1

Phn I
M u
I. t vn
Trong khi s an ton ca con ng-i i vi thc phm bin i
gen vn cn ch-a -c khng nh chc chn th vic nghin cu h
thng chuyn gen hiu qu cho cc loi hoa cy cnh t chuyn
-c cc gen mong mun to ra cc c tnh mi l (mu sc, cu trc
hoa, h-ng thm, tui th ca hoa cm, cc c tnh khng su v
bnh hi, chng chu vi iu kin bt thun.) lun l mong mun
ca cc nh chn to ging.
Lilium thuc h liliaceae, c khong 85 loi phn b ch yu cc
vng n i v cn nhit i thuc pha bc bn cu lily l mt trong cc
loi hoa p ang rt -c -a chung trn th tr-ng hoa hin nay. Vi
kiu dng p sang trng, mt s chng loi c h-ng thm quyn r v
bn hoa rt cao (915 ngy), d thu hoch, bo qun trn th gii
lilium cng vi tulip, freesia l ba loi hoa dang thn c ch yu, quan
trng trong ngnh hoa th-ng mi, chim 24% gi tr sn phm hoa
th-ng mi (Robinson v Pizarobady, 1993) Vit Nam hin nay, hoa
lily -c trng thnh cng nhiu tnh n-c ta nh- Lm ng, H
Ni v hu ht cc tnh min Bc v hiu qu kinh t rt cao, c th
xut khu quy m ln. Tuy nhin iu kin kh hu nng m, m-a
nhiu ca n-c ta to iu kin rt thun li cho cc loi c di pht
trin y l mt trong nhng yu t hn ch ch yu n sn xut
hoa lily. Chnh c di lm cho gim nng xut, phm cht, c bit n
cnh tranh dinh d-ng vi cy. c th tr b c th cn tn nhiu
cng lao ng, gim bt cng lao ng con ng-i phi dng n
cc loi thuc tr c nh-ng chnh iu ny gy nh h-ng khng nh
n cy hoa lily. c th va tr -c c m li khng gy nh h-ng
n cy th vic nghin cu chuyn gen khng thuc tr c PPT vo
cy hoa lily l i hi ca sn xut , xut
Hoa lily thuc Liliaceae l mt trong nhng loi hoa p v c gi
tr trn th tr-ng hin nay. Cc n lc chnh trong nghin cu chuyn
gem vo cy lily -c thc hin bi cc ph-ng php chuyn gen trc
tip nh- bn gen (Nishihara v cng s, 1993; Sanford v cng s ;
2
1993 ; Wilmink v cng s, 1995 ; Tsuchiya v cng s, 1996), xung
in (Miyoshi v cng s, 1995) v gn y to -c cy Lilium
longiflorum chuyn gen nh ph-ng php bn gen. Trong mt s nm
gn y, s thnh cng trong nghin cu chuyn gen nh vi khun
Agrobacterium -c cng b trn mt s loi thuc h Liliace bao gm:
Asparagus officinalis (Kiasaka v Kameya, 1998), Allium sativum (Kondo
v cng s, 2000), Allium cepa (Eady v cng s, 2000), Agapanthus
praecox (Suzuki v cng s, 2001) v Muscari armeniacum (Suzuki v
Nakano,2002). Gn y nht, Y. Hoshi v cng s nghin cu thnh
cng quy trnh to cy chuyn gen cho cy Oriental hybrid lily, Lilium
ev. Acapulco bng vi khun Agrobacterium.
Cc kt qu nghin cu chng t chng ta hon ton c th to
-c cy hoa lily.
Cc kt qu nghin cu cho thy, cc i t-ng thc vt c b gen
ln hoc l mc ti a bi ln nh- cy hoa cc, hoa ng tn s kh to
-c cy chuyn gen hn l cc loi c b gen hay mc ti a bi thp
hn (Elomaa et al, 1993; Robinson & Froozabady 1993). Hin ch-a c l
gii cho hin t-ng trn tuy nhin, c th do cc b gen nh hn th kh
nng cc gen l c th -c ci vo vng hot ng cao hn. (Elomaa et al
1993; Robinson & Froozabady 1993). S khng thnh cng trong biu
hin cc gen -c chuyn c th l kt qu ca s ng c ca DNA
(Eady et al. 1995a) hoc s metyl ho (Torres et al 1993) khng cho
php cc gen -c ci biu hin, chnh v vy vic nghin cu chuyn
cc gen chn lc hoc gen ch th tr-c khi nghin cu chuyn cc gen
c tnh trng mong mun cho cy hoa lily l cn thit v c tnh gi m
cho cc nghin cu tip theo.
Mc tiu ca ti l xy dng -c nhng khu c bn trong h
thng k thut chuyn gen khng thuc tr c cho cy hoa Lily nh vi
khun Agrobecterium tumefaciens.
t -c mc tiu ra trn chng ti cn han thnh cc yu
cu sau:
1. Xy dng h thng ti sinh hon chnh cho cy lily
2. Xc nh ng-ng gy cht ca PPT i vi mu vy c, lt ct
thn v nh sinh tr-ng ca cy hoa lily.
3
3. B-c u th nghim chuyn gen bar khng thuc tr c vo
cy hoa lily thng qua vi khun Agrobecterium tumefaciens.
4
Phn II
Tng quan ti liu
2.1 Gii thiu chung v cy hoa lily
2.1.1 Ngun gc
Cy hoa lily c ngun gc t Trung Quc, Nht Bn, Nam Triu
Tin, California (M) v mt s ni khc.
2.1.2 V tr phn Ioi thc vt
Trong h thng phn loi thc vt cy hoa lily -c xp vo nhm
cy mt l mm (Monocotyledones), phn lp hnh (Lilidae), b hnh
(Liliales), h hnh (liliaceae), chi lilium (theo D-ng c Tin, V Vn
Chi, 198).
Cy hoa lily l cy thn tho, thn dng hnh c vy. Thn th-ng
mc n c l. L lun c hnh mi mc hay hnh vch t khi rng, hnh
tim v mc xung quanh thn. Hoa l-ng tnh, kch th-c ln mc nch l
hay ngn, c rt nhiu mu sc khc nhau. Hoa c th mc ring l
hay thnh cm gm nhiu hoa, bao hoa 6 mnh dng cnh, nh 6, bu
hnh tr, u nhy hnh u, chia 5 thy. Qu nang c 3 gc v 3
nang, qu nang c nhiu ht.
2.1.3. Ging .
2.1.3.1 - Cc nhm ging trng trt
Cng tc to ging, ci tin ging hoa lyli -c thc hin trn 100
nm nay do nn ging rt phong ph. Trn th tr-ng v trong trng
trt th-ng thy 3 nhm: dng lyli thm, dng chu lai, dng lyli ph-ng
ng lai.
a. Dng lyli thm lai (the longi florum hybri!"
Gm cc ging -c sinh ra t lyli thm v lyli i Loanv cc ging
tp giao ca chng, cy cao t 120 140cm, mt s c th n 200cm,
l hp di hoc hnh kim s ra, gn vo thn, khng c cung l. N
rt to di t 12 18cm, khi v-n ra th hi trc xung, nm ngang ra,
chch ln hoc ng thng. Hoa c hnh loa kn, hoa trng tinh khit
v c v thm nng. Lyli thm t khi bng trng n khi ra hoa khong
12 14 tun, tu theo ging v nhit khng kh c bin ng nht
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nh. Thi gian x l lnh ph ng vi lyli thm ni chung t 4 6
tun nh-ng c thay i t nhiu tu theo ging v thi tit lc thu
hoch c. Cc ging trong n-c th-ng thy l: Hong hu tuyt(Sno
Queen), White Fox, Avita, Lilium formolongi. V ng xun Qung
ng trng rt nhiu dng lai ca ging ny.
b. Dng ch#u lai(the a!i!tic hybri!"
L nhm ging -c chn ra t mt s b m ca lyli Chu hoc
t cc dng lai ca chng .
cc b m l: quyn au xuyn bch hp, quyn au i to, hoa kn
triu tin, sn an. cy cao t 80160(cm) l hp di hnh kim s ra,
gn vo thn. n hoa thuc loi nh nht di 5,512,5(cm)
n hoa v-n ra theo cc h-ng ln, v-n ra ngoi v r xung, hnh dng
hoa cong cun li, hnh ci bt rt t c hnh hoa kn, mu sc rt
phong ph ch yu l mu vng, mu cam, hoa khng thm, t khi
trng n khi ra hoa t 816 tun, ging v nhit khng kh nh
h-ng n thi gian sinh tr-ng , thi gian x l lnh phi vi c ging t
68 tun, ging v thi gian v thu hoch ging nh h-ng rt ln n
thi gian x l lnh ph ng.
cc ging th-ng trng Trung Quc l: trung tm n-c(novar center)
mu vng, ging sa mc(cordclia) mu vng, pollyauma mu vng, Elite
mu cam, Brunello mu cam.
c- Dng lai $h%ng &'ng(the oriental hybri!"
gm cc ging thin h-ng, lc t(con h-u) cc ging c ngun gc t
hoa kn Nht Bn v cc ging thuc dng lai gia cc ging trn vi hoa
kn H bo cy cao 60240cm, l t -ng i to, hnh trng hoc hnh
kim s rng, l c cung r v khc vi dng loi hoa kn thm v lai
chu. ln ca n thuc loi hoa ln nht -ng knh t 1520 cm, n
a s mc thng rt t nm ngang. Hoa c hnh ci bt hoc hnh
sao, mu sc hoa rt phong ph mu , phn hng, v mu sc
trng l chnh, hoa c mi thm nng. t khi trng n khi ra hoa 1220
tun thay i tu theo ging v thi v. tnh khc nhau v thi tnh v
thu hoch cng nh h-ng ln ti thi gian s l lnh phi ng ni chung
khong 48 tun.
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cc ging trong n-c th-ng thy l: stargazer(sao hong tc), ging
nguyn soi(Acafulco), Sorbonne, Merostar, Siberiamaqssa, starfighter,
v tiber.
hin nay trn th gii ang m rng din trong cc ging dng lai thm
v lai chu (LA)v dng lai thm vi ph-ng ng (LO) dung ho
-u im ca hai ging
2.1.3.2- Cc ging trng trt ch yu
a-Dng lai loa ()n thm
Avito: trng vo v ng xun cy c th cao 120cm, thn cng, l di
hp, mu xanh m, l pha trn ngn, thng ln trn hnh dng p.
N to va di 1315 cm y, mp nm ngang hoc chch lu trn.
Hoa hnh loa kn, m to, p mt, cnh hoa xp cht, u cnh hi
cong ra ngoi, mu trng trang nh, l loi hoa ct tt, tnh chng chu
km, nhit hi cao n teo li, t l bi dc cao, thi gian sinh tr-ng
ngn li, cung hoa ngn.
SnoQueen cy cao 60115 cm cy t-ng i cng, l di hp, mu
xanh m, l pha trn cun li, n hoa di 1315 cm, nm ngang hoc
hi cong xung, ngoi hnh p, hoa n ming to, dng p, cnh hoa
xp t-ng i th-a,cnh ngoi cun ng-c li cnh pha trong hi cong,
mu trng l loi hoa ct t-n i p. tnh chng chu t-ng i yu
White fox cy cao, trng vo v ng thn cao ti 130 cm thn rt cng
l di hp, mu xanh m hi trc xung n tova 1315 cm, nm
ngang hi r xung, n to y mp, ngoi hnh p,hoa hnh loa kn,
ming to, hnh dng p, cnh hoa xp t-ng i kht cnh ngoi cnh
trong u cong ra ngoi mu trng, p, l ging lm hoa ct rt tt,
l ging rt -c -a chung , tnh chu nng km, nhit caon hoa
nhiu, t l bi dc cao, phm cht hoa km, gings ny t-ng i
mn cm vi nh sng yu.
Lilium formolougi l ging mi nhp vo n-c nm nay v pht trin rt
mnh c c sc l ging ct cnh trng bng cy con. cy cao to
100180 cm trng vo v ng c th cao ti trn 2m , thn rt cng, l
c nhiu dng, t hnh kim s dc n hnh trng, mu l t mu xanh
xm n xanh m, n t-ng i to t 1316 cm, a s thng ng,
rt t nm ngang n to mp, dng p, hoa hnh loa kn, ming khng

