TAP CHI SINH HQC, 2012. 34(3) 389-3')f)
N G H I E N C U U Q U Y T R I N H C H U Y E N G E N V A O C A Y DU'A H A U (Citrullus lanatus T h u m b . ) N g u y e n Thj T h a n h Nga'*, Ho M a n h T u d n g ^ P h a m Thj V a n S
N g u y e n T u d n g V a n ^ C h u H o i n g H a \ Le T r i n B i n h '
" ' ' D ^ i hpc Tay Bac, [email protected] '"Vi?n Cdng ngh? sinh hpc
TOM TAT: Trong nghien cihi nay, chiing toi da toi uu hda quy trinh chuycn gen hifu qua vao cay dua hau eiia Viet Nam thdng qua vi khuin Agrobacterium lumefaciens. Chiing vi khuan CV58 mang vector nhi phan pCB-gusplus chiia gen chpn Ipc khang kanamycin n/7///(neomycin Pliosphotransfcra.sc) va gen chi thi Gus-tnirun (^-Glucuronidase) dupe dung de chuan hoa. Vai heu chuyen gen la cic manh la mini 5- 7 ngay tudi dupe nhiem vdi vi khuan o ndng dp ODM>O 0,7 trong 30 phut. Sau 3 ngay ddng nudi cay. cac manh la mam dupe tii sinli tren mdi trudng chon Ipc MS co bo sung 1,5 ing/l BAP, 0.5 mg/l IBA, 200 mg'l kanamycin va 500 mg/l cefotaxime. Cac choi chuydn gen ra rc tren moi trudng MS co bo sung 0,2 mg/l IDA, 100 mg/l kanamycin va 250 mg I cefotaxime. Cd sy khac nhau dang kS vd hieu qua chuyen gen cua eac ddng dua hau khac nhau. Dong L2 (Fl TG939) cho hicu qua chuycn gen la 1,87%. tiep den la ddng 0 2 (F9 Tieu Long- Thing Long) l,Sfi"M>. cac dong dua hau LI (Fl Kinhkong) va D[ (cd ngudn goc Binh Thuan) cho hieu qua chuycn gen la 0% Kcl qua phan tich cay chuycn gen thdng qua bicu h i ^ cua gen gus va phan ling PCR vdi gen chpn Ipc nptll d i kli4ng dinh sy co m^t dn djnh cua gen dupe chuyen trong cay chuyen gen.
Tir khoa .Agrobaclerium, ehuyen gen, dua hau, gen gus, kanamycin.
Dua hau {Citrullus lanatus Thumb.) l i c i y trdng quan trpng thupc hp Bau bi, cd n ^ d n gdc tii chau Phi va N a m c h i u A. Q u i dua h i u cd gia tri dinh dudng v i thuong mai cao, cd the diing an true tiep, lam salad, nude ep, keo va an hat.
Gia ii I dinh dudng ciia dua h i u khdng chi bdi vi ngpl, m i t ctia n o ma cdn vi qua dua h i u chiia mot ham lupng Idn c h i t xo, nhieu loai vitamin khac nhau v i k h o i n g chat, hat dua hau rat giau chat beo v i protein [5, 13, 17].
Mdi trong nhung khd khan trong qua trinh san xuit dua hau l i c i y r i t de bi nhiem cac benh do \ ! khuan, virus hay do nam g i y ra [4, 9, 14], anh hudng Idn ddn n i n g s u i t va chat lupng qui.
Cho ddn nay, ngoii c i c bien phap truyen thdng nhu cai thien ky thuat canh tac, phun thudc phong v i trir sfiu bdnh thi chuyen gen de tao giong dua h i u khang benh la mpt irong nhiing hudng dang d u p e c i c nha khoa hpc quan t i m nghien ciru [9, 12, 18, 19].
6 Viet Nam, nhiing nghien ciiu vd dua hau tap trung chu ydu v i o cac bien phap ndng hpc nhim nang cao n i n g s u i t , chat lupmg dua hau [15], so sanh c i c chi tieu n i n g x u i t . chat lupng
ciia c i c gidng dua hien cd tai Viet Nam [16], nghien ciiu cac yeu td anh hudng den qua irlnh thu hoach v i b i o q u i n dua hau [8]. Mdt vai nghien ciiu iing dung nudi cay md trong viec nhan nhanh cac gidng dua hau phue vu cho cdng t i c gidng ciing da dupe tien hanh [6, 7, 10, 11]. Tuy nhien. cho den nay chua cd cdng bd nao ve chuyen gen v i o cay dua hau. Vi the, nghien ciiu nay dupe thyc hifn vdi muc dich xay dung dupe quy trinh chuyen gen vao cay dua hau de phyc vu cho cdng t i c gidng.
