Dung Hop Te Bao Tran

8/8/2019 Dung Hop Te Bao Tran

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NUI CY V DUNG HPNUI CY V DUNG HP

T BO TRNT BO TRN M uM u I. Nui cy t bo trnI. Nui cy t bo trn 1.1. Tch t bo trn1.1. Tch t bo trn 1.2. Phng php tch1.2. Phng php tch II. Cc yu t nh hng n sinh trng v phtII. Cc yu t nh hng n sinh trng v pht

trin cha t bo trn.trin cha t bo trn. 2.1. Mi trng nui cy.2.1. Mi trng nui cy. 2.2. iu kin nui cy.2.2. iu kin nui cy. 2.3. Mt nui cy.2.3. Mt nui cy. 2.4. ng dng ca t bo trn.2.4. ng dng ca t bo trn.


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III. Dung hp t bo trn.III. Dung hp t bo trn.

3.1. Cc phng php dung hp.3.1. Cc phng php dung hp. 3.1.1. Dung hp t pht (spontaneuouso3.1.1. Dung hp t pht (spontaneuouso

fusion).fusion). 3.1.2 Dung hp cm ng (inducd fusion)3.1.2 Dung hp cm ng (inducd fusion) 3.2. C s t bo hc ca dung hp t bo trn.3.2. C s t bo hc ca dung hp t bo trn. 3.3 La chn sn phm dung hp.3.3 La chn sn phm dung hp.


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M uM u

T bo trn l t bo thc vt b loi b vch bi 1T bo trn l t bo thc vt b loi b vch bi 1x l enzyme. l t bo t do, c lp, khng nhx l enzyme. l t bo t do, c lp, khng nhhng (v khng cn chu s tng quan trong hhng (v khng cn chu s tng quan trong hthng thc vt).thng thc vt).

Dung hp t bo trn l s hp nht ca cc t boDung hp t bo trn l s hp nht ca cc t bosma khng c thnh t bo ca cc c th hoc ccsma khng c thnh t bo ca cc c th hoc ccloi khc nhau v sau ti sinh cy lai t cc t boloi khc nhau v sau ti sinh cy lai t cc t bo dung hp. dung hp.

V sao li chn lai t bo trn?V sao li chn lai t bo trn?Lai hu tnh l phng php lai c bn nht to raLai hu tnh l phng php lai c bn nht to rabin d t hp thng qua dung hp giao t.bin d t hp thng qua dung hp giao t.


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--Tuy nhin lai hu tnh ch thc hin c gia ccTuy nhin lai hu tnh ch thc hin c gia ccc th trong 1 loi hay cc loi c quan h thn thuc.c th trong 1 loi hay cc loi c quan h thn thuc.Lai giu cc loi c quan h xa nhau thng rt khLai giu cc loi c quan h xa nhau thng rt khkhn v hng ro cn tr vic lai xa lm gim tnh hukhn v hng ro cn tr vic lai xa lm gim tnh hudng ca k thut lai trong vic tng ngun bin d didng ca k thut lai trong vic tng ngun bin d ditruyn cn thit cho vic ci thin cy trng.truyn cn thit cho vic ci thin cy trng.

--

Nui cy t bo trn c ch li:Nui cy t bo trn c ch li:+ Gip loi tr tnh bt th hu tnh, to cy lai hu+ Gip loi tr tnh bt th hu tnh, to cy lai huth.th.

+Gip chuyn nhng c tnh c li vo cy trng,+Gip chuyn nhng c tnh c li vo cy trng,t i hi phng tin phc tp, t tn km, nhanh vt i hi phng tin phc tp, t tn km, nhanh v

trc tip.trc tip.++ Bng thao tc di truyn t bo trn c th d

dng to ra cc t bo bin i gen. T bo vi kiugen bin i s c bo tn khi ti sinh t bo thnhcy hon chnh.


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I. Nui cy t bo trnI. Nui cy t bo trn1.1. Tch t bo trn1.1. Tch t bo trn1.1.1. Chn nguyn liu1.1.1. Chn nguyn liu

-- Thng s dng m tht l trng thnh nhng cyThng s dng m tht l trng thnh nhng cyc tnh trng sinh l tt. V l cy l ngun nguyn liuc tnh trng sinh l tt. V l cy l ngun nguyn liuthng dng v truyn thng cho k thut protoplast thcthng dng v truyn thng cho k thut protoplast thc

vt, do n cho php phn lp c mt s ln cc t bovt, do n cho php phn lp c mt s ln cc t botng i ng nht. Ctng i ng nht. Cng c th tchng c th tch t bo trn tt bo trn tm so, bao phn.m so, bao phn.

-- Ly l ngoi t nhin th phi t nhng cy khng bLy l ngoi t nhin th phi t nhng cy khng bx l vi thuc tr su, thuc dit c.x l vi thuc tr su, thuc dit c.--Nhng l c to ra trong mi trng nui cy invitroNhng l c to ra trong mi trng nui cy invitro

l vt liu l tng cung cp 1 lng ln t bo khol vt liu l tng cung cp 1 lng ln t bo khomnh.mnh.


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1.1.2 Phng php tch:1.1.2 Phng php tch:Trc khi thnh t bo b ph b, cc t bo cnTrc khi thnh t bo b ph b, cc t bo cn

c ngm trong dung dch lm n nh p sut thmc ngm trong dung dch lm n nh p sut thmthu v c iu chnh cn thn lin quan n khthu v c iu chnh cn thn lin quan n khnng thm thu t bo. Thng s dng mannitolnng thm thu t bo. Thng s dng mannitolhoc sorbitol 12hoc sorbitol 12--14% (W/v) duy tr tnh n nh14% (W/v) duy tr tnh n nhmng sinh cht.mng sinh cht.C 3 phng php tch:C 3 phng php tch:1.1.2.1. Phng php tch bng c hc1.1.2.1. Phng php tch bng c hc::Ct nh nhu m tht l ri nghin nh cho vo dungCt nh nhu m tht l ri nghin nh cho vo dung

dch co nguyn sinh nn c t bo cht b co nh lidch co nguyn sinh nn c t bo cht b co nh li

ngay lp tc cho vo dung dch phn nguyn sinh,ngay lp tc cho vo dung dch phn nguyn sinh,lm gin n 1 cch t ngt y phn nguyn sinhlm gin n 1 cch t ngt y phn nguyn sinhcht qua thnh t bo. Ta thu c t bo trn.cht qua thnh t bo. Ta thu c t bo trn.

