cÔng nghỆ tẾ bÀo thỰc vẬt

8/9/2019 CNG NGH T BO THC VT

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CNG NGH T BO THC VT ti: Nui cy to cy n bi t t bo sinh dcT VN

Trong chn to ging cy trng vic to ra c dng ng hp t tuyt i l mtvn rt c quan tm. Cc dng ny c to ra bng nhiu con ng khc nhau

nh: t phi, nui cy to cy n bi t t bo sinh dc Trong to cy n bi t tbo sinh dc l mt k thut mi c nhiu u im so vi cc k thut khc.KHI NIM CHUNG V TH N BI Cc th n bi l nhng c th thng l ca nhng loi nh bi hay a bi khcngun m trong t bo soma ca chng s lng nhim sc th bng na s lng NSTca loi khi u, trong mi cp NST tng ng n ch c mt NST. Cc cch hnh thnh th n bi:- Trinh sinh (gynogensis) v sinh sn n tnh c (androgensis).- Loi tr nhim sc th v gim nhim sc th soma.- Nui cy th n bi in vitro.Mt s c im ca th n bi.

Trong c th thc vt ch c th giao t (ht phn, non) l nhng t bo n bi.Nu chng pht trin thnh cy th cy c mc bi th n bi (n). th n bi th kiu hnh ca cy phn nh trung thc kiu gen. V vy th nbi l nguyn liu l tng cho cng tc chn ging cy trng.Kh khn ca vic to dng thun v hng khc phc. Trong chn ging thc vt to ra dng thun chng ngi ta tin hnh t thphn bt buc qua nhiu th h, vic ny tn rt nhiu thi gian. V vy vn t ra l lm th no trong mt thi gian ngn, chng ta c th tora c dng thun chng. Nm 1934, Stow pht hin ra s pht trin khc thng caht phn thnh nhng cu trc ging ti phi xy ra mt s loi thc vt Hyacinthus. Hin tng ny cho thy cc ht phn c kh nng phn chia hnh

thnh cc t bo mi hoc cc m khi c sinh trng trong cc iu kin thch hp vchng tip tc pht trin thnh cy n biu th ca vic to dng thun chng t nui cy bao phn, ht phn. Tnh ng hp t c c ch sau mt i nui cy trong khi chn dng thunthng thng phi mt 5 - 6 i t th phn. Cy lng bi ho tnh ng hp t tuyt i, trong khi t th phn thng thngqua nhiu i m vn cn tn d d hp t. S a dng di truyn qun th cy lng bi ho t nui cy bao phn ht phnln hn qun th t th phn to dng thun sau cc i t th. Nhng gen ln c th b che khut cc cy nh bi d hp t nhng cy nbi hoc lng bi ha t ht phn li c c hi biu hin ngay ra thnh kiu hnh. Cy n bi c th dng trong chn lc hi quy to ging chng bnh. Cy truyn lin tc cc dng callus t nui cy bao phn c th to ra bin dgiao t l nguyn liu cho chn ging.Kh nng ng dng ca cy n bi. Nghin cu di truyn v mi tng tc ca cc gen. To t bin mc n bi. To dng ng hp t tuyt i phc v cho cng tc chn ging cy trng.


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S cc hng ng dng n biTo cy n bi bng nui cy bao phn, ht phnNguyn l:

Da trn c s ca s sinh sn n tnh c (androgensis), ngi ta nui cy ccht phn n nhn (tiu bo t) tch ri hay cc bao phn c cha cc ht phn n nhn

trn mi trng dinh dng nhn to ph hp kch thch ht phn pht trin thnh cyn bi.Cc phng thc sinh sn v tnh c invitro to cy n bi Sinh sn n tnh trc tip (thuc l, c c dc).

Ht phn n nhn phi 1ncy 1n Sinh sn n tnh c gin tip (la, ng)

Ht phn n nhn m so 1n chi 1ncy1n Sinh sn v tnh c hn hp: qu trnh ny din ra tng t nh sinh sn v tnhc gin tip, nhng s to thnh m so ngn, kh nhn bit. V d: c chua.Cc phng php c bn

C 2 phng php c bn c s dng trong nui cy bao phn v ht phn l:

Phng php1: Cc bao phn c nui cy trn mi trng c agar hoc mitrng lng v s pht sinh phi xy ra trong bao phn.K thut nui cy

K thut ny bao gm nhng bc chnh nh sau: Chn bao phn: Bao phn thch hp nht c cha ht phn bt u t th t bo tn ngay sau ln nguyn phn th nht. Bao phn ca cc hoa u tin cho kt qu tthn bao phn ca hoa mun. X l n hoa: Cn x l nhit thch hp cc n hoa sau khi ct khi cy vtrc khi tch bao phn nui cy, nhm kch thch s phn chia ca ht phn v t to cy n bi.Cc yu t nh hng n kh nng to cy n bi trong nui cy bao phn, ht phn in

vitroTnh hnh nghin cu, thnh tu v hin trng.

Tnh hnh nghin cu:- 1964 Guka v Maheshwasi li hai nh khoa hc n ln u tin to thnhcng cy n bi qua nui cy bao phn cy c c dc.- K thut n bi c ng dng rt rng ri hu ht cc nc c cng nghchn ging trn th gii.- Cng ngh n bi c p dng i vi hu ht cc cy trng quan trng vmang lai hiu qu kinh t cao. i vi mt s cy nh la, ng, i mch, mng tythy l cng ngh khng th thiu c trong chn to ging.- nc ta t nm 1975 n nay to c nhiu loi cy trng bng nui cy

n bi nh ng, bp ci, thuc l.Thnh tu: Cc cy n bi c ngun gc ht phn c to ra 216 loi thuc 78 ging,31 h v nhiu loi khc cng c nghin cu thnh cng (Hu v Zhang, 1985). Thng qua nui bao phn to cc ging la thun nh Khao 85, Khao 1105,VH2. c bit thng qua chn dng t bo soma thu c cc ging DR1, DR2, DR3ang m rng ra qui m sn xut. Kt hp bin d t bo soma vi gy t bin to


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ging la KDM39. Trong nghin cu la lai ang p dng k thut lai xa, cu phi, tbin to dng TGMS v CMS mi.

Trong lnh vc nng nghip, cc kt qu nghin cu v cng ngh t bo - mphi thc vt gip chng ta nhanh chng to ra cc ging cy trng thun. Hng lotdng thun la (V2, MT4, DT26...) c to ra bng k thut n bi nui cy bao

phn v nui cy non. c bit, chng ta sn xut c dng la thun mang genequ nh gen bt dc c t bo cht, bt dc c nhn (gen TGMS, PGMS). i vi ng, to c 5 dng ng thun v hai t hp ng lai c trin vng.

K thut nui cy m t bo pht trin nhanh v ngy cng hin i m tim nng toln cho cho nui cy bao phn ht phn to cy n bi t to dng thun, ng ch l cc i tng c tm quan trng nh: la go, la mch, i mch, thuc l, ng,khoai tyu im ca vic to cy n bi bng nui cy bao phn v ht phn Nui cy bao phn: V bao phn c kch thc ln nn thao tc d dng. Mi trng nui cy n gin.

Nui cy ht phn : Ging to ra c tnh ng hp t cao. Pht sinh phi d dng trong qu trnh nui cy. To cy n bi thun li cho vic nghin cu di truyn. Tm li, nui cy bao phn, ht phn ra i lm gim thi gian, ng thi lmtng vt s lng cc c th n bi thu c.TO CY N BI BNG NUI CY NONCHA TH TINHKhi nim: To cy n bi t nui cy non cha th tinh l s kch thch t bo trng haycc t bo i cc,t bo km trong non pht trin v ti sinh thnh cy n bi.S hnh

thnh t non cha th tinh c gi l s sinh sn n tnh ci hay trinh n sinh c nhiu thnh cng trong nghin cu to cc cy n bi bng nui cy baophn. Nhng mt s loi cy nh hnh,hoa hng dng hay c ci ng thphng php ny t ra khng hiu qu. Chnh v vy trong nhng nm 70 cc nh nghincu tin hnh to cy n bi bng non cha th tinh v ginh c mt s ktqu ng k.Quy trnh to cy n bi bng non cha th tinhNhng nhn t nh hng n nui cy non cha th tinh K thut nui cy non cha th tinh cn gp nhiu kh khn v phc tp do victch non kh v d gy tn thng

Nhm tng hiu qu ca qu trnh ny ngi ta ang tp trung nghin cu cc yu

t nh hng n qu trnh nui cy to cy n tnh mong mun Kiu gen ca cy m l mt trong nhng nhn t quan trng nht i vi qu trnh nui cy Ngi ta nhn thy mi mt kiu gen c phn ng khc nhau trong qu trnh sinhtrng nn phi xc nh quy trnh ring cho tng loi cy c th ca tng kiu gen nhn t ny c nghin cu trn nhiu i tng khc nhau nh la,hoa ngtin. Giai on pht trin ca non


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Giai on pht trin ca non c ngha c bit i vi vic to th n bi Ti phi thnh thc l giai on ph hp cho hiu qu cao trong nui cy non Tuy nhin vic xc nh giai on pht trin ca th giao t ci l rt phc tp vti phi nm trong bu qu do kh quan st trc tip xc nh c th phi sdng cc phng php t bo hc nh tch ti phi,nhum mu bng lt c mng

Mi trng nui cy Cc mi trng nh MS, B5, MF thng c s dng lm mi trng nui cynon cha th tinh Mi trng ny ch yu dng c cm ng c s trinh sinh cn thit th phi b sung cc cht iu tit sinhtrng thc vt Ngoi ra nng ng cng l mt yu t cn c quan tm,nng phi phhp cho tng loi cy Trong nhiu trng hp vic b sung thm mt s cht ph gia nh nc da ctc dng to callus pht sinh phi v ti sinh cy iu kin nui cy

Qu trnh nui cy thng c duy tr nhit n nh 25-28 i vi a s loi thng giai n u ca qu trnh nui cy tin hnh trongiu kin ti, giai on ti sinh cy yu cu nh sng 2000-3000 luxNhng thnh tu t c trong phng php to cy n bi t non cha th tinh T l to cy n bi theo phng php ny c nhiu kh quan nh hnhv c ci ng :5-20%, du tm l 3-6% Cy ti sinh t b bch tng ngay c h ha tho,trong khi nu nui cy bng baophn v ht phn c t l bch tng 60-90% cc loi cy ng cc VN th bin php ny tng i n gin v d thnhcng nh cy ngSo snh

Ging nhau: u dng giao t to t bo n bi. u nhm to ra nhng cy n bi qua s cm ng pht sinh phi t nhng phnchia lp li ca cc bo t n bi,cc tiu bo t, cc ht phn non.Khc nhau Phng php nui cy ht phn tch ri to t bo n bi cn phi tch ri ccht phn khi bao phn khi nui cy. Cc ht phn ny thng c nui cy trn mitrng lng km theo ch lc hay nui cy chm trong mi trng bn lng. Phng php nui cy bao phn c p dng trn nhiu i tng thc vt khcnhau: ng, la, thuc l nhng khng p dng trn cc cy hnh, c ci ng, hoahng dng.

Phng php nui cy non cha th tinh ch yu trn cc i tng hnh, c cing, ng, cc cy ng cc. Tuy nhin k thut ny cn gp nhiu kh khn v phc tpdo vic tch vch t bo trng rt kh v d gy thng tn do vy ngi ta ang tptrung nghin cu cc yu t nh kiu gen cy m, giai on pht trin ca ti phi, ch x l nhit , mi trng nui cyHin trng Hin ti ngi ta mi nui cy ht phn trng thnh to cy n bi thnhcng qua con ng phi ho, hoc to thnh m so, t to cy n bi. Cn nui


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cy ht phn non cha t c nhiu hiu qu, hin ti ch c mt s kt qu v nui cyht phn non thnh cng nh nui cy ht phn non cy Trillium electum (Saparov vcs,1955), cy hnh Allium cepa (Vasil,1959), cy Atropa belladonna (Bajaj,1974). Hu ht cc cy ng cc v cc cy h u nui cy bao phn rt kh thnh cnghoc t l thnh cy thp, t l cy bch tng cao.

Nhiu gen c kh nng thnh cy thp thng khng c ng dng trong nuicy bao phn nhng c khi gen li c gi tr kinh t cao. V d cy bao phn ging laJaponica d thnh cng hn hn ging la Indica, tuy nhin ging Indica li c vai trquan trng hn nhiu ging JaponicaNui cy bao phn thuc lNguyn liu v thit b: N hoa thuc l (Nicotiana tabacum) Nc ct 2 ln v trng Etanol 90% Dung dch hipoclorit natri 10% (V/v) Thuc nhum acetocacmin

Agar a petri Mi trng MS hoc mi trng NN Knh hin vi gii phu Dao, ko, panh,... Dng cho nui cy Mt s ha cht pha mi trng lng: saccarozo, KNO3,Tin hnh Thu hi cc n hoa ca thuc l khi chng c trng hoa di 21-23mm (thi imcc ht phn thuc l hon thnh phn bo nguyn phn u tin) X l lnh cc n hoa khong 12 ngy 7-8oC, sau kh trng b mt n hoabng etanol 90% trong 30s, sau chuyn chng sang cc a petri cha dung dch

hipoclorit natri 10% trong 10 pht Ra cc n hoa vi ln trong nc ct 2 ln v trng. Dng dao, kim v panh giiphu cn thn n hoa v ly ra cc bao phn (c th s dng knh hin vi gii phu htr cho qu trnh thao tc Ly 1 bao phn ca mi n hoa v p trong acetocacmin xc nh giai onpht trin ca ht phn. Nu ht phn giai on pht trin thch hp th cc bao phncn li ca n hoa c dng trong nui cy Cc bao phn c th c nui cy theo 2 cch: Trn mi trng MS c (c 0,6-1,0%(W/v) agar) Nui cy ni trn mi trng lng gm: KNO3-950(mg/l); NH4NO3- 825(mg/l);MgSO4.7H2O- 185(mg/l); CaCl2- 220 (mg/l);KH2PO4-85 (mg/l); FeSO4.7H2O-27,8 (mg/l); Na2EDTA-37,3 (mg/l);saccarozo-20000 (mg/l)6. Qu trnh nui cy c duy tr nhit 25oC trong ti hoc nh sng yu.Cng nh sng thch hp khong 300lux. Sau 4-6 tun nui cy, cc cy con s xuthin t mt s bao phn.