to cnh hoa xp y, cnh nghing ra ngoi rt t gp cong cun, c
kh nng chu nng l ging ct cnh tt.
ging ny l ging lai gia hoa kn thm vi hoa kn i loan, qua chn
lc bi d-ng n nay hnh thnh mt nhm nhiu ging, chia ra loi
hnh trng bng cy con, loi hnh trng bng c v loi hnh kim
dng, cc ging th-ng thy l : Ngn h , Bch phong, bch m, tuyt
sn.
Dng loa kn chu nhit l dng do trung tm nghin cu khoa hc vin
nng nghip trng khi to ra mi y c th trng vo v h thu khu
vc hoa nam, t l ra hoa v phm cht hoa ct u cao.
b-Dng lai loa ()n ch#u
Nove cento: l ging trng trong n-c nhiu nm, sinh tr-ng kho chng
bnh mnh khng mn cm vi nh sng kh nng thch ng rng cy
khng cao khong 110 cm nhit , nh sng nh h-ng ln n chiu
cao cy, nhit qu cao, qu thp, nh sng mnh u c ch pht
trin chiu cao. L ging tt trng trong chu v ct cnh, thn cnh
rt cng, l di hp, l pha trn ngn, nm ngang, mu xanh m. N
to v ngn di ,58,4 cm nhiu n c mt s nhnh th hai c nh d
ra hoa, hoa hnh sao, t-ng i nh, cnh hoa ngn v dy hi cong
ng-c li, xp kht nhau. Hoa mu vng kim u hoa p, thi gian
sinh tr-ng ngn. vng ng bng Chu Giang trng vo v xun 10
tun ra hoa.
Cordelia: trng vo v ng xun cy cao 110 cm, thn cnh cng, c
gc cnh, l hnh kim x, nm ngang hi r xung, l xanh bng, hi b
chy. n di 8,510 cm t-ng i nhiu n c cnh cp hai t-ng i r.
hoa hnh sao, ming rng, cnh dy,pha gia v d-i rng , pha trn
t-ng i di hi cong ng-c li. Gc cnh c mt t m nh mu nu,
t gc cnh ko di n gia cnh c vt m mu vng cam . cnh
hoa xp th-a, tch ri nhau, thi gian sinh tr-ng ngn. ng bng
Chu Giang trng vo v ng xun t 1014 tun ra hoa khng mn
cm vi nh sng yu .
Pollyanna: qung ng th-ng gi l ly ly vng. dng cy dng hoa mu
sc hoa rt ging Cordelia nh-ng c rt nhiu tnh trng hn cordelia l
mt ging trong dng lai chu mu vng -c -a chung nht, c din
8
tch trng ln nht. ging ny sinh tr-ng kho cy cao n 120 cm l
xanh t-i hi h-ng ln trn. n t-ng i to , t nhnh th hai chng bnh
chy l v bnh mui tro mnh. thi gian sinh tr-ng t-ng i ngn.
vng ng bng Chu Giang trng vo v ng xun. sau 1012 tun ra
hoa c sc chu nng nht nh.
Elite: cy c th sinh tr-ng kho, cao 110120 cm , thn cnh cng, l
di, hnh kim, mu xanh ti, mc ngang, n mu cam, di 810 cm,
khng c cung cp hai. hoa hnh cc nng, hnh sao, t-ng i to,
cnh hoa t-ng i dy, xp kht nhau, cnh hoa mu cam, gc c
mt t m en nh thi gian sinh tr-ng ngn. ng bng chu Giang
trng vo v ng xun sau 1013 tun ra hoa. ging ny vng m v
nni khng kh b nhim bnh mi tro rt nng, c th dn n
hu dit. rt mn cm vi nh sng yu.
Brunello: cy sinh tr-ng kho, cao 110 cm thn to cng, l di hnh
km s mu xanh m, bng. n di v to mu cam, di 8,511 cm c
nhnh cp hai. hoa hnh sao to, cnh dy pha ngn hi cong, xp kht
nhau, cnh mu qut sng, u, khng c m, vt. thi gian snh
tr-ng ngn, vng ng bng Chu Giang gieo trng vo v ng xun
sau 911 tun ra hoa, dng mu cam ca ging ny c tnh chng chu
mnh khng mn cm vi nh sng yu l ging tt. cm nhim bnh
mui tro nh.
dng lai chu, ngoi cc ging mu vng, mu camcn c mu ,
mu phn hng, mu trng v nhiu mu nh- ging: Amarono, Phanro
nh-ng trung quc t thy .
c- Dng lai $h%ng &'ng.
Stargazer(loa kn la, hong tc.)
cy cao 60100 cm , thn to, cng, l hnh kim s, hnh trng di 910
cm, rng 3 cm l chch ln trn, n hoa to, di t 1113 cm, mp, thng,
dng p. hoa hnh sao, p, cnh hoa xp t-ng i kht, cnh trong
cnh ngoi cong ra pha ngoi, mp l l-n sng t gia cnh tr ln c
git nh- git sa ni ln , ,mu cnh , c vin trng hp. y l
ging ct cnh v trng trong chu tt, -c trng rng ri pha nam
trung quc, l m,t trong nhng ging ch lc trung quc. tnh chng
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chu kh, khng mn cm vi nh sng yu.Cn l-u hin t-ng kh u
l. Hoa nam thi gian sinh tr-ng t 90110 ngy.
Akapulco: cy cao to, 100120 cm thn cnh cng l di hnh kim, ngn
l ko di,mc ngang. Thn v-n di nhanh, khong cch cc l th-a. n
hoa to mp di 1315 cm. Hoa hnh sao hoc hnh cc nng, p,
u ngn cnh hi cong ra ngoi, mp l-n sng. T gia cnh tr ln c
git lmu ni ln cnh hoa mu sng u n. y l ging hoa
ct tt , n v hoa c dng p, mu sc p, -c trng nhiu
pha bc Trung Quc nhit thp d lm n bin hnh. Hoa nam
trng vo v thu ng thi gian sinh tr-ng 8090 ngy.
Siberia: cy cao 100110 cm, thn cnh cng l hnh kim s, mc ngang
hoc trc xung, n hoa to mp, p mt di 1214 cm. Hoa hnh sao
cnh cong lt ra ngoi, mp cnh l-n sng cnh hoa p mu trng.
Ging ny tnh chng chu mnh l loi ging hoa ct cnh rt qu, gi
cao.
Sorbone: cy cao 80100 cm, thn cnh cng, l di hp hnh kim di
1213 cm, rng 3,33,5 cm, h-ng ln pha trn, n hoa to di1213 cm,
mp p. Hoa hnh chn nng, ming rng, cnh trong, ngoi mc
ngang hoc hi cong ra ngoi, t gia cnh tr xung c git sa mu
, cnh hoa mu phn hng, mp cnh c vt trng hp.(bn dch
hoa lily)