PHUCfNG PHAP NGHIEN CUU V^t lieu
Hat dua h i u ciia c i c gidng lai H i e M y nhin F l Kinhkong (LI), F l TG939 (L2), con lai d the he F9 (cho tu thy p h i n ) ciia ddng 1 co nguon gdc Binh Thuan (qui c6 v6 den, tr6n,_to, rupt d d - D l ) va dong 2 cd ngudn gdc tii giong Fl-Tidu Long-Thing Long (qua cd vd xanh, hinh tning, to, rupt dd-D2) dupe su dung trong nghien ciiu nay,
Q u y t r i n h chuyen gen
Chiing Agrobacterium lumefaciens C58
Nguyen Thi Thanh Nga el al.
chiia vector pCB-gusplus dupe nudi lie qua dem trong mdi trudng LB Idng c6 b6 xung 50 mg/l rifamycin, 50 mg/l kanamycin d 28"C. Siu ndng dp vi khusiii di dupe thti nghi?m (0.0; 0.3;
0,5; 0,7; 0,9; 1,1) dc dinh gii anh hudng cua chiing dcii hiC'u qiui qud trinh chuycn gen vio 30 manh la m^m ciia cic ddn^ dua hau nghiSn ciiu,
\'i khuin dupe thu nh^n bing ly tam vdi vin t6c 4500 v/p trong 15 phiit va sau dd hda tan khuin uong 50nil iiidi truong MS m bd xung 200 |iM AS. 1.5 mg/I BAP, 0.5 mg/l IBA d^ tien hinh bicn nap (dung djch hda tan khuin),
Dc tim ngudng chpn Ipc thich hpp. cic choi dua hau D2 (khdng chuycn gen) dupe nudi trdn mdi trudng cd bd sung cac n^ng dp kanamycin kh;ic nhau (0. 50. 100, 150, 200 mg/l) d^ dinh gii anh huong ctia cic nong dp chit chpn loc kanamycin den khi ning sinh trucmg, phit trien cua chdi dua hau
Hat dua hiu dupe nay mim tren mdi trudng MS trong loi a 28"C, Sau 5-7 ngiy, cic minh li mam dupe cit thinh nhiing minh cd kich thudc khoing I -2 mm^ sir dyng diu ludi dao de t^o them cic vcl thuong tren mit la roi ngam vio dung djch hoa tan khuan irong tir 15,30 den 45 phiit. Tiep theo, cic manh la dupe thim khd va chuyen vao mdi trudng ddng nudi ciy (MS cd bd xung 3% sucrose, 200 |iM AS, 1,5 mg/l BAP, 0,5 mg/l IBA. (},8% agarose). Sau 3, 5, 7 ngiy. cic minh li mim dupe nia khuin trong nuoc cat khu triing cd bd xung 500 mg/l cefotaxime, thim khd vi chuyen vio mdi trudng tai sinh (MS bd xung 3% sucrose, 1,5 mg/l BAP, 0,5 mg/l IBA, 0,8% agarose, 200 mg/l kanamycin vi 500 mg/I cefotaxime), Vipc lii sinh chdi tir cac minh li mim dupe thyc hien d 25 ± 2 ^ vdi che dp chieu sing 8/16 h vi cudng dp chicu sang 2000 lux. Sau 6 tuin, cac chdi hoac cym chdi thu dupe dupe chuyen sang mdi trudng tao re hoac mdi trudng keo dii chdi (MS bd xung 0,1 mg/l IBA, 0,5 mg/l GA3, 150 mg/l kanamycin, 500 mg/l cefotaxime) irong 2- 3 tuan, tiiy thupc vao dp Idn ciia chdi.
Cac choi tai sinh dupe chuyen sang mdi trudng cim ling ra re (MS bd xung 0,2 mg/l IBA, 100 mg/l kanamycin, 250 mg/l cefotaxime) frong 2-3 tuan. Cac cay hoin chinh sd dupe chuycn sang gia the triu hun ngoii nhi kinh trudc khi tien hanh danh gii ciy chuyen gen.