Khi m bao phn t 3mm th tch cy con ra khi m v chuyn sang mi trngto r (c th dng mi trng MS vi thnh phn khong gim 1 na). Cng nhsng cao hn thi gian chiu sng khong 10-12h/ngy


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ti: Bo qun ngun gen thc vt in vitro

MC LC

I. M u

II. Ni dung1. Bo tn ngun gen thc vt2. S lc v cc phng php bo tn ngun gen3. Cc cch bo qun ngun gen thc vt in vitro4. u th v hn ch ca k thut bo qun ngun gen thc vt in vitro5. Kh nng ng dng ca bo qun ngun gen thc vt in vitro6. Phng php bo qun ngun gen invitro i vi ging hoa Lilium

Formolongo.

III. Kt lunM u Bo qun ngun gen thc vt l duy tr s phong ph v a dng di truyntrong loi, gia cc loi v trong h sinh thi ni chung nhm nh gi, khai thc sdng cc ngun gen c gi tr phc v li ch con ngi. Hin nay c nhiu phng php bo qun ngun gen thc vt, song bo qunngun gen thc vt in vitro l gii php cng ngh c trin vng, nht l vi cc cynhn ging v tnh v cc ht mc sc ny mm nhit v m thp.

V vy, chng ti tin hnh tm hiu ti: Bo qun ngun gen thcvt in vitroNi dung1.1. Mc ch

Bo v s a dng sinh hc, th hin 3 mc:

+) S a dng ca cc h sinh thi+) a dng ca cc loi+) S a dng di truyn hay s a dng ca cc ngun gen trong loi

nh gi, khai thc s dng cc ngun gen c gi tr phc v con ngi.1.2. ngha L mt vic lm cp thit v thng xuyn phc v nhim v trc mt vlu di ca cng tc ci thin ging, va gp phn quan trng vo cng tc bo tnthin nhin, bo v s a dng sinh hc. Bo tn ngun gen ngn chn s mt mt cc gen, cc phc hp gen ngnchn s tuyt chng ca cc ni a l, cc xut x v trong trng hp cc oan

l s tuyt chng ca loi.2. Cc phng php bo tn ngun gen t trc n nay2.1 Bo tn ngoi vi (ex - situ conservation)

y l hnh thc bo tn ch yu hin nay trn th gii, mt hnhthc bo tn ngoi phm vi c tr t nhin ca cc loi.

Gm :- Bo qun ht- Trng ngoi rung


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- Bo qun in vitro2.1.1. Bo qun htGm: Bo qun ngn hn (ht ging c lm kh ti m 9%, thi gian 5 nm) Bo qun trung hn ( m ht 7%, bo qun trong dng c bao gi v kho

chuyn dng m 10%, t0 l - 1 n - 50C) Bo qun di hn ( m ht 3%, ng trong hp kim loi, bo qun trongkho lnh su, t0C l - 150C n - 200C, thi gian t 20 - 30 nm).2.1.2. Bo qun trn ng rung

i tng: cy lu nm nh cy n qu, cy cng nghip, cy thuc,cy sinh sn v tnh, hu tnh khc

u im: D tip cn nghin cu, nh gi v s dng

Nhc im: D mt mt do iu kin khng thun li, i hi din tcht ai ln v ngun nhn lc2.1.3. Bo qun in vitro

i tng bo qun in vitro:- Vt liu sinh sn v tnh (chui, da, mt, khoai s, khoai lang)- Cc loi cy c ht recalcitrant - ht kh bo qun (xoi, mt, na, su ring, nhn,

vi v cy c mi)- Cc vt liu dng nhn nhanh phc v cc chng trnh chn to v nhn

ging, ht phn v ngn hng DNA2.1.3. Bo qun in vitroGm

Bo qun ngn hn: Thc vt c pht trin bnh thng trong iu kin vtrng ca phng th nghim, vt liu c bo qun cung cp cho cc nhu cuchn to ging v nghin cu ca mi c s.

Bo qun trung hn (bo qun bng sinh trng chm) th tc sinhtrng ca vt liu c lm gim mt cch ng k bng cch gi nhit nhsng thp hoc gim nng oxy.=> C 2 phng php ny p dng cho vt liu l: chi, m phn sinh v cy con.2.1.3. Bo qun in vitro

2.1.3. Bo qun in vitro- Bo qun di hn: Bo qun di hn l bo qun trong hoc trn b mt

nit lng (- 1760C n - 196C), vi iu kin ny mi qu trnh sng b nh chhon ton.

i tng: m so, t bo ht phn, phi ca mt s loi thc vt2.2. Bo tn ni vi (bo tn ti ch, in-situ conservation)

L bo tn ngun gen trong mi trng sinh sng. i tng: bt k thc vt no, ch yu l cc loi t tin ca cy trng, ccloi hoang di c quan h gn gi vi cy trng. Vi 1 s ging a phng c hnh thnh do qu trnh chn lc v trngtrt lu i ti 1 a phng cng c th bo qun ti ch trn ng rung ca nngdn hoc bo qun da vo cng ng.3. Cc cch bo qun ngun gen thc vt in vitro


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y l gii php cng ngh c trin vng trong vic bo qun ngun gen thcvt, nht l vi cc cy nhn ging v tnh v c ht kh bo qun c nhit v m thp.

Trong vng 20 nm gn y, cng ngh ny c pht trin rng v pdng vi hn 1000 loi khc nhau (F.Engelmann, 1997).

Gm:+ Bo qun sinh trng chm

+ Bin php ngng sinh trng tm thi3.1. Bo qun sinh trng chm

Gm bo qun ngn hn v trung hn

Vt liu thch hp l chi, m phn sinh v cy con

gim tc sinh trng ca cy trong ng nghim, ngi ta thay imt s yu t:

+ H thp nhit nui cy, a thm cc cht c ch sinh trng+ Cht lm tng p sut thm thu

+ V lm ngho thnh phn dinh dng trong mi trng nui cy

3.2. Bin php ngng sinh trng tm thi

y l phng php bo qun di hn cc vt liu trong hoc trn b mtnit lng (- 1760C n - 1960C ).

Bo qun ng lnh trong, trn nit lng c th c chia ra cc cng onsau:+ Tin sinh trng, x l vi 5 - 10 % proline, 3 - 6 % manitol.+ X l bo v chng ng bng.

3.2. Bin php ngng sinh trng tm thi a vo bo qun trong hoc trn mt nit lng (- 1960C 1580C).

Lm tan tuyt, x l nhit 35 400C trong iu kin phng th nghimbnh thng.

Phc hi sinh trng, ti sinh cy.3.2. Bin php ngng sinh trng tm thi

Bin php ngng sinh trng tm thi p dng thnh cng cho cc itng:

+ M so+ T bo ht phn

+ M phn sinh V d: ngi ta nghin cu m phn sinh ca khoai ty v a vo boqun ng lnh cho t l phc hi ti sinh cao xp x 36%.4. u th v hn ch ca k thut bo qun ngun gen thc vt in vitro

u im: m bo an ton v sch bnh cao, c kh nng to qun th cyng nht vi s lng ln.


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Bo qun c lu di ngun gen thc vt trong iu kin mi trng bnngoi khng cho php nhm phc v cho cng tc chn ging.

S dng cc b phn nh nh ht, chi, m,... nn c th tit kim c khnggian bo qun.

u im

Hn ch kh nng mt ngun gen, nht l cc ngun gen c nguy c xi mncao, cc loi c nguy c b tuyt chng

Vi phng php bo qun siu lnh c th bo qun c lu di vi slng ln v n nh

Kh nng ti to, phc hi cc ngun gen bin mt trong t nhin

Hn ch

Cha c nhng nh gi y v mi tng tc gia yu t mi trng

(thnh phn dinh dng trong mi trng nui, nhit , cht c ch sinh trng). C kh nng to ra bin d Soma vi tn s bin d khc nhau v t lp li

Chi ph bo qun ln, i hi trnh k thut cao v trang thit b hin i

Cha xc nh c khong thi gian ti a mt cch an ton trong qutrnh bo qun bng sinh trng chm cho tng loi cy trng5. Kh nng ng dng ca bo qun ngun gen thc vt in vitro

Ngy nay ngi ta phn bit c bn nhm i tng m chng cth bo qun invitro c li Nhm vt liu nui cy bo qun invitro bt buc bao gm cc nhn ging vtnh, cc loi cy c ht recalcitrant Nhm vt liu bo qun trong iu kin invitro gm cc dng c nht vnguyn liu c lm sch bnh. Vt liu bo qun invitro c th c li trong tng thi im chng hn nhnnhanh mt kiu gen, phc v chng trnh to ging hoc kho st nh gi trongiu kin invitro. Vt liu cn bo qun invitro c lin quan n k thut gen v sn xut cccht th cp.V d: Phng php bo qun ngun gen invitro i vi ging hoa Lilium xformolongo

i tng nghin cu: Lilium x formolongo

Phng php nghin cu:- Thu thp mu- Tch v nui cy vy c- Cy chuyn v bo qun ngun gen

Mi trng nui cy:Trong th nghim s dng mi trng vo mu l MS + 30g

saccarose/l + 6.5g agar/l, 1ml NAA, pH l 5.7

Mi trng cy chuyn: MS + 30g saccarose/l + 6.5g agar/l, pH l 5.7


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Tin hnh:Mu sau khi thu thp c a v phng th nghim, tin hnh kh

trng mu.Ha cht dng v trng : Clorua thy ngn vi nng s dng l

0.1 1% (W/v) trong thi gian t 10 15 phtSau khi mu c kh trng, tin hnh vo muThi gian t khi vo mu ti khi cy ra r l 3 tunCy chuyn nhiu ln vi mi trng MS + 30g saccarose/l + 6.5g

agar/l, pH l 5.7Cy chuyn ti khi cy t tiu chun nht nh, tng nng ng

ti 60g saccarorose + 6.5g agar/l, pH = 5.7

Tin hnh bo qun trong phng nui:+ Nhit phng nui: 100C

+ Thi gian chiu sng: 16h+ Cng chiu sng: 1800lux+ m phng nui: 50 60%

Kt qu:Nh tng nng ng t 30g/l n 60g/l kt hp vi nhit phng

nui thp, thi gian v cng chiu sng thp, thi gian cy chuyn gingLilium Formolongo tng t 4 tun ln n 10 tun.

III. Kt lunBo qun invitro c bit c gi tr vi nhng cy trng sinh sn hu

tnh, nhng ht ca chng khng th bo qun c m , nhit thp v cytrng nhn ging v tnh, vic duy tr chng trn ng rung ang c nhiu nguy cmt ging.

nc ta, cng tc bo qun invitro bc u tin hnh c kt qui vi mt s cy trng nhn ging v tnh trong nng nghip. Nhng nhu cu phttrin s dng, bo qun invitro rt ln. Do cn phi la chn mt chin lc boqun thch hp, s dng nhng thnh tu ca cng ngh sinh hc, pht trin nhnggii php mi, nui cy bo qun invitro h tr cc phng php c truyn nhngn hng gen ht v trn ng rung nhm bo qun tt hn ngun ti nguyn cachng ta.

Nui cy t bo trn

M u


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Cc t bo thc vt c tnh ton nng,c th nui cy, iu khin s pht sinh hnhthi ca chng cho ti thnh mt cy hon chnh. C th ni, ni dung bn trong v tbo trong vt cht chnh l cc thng tin di truyn cha trong nhn ca t bo quytnh mi ng hng ca qu trnh thc hin tnh ton nng ca t bo. V th,honton 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 cunhng tnh cht vt l ca mng sinh cht n nhng nghin cu nhp bo v hp thu ccphn t, cc bo quan v vi sinh vt.Protoplast1.Khi nim v Protoplast

Protoplast l cc t bo trong c thnh t bo c loi b v mng t bo chtl 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 bom 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 dinhdng th sau 5-10 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 trnhny 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 phit 1 t bo c lp. c bit bit c s sp xp cc si Xenluloz xc nh hng kodi 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 gipto ra cc c th lai mang cc c im di truyn ca cc b m c ngun gen khc xanhau 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 thngyu cu 1 s thay i do bn cht ca t bo trn. Cc thay i thng lin quan n siu chnh nng mui v c, thm vo cc hp cht hu c, vitamin, ng mbo 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 thnht 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 thrt 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 thtl thuc l, ci du Bng thao tc di truyn t bo trn c th d dng to ra cc tbo bin i gen. T bo vi kiu gen bin i s c bo tn khi ti sinh t bo thnhcy 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 bothc vt m trc kia thng b v t bo ngn cn.