+u im: Dng c n gin+u im: Dng c n gin

+Nhc im: Kh phc tp, hiu qu thp.+Nhc im: Kh phc tp, hiu qu thp.


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1.1.2.2.Phng php tch bng enzyme:1.1.2.2.Phng php tch bng enzyme:Dng enzym pectinase, hemicelolase, cellulase. Dng enzym pectinase, hemicelolase, cellulase.

ph v thnh t bo c th dng ring l hoc kt hpph v thnh t bo c th dng ring l hoc kt hptu vo tng loi t bo.tu vo tng loi t bo.-- u im: Thu c lng ln cc t bo trnu im: Thu c lng ln cc t bo trn

--Cc bc tin hnh nh sau:Cc bc tin hnh nh sau:

+Thu mu v v trng b mt l.+Thu mu v v trng b mt l.+ Ngm trong dung dch thm thu ph hp.+ Ngm trong dung dch thm thu ph hp.+Loi b biu b mt di hoc ct mu l thnh+Loi b biu b mt di hoc ct mu l thnh

cc lt mng, to thun li cho s xm nhp ca cccc lt mng, to thun li cho s xm nhp ca ccenzym.enzym.

+ X l hn hp enzym.+ X l hn hp enzym.+Tinh sch v thu nhn cc t bo trn.+Tinh sch v thu nhn cc t bo trn.+Chuyn t bo trn vo cc mi trng nui cy+Chuyn t bo trn vo cc mi trng nui cy

thch hp.thch hp.


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1.1.2.31.1.2.3. Phng php hn hp:. Phng php hn hp:--S dng phng php c hc thu c 1 lng lnS dng phng php c hc thu c 1 lng ln

cc t bo t do sau cc t bo t do sau dngdng enzyme thng c s dngenzyme thng c s dng tch thu c t bo trn. Thnh t bo cu to tch thu c t bo trn. Thnh t bo cu to gmgmcellulose, hemicellulose, pectin v mt lng t hn lcellulose, hemicellulose, pectin v mt lng t hn lprotein v lipit. V vy cn mt lng hn hp c 3 loiprotein v lipit. V vy cn mt lng hn hp c 3 loienzym cellulaza, pectinaza hemicellulaza nhng t lenzym cellulaza, pectinaza hemicellulaza nhng t lkhc nhau.khc nhau.

--Mt s loi enzym thng dng:Mt s loi enzym thng dng:

cellulosecellulose hemicellulosehemicellulose PectinazaPectinazaOnozukaOnozuka Rhozym HPRhozym HP--150150 MacerazaMaceraza

CellulysinCellulysin HemicelluloseHemicellulose Macerozym RMacerozym R--1010

MeicelazaMeicelaza PectolyazaPectolyaza


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1.1.3.1.1.3. Xc nh cht lng ca t bo trnXc nh cht lng ca t bo trnCc t bo trn u c dng hnh cu khi quan st diCc t bo trn u c dng hnh cu khi quan st di

knh hin vi.knh hin vi.--Bc 1: Xc nh xem cn thnh t bo hay khng?Bc 1: Xc nh xem cn thnh t bo hay khng?

Dng calcofour xc nh .Calcofour bm vo ccDng calcofour xc nh .Calcofour bm vo ccphn t cellulose v gy ra pht nh sng hunh quangphn t cellulose v gy ra pht nh sng hunh quangmu xanh rc r khi soi di tia cc tm.mu xanh rc r khi soi di tia cc tm.Nu cc t bo b loi b hon ton thnh t bo, hinNu cc t bo b loi b hon ton thnh t bo, hin

vi trng c mu ti thm, cc t bo trn s khng nhnvi trng c mu ti thm, cc t bo trn s khng nhnthy c ngoi tr s t pht nh sng hunh quang thy c ngoi tr s t pht nh sng hunh quang ca cc lp th.ca cc lp th.

--Bc 2: Xc nh kh nng sng st ca t bo trnBc 2: Xc nh kh nng sng st ca t bo trnBi v quy trnh tchBi v quy trnh tch t bo trn thng lm tn thngt bo trn thng lm tn thnghoc lm hng mt t l t bo nn khng th thiu bchoc lm hng mt t l t bo nn khng th thiu bcny.ny.


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C thC th quan st trc tip huyn ph t bo trnquan st trc tip huyn ph t bo trndi knh hin vi vt knh 20x v 40x.di knh hin vi vt knh 20x v 40x.T bo cht ca nhng t bo xut hin dngT bo cht ca nhng t bo xut hin dng

dng chy hoc chuyn ng trn, trng nh nhngdng chy hoc chuyn ng trn, trng nh nhnght nh trong nguyn sinh cht l nhng t boht nh trong nguyn sinh cht l nhng t bosng.sng.

Ngc li l nhng t bo gn nh b cht do mngNgc li l nhng t bo gn nh b cht do mngnguyn sinh cht b ph hu.nguyn sinh cht b ph hu.-- Bc 3: Xc nh sc sng ca t bo trn:Bc 3: Xc nh sc sng ca t bo trn:

+Dng knh hin vi hunh quang kt hp vi+Dng knh hin vi hunh quang kt hp vinhum xanh evan.nhum xanh evan.

Trn 2 ml dung dch thuc nhum 1% + 10 ml miTrn 2 ml dung dch thuc nhum 1% + 10 ml mitrng tch t bo trn.trng tch t bo trn.Ly 0,25 ml huyn ph t bo trn v a vo 1 mlLy 0,25 ml huyn ph t bo trn v a vo 1 ml

hn hp dch nhum trn, trong 15 pht,hn hp dch nhum trn, trong 15 pht,


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+Quan st di knh hin vi hunh quang.+Quan st di knh hin vi hunh quang.