Klcm tra bleu hl$n ctia g e n i u s
Bieu hi§n l^m thdi hay dn djnh ciia gen gus dupe phin tfch theo phuong phip ciia Jefferson (1987J. Cic minh li mam sau 3 ngiy dong nudi ciy vdi vi khuan Agrobacterium hoac cac bp ph$n ctia choi dua hiu dupe hinh thinh fren mdi trudng chpn Ipc dupe ngfim frong dung djch X-gluc (Tris/NaCl pH7,2 + 10 mg/ml X-gluc + 10%
Triton X-100) vi ti d nhipt 37"C trong 24-36 h Manh li ho^c ind sC dupe tiy het diq) lyc nhieu Ian vdi con 70% vi quan sit dudi kinh hien vi quang hpc. Mau dupe chuyen gen gus sS cd mau xanh cham d$c tnmg. Miic dp bieu hi$n cua gen gus dupe dinh gii thdng qua miic phin bo vi dp dim ciia miu xanh chim tren cac md kiem tra.
Phin tfch ciy chuyen gen thong qua phin frng PCR
Phin ling PCR ctia cac ddng dua hau chuyen gen dupe ihyc hien vdi cap mdi die hipu cho gen chpn Ipc npill. Trinh ty cip mdi sir dyng la: 5'-CGGCAACAGGATTCAATCTT- 3' vi 5'.AAGGGACTGGCTGCTATTGG-3', kich thudc do^n gen nptll nhin len duoc li 963 bp. Phan ting dupe tien hanh vdi chu ky nhipt nhu sau; mpt chu ky 94''C trong 4 phiit, 30 chu ky 94'*C trong 1 phiil, 5 2 ^ irong 45 giay, 72"C trong I phut. Sin phim PCR dugc dien di tren gel agarose 0,8%. nhupm vdi ethidium bromide vi quan sal dudi den UV.
K^T QUA VA THAO LUAN
Dinh gia kha ning song sot cua cay dua hau tren mdi trudng chiia chat chpn Ipc kanamycin
Vipc chpn Ipc va xic djnh nhiing ci the mang gen chuycn li khfiu rat quan trpng trong qui trinh chuyen gen. De chpn Ipc cd hieu qui, can xac dinh dupe ndng dp chit chpn Ipc phd hpp giiip cho vi?c loai bd phin Idn nhiing ca the khdng dupe chuyen gen, ddng thdi giu lai cic ci the dupe chuyen gen. Trong nghien ciiu niy, chiing tdi su dyng vector pCB-gusplus chiia gen chpn Ipc nplll khing khing sinh kanamycin.
Chiing toi di tien hinh thii nghiem inh hudng cua cac ndng dp kanamycin 0, 50, 100.
150, 200 mg/l ddn phit trien ciia cay dua hiu D2 trong 6 tuan nhim xic djnh ngudng nong dp phu hop cho viec chpn Ipc cfiy dua hfiu chuydn gen.
TAP CHI SINH HOC 2012, 34(3); 389-396 Bdng 1. Anh huong ciia kanamycin den s\r phat trien ciia choi
Nong dp kanamycin (ms/1)
0 50 100 150 200
So choi cam ijrng 30 45 45 42 48
So choisong 30 23 6 3 1
So choi chet 30 22 39 39 37
Ty le song sot (%) 100 51,1 13,3 7,1 2,1 KSt qua dupe trinh bay d bing 1 cho thay,
kanamycin cd inh hudng ro ret den kha ning sdng sot, sinh trudng, phit trien cua cic choi dua hiu dupe nudi cay Ngay nong dp 50 mg/l da CO gin 50% ciy bi chel sau 6 tuan nudi cay va tv le niy la 2,1% vdi ndng dp 200 mg/l, Trong mdt vai cdng bd \c chuycn gen vio dua hiu, ndng dp kanamycin dupe sii dyng giao ddng tir 20 mg/l ddn 125 mg/l [1, 9, 12]. Theo Bnikhin et al. (2000) [2], ngudng nong dd chit chpn loc phii hpp li khi nd loai bd dupe khoing 90% cac ciy khdng chuycn gen. Can cii vio ket qua tren, chiing tdi lya chpn kanamycin 1()0 mg/l li ngudng chpn Ipc cho cac dong dua hau chuyen gen ciia Vict Nam.
