ni m n c a vo. - Trong khi , DNA ca virus khi xm nhp vo genom cy ch li khng lin kt vi genome m chuyn t t bo ny sang t bo khc ngoi tr m phn sinh (meristem) [2]. 1.3.2. Phn ng ca t bo vi qu trnh chuyn gen Mc ch chnh ca chuyn gen l a mt on DNA ngoi lai vo genome ca t bo vt ch c kh nng ti sinh cy v biu hin n nh tnh

trng mi. Nu qu trnh bin np xy ra m t bo khng ti sinh c thnh cy, hoc s ti sinh din ra m khng km theo s bin np th th nghim bin np cha thnh cng. rt nhiu loi thc vt, iu kh khn l phi xc nh cho c kiu t bo no trong cy c kh nng tip nhn s bin np. Ht phn hay t bo non sau khi c bin np c th c dng to ra cy bin np hon ton, thng qua qu trnh th tinh bnh thng. Ht phn thng c coi l nguyn liu l tng gy bin np. Trong khi , vic bin np gen vo hp t in vivo hay in vitro li gp nhiu kh khn. Trong trng hp ny, ngi ta thng phi kt hp vi k thut cu phi. Vic bin np gen i vi cc t bo n ca cc m phc tp nh phi hay m phn sinh thng cho ra nhng cy khm. T nhiu thp k qua ngi ta bit rng, tnh ton th ca t bo thc vt to iu kin cho s ti sinh cy hon chnh in vitro qua qu trnh pht sinh c quan (hnh thnh chi) hay pht sinh phi. Cc chi bt nh hay phi c hnh thnh t cc t bo n c hot ha l nhng b phn d dng tip nhn s bin np v c kh nng cho nhng cy bin np hon chnh (khng c tnh khm) [2]. 1.3.3. Cc bc c bn ca chuyn gen K thut chuyn gen thc vt l s chuyn mt on DNA l, thng l mt gene c chc nng m ho cho mt thng tin hay c im c li nht nh vo t bo thc vt nh kh nng khng su hi, khng virus hay khng thuc tr c. Cy ti sinh t t bo chuyn np c gene l c lng vo genome, biu hin ra kiu hnh va di truyn n nh c gi l cy chuyn gene. Gene l c th c ngun gc t vi sinh vt thc vt, ng vt, thm ch gene tng hp. Qu trnh chuyn gene bao gm nhiu bc: xc nh v phn lp gene c ch, nhn gene, chuyn gene vo t bo thc vt, ti sinh t bo chuyn np thnh cy hon chnh v nh gi s biu hin ca gene.

Trong cc bc trn, k thut chuyn gene ng mt vai tr quyt nh i vi kt qu chuyn gene. Chuyn gene vo t bo thc vt c th thc hin gin tip thng qua vector hay trc tip. Sau y l v d v s chuyn qua thng qua Ti-plasmid ca vi khun Agrobacterium tumefaciens. A. tumefaciens l vi khun gy khi u cy hai l mm v phn ng hnh thnh khi u l kt qu ca s kin chuyn gene t nhin. Mu cht ca s hnh thnh khi u l Ti-plastmid (Ti: tumor inducing) ca vi khun cha cc gene m ho sinh tng hp cc horrmon (auxin v cytokinin) chu trch nhim cho s hnh thnh khi u. Cc gene ny nm trong vng T-DNA (transfered DNA) ca plasmid. Khi plasmid xm nhp vo t bo ca cy, cng T-DNA c chuyn vo genome. on T-DNA l yu t di truyn vn ng trong qu trnh chuyn gene. ngha ca Agrobacterium trong k thut di truyn l kh nng chuyn on DNA vo t bo thc vt. Li dng phng thc chuyn gene t nhin cc nh khoa hc thc vt da vo k thut phn t iu khin T-DNA thit k h vector bng cch lng on DNA cn chuyn gn vo cng T-DNA ca vi khun, sau cho cy nhim cc vi khun cha plasmid bin i. ly nhim ngi ta nui cy t bo trn thc vt, t bo n trong trong mi trng lng, hoc t m cy trong dung dch huyn ph cha vi khun c plasmid bin i trong mt thi gian nht nh. Qu trnh chuyn np thng qua - Phn lp gene c ch t th cho (DNA l) v nhn gene - Loi b T-DNA khi Ti-plasmid ca cc dong vi khun chn lc - Chuyn DNA l vo Ti-plasmid cng vi promotor v mt gene ch th c kh nng chn lc d dng (v d gene uidA ca vi khun m ho glucuronidaza thng gi l gene GUS, hay gene khng khng sinh). - a plasmid bin i vo t bo thc vt - Chn t bo c chuyn gene Agrobacterium gm cc bc sau y:

- Ti sinh t bo c chuyn gene - Chn cy biu hin c chuyn (cy chuyn gene)

