TIU LUN NUI CY M T BO THC VT ti: Nui cy t bo trn G/V hng

dn: PGS.TS Nguyn Th L Anh Nhm thc hin : Trn Th Chin L Th Cm L Phan
Th Thanh Phm Thu Trang Nguyn Th Tuyt M u Cc t bo thc vt c tnh
ton nng,c th nui cy, iu khin s pht sinh hnh thi ca chng cho ti
thnh mt cy hon chnh. C th ni, ni dung bn trong v t bo trong vt
cht chnh l cc thng tin di truyn cha trong nhn ca t bo quyt nh mi
ng hng ca qu trnh thc hin tnh ton nng ca t bo. V th,hon ton c
th nui cy to cy hon chnh t khi nguyn sinh cht cha nhn ca t bo. T
y a n khi nim nui cy t bo trn thc vt. T bo trn c s dng rng
ri trong nhiu lnh vc th nghim t nghin cu nhng tnh cht vt l ca mng
sinh cht n nhng nghin cu nhp bo v hp thu cc phn t, cc bo quan v
vi sinh vt. Protoplast 1.Khi nim v Protoplast Protoplast l cc t bo trong c
thnh t bo c loi b v mng t bo cht l lp ngoi cng nht trong t bo.
Protoplast c th thu c bng enzyme lytic c th loi b vch dung hp. T
bo trn c th c to ra bng nhiu cch: t dch huyn ph t bo, t bo m
so hoc t m ti nguyn trng nh l qua tc ng ca cc enzym Pectinase
phn hy pectin, cellulas phn hy hemicellulose. Cc t bo trn nu trn mi
trng dinh dng th sau 510 ngy s to vch t bo v phn chia.Cc t bo
trn, thm ch khc loi,c th kt hp vi nhau to t bo lai v quan trnh ny gi
l s dung hp t bo trn. 2. Ti sao cn phi nui cy t bo trn ? Nui cy t
bo trn s m ra 1 m hnh hp dn theo di qu trnh sinh phi t 1 t bo c
lp. c bit bit c s sp xp cc si Xenluloz xc nh hng ko di t
bo, v tr v hng ca mt phng phn chia. So vi cc k thut nui cy m t
bo khc, nui cy dung hp t bo trn gip to ra cc c th lai mang cc c
im di truyn ca cc b m c ngun gen khc xa nhau m lai hu tnh khng th
thc hin c. 3.So snh c im nui cy t bo trn vi nui cy cc t bo vn
cn thnh t bo: So vi quy trnh nui cy m t bo bnh thng th nui cy t
bo trn thng yu cu 1 s thay i do bn cht ca t bo trn. Cc thay i
thng lin quan n s iu chnh nng mui v c, thm vo cc hp cht hu
c, vitamin, ng m bo kh nng thm thu v cht iu tit sinh trng
kch thch s phn chia t bo. Nui cy t bo trn c nhiu u th hn so vi nui
cy cc t bo vn cn thnh t bo u th ca k thut nui cy v tch t bo
trn l t bo khng c mng cng, trng thi n bo, mt t bo thu c
trn mt mt n v th tch mi trng c th rt cao (t 106 t bo/ 1ml mi
trng). T bo trn mt s cy trng c kh nng ti sinh rt mnh, v d t bo
m tht l thuc l, ci du Bng thao tc di truyn t bo trn c th d dng
to ra cc t bo bin i gen. T bo vi kiu gen bin i s c bo tn khi ti
sinh t bo thnh cy hon chnh. iu ny rt kh thc hin cc t bo vn cn
thnh t bo. Nui cy t bo trn cho php kh nng bin np cc gen thun li
vo t bo thc vt m trc kia thng b v t bo ngn cn. Nui cy t bo trn
cho php kh nng dung hp t bo gn hai t bo trn li thnh mt t bo vi
hai b thng tin di truyn ca hai t bo to nn mt th lai v tnh m khng cn
hiu bit chnh xc v s lin h gia cc gen, t tn km, nhanh,trc tip v p
dng cc k thut chuyn gen( bm AND, ha thm, in thm)gip loi tr tnh