Anh hudng cua nong dp vi khuan (ODjoo) den hieu qui chuyen gen
Cic minh li mfim (30 minh cho moi ddng)
dupe ngam trong sau ndng dp vi khuan Agrobacterium OD(m 0,0; 0,3; 0,5, 0,7; 0,9, 1,1 trong thdi gian 30 phiit. Sau 3 ngay ddng nudi ciy, cic minh la mam dupe nhupm vdi dung dinh X-gluc va danh gii bieu hien tam thoi ciia gen gus Irong md bien nap. Ket qua the hien tren hinh 1 cho thay, ndng dp vi khuan anh hudng dang ke den hi?u qua chuycn gen. Hi^u qui chuydn gen tang tir 2 den 4 lan khi lang nong dd vi khuan tir OD = 0,3 den OD = 0,7.
Nong dp vi khuan ODfioo,^ 0,5 va 0.7 la thich hpp nhit cho dong dua hiu L2 va li 0,7 va 0,9 cho ddng dua hfiu D2 vdi tan sd chuydn gen dat ty le lin lupt la 86.67 vi 93,33%. Vdi cac dong cdn lai, mac dii ty le bieu hien tam thdi cua gen gus khdng cao bing nhung d ndng dp khuan ODdoo = 0,7 cung cho ket qua cao hon cac ndng dp con lai, vi vay chiing tdi lya chpn ndng dd vi khuin nay cho cic thi nghiem tidp theo
^ ,
¥ i
• OD = 0
> OD =0,?
O D ^ l . l
Hinh 1. Anh hudng ciia ndng Anh hudng ciia thdi gian bien nap den hl§u qui chuyen gen
Dd xac dinh thdi gian liy nhilm tdi uu cho viec chuydn gen, cic minh li mam (30 mau/cdng thuc) dupe lay nhilm vdi vi khuan trong 15, 30 vi 45 phut d nhiet dp phdng vdi ndng dp vi khuin (ODfioo) = 0,7. Sy cd mat tam
do vi khuin (ODeoo) ddn bieu hien Gus
thdi ciia gen gus duac phan tich sau 3 ngiy ddng nudi cay. Kdt qua cho thiy, thdi gian liy nhiem 30 phiit cho ty le bilu hien gus cao nhat d dua hau D2 (93,33%) vi L2 (86,67%) (hlnh 2). Thdi gian liy nhilm keo dai 45 phiit dudng nhu khdng cd tac dung, chi lam tang ly le bieu hien gen gus d dua hau Dl va D2 len 0,53 vi 0,54%, nhung lai lam giam ty le niy d dua hau 391
Nguyen Thi Thanh Nga et al.
LI (6,1%) va L2 (0,73%) so vdi thdi gian l i y 45 phiit cQng khdng cd sy khic bipt d i n g kl. Vl nhiem 30 phiit. Miic dO bicu hipn gen gus d cic* v§y, thdi gian Ifiy nhihn 3 0 phiit l i ngudng ddlii; tlii.i li:"ni kill (lirac lay iiliicin Irmig M) \\i dinrc CIUHI IIL- cliiiycn gen vim dua hau.
ill
Hint: ; Anh liinrriu aij llim ili:m l.iy nliicin clcii bleu lllC-n Cius
v$t nhir CUEl chil thii hail troi giai l i I tho ciia nuo
llu.
\ . l ' i ; :>
qu;i ly u ni:h
; ' i ; i
I L cu U rinh i. il cm.