Hnh 1.2. To ging la Bt bng phng php sng bn gene (particle bombardment method). mt s loi cy d nui cy nh khoai ty v c chua, cc mu l ct ri c nhng vo dung dch cha vi khun trong mt thi gian ngn. Sau cc mu l c a vo mi trng dinh dng. Trong khong thi gian vi khun tip tc sinh trng v xm nhp vo cc t bo l ri to ra mt s t bo chuyn gene. V ch mt phn nh t bo c chuyn gene nn cn phi tin hnh chn lc. Vic chn lc thng thng da vo gene chn lc, l gene khng khng sinh hay khng thuc tr c. Sau cc mu l c chuyn vo mi trng khc cha mt cht khng sinh dit Agrobacterium cn st li v mt cht khng sinh khc hay thuc tr c loi tr cc t bo khng chuyn gene. Cc t bo c chn c chuyn sang mt lot cc mi trng
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dinh ti sinh cy. Kt qu ti sinh v to cy chuyn gene ph thuc vo loi cy. Phng php chuyn gene thng qua Agrobacterium c p dng rt thnh cng nhiu loi cy hai l mm nh thuc l, khoai ty v c chua [2]. 1.4. Lch s pht trin, tim nng v hn ch ca k thut gen * Lch s pht trin: Lch s pht trin cng ngh gen ca thc vt chc chn c rt nhiu s kin quan trng. y ch nu ln nhng mc c ngha c bit nhm lm r s pht trin rt nhanh ca lnh vc ny: - Trc ht, vi khun t Agrobacterium tumefaciens c s dng lm phng tin vn chuyn DNA. Bnh thng vi khun ny to nn khi u thc vt. Mt phn nh ca Ti-plasmid c trong vi khun ny, c gi l T-DNA, c vn chuyn t Agrobacterium vo cy hai l mm. Nm 1980, ln u tin DNA ngoi lai (transposon Tn7) c chuyn vo thc vt nh A. tumefaciens, tuy nhin T-DNA vn cha c thay i. Nm 1983, nhiu nhm nghin cu bin i T-DNA v a DNA ngoi lai vo, to ra tnh khng mt s cht khng sinh. Ngoi ra, cc gen to khi u c ct ra. DNA ngoi lai cng vi phn cn li c chuyn vo thc vt v c bin np. Thnh cng ny nh nghin cu chnh xc con ng ly nhim ca A. tumefaciens trc v kh nng ca h thng chn lc i vi thc vt. T kt qu thnh cng u tin ny s lng cc loi c bin np ngy cng tng. Lc ny c thm nhiu phng php khc bin i gen: - Nm 1984, bin np bng t bo trn (protoplast) ng c thc hin. y thnh t bo c phn gii bng enzyme, xut hin t bo trn. Nh polyethylene glycol (PEG) hoc xung in (electroporation) m DNA c a vo t bo trn. - Nm 1985, ln u tin cy bin i gen c m t c tnh khng thuc dit c. Mt nm sau, ngi ta thnh cng trong vic to ra thc vt khng

virus. Nm 1996, cc th nghim v cy bin i gen c php a ra ng rung. - Nm 1987, phng php bin np phi sinh hc c s dng. y t bo thc vt c bn ph bng cc ht vng hoc wolfram bc DNA ngoi lai. Nh phng php ny m s bin np thnh cng cc cy mt l mm quan trng nh la (1988), ng (1990) v la m (1992). Cng trong nm 1987, c chua v thuc l khng cn trng lm cho cng ngh gen t c mt bc pht trin quan trng hn. Mt thnh cng quan trng khc l iu khin c qu trnh chn c chua, sau ny c tn l FlavrSaver. Nm 1994, ln u tin c chua bin i gen c bn trn th trng. - Nm 1989, khng nhng thnh cng trong vic chuyn cc gen m ha cc khng th vo thc vt, m ngi ta cn to nn cc sn phm gen ny nh mong mun. Kt qu ny m ra mt kh nng hon ton mi m cho vic sn xut vaccine v c kh nng chng bnh thc vt. - Nm 1990, thnh cng trong vic to ra cy bin i gen bt dc c, khng c kh nng to ht phn. Loi cy trng ny c ngha ln trong vic sn xut ht ging. - T nm 1991, thnh phn carbohydrate ca thc vt c bin i v nm 1992 l cc acid bo. Cng nm , ln u tin thnh phn alkaloid mt loi c c ci thin, l mt bc quan trng i vi thc vt trong vic tng hp nhm hp cht ny. Nhng thc vt ny c ngha ln i vi vic thu nhn dc liu. Sau khi thc vt bin i gen ny xut hin, cht nhn to phn gii sinh hc c tng hp. iu ny cho php chng ta hy vng rng, trong tng lai s c nhng thc vt c c tnh mi, c s dng nh l cc bioreactor thc vt sn xut nguyn liu ti sinh.

- Nm 1994, c chua Flavr SavrR l cy trng u tin bin i gen v qu ca n c a ra th trng. Nm 1998, trn th gii c 48 ging cy trng bin i gen v sn phm c th trng ha. Nm 1999, cy la bin i gen c a ra vi 7 gen c bin np. n u nm 1999, trn th gii c khong 9.000 th nghim ng rung cho php, trong khong 1.360 l EU. Cui cng, l mt s nhn xt v vic th trng ha cy bin i gen trong nng nghip. Cho n nm 1999, din tch gieo trng trn th gii t hn 40 triu ha. Trong 20% l ng, 50% l u tng v 1/3 din tch bng l M. Ngoi ra c hn 70% din tch ci du Canada c trng vi ging bin i gen. Khong 90% thc vt bin i gen chng chu thuc dit c hoc su bnh hi. Cn ch rng, M sn phm u tng c trong hn 20.000 loi thc phm khc nhau. iu ny cho thy rng, cng ngh gen nh hng n sn xut thc phm ca chng ta [1]. * Tim nng: - K thut chuyn gen cho php a vo cy trng mt gen l c phn lp t mt ging khc c quan h h hng vi n hoc t mt c th thc vt bt k khc, thm ch t ng vt hoc vi sinh vt. V vy, lc n lm xut hin nhng tnh trng hon ton mi, thm ch cha c trong t nhin. - Nh k thut chuyn gen ngi ta c th to ra nhng ging cy trng cha nhng hp cht c bit, nh cc cht hu c dng trong cng nghip, thc phm, y t ... V d nh cy ci du c chuyn gen c kh nng phng chng bnh tim mch. - T mt gc nht nh m xem xt th, nhiu cy trng c chuyn gen khng su bnh c hiu qu tch cc phng chng nhim mi trng do vic s dng cc ha cht c hi trong nng nghip.

