Tgp chi Cong ngh? Sinh hge 12(3): 523-530,2014
NGHI£:N cut! TOI UU H6A QUI TRINH CHUYDN GEN VAO LAT CAT VAY CU CAY
L I L Y B A N G AGROBACTERIUM TUMEFACIENSBui Thi Thu Huomg'-^, Nguyen Thi T h i m ^ T r i n h Khde Q u a n g ^ Phgm Bich Ngoc', Le TrSn Binh' 'Vi?n Cong ngh? sinh hpc, Vi?n Hdn lam Khoa hge vd Cong nghe Viet Nam
'Hge Vi?n Nong nghigp Vi?t Nam. Bg Nong nghiep vd Phdt triin nong thon
^Vi?n Nghien ciru Rau qud. Vien Khoa hge Nong nghiip Viet Nam Ngdy nhgn bdi: 14.4.2014
Ngay nhgn dang: 23.6.2014
T6M TAT
Nham edp phan ndng cao hifu qua cua qua trinh tao giong hoa lily mdi bang cong nghf gen, bao cao nay trinh bay ket qua nghien ciiu mpt s6 yeu to anh hudng den hieu qua chuyen gen vao lat cat vay cii lily thong qua vi khuan Agrobacterium tumefaciens mang gen chi thi Gus. Cac yeu to chuyen gen dugc nghien ciiu bao gom xdc djnh nong dg khang sinh cefotaxime thich hgp de dif t khuan ma khong gay anh hudng den su tai sinh ciia mlu cay, ngudng gdy chit mau ciia khang sinh chgn loc hygromycine (lam co sd chgn Igc cay chuyen gen), chiing vi khuan phii hgp, mgt df vi khuSn, thdi gian dong nuoi cay giiia mlu lat cit vay cii va dich khuSn.
Ket qua da toi uu dupc qui trinh chuyen gen nhu sau' sii dyng chiing vi khuan EHA105 mang vector nhi thi pCAMBIA1301 chiia gen Gus va gen hpt ma boa enzyme hy^omycme phosphotransferase vdi nong dg djch khuan (tinh theo ODMO) tir 0,6 den 0,9; lat cat vay cii duac lac ciing dich vi khuan tren khoang 30 phut, sau do dugc nuoi cay tren mdi trudng d6ng nu6i c3y trong 3-5 ngay; mau lat cat sau dong nuoi cay dugc nia khuan va nuoi cay tren moi trudng tdi sinh bo sung 250 mg/l cefotaxime. Cay dang cii chuydn gen dugc chgn Igc budc diiu tren moi tnrdng chgn Igc co khang sinh 75 mg/I hygromycine. Vdi cac dieu kien tren, hi?u qua chuyen gen vdo lily thu dugc tuong doi cao, vdi hp so chuyen gen la 8,8% (0,088 dong / lat cat vay cu).
TirkhdaA. tumefaciens, chuyengen, Gus. latcatvdycii. lily
Lily (ten khoa hpc la Lilium thupc hp Liliaceae), Id mpt trong cdc lodi hoa dpp dang rat dupc ua chupng tren thj trudng hoa hi?n nay. Vdi kieu dang dpp, sang trpng, mpt s6 chung logi cd huong thom quyen ru vd dp ben hoa rat cao. Tuy nhien, cdy lily cd phd trdng h?p do cd xudt xii d nhihig vung dn ddi. Ben cgnh dd, vdi nhu cdu phat triln ngudn gidng hoa lily mot each chil dpng, cd ndng sudt va chat lupng cao, dgc biet la thich nghi vdi dieu kipn thdi tilt, dich benh hay sinh thai khde nhau thi viec cdi tiln vd tgo giong cdy trdng mdi dang trd nen cap thiet. Ky thugt chuy6n gen vdo lily da dupc nghi£n cihi tren the gidi va mdi gdn ddy ngudi ta bdt ddu th\rc hi^n chuyen gen bang vi khudn Agrobacterium (Hoshi et al, 2005). Sau do, d Viet Nam ciing da cd mgt vdi nghien ciiu tgo cdy lily chuyen gen (chii ylu Id gen chi thi) nhd vi khuan da dupc thpc hipn. Cac ket qud thu dupc dang diing d v i ^ xay d^ng mpt quy trinh chuyen gen vao lily nhd vi khuan Agrobaclerium tumefaciens vi cac cdng bd mdi chi thu dupc nhung kit qud bieu hien gen budc
dau vdi hieu qua chua cao. Nguyen nhan mpt phdn ldn la do cay lily la cay mdt la mam khdng phai vat chil cua vi khuan A tumefaciens nen cdng viec nay gap nhieu khd khan. Cac nha nghien ciiu tiing budc chuan hda chung vdi timg dieu kien nhu cac ylu td mdi trudng dinh duong, chdt ddn du vi khudn (AS), thdi gian ddng nudi cay, vat lieu biln ngp . (Ogaki et al. 2008; Li et al. 2008; Thao el ai, 2008; Anh et al. 2009; Azadi etal. 2010; Yue etal. 2012). Chuih vi vgy, viec hoan thien quy trinh chuyen gen can tilp tuc dupc nghien ciiu nham phuc vu cho cdng tdc chpn tgo gidng hoa lily mdi. Bdi bdo ndy Uinh bdy ket qua nghien ciiu dnh hudng cua mdt sd yeu td den viec chuyen gen chi thj Gus vao lat cat vay cu lily in vitro.