2.1.4 c tnh sinh vt hc hoa Iy Iy
*+c tr%ng h,nh thi.
Ly ly l cy thn tho lu nm, thn cao t 50200 cm. Phn d-i
t l thn vy, mu trng hoc mu hng nht, pha ngoi khng c
mng bao bc, nn gi l thn vy trn (khng v, thn vy l c ging
trng.)
B phn trn mt t l thn, l ,v hoa,qu, l phn th-ng phm ca
lyli. R lyli c hai tng, r mc gc ca thn vy gi l r gc, to,
mm, c ngay trn c ging hoc mc ra ngay sau khi trng. R mc
ni tip gip gia c v thn trn mt t gi l r thn. a s ging
sn xut nhng thn vy( c) mc ra t r ny lm thc liu nhn
ging.
10
L lyli th-ng mc cho nhau , hnh di, hnh trng.khng c
cung hoc cung rt ngn, mt s t ging nch l c mm, c th
dng nhn ging.
Hoa lyli to mc ring r mc thnh chm hoc thnh cm trn nh ngn
thn.Mi c ch c mt cnh hoa, mm thn ca lyli b rng i, hoc
sau khi ngt ngn khng th mc ra thn mi, cngkhng th hnh
thnh cnh hoa khc. Nu mun ct hoa th khng -c bm ngn, hoa
c hnh loa kn, hnh phu, hnh sao, hnh ci chn nng. Mu sc
hoa rt phong ph, p c mu trng, mu vng lc, mu phn hng,
mu cam, mu , mu tm v nhiu mu. Mu cnh th-ng l n
sc, hoc c m mu nu, mu tm mt s t c h-ng thm.
Cnh hoa su ci, hai vng, mi vng ba ci, nh c su ci, vi nh di,
ti phn to c mu tm, ,mu vng. gia c trc hoa nh u phnh
to.
lnh cng di th ra hoa cng sm.(bn dch hoa lily)
2.1.5 Gi tr kinh t v gi tr s dng.
Loa kn c hoa to mu sc phong ph, h-ng thm l loi hoa
-c th gii -a thch, l loi hoa c l-ng l-u thng ln trn th tr-ng th
gii, sn l-ng ng hng th 4 th 5 ca hoa ct.
Loa kn t-ng tr-ng cho s tt lnh, on kt, m mn nn cng -c
ng-i Trung Quc -a chung v tr thnh loi hoa pht trin mnh, sn
l-ng v gi tr ngy cng cao.(bn dch hoa lily)
Hoa t-i l mt sn phm hng ha c bit ca ngnh Nng
Nghip. Hin nay trng hoa tr thnh mt ngnh sn xut pht trin
khp th gii. Trong hoa loa kn ng gp mt phn khng nh. Vi
-u th rt phong ph v hnh dng, mu sc, h-ng thm. Hoa loa ken
c gi tr kinh t cao trong sn xut ln mt tinh thn.
Bn cnh gi tr phong ph v tinh thn th hoa loa kn cn -c
s dng tch chit v tinh ch du thm phc v cho mt s ngnh
cng nghip, m phm, bnh ko. i vi y hc, loi ny cng c mt gi
tr nht nh. Loi hoa bch hp L.broniFEbron c tc dng cha
bnh, c th s dng lm thuc.
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Vi gi tr nh- vy hoa loa kn ha hn mang li mt ngun doanh
thu ln cho ngnh sn xut, kinh doanh mt hng ny. Hin nay, trn
th tr-ng Vit Nam, cc loi hoa loa kn ang -c bn ph bin t
10.000 n 15.000 ngcnh (theo iu tra ca thi bo kinh t). Nh-
vy u t- vn vo pht trin kinh doanh mt hng ny s em li cho
sn xut li nhun rt cao.
2.1.6 Bnh v cch phng tr:
a, Phng tr su bnh
trung Quc cc bnh th-ng thy l: bnh mui tro, su th-ng
thy l rp
b, Phng tr bnh
Trng im l bnh kh l v mui tro
Vi bnh kh l: sau khi trng 30 ngy bt u c bnh, bnh
tp trung phn gia l v nh ngn lm cho l bin nu, sau khi kh
th cun li bin thnh mu nu en. N b hi thi k u bin thnh
mu nu vng n mu nu en ri b rng, c th p dng cc bin
php phng hoc gim bnh.
Trong thi gian ra n dng l-i cn quang che bt nh sng, h bt
nhit v khng ch m, dng tay tch cc l b bnh cho n l ra
gim bnh ly sang n.
Vi bnh mui tro th phun thuc phng: bch khun thanh,
Dithan..ng thi qut thng gi .
2.2. C!"#$ G%$ T&C '(T
2.2.1. Chuyn gen thc vt v nhng Ii ch i vi ci to ging
cy trng
Vo nhng nm u ca thp k 80, cc nh khoa hc khm ph ra
vic chuyn gen hay cn gi l bin np gen vo thc vt to ra c
tnh di truyn mi nh- khng bnh hay vt gy hi. Nhiu k thut chuyn
gen ra i, -c hon thin v p dng rng ri nh-: bn gen trc tip, thng
qua Agrobacterium, nh vo xung in hoc ho hc. Trong , 2 ph-ng
php u t ra hu hiu hn c. Vo nm 1983, cy bin np gen u tin
ra i l cy thuc l khng khng sinh. Nm 1985, mt s cy chuyn gen
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khng su, bnh virus v bnh nm ln lu tin -c -a ra th nghim
ngoi ng rung.
n nay, s bin np gen vo cy trng khng cn l vn phi
tranh ci na m tr thnh k thut thng dng trong to ging cy trng.
c hn 50 loi gen -c chuyn vo cy trng v t nht khong 400 loi
-c kim tra ngoi ng rung. i vi cc loi cy rau, chuyn gen thnh
cng c chua, c rt, khoai ty, rau dip, cn ty, sp l, d-a chut, du
ty, ci xanh, ci bp, mng ty,. cc cy trng ng cc nh- la n-c, la
mch, la m, ng v c nhng cy cng nghip nh- cy bng vic nghin
cu chuyn gen cng thu -c nhiu kt qu kh quan 1.
Nhng cy trng -c chuyn gen vn ging cy trng truyn thng
nh-ng chng c thm mt s c im -c ci thin. V vy ng dng k
thut chuyn gen thc vt nhm ci to ging cy trng em li nhiu li
ch r rt trong pht trin sn xut nng nghip v phc v i sng con
ng-i. C th im nhng li ch nh-:
- Tng sn l-ng
- Gim chi ph sn xut
- Tng li nhun nng nghip
- Ci thin mi tr-ng
Ngy nay, cc nh khoa hc ang h-ng ti to nhng cy chuyn gen
th h th 2 c c im tng gi tr dinh d-ng hoc c nhng tnh trng
thch hp cho cng nghip ch bin. Nhng li ch ny h-ng trc tip hn
vo ng-i tiu dng nh-:
- La go giu vitamin A v st
- Khoai ty tng hm l-ng tinh bt
- Vacxin n -c ng v khoai ty
- Nhng ging ng trng -c trong iu kin ngho dinh d-ng
- Du n c li cho sc kho t u nnh v ci du no1
2.2.2. Nhng thnh tu chuyn gen thc vt trn th gii v trong n-c
Trn th gii, din tch trng cy chuyn gen tng t 1, triu ha nm
1996 ln 11 triu nm 199, 2,8 triu nm 1998, 39,9 triu nm 1999 v ti hn
44 triu nm 2000. Cc quc gia trng cy chuyn gen gm c Achentina, c,
13
Bungary, Canada, Trung Quc, Php, c, Mexico, Rumani, Ty Ban Nha,
Nam Phi, rgoay v M 9. Hin nay, nhng sn phm l-ng thc, thc phm
do cng ngh sinh hc to ra c mt trn th tr-ng.
Cc thnh tu ca bin np gen thc vt tp trung vo mt s h-ng
nh-:
- Chu cht dit c
Ng-i ta dng gen tng hp hp EPSP chuyn vo cy trng chu
-c Glyphosphat. Nhiu loi thc vt bin np v ang -c th nghim
nh- c ci -ng, u t-ng, nho. ci ht du, bng, c chua v thuc l.
Cy u t-ng chuyn gen chu thuc dit c khng ch c di tt
hn, gp phn nng cao hiu qu ca cc trang tri nh ti -u ho nng sut
v s dng hiu qu t trng trt, tit kim thi gian cho nng dn. Ging
mi ny hon ton ging cc ging u t-ng khc v dinh d-ng, cu to v
ph-ng thc ch bin thnh thc phm v thc n gia sc. -c trng nhiu
Achentina, c, Braxin, Canada, E, Nht bn, Hn Quc, Mexico, Nga, Thy
in, M v ruguay.
Ci du chu thuc dit c -c trng ti c, Canada v M.
- Khng bnh virus
ang nghin cu cc gen khng bnh virus ca thc vt. Ngoi ra ang
nghin cu kh nng khng virus bng bin np gen t ng vt.
u -c chuyn gen ca virus m ho cho protein v ca virus m
vng. Protein ny to cho cy kh nng t bo v chng li bnh m vng.
Mt gen t ngun bnh -c s dng khng li chnh n.
Cy khoai ty -c chuyn gen gip khng virus gy xon l, cy b
chuyn gen gip khng virus gy bnh.
Khng cc o!i g" h#i
Chuyn gen c t t $acius thuringiensis (BT) to ging chu
cn trng c hi. Cy bng chuyn gen Bt khng su c qu v n l, -c
trng ph bin nhiu n-c nh- Trung quc, Nam phi, Achentina,... Cc cy
bng, la mang gen chuyn ny c kh nng chng chu su c thn rt
tt.
Ch%ng chu cc bnh nm
14
ang phn lp gen 1,3glucanase (m ha enzym phn hy thnh t
bo ca cc mm bnh nm) t thc vt khng bnh chuyn vo cc
loi mn cm.
- &ha" '(i th!nh cc a)it b*o
Ng-i ta lm tng hm l -ng axit bo n khng bo ha v cc thnh
phn du thc vt. Cy u t -ng chuyn gen axit oleic c hm l -ng axit
oleic cao, mt axit bo c mt lin kt khng no. Theo cc nh dinh d-ng th
cht bo khng no -c xem l tt hn so vi cht bo no, -c tm thy
tht b, ln, phomat v mt s thc n th-ng ngy khc.
Ci du chuyn gen c hm l -ng Laurate cao, -c dng trong cng
nghip thc phm lm lp ph ngoi ko chocolate, bnh ngt, lp kem,
b. -c trng Canada v M. Ci du c hm l -ng axit oleic cao -c trng
Canada.
- +!m ch,m ch-n .u/ 0 c! chua
Cy c chua mang mt gen chuyn lm chm qu trnh lm mm qu
t nhin khi qu chn. y l loi thc phm chuyn gen u ti n -c sn
xut cc n-c pht trin. Ging c chua ny c thi gian l -u trn gi bn hng
di hn.
Ngoi ra ging chuyn gen c cc c tnh nh-:
- 1i2u ch3nh sinh t(ng h45 tinh b6t '7 !m ch8 m9c tinh b6t trong s/n
5h:m;
- C/i thin cht <4ng '#m t-ch =" trong h#t;
- &>ng h!m <4ng vitamin A trong h#t g#o;
n-c ta, mt s phng th nghim ln v ang i su vo cng tc
ci thin ging cy trng bng k thut chuyn gen. Ti phng Cng ngh
t bo thc vt, Vin Cng ngh Sinh hc, cc b-c c bn trong giai on
chun b -c thc hin. l: (1) Thu thp v ct gi cc ngun gen c
gi tr nh- gen CryA(b), CryA(c), gen c ch tryspin tr su, gen ?a@A
chng bc l vi khun, gen chu lnh, gen protein giu tryptopha; (2) Thit k
cc vector mang gen chuyn v th nghim thnh cng cc k thut chuyn
gen: gin tip thng qua vi khun Agrobacterium tumefaciens, trc tip bng
sng bn gen. n nay, nhiu dng la chuyn gen Xa21, CryA(c), u
chuyn gen chn chm v khng virus m vng -c to ra. Cc dng
15
cy chuyn gen ny s -c -a vo phn tch phn t; (3) Chun b thit
b tin hnh cc k thut sinh hc phn t phc v vic nh gi theo di
cy chuyn gen nh- PCR, lai Southern, RFLP, AFPD. Nh- vy, c th tin
hnh chuyn gen v -a k thut ny vo cng vic to ging cy trng Vit
Nam 1.
2.) $ghi*n c+u t,i -inh v chuy#n g.n t/*n cy hoa
lily
2.3.1 Byung Joon Ahn, Young Hee Joung, Kathryn K. Kamo,
2004. J Transgenic PIants of Eastr IiIy (LiIium IongifIorum) with
Phosphinothricin Resitance. J PIant BiotechnoIogy (2004) VoI. 6
(1).pp.9-13.
Tm tt
Qa trnh chuyn gen v biu hin ca gen uidA -c s dng
trong nghin cu ti -u ho cc thng s khi chuyn gen bng sng bn
gen cho huyn ph t bo ca hoa Easter lily, Lilium longiflorum. S
dng ht vng cho s biu hin ca gen uidA cao hn so vi ht n
bng tungsten. Tin x l dung dch huyn ph t bo tr-c 3 gi bng
osmoticum (0.125M) cho hiu qu biu hin ca uidA tng 1.5 ln. S
dng p lc 1550psi, khong cch 6cm c kt qu tt nht so vi cc p