bt th hu tnh, to cy lai hu th Gip chuyn nhng c tnh c li vo cy

trng, t i hi phng tin phc tp. T bo lai thu c t vic dung hp hai t
bo trn c ti sinh v thnh mt cy lai. Qu trnh ny xy ra t bo nn gi l
lai t bo v thng qua t bo soma nn gi l lai soma hay lai v tnh t bo. T
phng php ny ra phng php lai xa gia cc loi iu khng th thc hin
bng phng php lai hu tnh thng thng. Tuy nhin qu trnh nui cy
protoplast cn tn ti mt s tr ngi l: Qu trnh c lp nui cy phi hon
thin. Cha c phng php hiu qu tuyn chn cc sn phm ph hp. 4. K
thut tch t bo trn: 4.1. Phng php c hc: Phng php ny da trn c s
ph cc mi lin kt ca m bng cc dao sc nhn ( sharpedged knife) v gii
phng cc protoplast ring r. Phng php ny cho hiu sut thp. 4.2. Phng
php enzyme: ph v thnh t bo ngi ta thng s dng cc enzyme chit
xut t cc sinh vt cha nhiu enzyme phn gii thnh vch t bo nh: nm, c
v mi. Cc enzyme ny c thng mi ha theo cc phng php khc nhau
vi mc tinh khit khc nhau Hn hp enzyme thng dng l enzyme celluloza
v macerozim c chit xut t nm Trichdearina virde v Aspergillus niger v
c sn xut cng nghip. Nng dung dch enzyme s dng ty thuc i
tng. Cc hn hp enzyme thng c s dng pH 5,5 5,8 trong 38h. Ngoi ra
protoplast khng b v sau khi thnh cellulose b phn hy ngi ta phi b sung
nhng cht tng p lc thm thu vo dung dch enzyme duy tr cn bng thm
thu gia ni bo v mi trng bn ngoi. Cc dung dch thng dng l dung dch
ng manitol, sorbitol. Nng s dng khong 0,3 0,7M ty theo i tng thc
vt. So vi phng php c hc th phng php ny c hiu qu cao hn rt nhiu.
Phng php enzyme cho php tch c hng gram protoplast. C th thu c t
1 gam l c luzec hoc khoai ty l 612 triu t bo trn. V protoplast thc cht l
t bo trn khng c thnh cho nn c th tch c t nhiu ngun khc nhau nh
cc b phn ca cy ( r, l, ht phn), callus, t bo n Xc nh cht lng t
bo trn Sau khi ph v t bo vn c nhng mnh thnh t bo cn xt li lm nh
hng n nhng nghin cu sau ny Cch tt nht pht hin thnh t bo l
dng calcofluor, mt ha cht s bm vo phn t xenlulozo v gy ra pht nh
sng hunh quang vi mu xanh rc r khi soi di tia cc tm. Nu cc t bo trn
b loi b hon ton thnh t bo hin vi trng c mu ti thm cc t bo trn
s khng nhn thy c ngoi tr s t pht nh sng hunh quang ca cc lp
th Cn mt phng php khc l dng knh hin vi hunh quang kt hp vi
nhum xanh Evan xc nh sc sng ca t bo trn. Nhng t bo cn nguyn
vn s ngn khng cho thuc nhum xm nhp vo nguyn sinh cht ngc li cc
t bo c mng sinh cht b mt chc nng s bt mu xanh v chng khng th
sinh trng c 5. Nui cy t bo trn Cc yu t nh hng n qu trnh nui
cy Thnh phn ca mi trng nui cy lng hay c ty thuc vo vt liu thc
vt.Mi trng ny c thm Auxin v Xitokinin gip s ti to vch v cc ln
phn chia u tin. m bo cc yu t dinh dng: axit amin, polyamin,
Hydrolysat Cazein, nc da, mch nha m bo iu kin nhit , PH, nh
sng,p sut thm thu. Trong ln t nhn i u tin, mi trng phi c p
sut thm thu cao, Auxin, Xitikinin thch hp, nh sng yu. Sau cn gim p

sut thm thu bng cch pha long mi trng gip cho s tng trng t bo.