Tli dd xac dinh, lua chpn va tdi uu hda mpt sd dieu kien nham nang cao hieu suat chuyen gen vao lily.
VAT LIEU VA PHU'ONG PHAP V a t li^u
Vay cu lily in vitro gidng Lilium Oriental Hybrid Belladonna do Vien Nghien ciiu Rau qud cung cap. Vi
Biii Thi Thu Huong eraj;
khudn A. tumefaciens gom cdc chimg LBA4404, C58- PMP90, C58-pGV2260 va EHA105 diu chiia plasmid pCAMBIA1301 mang gen Gus ma hda cho fl- glucuronidase. gen hpt md hda cho hygromycme phosphotransferase do phdng Cdng nghe te bao thuc vdt, Vi?n Cdng n g h | sinh hoc cung cap.
P h u t m g phdp nghiSn cihi
Phuang phdp xdc dinh nong dp khdng sinh thich hgp cho chuyen gen vdo lily
Trong cdc nghien ciiu ve chuyen gen bang vi khudn A. tumefaciens vao thuc vgt, sau bien nap vd ddng nudi cdy, viec xdc dinh ngudng khdng sinh phii hpp d l di^t khudn md it anh hudng den khd nang tdi smh cua mau va chpn lpc cay chuyen gen anh hudng true tiep tdi hipu suat chuyen gen nen can thiet phdi dupc tim hieu, nghien ciiu sdu.
Xdc dinh nong dg cefotaxime ihich hap de diet khudn a mdu sau bien ngp
Lat cdt vdy cii day khoang 3-5 mm ciia vdy cu (8 tudn tu6i) cd the tdi sinh tgo cii con tdt trong mdi trudng MS (Murashige, Skoog, 1962) + 7,5 g/l agar + 60 g/l saccarose (Huong et al. 2013). Tuy nhign, sau khi bien ngp, trong mau thuc vdt cdn vi khudn nen phdi bd sung khang sinh cefotaxune vao mdi trudng tdi sinh de di^t khudn vdi cac ndng dp khde nhau (0, 125, 250, 375 va 500 mg/l). Xdc dinh khd ndng tdi sinh ciia mdu nudi cay sau thdi gian 4 tuan vdi cac chi tifiu nhu ty le mau sdng sgch (%) (phan tram sd Idt cdt vay cii sdng sdt, khdng bi nhiem khudn so vdi tdng sd lat cdt vdy cu nudi cay), ty le mdu tgo cu (%) (phan tram sd Idt c5t vay cu tgo cu so vdi tdng sd lat cdt vdy cu nudi cay) va he sd nhdn cii (sd cu hinh thdnh tnmg binh cua mot Idt cat vay cu nudi cdy).
Xdc dinh nong dg hygromycine thich hgp de chpn lpc mdu cu liiy chuyin gen
Cu nhd lily in vitro (khoang 4 tudn tudi) dupc cay tren mdi trudng MS + 60 mg/l saccarose va bd sung hygromycine d cac ndng dp khac nhau (0, 25, 50, 75 vd 100 mg/l). Sau 4 tudn, mlu nudi cdy dupc quan sat va danh gid dua tren cac chi tieu nhu ty Ip mau sdng (%) (phan tram sd cu sdng sdt so vdi tdng s6 cu nudi cay), tJ- I^ mdu xudt hign la (%) (phdn trdm sd cu hinh thanh la so vdi tdng sd cu nudi cay) vd ty IS mau xuat hien re (%) (phan tram s6 cu hinh thanh re so vdi tdng sd cu nudi cdy).