lc 1100,1200,1800 psi. S biu hin tm ca uidA xut hin 493 t
boa petri. chuyn gen bn vng, huyn ph t bo ca Lilium
longiflorum -c bn vi plasmid bao gm cucumber mosaic virus (CMV)
replicase -c iu khin bi Act promoter v gen bar -c iu khin
bi CaMV promoter, 10 cy ti sinh -c phn tch bi PCR, 2 trong
10 cy -c khng nh chuyn gen nh lai Southern, 2 cy
chuyn gen -c chuyn c lp trong c 1 cy mang gen bar mt
cy mang c hai gen bao gm CMV replicase v bar gen. Cc cy ny
-c xc nh l c kh nng khng PPT nng 1000 mgl giai
on n hoa chng t gen bar -c biu hin ton b l khi -c
iu khin bi CaMV 35 S promoter.
2.3.2 Y.Hoshi, M. Kondo, S.Mori, Y.Adachi, M.Nakano,
H.Kobayashi, 2004. Production of transgenic IiIy pIants by
Agrobacterium- mediated transformation, PIant CeII Rep (2004)
22:359-364
16
Tm tt
H thng to cy chuyn gen nh vi khun Agrobacterium cho
cy Oriental hybrid lily Lilium cv. Acapulco -c nghin cu. Callus
xut pht t ng phn -c ng nui cy vi vi khun Agrobacterium
dng EHA101plG 121 Hm, cha vc t mang cc gen nptH,
h"g
r
,
uidA trong vng TDNA. thu -c 6 dng khng hyg t hn 200 callus
bng cch lm tn th-ng callus vi giy rp (sandpaper) tr-c khi ng
nui cy, callus -c ng nui cy trn mi tr-ng MS khng c
NH
4
NO
3
, s dng Hyg trong mi tr-ng chn lc. Cc dng ti sinh khng
hyg to chi sau pht trin thnh cy khi chuyn sang mi tr-ng MS
khng b sung cht iu tit sinh tr-ng. Tt c cc cy -c kt lun
chuyn gen nh GS histochemical assay v phn tch inverse PCR.
2.3.3 Sakae SUZUKI anh Masaru NAKANO, 2002,
Agrobacterium- Mediated transformation in LiIiaceous ornamentaI
pIant. JARQ 36(3), 119-127(2002) http://www.jircas.affrc.go.jp
Tm tt
nghin cu chuyn gen cho 3 ging cy thuc h Liliace, Lilium
formosanum, Agrobacterium -c s dng, c ba dng uu ch-a
binary vector mang gen npt, hpt v gus trong vng TDNA. Vi Lilium
formosanum, khng thu -c m hoc cy chuyn gen sau khi ng
nui cy vi vi khun Agrobacterium mc d c s biu hin tm ca
gen gus trong callus trong qu trnh ng nui cy. Tuy nhin, thu
-c mt s cm t bo c kh nng khng hygromycin ca ging
Agapanthus praecox ssp. Orientalis v Muscarri armeniacum trn mi
tr-ng c b sung hygromycin, Callus khng hygromycin ti sinh to
cy hon chnh qua phi soma, hu ht trong s chng -c xc nh
l cy chuyn gen da trn php th GS histochemical assay v phn
tch PCR. Bng lai Southern pht hin -c 15 copy ca gen trong
b gen ca cy chuyn gen ca 2 ging trong hu ht l c 1 hoc
2 copy. H thng chuyn gen vo Muscarri armeniacum v Agapanthus
praecox ssp. Orientalis c th l mt cng c pht trin trong cc
nghin cu v sinh hc phn t.
2.3.4 A.Mercuri, L. De Benedetti, S. Bruna, R. BegIiano, C.
Bianchini, G.FogIia, T.Schiva. Agrobacterium- Mediated
1
transformation with roI genes of LiIium IongIirum Thunb. ISHS
Acta HorticIuturae 612
Tm tt
nghin cu thnh cng h thng chuyn gen cho +iium
ongiforum Thunb ging Sno Queen. Callus pht sinh phi xut pht
t vi nhu v cung -c cy v ng nui cy trong ngy vi
huyn ph ca vi khun Agrobacterium LBA 4404 mang binary vect
pBin 19 bao gm nptH gen -c iu khin bi nos promoter v cc
on gii hn TDNA EcoR 15, bao gm ORFs 10,11 v 12 t-ng ng vi
rol A, B v C -c ct t Agrobacterium rhizogenes pRi 1855. Cc chi
chuyn gen -c ti sinh trn mi tr-ng chn lc v d dng nhn
nhanh, ra r v chuyn ra t. C s a dng v kiu hnh ca cc
dng chuyn gen.
2.3.5 A. Lipsky, A. Cohen, R. Barg, S. Shabtai, Y. SaIts, V.
Gaba, K. Kamo, A. Gera v A. Watad DeveIopment of LiIium
IongifIorum ceII cuItures of high competence for transformation by
particIe bombarment and of high embryogenic capacity
Tm tt
c th to -c cy chuyn gen bn vng cho +iium
ongiforum Thumb.vn. Sno Queen cn thit lp h thng ti sinh
c -c callus (xut pht t cc on vy c) c ng u v hnh
thi cao trong mi tr-ng lng. Callus xut pht t vy c c ng
u thp sau 6 thng cy chuyn . Nghin cu chuyn gen -c thc
hin bng s dng thit b bn gen Finertype bombardment trn callus
c 3 hoc 10 thng -c duy tr trong mi tr-ng lng iu kin bng
ti. Cc ht vng -c bc bi plasmid pBQ3 gen GS bao gm gen
uidA -c iu khin bi BQ3 promoter. Th nghim -c lp li 3
ln mi ln mt a, 10000 n 15000 im biu hin ca gen gus.
Callus nui cy trn mi tr-ng lng cho php thu -c cy chuyn gen
bn vng cao hn t 500 ln so vi mi tr-ng c. ti sinh -c
81.63. cy 1g callus t-i t callus nui cy trong mi tr-ng lng lc
c chiu sng. u giai on ti sinh c s hnh thnh phi soma
mc cao. Kanamycin c hiu qu chn lc tt hn so vi bialaphos.
thu -c 10 cy chuyn gen bn vng ca Lilium longiflorum Thumb.
cn. Sno Queen. Gn y nht, cc tc gi sn xut -c hng trm
18
cy chuyn gen khc nhau s dng pCAMBA2301 bao gm gen rol B
d-i s iu khin ca Lat 52 promoter chng li s pht trin ca
phn hoa. Gen gus -c biu hin rt mnh trong cc cy chuyn
gen. Cc phn tch v gen ca nhng cy ny ang -c tin hnh.
2.4 k thut chuyn gen vo thc vt.
2.4.1 khi nim v chuyn gen
k thut chuyn gen l k thut -a mt hay nhiu gen l -c
thit k dng ADN ti t hp vo t bo ch cy trng ni ring v
cc vi sinh vt ni chung (vi sinh vt, ng vt) lm cho gen l c th
tn ti dng Plasmid ti t hp hoc gn vo b gen t bo ch. Trong
t bo ch cc gen ny hot ng tng hp nn cc protein c tr-ng
dn ti vic xut hin cc c tnh mi ca c th chuyn gen.
2.4.2 S ti sinh c th thc vt - c s thnh cng ca
chuyn gen
2.4.2.1 C s khoa hc v kh nng ti sinh ca thc vt
a, Tnh ton nng ca t bo.
Haberlandt (1902), ln u tn quan nim rng mi t bo bt
k ca mt c th sinh vt a bo u c kh nng tim tng pht
trin thnh mt c th hon chnh. Theo quan im ca sinh hc hin
i th mi t bo ring r phn ha u mang ton b thng tin di
truyn cn thit v ca c sinh vt . Khi gp iu kin thch
hp, mi t bo u c th pht trin thnh mt c th hon chnh.
l tnh ton nng ca t bo. tnh ton nng ca t bo m Haberlandt
nu ra chnh l c s l lun ca ph-ng php nui cy m t bo thc
vt. Cho n nay con ng-i hon ton chng minh -c kh nng ti
sinh ca mt c th sinh vt hon chnh t mt t bo ring r. 0.1.
G.C.Philips, 199.
b, S phn phn ha v phn ha ca t bo.
C th thc vt tr-ng thnh l mt chnh th thng nht bao
gm nhiu c quan chc nng khc nhau, -c hnh thnh t nhiu loi
t bo khc nhau. Tuy nhin tt c cc loi t bo u bt ngun t
mt t bo u tin (t bo hp t). giai on u, t bo hp t
tip tc phn chia hnh thnh nhiu t bo phi sinh ch-a mang chc
19
nng ring bit (chuyn ha). Sau t t bo phi sinh ny chng -c
tip tc bin i thnh t bo chuyn ha c hiu cho cc m, c
quan c chc nng khc nhau.
S phn ha t bo l s chuyn cc t bo phi sinh thnh cc
t bo m chuyn ha, m nhm cc chc nng khc nhau. V d: m
du lm nhim v quang hp, m b lm nhim v bo v, nhu m lm
nhim v d tr, m dn lm chc nng dn n-c v dinh d-ng.
Qu trnh phn ha t bo c th biu th:
Qu trnh phn ha
T bo phi sinh T bo chuyn ha
Qu trnh phn phn ha
Tuy nhin khi t bo phn ha thnh cc t bo c chc nng
chuyn, chng khng l hon ton mt kh nng bin i ca mnh.
Trong tr-ng hp cn thit, iu kin thch hp, chng li c th tr
v dng t bo phi sinh v phn chia mnh m. Qu trnh gi l qu
trnh phn phn ha t bo, ng-c li vi s phn ha t bo. v bn
cht th s phn ha v phn phn ha l mt qu trnh hot ha, c
ch cc gen. Ti mt thi im no trong qu trnh pht trin c th,
c mt s gen -c hot ha (m vn tr-c nay b c ch) cho ta
tnh trng mi, cn mt s gen li b nh ch hot ng, iu ny sy
ra theo mt ch-ng trnh -c m ha trong cu trc ca mt t bo
khin qu trnh sinh tr-ng pht trin ca c th thc vt lun -c hi
ha.
Mt khc, khi t bo nm trong mt khi c th th-ng b c ch
b cc t bo xung quanh khi tch ring tng t bo hoc gim kch
th-c ca khi m s to iu kin thun li cho s hot ha cc gen ca
t bo.
Qu trnh pht sinh hnh thi trong nui cy m, t bo thc vt
thc cht l kt qu ca qu trnh phn ha v phn phn ha ca t
bo. k thut nui cy m t bo cho n cng l k thut iu khin
s pht sinh hnh thi ca t bo thc vt (khi nui cy tc ri trong iu
kin v trng) mt cch nh h-ng da vo s phn phn ha v phn
ha ca t bo trn c s tnh ton nng ca t bo thc vt. iu
khin s pht sinh hnh thi ca m nui cy, ng-i ta th-ng b xung
20
vo mi tr-ng nui cy c hai nhm cht iu tit sinh tr-ng thc vt
l auxin v cytokinin.
Ty theo mc ch nghin cu t ra nh- to m so, to chi
ph, to r, to phi v tnh hay cho mc chi trc tip t m nui cy
m mi tr-ng thch hp l cn thit nht (Baa v cng s, 195).
2.4.2.2 Vai tr ca vic xy dng h thng ti sinh.
Trong k thut chuyn gen vo c th thc vt, th nhng i
t-ng thng -c s dng chuyn gen l: M l, m thn, m callus
hay nhng t bo tch ri nhng vt liu ny sau khi -c bin np
thnh cng, cc t bo dung np nhng gen ngoi lai, ta phi tm cch
lm cho cc t bo ny ti sinh -c thnh cy hon chnh bng cch
t n vo mi tr-ng ti sinh ph hp. Nu nh-, s ti sinh ny thnh
cy hon chnh khng thnh cng th tt c nhng vt liu -c
bin np s tr nn v ngha. Chnh v vy s thnh cng ca k
thut chuyn gen ph thuc vo s ti sinh ca t bo. do vy, vic la
chn v iu chnh mi tr-ng nui cy m v t bo thc vt ng
vai tr ht sc quan trng, mang tnh quyt nh n s thnh cng
hay tht bi ca th nghin bin np, bn cnh , vn s dng m
hay t bo thch hp bin np cng l mc tiu nghin cu khng
km phn quan trng (potrykus, 1991).
amesF.thutchinson, Daniel senegger, Savitri Nadesan, Neil Smith
and Piter Waterhouse, (2000) nhn thy: c th to -c ging mi
nh ci bin di truyn mt cch c hiu qu th phi m bo 4 yu
cu sau:
H thng ti sinh thch hp ti sinh -c cy chuyn gen:
H thng vecter thch hp c th -a -c cc gen vo DNA:
Cc gen thch hp -c nhn rng v xc inh cc c tnh:
Cc ph-ng php nh gi cc cy chuyn gen trong phng
th nghim, nh knh v ng rng (amesF.thutchinson, Daniel
senegger, Savitri Nadesan, Neil Smith and peter Waterhouse,2000)
2.4.3 i c-ng v cc ph-ng php chuyn gen:
Ta c th phn chia thnh hai nhm ph-ng php chuyn gen chnh l:
