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Xây dựng hệ thống tái sinh vitro cây ngô Zea mays L. và bước đầu ứng dụng trong chuyển nạp Gen gạo protein giàu sắt nhờ vi khuẩn Agrobacterium tumefaciens

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Ngô (Zea mays L. là cây lương thực quan trọng trên thế giới cũng như ở nước ta. Ngoài việc dùng làm lương thực cho con người, ngô còn được dùng làm thức ăn trong chăn nuôi gia súc, gia cầm, làm nguyên liệu cho công nghiệp sản xuất rượu, cồn, tinh bột... Ngô là nguồn xuất khẩu thu ngoại tệ lớn đối với một số nước như Mỹ, Pháp Argentina, Trung Quốc... Trong nghiên cứu cấy mô và chuyển gen, các nhà khoa học thường dùng vật liêu phôi non (10-20 ngày sau thụ tinh) để nghiên cứu do có khả năng đáp ứng cao với nuôi cấy (tỷ lệ tạo mô sẹo sinh phôi non và cần nhiều thời gian, công sức cho công việc tách lấy phôi....

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Nội dung Text: Xây dựng hệ thống tái sinh vitro cây ngô Zea mays L. và bước đầu ứng dụng trong chuyển nạp Gen gạo protein giàu sắt nhờ vi khuẩn Agrobacterium tumefaciens

Tph^V: CdNG NGHg GEN THgC VAT 339<br /> <br /> <br /> <br /> <br /> XAY D V N G H E T H O N G TAI SINH in vitro CAY NGO Zea<br /> ^mays L. VA BlTOC DAU iTNG DUNG TRONG CHUYEN<br /> [p..<br /> NAP GEN TAO protein GlAU SAT NHd VI K H U A N<br /> Agrobacterium tumefaciens :/<br /> <br /> Nguyen Thi Phirong Nam, Le Tan Dure, Pham Du-c Tri, Nguyin Hiru Tam<br /> Nguyen Hihi Ho, Nguyen Van Uyen<br /> Phdng Cdng nghe gen thyc vat, Vien Sinh hgc Nhiet ddi<br /> <br /> <br /> MdDAu<br /> Ngd (Zea mays L.) Id cdy luong thyc quan frgng fren thi gidi ciing nhu d nude ta.<br /> Ngoai viec dimg lam luong thyc cho con ngudi, ngd cdn dugc dimg lam thiic an frong<br /> chdn nudi gia siic, gia cam, ldm nguyen lieu cho cdng nghiep sdn xuat mgu, cdn, tinh<br /> bpt... Ngd Id ngudn xuat khdu thu ngoai te Idn ddi vdi mgt sd nude nhu My, Phdp,<br /> /Argentina, Tnmg Quoc...<br /> f- Trong nghien ciiu nudi cdy md va chuyen gen, cdc nhd khoa hgc thudng diing vat<br /> lieu phdi non (10-20 ngdy sau thu tinh) [3,4,5,6,16,18,22] dl nghien ciiu do cd khd ndng<br /> dap ling cao vdi nudi cdy (ty le tao md seo sinh phdi vd tdi sinh cao). Tuy nhien, thudng<br /> khd chii ddng frong viec thu phdi non vd can nhieu thdi gian, cdng sire cho cdng viec<br /> tach Idy phdi. De chii ddng hem trong nghien cim, chiing tdi dimg ddt thdn (nodal<br /> section) vd choi dinh cdy in vitro (cd ngudn gdc tir hat moi tmdng thdnh) nhu nguyen<br /> lieu diing cho nghien cim nudi cay md vd de tao md te bdo thich hgp diing chuyen nap.