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Nui cy t bo trn cho php kh nng dung hp t bo gn hai t bo trn lithnh mt t bo vi hai b thng tin di truyn ca hai t bo to nn mt th lai v tnhm khng cn hiu bit chnh xc v s lin h gia cc gen, t tn km, nhanh,trc tipv p dng cc k thut chuyn gen( bm AND, ha thm, in thm)gip loi tr tnhbt 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 mtcy lai. Qu trnh ny xy ra t bo nn gi l lai t bo v thng qua t bo soma nngi l lai soma hay lai v tnh t bo. T phng php ny ra phng php lai xa giacc 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 l p nui c y ph i hon thi n. Cha c phng php hi u qu tuy n ch n cc s n ph m ph h p.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 scnhn ( sharp-edged knife) v gii phng cc protoplast ring r. Phng php ny chohiu sut thp. 4.2. Phng php enzyme:

ph v thnh t bo ngi ta thng s dng cc enzyme chit xut t ccsinh vt cha nhiu enzyme phn gii thnh vch t bo nh: nm, c v mi. Ccenzyme ny c thng mi ha theo cc phng php khc nhau vi mc tinhkhit khc nhau

Hn hp enzyme thng dng l enzyme celluloza v macerozim c chit xutt 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 sdng pH 5,5 5,8 trong 3-8h. Ngoi ra protoplast khng b v sau khi thnh cellulose b phn hy ngi taphi b sung nhng cht tng p lc thm thu vo dung dch enzyme duy tr cn bngthm thu gia ni bo v mi trng bn ngoi. Cc dung dch thng dng l dungdch ng manitol, sorbitol. Nng s dng khong 0,3 0,7M ty theo i tngthc 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 1gam l c luzec hoc khoai ty l 6-12 triu t bo trn.

V protoplast thc cht l t bo trn khng c thnh cho nn c th tch c tnhiu ngun khc nhau nh cc b phn ca cy ( r, l, ht phn), callus, t bo nXc nh cht lng t bo trn Sau khi ph v t bo vn c nhng mnh thnh t bo cn xt li lm nh hngn nhng nghin cu sau ny

Cch tt nht pht hin thnh t bo l dng calcofluor, mt ha cht s bmvo phn t xenlulozo v gy ra pht nh sng hunh quang vi mu xanh rc r khi soi


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di tia cc tm. Nu cc t bo trn b loi b hon ton thnh t bo hin vi trngc mu ti thm cc t bo trn s khng nhn thy c ngoi tr s t pht nh snghunh 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 khngcho thuc nhum xm nhp vo nguyn sinh cht ngc li cc t bo c mng sinh chtb mt chc nng s bt mu xanh v chng khng th sinh trng c5. 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 thcvt.Mi trng ny c thm Auxin v Xitokinin gip s ti to vch v cc ln phnchia 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 cchpha 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, tngnng Auxin.Nui cy t bo trn chia lm 2 giai onGiai 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 phnchia 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 tm 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 thuCc yu t nh hng n sinh trng, pht trin ca t bo trn


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Nui cy t bo trn thng yu cu 1 s thay i so vi quy trnh nui cy mbnh thng do bn cht ca t bo trn.

Cc thay i thng lin quan n s iu chnh nng mui v c, thm vocc hp cht hu c, vitamin, ng m bo kh nng thm thu v cht iu titsinh trng kch thch s phn chia t bo.

6. Dung hp t bo trn C th ni vic dung hp t bo trn v ti sinh thnh cy lai t t bo trn l 1trong 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 phngphp 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 vonhau (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 nngi 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 ccprotoplast tht l i vi actinomycin D

C 2 phng php dung hp t bo trn:

+ Dung hp bng ha cht

+ Dung hp bng inDung hp bng ha cht

X l bng NaNO3

Nm 1970, Power v cng s dng NaNO3 (0,25 M) kch thch dung hp haiprotoplast. Carlson v cng s (1972) cng dng phng php ny sn xut cy laisoma u tin (Nicotiana glauca N. langsdorffii). Tuy nhin, phng php ny cho hiusut thp v NaNO3 khng thch hp vi t bo b khng bo ha mnh nh protoplast tnhu m l.

X l bng PEGThng 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 sthnh cng ca th nghim dung hp. PEG c trng lng phn t thp (~ 100) khng thto ra mt s dnhcht 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 caccprotoplast.

Qu trnh dung hp s c ci thin hn nu xy ra trong mi trng kim (pHt 8- 10) v khi c b sung CaCl2 (50-250 mM).

Sau khi x l bng tc nhn dung hp, cc protoplast c nui cy theo phngthc chun.


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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 PEGDung hp bng in

Phng php ny n gin hn, nhanh hn v hiu qu hn dung hp bng hacht. iu quan trng hn c l dung hp bng in (electrofusion) khng gy c ivi 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 votrong t bo thc vt, k thut ny lm tng s quan tm v vic ng dng dung hpbng 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 xungin cao (750-1000V) trong 1 thi gian rt ngn(1-200 mili giy) vng tip xc gia 2mng t bo s b v, 2 t bo trn ha nhp vo nhau-qu trnh dung hp s xy ra.7. Trin vng ng dng ca k thut nui cy t bo trn v lai t bo somaMc d nhng kh khn v mt k thut lm hn ch tim nng s dng t bo trn tuynhin 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 tbo trn c ti to nhanh chng thnh t bo mi v qu trnh pht trin ny a ramt 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, tith,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 ccthao tc tng t nh i vi cc vi sinh vt qua c th la chn dng t bin v tchdng 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 tcao,nhng kh nhn ging bng phng php thng thng.

Cng nh ny lm nhn nhanh ging v kt hp lm sch virus. Nghin cu toging khoai ty khng bnh virus ca GS.TS Nguyn Quang Thch trng HNN HNi.

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


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cy khoai ty vi t bo cy c chua. Kt qu l to ra c cy lai P-ma-t m trn mtt 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 mlai hu tnh khng thc hin c. K thut dung hp t bo trn cho php m rng ngun gen ca cc loi thc vtto 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 lcchng c di hoc chng nm bnh.

To iu kin thun li cho s nghin cu v sinh l t bo: tnh thm ca mng, vnchuyn 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. Saukhi phn chia protoplast, thnh t bo c ti sinh tng s pht trin callus v tiptheo 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 choc 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) vmt s h khc, nhng thnh cng h ha tho (Poaceae) l h ca cc cy trng ngcc chnh cn rt hn ch.

Potrykus (1980) tho lun rt k v vn ny. Theo Potrykus c nhng chtiu 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.


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- 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 bo3. 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 trnQuy trnh nui cy Protolast t l ca cy thuc l.9.1. Nguyn liu thc vtL cy:L l ngun nguyn liu thng dng v truyn thng cho k thut protoplast thc vt don 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 npgene. L ca cy thuc l in vitro c chn dng lm nguyn liu tch protoplast. Sdng 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 %


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+ pH mi trng ~ 5,8

Dung dch ny c kh trng bng mng lc Millipore loi c ng knh l lc0,2-0,25 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 tinhv 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 mldung dch enzyme tch protoplast. Bc cc ong bng giy parafilm, ghi nhn v ttrong 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) ttrong cc loi 50 ml. B sung thm 3 ml dung dch ra (PI + 10% mannitol) vo cc cha cc mnh lvn, lc mnh, v lc tip trn li lc ri chuyn vo hn hp protoplast-enzyme cabc trn. B sung thm 2 ml dung dch ra ra proptoplast ht khi li lc l. Chuynhn hp protoplast-enzyme (tng cng 15 ml) vo tube ly 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 dchtch (PI + 20% sucrose), thm 1ml dung dch ra ln trn b mt dung dch tch vprotoplast sao cho khng lm ha ln hai dung dch, tt nht l to thnh hai pha (nh tt 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 chuynn sang mt tube ly tm sch, thm 10 ml dung dch ra v ly tm li. Protoplast s lngxung y tube v c dng vin.

Ra protoplast thm mt ln na trong 10 ml dung dch ra. Loi b phn ni trnmt 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 tbo/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 baChuyn sang nui cy nh sng yu (10-20 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 2ngy. Ngy th nm


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Chuyn sang nui cy cng nh sng cao hn (50-75 mol/sec/m2) trong 2ngy bng cch b lp vi tha ra.

Ngy th byTa 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 100-200 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 khnng khng bnh. Vic gii thiu v quy trnh s dng enzyme c lp t bo trn thcvt (Cooking 1960) a ra mt b mt mi y ha hn cho t bo thc vt v nuicy m, m ra mt lnh vc mi trong sinh hc t bo thc vt. iu lm cho t bo trnc tc ng mnh nh mt h thng th nghim l h enzyme phn hy vch t bolm l ra b mt mng t bo nh l mt ro cn gia mi trng bn ngoi v thnhphn 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 mkhng th thc hin c khi b bao ph bi vch t bo. T bo trn c s dng rngri trong nhiu lnh vc th nghim t nghin cu nhng tnh cht vt l ca mng sinhcht (Ruesink 1973) n nhng nghin cu nhp bo v hp thu cc phn t (Willison etal. 1971), cc bo quan (Potrykus 1975) v vi sinh vt (Davey v Power 1975).

K thut th phn v nui cy phi invitro Th phn (pollination) l s tip nhn cc ht phn t bao phn ti nm nhy thc hin th tinh hoa. thc vt c hai phng thc th phn l th phn cho v t

th phn Th tinh (fertilization): th c v t th tinh l s k t h p c a hai giao t c v ci (tinh trng v non) thnh h p t (phi, bo t ho c h t) l c trng c bn c a sinh s n h u tnh, sinh s n l ng tnh. th c v t h t kn c phng th c th tinh kp. Phi l mt nhm t bo c kh nng pht trin to thnh c th hon chnh l phapht trin trung gian gia hp t hay t bo soma v bo t th. C hai loi phi: phi hu tnh & phi v tnh ( phi soma) Phi hu tnh : l phi trong ht c to ra do th tinh gia t bo trng v giaot c (do lai hoc t th) cn gi phi hp t. Phi soma: l phi c pht sinh t t bo sinh dng 2n ch ca b hoc m v

t bo y c cu trc nh mt phi gi l phi v tnh ( phi soma). Th phn invitro l thc hin qu trnh th phn trong ng nghim khng phthuc vo c th m. Nui cy phi (cu phi) l s tch ri v nui cy invitro phi hp t thnhthc hoc cha thnh thc thnh cy hon chnh.II. K thut th phn invitro Mc ch iu kin c th th phn in vitro


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Cc bc tin hnh Cc phng php th phn in vitroII.1. Mc ch phi tin hnh k thut ny khc phc s bt hp trc khi th tinh. Th phn, th tinh iu kin in vitro to ra c hi sn xut cc phi lai gia cc

loi thc vt khng th lai bng cc phng php gy ging cy trng truyn thng.II.2. iu kin c th th phn in vitro iu kin c bn l phi nui cy thnh cng bu qu hay non phn lp (nontrn) ca cy m. Ch ng iu khin qu trnh ny mm ca ht phn cy b trong mi trngnui cy v trng. Tin hnh th phn s th tinh din ra v nui cy hp t pht trin thnh philai trn mi trng dinh dng v trng.II.3. Cc bc tin hnh Ly n ca hoa m thi im trc khi n hoa 2 ngy, kh trng, tch lybu qu hay l non nui cy invitro.

Ly n hoa ca cy b vo ngy n hoa, kh trng, tch ly bao phn v trong iu kin v trng n khi chng tung phn. Ly ht phn rc trc tip ln mt ct ca bu qu hay l non th phnin vitro. Khi non th tinh, chng hnh thnh hp t v to phi. Cc phi lai in vitrothng b qua giai on ng, ngh v mc thnh cy in vitroII.4. Cc phng php th phn in vitro

II.4.1. Th phn bng phng php ct ngn vi nhy L phng php d v hiu qu. Nm nhy v 1 phn hoc ton b vi nhy cahoa c ct ngn, sau ht phn ca cy b c th trc tip ln vi nhy ct ngn

v kt qu l c rt nhiu ng phn ko di c ti bu nhy. Nhc im: s lng ht trong 1 qu t. C th do ng phn gp kh khn trongkhi m xuyn qua vch bu.II.4.2. Th phn bng phng php ghp vi nhy Ht phn cn th c gi trn 1 nm nhy thch hp v ny mm, 1 ngy sauvi nhy v 1/3 bu ca hoa cha ht phn c ct v ghp ln 3/4 bu nhy ca hoacy m cn th phn. Theo Vantuyl v cng s(1991) th mt ngy sau khi gi ht phn vi nhyc ct ngn cch trn bu 1-2mm v c gn ln bu nhy ca hoa cy m. Bu vvi nhy ngoi ng rung c kt hp vi nhau = ng ni cn trong invitro ch cn sdng agar c nh l .