dlfOV hi
r •^'
2. ."^
ijni CU.1 gcii CO
^o^^^. ini.-.i cay giC 11,111 \,:u<ha,'icnu,
iiiili liuaiii: dang k chuycn gen Trong I'T! gian doii^ nuoi Manh la mam cua 11 nap \'i'ri vi khuai pliiil va dirac dnng riud . 4 \
\ a i gen gus anh
a va la c dc
-t^lu cay
Ciic licii llurc nui iMiMg
liivu qua n ciru cua ling duoc dong dua ( O D M . I ) = 0.7 cay a 5 ngay. Sau do. nhuom X-gluc dc danh gid
gus Kcl qua phan (liinh 3) cho ihiiy,
hudng khdc nhau bicu ticli thai
ircn Irong thoi
cac miinli hicn tam biC-u hicn gian ddng cac dong
iigliicii ciru. Ty 1^ bieu liiv'ii gen gus cao cho (long 1)2 Vii L2 (tren 90%) la sau ihm gian ddng nudi ciiy 3 liojc 4 ngay Trong kin do, ddi vdi LI la 2 ngay va Dl lai khong cd bicn ddng neu lang tir 2 ngay len 4 hoac 5 ngay, Tuy nhicn. ddi viVi dong CO phan irng lot vdi .•igrobacleritim nhu D2 va 1.2 mac dii ly li; bicu liicn lam Ihdi cua gen gus cao nhat d 4 ngay nhung nguy co phiit Iricn qua miic ciia .Agrohiiclvrium a moi irudng chpn Ipc lai cao, Vi the chiing idi da lua clinn ngudng 3 ngay ddng nuoi cay de chuyen gen \ a o hai Jong tlua hau tren.
Hinh 4. Anh huong cua thdi gian ^ nhiem dgn tiieQ^^M gus A. ODtoo = 0,7; Ihdi gian lay nhiem 30 phiit; B. ODtoo = 0,7; thdi gian lay nhilm 45 phut;
C. ODsm = 0,3; thdi gian lay nhiem 30 phiit; ODsoo = 0,9; thdi gian liy nhiem 30 phut '
TAP CHI SINH HQC, 2012, 34(3): 389-396 Anh hudng cua kieu gen ddng gdc din bieu
hiSn tam thdi ciia gen gus
Kel qui bieu hipn lam thdi ciia gen gus trong cic minh li sau 3 ngiy ddng nudi cay cho thiy, kha ning Ifiy nhilm vi khuin vio mau nghien ciru li khac nhau rd r^t ddi vdi tirng dong. Trong cic ddng nghidn ciiu, ddng dua hiu D2 cho hi^u qui biln n^p la cao nhat, vdi
93,33% cac minh la c6 bilu hien t^m thdi gen gus, d^ng thdi miic dp bieu hidn ciia gen gus d D2 cung cao ban cic dong cdn lai (bing 2). Ty le bieu hipn gus t^m thdi thip nhit d LI va mire dp bieu hipn tam thdi ciia gen gus lai thip nhit d ddng Dl. Sy khac bipt ve miic dp Ifiy nhiem vi bieu hi^n ciia gen gus nhu tren cd the la do ylu t6 di truyen quylt dinh.
Sang 2. Ket qui bieu hipn tgm thdi ciia gen gus Lo thi nghiem
Dl D2 LI L2
So mau kiem tra 30 30 30 30
So mau {+) gus 23 28 20 26
Tyle(%) 76,67 93,33 66,67 86,67
Muc dp bieu hien (+) (+++)
(++) (+++) Phin tich va danh gii cay chuycn gen
Sau khi tdi uu cac yeu to chii yeu inh hudng din qui trinh chuyen gen tgm tlidi d dua hiu, chiing tdi co duac quy trinh chuyen gen nhu sau La mim 5-7 ngiy tudi dupe cat thinh nhiing manh nhd cd kich thudc khoing 1-2 mm^, rdi ngim vao dung dich hda tan khuin trong 30 phiiL Sau dd, cac minh li dupe thim khd vi chuyen vio mdi trudng dong nudi cay (MS cd bd xung 3% sucrose, 200 pMAS, 1,5 mg/l BAP, 0,5 mg/l IBA, 0.8% agarose). Sau 3 ngii^, cic mirJi la mim dupe nia khuan frong nude cat khii trung CO bd xung 500 mg/l cefotaxime, thim khd va Bdng 3 Kdt qui bien nap gen gus vao dua hau
chuyen vio mdi trudng tai sinh (MS bd xung 3%
sucrose, 1,5 mg/l BAP, 0,5 mg/l IBA, 0,8%
agarose, 200 mg/l kanamycin vi 500 mg/l cefotaxime). Sau 6 tuan, cic chdi hoac cym choi thu dupe se dupe chuyen sang mdi trudng tao re (MS bd xung 0,2 mg/l IBA, 100 mg/l kanamycin, 250 mg/l cefotaxime) hoac mdi tnrdng keo dai chdi (MS bd xung 0,1 mg/l IBA, 0,5 mg/l GA3, 150 mg/l kanamycin, 500 mg/l cefotaxime) trong 2-3 tuin tiiy thudc vao dp Idn ciia chdi. Cac ciy hoin chinh se dupe chuyen sang gii the trau hun ngoii nha kinh trudc khi tien hinh danh gia ciy chuyen gen.