Khi m so c hnh thnh cn chuyn chng vo trong mi trng rn cha Auxin
nng thp hn v Xitokinin cao hn. Sau cng kch thch ra r cn loi
Xitokinin, tng nng Auxin. Nui cy t bo trn chia lm 2 giai on Giai on 1
T t bo trn tao thnh t bo, phn chia to thnh microcallus. Sau mt thi gian
nui cy mt n hai tun cc t bo trn ti to v v phn chia to nn cc
microcallus. iu kin nui cy Nui trong mi trng lng lc Lp nui tr dng:
t bo trn, lp xp c kh nng thm t di ln, callus t m t bo m t tch
t bo trn,agar. Nui trong iu kin nh sng yu, nui ti. nh sng thm thu
ng trng.Thi gian nui t 1 n 2 tun. Giai on 2 Microcallus thnh callus n
nh hnh thnh pht sinh c quan. Chuyn cc microcallus ln mi trng cng,
chng s to thnh cc m so. T chuyn sang mi trng ti sinh chi v cy
hon chnh. iu kin nui cy. Nui cy trn mi trng c Ch ti nh sng v
quang chu k C cht iu tit sinh trng cho qua trnh ti sinh cy Loi b cht
gy nh sng thm thu Cc yu t nh hng n sinh trng, pht trin ca t
bo trn Nui cy t bo trn thng yu cu 1 s thay i so vi quy trnh nui cy
m bnh thng do bn cht ca t bo trn. Cc thay i thng lin quan n s
iu chnh nng mui v c, thm vo cc hp cht hu c, vitamin, ng
m bo kh nng thm thu v cht iu tit sinh trng kch thch s phn
chia t bo. 6. Dung hp t bo trn Dung hp l hin tng ct t mng sinh cht
ni tip xc gia 2 t bo trn khc loi do tc ng ca cc nhn t bn ngoi. Sau
l s ti t chc cc mng ban u thnh 1 v bao ly t bo cht v 2 nhn cha
m. C th ni vic dung hp t bo trn v ti sinh thnh cy lai t t bo trn l 1
trong nhng thnh tu tuyt vi ca k thut nui cy m t bo. Bng phng php
ny ra phng php lai xa gia cc loi iu m khng th thc hin bng cc
phng php lai hu tnh thng thng.T bo trn l nhng t bo khng c thnh
t bo. Chnh v th chng c th ha ln vo nhau (dung hp) v thnh 1 t bo lai
mang trong mnh vt cht di truyn ca c 2 t bo. T bo lai ny c ti sinh v
thnh 1 cy lai. Qu trnh ny xy ra t bo nn gi l lai t bo v thng qua t
bo soma nn gi l lai soma hay lai v tnh t bo S chn lc cc th lai soma
bng cch ng dng s mn cm khc nhau ca cc protoplast tht l i vi
actinomycin D C 2 phng php dung hp t bo trn: + Dung hp bng ha cht
+ Dung hp bng in Dung hp bng ha cht X l bng NaNO3 Nm 1970,
Power v cng s dng NaNO3 (0,25 M) kch thch dung hp hai protoplast.