II.4.3. Th phn cho gi non Bu c ct theo chiu dc thnh nhiu ming vo ngy nm nhy tit ra dchnhy hoc 1-2 ngy sau . Mi 1 ming s cha 1 gi non v 1 hng non, c th lihoc khng li vch bu. Mt lng ln ht phn cn th c t theo gi non. kch thch ht phn ny mm v m xuyn vo non c th t vo mi trng nui cy1 hay 2 vi nhy. C th nui cy ngoi sng hay trong ti. Non ny mm sau 5-7 tun th phn vtip tc c th phn


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Th phn bn trong bu (intraovarian pollionation): c vi nhy hoc 1 phn can c th c tch ra v ht phn hoc c t trn b mt vt ct bu qu hoc chuynqua l trn thnh vi nhy n bu quIII. K thut cu phi Mc ch

Nui cy phi hu tnh Cc yu t nh hng ng dngIII.1. Mc ch Khc phc s bt hp sau th tinh. Khc phc s bt hp gia ni nh v phi khi lai xa. To cy n bi (la m x ng, la m x yn mch dn n s loi b mt bnhim sc th). Ngn nga s thui cht phi nhng loi qu hch chn (o, mn, m). Ph ng ngh, rt ngn chu k to gingIII.2. Nui cy phi hu tnh

S pht sinh phi hu tnh:-Xy ra qu trnh th tinh gia ht phn v non hnh thnh hp t-Hp t tri qua qu trnh nguyn phn lin tip to thnh phi Nui cy phi hu tnh gm-Tch phi-Nui cy phi to cy.

III.2.1. Tch phi Phi hu tnh c hnh thnh trong mi trng v trng ca non v m buhoa.

- mt s loi phi c kch thc ln, thun tin cho qu trnh tch phi (cy hu), nhng mt s loi hoa kh tch phi (hoa lan_ht c kch thc nh, v ht tiu

gim v thiu ni nh)- thc vt hoa dng chm, m non thng xp nh ca chm hoa Trong qu trnh thu nhn phi cn hn ch s tn thng ca dy treo phi. Dytreo phi c kch thc nh v cu trc mng manh nn kh tch n nguyn vn cng viphi a vo nui cy. Khng c dy treo phi lm gim ng k t l hnh thnh cycon t nui cy phi nonIII.2.2. Thnh phn mi trng nui cy

Sau khi tch t ht phi c nui cy trong mi trng cha: Mui khong Cht iu ho sinh trng Ngun carbon

Aminoaxit v thnh phn hu c phc hpMui khong Mi trng vi hm lng cao cc ion K+, Ca2+ m bo cho t l sng cao caphi v kh nng thc y sinh trng tt i vi phi ca mt s loi thc vt.Cht iu ho sinh trng Auxin v cytokinin khng c s dng nhiu trong nui cy phi do chng cmng to callus. nng rt thp (0.01 mg/ l) GA3 kch thch s ny mm sm ca phi.


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Ngun carbon ng: l thnh phn khng th thiu trong mi mi trng nui cy, v nl ngun cung cp Cacbon ch yu. Trong nhiu trng hp ng sucrose cho ktqu tt hn cc ng khc. Nng sucrose c th dng t 0.5% n 18%. Glucose v sucrose ngoi vai tr dinh dng, cn c kh nng duy tr p sut

thm thu ca mi trng. Phi trng thnh sinh trng kh tt nng thpnhng phi non i hi nng sucarose cao hn.Aminoaxit v thnh phn hu c phc hp Glutamin l a.a hiu qu nht i vi nui cy phi nhiu loi thc vt. Cazein thu phn(CH) (Ziebur & Brink, 1951) l hn hp cc a.a c s dngrng ri trong nui cy phi. Lm tng kch thc v t l phn ho phi. Ngoi ra mt s cht t nhin nh nc da (Overbeek, 1942), nc chit malt(Blackeslee & Satina, 1944), l nhng cht m khi c b sung vo mi trng nui cys em li hiu qu cao hn v trong cc cht ny c cha nhiu vitamin, DNA, RNA,v mt s cht iu tit sinh trng khcIII.3. Cc yu t nh hng

pH mi trng Nhit nh sngIII.3.1. pH mi trng Cc phi tch ri sinh trng tt trn mi trng c pH 5.0-7.5 y l phm vi pH ca dch non (6.0)III.3.2. Nhit 252C thch hp cho sinh trng v ny mm ca phi. Nhit ti u cho nui cy phi c th khc nhau gia cc genotype trong cng1 loi VD: LoiZamia,Phaseolus, bng: 27-30C

Loi laiBrassica, la, la mch: 17-22CIII.3.3. nh sng Khi nui cy phi cha trng thnh ca la mch, lanh, cc c th loi laiAlliumtin hnh trong ti trc khi chuyn sang iu kin sng ny mm.III.4. ng dng ca nui cy phi hu tnh Thu nhn th n bi Kim tra nhanh sc ny mm ca ht Nhn ging cc cy him Th phn trong ng nghimIII.4.1. Thu nhn th n bi Nui cy phi hu tnh thu nhn cc thn bi thng qua qu trnh loi b trc tip NST sau khi lai xa. T hp gia hai loi

cng khc xa nhau th s o thi hon ton NST n bi ca mt loi cng d xy raIII.4.2. Kim tra nhanh sc ny mm ca ht

Nui cy phi hu tnh c dng kim tra nhanh kh nng ny mmca ht, c bit l cc ht ging c sc sng km v ht sau thi gian bo qun di.Phng php ny cho chnh xc caoIII.4.3. Nhn ging cc cy him


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Ht ca mt s cy rt kh ny mm trong iu kin t nhin, v vylm gim kh nng sinh sn hu tnh ca chng. Bng cch nui cy phi tch ri thu c rt nhiu cy con. p dng vi loi m ht c sc sng km, ht khngc ni nh hoc t ni nh.

VD: i vi cc loi phong lan ht ca chng rt nh v khng c ni

nh v vy kh nng ny mn ca ht l rt thp v vy bng phng php nui cyphi ta s lm tng t l ny mm ca phong lan.III.4.4. Th phn trong ng nghim Mt s ro cn i vi qu trnh th tinh: Ht phn khng c kh nng ny mm trn nm nhy loi khc ng phn khng tip cn c non vo thi im thch hp V ng phn trong vi nhy Nhng ro cn c th c vt qua bng k thut th phn trong ng nghimIV.1. V d v th phn invitro v cu phiTh phn invitro cu phi trong lai to ging hoa Lily Vit Nam Hin nay, Vit Nam lily c xp vo loi hoa cao cp, tim nng th trng rt

ln. Tuy nhin cc ging hoa lily c mt trn th trng Vit Nam hin nay ch yu lnhp ni t Trung Quc, Nht Bn v H Lan vi gi thnh cc cao, thm vo mt sging nhp ni t Trung Quc rt nhanh b thoi ha do cht lng ging km Vn t ra l chng ta phi nhanh chng ch ng v ging i vi loi hoaqu ny.IV.1.1. Th nghim th phn invitro Chn n hoa trc khi n 1-2 ngy, kh trng: ra sch bi t bng nc thngtrc khi a vo bung cy, kh trng bng cn Ethanol trong 30giy, trng li = ncct v trng, tip tc kh= HgCl2. 0.1% trong 10 pht, lc u mu, trng li bng ncct v trng 3 ln.

Tch cnh hoa: -Hoa b: tch ly bao phn gi trong iu kin v trng.-Hoa m: gi ton b c quan sinh sn ci: bu nhy, vi nhy v nm nhy. Chng ct ng thng trong ng nghim hoc bnh, trn nn mi trng th phn Th phn invitro: c th s dng- K thut th phn ct vi nhy: ct ht vi nhy ht phn c th ngay ti im ct.- K thut ghp vi nhy: Ht phn b c th trn u nm nhy ca hoa b, 1 ngy

sau vi nhy ca b th phn c ct pha trn bu 2mm v gn vo bu nhy nh 1ming agar.- K thut th phn gi non: Ht phn b c thu trc tip vo hng gi non c

tch t bu hoa m.IV.1.2. Th nghim cu phi Sau khi th phn invitro cho cc t hp, tin hnh cu phi vo cc thi imkhc nhau bng k thut nui cy lt ct bu nhy. Qu c ct thnh nhng lt mngthnh 2-3mm v nui cy trn mi trng trn mi trng lt ct bu nhy: MS b sungthm 1mg/l -NAA v 90g/l sucrose. H t c hnh thnh v i t l phi trong h t cao. Sau pht tri n thnh cyhon ch nh.IV.2. Th nghim cu phi hu tnh khoai ty


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1. Thu hi cc qu khoai ty trng thnh, khi qu cn mu xanh, khong 21 n 28 ngysau khi th phn2. Kh trng b mt qu bng etanol 90% trong 30s3. Ngm qu trong hn hp dung dch hypoclorit canxi 5% v nc ct v trng trong 15pht

4. Trong t cy v trng, tin hnh tch ht t qu v chuyn sang a petri5. Bc v ht v tch ri phi6. Cy phi vo a petri cha sn mi trng dinh dng v nui nhit t 18-22C vi nh sng yu7. Cc phi c tch tt t ht s pht trin dn thnh cy khoai ty con sau c thchuyn ra trng trong vn m

Nhn chung, ngun gen khai thc t cc loi lily l v cng a dng, to iu kinthun li cho qu trnh chn to ging. Vic pht trin cc phng php th phn v cuphi thch hp s l cng c hu ch to ra nhiu ging mi vi nhiu c tnh qu.

Bin d dng t bo soma trong qu trnh nui cy invitro

I. Mt s khi nim v bin d dng soma Bin d Bin d t bo soma So snh bin d dng soma vi t binII. Phn loi bin d dng soma Bin d kiu gen Bin d kiu hnh

III. Nguyn nhn gy bin d dng somaIV. C ch to bin d somaV. u im v nhc im ca bin d dng soma trong qu trnh nui cy invitroVI. Chn lc bin d dng somaVII. Kh nng ng dng v trin vngVIII. PHN LP V CHN LC T BIN DU TM BNG K THUT NUICY INVITRO.1.1 Bin d: Bin d l nhng bin i mi m c th sinh vt thu c do tc ng ca ccyu t mi trng v do qu trnh ti t hp di truyn. Bin d to nn s a dng v cng ln cc c th sinh vt, l nguyn nhn cbn ca tin ho v l ngun nguyn liu cho chn ging.1.2 Bin d dng soma Bin d dng soma (somaclonal variation) l khi nim dng ch tt c cc bind th hin cc t bo, m nui cy v cy c ngun gc t nui cy m (Larkin vScowcropt, 1981). Bin d dng soma cn c gi l bin d dng v tnh.


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Bin d ny c quan st nhiu loi cy trng nh thuc l, khoai ty, cchua, ma, h ci bao gm y cc tnh trng nng hc nh chiu cao cy, s nhnh,thi gian sinh trng cng nh cc tnh trng ha sinh khc. Bin d kiu gen (genetic or heritable variation) Bin d kiu hnh (epigenetic hay phenotypic variation)

L cc bin d c kh nng di truyn, xy ra vi t l rt thp v khng c tchthun nghch Bao gm 3 loi: t bin h gen, t bin NST v t bin gen (Larkin et all,1989) L cc bin i v s lng NST. Loi ph bin l s sai khc v s lng NSTnh a bi, d bi, hay th khm. Cc loi c bi th cao v nhiu v s lng NSTcao d b bin i hn cc loi c mc bi th thp v t NST. (Creissen and Karp,1985). Bin i ny xy ra thng xuyn trong nui cy t bo, c bit trong nui cy tbo trn. Nhng bin i ny c th xy ra ngay t giai on u ca qu trnh nui cy, dos phn tch NST khng bnh thng nhng chu k t bo u tin.

Hin tng khuych ai gen cung lam thay i h gen. Vi du nh co linh lng a thu c dong t bao co mc khang thuc thuc tr cphotphinotrixin tng 20 ln so vi bnh thng. Thuc ny c tc ng c ch enzymeglutaminsythetase. Phn tch cho thy, gen kim tra ezyme ny c khuch i 4 -11bn, lm tng hot tnh phin m ln 8 ln L cc bin i v cu trc NST, cc thay i ny c th bao gm cc hin tngnh: Mt on, o on, thm on hay nhn on (To ra cc NST ln hn), chuynon v cc bin i trong qu trnh gim phn. Nhng bin i ny c th nh hng ti kiu hnh R0 v cc th h tip theo. L cc bin i mc phn t: s thay i ca mt cp base, thay i v slng bn sao ca mt trnh t c th, s thay i trong th hin ca cc nhm a gen

hay s th hin ca cc gen nhy(Transposable elements).S xut hin cc t bin nyv c bn mang tnh ngu nhin Nhng tnh trng t bin gen thu c nhng cy ti sinh R0 v cng c ditruyn cho cc i sau. Cc bin d kiu hnh thng lin quan n s thay i trong qu trnh th hinca mt gen nht nh. in hnh l cc qu trnh khuch i v methyl ha gen. Cc bind kiu hnh thng xuyn xut hin cc cy ti sinh sau nui cy nh l kt qu ca ccphn hi v mt sinh l. Cc thay i v kiu hnh c th l tm thi, khng c tnh di truyn v c thphc hi trng thi ban u. Tuy nhin chng c th duy tr trong sut chu k sng cacc cy ti sinh Nguyn nhn: C lin quan n mt vi thay i trong qu trnh biu hin gen. V d, hin tng tng mnh m kh nng sinh trng ca cc cy ti sinh khitrng trn t. Biu hin ny c th lin quan n vic tr li ca trng thi tr ho hocqu trnh loi b virus khi ngun mu cy ban u Cc v d khc nh: hin tng ra hoa sm, bch tng, cc thay i v hnhdng, mu sc cnh hoa, hnh dng l v chiu cao cy... Bt k mt yu t c kh nng c th dn n cc thay i di truyn u cxem nh l mt nguyn nhn gy ra cc bin d ny.


