Tph^V: CdNG NGHg GEN THgC VAT 339<br />
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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 />
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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 />
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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 />
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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 />
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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 />
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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 />
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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 />
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KET QUA VA T H A O L U A N<br />
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Ve nudi cdy md:<br />
- Nudi cdy dot thdn:<br />
9<br />
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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 />
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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 />
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: 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 />
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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 />
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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. The Plant Cell 4: 1495-1505.<br />
6. Danson, J. W., M. Lagat, M. Mbogori, 2006. Screening fropical maize lines for tiie<br />
production and regeneration of friable and embryogenic type II callus. African<br />
Joumal of Biotechnology 5(230): 2367-2370. ...<br />
7. Fennell, A., R. Hauptmann, 1992. Elecfroporation and PEG delivery of DNA into<br />
maize microspores. Plant Cell Reports 11: 567-570.<br />
8. Frame, B. R. et al., 2002. Agrobactemm tumefaciens-mediatedti-ansformationof i<br />
maize embryos using a standard binary vector system. Plant Physiol. 129: 13-22.<br />
9. Fromm, M. E., L. P. Taylor, V. Walbot, 1986. Stable fransformation of maize after A<br />
gene fransfer by elecfroporation. Nafrue 319: 791-793.<br />
10. Goto, F., T. Yoshihara, H. Saiki, 1998. fron accumulation in tobacco plants'^<br />
expressing soybean ferritin gene. Transgenic Research 7: 173-180.<br />
11. Goto, F., T. Yoshihara, N. Sh'igemoto, S. Toki, F. Takaiwa, 1999. 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 />
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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. Factor influencing<br />
gene delivery into Zea mays cells by high-velocity microprojectiles.<br />
Bio/Technology 6: 559-563.<br />
17. Laursen, C. M., R. A. Krzyzek, C. E. Flick, P. C. Anderson, T. M. Spencer, 1994.<br />
; Production of fertile fransgenic maize by elecfroporation of suspension culmre<br />
. cells. Plant Molecular Biology 24: 51-61.<br />
18. O'Kennedy, M. M., J. T. Burger, D. K. Berger, 2001. Transformation of elite white<br />
maize using the particle inflow gun and detailed analysis of a low-copy integration<br />
,. event. Plant Cell Reports 20: 721 -730.<br />
19. Planckaert, F., V. Walbot, 1989. Transient expression after elecfroporation of<br />
' protoplasts derived from embryogenic maize callus. Plant Cell Reports 8: 144-147.<br />
20. Sidorov, V., L. Gilbertson, P. Addae, D. Duncan, 2006. Agrobacterium-mediated<br />
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<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 />