<br /> ^ -^. Trtrdc day, chgn t^o gidng ngd dugc thyc hi?n thdng qua phucmg phdp lai tao, ^dy<br /> dpt bien. Hien nay, cdng nghe sinh hgc da cd nhieu ddng gdp quan frgng frong tao gidng<br /> c\i thi la nhd cdng nghe gen nhilu pidng ngd chuyen gen vdi ddc tmh quy da dugc tao<br /> ra vd dugc thuong mai hod nhu ^idng ngd khang sau, khang thudc trir cd... Theo cac<br /> nhd nghien cim, cdng nghe chuyen nap gen ngay cang cd vi tri quan frgng fron^ cdng<br /> tac gidng ngd ddc biet frong tinh frang vdn gen ty nhien cay ngd cdn nhieu ban che.<br /> Sy thilu hut dinh dudng sdt la mdt vdn de gay dnh hudng nghiem frgng den khodng<br /> 30 % ddn sd thi gidi. Sy thieu mdu do thieu hut sat cd dnh hudng xdu hoac rat xau den<br /> tre em, thai phu vd phu nir sau khi sinh. C) nude ta, theo dieu fra tai cdc vimg ngheo ty le<br /> tre thilu mdu, thilu sdt len tdi horn 70%, chii yeu do bira dn thieu chdt dinh dudng va<br /> ngheo thdnh phan; sy thilu sdt cd tac ddng xau den sy phdt frien ve the chat vd tri tue<br /> cua fre. Mac dii viec bd sung sat vao thyc pham hoac khuyen cao sir dung thudc da td ra<br /> c6 hieu qud nhdm kilm sodt nan tiiieu hut sdt nhung cdc bien phdp ndi tren khd cd the<br /> dp dung nhanh chdng tai cdc nude cdn ngheo nan lac hdu vi chi phi phdt sinh cao va sd<br /> lugng cdc chuong trinh chdm sdc sire khoe ban dau cdn ban che. Mdt sd cay ho Dau,<br /> cay cdi spinach... dugc bilt chiia ham lugng sat cao nhung ciing chira nhieu acid oxalic<br /> va cac hgp chdt cd nhilu acid phytic (phytate-like substances) gay ban che hap tiiu sat.<br /> 390 HQi nghi KHOA HOC VA C O N G NGHg 2007<br /> <br /> <br /> Ferritin - protein dy trii sdt dugc phdn bd rdng rai frong ddng vat, tiiyc vat vd vi<br /> khudn. Ferritin cua thyc vat gom 24 tiiu phdn (subunit) cd the chira tii 4.000 - 4.500<br /> nguyen tir sdt d khoang trung tdm phdn tir (central cavity) [10,11]. Cac doan cDNA<br /> ferritin thyc vat da dugc ddng hda de nghien ciiu tir nhieu ngudn khac nhau nhu fry<br /> dudi la mam dau nanh Glycine max [10], hat non dau Pisum sativum (pea), la non dau<br /> Vigna unguiculata, hat dau Phaseolus vulgaris (French bean)... Ferritin cd hai nhiem<br /> vu chinh trong te bao sdng; mdt Id, cung cap sat de tdng hgp cac protein chiia sdt nhu<br /> ferredoxin va cac cytochrome, hai la, chdng cac tdc hai gdy ra bdi cac gdc tu do tao ra<br /> bdi sy tuong tac sat / dioxygen. Cdc nghien ciiu gdn ddy cho thdy ferritin cd the dugc sii<br /> dung de xir ly cac tmdng hgp benh thieu mdu d chudt. Cdc phat hien fren cho phep cac<br /> nhd khoa hgc dua ra cac nghien ciiu, bang con dudng cdng nghe di tmyen, tao ra cdc<br /> cay ngii cdc chuyen gen gidu sat nham gdp phan nhdt dinh vdo viec gidi quyet nan thieu<br /> ' 9 9<br /> <br /> <br /> hut sat d con ngudi vd cho den nay, ngudi ta da tao ra dugc mgt sd ddng liia indica va<br /> japonica vdi ham lugng sdt cao hon gdp nhieu lan [11].