Carlson v cng s (1972) cng dng phng php ny sn xut cy lai soma
u tin (Nicotiana glauca N. langsdorffii). Tuy nhin, phng php ny cho hiu
sut thp v NaNO3 khng thch hp vi t bo b khng bo ha mnh nh
protoplast t nhu m l. X l bng PEG Thng s dng poly ethylenglycol(PEG 5
25%) l cht c tc dng dnh kt t bo trn d dung hp chng. Nng v trng
lng phn t ca PEG quyt nh s thnh cng ca th nghim dung hp. PEG c
trng lng phn t thp (~ 100) khng th to ra mt s dnh cht chc chn,
trong khi PEG trng lng phn t 6000 cho hiu qu dung hp cao hn. X l PEG
cng vi pH/Ca2+ c hiu qu tng tn s dung hp v kh nng sng ca cc
protoplast. Qu trnh dung hp s c ci thin hn nu xy ra trong mi trng

kim (pH t 8 10) v khi c b sung CaCl2 (50250 mM). Sau khi x l bng tc nhn
dung hp, cc protoplast c nui cy theo phng thc chun. PEG c 2 tc dng:
+ Cung cp mt cu ni Ca2+ c th lin kt cc b mt mng vi nhau + Dn
n s ri lon tch in b mt mng trong sut qu trnh ra gii. Dung hp t
bo trn bng x l PEG Dung hp bng in Phng php ny n gin hn, nhanh
hn v hiu qu hn dung hp bng ha cht. iu quan trng hn c l dung hp
bng in (electrofusion) khng gy c i vi t bo nh thng thy cc
protoplast hoc cc th d nhn c x l bng PEG. Ngi ta dng cc xung
in (electric pulses) a trc tip DNA ngoi lai vo trong t bo thc vt, k
thut ny lm tng s quan tm v vic ng dng dung hp bng in vo lnh
vc di truyn t bo soma. Cch tin hnh a dung dch hn hp t bo trn(2 bn
cc c thit k trong cc hp dung hp), cc t bo trn s ln lt sp xp thnh
chui nm gia 2 bn cc. Khi c 1 xung in cao (7501000V) trong 1 thi gian rt
ngn(1200 mili giy) vng tip xc gia 2 mng t bo s b v, 2 t bo trn ha
nhp vo nhauqu trnh dung hp s xy ra. 7. Trin vng ng dng ca k thut
nui cy t bo trn v lai t bo soma Mc d nhng kh khn v mt k thut
lm hn ch tim nng s dng t bo trn tuy nhin t bo trn vn c ng
dng trong mt s lnh vc nghin cu: Sau khi loi b thnh t bo v c nui
cy trn mi trng thch hp nhng t bo trn c ti to nhanh chng thnh t
bo mi v qu trnh pht trin ny a ra mt h thng l tng cho nghin cu
sinh tng hp thnh t bo (Willison v Cocking, Grout 1973) Cc t bo trn c kh
nng tip nhn cc vt liu t bn ngoi a vo trong t bo do t bo trn l
i tng thch hp cho cc nghin cu a nhn lp th, ti th,DNA, Plasmit vo t
bo (Dodds v Bengochea 1985) Qun th t bo trn c th c xem nh mt h
thng t bo n v bi vy cc thao tc tng t nh i vi cc vi sinh vt qua
c th la chn dng t bin v tch dng qun th t bo thc vt (Evans v
Cocking 1977) Mt s ng dng khc : Sn xut cc dng b m phc v sn xut
ht lai. Sn xut cc hp cht th cp qua nhn sinh khi t bo trong mi trng
lng (nui lc, nui trong bioreactor). Cng ngh nui cy t bo trn ng dng cho
nhng cy c gi tr kinh t cao,nhng kh nhn ging bng phng php thng
thng. Cng nh ny lm nhn nhanh ging v kt hp lm sch virus. Nghin cu
to ging khoai ty khng bnh virus ca GS.TS Nguyn Quang Thch trng HNN
H Ni. Bo qun ngun gen. . Nh k thut dung hp ngi ta c th lai to gia
hai loi thc vt khc nhau, thm ch c cc loi thuc nhng chi khc nhau. in
hnh nht l vic dung hp t bo cy khoai ty vi t bo cy c chua. Kt qu l
to ra c cy lai Pmat m trn mt t cho qu c chua cn di mt t cho