Cc ph-ng php chuyn gen trc tip:
21
- Dng PEG (Polyetylen glycol).
- Dng sng bn gen (gen gun).
- Dng in xung (electropration).
- Dng vi tim (microinection).
- Dng siu m (microaves).
- Dng silicon carbide.
- Dng in di.
- S dng ng phn (pollentube pathays).
Cc ph-ng php chuyn gen gin tip: y l ph-ng php chuyn
gen m cc on DNA khng -c -a trc tip vo t bo hoc m m
phi thng qua cc vector mang, cc vector ny th-ng l cc plasmid ti
t hp hoc c th l DNA ca virus. Hin nay -c s dng nhiu
nht l hai ph-ng php sau:
- Chuyn gen nh vi khun: th-ng s dng hai loi vi khun t l
Agrobacterium tumefaciens v Agrobacterium rhizogens.
- Chuyn gen nh virus: c hai loi th-ng s dng l: Gemini virus
v caulimosaic virus.
Sau y chng ta s tm hiu v mt s cc ph-ng php -c s dng
ph bin:
2.4.1 Chuyn gen bng PEG (phoIyethyIene gIycoI).
PEG l cht c i lc ln vi n-c. nng cao, PEG l cho DNA khng
cn trng thi ha tan m kt dnh vo mng nguyn sinh ca t bo
sau b t bo nut theo c ch ca Amip. C nhiu cht c i lc
ln nh- PEG nh-ng PEG l cht t gy c cho t bo nht nn -c s
dng ph bin.
Chyn gen qua PEG l mt ph-ng php thc hin kh n gin m li
hiu qu cao. Trong cng mt th nghim ta c th x l mt l-ng kh
ln t bo, c th thu -c hng nghn c th chuyn gen ring bit.
Hn na li khng c nhng hn ch v dy vt ch nn -c s dng
ph bin nghin cu cc c ch iu ha s biu hin gen.
22
Nng ion Mg hoc Ca trong hn hp , trng l-ng phn t v
nng PEG l nhng thng s quan trng nht tc ng n hiu
qu ca ph-ng php ny.
2.4.2 Chuyn gen bng k thut vi tim (Micro injection).
Qua knh hin vi, DNA plasmid c th -c tim vo t bo vi
sinh vt v protoplast i vi t bo thc vt d-i p lc cao tin
hnh chuyn gen. Ng-i ta thc hin chuyn gen thnh cng kh
nhiu i t-ng thc vt, vi sinh vt cng nh- ng vt bng ph-ng
php ny.
Tuy nhin, ph-ng php ny t -c s dng phng th nghim
v thao tc vi tim d-i knh hin vi i hi thit b vi thao tc cc nhy,
rt t tin. S thnh cng ph thuc nhiu vo s kho tay v kin
nhn ca ng-i thao tc.
2.4.3 Chuyn gen bng xung in:
Ph-ng php ny s dng nhng xung in ngn trong mt in
tr-ng cc mnh to mt l thng tm thi trn mng t bo lm tng
kh nng xm nhp ca DNA vo t bo.
Protoplast hoc cc t nguyn vn -c t gia hai tm kim loi
cch nhau 14 mm trong mt cuvette bng nha. in th cao, xung
in to cc l tm th (c 30 nm) trn mng protoplast v DNA bn ngoi
c th xm nhp vo bn trong t bo.
Xung in t lu -c dng bin np v hp nht cc t bo
trn. So vi ph-ng php bn ht, ph-ng php ny hiu qu c phn hn
ch hn, nh-ng i vi nhng m d hp thu DNA nh xung in th
s dng ph-ng php ny nhanh, n gin v r tin hn.
2.4.4 Chuyn gen bng ph-ng php bn gen (BioIistic hoc
Bombardement).
Ph-ng php s dng sng bn gen do ohn Stanford i hc
Comell pht minh (Klein v ctv, 198). y l ph-ng php trong cc vi
n c tc cao -c s dng chuyn cc axit nucleic vo cc t
bo sng. Ph-ng php ny rt c hiu qu trong vic chuyn DNA l
vo t bo thc vt.
23
Ph-ng php ny c kh nng x l i vi cc m bit ha v c
th bin np vo cc loi kh chuyn gen nh-: u t-ng, bng, ng,
la. S biu hin gen tm thi -c chng minh nhiu loi khc
nhau.
Hiu qu ca ph-ng php ph thuc vo nhiu yu t:
- Bn cht ca m ch: Phi c kh nng bin np v d ti sinh.
- Bn cht, cc tnh cht ha hc v vt l ca cc ht kim loi dng
mang DNA l, vic chun b v gn DNA vo cc ht mang.
- Cc yu t v mi tr-ng nh-: nhit , m, chu k sng.
u im ca ph-ng php:
- D s dng.
- Bn mt ln c th chuyn np vo rt nhiu t bo.
- Gen -c bc ngoi ht kim loi vn c hot tnh sinh hc cn
thit.
- Cc t bo, m cn chuyn rt phong ph.
- im ti ca ht kim loi c th l trn b mt hoc su bn trong
ca m.
Nh-c im ca ph-ng php:
- Nhiu t bo b ph hy.
- To ra nhiu bn sao ca DNA, khi kt gn d lm xo trn h
thng di truyn ca m ch.
- Vic kt gn bn vng thp.
- Cng vic chun b vt liu tn nhiu th gian.
- Thit b t tin.
2.4.5 Chuyn gen qua trung gian I cc virus vector:
Do virus c kch th-c nh, cu trc n gin ch gm v protein
v li DNA hoc RNA li c kh nng xm nhim t nhin vo cc vt
ch nn ng-i ta ngh n vic s dng chng lm vector chuyn
gen.
C hai loi virus th-ng -c s dng lm vector chuyn gen l:
24
- Gemini virus: c ph k ch ng (c c cy ha tho)
- Caulimosaic virus: virus hi cy h ci, c kh nng ti v chuyn
gen tt, c promotor mnh.
Tuy nhin ph-ng php ny vn cn t -c s dng do nhng gen
-c chuyn nh virus khng di truyn qua ht nn khng di truyn
cho cc th h sau -c. Mun di truyn -c cho cc th h sau
phi tin hnh nhn v tnh nn khng phi loi cy no cng thc
hin -c. Mt khc vic la chn cc chng virus phi tha mn mt
s yu cu sau:
- Cu trc di truyn phi l DNA.
- Khng c tnh c hoc tnh c thp.
- Kh nng ti ln.
- C ph k ch rng.
2.4.6 Ph-ng php chuyn gen qua trung gian Agrobacterium.
Vi khun Agrobacterium l mt tc nhn chuyn gen rt hiu qu
trn i t-ng thc vt. C hai chng -c s dng ph bin l:
- Agrobacterium tumefaciens: vect mang l Tiplasmid.
- Agrobacterium rhizogens: vect mang l Riplasmid.
Tr-c y ng-i ta cho rng ph-ng php chuyn lp gin tip ny ch
thc hin c hiu qu i vi cy hai l mm v trong t nhin cc vi
khun ny khng c kh nng gy bnh cy mt l mm. Tuy
nhin, gn y nhiu nh khoa hc tin hnh chuyn gen thnh
cng trn i t-ng cy mt l mm nh-: la, ng. nh
Agrobacterium. Chnh v vy ph-ng php ny -c s dng kh ph
bin hin nay. c bit n-c ta y c th coi l ph-ng php
chuyn gen ph bin nht do n khng i hi cc thit b t
tin m li c hiu qu cao.
2.5 Cc h-ng to cy chuyn gen.
2.5.1 Tnh hnh chung v cy chuyn gen.
Ng-i ta bt u tin hnh chuyn gen vo i tng thc vt t
nm 1982, tip theo nhiu ging cy trng mi -c to ra bng
ph-ng php chuyn gen v ph bin vo sn xut t nm 1996. Din
25
tch gieo trng cy chuyn gen tng nn khng ngng: 2,8 triu ha nm
1996, 12,8 triu ha nm 199, 2, 8 triu ha nm 1998, ring M nm
1999 t 28, triu ha (15 triu ha l u t-ng, 10,3 triu ha l ng,
3,5 triu ha l bng chim 2% din tch trng cy chuyn gen trn
ton cu). Tp trung mt s n-c nh-: M, Canaa, Trung Quc. theo
tng kt ca cng ty cng ngh sinh hc M, Monsanto th vn
cy chuyn gen c 18 nm kinh nghim, s dng i vi 56 loi cy
45 n-c. Tc pht trin cc ging cy trng chuyn gen rt nhanh, li
nhun -a li t cc sn phm ny rt ln: -c tnh n nm 2010 th
tr-ng thc phm bin i gen s tng gp tm ln so vi hin nay, tc
khong 25 t SD. Trong s cc cy trng chuyn gen, cy -c
chuyn gen khng su bnh -c s dng kh ph bin c bit l vi
cc cy cng nghip nh-: bng, thuc l. cy l-ng thc nh-: ng, la.
T nm 1986 n 1993 c ti 96 th nghim i vi vic chuyn gen
khng su cho cy trng (34 th nghim khoai ty, 19 th nghim i
vi thuc l, 24 th nghim i vi ng, 16 th nghim i vi c chua, 3
th nghim i vi ht cha du) (Plant Molecular Genetics Monica A.
Hughes). Nhng ging cy khng su ny th s c hiu qu nn
em li nhiu li nhun do khng phi s dng thuc tr su nng sut
cht l-ng li tng: nm 1996 li nhun n-c My do bng Bt em li l 60
triu SD, do ng Bt em li l 200 triu SD. Do nhng li ch to ln
nh- vy nn cng ngh chuyn gen cho thc vt ngy cng -c hon
thin, cc ph-ng php chuyn gen ngy cng phong ph. Hng lot cc
ph-ng php chuyn np gen vo thc vt -c thng bo thnh cng.
C nhng ph-ng php rt phc tp, phi s dng n nhng k thut
cao nh-ng cng c nhng ph-ng php kh n gin.
2.5.2 Cc h-ng chnh to cy chuyn gen:
To cy chng chu bnh virus nh chuyn gen:
Bnh virus l bnh khng cha -c v gy tn hi ln n nng
sut cng nh- cht l-ng ca cy trng. Do h-ng nghin cu ny
rt quan trng.
a s virus hi cy c cu trc gm mt v bc protin v phn li l
axit nucleic (AND, ARN). Gen m ha cho protein v -c gi l cc
gen CP (Coat protein genes). Gio s- Beachy vin nghin cu
SCRPP ti Sandiego (My) l ng-i u tin nhn thy nu chuyn
26
gen CP vo b gen ca cy th cy tr ln khng hon ton vi loi
virus t-ng ng. C ch ca hin t-ng ny cn ch-a -c sng t tuy
nhin ng-i ta gi nh s d- tha protein v virus nh- tn hiu
ngng tng hp phn tng hp axit nucleic ca virus. Chnh v th
khi c mt sn protein v do gen CP sn sinh, virus mi xm nhim
s khng th nhn ln -c. Li dng tnh cht ny ng-i ta tin
hnh chuyn gen m ha qu trnh tng hp protein v virus vo cy
gip cy chng chu bnh virus. H-ng chuyn gen ny rt
thnh cng i vi cy h c: thuc l. khoai ty, c chua, u .
To cy khng su:
Su l mt tc nhn gy hi rt ln cho ngnh nng nghip. Hng
nm mt l-ng ln thuc tr su ha hc -c s dng hn ch
cc dch hi cy trng. Nh-ng bin php ny c hi cho sc khe con
ng-i v nhim mi tr-ng nn cn thay th bng mt ph-ng php
khc an ton hn.
T u th k 20 pht hin vi khun t Bacillus thuringens
c kh nng tng hp c t c bn cht protein gi l Bt toxin, cht
ny c kh nng dit su non ca cn trng m khng nh h-ng n
ng vt c x-ng sng (Nguyn vn yn, 199) 10. Cc protein
c t th-ng dng tinh th protein hnh qu trm gi tt l Cry.
Hin nay pht hin -c trn 50 loi t Cry n CryX. Cc Cry
khc nhau c c ch gy hi khc nhau. Theo thng k cho thy 525
loi su thuc 13 b cn trng c th b nhim bnh do B.
thuringiensis gy ra: B cnh vy (318 loi), B hai cnh (59 loi), B
cnh mng (5 loi), B cnh cng (34 loi).
Nm 1985 trnh t nhng gen m ha cho vic to protein tinh th
c ln u tin xc nh (Adang v cs, 1985) 13. Nhng th
nghim u tin v chuyn gen m ha protein tinh th -c thc
hin nh trung gian Agrobacterium tumefaciens -c thc hin trn
cy thuc l v cy khoai ty (Adang v cs, 198) 14. Tip
Vector mang trnh t m ha cho tin c t CryA(a), CryA(b),
CryA(c) -c chuyn vo cy thuc l (Barton v cs 198) 15.
Nhiu nghin cu cho thy rng vic chuyn gen Cry c sa cha
so vi nguyn bn cho hot tnh khng su cao hn nhiu. Cy bng
chuyn gen Cry ci tin th hin tnh khng cao vi loi su xanh
2
nguy him (Perlak v cs, 1993) 19. Trn cnh ng cy c ci
chuyn gen CryA(b) ci tin khng li hon ton su hi c v
su hi l (Wilson v cs, 1992) 20. Nh- vy c th thy h-ng nghin
cu ny c rt nhiu tim nng ng dng vo thc tin sn xut l