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- S dng k thut gen l con ng gy to ra nhng ging cy trng c nhng tnh trng mong mun ch vi mt thi gian ngn v trong mt phm vi nghin cu hp [3]. * Hn ch ca k thut gen: - Cho n nay, s lng cc gen kim sot cc tnh trng nng - sinh hc qu, c bit l cc tnh trng s lng cn cha nhiu. - Cc quy trnh chuyn gen cha tht hon thin. - Kin thc cng nh cc phng php nghin cu v s biu hin ca gen sau khi c bin np cn cha hon chnh. - K thut nui cy m - t bo v ti sinh cc cy c chuyn gen cho chng c hu th cao vn cn nhiu hn ch [3]. .

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CHNG 2. K THUT CHUYN GEN TO CC CY TRNG KHNG SU 2.1. C s ca k thut chuyn gen to cc cy trng khng su 2.1.1. Nhng thit hi do su v cn trng gy ra Su v cn trng c th c chia lm hai loi da trn phng thc s dng thc n ca chng: - Cn trng chch ht: b ry, rp, b xt. - Cn trng n cc b phn khc nhau ca thc vt: c thn, c r, cun l, c qu, n l. Tt c cc loi ny u lm gim nng sut v cht lng sn phm. S mt mt v cht lng thng xuyn hn v mc cao hn s mt mt v sn lng. Mt ging nhim su thng a n cc hu qu sau: - Gim s pht trin ca cy trng - Ph hy l, thn, cnh, n hoa, hoa, chi v tnh, qu v ht, - Lm gy cy Mc thit hi trc ht ph thuc vo cng tn cng ca cn trng v mc nhim ca k ch. Cn trng cn c th gy tc hi gin tip, rt nhiu loi cn trng l vt truyn bnh virus. S chn thng do cn trng gy ra to iu kin cho nm v vi khun pht trin [2]. 2.1.2. C ch ca tnh khng su Tnh khng su c chia lm 4 nhm: Khng a thch, Khng sinh, Chng chu, C ch trnh. * C ch khng a thch: K ch to ra s khng hp dn cho su v cn trng, chnh l s khng thch ng cho vic to vng sng v vng trng. Kiu khng ny hn ch kh nng tn cng ca su hi hoc s khng chp nhn tn cng. S khng chp nhn xut hin khi cn trng khng dng thc n ca k ch hoc k ch khng