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Cc yu t ny chia lm ba nhm: sinh l, di truyn, ho sinh. Hai nguyn nhn chnh gy bin d dng soma l: Tnh khng ng nht di truyn ca cc t bo soma ca mu cy ban u (s adng di truyn t nhin ca cc t bo nui cy ). Tc ng ca cc yu t bn ngoi trong qu trnh nui cy in vitro (sinh l, ha

sinh). Cc mu cy c ngun gc t mt dng n tnh, t ht hay cy con c xemnh l ng nht v mt di truyn v khi ly mu c th c kiu hnh ging nhau. Tuynhin cc mu cy ny thc t li bao gm nhiu loi t bo khc nhau nh l phloem,xylem, nhu m Nhng t bo ny c mc bi th khc nhau. Ni cch khc, c sa dng t bo gia cc loi t bo trong cng mt mu cy. S a dng ny c gi la bi v tnh (Polysomatic). Ngoi ra nhiu thc vt tn ti dng th khm. Chng cha nhng lp t bohoc m c cu trc di truyn khc nhau c pht trin t meristem c cha lp hay bphn m b t bin. Hin tng ny c bit ph bin cy thn g. Loi mu cy.

Cc loi mu cy khc nhau thng th hin mc bin d khc nhau.Cc mu cy c ngun gc t cc th tin chi nh chi nch, chi nh hoc meristemthng c mc bin d thp hn khi s dng cc mu cy c ngun gc khng phinh sinh trng nh l, r hay protoplast. Kh nng xy cc bin d soma cn ph thucvo kiu gen cng nh tui cy m. Cc dng gi hn thng n cc bin d sn c mc cao hn cc dng tr hn. Cc loi c bi cng cao v s lng NST cng nhiuth c tnh bin d cng cao Phng thc nhn ging in vitro:

Cc phng thc nhn ging khc nhau s cho t l xut hin cc bin dv tnh khc nhau. Nhn chung nu chi bt nh c ti sinh t mt t bo th c hi xut hin cc bin d soma thng l ln hn rt nhiu t cc chi c ti sinh t nhiu

t bo. Cc qu trnh nui cy callus, huyn ph hoc protoplast do thng c nhiubin d soma3.2. Tc ng ca cc yu t bn ngoi trong qu trnh nui cy Loi v nng cht iu tit sinh trng s dng

2,4 D hoc 2,4,5 T thng gy ra cc sai khc trong cy ti sinh. V d nhcc cy du da ti sinh t callus Cho cc m nui cy di ngy trong mi trng chacc auxin mnh nh nui cy di ngy trn mi trng c cha 2,4 D c t l rt ln ccbin d khi trng trn ng rung Thi gian nui v s ln cy chuyn

Vic nui cy di ngy trong iu kin in vitro cng nh tng s ln cychuyn cng s lm tng kh nng xut hin cc bin d soma

Nguyn nhn: do s thay i ca cc kiu methyl ha bnh thng caDNA genome.5.1.Nhc im: Mt s bin d dng soma khng n nh (c th ch l do mc biu hin camt gen) v khng c di truyn cho i sau Mt s bin d dng soma li kt hp vi mt hay mt s tnh trng khng mongmun nh lch bi, bt dc. L mt nhn t cn tr ca qu trnh nhn ging v tnh.


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5.1. Nhc im: Vic ti to cy hon chnh ch xy ra mt s kiu gen nht nh v cc kiu genny c th c t ngha hoc khng c ngha i vi nh chn ging. Nu s dng dng t bo nui cy lp li nhiu ln th t l ti sinh thnh cygim

5.2. u im Cng lc kho st c mt khi lng ln hng triu t bo trong mt khnggian mi trng hp, ng nht v cng chu mt p lc chn lc ch nh thch hp, ddng tin hnh theo qui trnh cng nghip. To ging mi c cc c tnh mong mun mt cch nhanh chng thng qua nuicy m kt hp vi chn lc dng t bo. Gip nh khoa hc c th nghin cu mt loi nhit i vng n i hocngc li v iu kin mi trng c th c th to ra rt d dng trong phng thnghim bt c u.5.2. u im C tim nng nht nh trong cng tc ci tin ging cy trng, c bit l cc cy

lu nm vn b cn tr bi nn di truyn hp v chu k ti sinh di. Cc bin d sinh ra trong nui cy m khng gp phi nhng cn tr v mt xhi, o c nh cc cy trng chuyn gen. Phn lp cc bin d c ch kiu hnh l tng song cn thiu mt s tnh trngmong mun. Phn lp cc bin d c ch sn xut cc hp cht s cp v th cp. Phn lp cc bin d c ch vi kh nng khng bnh, chng chu stress tt hn. To cc bin d di truyn khng qua lai hu tnh nhng dng u t Cc bin d soma sinh ra do cm ng l con ng tt nht ci to cc cy lunm.Qui trnh chung:

Nui cy TB xma 2n NST trn mi trng nhn to c nh hng . Cc TB phn chia thnh nhiu dng bin d c b NST thay i v biu hin caohn mc bnh thng (bin d dng xma). Chn lc v pht trin cc dng TB c bin d theo mong mun thnh c th mi7.1. Nguyn tc chn lc t bo:7.1.1. Chn trc tip:

Thng qua u th v sinh trng hay s khc bit thy c v mu sc cth chn c t qun th t bo.

H thng t bo hay c s dng l cc t bo dch huyn ph hoc khicallus.

iu kin chn lc l cc c t vi nng khc nhau gy tc ng trctip ln sinh trng ca t bo. Thng thng ngi ta trn t bo v mi trng thchcha c t v chn nhng t bo sng st phn chia thnh khun lc m so hoc cytrc tip ln mi trng chn lc cha c t

y, c im ca dng c chn l kt qu biu hin khuyt tt ca tbo. Th d in hnh l chn dng thiu Enzyme nitrate reductase (NR). Trn mi trngcha ClO3- nhng t bo c NR s dng ClO3- nh NO3- v kh thnh ClO2-, ClO2- tcdng nh mt c t cho nn ch c nhng t bo khng c NR mi sng st trn mitrng chn lc


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7.1.3. Chn tng thCc t bo d dng thc vt thng c chn bng phng thc x l

t bin v nui trn mi trng c cha yu t dinh dng cn thit, cc yu t ny ckhi li chnh l yu t gy t bin. VD: t bin ln chu c S-2-aminoethyi cysteinexut hin sau khi x l phi nui cy.

7.2.1. Chn lc trong iu kin khng c tc nhn chn lcCc t bo v callus khng x l sinh trng trong nui cy in vitro ccthi k khc nhau trn mi trng khng cha tc nhn chn lc (c t hoc cc cht cch),c cm ng phn ha cc cy hon chnh. Cc cy ti sinh s c trng trnng rung chn lc cc bin d. Bng phng thc ny ngi ta thu c cc bind dng soma ca cc cy trng khc nhau.

V d:1. Vi Cy ma ng (Saccharum officinarum) ngi ta chn c cc

cy khng bnh mc sng (downey mildew), bnh Fiji (do virus apid transmitted) trnging ma Pindar, hoc ci thin mt s gi tr nng hc ca ging ma Q10 khng bnh

dm mt. (Do Helminthosporium sacchari).2. Vi cy khoai ty.Shepard v cng s (1980) ti sinh mt s ln cy t protoplast t bo

tht l ca ging Russet burbank v thng bo cc bin d thu c trong qun thprotoclones. Mt trong s chng khng c bnh thi sm (early bright do Alternariasolani) hoc thi mun (late bright do phytophthora infestans)

3. Vi cy c chuaEvan v cs phn lp cc dng soma ca c chua bng cc bin d hnh

thi l cc t bin ln ca tnh bt dc khng nm Fusarium oxysporium mt cung l,kh nng lc ha ca l, mu sc ca qu v hoa.

Theo phng php ny cc dng t bo bin d c sng lc t nui cy nh vokh nng sng st ca chng khi c mt cc c t/cht c ch trong mi trng dinhdng, hoc di cc iu kin stress ca mi trng. Cc bin d c th thu c bngcch chn lc trc tip, gin tip. S phn lp c tin hnh trong nui cy dch huynph hoc bng cch dn tri t bo n/protoplast.

Cc hng chn lc bao gm:7.2.2.1. Chn lc dng khng bnh S dng cc yu t chn lc nh phytotoxin chn lc kh nng khng vi nm,vi khun hoc virus. Tuy nhin, li ko theo nhng c tnh khng mong mun khc. V d chn lclin tip vi mt phytotoxin lm cho khoai ty khng bnh nhng c khoai nh i v chtlng dinh dng ca c khoai kem hn Thng thng ngi ta s dng trc tip nm, vi khun lm yu t chn lc pht hin dng t bo soma khng bnh, t cc dng ny to ra cc cy khng bnh tngng.7.2.2.2. Chn dng khng thuc dit c

Nui cy Protoplast trn mi trng c cc cht dit c khc nhau cm ng tbin to ra cc dng t bo chng chu sau ti sinh thnh cy


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7.2.2.3. Chn dng chng chu vi cc stress ca mi trng Nhiu vng c mn, phn, kim loi nng kh cao km hm s pht trin cacy trng Cn to cc dng chng chu. ti sinh thnh cng cy thuc l khngNaCl v nhiu dng t bo ca nhiu ging cy trng khc nhau c th chng chu nng mui cao.

to cc dng chu lnh bng cch nui cy t bo ca cy Nicotiana sylvestristrong iu kin nhit d thp.Mt s hng chn lc quan trng khc Chn dng khng khng sinh Chn dng khng cc ng ng base ca DNA Chn dng khng cc acid amin c hiu Chn dng sn sinh cc sn phm th cpMt s thnh tu

Ging la CR203 c nhiu gen qu nh tnh chng chu ry nu Biotip 1v 2, tnh thch ng rng, nng sut n nh v phm cht go kh..Mt s thnh tu

Ging la DR2 ( nm 2000 ) c to ra t dng t bo bin b xma ca ging la CR203, dng ny c tch v ti sinh thnh cy. Ging la DR2 c ng u rt cao, chu kh hn tt, nng sut trung bnh t 45-50 t/haMt s thnh tu

Ging la CM64 c ngun gc t IR64, do Vin La ng bng sng CuLong ci tin thng qua cng ngh sinh hc nui cy m, to bin d t bo soma. Tngc ca n l OM3005-6

To ging la chu hn c nng sut cao caTrng H KH Hu

PHN LP V CHN LC T BIN DU TM BNG K THUT NUI CYINVITRO(Nguyn Vn Vinh)

I. t vn Phn lp v chn lc t bin l mt trong nhng cng on i hi mt nhiucng sc, thi gian v tin ca i vi cc cy cho qu, ht thng qua con ng gy t bin thc nghim ,chunhgs s phn ly theo nh lut di truyn Menden, v th phi qua nhiu th h vinhiu cng on nh giao ring r tng dng, trng cy bit lp mi hy vng thu ct binI. t vn

Bng con ng phn lp v chn lc thng qua k thut nui cy invitro, khngnhng thu hp c khng gian nghin cu m thi gian thu nhn t bin cng ngnhn nhiu so vi con ng in vivo. Trong nghin cu gy to t bin du tm, ngoi x l cy invitro chng ti cnx l c hom v thng qua k thut phn lp v chn lc chng ti thu c cc tbin c gi tr.II. I TNG V PHNG PHP NGHIN CU1. i tng v vt liu nghin cu


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Hom du hai ging Bu en v VA186 c x l 5 Krad da vo kt quxc nh nhy cm bc x gama ca cy du tm, sau c trng ngoi vn thnghim. Khi hom mc chi, chng ti ct v chuyn sang nui cy invitro thng qua ktqu nhn ging v tnh du tm bng phng php nui cy invitro vi mi trng tocm chi c thnh phn MS, NAA 0,5 1,5 mg/l v BAP 1,5 5 mg/l. Thnh phn mi

trng to r gm MS, IBA 1 - 3 mg/l v NAA 1 mg/l phn lp v chn lc t bin2. Phng php nghin cu Nui cy ton b chi hom t hom du chiu x t M1V1 n M1V4, theo di khnng sinh trng, tc ra l, hnh dng l v cc c im sinh hc khc, so snh v ichng tm kim t bin.III. KT QU V THO LUN

Sau khi chiu x 500 hom du ca mi ging bu en v VA186, nui cy invitrot M1V1 n M1V4, kt qu c trnh by cc bng sau:

Trong tng s 12654 cy bu en liu 5 Krad, th h M1V4 c 1654 cy t bin , tn st bin l 0,13. y ta thy t bin l bch tng thp nht, c 112 cy, t tn s

0,009; t bin l x thy cao nht, c 204 cy, t tn s 0,016. qua theo di qun tht bin ca ging bu en chiu x liu 5 Krad, chng ti nhn thy 3 dng thp cy:B16, B17 v B18 c tim nng nng sut rt tt

Qua bng 2 cho thy: Trong tng s 11558 cy du ging VA186, liu 5 Krad c 1452 cy t bin,tng tn s t bin l 0,125. Trong s cy t bin, dng t bin l bch tng thp nht,c 98 cy; dng t bin thp cy cao nht, c 231 cy. Trong s nhng cy t bin thu c chng ti nhn thy c 3 cy t bin l tol VA12, VA15 v VA18 t ging VA186 rt c trin vng v tim nng nng sut caohn ging chng.