Phuongphdp chuyin gen vdo Idt cdt vav cu nk^a
khuan A. tumefaciens r Chudn bf djch khudn J
Bon chiing vi khudn A. tumefaciens ife LBA4404, C58-PMP90, C58-pGV2260 va EHA105 dupc nudi hogt hda trfin mdi trudng gom 10 ^ Tripton + 10 mg/I NaCI + 5 g/l cao nam men vdbS sung cac khang sinh chpn loc phii hpp vdi timg lo^j khuan, Idc 200 vdng/phiit trong 24 h d 28°C. Sau 86, dich vi khudn dupc li tara thu smh khdi d tic dJ 5000 vong/phiit d 4 ^ trong 15 phiit, hda tan cSn khudn bdng mdi tnrdng HMS ldng + 3 % saccarose, pH = 5,8 cd bd sung 100 p M acetosyringone (AS)%
thu dupc dich huyen phii vi khuan (Ogaki et K
2007). j Chudn bi mdu Idy nhiem
Vay cii lily in vitro dupc sir dung ldm vat li^
chuyen gen do vay cu cd kha nang tai sinh cao, de hinh thdnh cdc cu bat dinh va cd the su dgng de chuyfin gen khd hiiu hidu (Simmonds, Ciumning, 1976; Thao el al, 2008). Vdy cii dupc tdeh ra, cai thanh timg lat manh nhd (khodng 2 d i n 3 mm) trong td cay vd trung.
Biin ngp vd dong nudi cdy
Cdc lat cdt vdy cu dupc dua vao dich huyen phu te bdo vi khudn A. tumefaciens (cd nong dp xac dinh tuy thi nghipm), lac nhe trong 30 phiit, thdm kho bang giay tham vd triing vd sau dd dupc nu6i c!^
vao mdi trudng ddng nudi cdy (MS + 3 % saccarose + 100 pM AS + 10 mM Mes + 2 g/l gellan, pH = 5,8) (Ogaki et ai, 2007), trong dilu kien tdi a nhi^
dp 25"C, thdi gian ddng nuoi cay tii 1 din 7 ngdy t&y thupc vdo cong thiic thi nghifim (Hoshi et ai, 2005;
Li etal. 2008; Anh etal. 2009; Azadi elai, 2010;
Yue era/., 2012).
Tdi sinh. sdng Igc cdy chuyin gen
Mau sau khi dupc bien ngp vd ddng nuoi cay dupc rira khuin 3 ldn bdng dung dich Y2 MS c6 bo sung cefotaxime 500 mg/l, thdm khd va chuyin sang mdi trudng tdi sinh cd khdng sinh diet k h u ^ (MS + 6% saccarose + cefotaxune (ndng dp thich hpp tiiy thi nghidm) + 7,5 g/l agar, pH = 5,8). Sau khodng 2 din 4 tudn, cac manh vdy cii bdt ddu phdt sinh cac cii lily nhd dupc chuyin sang moi trudng cd khang sinh chpn lpc cdy chuyin gen (MS + 6% saccarose + hygromycine ndng dp thich hpp tiiy thi nghipm) ;f 7,5 g/l agar, pH = 5,8) dgt trong dilu kien tdi d nhJel dp 25''C khodng 4 d i n 6 tudn.
Tgp chi Cong ngh? Sinh hge 12(3): 523-530,2014 Xdc djnh khd ndng chuyen gen vdo lat cdt vdy cu lily
Xdc dinh anh hudng ciia cac ylu td den hieu qud chuyin gen qua cdc chi tieu nhu hp sd nhdn cu (sd cii hinh thdnh trung binh cua mot lat cdt vay cu sau 4 tuin nudi cdy trong mdi trudng tdi sinh cd cefotaxime); ty 1? mau sdng sdt tren mdi tiudng chpn lpc (%) (phdn trdm sd cu song sdt so vdi tdng sd cu (da hinh thdnh trong mdi tnrdng tdi sinh) sau 4 tudn nudi cdy trong mdi tnrdng chon lpc cd hygromyxine), ty Ip ddng duong tinh vdi X-gluc (%) (phdn trdm s6 cdy in vitro (dgng cii) cd it nhat mdt didm bdt ky cd mau xanh chdm dgc trung so vdi tdng sd cay khi chiing dupc nhupm vdi X-gluc 1%^ (phuang p h ^ ddnh gid svr bilu hi^n tgm thdi va ben vttng ciia gen Gus trong cac mdu bien ngp theo Jefferson (Jefferson e/o/.,1987). Hp sd chuyen gen dupc tinh bdi sd ddng (cii in vitro) ducmg tinh vdi Xgluc dupc tgo ra trung binh ciia mdt Idt cdt vay cu biln ngp.
KET QUA VA THAO LUAN
Xdc djnh nSng dS kbdng sinh thich h o p cho chuyin gen vdo lily
Anh hudng cua khdng sinh cefotaxime den khd ndng tdi sinh cu cda lat cdt vdy cd dd bien ngp
Kit qud nghien ciiu cho thdy su sai khac ve khd ndng tdi smh cu sau 4 tudn nudi c^y lat cdt vdy cii lily nudi cay. O mdi trudng khdng bd sung cefotaxime, ty le mau sdng sgch vi khudn Id 0%.