Nhng t bo cn nguyn vn s ngn khng cho thucNhng t bo cn nguyn vn s ngn khng cho thucnhum xm nhp vo nguyn sinh cht, cnhum xm nhp vo nguyn sinh cht, cnn nhng tnhng tbo c mng nguyn sinh cht b mt chc nng s btbo c mng nguyn sinh cht b mt chc nng s btmu xanh v chng khng th sinh trng c.mu xanh v chng khng th sinh trng c.

+Kim tra t l sng ca t bo trn:+Kim tra t l sng ca t bo trn:S dng fluorescein diacetat (FDA) th tch 0,8mlS dng fluorescein diacetat (FDA) th tch 0,8ml

dung dch vi 20ml mi trng cha t bo trn.dung dch vi 20ml mi trng cha t bo trn.Khi soi di nh sng t ngoi, t bo trn cn sng sKhi soi di nh sng t ngoi, t bo trn cn sng s

pht nh sng hunh quang mu xanh, nhng t bo trnpht nh sng hunh quang mu xanh, nhng t bo trnc mng sinh ct b ph hu s c mu sm hoc muc mng sinh ct b ph hu s c mu sm hoc muti.ti.


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1.1.4. Phng php nui cy t bo trn1.1.4. Phng php nui cy t bo trnCc t bo trn sau khi c tch t mu thc vt sCc t bo trn sau khi c tch t mu thc vt s

c lm long trong mi trng lngc lm long trong mi trng lng--Xc nh mt t bo trong bung m hng cu.Xc nh mt t bo trong bung m hng cu.+ Mt thch hp cho nui cy t bo trn l10+ Mt thch hp cho nui cy t bo trn l1044 --101066

t bo/ml mi trngt bo/ml mi trng+Trong iu kin nui cy thch hp thnh t bo mi+Trong iu kin nui cy thch hp thnh t bo mi

c ti to sau khong 48c ti to sau khong 48--96 gi nui cy.sau cc t96 gi nui cy.sau cc tbo s bt u phn chia v hnh thnh m so.khi cbo s bt u phn chia v hnh thnh m so.khi cchuyn sang mt mi trng mi cc t bo m so schuyn sang mt mi trng mi cc t bo m so spht sinh phi(sau khong 3pht sinh phi(sau khong 3--4 tun nui cy) ri pht4 tun nui cy) ri phttrin thnh cy hon chnh.trin thnh cy hon chnh.--u im:u im:Nui cy trong mi trng lng c sNui cy trong mi trng lng c s

dng nhiu do c th iu chnh c mt t bo vdng nhiu do c th iu chnh c mt t bo vp sut thm thu.mt s loi thc vt t bo trn khngp sut thm thu.mt s loi thc vt t bo trn khngth phn chia trn mi trng c.th phn chia trn mi trng c.


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II. Cc yu t nh hng n sinh trngII. Cc yu t nh hng n sinh trngv pht trin ca t bo trnv pht trin ca t bo trn

2.1. Mi trng nui cy2.1. Mi trng nui cy- Trong qu trnh phn lp v nui cy, cc t bo trn

cn c duy tr cn bng p lc thm thu trnh

hin tng u trng hoc nhc trng dn n tnhtrng t bo trn b v hoc teo li.

-Cc cht iu chnh p lc thm thu trong mi trngnui cy t bo trn v trong hn hp enzyme l:

sorbitol, mannitol, glucose, hoc sucrose.Cc t bo trn s sinh trng n nh hn trong dung

dch c tng nh p lc thm thu.


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- trnh gy c v thc y s phn bo th nng trnh gy c v thc y s phn bo th nng nitratamon = nitratamon = -- hm lng nui cy m ca t hm lng nui cy m ca t

bo bnh thng.bo bnh thng.Cc mi trng nui m v c quan bnh thng cCc mi trng nui m v c quan bnh thng c

nng Ca t 0,5nng Ca t 0,5--0,3mM v nhng nng thp0,3mM v nhng nng thp

ny, cc t bo trn b ngng kt v ho nu nhanhny, cc t bo trn b ngng kt v ho nu nhanhchng.chng.

Hm lng Ca phi tng ln 14Hm lng Ca phi tng ln 14--40mM s thc y40mM s thc yphn bo sm, tng tnh ng b t bo s lm gim sphn bo sm, tng tnh ng b t bo s lm gim s

ngng kt v ho nu ca t bo trn trong giai onngng kt v ho nu ca t bo trn trong giai onsm ca nui cy.sm ca nui cy.

Thnh phn Ca cung cp trong mi trng MS dngThnh phn Ca cung cp trong mi trng MS dngCaClCaCl22.2H.2H22O, Ca(HO, Ca(H22POPO44))22


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--Thnh phn ha c nh inositol, a.nicotinic, biotin,Thnh phn ha c nh inositol, a.nicotinic, biotin,pyridoxy, thiamin, glyxin, axit folic, c b sung vopyridoxy, thiamin, glyxin, axit folic, c b sung vomi trng nui cy.mi trng nui cy.

Trong cazein thu phn,DTrong cazein thu phn,D--Ca pantothenat, cholin,Ca pantothenat, cholin,

chlorit,xystein, a.malic, axit ascobic, adenin sulfatchlorit,xystein, a.malic, axit ascobic, adenin sulfatthng c a vo mi trng nui cy 0.01thng c a vo mi trng nui cy 0.01 --10mg/l y nhanh s sinh tng hp v thc y s10mg/l y nhanh s sinh tng hp v thc y sphn bo,phn bo,

a s cc thnh phn ny khng c s dng sau khia s cc thnh phn ny khng c s dng sau khit bo trn c ti to thnh t bo mi v phn chia.t bo trn c ti to thnh t bo mi v phn chia.