rt ln.
To cy khng thuc tr c:
Thuc dit c tc ng ln cy trng bng nhiu con ng: Qua con
-ng quang hp, qua con -ng tng hp axit amin, cht bo hoc
tng hp cc hoc mn sinh tr-ng thc vt.
Basta l mt loi thuc tr c sinh hc c d-c cht chnh l
phosphinothricin (PPT0 tc dng ln c ch tng hp axit amin c th
l enzim Glutamin synthase (GS), (Monica A.Huges, 199519. Loi
thuc tr c ny c tc dng tiu dit khng chn lc nn khi s dng
tiu dit th ng thi cng nh h-ng n cy trng. Gen bar
khng c trong thc vt, -c phn lp t x khun Streptomyces
hygroscopicus m ha cho vic hnh thnh enzim phosphinothricin
acetyltransferase (PAT) c tc dng lm mt c tnh ca PPT
(Nguyn vn yn, 1998)8, -c nghin cu chuyn vo cy to
cho cy to cho cy c kh nng khng -c PPT. Gen bar th-ng -c
chuyn km cng vi cc gen hu dng khc nh- gen khng su. vi
vai tr va nh- mt gen chn lc va nh- mt gen hu dng to ng
thi tnh khng thuc tr c cho cy.
Thuc tr c tc ng ln s quang hp ca cy bng cch c ch
enzim quang hp, lm ngng tr qu trnh quang hp lm cho cy b
cht. Hin nay thuc tr c ph bin, c hiu c hiu qu cao,
khng c vi mi tr-ng l cht Glyphosat: cht ny tc ng ln
enzim bin i sn phm th cp ca quang hp Enolpyruvat
sikimat phosphat synthetaza (EPSPS). Nu enzim ny b hng s
nh h-ng n s tng hpc cc hp cht mch vng nh-: axit sikimic
tin cht to axt amin mch vng, cc cht kch thch sinh
tr-ng. lm cho cy b cht. Loi thuc dit c ny cng c tc
dng tiu dit khng chn lc cho nn khi s dng cc loi cy trng
cng b nh h-ng. Hin nay phn lp gen m ha gen tng hp
enzim EPSPS t vi khun Salmonella typhimurium v Escherichia
coli c hot tnh tng hp rt cao chuyn vo cy lm tng hot tnh
28
ca enzim ln trn 20 ln v cy chiu -c nng thuc dit c
cao hn 4 ln (Monica A.Huges, 1995)19.
y l h-ng nghin cu hin ang -c nghin cu rng ri. theo
thng k t nm 1986 n 1993 tng cng c 214 th nghim
chuyn gen khng thuc tr c cho 5 loi cy in hnh sau: 16 th
nghim vi khoai ty, 94 th nghim vi cy c ht cha du, 29 vi
thuc l, 54 vi ng, 21 vi c chua (Monica A.Huges, 1995)19.
Chuyn gen to mu sc cho hoa: hoa l mt sn phm rt c gi tr kinh
t !ut khu" #oa $%&c '(ng $ trang tr) n*n rt c+n c s, phong ph-
$a 'ng . m+u sc" /uy nhi*n h%0ng nghi*n c1u ny kh ph1c tp
hi2n nay c3n ch%a t4m hiu ht" 5t i ) '6: chuyn gen t7ng h&p sc t8
!anh 9elphini'ium 9aniella o hoa h:ng to ra hoa h:ng c mu !anh
'a tr;i< chuyn gen t7ng h&p sc t8 antocyan c=a ng> cho rt nhi.u loi
hoa to ra nhi.u mu sc khc nhau"
Chuyn gen t7ng h&p cc protein $ng ?t o th,c ?t: to protein $ng
?t phi th>ng @ua chAn nu>i t8n kBm hCn so 0i chAm sc cDy tr:ng n*n
ng%;i ta $E chuyn gen mE ha cho i2c t7ng h&p cc protein $ng ?t
o th,c ?t $ Ft cDy tr:ng t7ng h&p l*n ch-ng" #i2n ng%;i ta $E
chuyn gen t7ng h&p hai loi Grotein l enkeplain Hmt loi protein th+n
kinhI alFumin huyt thanh"
Chuyn gen lm thay $7i phm cht n>ng sn: ng%;i ta tin hnh chuyn
cc gen nh%: gen to m(i thCmJ gen mE ha cho i2c t7ng h&p - carotenJ
o l-a $ to ra cc gi8ng l-a giu - carotenJ chuyn gen t7ng h&p cc
protein ch1a nhi.u a!it amin c l%u huKnh o cL chAn nu>i cMu $ tAng
l%&ng l>ng thu $%&c" chuyn gen t7ng h&p cc protein c ngNt cao o
cDy An @uO
2.6 Phng php chuyn gen cho i tng thc vt.
2.6.1 Chuyn gen trc tip vo t bo thc vt
/ Fo th,c ?t c mt $Pc $im l c mt l0p thnh t Fo rt Qng chc
n*n rt kh tc $ng tr,c tipJ th%;ng cc ph%Cng php chuyn gen tr,c
tip tr*n HtrM ph%Cng php sR '6ng s-ng Fn genI $.u $%&c p '6ng ph7
Fin 0i protoplats $Dy l thu?t ngQ $ chS t Fo th,c ?t $E $%&c tch FL
l0p thnh t Fo F*n ngoi chS c3n li mng nguy*n sinh ph+n trong
c=a t Fo" 5ng nguy*n sinh cho phBp protoplat c th hp thu o t
29
Fo cc $i phDn tR theo cC ch c=a amipJ khi $1ng cnh nhau ch-ng c
th h3a lm mt gNi l 'ung h&p" T&i '6ng $Pc t)nh ny c=a protoplast
ng%;i ta tin hnh chuyn tr,c tip cc $on 9UV o $ to ra cc c th
m0i HUguyWn An XynJ YZZ[I\]^"
2.6.2 Chuyn gen vo t bo thc vt !u" trung gi"n # vi $hu%n
VgroFacterium tume_acients"
/rong khi cc ph%Cng php chuyn gen tr*n kh ph1c tpJ $3i hLi cc
thit F $t ti.n th4 chuyn gen o t Fo th,c ?t @ua trung gian
VgroFacterium li tL ra rt c hi2u @u m kh>ng c+n t0i cc thit F ph1c
tp HUguyWn An XynJ YZZ[I\]^"
VgroFacterium l mt loi i khun c 'ng h4nh @ueJ gram Dm thuc hN i
khun `hiaoFraeae" Ch-ng $%&c phDn loi theo $Pc t)nh gDy F2nh th,c ?t nh%
sau: V"tume_acients gDy F2nh s+n cDyJ V"rhiaogenes gDy F2nh rW l>ng
V"ra'ioFacter kh>ng $c" Ch-ng c kh nAng sDm nh?p o cDy @ua t th%Cng
gn mt $on 9UV c=a ch-ng o F gen c=a t Fo th,c ?tJ $Dy l nhQng
gen mE ha cho i2c h4nh thnh kh8i u t7ng h&p ra nhQng cht 'inh '%bng
$Pc Fi2t gi-p cho i khun pht trin" Ug%;i ta $E t4m thy nhQng a!it amin l
m th,c ?t kh>ng c kh nAng t7ng h&p nh%: octopineJ nopalineJ agropine
succinamopine trong kh8i u" Hcohn 9raperJ YZZdI\Ye^" 5Pt khc kh8i u tAng
tr%fng kh>ng c+n c cc cht k)ch th)ch sinh tr%fng th,c ?tJ nh% ?y cho phBp
kt lu?n $E c mt s, Fin $7i nht $nh trong F my 'i truy.n c=a t Fo th,c
?t ti $Dy" Ug%;i ta $E pht hi2n ra t)nh gDy F2nh c=a nhQng i khun ny ph+n
l0n l 'o cc gen f plasmi' tip h&p dgg-dhg kF @uy $nhJ f V"tume_acients gNi
l /i-plasmi'J f V"rhiaogenes gNi l `i-plasmi'"
/i-plasmi':
/i-plasmi' l 9UV 3ng kBpJ tr3n c cu tr-c $%&c chia thnh F8n (ng ch)nh"
iua nhi.u nghi*n c1u cho thy c hai (ng ch)nh c tc $ng tr,c tip $n s,
h4nh thnh kh8i u l (ng /-9UV H/rans_erre' 9UVI (ng ir HirulenceI
c3n hai (ng c3n li th4 ph6 trch @u tr4nh tip h&p ti Fn /i-plasmi' trong
i khun" /rong @u tr4nh h4nh thnh kh8i u $on /-9UV sj $%&c chuyn o t
Fo th,c ?t gn o Fi gen nhDn c=a t Fo th,c ?t" k(ng /-9UV c k)ch
th%0c khong dl kF $%&c gi0i hn Ffi tr4nh t, lPp li khong dhF"p gNi l
(ng F; tri F; phi /i-plasmi' $%&c chia thanh hai loi ch)nh mctopine
Uopaline /i-plasmi'" Hcohn 'raperJ YZZdI\Ye^
30
/in tr4nh gn o c=a i khun tin tr4nh 'i chuyn 9UV c li*n @uan
$n hi2n t%&ng tip h&p" UhQng t Fo cDy F th%Cng tit ra nhQng phDn tR c
t)nh cht t)n hi2uJ lm cho gen f (ng ir c=a /i-plasmi' trf l*n hot $ng"
UhQng cht k)ch th)ch ny l cc h&p chDt phenol c=a th,c ?t nh%:
acetosyringone HVnI -hy'roaceto syringone Hm#-VnI" k(ng ir Fao g:m
cc gen irVJ oJ CJ 9J p q" Grotein irV c phDn tR kh8i l ZdgggJ 0i churi
mE kt th-c l cc a!it amin %a n%0c c nhi2m 6 'i chuyn phDn tR t)n hi2u Vn
tM m> F th%Cng o t Fo c=a VgroFacterium" Vn $%&c !em nh% hot $ng c
t)nh hr tr& 0i protein irq $ k)ch th)ch s, th hi2n cc gen iroJ CJ 9J p $ ct
/-9UV ra khLi /i-plasmi' $%a n o t Fo cDy" Hi2n l-a sonCT-#i tho
t?p hunI\h^"
T&i '6ng t)nh cht nyJ ng%;i ta tin hnh thay th $on /-9UV Ftng
mt $on gen m0i m ng%;i ta mu8n chuyn o t Fo th,c ?tJ chuyn np li
o i khunJ nu>i cy chung m> t Fo th,c ?t 0i i khun" nau mt th;i gian
nht $nh ta tin hnh nu>i cy ti sinh m> th,c ?t tr*n m>i tr%;ng c cht chNn
lNc $ nh?n Fit cc c th $E $%&c chuyn gen cht khng sinh $ ti*u 'i2t i
khun"
s th,c hi2n @u tr4nh chuyn gen phi c $%&c ector chuyn gen" /uy
nhi*n /i-plasmi' hi t6 $= cc y*u c+u c=a mt ector chuyn gen nh%ng n li
kh>ng $%&c sR '6ng tr,c tip 'o:
- K)ch cb l0n kh thao tc"
- Kh>ng c nhQng $im ct $Pc hi2u"\h^"
Ch)nh 4 ?y ng%;i ta phi tin hnh sRa chQa /i-plasmi' tr%0c khi sR '6ng"
#i2n nay c hai loi ector $ang $%&c sR '6ng:
- Co-intergrate etor Hector $:ng t%Cng tcI:
#2 th8ng ny $+u ti*n c+n c d plasmi': mt Glami' nhL c kh nAng t, nhDn
l*n trong p"Coli gNi l ector trung gianJ plasmi' th1 hai l /i-plasmi' $E
$%&c loi FL $on /-9UV thay o $ l mt $on 9UV t%Cng $:ng 0i
ector trung gian ni tr*n" ng%;i ta F8 tr) tr*n ector trung gian cc gen chS
thJ chNn lNc mt $im Cloning $ c th chun cc 9UV mong mu8n o
$" kector trung gian $%&c nhDn l*n trong p"Coli sau $ $%&c chuyn trf li
o t Fo V"tume_acients Ftng tip h&p" /rong t Fo i khun
V"tume_acients sj !y ra @u tr4nh ti t7 h&p giQa ector trung gian /i-
plasmi' to thnh mt Glasmi' $:ng nht - $Dy ch)nh l ector $:ng h&p
31
nhtJ hay Co-intergrate etor" 9o t+n s8 $%&c $%a trf li VgroFacterium rt
thp HtM Yg
h
- Yg
e
I n*n ector ny )t $%&c sR '6ng" Hcohn 'aperJ YZZdI\Ye^"
- oinary ector Hector kBpI:
/r*n cC sf (ng ir hat $ng theo m> h4nh trans" 9o $ (ng ny c th $%&c
F8 tr) tr*n mt Glasmi' khc" Glasmi' ny $%&c gNi l vhelperw /i-plasmi'" x
vhelperw /i-plasmi'J ton F (ng /-9UV $E F ct FL" Uh% ?y FDy gi; ng%;i
ta c mt Glasmi' kh>ng c $on /-9UV nh%ng c (ng ir H#elperI" Glasmi'
kh>ng c gen ir nh%ng li mang $+y $= cc macker chNn lNc tr*n (ng /-
9UV $%&c thit k mt $im clone l tr) m ti $ ta c th ci o cc $on
9UV mong mu8n" Khc 0i co-intergrate ectorJ Finary ector c+n c h2 th8ng
hai Glasmi'" #2 th8ng ny cho hi2u @u chuyn gen cao hCn n*n hi2n nay $%&c
sR '6ng ph7 Fin hCn"
yu $im c=a ph%Cng php ny l:
- C hi2u @u cao"
- Cc F%0c tin hnh $Cn ginJ 'W th,c hi2n"
- Kt gn o genom c=a t Fo cDy f nhQng tr) ngzu nhi*nJ s8 Fn sao
kt gn )t hCn tr%;ng h&p chuyn gen tr,c tip"
Uh%&c $im c=a ph%Cng php ny l:
- #i2u @u c=a chuyn np t(y thuc o loi cDy tr:ng kh nAng ti sinh
c=a t Fo chuyn np
- #i2u @u c=a chuyn np c3n ph6 thuc o tu7i cDy giai $on sinh l{
c=a t Fo"\h^"
s8i 0i chuyn gen ot f n%0c ta hi2n c|ng sR '6ng ch= yu l ph%Cng php
ny" qen ot l $on 9UV mE ha cho s, t7ng h&p cc protein $c t8 trong t
Fo i khun oacillus thuringensisJ $%&c pht hi2n nghi*n c1u tM nAm YZYh
HUguyWn An XynJ YZZeI\Yg^" ki khun ot $%&c chia ra nhi.u n3i HnerotypeI c
t)nh $c rt chuy*n t)nh f nhQng nhm sDu khc nhau"
Hnguyn bo ngc ti)