Tuy nhien, mpt sd mdu hi nhiem khudn ndy (10%) van cd bieu hidn phdt sinh cu nhung vdi he sd nhdn cO rit thdp (0,02 cii/ldt). Hon nua, nhiing cu ndy rat nhd (kich thudc khodng 0,01 mm^ - chi la nhiJng dilm mau trdng nhd ten tren be mat lat cat) (s6 li^u khdng t h i hien d bdng). Vdi moi trudng nuoi cdy cd bo sung khdng sinh nay thi ty Ip mdu sdng sgch vi khudn cao ( 7 5 % d mdi trudng cd 125 mg/l cefotaxime va 100% vdi mdi trudng cd lupng cefotaxime la 250; 375 hay 500 mg/l). Tuy nhien, d ndng dp cefotaxime cdng cao thi ty le m l u tgo cu vd h$ sd nhdn cii la thdp ddn (vdi mdi trudng bd sung cefotaxime d ndng dp 125 mg/I cefotaxime thi 86,5% m l u tgo cii va cd 1,8 cii/ldt; ndng dp 250, 375 vd 500 mg/l cd 69,5%, 4 4 % vd 32% m l u tgo cii vd trung binh tgo 0,81; 0,15 va 0,14 _cu/ldt, tuong ling. Mgc dii ty le mdu tgo cu va he sd nhan cii ciia m l u trong mdi trudng cd hb sung 250 mg/l cefotaxime It hon d mdi trudng cd 125 mg/l cefotaxime nhimg 100% mdu sgch vi khudn va cu con mdi hinh thdnh cd kich thudc to hem cd (Hinh l . B a n g I).
Moi truong nin: MS + 7,5 g/l agar + 60 g/l saccarose
Ket qua tren chiing td rang, khi khdng su dung hodc sii dung khdng sinh vdi ndng dp thap (125 mg/l), vi khudn (vdn cdn trong lat cdt) dd lie che su phat trien cua mau; cdn sii dung khdng sinh vdi ndng dp cao (> 375 mg/l), mac dii vi khudn hi tieu diet nhung khdng sinh Igi uc c h i mgnh kha ndng tdi sinh cua lat cat vay cu lily. Sir dung ndng dp khdng sinh cefotaxime 250 mg/l la thich hop nhat, hipu qua diet khudn cao (100%), khd nang tdi sinh mau kha cao (69,5%). Mpt sd tac gid da cho rdng, vipc sir dyng Cefotaxime 400 mg/I hay 500 mg/l da dipt khudn rdt tdt va khdng gdy tdn thuong mdu cay; tuy nhien hieu suat chuyen gen thu dupc khdng cao (khodng 2,5% - 4%) do cefotaxime ham lupng cao cd dnh hudng Idu dai den kha nang chuyen gen vdo lily cua vi khudn hay kha ndng sdng ciia mau chuyen gen.
Anh hu&ng cua hygromycine den kha nang phdt triin cda cay lily in vitro dgng cu
Moi loai cay cd dp man cdm kbdc nhau vdi khang sinh ndi chung vd vdi hygromycine ndi rieng.
Cdc mau chuyin gen mang cdu tnic vector cd gen khang hygromycine cd thi sdng sdt trong mdi trudng cd bd sung khang sinh ndy. Tuy nhi5n, nudi mdu tren mdi trudng chpn lpc cd ndng dp hygromycine qua cao s6 lam mdu bj chit hoac sinh trudng bi lie che.
Ngupc lgi, neu nudi mdu tren mdi trucmg chpn lpc cd nong dp khang sinh thdp se khd chpn lpc dupc cdy chuyen gen. Nhu vgy, xac dinh ndng dp khdng sinh thich hpp cho chpn lpc m l u chuyen gen, cu lily in vitro 4 tudn tudi da dupc kilm tra miic dp man cam mdi trudng tdi sinh bd sung hygromycine d cac ndng dp khac nhau. K i t qud thi hi6n nhu sau: (Bang 2, Hhih2).
Moi trucmg nin: MS -^ 7,5 g/l agar + 60 g/l saccarose
d mdi trudng bd sung hygromycine 0; 25 va 50 mg/I ty le mdu sdng tuong iing Id 100; 50,0 vd 10,3%. Ngoai ra, cac mdu nghifin ciiu ndy cd su xudt hipn Id va re, ty le nghich vdi su tang ndng dp khdng sinh, cu the, ty le mdu xuat hi?n Id la 77,3; 56,3, 27,6% va ty le mlu xudt hien r l la 100; 87,5; 51,7%.