c khoai ty (!). y l s dung hp 2 t bo trn khc loi to t bo lai cha b
NST ca 2 t bo gc. To c cy lai t t bo khoai ty & c chua. Nui 2 dng
t bo sinh dng khc loi trong cng 1 mi trng, c s kt dnh ngu nhin ca
2 hay 1 s t bo khc loi ca t bo lai cha b NST ca 2 t bo gc tng t l
kt dnh keo hu c xung in cao p.Dng hcmn kch thch t bo lai pht trin
thnh cy lai. Vi phng php ny c th to ra nhng c th c ngun gen khc
xa nhau m lai hu tnh khng thc hin c. K thut dung hp t bo trn cho
php m rng ngun gen ca cc loi thc vt to ra cc dng t bo sn xut mi

mang cc c tnh di truyn u vit ca c b v m Nhiu cy lai c to ra bng

phng php dung hp t bo c c nng lc chng c di hoc chng nm
bnh. To iu kin thun li cho s nghin cu v sinh l t bo: tnh thm ca
mng, vn chuyn cc cht ha tan, ion, c ch hot ng ca hoocmon thc vt
Mt ng dng y trin vng khc ca nui cy t bo trn l vi nhn ging thc
vt. Sau khi phn chia protoplast, thnh t bo c ti sinh tng s pht trin
callus v tip theo l cy hon chnh nh thc vt c th c nhn ln nhiu
ln. Nui cy t bo trn i hi s sinh trng ca protoplast trn mi trng c
hoc lng. T cc protoplast c phn lp c th c s dng : Bin i
thng tin di truyn ca t bo thc vt To ra cy lai v tnh thng qua dung hp t
bo trn Nghin cu s xm nhim ca virus thc vt v nhng vn khc. 8.
Tn ti ca k thut protoplast K thut protoplast thu c thnh cng cc
loi thuc h c (Solanaceae) v mt s h khc, nhng thnh cng h ha tho
(Poaceae) l h ca cc cy trng ng cc chnh cn rt hn ch. Potrykus (1980)
tho lun rt k v vn ny. Theo Potrykus c nhng ch tiu sau lin quan
n kt qu nui cy v ti sinh t protoplast tim lc in vitro. 1. Phn lp C s di
truyn ca t bo (kh nng tim tng ca t bo trong nui cy in vitro). Tng
tc t bo trong cy. S phn ha trong qu trnh pht trin c th. Ngun gc c
quan v cy hon chnh. Trng thi sinh l ca t bo. Tc ng ca qu trnh phn
lp. Tc ng ca trng thi phn lp. 2. iu kin nui cy Nhu cu dinh dng.
Nhu cu v phytohormone. iu kin vt l ca nui cy. Cc yu t c ch c th
xut hin. Tc ng ca mt qun th t bo 3. S phn bo Sinh tng hp
thnh t bo. iu khin sinh tng hp thnh t bo. Chc nng ca thnh t bo
i vi phn bo. iu khin s phn phn bo. iu khin s phn chia nhn.
iu khin phn bo. 4. S phn ho iu khin phn ha t bo. Tng tc t bo
trong nui cy. iu khin v c ch to kiu m. iu khin qu trnh to c quan,
phi. 9. Quy trnh c th minh ha vic nui cy v dung hp t bo trn Quy trnh
nui cy Protolast t l ca cy thuc l. 9.1. Nguyn liu thc vt L cy: L l
ngun nguyn liu thng dng v truyn thng cho k thut protoplast thc vt do
n cho php phn lp c mt s ln cc t bo tng i ng nht. Phng php
c bn tch protoplast t l cy: Kh trng mu l. Ngm mu l trong dung dch
thm thu t bo co nguyn sinh cht. Tch lp mt di l. Ngm mu trong
hn hp enzyme. Tinh sch t bo trn. Nui cy t bo trn trong mi trng thch
hp hoc dung hp hay chuyn np gene. L ca cy thuc l in vitro c chn
dng lm nguyn liu tch protoplast. S dng cc l c tch trong ngy. Cc
bc phn lp protoplast t l cy 9.2. Chun b mi trng Dung dch enzyme tch
protoplast: + Onozuka cellulase R10 0,5 % + Onozuka macerozyme R10 0,1 % +