32

Phn III

$0i 1ung v 2h34ng 2h,2 nghi*n c+u


'(T 5I! ' P67$G P,P $GI8$ C+!

9. :;I T6<$G =9 5I5"
Vt Iiu:
- Orienta Bberia hoa trng

Ph-ng php nghin cu
BC dDng 5h<Eng 5h5 nghiFn c9u nuGi
c" mG hin h!nh
Ph<Eng 5h5 chu"7n gen bHng vi Ihu:n Agrobacterium tumefaciens
Ph<Eng 5h5 nuGi c" vi Ihu:n
Ngun vi khun: Vi khun -c l -u gi trong t lnh 20
0
C 80
0
C
M'i tr%-ng nu'i c.y (hu/n 01
pha 1 lt mi tr -ng AB cn hp tit trng 5 g glucose v 15 g
bactoagar trong 900ml n-c ct. Sau khi hp xong cho vo dung dch
50ml ca 20x AB buffer v 20x AB salts -c hp tit trng. B sung
kanamycin c nng 50mgl. Kanamycin -c b sung khi nhit
mi tr-ng khong 50
0
C. Mi tr-ng vo a petri, 25mga.
20x AB buffer (cho 1 lt): 30 g K
2
HPO
4
v 4 g NaH
2
PO
4