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c thc n ph hp cho cn trng. C ch khng a ny c lin quan n nhiu thuc tnh hnh thi sinh l hoc sinh ha ca cy ch. * C ch khng sinh: Cc cht khng sinh c tc dng khng li s n cy ch ca su bo tn s pht trin v ti sinh ca cy trng. Trong mt s trng hp, cn trng b tiu dit khi chng tn ph cy trng. Cht khng sinh c lin quan n: Thuc tnh hnh thi, c tnh sinh l, c tnh ha sinh, c th l tng hp ca 3 c tnh trn. * C ch trnh: Cy trng trnh su cng nh trnh bnh, khng phi l tnh khng thc. Tuy vy n c nh hng nh l tnh khng thc trong vic bo v cy trng khi s ph hoi ca cn trng v su hi. Tt c cc trng hp trnh su thc cht l hin tng cy ch khng trong giai on khi cn trng ang nh cao pht trin. V d: ging bng ngn ngy c th trnh c s ph hi ca su ph hoi vo cui v trng. C trng hp, s pht trin trong iu kin khng ph hp cho s pht trin ca su v cn trng. V d: sn xut khoai ty ht l trnh s ph hi ca rp [2]. * C ch chng chu: Tnh chng chu su cng nh tnh chng chu bnh. Ging chng chu su cng b tn cng mc ging nh ging nhim nhng ging khng vn cho nng sut cao hn. Trong mt s trng hp, ging khng phc hi nhanh hn sau khi b cn trng tn ph. Tnh khng cn trng th hin lin quan n cc thuc tnh hnh thi, sinh l hoc ha sinh ca k ch. 2.1.3. Bn cht di truyn Tnh khng su c th do n gene iu khin vi mt s lng ln cc c th c hiu lc, c th do nhiu gene iu khin c tc dng cng tnh hoc do
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gene t bo cht. Da trn c s ny, ngi ta s dng k thut tch DNA, tch cc gene khng su qu him v thc hin bin np cc gene ny vo cc ging cy trng bng phng php lai t bo trn, phng php xung in hoc sng bn gene ... to ra cc cy trng c kh nng khng su. y chnh l c s khoa hc ca phng php chuyn gen to ra cc cy trng khng su. 2.1.4. Nhng kh khn ca vic to ging khng su + Trong mt s trng hp chn c ging khng su ny li b cc su khc gy hi. iu ny c th do thuc tnh ca cy ch, gene khng tri mt loi su ny c th lin kt vi gene khng khng loi su khc. + Trong mt s trng hp chn to ging khng su lm gim cht lng sn phm, thm ch khng th s dng c + Trong trng hp gene khng su ch c cc loi hoang di. Vic chuyn gene bng phng php lai gia cc loi l rt kh khn v thng c s lin kt gia gene khng vi cc gene bt li khc. Thng ging hoang di mang gene khng li cho nng sut thp v cht lng km. + Sng lc tnh khng su l mt khu v cng kh khn trong cng tc to ging. i hi phi c s phi hp cht ch gia cc nh to ging vi cc nh cn trng hc, cc nh bnh hc, ha sinh, sinh l. + Chn to ging khng su l mt chng trnh di hn cn phi c u t thch ng v ti chnh v cc mt khc [2]. 2.2. Cc gen c chuyn vo cy to cy trng khng su 2.2.1. Cc ngun gene khng su Ngun gene khng su c th tm thy : + Cc ging cy trng + Ngun ti nguyn di truyn ca cc loi + cc loi hoang di c quan h huyt thng vi cy trng 2.2.2. Mt s gen khng su c chuyn vo cy trng
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Hin nay, to cc cy trng c kh nng khng su, gen c cc nh nghin cu tp chung chnh l gen Bt ca vi khun Bacillus thuringiensis. Vi khun ny sn xut ra cc protein kt tinh rt c i vi u trng ca cn trng nhng khng gy c i vi ng vt c xng sng. Tinh th protein c t ca vi khun ny khi vo mt cn trng s b cc enzym protease phn gii thnh cc on peptit, trong c on khi lng khong 68.000 dalton cha khong 1200 axit amin lm hng rut cn trng. Cc gen m ha c t ny nm trn plasmid ca vi khun, c gi chung l Cry (crystal). Nm 1911, nh nghin cu c, Ernst Berliner pht hin ra mt vi khun c c tnh i vi su nhy v t tn l Bacillus thuringiensis (Bt). Nm 1981, sau khi cc nh nghin cu M tch c gen u tin lp m cho mt c t ca Bt, ngi ta bit tch tng gen Bt a vo b gen n bi ca t bo thc vt, nhm to ra cy t t bo ny, bng cch thu c nhng loi cy chuyn gen th h 1, tit c t chng tng loi cn trng. T , mt s phng th nghim to ra cc ging cha gene Bt (cry1Ab, 1Ac 1Aa, 2A, 1B, hay kt hp cc gene ny) khng li cc cn trng thuc b cnh vy. Nm 1978, cy thuc l v c chua chuyn gen Bt ra i, nm 1990 l ng v nm 1993 l la chuyn gen Bt ... Cho n nay, trn th gii hin c ti 50 triu hcta trng cy chuyn gen loi ny, gip lm gim thuc tr su mt cch ng k.

Hnh 2.1. Vi khun BT v Gen c t Bt 2.3. Cc phng php chuyn gen

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C hai hnh thc chuyn gen ch yu l chuyn gen trc tip v chuyn gen gin tip. 2.3.1. Cc phng php chuyn gen gin tip 2.3.1.1. Chuyn gen gin tip nh Agrobacterium tumefaciens * Nguyn l chung: S dng vi sinh vt t Agrobacterium (l vi khun Gram m) chuyn gen thc vt l phng php hiu qu v ph bin nht hin nay nh vo kh nng gn gen ngoi lai vo h gen thc vt mt cch chnh xc v n nh. S chuyn gen thc vt dng A. tumefaciens da trn nguyn l nh hnh sau:

Hnh 2.2. S chuyn gen thc vt dng A. tumefaciens Theo , Agrobacterium mang plasmid gy khi u thc vt (tumour inducing plasmid- Ti plasmid), plasmid ny cha cc gen vir v vng gen cn chuyn (T DNA). Gen quan tm c chn vo vng T-DNA. Cc t bo tn thng thc vt tit ra hp cht bo v, hp cht ny kch thch s biu hin gen vir Agrobacterium. Vir protein to ra T-strand t vng T-DNA trn Tiplasmid. Sau vi khun gn vo t bo thc vt, T-strand v mt s protein vir chuyn vo t bo thc vt qua knh vn chuyn. Trong t bo thc vt, cc protein vir tng tc vi T-strand to ra phc h (T- complex). Phc h ny vo