LTN WT Dl D2 LI L2
MTN 30 142 162 167 107
SSCL 0 67 97 81 59
MTC 0 21 48 31 41
TSC 0 29 57 43 54
CRR 0 5 11 8 12
(+) GUS 0 0 3 0 2
(+) PCR 0 0 3 0 2
TLCG 0 0 1,85
0 LTN. Lo thi nghiem; MTN. So mau thi nghiem; SSCL. Mlu song sau chpn loc; MTC. Sd mau tao cho.; TSC.
Ting s6 chdi; CRR. S6 chdi ra rl; (+) GUS. S6 chdi bilu hicn gus; (+) PCR. So choi (+) vdi cap moi dac hi?u ciia gea npdl; TLCG. Ty le chuyin gen (%)
Ket qua chuyin gen dupe thi hien d bing 3 cho thiy, 4 ddng dua hau cho hieu qua chuyen gen dn dinh rit khic nhau. Sau 4 tufin nudi ciy, ty le cic minh la mam song sdt tren mdi trudng chpn Ipc giao ddng tir 47,18% (Dl) den 59,88%
(D2); ty 1§ cic minh la mim tii sinh thinh chdi
til cac minh sdng sdt tren mdi trudng chpn Ipc giao ddng tir 31,34% (Dl) din 69,49% (L2).
Tdng sd chdi thu dupe d 4 ddng dua hau nghien cim giao ddng tir 29 (D2) din 57 (D3) chdi, ty le tao rl tir cic chdi thu dupe giao ddng tii 17,24% (Dl) din 22,22% (L2). Sau khi nhupm 393
Nguyen Thi Thanh Nga et al X-gluc thu dupe 5 ddng c i y (+) vdi gus, trong
dd 3 ddng dupe tii sinh ttr D2 v i 2 ddng lii sing tir L2. Ty 1? chuycn gen d ddng D2 li 1,85% v i o L2 la 1,87%. Ddng D l v i LI khdng tpo dijpc c i y chuycn gen Kcl qui n i y m^t lin niia khfing djnh khi ning chuycn gen d c i c ddng khic nhau li khdng giong nhau.
C i c c i y dupe chuyin gen 6n dinh deu cho
phin ling duong tinh vdt gen gus. Muc dp bieu hipn cila gus khi m^nh, m i u xanh d i m xuat hi$n d c i rl, thin cfiy, li cfiy, dinh choi, hoa. Ket qui di$n di s i n phim PCR vdi c$p moi d i e hi?u ciia gen nplII d 5 ddng dua hau chuyin gen cho thay, cd sy xuit hi§n m^t vpt D N A vdi kich thudc xap xi lOOObp tuong ting vdi kich thudc ly thuyet ctia doan gen nptll dugc nhfin bin (hinh 5).
Ilnih .•> lluih anil cliuvcn gen cay dua liau
' t.ic nianli l,i mam sau .1 ngay di'ing nudi ciy; B. Bicu hicn gus ben \Trng a cay I ill >.-,m pham I'CK 5 iliiiig ciiy chuycn gen gus vai cap moi dac hieu cua gc-n nptU ing dua hau cluiyen gen gus ihu dime; (+). dcii chimg dutmg).
I iicu qua cliuycn gen v;io dua hau phy Ihudc o rat nhicu ycu id nhu kieu gen, chiing vi i.ian, \ector sir dung va cac budc trong qud
;,li chuyen gen.... Gen gus da duoc diing de lai boa quy trinh chuyen gen \ a chuyen iiih cong tren nhicu gidng dua hau khdc nliau :;i suai chuyen gen duoc (hdng hao dao dong
PT
li' 7 , ' V « ' ^
m^
tir 0% den 10.28% [14, 3, 9], Quy trinh chuyen gen ciia chiing tdi dp dung tren cac dong dia phuong ciing ihu dupe kct qua tucmg lu va vi vay, quy Irinli nay cd the ling dung d e chuyen gen muc tieu khdc nhau vao hai dong dua hau D2 vd L2. Quy trmh chuycn gen d u a c Irinh bay o liinh ('.