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--ng cung cp l ngun C v cht n nh p sutng cung cp l ngun C v cht n nh p sutthm thu nh manitol,sorbitol (0.3thm thu nh manitol,sorbitol (0.3--0.7M) trong khi0.7M) trong khi

sacarozo v glucozo l ngun C hm lng t 0.2sacarozo v glucozo l ngun C hm lng t 0.2--0.6M. Nng ng c gim dn sau khi cc t0.6M. Nng ng c gim dn sau khi cc tbo bt u phn chia.bo bt u phn chia.-- Cc cht iu ho sinh trng l rt cn thit choCc cht iu ho sinh trng l rt cn thit cho

trong nui cy t bo trn. Loi v nng s dng ltrong nui cy t bo trn. Loi v nng s dng lkhc nhau gia cc loi v kiu ti sinh. NAA 2,4khc nhau gia cc loi v kiu ti sinh. NAA 2,4--D cD cnng t 0,45nng t 0,45 10,7um, trong khi cytokinin nh10,7um, trong khi cytokinin nhzeatin v benzyl amino purin c dng t 2zeatin v benzyl amino purin c dng t 2--5um.5um.

VD:i vi cc t bo trn tht l ca ng cc vu th mannitol hoc sorbitol l cc nhn t n nh plc thm thu thch hp hn c, trong khi sucrose lithch hp hn glucose hoc mannitol trong nui cyprotoplast ca khoai ty, u hoa (sweet pea), tc


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mch (brome grass) v sn. Trong nui cy dch huynph thuc l, galactose v fructose c s dng

iu chnh p lc thm thu.2.2. iu kin nui cy2.2. iu kin nui cyKhi cha hnh thnh thnh t bo rt nhy cm viKhi cha hnh thnh thnh t bo rt nhy cm vi

nh sng v vy chng c nui cy trong ti hoc nhnh sng v vy chng c nui cy trong ti hoc nh

sng yu cho n khi tng hp xong thnh t bo sau sng yu cho n khi tng hp xong thnh t bo sau chuyn dn ra nui mc nh sng cao hn. nh sngchuyn dn ra nui mc nh sng cao hn. nh sngtrng v xanh th tt hn so vi nh sng trong nuitrng v xanh th tt hn so vi nh sng trong nuicy t bo trn. Giai on hnh thnh m so th a racy t bo trn. Giai on hnh thnh m so th a ranh sng bnh thng .nh sng bnh thng .

Qu trnh nui cy t bo trn c duy tr nhit Qu trnh nui cy t bo trn c duy tr nhit t 20t 20--252500CC2.3. Mt nui cy2.3. Mt nui cy

Mt nui cy c nh hng n sinh trng v phtMt nui cy c nh hng n sinh trng v phttrin ca t bo trntrin ca t bo trn


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VD: Nui cy thuc l mt 5.10VD: Nui cy thuc l mt 5.1044 tb/ml cho hiutb/ml cho hiuqu tt nht. Khi gim cn 5.10qu tt nht. Khi gim cn 5.1055 tb/ml t bo trntb/ml t bo trn

khng phn chiakhng phn chia1.3.ng dng ca t bo trn1.3.ng dng ca t bo trn--Chn dng t boChn dng t bo--Dung hp protoplast lai v tnh t bo thc vtDung hp protoplast lai v tnh t bo thc vt

--Bin np di truyn, a cc c quan t, virut, DNABin np di truyn, a cc c quan t, virut, DNAngoi lai vo t bo thc vt.ngoi lai vo t bo thc vt.

--L h thng l tng cho nghin cu sinh tng hpL h thng l tng cho nghin cu sinh tng hpthnh t bothnh t bo

1.3.1.u im1.3.1.u im--Thao tc thc hin thun liThao tc thc hin thun li--c th thao tc t bin v tch qun th t bo thcc th thao tc t bin v tch qun th t bo thc

vt, chn c dng t bo ring r.vt, chn c dng t bo ring r.


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--C th chuyn gen c d dng hn vo h gen nhnC th chuyn gen c d dng hn vo h gen nhnhoc vo cc bo quan.hoc vo cc bo quan.

--To ra nhng sinh vt mi bin i gen ,to th lai tTo ra nhng sinh vt mi bin i gen ,to th lai tbo cht hoc th lai gen.bo cht hoc th lai gen.--S dng t bo trn ring r cho php loi b mi tngS dng t bo trn ring r cho php loi b mi tng

tc vi cc t bo bn cnh v nhng thay i di truyntc vi cc t bo bn cnh v nhng thay i di truync iu kin biu hin r rng hn.c iu kin biu hin r rng hn.

1.3.2.Nhc im1.3.2.Nhc im::--Mi ch thnh cng h c solanacea, hMi ch thnh cng h c solanacea, h

Brassiccaceac..Brassiccaceac..--Cn h ho tho Gramineae l h ca cc cy trngCn h ho tho Gramineae l h ca cc cy trng

ng cc cn hn chng cc cn hn chVD: Cy la l i tng thnh cng nht trong nui cyVD: Cy la l i tng thnh cng nht trong nui cy

protoplast nhng vn ch hn ch cc ging thucprotoplast nhng vn ch hn ch cc ging thucloi ph. Japonica ,cn cc ging la thuc di chiloi ph. Japonica ,cn cc ging la thuc di chi


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indica vn kh ti sinh thnh cng v trong s cy ti sinhindica vn kh ti sinh thnh cng v trong s cy ti sinhc t l bt th thng rt caoc t l bt th thng rt cao

--Cch dung hp c th to c ging mi do s kt hpCch dung hp c th to c ging mi do s kt hpnhng tnh trng tt ca hai loi tuy nhin cch dung hpnhng tnh trng tt ca hai loi tuy nhin cch dung hpny thng c s loi bt NST trong phn bo v khngny thng c s loi bt NST trong phn bo v khngn nh .n nh .

--cho n nay ngi ta ch nhn c cy pomato l cy laicho n nay ngi ta ch nhn c cy pomato l cy lai

gia c chua v khoai ty v phi gi ging kh khngia c chua v khoai ty v phi gi ging kh khntrong iu kin th nghim.trong iu kin th nghim.


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1.4 Th nghim tch v nui cy t bo trnthuc l

1.4.1. Nguyn liu thc vtL ca cy thuc l in vitro c chn dng lm nguynliu tch t bo trn. S dng cc l c tch trong ngy.1.4.2. 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 ~ 5,8Dung dch ny c kh trng bng mng lc Milliporeloi c ng knh l lc 0,2-0,25 m.