16

nhn t bo thc vt, T-DNA chn vo b gen thc vt v biu hin gen quan tm C th nh sau: Phng php ny s dng Ti plasmid ca A.tumefaciens hoc Ti plasmid ca Arhizogenes lm vect a ADN vo t bo. Ti plamid gm hai thnh phn: T- DNA v vng Vir. T- DNA c th chuyn t Agrobacterium vo t bo thc vt, chnh nh vy m c th gn gen mun chuyn vo T- DNA a vo t bo. T- DNA cha gen iu ho sinh trng cc b (auxin, cytokinie), gen tng hp cc cht Opine v gen gy khi u. on cui c 25 cp baz lp li, bt k on no trong vng ny cng c th xm nhp vo phn t ADN thc vt. Trong plasmid v tr ca T- DNA c gii hn bi b tri v b phi. Vng Vir ph trch kh nng ly nhim. Chng gm 6 nhm t VirA n VirG v VirE c tc dng lm tng tn s bin np. thit k vector dng trong bin np: trc ht l ct b gen iu ha sinh trng cc b, sau gn thm gen ch th (khng thuc hoc cht dit c) cng vi on iu khin ph hp chn cc th bin np v cui cng gn gen cn bin np. Cc plasmid ca Agrobacterium c s dng trong cng ngh gen thc vt gm c 2 loi: vector lin hp v vector nh th. - Vector lin hp (Cointegrated vector): Vector lin hp da trn s ti t hp ca 2 plasmid. Mt vector lin hp gm c: v tr ghp cc gen quan tm, thng v tr ny c ngun gc t plasmid ca vi khun E.coli. Gen ch th khng sinh gip cho chn lc trong vi khun E.coli, Agrobacterium v gen chn lc hot ng c t bo thc vt. Nh vy vector lin hp c hnh thnh t mt plasmid mang trnh t ADN mong mun v plasmid kia c cha vng vir gen v vng b lp li ca T-ADN. Sau ti t hp, mt vector lin hp c to ra v dng cho chuyn gen vo thc vt. - Vector nh th (Binary vector): Vector nh th l h thng dng 2 plasmid ring bit: mt cung cp T-ADN mt c tnh gy khi u, plasmid kia l Ti
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plasmid c kh nng thm nhp vo t bo thc vt (mang cc gen vir). Plasmid th nht mang gen chn lc thc vt v gen s c ghp vo b gen thc vt. Khi plasmid ny c a vo chng Agrobacterium c cha Ti plasmid, nhng sn phm mang gen vir c th a T-AND vo t bo thc vt, mc d T-ADN nm trn phn t ADN khc [1]. * u im - Gen t b o thi K thut chuyn gen thc vt & ng dng - S lng bn sao t hn. Do trnh c hin tng c ch ln nhau v cm lng ln nhau. - Tn ti bn vng trong c th thc vt do s ph thuc cht ch vo h thng protein Vir, cn nhng phng php khc gen mc tiu c ti t hp chuyn bit nh hai trnh t IS hai u nhng d dng b tch ra ngay sau . * Nhc im - C ph tn cng gii hn - Gy khi u cho c cy kha t v cy hai l mm nhng li khng gy khi u cho cy mt l mm (do cy mt l mm l cy thuc nhm tin ho nht, n tch ly nhiu c ch khng bnh hn cy hai l mm nh khi b thng cc t bo c xu hng ha g ch khng phn chia mnh ti to hoc tip hp cht phenol nh cy hai l mm...) 2.3.1.2. Chuyn gen gin tip nh virus Ngoi vic s dng vi khun, ngi ta cn s dng virus lm vector chuyn gen vo cy trng. Chuyn gen nh virus c th thun li do virus d xm nhp v ly lan trong c th thc vt v ng vt ng thi virus c th mang on ADN ln hn nhiu so vi kh nng ca plasmid. Tuy nhin, virus lm vector chuyn gen cn phi c cc tiu chun sau: - H gen ca virus phi l ADN

18

- Virus c kh nng di chuyn t t bo ny sang t bo khc qua cc l vch t bo - C kh nng mang c on ADN (gen) mi, sau chuyn gen ny vo t bo thc vt - C ph k ch rng (trn nhiu loi cy) - Khng gy tc hi ng k cho thc vt i chiu cc tiu chun trn, hin nay c hai loi virus c s dng lm vector chuyn gen l caulimovirus v geminivirus. Tuy nhin, vic s dng virus chuyn gen thc vt cn t c s dng v ADN virus kh ghp ni vi h gen ca thc vt. 2.3.2. Cc phng php chuyn gen trc tip 2.3.2.1. Chuyn gen bng sng bn gen (gene gun)

Hnh 2.3. Sng bn gen v s nguyn l hot ng ca sng bn gen * Nguyn l

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Ngm nhng vin n nh (vi n) bng vng hoc tungsten c kch thc cc nh, ng knh khong 0,5 - 1,5 m vi dung dch c cha on ADN ngoi lai cn chuyn vo t bo thc vt. Cc vi n ny c lm kh trn mt a kim loi mng c kch thc 0,5 - 0,9 cm. a kim loi ny c gn vo u mt vin n ln (macroprojectile) bng nha, hoc vt liu nh. Vin n ln c kch thc va kht u nng sng bn gen. Khi bn, p sut hi s y vin n i vi tc cao. Ti u nng sng, vin n ln s b cn li bi mt li thp mn, cn cc vin n nh (vi n) vn tip tc di chuyn vi tc ln ti 1300 m/giy n i tng bn ri ri xuyn vo t bo. Sau khi bn, tch cc m, t bo v nui cy invitro ti sinh cy. Cc gen cn chuyn c th ti t hp vo h gen ca cy, t to thnh cy chuyn gen. Ch c mt t l no ca t bo mang gen chuyn do vy cn phi chn lc. Ngi ta thng dng kh nn l helium p lc cao bn gen. * u im: - Thao tc d dng, bn mt ln c nhiu t bo - Nguyn liu bn a dng (ht phn, t bo nui cy, t bo m ha v m phn sinh) * Nhc im: Tn s bin np n nh thp v th theo Potrykus th u vit ca phng php ny l nghin cu cc gen tm thi. Tuy nhin, nhng nm gn y phng php ny thnh cng trn la, ma, u , bng khng nh tnh u vit ca phng php ny. 2.3.2.2. Chuyn gen bng xung in (electroporation) * Nguyn l: Trong cng ngh di truyn thc vt, ngi ta s dng phng php xung in chuyn gen vo protoplast thc vt. in th cao, trong thi gian ngn c th to ra cc l trn mng t bo trn (protoplast) lm cho ADN bn ngoi