Hinh 6. Quy trmh chuyen gen v i o c i y dua h i u
A. Hat nay mim tren moi truong MS, B, Manh li mim trfin mdi trudng ddng nudi ciy; C. Chdi hinh thanh sau 7-10 ngay tren mdi tnidng tai sinh; D. Choi hinh thanh sau 4-6 hiin tren mdi trudng tii sinh; E. Chpn loc chdi tren mdi trudng ra re; F Cay con trdng tren gia Ihl triu hun.
TAP CHi SINH HOC 2012, 34(3): 389-396 KtT LUAN
Di nghidn cdn thinh cdng guy trinh chuyin gen gus vao ciy dua hau cd n^udn goc Vi?t Nam thdng qua chiing vi khuan Agrobacterium lumefaciens CV58 chiia vector pCB-guslus. {)uy trinh niy cd thi dupe sii dyng cho vi?c chuyin gen myc tieu mang muon vao dua hau Vipt Nam.
TAI LIEU THAM KHAO
1. Akashi K., Morikawa K., Yokota A., 2005.
Agrobacterium-mediatcd transformation system for the drought and excess light stress-tolerant wild watermelon (Citrullus lanatus). Plant Biotechnol., 22(1): 13-18.
2. Brukhin V., Clapham D., Elfstrand M., Amol S. v., 2000. Basla tolerance as a selectable and screening marker for transgenic plants of Norway spruce. Plant Cell Rep., 19:889-903.
3 Cho M. A., Moon C. Y.. Liu L. R., Choi P.
S., 2008. Agrobacterium - mediated transformation in citrullus lanatus. Biologia Plantarum, 52(2): 365-369.
4. Compton M. E., 1999. Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon.
Plant Cell, Tiss. Org. Cult., 58: 185-188.
5. Compton M. E., Gray D. J., Gaba V. P., 2004. Use of tissue culture and biotechnology for the genetic improvement of watermelon. Plant Cell, Tiss. Org. Cult., 77:231-243.
6. Lam Ngpc Phuong, Nguyen Bao Ve, Dd Thi Trang Nha, 2005. Nhan chdi dua hiu lam bpi {Citrullus vulgaris Schrad.) tir chdi dinh tren mdi trudng MS co cytokinin vi auxin. Tap chi Ndng nghiep va Phat trien ndng tiidn, 2; 30, 31 & 35.
7. Lim Ngpc Phuong, Nguyen Bao V?, 2006.
Anh hudng cua chit dilu hda sinh trudng thuc vat IBA, BA va than boat tinh den sy tao re ciia choi dua hau tam bpi in vitro {Citrullus vulgaris Schrad). Tap chi Ndng nghiep va Phit iriln ndng thdn, 2: 39-44.
8. Le Vin Viet Min, Trfin Qudc Huy, 2008.
Nghien ciiu keo dai thdi gian bao quin dua
hau tuoi cit mieng bing phuong phap xii ly ozone. T^p chi phit trien Khoa hpc vi Cdng nghe. 9(11): 77-82.
9. Li J., Tang Y., Qin Y., Li X., Lt H., 2012.
Agrobaclerium-mediated transformation of watermelon {Citrullus lanatus). Afr. J.
Biotechnol., 11(24): 6450-6456.
10. Nguyen Thj Phuong Thio, Ninh Thi Thio, Vu Thj Ha, 2010. Nghien cihi nhan nhanh in vitro ciy dua hau {Citrullus lanatus). Tap Chi Khoa hpc vi Phit trien. Trudng Dai hpc Ndng nghi?p Ha Npi, 3(8): 418-425.
11. Nguyen Thj Thanh Nga, Nguyin Tudng Vin, Chu Hoang Hi, Le Trin Binh, 2010.
Nghien ciiu quy trinh tii sinh in vitro cay dua hiu {Citrullus lanatus Thumb.). Tap chi Cdng nghp sinh hoc, 8(3B): 1353-1358.