Tren mdi trudng khong bd sung hygromycine va bd sung vdi ndng dp thdp (25 mg/I), d gdc cu nhd in vitro ban ddu cdn cd su xuat hien thfim cu radi, dieu nay it h o | c khdng thdy d cdc dia thi nghipm cd ndng dp khdng sinh cao hon 50 mg/l (Hinh 2) (sd lieu khdng bieu hien d bdng). Vdi mdi trudng cd ndng dp hygromycine ldn (75; 100 mg/l), cdc cii lily in vitro 525
Biii Thi Thu Huong ej deu ngimg sinh trudng va cd hien tugng chet, ddng
thdi ciing khdng thdy su xudt bien la hay re (ty Ie mdu sdng, ty le mau xuat hien Id va ty Ie mau xudt hien re deu la 0%).
Nhu vdy, tu k i t qua nghien cihi ndy chi ra rang, cd tbi sii dung ndng dp 75 mg/l hygromycme du chpn lpc cu lily chuyen gen.
Bang 1. Anh hudng dJa Cefotaxime din kha nang tai sinh cii con ciia lat cdt vdy cii dd bi^n ngp.
Cefotaxime (mg/l) 0 125^ 2S0 375 Ty Ig mlu song sgch khuan (%)
Ty Ig mlu tao cii (%) Hg s6 nhSn cii (lln)
0.0 10,0 0.02
75.0 86,5 1.2
100,0 69,5 0.81
100.0 44.0 0,15
100,0 32.0 0,14
-V«''..,l.,.
Hinh 1. Sg tii smh cii tO ISt cat vdy cCi da bien ngp sau 4 tuan nuoi cay trong moi truPng tii smh.Hlnh la Mau 161 cit viy cQ tmng moi tnrang tai sinh khdng b6 sung celotaxime; Hinh lb, lc, Id. Ie. Miu lit cit viy cO trong mdi trucmg tai smh bo sung cefotaxime 125; 250; 375; 500 mg/l, tuang Cmg.
Bang 2. Anh hudng cCia Hygromycine den kha n Hygromycine (mg/l)
T91$ mdu s6ng (%) T^ 1$ mdu xu^t hign la (%) TJ 1$ mlu xudt hign re (%)
50,0 56,3 87,5
10,3 27,6 51,7
Hnh 2. Khd nang sinh hudng cu lily invitro sau 4 tuin nuoi cay trong mfli trudng c6 b6 sung Hygromycine. Hinh 2a: MiusH tmng moi truong tii sinh khong bo xung hygromycine.M/i/j 2b,2c,2d.2e: Miu cO in vitro trong mdi tnrdng 1)6 xunj) hygromycine 25; 50; 75; 100 mg/l, tucmg ung.
Xdc dinh chiing A. tumefaciens thich h p p cho chuyen gen
De ndng cao hieu qud chuyen gen thdng qua A.
tumefaciens, ddi vdi mdi loai thuc vdt cdn xdc dinh dupc chiing A. tumefaciens thich hpp. Bdn chiing A.
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tumefaciens LBA4404, C58-PMP90, C58-pGV226l) vd EHA 105 da dupc khdo sat khd ndng chuyin gea vao gidng lily Belladonna. K i t qua nghien cihi thu dupc (Bang 3) cho thdy, khi cdc Idt vdy cii (dd dirpc biln ngp bdi cac chiing vi khudn) d i u cd hp s i nhjn cu tuong ddi bdng nhau (tu 0,68 - 0,72 cii/lat), toy
Tgp chi Cong ngh? Sinh hge 12(3): 523-530,2014
nhien ty Ip man sdng sot tr€n mdi trudng chpn lpc vd ty 1^ mSu duong tinh vdi X-gluc khac nhau. Chung C58 - PMP90 vd C58 - pGV2260 cd khd ndng chuyin gen vao lat cdt lily thdp gdn nhu nhau, thi hipn sd ddng hinh thdnh va ducmg tinh vdi X-gluc ciia mpt lat cat bien ngp la 0,038 vd 0,033; mac dii chiing C58 - pGV22260 khi xdm nhdp vao mdu khien khodng 8,79% mdu sdng sdt tren mdi trudng chpn lpc, cao hon chiing C58-PMP90 (cho ty le khodng 5,43% mau sdng sdt). ChiSng LBA4404 cho
Bang 3. Khd nSng chuy4n gen ciia c i c chiing A tumefaciens vdo lily.
hieu qud chuyen gen khd cao, thd hi€n ty le mau sdng sdt trong mdi trudng chpn lpc la khoang 9,29%
va he sd chuyen Gus Id 0,060. Mlu dupc bien ngp vdi chiing EHA 105 cd ty le mau sdng sdt vd duong tinh Xgluc cao nhat, dat tnmg binh Id 10,08 % va 6,7
% (0,067). Chung vi khuan nay gdn day cung dupc mdt so nha nghien ciiu de chuyen gen vao lily nhu M. Ogaki etai, (2007), Yue etal, (2012). Nhu vdy, chimg EHA 105 da dupc chpn de thuc hien cac thi nghiem tiep theo.