Mannitol 13,0 % + pH mi trng ~ 5,8 Dung dch ny c kh trng bng mng
lc Millipore loi c ng knh l lc 0,20,25 m. 9.3. Tin hnh Ngy th nht Cc
mu l thuc l in vitro c loi b ht gn l v t ln a petri thy tinh v
trng. Dng dao cy bm nh cc mnh l. Cho cc mnh l bm nh vo cc
ong v trng loi 50 ml b sung 10 ml dung dch enzyme tch protoplast. Bc cc
ong bng giy parafilm, ghi nhn v t trong ti qua m trn my lc tc
thp (khong 40 vng/pht). Ngy th hai Dng micropipette chuyn dch

protoplast ln li lc v trng ( = 65m) t trong cc loi 50 ml. B sung thm 3

ml dung dch ra (PI + 10% mannitol) vo cc cha cc mnh l vn, lc mnh, v
lc tip trn li lc ri chuyn vo hn hp protoplastenzyme ca bc trn. B
sung thm 2 ml dung dch ra ra proptoplast ht khi li lc l. Chuyn hn hp
protoplastenzyme (tng cng 15 ml) vo tube ly tm loi 15 ml v ly tm 50g
trong 10 pht. Loi b phn dch ni pha trn, ti huyn ph tiu th bng 10ml
dung dch tch (PI + 20% sucrose), thm 1ml dung dch ra ln trn b mt dung
dch tch v protoplast sao cho khng lm ha ln hai dung dch, tt nht l to
thnh hai pha (nh t t dung dch ra bn thnh tube trnh bn tung to ln dch
ra bn di), ly tm 50g trong 10 pht. Cc protoplast sng st s nm trn b
mt dung dch . Dng micropipette ht lp protplast mu xanh lc ni trn tube ly
tm v chuyn n sang mt tube ly tm sch, thm 10 ml dung dch ra v ly tm
li. Protoplast s lng xung y tube v c dng vin. Ra protoplast thm mt ln
na trong 10 ml dung dch ra. Loi b phn ni trn mt v ti huyn ph
protoplast trong khong chng 1 ml mi trng nui cy protoplast (PC). t mt
git protoplast trn bung m hng cu v c lng mt protoplast (s lng
t bo/s m 10.000). Nui cy protoplast bng cch pha long 50.000 t
bo/ml mi trng nui cy protoplast v chuyn sang mt a petri v trng. Bc
li, ghi nhn v nui cy qua m trong ti 28oC. Ngy th ba Chuyn sang nui
cy nh sng yu (1020 mol.sec/m2 hoc bc mt lp vi tha di n hynh
quang nh sng trng, vi chu k chiu sng 16 gi, nui trong 2 ngy. Ngy th
nm Chuyn sang nui cy cng nh sng cao hn (5075 mol/sec/m2) trong
2 ngy bng cch b lp vi tha ra. Ngy th by Ta c c kt qu ca vic nui
cy protoplast, s t bo phn chia t tng s t bo nui cy ban u, trong mt
mu c 100200 protoplast nui cy. C du hiu ca s nhim bn khng. KT LUN
K thut nui cy v dung hp t bo trn bt u c ng dng rng ri to
ra cc cy trng mi hoc c sn lng hay cht lng cao hn, hoc l c thm
kh nng khng bnh. Vic gii thiu v quy trnh s dng enzyme c lp t bo
trn thc vt (Cooking 1960) a ra mt b mt mi y ha hn cho t bo
thc vt v nui cy m, m ra mt lnh vc mi trong sinh hc t bo thc vt.
iu lm cho t bo trn c tc ng mnh nh mt h thng th nghim l h
enzyme phn hy vch t bo lm l ra b mt mng t bo nh l mt ro cn
gia mi trng bn ngoi v thnh phn bn trong t bo. S tip cn n mng
sinh cht c ngha l th nghim c th c thit lp nghin cu v thao tc
trn cc thuc tnh ca mng t bo iu m khng th thc hin c khi b bao
ph bi vch t bo. T bo trn c s dng rng ri trong nhiu lnh vc th
nghim t nghin cu nhng tnh cht vt l ca mng sinh cht (Ruesink 1973)
n nhng nghin cu nhp bo v hp thu cc phn t (Willison et al. 1971), cc
bo quan (Potrykus 1975) v vi sinh vt (Davey v Power 1975).