<br /> Nham tiep can hudng tao gidng dimg phuong phap cdng nghe sinh hgc, d nghien<br /> cim nay, chiing tdi neu ndi dung nghien ciai xay dyng h? thdng tdi sinh cay tir md ddt<br /> than va chdi dinh va budc ddu iing dung trong chuyen nap gen tao ferritin nhd vi khudn<br /> Agrobacterium tumefaciens vdi hy vgng cdi thien ham lugng sat d cay ngd.<br /> <br /> VAT LIEU VA PHU'ONG P H A P<br /> Giong ngo:<br /> Sir dung gidng ngd Nep dia phuong d tinh Tiln Giang.<br /> Phuang phdp:<br /> Khir triing hat tuoi<br /> Bdc bd la bao ben ngoai qua ngd tuoi de Id hat ngd ben frong. Tdch lay hat bdng tay<br /> va khir triing hat bang hypochlorite Na 50% (v/v) frong 20 phut. Rira sach bdng nude<br /> cat nhieu idn vd cay hat tren mdi tmdng MS khdng chdt sinh tmdng.<br /> Ky thuat tdch nudi choi dinh<br /> Diing dao md cdt bd phdn tren vd dudi dot than cdy ngd in vitro phdt triln tir hat<br /> (sau khoang 1/2 thang nudi cay). Tdch bd phan md bao phia tren ddt than dl Id dinh<br /> sinh trudng. Cung bdng dao md cdt Idy choi dinh 0,4 x 0,4mm vd dat vao mdi trudng<br /> nudi cay.<br /> Mdi truomg nudi cdy mo<br /> Mdi tmdng cdy hat sau khir trimg: MS (Murashige-Skoog, 1962) khdng chdt sinh<br /> trudng.<br /> Mdi tmdng nudi cay ddt thdn cay in vitro:<br /> 1. MS + 0,5mg/l 2,4-D + 1 mg/l BA<br /> 2. MS + 0,5mg^ 2,4-D + 2,2g/l picloram + \,2>%oj\ L - proline + 3,4mg/l nifrate bac<br /> pfijn V: CdNG NGHg GEN THgC VAT ^91<br /> <br /> <br /> Mditiordngnudi cdy chdi dinh (kich thudc 0,4 mm) than cdy in vitro: .. •..: i,;-^-,.,. .-..;-A»^i<br /> ,. 3.MS + 0-0,05mg/lBA ^[,'- - '.^ ,:-<br /> 4. MS + 0,05mg/l NAA+l-2mg/l BA .••",<br /> 5. MS + 0,5mg/l 2,4-D + lmg/1 BA<br /> Mdi tiidng tao vudn cdy tii cum chdi (mdi tiidng sd 6): MS + 0,lmg/l NAA + lmg/1 BA<br /> Mdi tmdng nudi cay: MS khdng chdt sinh tmdng.<br /> Dieu kien nudi cdy: 9 gid chieu sdng/ngdy, nhiet do 25-28°C.<br /> Dong vi khudn va moi tru&ng nuoi cay:<br /> ' • Sir dung ddng vi khuan Agrobacterium tumefaciens LBA 4404 chiia plasmid pITB-<br /> FERRITIN (do TS. Nguyen Hiiu Tdm thiet ke) mang gen tao protein gidu chdt sdt<br /> (ferritin) va gen khdng hygromycin hph.<br /> |ri' Mdi tmdng giir gidng vi khudn Id mdi tmdng LB cd 50mg/l kanamycin va 25mg/l<br /> stteptomycin. De nhdn gidng cho nghien ciiu chuyen gen, vi khudn dugc nudi cdy ldc<br /> qua dem frong mdi tmdng khodng AB (Chilton vd cs., 1974) cung chiia cdc khdng sinh<br /> nhu tren. Nhiet do nudi cdy: 25- 28°C.