i L i n t h i nghidm
S i m l u s 6 c u c o n 1 ngp h i n h
H # s 6 n h a n
S6 c u T y le m a u g g j , ^ „ g j , ^ g He sS c h u y i n gen s o n g s d t s d n g s o t ^^^ ^ ^ ( d o n g diFO^ng t i n h H * . £4> t h i n h / , . M \ ( c u / l a t sau c h p n sau c h o n X g l u c / l a t c a t b i l n (VA cii) t h i n h (cu) J^^^j , ^ ^ j ^ ^ j i^^^^j x g l u c (dong) ^ ^ ^ j
75 50
10,88±0,6 13
C58- PMP90
C58- pGV226
LBA 4404
Xdc djnh n6ng dp djch khuSn A. tumefaciens thfch hQp dl chuyin gen
Nlng dO vi khuin trong m$t don vi the tich khi lay nhilm cung ddng vai trd quan trpng mang lgi hi^u qua cao cho chuyin gen. Thi nghiera d ddy dupc hh tri vdi 4 radt d$ tl bdo vi khuan chiing EHA105 tren tuong iing vdi ODeoo lan lu0 Id 0,3;
0,6; 0,9 vd 1,2. Kit qud thu dupc (Bdng 4) cho thdy mgt dp vi khuin d ODsoo tir 0,6 din 0,9 cho he so nhdn cu thap (0,65 - 0,66) nhimg ty le mau sdng sdt tren mdi trudng chpn lpc cao (khoang 11,49 den 12,18% ) va hp sd chuyin gen khd cao tuong ung la 0,068 vd 0,055. Vdi ndng dp vi khudn d ODeoold 0,3 ho§c 1,2 cho hipu sudt chuyen gen thap hon ban vdi hp so ndy Id 0,046 vd 0,036; mde dCi ty Ip mlu sdng sdt tren mdi trudng chpn lpc khd cao (khodng 7,78 - 8,13 %). Budc dau, ta cd thi kit lugn rdng dich khudn cd ODeoo^ 0,3 la qud thdp, lupng khudn it cho vipc biln ngp vdo mau; cdn ODeoo =1,2 lgi qua cao.
lupng sd Iupng vi khuan ldn nen chung cd the da chit trong cgnh tranh dmh dudng va khdng gian sdng hgn che. Nhu vgy, dich khuan diing de lay nhiem cho lily cd OD^oo trong khodng 0,6 den 0,9 la thich hpp.
Ket qua nay tuong iing vdi nghien cuu ciia Li et al.
(2008) khi lac mau md seo lily trong dung dich huyen phu te bao kha bien cd OD^oo la 0,8.
Xdc dinh thdi gian ddng nuoi cay thich hpp de chuyen gen
Thdi gian dong nudi cay giiia vat lieu thuc vat vd vi khudn A. tumefaciens la mpt trong nhirng yeu td anh hudng dang ke den hieu qud cua qud trinh chuyen gen. Cdc lat cat vay cu dupc lay nhiem vdi dich vi khuan (ODeoo = ^fi) trong 30 phiit va dupc ddng nudi cdy trong thdi gian 1, 3, 5 vd 7 ngdy. Ket qud (Bang 5) cho thay, vdi thdi gian ddng nudi cay la I ngay, he sd nhan cu cao nhat dgt 0,83 cii/ldt, cdn khi ddng nudi cdy vdi thdi gian nhieu hon (3, 5, 7 ngdy), he sd nhdn cii thdp ban (0,74 - 0,76 cii/ldt).
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Tuy nhien, vdi thdi gian ddng nudi cdy ngan (I ngdy), hieu qud chuyen gen vao mlu khdng cao, thi hien ty Ie mau song sdt trong mdi trudng chpn Ipc t h ^ (khoang 3,75%) va ty Ie ddng hinh thdnh duong tmh Xgluc ciing thdp Id 2,4 % (0,024).
Ngupc lgi, vdi thdi gian ddng nuoi cay dai (7 ngay), ty Ie mdu sdng sdt trong mdi tnrcmg chpn Ipc khd cao (7,37%) va ty le mau tao thdnh duong tinh vdi Xgluc la 3,9 % (0,039). Dii vdy, thdi gian ddng nuoi cdy 7 ngdy cho hi?u qud chuyen gen khdng
cao bdng thdi gian ddng nudi cdy Id 3 hoac 5 n_
Cd thi qua trinh ddng nudi cdy qud ddi neni^
khuan sinh trudng qua mirc n£n tic che su phat tri^
ciia cdc tl bao lily da dugc chuyen gen. Thdi ^ ddng nudi cay tir 3 den 5 ngdy cho hieu suat d ^ gen cao nhdt, ty le mdu sdng sdt trong radi t chpn lpc khoang tir 11,31 - 12,78% vd vdi h^n chuy€n gen (sd ddng hinh thanh, duong tinh voi X- gluc ciia mdt lat cat vdy cii biln ngp) la 0,085 vi 0.088.