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mi trng c th xm nhp vo bn trong t bo. Ngi ta chun b protoplast vi cc plasmid ti t hp mang gen mong mun cn chuyn vo thc vt. Dng thit b xung in to in th cao (200 400 V/cm) trong khong thi gian 4 - 5 phn nghn giy. Kt qu lm mng t bo trn xut hin cc l thng tm thi gip cho plasmid c th xm nhp v gn vo h gen ca thc vt. Qu trnh c thc hin trong cuvett chuyn dng. Sau qu trnh xung in, em protoplast nui trong mi trng nui cy thch hp, mi trng chn lc tch cc protoplast c bin np. Tip theo l nui cy invitro, ti sinh cy v chn lc cy chuyn gen. i vi protoplast phng php xung in c kt qu khch l nhng cha chng minh chc chn cho s bin np. Hn na, vic nui cy protoplast mt s loi cy vn cn gp kh khn [1]. 2.3.2.3. Chuyn gen bng vi tim (microinjection) Phng php ny s dng vi kim tim v knh hin vi a ADN nhng t bo nht nh, nhm to ra cc dng bin np t protoplast v cy bin np khm t phi pht trin t ht phn. * u im: - C th ti u lng ADN a vo t bo - Quyt nh c a ADN vo loi t bo no - C th a mt cch chnh xc thm ch vo tn nhn v c th quan st c - Cc t bo c cu trc nh nh ht phn v t bo tin phi mc d hn ch v s lng cng c th tim chnh xc - C th nui ring l cc t bo vi tim v bin np c vo mi ging cy * Nhc im: - Mi ln tim ch c mt pht tim v ch vi mt t bo - Thao tc trong khi lm i hi chnh xc cao 2.3.2.4. Chuyn gen nh k thut siu m
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K thut siu m dng chuyn gen vo t bo trn protoplast. Sau khi to protoplast, tin hnh trn protoplast vi plasmid ti t hp mang gen mong mun to hn hp dng huyn ph. Cm u siu m ca my pht siu m ngp trong hn hp huyn ph su khong 3mm. Cho my pht siu m vi tn s 20KHz theo tng nhp ngn, mi nhp khong 100 mili giy. S nhp khong t 6 - 9 nhp vi tng thi gian tc ng t 600 - 900 mili giy. Sau khi siu m, em protoplast nui trong cc mi trng thch hp, chn lc tch cc protoplast c chuyn gen. Nui cy invitro ti sinh cy. Chn lc cy v a ra trng mi trng ngoi [1]. 2.3.2.5. Chuyn gen bng phng php ha hc Chuyn gen bng phng php ha hc l phng php chuyn gen vo t bo protoplast nh cc cht ha hc nh polyethylen glycol (PEG). Khi c mt PEG, mng ca protoplast b thay i v protoplast c th thu nhn ADN ngoi lai vo bn trong t bo.Phng php chuyn gen bng ha cht c th p dng vi nhiu loi thc vt nhng kh nng chuyn gen vi tn s chuyn gen rt thp. Tuy nhin, vi kh nng to ra s lng ln protoplast, do vy khc phc c hn ch ca phng php ny [1]. 2.3.2.6. Chuyn gen trc tip qua ng phn (pollen tube) Phng php chuyn gen qua ng phn l phng php chuyn gen khng qua nui cy m invitro. Nguyn tc ca phng php ny l ADN ngoi lai chuyn vo cy theo u ng phn, chui vo bu nhy ci. Thi gian chuyn gen vo lc ht phn mc qua vi nhy v lc bt u a tinh t vo th tinh, tt nht l s chuyn gen xy ra ng khi qu trnh th tinh non v cho t bo hp t cha phn chia. Nh vy, s chuyn gen ch xy ra mt t bo sinh dc ci duy nht v khi ti sinh cy s khng hnh thnh th khm. - Sau thi gian hoa n 1- 2 gi, ct 2/3 hoc 3/4 phn trn ca hoa la

22

- Sau khi ct hoa, dng ng mao qun nh c ng knh 0,2mm a dung dch ADN ti t hp mang gen mong mun vo u ng nhy b ct (nng ADN ti t hp khong 50 g/ml) - Bao bng la li, ch la chn thu hi. - Phn tch v xc nh kt qu chuyn gen th h sau. 2.4. Thnh tu to cc cy trng khng su bng k thut chuyn gen Hin nay, trn th gii c ti 50 triu hcta trng cy chuyn gen khng su, Cc phng th nghim to ra nhiu ging cha gene Bt (cry1Ab, 1Ac 1Aa, 2A, 1B, hay kt hp cc gene ny) khng li cc cn trng thuc b cnh vy, gip lm gim thuc tr su mt cch ng k. Cc cy trng quan trng nht c chuyn gen Bt M l bng (46% s th nghim), u nnh (17%), c chua (16%), khoai ty (9%), ng (8%), thuc l (4%). Cc thnh tu tiu biu trong lnh vc ny c th k n nh: * Chuyn gen cry1Ab vo cy bng vi G.A. Khan v ctv. thuc Vin nghin cu bng vi ca Pakistan, chn thnh cng gen cry1Ab t vi khun sng trong t vo ging bng vi MNH-93 ca Pakistan thng qua k thut bn gen. Transgene hp nht vo trong genome v th hin qua xt nghim PCR v phn tch Dot blot. S lng Bt protein cng c khng nh, chng bin thin t 0 n 1,35% protein tng s. Transgenic plants c trng trong nh knh an ton sinh hc v trn ng rung nh gi tc ng trn ng, v kt qu cho thy cc dng transgenic th hin mc tnh khng su b cnh vy t 40 n 60%. Dng cc ging bng chuyn gen Bt kim sot hon ton su c qu (bollworm) v Spodoptera, c th tit kim c 1.161 triu USD tin thuc su [7].