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1.4.3 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 dung dchenzyme tch protoplast. Bc cc ong bng giyparafilm, ghi nhn v t trong ti qua m trn my

lc tc thp (khong 40 vng/pht).* Ngy th hai: Dng micropipette chuyn dch t bo

trn ln li lc v trng ( = 65m) t trong cc loi50 ml. B sung thm 3 ml dung dch ra (PI + 10%

mannitol) vo cc cha cc mnh l vn, lc mnh, vlc tip trn li lc ri chuyn vo hn hp protoplast-enzyme ca bc trn. B sung thm 2 ml dung dchra ra t bo trn ht khi li lc. Chuyn hn hpprotoplast-enzyme (tng cng 15 ml) vo tube ly


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tm loi 15 ml v ly tm 50gtrong 10 pht.+Loi b phn dch ni pha trn, ti huyn ph tiu th

bng 10ml dung dch tch (PI + 20% sucrose), thm 1mldung dch ra ln trn b mt dung dch tch v t bo trnsao cho khng lm ha ln hai dung dch, tt nht l tothnh hai pha (nh t t dung dch ra bn thnh tubetrnh bn tung to ln dch ra bn di), ly tm 50gtrong 10 pht.

Cc t bo trn sng st s nm trn b mt dung dch.+Dng micropipette ht lp t bo trn 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, t bo trn s lng

xung y tube v c dng vin.+Ra t bo trn thm mt ln na trong 10 ml dungdch ra.


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+ Loi b phn ni trn mt v ti huyn ph t bo trntrong khong chng 1 ml mi trng nui cy t bo trn

(PC). t mt git t bo trn trn bung m hng cuv c lng mt t bo trn (s lng t bo/s m 10.000).

Nui cy t bo trn bng cch pha long 50.000 tbo/ml mi trng nui cy t bo trn v chuyn sangmt a petri v trng. Bc li, ghi nhn v nui cy quam trong ti 280C .* Ngy th ba:

Chuyn sang nui cy nh sng yu (10-

20mol.sec/m2 hoc bc mt lp vitha di n hynhquang nh sng trng, vi chu k chiu sng 16 gi, nuitrong 2 ngy.


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* Ngy th nm:

Chuyn sang nui cy cng nh sng cao hn (50-

75 mol/sec/m2) trong 2 ngy bng cch b lp vi tha ra.

* Ngy th by Ghi li kt qu ca bi hc nui cy t botrn, s t bo phn chia t tng s t bo nui cy ban u,

trong mt mu c 100-200 t bo trn nui cy. C du hiuca s nhim bn khng.


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Cc bc nui cy t bo trnCc bc nui cy t bo trn


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III. Dung hp t bo trn v lai t bo xomaIII. Dung hp t bo trn v lai t bo xomaDung hp l hin tng ct t mng sinh cht niDung hp l hin tng ct t mng sinh cht ni

tip xc gia 2 t bo trn khc loi do tc ng ca cctip xc gia 2 t bo trn khc loi do tc ng ca ccnhn t bn ngoi. Sau l s ti t chc cc mng bannhn t bn ngoi. Sau l s ti t chc cc mng banu thnh 1 v bao ly t bo cht v 2 nhn cha m.u thnh 1 v bao ly t bo cht v 2 nhn cha m.

Khi 2 hoc nhiu t bo trn dung hp ngu nhin sKhi 2 hoc nhiu t bo trn dung hp ngu nhin s

to mt khi t bo trn, trong nhn ca chng c thto mt khi t bo trn, trong nhn ca chng c thkt hp hoc tn ti ring r. Nu hai nhn khc nhau skt hp hoc tn ti ring r. Nu hai nhn khc nhau sto ra th lai d nhn ngc li c th lai ng nhn. Sto ra th lai d nhn ngc li c th lai ng nhn. Skt hp nhn trong th d nhn hnh thnh t bo trn laikt hp nhn trong th d nhn hnh thnh t bo trn lai

thc s gi l th d nhn. Nu 1 trong 2 nhn b othc s gi l th d nhn. Nu 1 trong 2 nhn b othi, nguyn sinh cht ca 2 t bo trn s dung hp tothi, nguyn sinh cht ca 2 t bo trn s dung hp tora th lai t bo cht cybrid (Cytoplasmid hybrid)ra th lai t bo cht cybrid (Cytoplasmid hybrid)


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33..11. Cc phng php. Cc phng phpdung hpdung hp33..11..11. Dung hp t pht. Dung hp t pht::--KhKhi nii nimm:: ll hihin tn tngng ccc tc t b bootrtrnn c cnh nhau dung hnh nhau dung hp vp vi nhau.i nhau.

--

c ic imm:: TrongTrong qu trqu trnh phn cnh phn cttththnh tnh t b bo bo bng enzymng enzym,, ccc tc t b bootrtrn n cc hhnhnh ththnh tnh t nhnhng tng t b bootitipp gigip vp vi nhaui nhau cc thth dung hdung hppvvi nhau qua ci nhau qua cu nu ni sinh chi sinh chtt.. ViVicc

loloii b thb thnh tnh t b bo o l lm chom cho ccccccu nu ni sinh chi sinh cht git giaa ccc tc t b boo giginnnn to lnto ln.. ChCht nguyn sinh v cct nguyn sinh v cc


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c quan tc quan t hai hay nhihai hay nhiu tu t b boo cc thth lin thng sanglin thng sangvvi nhaui nhau hhnhnh ththnh thnh th dung hdung hpp

--SS dung hdung hp tp t ph phtt ch xch xy ra trongy ra trong phphm vim vi 11 loloiittcc ll nhnhng tng t b boo ccngng loloii c cng nhau trongng nhau trong ququtrtrnhnh ttchch.. HiHin tn tngng nny hiy him gm gp dop do cc ss t tch ich innm trn bm trn b mmtt mmng tng t b bo tro trnn llm chom cho ccc tc t b bo o yy

nhau ranhau ra.. yy ll nguyn nhn ngnnguyn nhn ngn ccnn ccc tc t b bo tro trnntt kkt ht hp vp vi nhau.i nhau.