<br /> Quy trinh chuyen gen:<br /> Cdc cum md dang frong qud trinh tdi sinh (kich thudc 0,5 x 0,5mm) hinh thdnh tir<br /> dot thdn hoac/va chdi dinh tren mdi tmdng cd 0,5mg/l 2,4-D + lmg/1 BA (mdi tmdng<br /> so 1) dugc dimg lam vdt lieu chuyin gen.<br /> Tom tdt quy trinh chuyen gen:<br /> 1. Nudi cdc cum md fren mdi frudng sd 1frongtiidigian 2 ngdy.<br /> 2. Tao vlt tiiuong bdng cdch chdm ddu ludi dao mo nhgn vdo md, lay nhiem md vdi<br /> vi khudn trong thdi gian 10 phiit. Vdt mau ra, tham bdt dich vi khudn tiiira bdng<br /> "' gidy lgc.<br /> 3. Nudi chung md vdi vi khudn frong 2 ngdy (sir dung mdi frudng so 1 cd bd sung<br /> acetosyringone 100 pM).<br /> 4. Rira that sach vi khudn bdng nude cdt va bdiig dung dich cefotaxime 500mg/l (kit<br /> hgp ldc nhe).<br /> 5. Vdt mau ra, thdm rdo nude va cdy md fren mditimdngchgn lgc: mditimdngsd 1 cd<br /> nong do hygromycin 20mg/l hygromycin, 500mg/l cefotaxime. Thdi gian chgn lgc<br /> 15 ngdy.<br /> 6. Tilp tuc chgn lgc tl bao them 3 chu ky (15 ngdy / chu ky) fren mdi frudng chgn lgc<br /> nhu tren nhung cd 30mg/l hygromycin. . ,^ ,.,<br /> 7. Cay tmyen cdc md sang mdi tmdng sd 6 ciing bd sung 30mg/l hygromycin, thdi<br /> gian nudi 20-30 ngdy. ^, ..^^.^ ^ ^ .-:.ii^ t^ibf.<br /> 392 . H^i nqhj KHOA HQC VA C O N G NGHg 20^"<br /> <br /> <br /> <br /> Kiem tra cay chuyen gen:<br /> 1. Tach cay va cay cay sang mdi tiirdng MS khdng chdt sinh timdng cd 30mg/r<br /> hygromycin, thdi gian theo ddi sy sinh tmdng khodng 15 ngdy. , J<br /> 2. PCR: Sy hien dien ciia gen hph dang dugc kilm tra dimg cap mdi dac hieu: HI:'<br /> CGTCGTTCGAGAAGTTTC, H2: TACTTCTACACAGCCATC; chuong frinh<br /> nhiet: 94°C: 5 phiit; 35 chu ky (94°C: 1 phut, 62°C: 1 phut, 72°C: 1 phiit); 72°C: 5-<br /> phiit; khuech dai doan DNA 800 bp.<br /> <br /> KET QUA VA T H A O L U A N<br /> <br /> Ve nudi cdy md:<br /> - Nudi cdy dot thdn:<br /> 9<br /> <br /> <br /> Trong nghien ciiu nay, ndi chung cd sy hinh thdnh hai loai he thdng md khac nhau: \<br /> mdt la, md seo cd khd ndng sinh phdi, hinh thanh fren cd hai mdi trudng sd 1 va 2, v6i \<br /> ty le cao hon tren mdi tmdng sd 2 (60%); hai la, md seo mang phac the chdi (dang frong<br /> qua trinh tdi smh) chi hinh thanh fren mdi tmdng sd 1 vdi ty le khodng 52 %; va lihyi<br /> vay tren mdi tmdng sd 1 cd sy hinh thanh cd hai loai md ndi fren. Theo chiing tdi, c6 s^<br /> hinh thanh hai loai md ndi fren tren mdi tmdng sd 1 la do mdi tmdng nudi cay cd m^t<br /> lugng auxin 2,4-D nhat dinh thiic day sy tao md seo va cd cytokinin BA tao sy phan hoil<br /> chdi. Thdnh phan mdi tmdng sd 2 cd acid amin L - proline vd tac nhan lie che hinh<br /> thdnh ethylen la nitrate bac, theo mdt sd tac gia, can thiet cho sy hinh thanh md s?o c6j<br /> khd nang sinh phdi ddi vdi cay ngd vdi tan sd cao.