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Hnh 2.5. Bng chuyn gen Bt v qu i chng. * Ng chuyn gen Bt: Ng chuyn gen hin ang c con ngi canh tc c tn l ng Bt c th t n to ra cc protein dit vi khun c trong t c tn l Bacillus theringiesis, v trit tiu cc loi su c bp M, nm 1996 ging ng chuyn gen Bt vi c tnh khng su cn gi c trng trn din tch 2 triu ha nhiu bang. Cng vi ng, c quan gim st an ton sinh hc APHIS thuc B Nng nghip M cng cho php a vo sn xut cc ging c chua, bng, u nnh, da, u ... chuyn gen khng su. Li ch thu c do tit kim thuc tr su i vi cy ng chuyn gen Bt c xc nh khong 1094 triu USD.

Hnh 2.6. Cy Ng chuyn gen Bt v ging i chng * Chuyn gen to la c kh nng khng su:

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Mt s phng th nghim to ra cc ging cha gene Bt (cry1Ab, 1Ac 1Aa, 2A, 1B, hay kt hp cc gene ny) khng li cc cn trng thuc b cnh vy. Cc th nghim u tin trn ng rung v ging la Bt do Trung Quc thc hin vo nm 1998. Tuy nhin cha c ging la Bt no c a ra thng mi ha cho n nay. Cui nm 2009 Trung Quc lm th tc cng nhn cho phng thch 2 ging la Bt Huahui No.1 v Bt Shanyou 63 a ra i tr vo 2012.

Hnh 2.7. La chuyn gen Bt MH63 v ging i chng Ging la chuyn np gen Bt MH63 (T) vn pht trin rt tt trong khi ging MH63 bnh thng (C) b nhim su cun l nng. K bn l ging Bt MH63 (T) khng su c thn trong khi ging MH63 bnh thng (C) b nhim rt nng. Bng 2.1. Cc loi cy trng chuyn gen khng su c thng mi ha (Ngun d liu c bn: C s d liu v GM ca Agbios - 2009). Cy trng Bng Khoai ty Ng Hng Dng S ging chuyn gen 4 14 3 2 Nc s dng Achentina, xtralia, Trung quc, Canada, Nht Bn, Mxic, Philippine, Nam Phi, M. xtralia, Canada, Philippine, M. Achentina, xtralia, Canada, EU, Nht Bn, H Lan, Philippine, nam Phi, Thu s, M. Canada, Nht bn, Philippine, Hoa k
25

C tm

n , Braxin, Canada, Nht Bn, Mxic, Philippine, Nam phi, Thu s, Anh quc, M.

CHNG 3. QUY TRNH TO CY THUC L CHUYN GEN KHNG SU VIP3A 3.1. Gii thiu chung
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Thuc l (Nicotiana tabacum L) l cy cng nghip ngn ngy c gi tr kinh t cao. Sn phm thu hoch l l ti, phi thng qua giai on ch bin c trng (sy, hong, phi) mi c th dng l lm nguyn liu sn xut thuc l. Ngoi ra cy thuc l cng c s dng lm nguyn liu sn xut nicotin, dng lm ch phm tr su, axit hu c, ht dng chit xut du thc vt. Nghin cu chn to ging thuc l theo cc hng khc nhau c quan tm cc quc gia ln nh M, Brazil, Trung Quc, Zimbawe Mt trong nhng hng chn to ging c quan tm hin nay l ng dng cng ngh bin i di truyn chn to ging khng su bnh hi chnh nh khang cac bnh virus khm l thuc l (TMV,CMV), virus xon l thuc l (TLCV) hoc khng su (su xanh, su xm...). Ngoi khuynh hng to tnh khng su ca cy bng gen cry m ha cho s to ni c t trong pha sinh bo t ca vi khun Bacillus thuringensis (Bt), gn y cc nh khoa hc pht hin cc gen vip (vegetative insecticidal protein - cc protein sinh dng dit cn trng) m ha cho cc protein trong pha dinh dng ca chu trnh pht trin vi khun Bt v Bacillus cereus (Bc), chit tch, nhn bn, xc nh trnh t v th hot tnh sinh hc. Kt qu cho thy cc protein Vip c hot lc v ph tc dng dit cn trng cao hn rt nhiu ln cc protein do gen cry m ha. Gen vip3 to protein c hot tnh khng su xm cao gp 260 ln so vi protein cry IA v c ph hot ng rng nh dit c su xanh hi ng, su xanh hi thuc l [6]. Vit Nam, cy thuc l l mt trong nhng cy cng nghip ngn ngy c gi tr kinh t cao. Vn su bnh hi trong sn xut l mt trong nhng nguyn nhn gy gim nng sut v cht lng ca nguyn liu, do vy ng dng cng ngh bin i di truyn chn to ging khng su bnh hi l mt trong nhng hng i gii quyt vn . Vin Cng ngh Sinh hc bc u thnh cng trong vic chuyn gen vip3A vo cy thuc l, tao ging cy thuc l chuyn gen khang su.