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3.1.2. Dung hp cm ng

3.1.2.1. X l bng NaNO3Nm 1970, Power v cng s dng NaNO3 (0,25 M)kch thch dung hp hai protoplast. Carlson v cs (1972)cng dng phng php ny sn xut cy lai soma u

tin (Nicotianaglauca N.langsdorffii). Tuy nhin,phng php ny cho hiu sut thp v NaNO3 khng thchhp vi t bo b khng bo ha mnh nh t bo trn tnhu m l.


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3.1.2.2. X l bng PEG

-Tc nhn kch thch dung hp l PEG (polyethylen glycol).

+Khong 0,6 ml dung dch PEG (ha tan 1 g PEG mol. wt. 1500trong 2 ml glucose 0,1 M, CaCl2 10 mM, v KH2PO4 0,7 mM) lmthnh mt git protoplast v chuyn ln a petri.

+Sau khi y np, t bo trn trong dung dch PEG c nui nhit phng trong 40 pht, pha long dung dch PEG bng cch

b sung 0,5-1 ml mi trng nui cy t bo trn sau mi 10 pht.+ Ra t bo trn vi dung dch khng c tc nhn dung hp bngcch ly tm v cc t bo trn c treo tr li trong mi trngnui cy.

-Nng v trng lng phn t ca PEG quyt nh s thnhcng 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


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T bo x l bng PEGT bo x l bng PEG

trng lng phn t 6000 cho hiu qu dung hp caohn. PEG cng vi pH/Ca2+ c hiu qu tng tn s dunghp v kh nng sng ca cc protoplast. Sau khi x l

bng tc nhn dung hp, cc protoplast c nui cytheo phng thc chun. PEG c 2 tc dng: hoc cungcp mt cu ni Ca2+ c th lin kt cc b mt mngvi nhau hoc dn n s ri lon tch in b mt mng

trong sut qu trnh ra gii.


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3.1.2.3. X l vi nng ion Ca3.1.2.3. X l vi nng ion Ca+2+2 cao v pH caocao v pH caoHn hp t bo trn tch t tht l c trong miHn hp t bo trn tch t tht l c trong mi

trng kim mnh c 0,05M CaCLtrng kim mnh c 0,05M CaCL22, 0,4M 9% (W/v), 0,4M 9% (W/v)manitol, pH cao (8manitol, pH cao (8--10) v ly tm nh 50g trong 3 pht,10) v ly tm nh 50g trong 3 pht,tip theo em trong nc nhit 37tip theo em trong nc nhit 3700C trong 45 pht.C trong 45 pht.Bng phng php ny Keller v Melcher (1973) dungBng phng php ny Keller v Melcher (1973) dung

hp thnh cng t bo trn tch t m du ca 2 dng thuchp thnh cng t bo trn tch t m du ca 2 dng thucl. Tuy nhin pH cao c th gy c cho t bo trn ca mtl. Tuy nhin pH cao c th gy c cho t bo trn ca mts loi thc vt.s loi thc vt.


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3.1.2.4. Dung hp bng inPhng php ny n gin hn, nhanh hn v hiu qu hndung hp bng ha cht.

- Dung hp bng in (electrofusion) khng gy c i vit bo nh thng thy cc t bo trn hoc cc th d

nhn c x l bng PEG.Ngi ta dng cc xung in (electric pulses) atrc tip DNA ngoi lai vo trong t bo thc vt, k thutny lm tng s quan tm v vic ng dng dung hp

bng in vo lnh vc di truyn t bo soma.- Cc nh khoa hc a ra mt protocol c th s dng

rng ri.


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Hnh 6. Cc tbo trntht l ca cy thuc l xpthnh chui ngc trai dinh hng ca trng AC(100 V/cm, 0,6 MHz)

Cc th d nhn hnh thnh nhdung hp bng in phn chia

trong mi trng nui cy v ckh nng ti sinh chi hoc cy laisoma, bao gm:Nicotiana tabacum(+) N. tabacum,N. plumbaginifolia

(+) N. tabacum,N.glauca (+) N.langsdorfii, v Solanum tuberosum(+) S. phureja.

Mt s t hp lai protoplast

hnh thnh callus nh:Brassicanapus(+)B. napusv Solanumbrevidens(+) N. rustica.


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3.2. C s t bo hc ca dung hp3.2. C s t bo hc ca dung hp--Dung hp 2 t bo trn mc ch to thnh 2 th laiDung hp 2 t bo trn mc ch to thnh 2 th lai

hn hp v xy ra cc hin tng nh sau:hn hp v xy ra cc hin tng nh sau:* Dung hp t bo cht v c quan t nhng khng* Dung hp t bo cht v c quan t nhng khngdung hp nhn.dung hp nhn.

*

Dung hp tt c.*

Dung hp tt c.Nu dung hp ch xy ra t bo cht v c quan t ccNu dung hp ch xy ra t bo cht v c quan t ccnhn vn gi nguyn th to thnh th lai t bo chtnhn vn gi nguyn th to thnh th lai t bo cht(Cybrid). Cn trng hp t bo cht v nhn c(Cybrid). Cn trng hp t bo cht v nhn c

dung hp th to t bo lai thc s (Hybrid)dung hp th to t bo lai thc s (Hybrid)-- Dung hp t bo trn bao gm 3 pha:Dung hp t bo trn bao gm 3 pha:


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**Pha 1Pha 1: Pha kt dnh: Pha kt dnhMng sinh cht ca 2 hay nhiu t bo trn tin st nhau.Mng sinh cht ca 2 hay nhiu t bo trn tin st nhau.

**

Pha 2Pha 2: Pha Dung hp mng ti nhng vng nh lin kt: Pha Dung hp mng ti nhng vng nh lin ktcht dn n hnh thnh t bo cht lin tc hoc cu giacht dn n hnh thnh t bo cht lin tc hoc cu giacc t bo trn.cc t bo trn.