<br /> <br /> - Nudi cdy chdi dinh:<br /> Tren mdi tiudng cd 0,05mg/l NAA va 0, lmg/1 BA, tdt cd cac choi dmh deu phat triral<br /> thdnh cdy don. Khi giir nguyen lugng NAA va cd lugng BA tang cao hon (lmg/1), cd sy hinhJ<br /> tiianh cum chdi vdi ty le khoang 35% tuong ty nhu he tiiong cum chdi d kit qua nghien ciiui<br /> tmdc day [3]. Tren mdi tmdng cd 0,5mg/l 2,4-D vd lmg/1 BA, khodng fren 50% sd chdi dinh<br /> nudi cay hinhtiianhcd hai loai md nhufrongtmdng hgp nudi cdy ddt tiian. \<br /> .1<br /> Trong nghien cuu nudi^ cdy md cay ngd, fren thi gidi ciing nhu frong nude, cd mQt I<br /> so vat lieu phd bien dimg dl tao md seo la phdi non [4,6,18], md thuan (scutellum) hoSc<br /> ddt thdn phdi tiidng tiianh [14,20]... Phdi non dugc sir dung nhilu do kha ndng dap<br /> img cao ddi vdi sy hinh thanh md seo phdi hod ciing nhu tai sinh. Tuy nhien, viec thu<br /> phdi non phu tiiudc vao tiidi vu, gay frd ngai cho nghien cuu. Do vay, frong nghien ciiu<br /> nay, tiieo hudng cua cac tac gid Sidorov va cs., 2006 [20], chung tdi sir dung ddt th3n<br /> cay in vitro lam nguyen lieu tao md cdn thilt. Viec dimg nguyen lieu nay mang tinh chij<br /> ddng cao va nhan thay md ciing cd dap ung kha tot vdi nudi cdy.<br /> <br /> Ve chuyen gen:<br /> Qua sir dun;<br /> dung md seo dang trong qua trinh tai sinh dk chuyin nap gen bdng p h ^ ^<br /> phap dimg vi khuan Agrobacterium tumefaciens, ch'ing tdi budc ddu r i a n dugc^2<br /> PhAn V: CdNG NGHg GEN THgC VAT<br /> 393<br /> <br /> md cd khd ndng sinh tmdng tot fren mdi tmdng cd 30mg/l hygromycin. Thyc tl nghien<br /> ciiu cua chiing tdi cho thdy cd vdn de khd khan frong viec sir dung md seo phdi hod dl<br /> nghien ciiu chuyen gen dimg vi khudn la md seo sau khi xir ly vi khudn, nudi chung, rira<br /> va cay chgn lgc thudng trd nen nhao vd chet vdi ty le rdt Idn. Chiing tdi dang tilp can<br /> hudng sir dung phuong phdp ban gen nhdm loai bd mdt sd yeu td cd thi gdy dnh hudng<br /> xdu fren md nhu thao tdc co hgc xir ly vi khudn, rira, thdm khd md... vd ciing cd thi sy<br /> khd khdn tren ddy la do yeu td gidng. Khd ndng tao md seo phdi hod vdi tdn sd thap<br /> cQng nhu duy tri nd frong thdi gian ddi ma khdng gidm khd nang tdi sinh da khiln mdt<br /> s6 tdc gid tren the gidi tiep can phuong phdp diing chdi dinh (intact tisue) dl nghien ciiu<br /> chuyen gen [12,13]. Diem thuan Igi ciia chiing tdi khi sii dung he thdng md seo dang<br /> frong qud trinh tdi sinh Id loai md ndy khdng gidm khd ndng tai sinh frong thdi gian dai<br /> cdy tinyen. Dugc biet den nay, da cd nhilu phuang phdp chuyin gen dugc dp dung tren<br /> doi tugng cdy ngd nhu bdn gen [16,18,21,22], dimg vi khudn Agrobacterium<br /> tumefaciens [12,13,15, 20], dimg xung dien [5,7,9,17,19] nhung hien nay chi hai<br /> phuong phap ban gen vd diing vi khuan Id cdn dugc dp dung nhieu.