KT LUNVi kt qu nghin cu t c, chng ti rt ra nhng kt lun sau: Bng con dng phn lp v chn lc t bin invitro trong phng th nghim vvn m rt ngn thi gian nghin cu t 6 nm xung cn 2 nm. chn lc c 3 dng t bin thp cy l B16, B17 v B18 t ging bu enc tim nng kinh t hn so vi i chng. Tng t cng thu c 3 dng t bin l toVA12, VA15 v VA18 t ging VA186 rt c trin vng v nng sut.Thanks so much for listening!Tiu lun: Cng ngh nui cy m t bo thc vt ti:Nui cy huyn ph t boT VN Bn thn mi t bo thc vt l mt n v c lp, n cha ng tt c nhngthng tin di truyn c trng ca c th t n sinh ra. Cho nn mi t bo c th xydng li ton b c th mi nh tnh ton nng. Thc vt bc cao la mt nguncung cp cac hp cht hoa hoc va dc liu rt quan trong. Tuy nhin trongnhng nm gn y san lng cac thc vt o rt kho am bao mcn nh do hu qua cua mt s yu t nh: - iu kin t nhin khng thun li.


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- Chi phi lao ng ngay cang tng. - Kho khn ky thut va kinh t trong trng trot. Phng phap nui cy huyn ph t bao (dch lng) ca thc vt co khanng gop phn giai quyt nhng kho khn trn.NI DUNG

Khi nim nui cy huyn ph t bo Lch s nghin cu Cc phng php nui cy c trng ca t bo nui cy huyn ph Cc ch tiu xc nh tc sinh trng ng dng ca nui cy huyn ph t bo Cng trnh nghin cu c th Kt lunI. KHI NIM Nui cy huyn phu t bo (cell suspension cultures): Phng thc nui tbao n (single cell) hay cum nhiu t bao (cell aggregate) trang thai l lngtrong mi trng long. Dch huyn ph c to ra do s nui cy mt mnh m so khng c kh nngbit ha, trong mi trng lng v c chuyn ng trong sut thi gian nui cy.I. KHI NIM Cc t bo tch ra khi m so v phn tn trong mi trng lng. Sau mt thi gian, dch huyn ph l mt hn hp cc t bo n, cc cm t bo,cc mnh cn li ca mu cy v cc t bo cht.

II. LCH S NGHIN CU 1949: Caplin v Steward nui cy t bo thc vt trn mi trng lng c

khuy. Muis (1954) v Nickell (1956) cho thy rng cc t bo thc vt c th sinhtrng c nh cc c th vi sinh vt. 1959, Melches v Beckman tch v nui cy thnh cng t bo n v huynph t bo.III. CC PHNG PHP NUI CY Ty iu kin v mc ch m c cc phng php nui cy t bo huyn phkhc nhau. Ch yu l phng php:Nui cy dch th ng.

Trong nui cy dch th ng c h thng cung cp kh (thi kh), cc kh uc v trng.III. CC PHNG PHP NUI CY3.1. Nui cy dch th ng3.1.1. Nui cy chm lin tc Cc t bo c tip xc vi mi trng dinh dng do chng c ngm hn vodung dch mi trng. S thng kh c thc hin nh my lc chy tc 100 - 150 vng/pht. Kha vo phi m bo v trng. Qu trnh thng kh cn ngn chn v lm gim s ktdnh ca cc t bo vi nhau.


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III. CC PHNG PHP NUI CY Theo Thomas v Davey (1975), nui cy huyn ph t bo c dung tch 25 ml, tc ph hp nht ca my lc l 100 120 vng/pht. Th tch ca mi trng lng cng phi ph hp vi kch thc bnh nui cy,thng chim 20% th tch bnh.

III. CC PHNG PHP NUI CY Cc nui cy quy m nh v trong nhng thi gian ngn, c th s dng mykhuy t tc 250 vng/pht v thi gian cho qu trnh nui cy thng t 10 15ngy. Sau , cc mu nui cy phi c cy chuyn sang mi trng mi m bos sinh trng v pht trin ca cc t bo.III. CC PHNG PHP NUI CY3.1.2. Nui cy chm tun hon Cc t bo c nhng chm vo mi trng dch th, xen k vi nhng khongthi gian c a ra ngoi mi trng. Qu trnh c thc hin nh s chuyn ng bp bnh ca cc bnh nui cy.III. CC PHNG PHP NUI CY Khi chuyn ng: Khi t bo u ny c a vo mi trng. Khi t bo u kia tip xc vi khng kh. Steward v cng s (1952) cng thit k nhng bnh nui cy c bit theo phngphp nui cy chm tun hon.III. CC PHNG PHP NUI CY3.2. Nghin cu nui cy huyn ph t bo bng Bioreactor: H thng nui cy c thng kh hoc c trao i kh. Mi trng nui cy c th d dng thay mi theo tng thi k.

T bo thc vt nhy cm vi stress hn vi khun nn h thng bioreactor chonui cy t bo thc vt yu cu thng xuyn ci thin h thng thng kh v h thngtrn mi trng. T bo thc vt cn nh sng cho quang hp nn cn c s lin kt gia bioreatorvi h thng chiu sng.Tng hp lng ln sinh khi toIII. CC PHNG PHP NUI CY Takayama v Miasawa l nhng ngi u tin s dng bioreactor vo nhnging cy trng: nhn c siu nh khoai ty, c ging hoa ly, hoa lan h ip. Cng ngh ny cho php nhn nhanh v hn cc ging cy trng nh thit bbioreactor hon ton t ng ha.

VD: 1 bioreactor vibro-mixer trang b vi cc ng silicone c kh nng sn xut100.000 phi v tnh ca cy trng nguyn trong 1lit dch huyn ph nu nh dung dch c t trn 1tm giy lc v pht trin trong 4 tunIII. CC PHNG PHP NUI CY Bioreactor s dng trong nui cy m t bo thc vt c ci tin t cc loibioreactor trong nui cy t bo vi sinh.III. CC PHNG PHP NUI CY Thun li:


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Th tch nui cy tng gip sn xut nhiu hn m khng cn nhng k thut caocp. Hu ht cc bnh bioreactor c thit k vi c ch khuy bng c hc hay thikh duy tr nui cy gn nh ng dng.III. CC PHNG PHP NUI CY

Khi thao tc nui cy lin tc, mi trng nui cy v mi trng vt l c thc kim sot thch hp cho sinh trng. iu ny khng th thc hin vi h thngnui cy bnh tam gic. Nhc im: i hi thit b hin i v t tin, vn hnh phc tp c bit l khuchng nhim cho huyn ph nui cy.IV. C TRNG CA T BO TRONGNUI CY HUYN PH Nui cy huyn ph t bo cn mt lng m so kh ln, xp x 2 3 g/100mldung dch mi trng (Helgeson, 1979).

Sau mt thi gian, dch huyn ph l mt hn hp cc t bo n, cc cm t bo,

cc mnh cn li ca mu cy v cc t bo cht.IV. C TRNG CA T BO TRONGNUI CY HUYN PH Mc tch ri ca cc t bo ph thuc vo c tnh ca cc khi t bo xp vc th c iu chnh bi thnh phn mi trng.

VD: trong nhiu trng hp, tng t l Auxin/Cytokinin s sn sinh nhiu khi tbo xp. Theo King v Street (1977), khng c mt quy trnh chun no cho nui cy huynph t bo.IV. C TRNG CA T BO TRONGNUI CY HUYN PH Sinh trng v pht trin ca t bo nui cy: Pha lag: bt u khi a m so vo mi trng, ko di cho ti ln phn bo utin. Pha tng tc: cc t bo bt u phn chia v s lng t bo tng dn. Pha hm s m: s lng t bo tng theo hm s m. Pha n nh: s lng t bo t cc i v khng i theo thi gian, s t bosinh ra (do phn bo) xp x s t bo cht i.IV. C TRNG CA T BO TRONGNUI CY HUYN PHIV. C TRNG CA T BO TRONG

NUI CY HUYN PH duy tr qu trnh nui cy, cc t bo cn c cy truyn vo giai on smca pha n nh. Thi im cy truyn c th da vo kinh nghim, ni chung l nn bt u khimt t bo cc i. Mt t bo cc i trong khong 18 25 ngy, vi huyn ph sinh trngmnh c th ngn hn, t 6 9 ngy.


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IV. C TRNG CA T BO TRONGNUI CY HUYN PH ln cy truyn u tin, dch nui cn c lc nhm loi b cm t bo ln,cc mnh t mu cy ban u, sau dng pipet ly dch cy truyn. Lng t bo em cy truyn phi ln m bo mt t bo, v khi thp

qu cc t bo s khng sinh trng c.VD: i vi t bo cy sung du (Acer pseudoplatanus) mt thch hp 9

15.103 tb/ml.IV. C TRNG CA T BO TRONGNUI CY HUYN PH Theo King (1980), nhng t bo tri qua qu trnh nui cy, sinh trng v traoi cht trong dch huyn ph gi l dng t bo. c im ca dng t bo: Kh nng tch t bo cao. Hnh thi t bo ng nht. Nhn r rng v t bo cht m c.

Nhiu ht tinh bt.IV. C TRNG CA T BO TRONGNUI CY HUYN PH Tng i t cc yu t mch. C kh nng nhn i trong 24 72h. Mt tnh ton nng. Quen vi cht sinh trng. Tng mc a bi th.V. CC CH TIU XC NH TC SINH TRNG5.1. S lng t bo Trc khi m, x l nhng cm t bo qua acid chromic, un nng 700C trong 5 10 pht, sau lm ngui v lc mnh trong vi pht. Pha long dch, nhum v m trn bung m hng cu. Kt qu: s lng t bo/ml dung dch nui cy.V. CC CH TIU XC NH TC SINH TRNG5.2. Th tch t bo Ly ngu nhin mt th tch dch nui cy, em ly tm tc 2000 vng/phttrong thi gian 5 pht. Thu t bo v em xc nh th tch.

Kt qu: S ml t bo/th tch mi trng nui cy. T l %.V. CC CH TIU XC NH TC SINH TRNG5.3. Khi lng ti v khi lng kh t bo Khi lng ti: Thu thp t bo trong mt th tch dch xc nh.


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Ra bng nc ct v trng Lm kh trong chn khng. Cn xc nh khi lng. Khi lng kh: Ly mt th tch mu xc nh.

Loi b phn ni, ra phn t bo trn giy lc Whatman. Sy kh trong 12h 800C n khi lng khng i.V. CC CH TIU XC NH TC SINH TRNG5.4. Xc nh thnh phn Protein Thu thp t bo, chuyn ln lc qua giy Whatman. Ra t bo trong ethanol 70% ang si. Lm kh vi Aceton ri chuyn vo dung dch NaOH 1M. un nng n 850C trong 1.5h. Lc v xc nh Protein thy phn trong dch theo phng php Lowry v cs

(1951).V. CC CH TIU XC NH TC SINH TRNG5.5. Xc nh ch s nguyn phn Huyn ph t bo c c nh trong hn hp Aceto-orcein : Acid Acetic (3:1)sau chuyn sang lam knh. Nh 1 git Aceto-orcein ln mu, h trn ngn la n cn, ngui trong 5pht. y mu bng Lamel, lm kh mu, quan st di knh hin vi quang hc vtknh 100. Xc nh cc k trong khong 1000 t bo.

T l % cc t bo ang phn bo gi l ch s nguyn phn.VI. NG DNG6.1. Sn xut sinh khi t bo v

cc hp cht th cp Sn phm ca nui cy huyn ph t bo thng c trng l nhng cht c gi trcao v cn lng nh. C th chia thnh cc nhm da theo cng dng: Hp cht th cp trong y hc. Hp cht th cp trong ha m phm. Hp cht th cp trong cng nghip.

VI. NG DNG Ti sao phi nui cy huyn ph t bo thu sinh khi? Tn ph mi trng v s xi mn di truyn. S cung cp ngun nguyn liu khng u n, khng vng chc, cht lngkhng n nh. Nhim bn do thuc tr su, bnh hi, cht phng x hay kim loi nng Nui cy huyn ph t bo khc phc c cc nhc im trn.


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VI. NG DNGNgoi ra, nui cy huyn ph t bo cn c mt s u im sau: Sn xut mt cch c nh hng, theo quy m cng nghip, khi lng sn phmln, khng ph thuc vo ma v. T l % khi lng kh cc hp cht th cp cao hn so vi nguyn liu thc vt

thu hi trong t nhin. Khng tn nhiu din tch gieo trng. Thu sinh khi t bo ln trong thi gian ngn.