**Pha 3Pha 3: Gin n ca cc cu nguyn sinh cht v hnh: Gin n ca cc cu nguyn sinh cht v hnh

thnh th ng nhn hoc d nhn hnh cu.thnh th ng nhn hoc d nhn hnh cu.Sau khi dung hp mng t bo cht ca 2 t bo trn s ktSau khi dung hp mng t bo cht ca 2 t bo trn s kthp vi nhau khong vi gi. Nu th d nhn lai t bohp vi nhau khong vi gi. Nu th d nhn lai t bocht 2 nhn c th phn bo ng thi trn cng 1 sicht 2 nhn c th phn bo ng thi trn cng 1 si

thoi v sc chung nhng cc nhim sc th khng i cngthoi v sc chung nhng cc nhim sc th khng i cngnhau. Kt qu to ra m khm sau c s thoi ho vnhau. Kt qu to ra m khm sau c s thoi ho vphn ly 1 nhn to ra nhng t bo cha t bo cht caphn ly 1 nhn to ra nhng t bo cha t bo cht cac 2c 2


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t bo trn nhng ch c 1 nhn.t bo trn nhng ch c 1 nhn.Th d nhn ch duy tr 1 vi th h t bo, sau xy raTh d nhn ch duy tr 1 vi th h t bo, sau xy ra

dung hp nhn gian k hoc ln nguyn phn ng bdung hp nhn gian k hoc ln nguyn phn ng bu tin.u tin.Ch c nhng t bo lai c to ra qua dung hp nhnCh c nhng t bo lai c to ra qua dung hp nhn

mi c kh nng tip tc pht trin.mi c kh nng tip tc pht trin.

--Trong cc t bo lai s hp nht hon ton genom t 2Trong cc t bo lai s hp nht hon ton genom t 2ngun thng khng xy ra. S o thi dn dn nhimngun thng khng xy ra. S o thi dn dn nhimsc th ca 1 trong cc dng cha m. Kt qu ca t bo laisc th ca 1 trong cc dng cha m. Kt qu ca t bo lail 1 t bo ca 1 loi. T bo lai ch dng trong thi gianl 1 t bo ca 1 loi. T bo lai ch dng trong thi gianngn, trong vng 1 vi th h.ngn, trong vng 1 vi th h.

VdVd: Lai dng t bo lai gia loi tng v thuc l thy: Lai dng t bo lai gia loi tng v thuc l thys phn bo khng bnh thng xy ra ln phn chia us phn bo khng bnh thng xy ra ln phn chia utin sau 1 thng nhim sc th thuc l him gp vtin sau 1 thng nhim sc th thuc l him gp vsau 7 thng ch cn li 1sau 7 thng ch cn li 1


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s nhim sc th ca thuc l cn tn ti trong th lai ngs nhim sc th ca thuc l cn tn ti trong th lai ngthi cc dng t bo lai c nhng c im ging vi ccthi cc dng t bo lai c nhng c im ging vi ccdng tng.dng tng.

3.3. La chn sn phm dung hp3.3. La chn sn phm dung hp--Sau khi dung hp, qun th t bo trn s gm c cc tSau khi dung hp, qun th t bo trn s gm c cc t

bo trn cha m, th ng nhn, th d nhn v cc snbo trn cha m, th ng nhn, th d nhn v cc snphm dung hp a nhn. V vy phi la chn sn phmphm dung hp a nhn. V vy phi la chn sn phm

dung hp t bo trn c lai 1 cch hon thin c v tdung hp t bo trn c lai 1 cch hon thin c v tbo cht v nhn.bo cht v nhn.--C th nhn bit sn phm dung hp da vo cc cC th nhn bit sn phm dung hp da vo cc c

im hnh thi, gen nh du, mi trng chn lc, dng tim hnh thi, gen nh du, mi trng chn lc, dng t

bo trn t cc loi m khc nhau hoc dng phng phpbo trn t cc loi m khc nhau hoc dng phng phpnhum bng cc cht pht hunh quang.nhum bng cc cht pht hunh quang.+Con lai xoma c th c phn bit nh nhng khc bit+Con lai xoma c th c phn bit nh nhng khc bit

t nhin gia cc dng cha m.Thng th sn phm dungt nhin gia cc dng cha m.Thng th sn phm dung

hp to ra chi mu tm .hp to ra chi mu tm .


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VDVD: Khoai ty lai, cy khoai ty di Solamum chacoense: Khoai ty lai, cy khoai ty di Solamum chacoensec cc t bo cha sc t anthocyanin mu tm nhngc cc t bo cha sc t anthocyanin mu tm nhngsinh trng rt km trong mi trng nui cy v khngsinh trng rt km trong mi trng nui cy v khngth ti sinh thnh c chi. Khoai ty trng Solamumth ti sinh thnh c chi. Khoai ty trng Solamumtuberosum mang cc c im tri ngc, c kh nngtuberosum mang cc c im tri ngc, c kh nngto chi nhanh. M so hnh thnh t sn phm dung hpto chi nhanh. M so hnh thnh t sn phm dung hpsinh trng mnh hn m so t cc dng cha m v tosinh trng mnh hn m so t cc dng cha m v to

ra cc chi mu tm .ra cc chi mu tm .+Cc con lai cng c th c pht hin qua mi trng+Cc con lai cng c th c pht hin qua mi trng

sinh trng chn lc.sinh trng chn lc.VDVD:: 22 loi thuc l Nicotiana glauca v N.langsdoffii uloi thuc l Nicotiana glauca v N.langsdoffii u

khng c kh nng sinh trng trn mi trng thiukhng c kh nng sinh trng trn mi trng thiuauxin. Nhng sn phm dung hp t bo trn caauxin. Nhng sn phm dung hp t bo trn ca 22 loiloili c kh nng t dng v auxin v sinh trng cli c kh nng t dng v auxin v sinh trng ctrn mi trng khng c auxin.trn mi trng khng c auxin.