Bang 4. Anh hudng cua ndng dg dich vi khuin khic nhau tdi khi ning chuyen gen vao lily.
Lin thi s6 mSu biln nghidm ngp (lit cit)
He s6 S& cii Ty Ig miu nhSn s6ng sdt song sot (cu/ lat sau chpn sau chpn ' cat) lpc (cO) lpc(%)
So dong Hf s6 dinnig tinh chuyin gen v6! Xgluc (ding diKj<ng tii (ddng) Xgluc/lit biln n 7.73±0.37 12
12.18±0,59 17
10.49±0,46 14
8,13±0,73 5
Bang 5. Anh hudng ciia thdi gian ddng nu5i d y den hieu qui chuyin gen vdo lily.
SS mlu So cu SScii
' ' . . .7 r:hnn Ifif.iV.^ Vnliif fHAnni dong nu6i . . . uisnn^p buiiiniin
cay (ngay) "»"'*™ (IMcIt) thanh (cu] chpn lpc(%) Xgluc (ddng)
3,75±1,07 3 12,78±0.50 11
11,31±1.02 11 7.37±1.44 5
Nhu vdy, viec ddng nudi cay vi khuan vdi lat cat vdy cii lily trong 3 den 5 ngay cd tfal thu dupc cay hly chuyen gen dgng cu in vitro hien qua hon cd. (Li
etal., 2008) ciing tien hanh ddng nudi cdy mlu mdSpO vd vi khuan trong 5 ngay thu dupc md spo co
bilu hien gen dat x ^ si 12%. Ben cgnh dd, Anh va
cpng su (2009) cung da ddng nudi cdy md spo ciy
hoa loa ken trSng va vi khudn Agrobacterium trong 3
Tgp chi Cong ngh? Sinh hpc 12(3): 523-530,2014 ngdy, budc d i u nhgn thdy sg bilu hien tgm thdi cua gen GFP tren md s^o cao (tren 50%). Cl day, chung tdi nhgn thay rdng, su bieu hipn tgm thdi gen Gus rdt cao (tren 70%) (Hinh 3) (s6 Iifu khdng bilu hien d bdng); song svr bi£u hifn gen Gus trong cdy in vitro tdi sinh dang cit d cac vi tri khac nhau khd bin vQng (giai dogn cii in vitro 8 - 1 2 tudn tudi). Tuy nhien, su bieu hien gen Gus nhln khdng ro d phia be mdt ngodi ciia cu tai sinh in vitro ma chi the hi§n d rapt vdi vi tri nhu md spo hinh thanh d gdc (Hinh 4A), tren re non (Hinh 4B), trfn thdn radra khi cat dpc cii (Hinh
4C)vd mdnh la (Hinh 4D). Knh 3. S\i bleu hien tam fliffi gen Gus tr§n be m^t cat vdy ci Illy d mau sau biln nap.
Hnh 4. Subllu hi^ngen Gus d cSc v| tri trfin cSy lily in vitro 8-12 tuan tuoi. Hinh4a,4b. 4c.4d-Sg xuit hi$n miu xanh chim cf$c tnmg Wi/ nhupm Xgluc trin md sgo hinh thinh i> g6c thin, tren re cay. tren thin mim vi trin minh li. tircmg Ong.
KfiT L U A N TAI LIEU THAM KHAO
Chiing EHA105 (ODsoo tir 0,6 den 0,9) mang vector pCAMBlABOl Id thich hpp dupc sii dung d l biln ngp vdo Idt cdt vdy cii lily. Sau khi lay nhilm vdi vi Idiuan, m l u dupc tiln hdnh dong nudi cay tir 3 din 5 ngdy, nia khuin vd nudi ciy trfn mdi trudng tdi sinh cd b l sung khdng sinh cefotaxime 250 mg/l cho ty If mdu sdng sdt cd khd ndng tdi smh cu con cao. Khi cii lily xuat hipn tren cdc Idt cdt vdy cii (khodng 2 - 4 tuan), c^y chuyen chiing sang mdi trudng chpn lpc cd 75 rag/l hygromycine. Vdi cac dilu kifn dupc xdc djnh trfn, hifu sudt chuyen gen vdo Idt cdt vdy cu lily Belladonna dgt 8,8 %. Ddy Id rapt ket qud thu dupc cd y nghia va trien vpng dp dgng d l i vdi chuyin cdc gen muc tifu vdo cdy hoa lily Belladonna ndi rieng va lily ndi chung, nhdra gdp mpt phan nhd cho cdng tdc chpn tgo gidng hoa lily mdi.