20x AB Salts (cho 1 lt): 20 g NH
4
Cl, 6 g MgSO
4
.H
2
O, 3g KCl,
3g CaCl
2
.2H
2
O v 50mg FeSO
4
.H
2
O.
LiIy Siberia
33
Th2nh $h3n m'i tr%-ng 451
Tripton (pepton) 10,0 glt
NaCl 5,0 glt
Cao nm men 10,0 glt
AS 20mglt
Kanamycin 50mglt
PH ,0
Nui cy 28
0
C, 24 gi, lc 200
vngpht.
(a) Mi tr-ng nui cy vi khun: mi tr-ng YEB lng, 20mgl AS .
khng sinh chn lc, nui 24 gi 28
0
C, 200 vngpht.
(b) Dch vi khun -c ly tm tc 3000 vngpht trong 20 pht
iu kin nhit phng v -c pha long trong mi tr-ng ti khi c
OD
600
0,3.
(c) Mi tr-ng pha long vi khun: MS (NH
4
NO
3
free), 20mgl AS, 30gl
sucrose, 10g/I gIuco, 10mM MES/I, 1mgl 2.4D, 0.2mgl BA, 1g/I
cassamino acid, 00mgl Lasparagine monohydrat, 00mgl L
glutamine, pH 5,2.
MGi tr<Jng nuGi c" mKu;
(a) Mi tr-ng ti sinh callus: MS, 30gl sucrose, 10gl gluco, 3gl phytagel,
1mgl 2.4D, 0.2mgl BA, pH 5,3 5,5.
(b) Mi tr-ng ng nui cy cho hoa lily: MS (NH
4
NO
3
free), 20mgl AS,
30gl sucrose, 10g/I gIuco, 3gl phytagel, 10mM MES/I, 1mgl 2.4D,
0.2mgl BA, 1g/I cassamino acid, 00mgl Lasparagine monohydrat,
00mgl Lglutamine, pH 5,2.
(c) Mi tr-ng ng nui cy cho hoa ng tin: MS, 30gl sucrose, 10g/I
gIuco, , 10mM MES/I, 1mgl 2.4D, 0.2mgl BA, 1g/I cassamino acid,
00mgl Lasparagine monohydrat, 00mgl Lglutamine, pH 5,2.
(d)
(e) Mi tr-ng dit vi khun: MS, 30gl sucrose, 10gl gluco, 3gl phytagel, 1mgl
2.4D, 0.2mgl BA, pH 5,2 5,5, 300mgl cefotaxim (c th s dng ti
400mgl).
(f) Mi tr-ng chn lc: MS, 30gl sucrose, 10gl gluco, 3gl phytagel, 1mgl
2.4D, 0.2mgl BA, pH 5,2 5,5, 300mgl cefotaxim, 50mgl kanamycin.
34
Ph-ng php nhim mu vi vi khun
Cch 1. Mu cy -c nhng vo dung dch vi khun trong thi gian t
5 pht sau vt ra, kh trn giy thm v trng.
Cch 2. Mu cy -c cy trn mi tr-ng ly nhim sau dng
micropipet ht dch vi khun trn nh vo mu, mi mu cy ch nh
10l dung dch vi khun.
Ph<Eng 5h5 rCa vi Ihu:n
Vi khun -c ra 23 ln bng n-c ct v trng hoc mi tr-ng MS
v trng sau -c trng li bng dung dch khng sinh cefotaxime nng
300ppm. Thao tc ra cn nhanh (trong vng 30 giy n 1pht mt
ln ra), chnh sc, trnh nhim tp.
Q!" T>?$ C!"#$ G%$ $@ 'I A!B$ AGROBACTERIUM '=
C" 5I5"
B-c 1. Nui cy vi khun v chun b huyn ph vi khun
- Nui vi khun trn mi tr-ng AB (28
0
C, 48 gi)
- Nui vi khun trn mi tr-ng YEB (28
0
C, 48 gi, 200 vngpht)
- Chun b mi tr-ng dng pha long vi khun, b sung 20mgl AS,
o OD
600
ly gi tr mu blank.
- Dch vi khun -c ly tm ly sinh khi t bo vi khun tc
5000 vngpht trong 10 pht iu kin nhit phng v -c pha
trong mi tr-ng pha long vi khun ti khi c OD
600
0,3.
B-c 2. Chun b mu cy
- Callus: Ct callus thnh cc lp c kch th-c 5 x 5 x 2mm (di, rng,
cao)
- Phn m thn: ct theo chiu ngang -c mu cy c kch th-c
0,5mm.
- Phn vy c: ct b phn l trn v ct b phn gc sau mu -c
ct thnh cc lp mng c chiu dy 0.51mm hoc khi m c dy
l 2 5mm.
- Mu cy -c t v ct pha d-i mi tr-ng ly nhim (mu ngp
trong mi tr-ng.
B-c 3. Nhim mu vi vi khun.
- Cch 1. Mu cy -c nhng vo dung dch vi khun trong thi gian t
5 pht sau vt ra, kh trn giy thm v trng.
- Cch 2. Mu cy -c cy trn mi tr-ng ly nhim sau dng
micropipet ht dch vi khun trn nh vo mu, mi mu cy ch nh
10l dung dch vi khun.
35
- Mu ro n-c trn giy thm v trng, sau -c cy vo mi tr-ng
ng nui cy
B-c 4. Nui cy mu
- Mu -c nui cy trong ti, 22 24
0
C trong ngy.
B-c 5. Xc nh kt qu
- Mu cy -c ra sch vi khun sau -c b vo cc ng effendoff
hoc vo ging ELSA. Ch c mu i chng m.
- B sung XGLC n khi ngp mu
- Ph kn bn mu bng giy nhm
- qua m nhit 3
0
C, iu kin ti
- Ht dch nhum
- B sung dung dch ph cu trc chlorophyll, lc nh, ngm khong
1560 pht (c th di hn). Thao tc ny c th tin hnh nhiu ln
n khi thy mu c mu trng (ht chlorophyll).
- Soi mu d-i knh lp quan st s nhum mu GUB.
Nu c s chuyn gen v gen -c biu hin th ti v tr
m t bo -c chuyn gen s c mu xanh chm c tr-ng.
Mu xanh chm c dng cc im c kch th-c nh.
Nu khng c gen -c biu hin th mu cy s khng c mu
xanh chm.
B-c 6. Chuyn mu qua mi tr-ng dit vi khun(mi tr-ng
phc hi)
- Mu cy -c ra sch vi khun, ro n-c trn giy thm v trng, sau
-c cy vo mi tr-ng dit vi khun.
- iu kin nui cy: 25
0
C, trong bng ti.
- Thi gian nui cy phc hi: 5 ngy.
B-c 7. Chuyn mu qua mi tr-ng chn Ic
- Mu -c cy chuyn nh k 2 hoc 3 tunln xang mi tr-ng
chn lc mi. Lp li thao tc cy chuyn trn mi tr-ng chn lc 3 4
ln.
- iu kin nui cy: 25
0
C, trong bng ti.
B-c 7. Cm ng to r cho cc chi ti sinh
- Mu -c cy chuyn xang mi tr-ng cm ng to r.
- iu kin nui cy: 25
0
C, chiu sng 16h.
B-c 8. Chn Ic cy chuyn gen bng HistochemicaI staning,
PCR v Southern bIot
Ni Dung Th Nghim
36
&h- nghim AL nh h-ng c thi gian ng nui cy n kh nng
chuyn gen ca vi khun Agrobacterium.
Nghim thc Thi gian ng nui cy ( ngy)
1 i chng (khng ly nhim vi vi khun)
2 5
3
4 10
5 15
&h- nghim @: nh h-ng ca ngun mu cy n kh nng
chuyn gen ca vi khun Agrobacterium.
Nghim
thc
Ngun mu cy
1 i chng (khng ly nhim vi khun)
2 callus
3 m l
4 m thn
5 lp mng vy c
6 vy c
nh sinh tr-ng

&h- nghim M: nh h-ng ca ph-ng php ct mu n kh nng
chuyn gen ca vi khun Agrobacterium.
Nghim
thc
Ph-ng php cy mu
1 i chng (khng ly nhim vi vi khun)
2
Mu -c ct trn giy thm, (khng nhng trong mi
tr-ng)
3 Mu ct trong mi tr-ng ng nui cy
4 Mu ct trong mi tr-ng ng nui cy pha long vi vi
3
khun
&h- nghim N: nh h-ng ca vic nui cy km mu sau khi
nhim vi vi khun vi m l cy thuc l n kh nng chuyn gen ca
vi khun Agrobacterium.
Mt s nghin cu cho thy : thuc l l cy c kh nng ti sinh to
chi, callus trn cc mi tr-ng nui cy m khng cn c nhng tc
ng nhiu v mi tr-ng cng nh- cht iu tit sinh tr-ng hay ch
nui cy. vic nghin cu nh h-ng ca ph-ng php nui cy km
vi m l cy thuc l nhm nng cao kh nng ti sinh ca mu cy
-c nghin cu v p dng v c kt qu rt kh quan trn mt s i
t-ng cy trng kh ti sinh.
Chnh v vy, mc tiu ca cc th nghim d-i y l tm hiu
nh h-ng ca vic nui cy km vi m l cy thuc l n kh nng ti
sinh ca mu cy lily sau khi ng nui cy vi vi khun.
Nghim thc Ph-ng php nui cy mu
1 i chng (khng ly nhim vi vi khun)
2 Mu cy
3 Mu cy m l thuc l
&h- nghim O: nh h-ng ca tin nui cy n kh nng chuyn
gen ca vi khun Agrobacterium.
Nghim
Thc
Thi gian tin nui cy (ngy)
1 2
2 3
3 5


Phn I'. AC oDch ' E& AiCn ACt QuF :Dt :3<c

K hoch thc hin ti: 5 thng.
38
Kt qu chnh d kin s t c: Chuyn -c gen khng PPT
vo mu cy lily trong phng th nghim.
39


2h$ v

Ti 5iu ThaG AhFo


1. Ti Iiu Ting Vit
Bo co khoa hc trang 85 ca nh xut bn khoa hc k thut
2. Ti Iiu ting Anh
Byung oon Ahn, Young Hee oung, Kathryn K.kamo, 2004.
transgenic plants of Eastr lily (lilium longiflorum) ith phosphinothricin
Resitance. . plant Biotechnolgy (2004). vol 6 pp913.
Y.Hoshi, M.kendo, S.Mori, Yadachi, M.Nakano. H.kobayashi,
2004. Production of transgenic lily plants by Agrobacteriummeiate
transformation, plant cell Rep (2004) 22:359364.
Sakae Suzuki and Masaru NAKANO, 2002 Agrobacterium
Mediated tranformation in liliaceous ornamental plants. ARQ 36, 119
12 (2002).
A. Mercuri, L.Debenedetti, S. Bruna, R. Begliano, c.Bianchini,
G.Foglia, T.schiva. Agrobacterium. Mediated transformation ith rol
genes of lilium longiflorum thunb. SHS Acta Horticluturae 612.
A. lipsky, Acohen, R.Barg, S.shabtai, Y.salts, V.Gaba, K.kamo,
A.Gera v A.atad Development of lilium longiflorum. cell cultures of
high competence for transfomation by particle bom barment anh of high
embryogenic capacity
nHar Park, HeeSung Park, 2002 lily pollen groth invitro and
Agrobacteriumnfiltration. .Plant Biotechnology (2002) vol 4pp. 151
154