VI. NG DNG Mt s hp cht th cp c sn xut t nui cy huyn ph t bo thcvt: Diosgenin: Tc dng chng vim; cha st, i but, i vng, thp khp, aulng, au dy thn kinh.- Vic nui cyDioscorea deltoidea Wall ex Griseb (cy C nu) trong bioreactor thu c nng sut diosgenin cao hn so vi hiu sut c c t nguyn liu t

nhin.- Trong nui cy t bo, diosgenin chim 7.8 % khi lng kh, trong khi chchim 2 % vi nguyn liu thc vt thu hi trong t nhin.VI. NG DNG Codein: Gim au v gim ho.- Papaver somniferum l ngun ca 2 hp cht rt quan trng trong y hc lmorphine v codein.- Sn xut morphin c thc hin bng vic nui cy huyn ph t bo t boPapaver somniferum.VI. NG DNG Steviol:- Cht to v ngt c bit n rng ri, thu nhn t cy Stevia rebaudiana.- Nui cy callus l giai on tin nui cy huyn ph t bo trong tch lysteviol.- Trong nui cy callus, hiu sut t ti 36.4 %.VI. NG DNG Taxol:- L mt nhm cht trong ha tr liu ung th, iu tr ung th bung trng, ung th v,ung th phi, khi u c tnh, v cc dng u bu khc.- Nui cy t bo cc loi Taxus c xem nh l mt phng php u th cung cpn nh ngun taxol v dn xut taxane ca n (T. cuspidata, T. baccata, T. mairei).VI. NG DNG Scopolamine v hyoscyamine:- L 2 loi tropane alkaloid c s dng tr co tht v gy m.- C nhiu trong cc cy thuc h Solanaceae.- Nui cy huyn ph t bo Scopolia japonica c b sung tropic acid (nh 1 tincht) th c th tng hm lng alkaloid ln 15 ln.VI. NG DNG Saponin v sapogenin:


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- L mt dc phm qu gi, c nhiu trong r cy nhn sm (Panax ginseng), ctc dng cha bnh ri lon tiu ha, chng suy nhc c th, tng cng sinh lc.- Nui cy huyn ph t bo cy P. ginsengang tin hanh trn quy m cngnghip.- y l mt trong cc i tng c cc nh khoa hc trn th gii tp trung

nghin cu nhiu nht.VI. NG DNG Betalaine:- L mt nhm cc cht nhum mu thc phm.- Bao gm betaxanthine (mu vng) v betacyane (mu ).- Thnh tu nui cy c quan cyBeta vulgaris, nui cy callus v huyn ph caPortulaca americana.VI. NG DNG6.2. Trong cng tc chn ging cy trng: S dng chn lc cc dng t bo mong mun qua cc test thanh lc.

Sau ti sinh cc t bo chn lc thnh cy hon chnh. Cy hon chnh s tr thnh vt liu khi u quan trng trong cng tc ging. Ngoi ra, tch protoplast hoc l ngun sn xut cc phi v tnh.VII. CNG TRNH NGHIN CU Nghin cu nui cy m so v dch huyn ph cy Bt ruiDrosera indica L.cho mc tiu thu nhn Plumbagin. (Quch Dim Phng). Nghin cu nui cy m so v dch huyn ph cy Bo tDrosera burmalniVahl cho mc tiu thu nhn 1 hp cht Anthraquinone. Quch Dim Phng ( tiNCCB ca H Quc gia TP HCM)VII. CNG TRNH NGHIN CU

Nghin cu nui cy m so v dch huyn ph cy Mn nh HngAlthaearosea L. nhm thu nhn Rutin trong iu tr tim mch. (Quch Dim Phng). Nghin cu ng dng cng ngh nui cy t bo h cyDrosera.sp thu nhncc hp cht Quinone c hot tnh sinh hc.(Quch Ng Dim Phng).VII. CNG TRNH NGHIN CU Thnh cng trong quy trnh to sinh khi t bo r sm Ngc Linh bng ng dngcng ngh Biomass Hc vin Qun Y. Cc nh khoa hc Vin Cng ngh sinh hc (Vin khoa hc v Cng ngh Vitnam) hon chnh quy trnh nhn nhanh sinh khi m ca cy Hong Lin gai(Berberis Wallichiarna DC) bng cng ngh t bo thc vt lm ngun dc liu snxut thuc becberin.

Tuy nhin, do nhng kh khn trong vic tm kim ti liu nn nhm chng em khnga ra c mt quy trnh sn xut da trn nui cy huyn ph t bo, m ch a rac mt cng trnh nghin cu giai on u:VII. CNG TRNH NGHIN CUNH HNG CA CHT IU HA SINH TRNG THC VT VNG SACCHAROSE LN DCH


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NUI CY HUYN PH T BODA CN CATHARANTHUS ROSEUSTc gi:Bi Vn L, Trng i hc Khoa hc T nhin, HQG-HCMNguyn Ngc Hng, Trng i hc Bn cng Tn c Thng

VII. CNG TRNH NGHIN CU1. T VN Da cn Catharanthus roseus.G.Don thuc h Trc o Apocynaceae l mttrong nhng dc liu cha nhiu alkaloid. T da cn ngi ta chit c cht cha ung th nh vinblastin v cha caohuyt p nh ajmalicin, serpentin. Tuy nhin hm lng ca nhng cht ny c trong cy l rt thp. Vic nui cyt bo cy da cn nng cao hm lng alkaloid mong mun c nhiu nc trnth gii nghin cu v ng dng vo sn xut.VII. CNG TRNH NGHIN CU

2. VT LIU V PHNG PHP M so mu xanh c nui trong iu kin chiu sng t l da cn in vitro 4thng tui c chuyn vo 100 ml mi trng cm ng trong erlen 250 ml. Mi trng cm ng gm mi trng MS + Vitamin Morel + Hormon tng trngthc vt (NAA hoc 2,4-D v Kin hoc BAP) + 30g/l ng sucrose. Sau t vo my lc 200 vng/pht nhit 25 2oC trong iu kin chiusng lin tc 16 gi/ngy.VII. CNG TRNH NGHIN CU Xc nh sinh khi bng phng php cn. Chit tch alkaloid ton phn t sinh khi t bo Da cn theo phng php caKutney v cng s (1983). Xc nh alkaloid ton phn bng phng php acid-baz - khan theo dc inVit Nam v dc in Anh.VII. CNG TRNH NGHIN CU3. KT QU V THO LUN3.1. Kho st nh hng ca s thay i nng cytokinin n s to sinh khiKt lun: bng trn cho thy mi trng ch b sung NAA c s to sinh khi thp hnso vi mi trng b sung NAA kt hp vi Kin. Mi trng K4 (MS + NAA (1mg/l) + Kin (0.5 mg/l) v K8 (MS + NAA (1mg/l)+ BAP (0.5 mg/l) cho sinh khi ti v kh u cao hn cc mi trng khc.3.2. nh hng ca cc cht iu ha sinh trng n vic to sinh khiKt lun: Mi trng c b sung 2,4-D to sinh khi ti nhiu hn so vi mi trng bsung NAA nhng trng lng kh li rt thp. Mi trng K3 v K4 c trng lng tithp hn mi trng K1 v K2 nhng cho sinh khi kh nhiu hn. Mi trng K3 chosinh khi ti v kh u cao hn mi trng K3 nhng xt v mt thng k, hai mitrng ny khng c s khc bit mc ngha = 0.01.3. KT QU V THO LUN


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3.2. nh hng ca cc cht iu ha sinh trng n vic to sinh khiKt lun: 2,4-D kch thch s phn chia t bo nhanh nhng li ph hy cy trc cht ch ca tbo lm cho t bo xp v gim i vic to cc sn phm th cp. Cytokinin cn thit chos hnh thnh cc hp cht th cp, khi kt hp cytokinin vi NAA gip duy tr s tng

trng cng nh to ra alkaloid cao.3. KT QU V THO LUN3.3. nh hng ca ng sucrose n vic to sinh khiKt lun: Nng ng tng dn t 20 - 60 gam/l sinh khi ti v kh thu c trongmi trng tng dn theo. Mi trng ti u cho th nghim v nh hng ca nng ng ln s to sinh khi l mi trng 5. Tuy nhin khi tng nng ng ln cao hn l 70 - 80 gam/lt th trng lngti v kh gim dn. Nng ng c nh hng n s to thnh cc sn phm thcp trong nui cy t bo. nng ng sucrose cao va phi khong 50 60 gam/lts kch thch to sinh khi v alkaloid.

3. KT QU V THO LUN3.4. nh lng alkaloid vinblastin v vincristin c trong l cy khng qua nui cy invitro v trong dch huyn ph t boKt lun: bng phng php nui cy dch huyn ph c b sung 2,4-D th lng vincristin vvinblastin khng c trong mu alkaloid ton phn cn dch huyn ph NAA + Kin cholng vinblastin thp hn so vi cy trng ngoi t nhin nhng cho lng vincristin khcao trong khi cy trng ngoi t nhin trong th nghim ny li khng c.VII. CNG TRNH NGHIN CU4. KT LUN: Tm c nng hormon ph hp: nng 1 mgl-1 -naphthaleneacetic

acid (NAA) v 0,5 mgl-1 kinetin (Kin) thu nhn c sinh khi v alkaloid ton phn caonht trong khi cng mi trng ny nhng thay th NAA bng 2,4-dichlorophenoxyacetic acid (2,4-D) cng nng thu c sinh khi v alkaloid tonphn thp. Nng ng ti u cho vic nui dch huyn ph t bo da cn: nng 60gl-1 cho lng sn phm l cao nht.VIII. KT LUN Tuy nhin, do c s vt cht c bn cho mt phng th nghim nui cy huynph t bo cn cao nn Vit Nam, hng nui cy huyn ph t bo ch dng li mcnghin cu.

Trong nhng nm gn y, Vit Nam thnh cng trong nhng quy trnh pdng cc phng php ca nui cy huyn ph t bo sn xut ra cc ch phm sinhhc, y hc

Trng i hc Nng Nghip H NiKhoa Cng ngh sinh hcNui cy meristem to cy sch bnh virus


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I. T VN

Bnh virus trn cy l mt bnh hi ph bin, nghim trng v gy thit hi lni vi ngi trng.

Nhng phng php phng cha bnh cy nhim virus thng thng t c hiuqu Phng php meristem to cy trng sch bnh c nhiu u im v c s dngph bin.II. NI DUNG Tc hi ca bnh virus i vi cy trng. Cc bin php k thut v c s to cy sch bnh virus. Cc k thut nui cy meristem. Cc phng php kim tra, nh gi bnh virus bin php nh gi bnh virus trnng rung Quy trnh sn xut cy c mi sch bnh virus nh k thut nui cy nh sinhtrng.1. Tc hi ca bnh virus: Virus c th lm gim nng sut, cht lng v sc sinh trng ca cy trng.

V d: virus gy bnh cun l khoai ty, c chua (Tomato yellow leaf curlvirus) v virus Y c th lm gim 95% nng sut ca cy trng.1. Tc hi ca bnh virus: Virus ly lan rt nhanh ch trong mt khong thi gian ngn c th xm nhim cmt vng rng ln gy thit hi v c th tht thu hon ton.

Virus c ly truyn qua cc th h thng qua cc c quan nhim virus nh c,ht ging, cc b phn sinh dng s dng lm ging.

Virus l mt loi bnh khng cha c. Khng c cc loi thuc hay ho chtc hiu cho vic phng tr cc loi bnh gy ra bi virus.

Chnh v th chn ging sch bnh virus ang c nghin cu rng ri trong c k thut chn ging cy trng sch bnh virus nh nui cy nh sinh trngmeristem.1. Tc hi ca bnh virus:1. Tc hi ca bnh virus:Papaya ringspot virus1. Tc hi ca bnh virus:D ly lan, ly lan rt nhanh.Papaya ringspot virus1. Tc hi ca bnh virus:

Virus l mt loi bnh khng cha c. Khng c cc loi thuc hay ho chtc hiu cho vic phng tr cc loi bnh gy ra bi virus.

Khng c cu to t bo.

Sng k sinh bt buc.


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Chnh v th chn ging sch bnh virus l vn bc thit v c quan tmnhiu.1. Tc hi ca bnh virus:1. Tc hi ca bnh virus:1. Tc hi ca bnh virus:

Trong nui cy m c hai cch chn ging sch bnh virus: Cch 1: Dng cc phng php chun on bnh virus.

Cch 2: Dng k thut lm sch virus bng nui cy meristem.2. Cc bin php k thut lm sch virus trong nui cy m:

Dng cc phng php chun on bnh virus thanh lc cc mu nhim bnhtrc khi a vo nui cy, s dng bin php nhn nhanh invitro nhn nhanh musch

Lm sch virus mu b nhim, sau khi to mu sch th tip tc s dngbin php nhn nhanh invitro nhn mu sch.

M phn sinh (meristem)2.1. Nguyn l ca k thut nui cy nh sinh trng meristem:

Virus sng k sinh v tn ti trong mi t bo sng, tuy nhin nhng nghin cuca White (1934), Limasset v Cornuet (1950) v Martin (1952) cho thy nhng tbo cng gn nh sinh trng th cng cha t virus hn.Gi thit v s khng tn ti virus meristem:The Mathews, Wang et al Hu a ra gi thuyt v s khng tn ti ca virus meristem nh sau:

Virus vn chuyn trong cy nh h thng dn, tuy nhin h thng ny khng ctrong m phn sinh nh

Trong s phn chia, cc t bo phn sinh nh khng cho php sao chp cc thngtin di truyn ca virus

H thng v hiu ho vng meristem mnh hn cc vng khc trong cy

Nng auxin, cytokinin cao nh sinh trng c th ngn cn qu trnh saochp thng tin ca virus.

2.2. Cc k thut lm sch virus in vitro

Nui cy meristem. Nui cy meristem kt hp x l nhit.

Nui cy meristem kt hp x l ha cht. Vi ghp.2.2.1. Nui cy meristem

Tch chnh xc meristem c kch thc nh hn 0,3 mm, nui cy chng trn mitrng dinh dng ph hp ti sinh thnh cy nguyn vn. Sau kim tra schvirus cy ti sinh bng cc phng php khc nhau thu nhn c cy sch bnhvirus.