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3.2. Quy trnh to cy thuc l chuyn gen khng su vip3A Cc nh khoa hc ca Vin Cng ngh Sinh hc thc hin thnh cng vic to cy thuc l chuyn gen khng su vip3A theo quy trnh sau:
Thit k mi PCR nhn on gen vip3A v tinh sch sn phm PCR La chn vector tch dng

Ghp ni on gen vip3A vo vector tch dng

Bin np plasmit ti t hp vo t bo kh bin E.coli DH5

Chn lc plasmit ti t hp

To vector chuyn gen mang gen vip3A

Bin np vector mang gen vip3A vo t bo A. tumerfaciens bng xung in

2
Chuyn cu trc mang gen vip3A vo cy thuc l thng qua vi khun A. tumefaciens

Nui cy m t bo

3
nh gi v mt hnh thi Kim tra bng sinh hc phn t

Hnh 3.1. Quy trnh to cy thuc l chuyn gen khng su vip3A

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Qu trnh to cy thuc l chuyn gen khng su vip3A c chia ra thnh 3 giai on chnh nh sau: (1). Thit k vector pBI121 mang gen vip3A (2). Kim tra v theo di kt qu chuyn gen (3). Chuyn gen vip3A vo cy thuc l. Vi quy trnh nh trn (hnh 3.1) cc nh khoa hc ca Vin Cng ngh Sinh hc - thuc Vin Khoa hc v Cng ngh Vit Nam, to thnh cng ging cy thuc l chuyn gen khng su vip3A. Thnh cng ny khng ch c ngha v mt kinh t m n cn mang mt ngha khoa hc v cng to ln, bi ngoi vic to thnh cng cy thuc l chuyn gen khng su a vo sn xut, tit kim kinh ph trong vic s dng thuc tr su ha hc, bo v mi trng, cy thuc l chuyn gen cn c coi nh cy m hnh, t cc nh khoa hc tip tc pht trin cc nghin cu chuyn gen, to ra nhiu ging mi c kh nng khng su cng nh cc cy chuyn gen mang cc tnh trng c li khc nh: kh nng khng virus, khng bnh, kh nng chng chu cc iu kin bt li nh: chu hn, chu nng, chu lnh hay cc tnh trng cm quang, tnh trng quy nh gi tr dinh dng ca cy

KT LUN Cng ngh to cy trng chuyn gen ni chung v cng ngh to ging cy trng chuyn gen khng su ni ring ngy cng pht trin. Cc nh khoa hc
29

 v ang to ra hng trm ging cy trng chuyn gen khc nhau mang nhiu c tnh qu C th ni, k thut chuyn gen to ra cc cy trng c kh nng khng su mang li li ch v cng to ln khng ch i vi con ngi m cn c i vi mi trng sinh thi. Tuy nhin vn cy trng chuyn gen (GMOsGenetically Modified Organisms) cn l vn tranh ci, thm ch cn gp phi s phn i gay gt t nhiu nh khoa hc. Sn phm chuyn gen b nhiu ngi tiu dng lo ngi. Cc sn phm chuyn gen phi c dn nhn nhng ngi tiu dng la chn. Nhiu nc cha cho php nhp khu cc sn phm chuyn gen cng nh cy trng chuyn gen. May mn l, cc nh Khoa hc khng nh vic s dng cy trng c gen khng su bnh s khng gy nhim mi trng, khng gy hi n qun th thin ch, gim chi ph sn xut, tng nng sut cy trng, tng thu nhp cho ngi sn xut v to ra sn phm an ton cho ngi tiu dng. y chnh l hng i ng n hng ti m bo pht trin nng nghip bn vng.

TI LIU THAM KHO TI LIU TING VIT

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1. Trn Quc Dung, Nguyn Hong Lc, Trn Th L, Cng ngh chuyn gen (ng vt Thc vt), Nxb i hc Hu, 2006. 2. Hong Trng Phn, Trng Th Bch Phng, C s di truyn chn ging thc vt, Nxb H Hu, 2008. 3. L Duy Thnh, C s di truyn chn ging thc vt, Nxb Khoa hc v K thut H Ni, 2000. 4. Nguyn Th Hi Yn, Bi ging Cng ngh sinh hc, Trng i hc Khoa Hc, H Thi Nguyn, 2010. 5. http://agrobiotech.gov.vn/tabid/38/MenuID/1/language/vi-VN/Default.aspx. TI LIU TING ANH 6. Singh, D.P. and A. Singh, Disease and Insect Resistance in plants. Science Publishers, 2005. 7. G. A. Khan, A. Bakhsh, S. Riazuddin and T. Husnain, Introduction of cry1Ab gene into cotton (Gossypium hirsutum) enhances resistance against Lepidopteran pest (Helicoverpa armigera), Spanish Journal of Agricultural Research 2011 9(1), 296-302.

MC LC
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