<br /> ' "itJ> Jl —y' ><br /> rf^sw-ri •<br /> I I.<br /> <br /> <br /> <br /> <br /> A,<br /> 00iSi^ ^'n<br /> 'tgfJijT JJjByJyjgttl<br /> <br /> <br /> <br /> <br /> : Hinh 1: Md s§o cd khd nan^ sinh phoi dung chuy4n gen. 2: Cum md khdng hygromycin.<br /> 3: Thip GUS du'ang tinh ddi vdi mo chdi. 4: K§t qua PCR du'cng tinh gen hph vdi bang<br /> ONA 800 bp. 5: K i t qud PCR du'vng tinh gen tao ferritin vdi bdng DNA 700 bp<br /> <br /> <br /> <br /> MfcTLUAN<br /> Qua nghien ciiu nudi cay ddt than, chdi dinh vd chuyen nap gen fren gidng ngd Nep<br /> tien Giang, chiing tdi cd mdt sd ket luan sau:<br /> Trong sd cdc mdi tmdng khdo sat, mdi tmdng thich hgp tao loai md vira mang khd<br /> ndng tdi sinh cao vira cd the dugc dimg cho nghien ciiu chuyen nap gen la mdi<br /> tmdng MS cd 0,5mg/l 2,4-D vd lmg/1 BA. Khd ndng tdi sinh cay ddi vdi loai md<br /> nay cd thi dugc duy tri frong thdi gian dai.<br /> • Chdi dinh qua nudi cay cd the phdt frien theo hudng tao dang md s?o mang phac<br /> thi choi hodc theo hudng tao cum chdi tuy theo loai chdt dieu hod sinh tmdng vd<br /> ndng dg dugc sir dung.<br /> • Qua chuyin gen nhd vi khuan Agrobacterium tumefaciens mang gen hph vd gen tao<br /> protein giau sdt (ferritm), budc dau chiing tdi da chgn dugc 2 ddng md ngd cd khd<br /> ndng sinh tmdng tot fren mdi tmdng cd 30mg/l hygromycin.<br /> L6ICAMON<br /> Cac tdc gid xin chdn thdnh cam on Chuong trinh Nghien ciiru co bdn da tdi frg cho<br /> j^ghien cim ndy.<br /> 394 HQi nghj KHOA HQC VA CdNG NGHg 2007<br /> <br /> <br /> <br /> TAI LIEU THAM K H A O<br /> <br /> 1. Trdn Thi Dung, Trdn Trung Hilu, 2000. Budc ddu chuyin gen vao cay bdp (Zea<br /> mays L.) nhd vi khuan Agrobacterium tumefaciens. Tap san KHKT Ndng Lam<br /> nghiep 2: 59-61, Tmdng DH. Ndng Lam T/p HCM. ' '"'^<br /> 2. Nguyen Hihi Hd, Le Tdn Dire, Nguyen Him Tam, Nguyen Vdn Uyen, 2003. Mgt s6<br /> nghien ciiu chuyen gen vao cay thudc Id (Nicotiana tabacum L.) va cay ngd (Zea<br /> mays L.). Tuyen tap Bao cao khoa hgc Hdi nghi Cdng nghe Sinh hgc todn qudc, Ha<br /> Ndi, 16-17/12/2003, fr. 1096-1101. NTid xudt ban Khoa hgc vd Kytiiuat,Hd Ndi. ,_^^<br /> 3. Nguyen Hiru Hd, Nguyen Vdn Uyen, Z. Y. Tkaschuc, L. V. Tkaschuc, 1995. Tao<br /> cum chdi cdy ngd (Zea mays L.) tir dinh sinh tmdng vd phdi hat nudi cay in vitro.