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TIỂU LUẬN NUÔI CẤY MÔ TẾ BÀO THỰC VẬTĐề tài
TIỂU LUẬN NUÔI CẤY MÔ TẾ BÀO THỰC VẬTĐề tài
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
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