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S to cy bng nui cy meristemTch meristem2.2.2. Nui cy meristem kt hp vi x l nhit

C s: nhit cao 36 370C th mt s loi virus khng c kh nng nhn ln.

u im: Bin php ny cho php c th tch meristem kch thc ln hn (0,5 1mm) gip cho vic tch v ti sinh cy thun li hn so vi phng php tch kchthc 0,3 0,5mm.S lm sch bnh virus cho cy khoai ty bng nui cy meristem kt hp vi x lnhit2.2.3. Nui cy meristem kt hp vi x l ha cht

C th nui cy meristem vi kch thc ln (0,5 1 mm) kt hp vi vic bsung vo mi trng nui cy cc cht khng virus to mi trng sch bnh.

Cht khng virus nh: 2-thiouracil, ribavirin, vidarabin lm tng kh nng khngca t bo, m thc vt v c ch s nhn bn ca virus.

Phng php ny c hiu qu cao trong vic to cy sch bnh thuc l, khoaity, loa kn.2-thiouracil

l dn xut ca pirimidin

c tc dng lm ri lon s tng hp bnh thng ca cc axt nucleic ca virusRibavirin

c cu to tng t adenosine, guanosine

gy t bin trong qu trnh ti bn RNA ca virus

Ribavirin 5'-mono(di, tri) phosphate c ch RNA polymerase ca virus

Vidarabine c hot tnh chng virus herpes, poxviruses, rhabdoviruses, hepadnarviruses

can thip vo s tng hp ca DNA ca virus

tng t nucleoside, c phosphoryl ho to ara-ATP cnh tranh vi dATP trongqu trnh tng hp DNA ca virus

2.2.4. Vi ghp- L k thut ghp cc m phn sinh nh ln cy gc sch v khng bnh trong iu kinin vitro sn xut cy sch virus.- K thut ny thng s dng vi cc cy thn g, c bit l h cam, chanh v meristem

ca chng khng th sinh trng v ti sinh chi khi nui cy trc tip trn mi trngnhn to.2.3. Nhn nhanh v duy tr tnh trng sch bnh.1. Cy trng nn c trng trong nh cch ly (nh mn, nh knh) khng cha cc ngunbnh v cc vector truyn bnh hay t nhin khng c cc mang virus.2. Cn loi tr thng xuyn cc ngun ly nhim, cn phng tr cc vt mang bnh (cntrng, tuyn trng).2.3. Nhn nhanh v duy tr tnh trng sch bnh.


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3. Phng trnh s ly nhim c gii trong qu trnh chm sc cy trng4. Thng xuyn chn lc, c th bng mt v qua cc phn tch chn on5. hon ton chc chn, nn duy tr vt liu sch bnh invitro.3. Cc phng php chn on bnh virus

Chn on bng mt.

Phng php chn on bng knh hin vi in t. Chn on bng cy ch th. Phng php huyt thanh. Phng php knh hin vi in t. Phng php ELISA. Phng php phn tch DNA.3.1. Phng php chun on bng mt

y l cc phng php chn on da trn cc triu chng bnh ca cy.

u im:n gin, t tn km.

Nhc im: Phi c kinh nghim khng d ln vi cc triu chng khc khng phi l bnh Phng php ny gp kh khn khi cy b nhim t hp virus. Ph thuc vo ngoi cnh, tui cy, c im ca ging.3.2. Chun on bng cy ch th

Cy ch th l cy khi b nhim virus s xut hin cc triu chng c trng.Phng php ny c tin hnh t nhng nm 1940.

Phng php th bng cy ch th lun c coi l phng php xc nh u tin

v cng l phng php nhy cm nht, tuy nhin kt qu xt nghim cng cn ph thuccc yu t khc na.3.2. Chun on bng cy ch th

u im:Nhy v chnh xc c th pht hin nng virus thp.

Nhc im: Thi gian chn on di, cn qu trnh bnh. Ph thuc vo iu kin th nghim. Phng php ly nhim phc tp. Tn cng chm sc cy ch th, mi gii truyn bnh, chi ph u t cao. Mt s bnh virus khng tin hnh c3.3. Chun on bng knh hin vi

Knh hin vi in t mang li nhng kt qu ng tin cy i vi xt nghim hnglot.

u im: Chnh xc, tin cy. n gin, nhanh chng.


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Nhc im: Chi ph cao s lng mu hn ch. Loi virus c chng minh cng ch l loi hnh a v hnh si. Nu virus tnti dng cu th rt kh pht hin v n kh ging cc c quan t ca t bo thc vt bnhthng.3.4. Phng php huyt thanh u im : Tnh c hiu cao xc minh nhanh s tn ti ca virus v phn loi chng. Ktqu thu c chm nht l sau 48 gi. Chi ph cho xt nghim thp. chnh xc cao. Nhc im: Cha sn xut c khng th i vi tt c cc loi virus v k c khi c huytthanh ri cng cha c th ni rng kt qu xt nghim hon ton bo m. Khng th xc minh c c tnh gy bnh ca tng loi virus i vi thc vt

ch C nhiu phng php huyt thanh khc nhau c ng dng phng php ktta git, xt nghim khuych tn agar gel hai chiu, xt nghim latex,xt nghim mindch hng tm...3.5. Phng php ELISA ( Enzymed linked immuno sorbent assay)

Enguall-Permann xut nm 1971-1972 cho lnh vc nhn y. Clarkm, Adams, Barbara (1976) xut cho chun on bnh virus thc vt.

Nguyn l chung:Da vo phn ng khng nguyn khng th nhng y khng th c lin kt vi

mt enzyme (enzymed linked). Phc khng nguyn khng th - enzyme d dng nhnbit mu do chnh enzyme xc tc.3.5. Phng php ELISA

Enzym thng dng l phosphataza kim. C cht ca phn ng l 4- nitrophenylphophat. Khi b tch phosphat s thnh -nitro phenol c mu vng.

u im:- Mi enzyme xc tc cho hng ngn phn t c cht nn tn hiu c khuch irt r.

- C th nh tnh, nh lng.

3.6. Phng php phn tch ADN

Xc nh trc tip s c mt li ARN (hoc ADN) ca virus. Phng php ny cho kt qu chnh xc nhng t tin, yu cu phi c trnh k thut cao.

S nguyn l:


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Chit tch ARN virus cDNA Phn ng lai vi ARN virus Nhn bit khi cDNAgn phn x hoc enzyme.4. ng dng thc t ca k thut nui cy meristem Nhn nhanh ging khoai ty sch bnh: cc cn b thuc b mn Sinh l Thc vtca Vin Sinh hc Nng nghip, trng H Nng nghip H Ni nghin cu hon thin

sn xut khoai ty ging sch bnh. Din tch trng khoai ty nc ta c thi gian t trn 11 vn4. ng dng thc t ca k thut nui cy meristem To ging hu sch bnh: u thng 12/2006, mt ging hu sch bnh t phngth nghim ca trng i hc Cn Th cho hoa m bo cht lng.4. ng dng thc t ca k thut nui cy meristem4. ng dng thc t ca k thut nui cy meristem5. Quy trnh to cy c mi sch bnh t nui cy meristem1. To cy u dng sch bnh (cy So) bng k thut vi ghp nh sinh trng:

Nguyn tc: da vo c im ly lan ca bnh vng l greening (VLG) v ccbnh virus, viroid ca cy c mi:

- Bnh ch lan truyn trong cy theo mch dn.- Bnh khng ly lan qua ht.5. Quy trnh to cy c mi sch bnh t nui cy meristem2. Chun b gc ghp ln 1:- X l ht gieo lm cy gc ghp: ly ht ca cc ging cam 3 l v bi chua,c bc sch v v kh trng bng dung dch Javel 1% trong 5 pht.- Ht c gieo trn mi trng thch c cha cht dinh dng (mi trng MS) trong ng nghim v t trong bung ti nhit 28oC.- Tiu chun cy gc ghp: chiu cao 10 - 12 cm, ng knh thn 1,5 - 2 mm.5. Quy trnh to cy c mi sch bnh t nui cy meristem3. Chun b nh sinh trng:

nh sinh trng c ly t cc chi non ca nhng cy ging gc ctuyn chn trn vn sn xut hay trong nh li cch ly. Sau khi thu chi non, ta nhngl to xung quanh, ch gi li phn ngn ca chi di khong 1 - 1,5 cm.5. Quy trnh to cy c mi sch bnh t nui cy meristem4. K thut vi ghp ( ghp ln 1):- Cy ging cn lm sch. Ta l xung quanh. Ct nh sinh trng. V tr t nhsinh trng bnh. Sau 10 ngy ta l, gc ghp cc chi non mc ra.- Cy gc ghp 15 ngy tui c ly ra khi ng nghim, ct ngn pha trncch c r 2 - 2,5 cm; r cc cng c ct bt ch li 4 - 5 cm.- Dng dao vi ghp v knh lp soi ni m ming ghp trn gc ghp, phi thntrng tng sinh g khng b tn thng.5. Quy trnh to cy c mi sch bnh t nui cy meristem- nh sinh trng l phn m phn sinh di khong 100 - 150 nm, dng dao limng ct v t nhanh vo v tr ghp trn gc ghp.- Cy con sau vi ghp c t trong ng nghim c sn mi trng lng (mitrng MS + ng saccaro). Cy c bo qun nhit 28oC, cng nh sng1000 lux trong 16 gi hng ngy bng n hunh quang.- Sau 1 tun dng knh lp kim tra xem chi ghp c sng khng. Nu chighp sng, ch 1 thng sau c thm 2 l mi, t tiu chun ghp ln th 2.


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5. Quy trnh to cy c mi sch bnh t nui cy meristem5. K thut ghp ln 2:- Sau khi ghp ln 2, cy c bao chm ti nilon khong 3 tun. Nu cy ghp sng,chuyn cy ra chu to v bo qun trong nh li chng cn trng.

- Nhng cy sau vi ghp c chm sc y v c gim nh bnh bng kthut PCR, ELISA.III. Kt lun:

Meristem vi nhng u im ca n c cc nh khoa hc p dng vo thctin sn xut.

Trn thc t to c nhiu cy sch bnh virus nhm tng nng sut v chtlng sn phm

ti: Nhn ging v tnh invitroKhi nim v nhn ging invitro

Nui cy m t bo thc vt l phm tr chung cho tt c cc loi nguyn liu

nui cy thc vt hon ton sch vi sinh vt trn mi trng nhn to, trong iu kin vtrng.

Nhn ging v tnh invitro l qu trnh sn xut mt lng ln cy hon chnh tcc b phn c quan nh: Chi, mt ng, vyc, thn l ca cy m ban u thng quak thut nc in vitro.2. Tnh cp thit ca phng php nhn ging v tnh in vitro:

T xa xa ng cha ta bi t nhn gi ng cy tr ng b ng phng php v tnhtruy n th ng gim, chi t, ghp,

Nhng cc bin php ngy cng bc l nhiu hn ch : ging c nhn nhiu

ln b thoi ha ging, mc nhiu bnh hn, nng sut gim, phn ly Bng bin php nhn ging invitro, vi cng ngh v trng cao khc phcc nhng hn ch trn, nng cao nng sut cht lng ca ging cy trng c v gi trnng hc v gi tr thng phm.

V vy vic nghin cu, pht trin nhn ging invitro ngy nay l rt quan trngcn c ch trng u t.3. u im ca phng php nhn ging invitro

C h s nhn rt cao

C th nhn ging quy m cng nghip, c bit i vi cc i tng kh nhn

bng cc phng php thng thng.

Ch ng ging trong sn xut

To ra cc ging cy trng sch bnh

VD: khoai ty, da4. Nhc im ca phng php invitro


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Chi ph cao hn so vi cc phng php nhn ging v tnh khc nn kh cnhtranh.

Mt s cy rt d b bin d khi nhn ging invitro.

Nhn ging invitro khng th p dng trn tt c cc i tng.II. C s khoa hc

Da vo tnh ton nng ca t bo thc vt. Theo quan im ca sinh hc hin i: tnh ton nng ca t bo thc vt t bo lt bo cha y lng thng tin di truyn cn thit ca c th thc vt.

S hnh thnh v pht trin ca phng php nui cy m t bo thc vt l ktqu ca nhng pht hin sau y trong lnh vc sinh l thc vt v di truyn hc phnt:

1. Tnh ton th (potipotency) ca m v t bo thc vt cho php ti sinh ccy hon chnh t m, thm ch t mt t bo nui tch ri; 2. Kh nng to cc cy n bi qua nui cy ti phn, t to cc dng nghp t tuyt i v nh rt ngn c chu trnh lai to;

3. Kh nng hp thu ADN ngoi lai vo t bo thc vt v kh nngchuyn gen gy bin i (transformation) thc vt do ADN ngoi lai nh cngngh gen (genetic engineering).

4. Kh nng nui cy cc t bo thc vt nh nui cy vi sinh vt dn n khnng ng dng di truyn phn t vo thc vt bc cao phc v cho cng tc toging;

5. K thut nui cy protoplast v kh nng dung hp protoplast, ti sinh cyhon chnh t protoplast lai (cybrid); 6. Kh nng loi tr virus bng phng php nui cy nh sinh trng, to ccdng v tnh sch bnh cc cy nhn ging v tnh.

7. Kh nng dng chi nch, cc th chi protocorm vo nhn ging v tnhvi tc cc nhanh mt s cy trng;

8. Kh nng s dng phng php nui cy phi khc phc hin tng btth khi lai xa;

9. Kh nng bo qun cc ngun gen bng nui cy trong ng nghim; 10. Kh nng tn tr cc t bo thc vt sng trong thi gian di v nhit thp m khng mt tnh ton th ca t bo.

III. CC BC THC HINBc o: chn lc v chun b cy m

Cy m phi l cy s

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