<br /> Tap chi Khoa hgc vd Cdng nghe 4: 6-9. -&<br /> 4. Pham Thi Ly Thu, Le Huy Ham, Pham Minh Thoi, Do Nang Vinh, 2003. Nghien<br /> cim xay dyng he thdng tai sinh sir dung cho bien nap gen cay ngd. Tuyen tap Bdo<br /> cao khoa hgc Hdi nghi Cdng nghe Sinh hgc todn qudc, Hd Ndi, 16-17/12/2003, fr.<br /> 820-825. Nhd xuat ban Khoa hgc vd Ky tiiuat, Hd Ndi.<br /> 5. D'Halluui, K., E. Bonne, M. Bossut, M. De Beuckeleer, J. Leemans, 19<br /> Transgenic maize plants by tissue elecfroporation. 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Iron fortificatio<br /> of rice seed by the soybean ferritin gene. Nafrire Biotechnology 17: 282-286.<br /> 12. Gould, J., M. Devey, 0. Hasegawa, E. C. Ulian, G. Peterson, R. H. Smitii, 19S<br /> Transformation of Zea mays L. using Agrobactemm frimefaciens and the she<br /> apex. Plant Physiol. 95: 426-434.<br /> 13. Grave, A. C. F., S. L. Goldman, 1986. The fransformation of Zea mays seedling<br /> with Agrobacterium frimefaciens. Plant Molecular Biology 7: 43-50.<br /> 14. Huang, X. Q., Z. M. Wei, 2004. High-frequency plant regeneration tiirough ca<br /> initiation from mafrire embryos of maize (Zea mays L.). Plant Cell Reports 22: 793-800.1<br /> Phin V: CdNG NGHg GEN THgC VAT 395<br /> <br /> <br /> 15. Ishida, Y., H. Saito, S. Ohta, Y. Hiei, T. Komari, T. Kumashiro, 1996. High<br /> efficiency fransformation of maize (Zea mays L.) mediated by Agrobacterium<br /> tumefaciens. Nature Biotechnology 14: 745-750.<br /> 16. Klein, T.M., T. Gradziel, M. E. Fromm, J. C. Sanford, 1988. 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Plant Cell<br /> Reports 12: 84-88.<br /> <br /> SUIMMARY<br /> Plant regeneration system and genetic transformation of<br /> maize (Zea mays L.) with the ferritin gene via Agrobacterium<br /> t tumefaciens<br /> ' ' Nguyen Phuong Nam, Le Tan Due, Pham Due Tri,<br /> Nguyen Huu Tam, Nguyen Huu Ho, Nguyen Van Uyen<br /> Institiite of Tropical Biology<br /> E'<br /> Nodal sections of 10 - 15 day old seedlings (Nep, Tien Giang province) were cut and<br /> cultured on the media: MS + 0.5mg/l 2,4-D + lmg/1 BA or MS + 0.5mg/l 2,4-D + 2.2<br /> g/1 picloram + 1.38g/l L - proline + 3.4mg/l silver nifrate for the formation of<br /> regenerating callus and/or embryogenic callus. Shoot apex culture (0.4mm in size) were<br /> also carried out. There was the formation of multishoot from shoot apex cultured on the<br /> MS medium containing NAA (0.05mg/l) and BA (l-2mg/l). The regenerating calli were<br /> used for genetic fransformation via the Agrobacterium tumefaciens strain LBA 4404<br /> with the aim to enhance the ferritin in the fransformed plants.<br />
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