S d bi i h h l h h l i dS d bi i h h l h h l i d


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S dng t bin sinh ho v la chn th lai xoma daS dng t bin sinh ho v la chn th lai xoma datrn s hnh thnh cc c im b xung.trn s hnh thnh cc c im b xung.VDVD: Khng sinh actinomycin D c dng pht hin: Khng sinh actinomycin D c dng pht hin

sn phm dung hp giasn phm dung hp gia 22 loi thuc chi Petunia. Cc t boloi thuc chi Petunia. Cc t bonui cy ca loi Petunia hybrida khng th sinh trngnui cy ca loi Petunia hybrida khng th sinh trngc khi c actinomycin D. Ngc li, nhng t bo cac khi c actinomycin D. Ngc li, nhng t bo caloi P.parodii sinh trng trn mi trng c actinomycin Dloi P.parodii sinh trng trn mi trng c actinomycin D

nhng khng th ti sinh c cy t m so nui cy.nhng khng th ti sinh c cy t m so nui cy.Nhng t bo va c th sinh trng khi c khng sinh vaNhng t bo va c th sinh trng khi c khng sinh vac kh nng ti sinh thnh cng l sn phm dung hp cac kh nng ti sinh thnh cng l sn phm dung hp ca 22dng t bo trn cha m.dng t bo trn cha m.

Khi khng c c im b sung hoc t bin r rt, cKhi khng c c im b sung hoc t bin r rt, cth dng phng php nhum ho cht v nhn bit quath dng phng php nhum ho cht v nhn bit quapht hin hunh quang. Cpht hin hunh quang. C 22 loi thuc nhum c sloi thuc nhum c sdng l fluorescein isothiocyanate (FITC) c mu xanh vdng l fluorescein isothiocyanate (FITC) c mu xanh vrhodamine isothiocyanate (RITC) c mu khi quan strhodamine isothiocyanate (RITC) c mu khi quan st

di knh hin vidi knh hin vi


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hunh quang. Sn phm dung hp ca c 2 s c muhunh quang. Sn phm dung hp ca c 2 s c muvng v c th tch ra nui cy trn mi trng thchvng v c th tch ra nui cy trn mi trng thch

hp cho qu trnh to m so v ti sinh.hp cho qu trnh to m so v ti sinh.3.4. Th nghim dung hp t bo trn khoai ty3.4. Th nghim dung hp t bo trn khoai ty

3.4.1 Nguyn liu v thit b3.4.1 Nguyn liu v thit b

_nguyn liu l cy khoai ty_nguyn liu l cy khoai ty

_ Du khong thuc nhum FITC v RITC (nng _ Du khong thuc nhum FITC v RITC (nng 3,0mg/ml, pha trong ethanol).3,0mg/ml, pha trong ethanol).

_ Mi trng PEG._ Mi trng PEG.

_ Mi trng ra c Ca_ Mi trng ra c Ca2+2+.._ Mi trng nui cy t bo trn v sn phm dung hp ca_ Mi trng nui cy t bo trn v sn phm dung hp ca

khoai ty.khoai ty.


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3.4.2. Tin hnh3.4.2. Tin hnh

_ Tin hnh tch t bo trn t 2 ging khoai ty khc_ Tin hnh tch t bo trn t 2 ging khoai ty khcnhau. Nhum qun th t bo trn th nht bng FITC,nhau. Nhum qun th t bo trn th nht bng FITC,v qun th t bo trn th 2 bng RITC.v qun th t bo trn th 2 bng RITC.

_ Ly 1 git nh du khong v trng cho vo giu a_ Ly 1 git nh du khong v trng cho vo giu apetri, t 1lamen v trng ln trn gipetri, t 1lamen v trng ln trn git du khong.t du khong.

__ Nh tng git mi trng PEG v xung quanh cc tNh tng git mi trng PEG v xung quanh cc tbo trn (5git) v thm 1 git na vo gia qun th tbo trn (5git) v thm 1 git na vo gia qun th tbo trn. Gi nhit phng trong vng 20bo trn. Gi nhit phng trong vng 20--25 pht.25 pht.

_ Thm mi trng ra cha Ca_ Thm mi trng ra cha Ca2+2+ vo 1 pha ca hn hpvo 1 pha ca hn hp

t bo trn: c 3 pht li thm t t 3 git v lp li thaot bo trn: c 3 pht li thm t t 3 git v lp li thaotc ny trong 20 pht. Trong khi pha i din dngtc ny trong 20 pht. Trong khi pha i din dngpipet loi b 3 git mi trng cha PEG v s kt dnhpipet loi b 3 git mi trng cha PEG v s kt dnht bo trn s xy ra trong qu trnh ra nyt bo trn s xy ra trong qu trnh ra ny..

Ch 3 i t i t i 1 h h h tCh 3 i t i t i 1 h h h t


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_ Cho 3 git mi trng nui cy vo 1 pha ca hn hp t_ Cho 3 git mi trng nui cy vo 1 pha ca hn hp tbo trn v 1 pha i din loi b 1 phn mi trngbo trn v 1 pha i din loi b 1 phn mi trngra, lp li thao tc thm 2 ln na. Tip tc ly 1 s gitra, lp li thao tc thm 2 ln na. Tip tc ly 1 s git

mi trng cho vo phn a xung quanh lamen duymi trng cho vo phn a xung quanh lamen duytr m, sau nui cy trong nh sng yu nhit tr m, sau nui cy trong nh sng yu nhit 252500C.C.

_Sau 2_Sau 2--7 ngy nui cy, cc t bo trn cn c kim tra7 ngy nui cy, cc t bo trn cn c kim tra

v s ti to thnh t bo, s phn bo v tn s dungv s ti to thnh t bo, s phn bo v tn s dunghp. Khi cc t bo phn chia v hnh thnh khi m sohp. Khi cc t bo phn chia v hnh thnh khi m soang tng trng, qun th t bo nui cy cn c lmang tng trng, qun th t bo nui cy cn c lmlong bng mi trng mi vi hm lng khong caolong bng mi trng mi vi hm lng khong caohn v c 0,6%agar (sau khong 2 tun nui cy)hn v c 0,6%agar (sau khong 2 tun nui cy)

_Sau hn 2 tun nui cy, c th quan st thy cm m_Sau hn 2 tun nui cy, c th quan st thy cm mso. Chng c th khc nhau v hnh thi, mu sc vso. Chng c th khc nhau v hnh thi, mu sc vkch thc. Nhng khi m so sinh trng nhanh hockch thc. Nhng khi m so sinh trng nhanh hocc khc bit r rt c th l du hiu ca con lai xoma.c khc bit r rt c th l du hiu ca con lai xoma.

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