Liii c i m a n : Cong trinh dugc thgc hi?n trong chuong Irinh ddo Igo liin sT tgi phong Cong nghe te bdo Thgc vgt - Vi?n Cong ngh? sinh hge. Vi?n Hdn ldm Khoa hge vd Cong ngh? Vi?l Nam.
Azadi P, Chin DP, Kuroda K, Khan RS, Mii M (2010) Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium. Plant Cell Tissue Organ Cult 101:201-209.
Huong Bui Thi Thu, Quang Trinh Khac (2013) Khd ndng tgo cu lily invitro cita mot so dong lily nhgp npi. Tgp chi Nong nghiep vo Phdt trien Nong thon, so 1: 13-21.
Hoshi Y, Kondo M, Kobayashi H, Mori S, Nakano M (2005) Agrobacterium-mediated transformation of Lilium longiflorum. Acta Hort 673 (2): 543-547.
Simmonds JA, Gumming BG (1976) Production of plantlets from bulb-scale callus cultures for increased propagation rates. Science Hortic 5: 161 -170.
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fiision: glucuronidase as a sensitive and versatile gene fiision marker in higher plants. EMBOJ6: 3901 - 3907.
Li Qiu-Hua, Hong Bo, Tong Zheng, Ma Chao, Guan Ai- Nong, Yu Jing-Juan, Gao Jun-Ping (2008) Establishment of regeneration system and transformation q/"Zm40l gene in Lilium longiflorum^L. formosanum Chinese Joumal of
11 H u o n g s Agricultural Biotechnology / Volume 5 /Issue 02 /August:
113-119.
Murashige T., Skoog, F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture.
Physiol Plant 15:473-479.
Thao Nguyen Thi Phuong, Thuy Nguyen Thi, Thach Nguyen Quang (2008). Developing an Agrobacterium- mediated transformation system for Lilium x formolongo using thin cell layer of bulb scales. Journal of Science and Development. April 2Q0Z: 123 -128.
Anh Nguyin Thi Ly, Son Dinh Trudng, Phucmg Nguyin Thi Thanh, Hoa Nguyen Thi (2009) Nghien cuu tdi sinh in
vitro vd chuySn gen Green Fluorescent Protein viol hoa loa ken (Lilium Longgiflorum) nha yi Mi Agrobacterium. Tap chi Khoa hoc vd Phdt trien 7 (:
121- 129.
Ogaki, M., Y. Furuichi (2008) Importance of ^ cultivation medium pH for successful Agroba mediated transformation of Lilium x formolongf. • Cell Rep 27: 699-705.
Yue Wang, Bemadette van Kronenbuig, Tiia MeimL Chris Maliepaard. Xiaohui Shcn, Frans Krens (2012|
Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars. Plant Cell Tiss Organ Cult 111;
113- 122.
O P T I M I Z I N G AGROBACTERIUM TUMEFACIENS T R A N S F O R M A T I O N E F F I C I E N a IN L I L Y T H R O U G H B U L B S C A L E S L I D E S
Bui Thi Thu Huong'*^-, Nguyen Thi T h i m \ Trinh Khac Quang^, Pham Bich Ngoc', Le Tran B u h ' 'institute of Biotechnology, Vietnam Academy of Science and Technology
' Vietnam National University of Agriculture. Ministry of Agriculture and Rural Development
^Vegetable and Fruit Institute. Vietnam Academy of Agricultural Sciences SUMMARY
In order to improve the gene transformation system for a lily vanety, this report presents some research -^
results that affected to gene transformation of Belladonna Hly bulb scales slides via Agrobacteriion ^ tumefaciens. There are some major factors influencing transformation efficiency have optimized such as appropriate concentration of cefotaxime could kill the bactena and unaffected the regeneration of sample, the threshold of hygromycine that help lo select of transgenic plantlets, suitable bacteria strain with proper ' •*
bactenal concentration and the suitable time of co-culture. The results show that the hly bulb scales shdes were immersed with the EHA105 sttain carrying binary pCAMBIA1301 vector that contains the Gus gene and hygromycine resistance gene at a concentration of bactenal suspension ODeoo from 0.6 to 0.9, around 30 minutes, then co-cultured for 3 days are the best. Continuously, samples were washed by sterilized fluid MS supplement with 500 mg/l cefotaxime and cultured on regeneration enviromnent supplement with 250 mg/l cefotaxime to regenerate new bulblets. Transgenic bulblets initially could be selected on selective antibiotics medium with 75 mg/l hygromycine. Under the above conditions, efficient of gene transformation to lily were obtained relatively high efiBciency, which is 8,8% (0,088 line / bulb scales slide).
Keywords: A. tumefaciens, bulb scale layers, gene transformation. Gus, lily
• Author for correspondence: Tel: +84-463284139; E-mail: btthuonshp(S).email. com 530