12. Park S. M., Lee J. S., Jegal S., Jeon B. Y., Jung M., Park Y. S., Han S. L., Shin Y. S., Her N. H., Lee J. H., Lee M. Y., Ryu K. H., Yang S. G., Ham C. H., 2005. Transgenic watermelon rootstock resistant to CGMMV (Curcumber green mottle mosaic virus) infection. Plant Cell Rep., 24: 350-356.
13. Sultana R. S., Bari M. A., 2003. Effect of Different Plant Growth Regulators on Direct Regeneration of Watermelon (Citrulus lanatus Thumb.). Plant Tiss. Cult., 13(2):
173-177.
14 Suralman F., Huyop F., Waguan A., Rahmat Z , Ghazali H., Parveez G. K. A., 2010. Cotyledon with Hypocotyl Segment as an Explant for the Production of Transgenic Citrullus vulgaris Schrad (Watermelon) Mediated by Agrobacterium lumefaciens. Biotechnol., 9(2): 106-118.
15. Trin Dinh Nguyin, Trin Thi Ba, 2008.
Nghien ctiu bien phip kj thuat tao trai dua hau hinh vuong phyc vu chung let. Tap ehi Khoa hpc Cdng nghe, D?i hpc Cin Tha, 9:
128-135.
16. Trin Thj Ba, Vo Thi B ich Thiiy, 2004. So sinh nang suit vi phim chat mot so gidng dua hiu tai ngoai thanh thinh phd Can Tha tir nim 2(101-2003. Tap chi Khoa hpc Cdng ngh§, D?i hpc Cin Tho, 2: 131-137.
Nguyen Thi Thanh Nga et al.
17. Wchner T. C , 2008, Watermelon, (p. 3 8 1 - 2011. Generation of transgenic watermelon 418). In: J. Prohens and F. Nuez, eds. resistant to Zucchini yellow mosaic virus Handbook of Plant Breeding. Vegetables I: and Papaya ringspot virus type W. Plant Asleraceae, Brassicaceae, Chcnopodiaceae Cell Rep., 30(3): 3 5 9 - 3 7 1 .
and Cucurbitaccae. Springer , 9 ^ h o n g W. H., Jie Z. P., Chen X. J., Huai Z., Sciencc+Busincss LLC, N e w York, NY, g h e n g Z. H., 2 0 0 3 . Virus Resistance in ''26 p. 17. Transgenic Watermelon Plants Containing a 18. Yang C. F . . Y u T , A„ Chiang C . H . , W u H . W M V - 2 Coat Protein Gene. J. G. G.,
W., Li C. M., Chen J. H., v i Yeh S. D„ 30(1): 70-75,
OPTIMISATION OF GENETIC TRANSFORMATION PROCEDURE FOR WATERMELON (Citrullus lanatus Thumb.)
Nguyen Thj T h a n h N g a ' , H o Manh T u o n g ' , Pham T h i V a n ^ Nguyen Tuong Van', Chu Hoang Ha^, Le Tran Btnh'
' " T a y Bac University
"'Institute of Biotechnology, V A S T SUMMARY
In this study, Ihe binary vector pCB-gusplus carrying nptll and gus - intron genes as selectable and reportable markers was used to optimize ^gz-ofcacrcrium-mcdiated gene transformation protocol of four Vietnam watermelon lines, Agrobacterium lumefaciens CV58 habouring pCB-guspIus in the concentration of ODhoo 0,6-0.8 was infected into 5-7 day cotyledons for 30 minutes and co-cultured for 3 days. The transformed leave materials was cultured in the selectable medium containing MS salts and vitamins supplemented with 1.5 mg/l BAP, 0.5 mg/l IBA, 200 mg/l kanamycin v i 500 mg/I cefotaxime. Regenerated shoots was rooted in MS medium added with 0.2 mg/l IBA, 100 mg/I kanamycin and 250 mg/l cefotaxime There was a significant difference in die transformation frequency among watermelon lines. Fl TG939 line showed the transformation frequency of 1.87%), following F9 Tieulong - Thanglong of 1.85%, Fl Kinhkong and F9 Binhthuan lines showed the yansfwrnation frequyency of 0%. The analysis of the obtained lines using GUS histochemical assay and PCR reaction with specific primers ointpW gene had conflrmed the presence of gus gene into S transgenic lines.
Keywords. Agrobacierium, Cilrullus lanatus, gene gus, genetic transformation, kanamycin.
Ngdy nhdn bdi: 25-2-2012
