Ch :

NUI CY M T BO THC VT TRONG IN VITRO

Lch s nui cy m t bo

Giai on khi xng( 1898- 1930):

Haberlandt(1902) l ngi u tin xut phng php nui cy m t bo thc vt chng minh tnh ton nng ca t bo da trn thuyt t bo ca Schleiden-Schwann

Giai on nghin cu sinh l(1930-1950):

Bt u vi cng trnh ca White(1934) nui cy thnh cng r c chua trn mi trng lng cha mui khong, glucose, dch chit nm men 1935 Thimann pht hin ra auxin(IAA) trong m thc vt. Nhiu nh nghin cu s dng IAA cng cc vitamin b sung vo mi trng nui cy thu c kt qu tt. Nhng nm 1940 nhiu cht iu ho sinh trng thuc nhm IAA c tng hp thnh cng v c s dng nhiu trong nui cy kt qu cho thy cht ny c tc dng kch thch to m so, phn chia t bo

Giai on nghin cu pht sinh hnh thi(1950-1960)

1954-1955 Skoog pht hin kinentin c tc dng kch thch s phn chia t bo.
1956 Skoog v Miller tm hiu nh hng ca t l Auxin/Cytokinin trong mi trng nui cy n s hnh thnh c quan v to c chi t l cy thuc l 1960 Bergman ti sinh t bo n thuc l trong mi trng lng

Giai on trin khai nui cy m t bo vo cng ngh sinh hc thc vt(1960 n nay)
1960 Cooking dng enzym cellulase phn hu v cellulose ca t bo thc vt thu c t bo khng v gi l t bo trn

1968 Nakata v Tanaka to c cy thuc l n bi bng cch nui cy bao phn

T 1977 Melchers dung hp t bo trn gia khoai ty v c chua thnh cng to ra cy lai khoai ty-c chua T nm 1980 hng lot nghin cu trong lnh vc cng ngh sinh hc c cng b Ngy nay nui cy m t bo khng nhng l c s quan trng ca cng ngh sinh hc hin i m cn l cng c quan trong trong chon to nhn ging, ng gp c s ly lun mi cho sinh hc hin i

KHI NIM

Nui cy m- t bo thc vt l k thut cho php nui cy d dng nhng t bo thc vt hay m phn sinh sch bnh trong mi trng nhn to thch hp to ra nhng khi t bo hay nhng cy hon chnh trong ng nghim.

C S KHOA HC CNG NGH NUI CY M- T BO Tnh ton nng :

- T

bo bt k ca c th simh vt no cng mang ton b lng thng tin di truyn cn thit v ca sinh vt ,khi gp iu kin thch hp mi t bo u c th pht trin thnh c th hon chnh

 Ty tng t bo, tng loi m, tng thi k sinh trng, pht trin m cc gen ph hp hot ng; cc gen khng cng hot ng nh nhau trong cc giai on pht trin ca c th (do c ch iu ha hot ng ca gen).

S PHN CHIA, PHN HO, PHN PHN HO CA CNG NGH NUI CY M-T BO Phn ha: 1 t bo,1khi t bophn ha to m c quan h c quan. Phn phn ha: khi cc t bo phn ha thnh cc m chc nng ring bit nhng vn c th quay v trng thi chc nng phi sinh ban u khi gp iu kin thun li.

S tr ho
-

Kh nng ra chi,r cc thnh phn khc nhau l rt khc nhau

- Trong nui cy invitro cc b phn non tr s ra chi,r tt hn cc b phn trng thnh

CC NHN T M BO THNH CNG TRONG CNG NGH NUI CY M- T BO THC VT:

m bo iu kin v trng, phng th nghim phi chuyn ha cao. Chn ng mi trng v chun b mi trng ng cch. Chn m cy, x l m cy thch hp trc v sau khi cy.

Cc iu kin cn thit ca nui cy m - t bo thc vt.

Phng th nghim. B tr theo s : Phng chun b v gi mi trng Phng cy Phng nui cy

Mi trng vt l.
-nh sng: Bao gm: + Cht lng nh sng + Thi gian chiu sng + Cng chiu sng -Nhit : Thch hp nht l 25 0C.

Mi trng ho hc
Hu ht cc loi mi trng nui cy m v t bo thc vt u bao gm: Cc loi mui khong a lng v vi lng, ngun cacbon, vitamin, cc cht iu ho sinh trng, cc nhm cht b sung, cht n

Cc nguyn t khong a lng.

-Nit (N): dng NO-3 v NH+4 ring r hoc phi hp vi nhau -Phtpho (P): thng dng ortophotphat hoc ng phtpho. -Lu hunh (S): dng mui SO4-2 vi nng thp -Cc ion Na+ v Cl- cn nng thp

Cc nguyn t khong vi lng.

-St (Fe): thiu st t bo mt kh nng phn chia -Mangan (Mn): Thiu Mn km phn bo. - Bo (B): Thiu B trong mi trng gy nn biu hin nh tha auxin v thc t Bo lm cho cc cht c ch auxin oxydase trong t bo gim. M nui cy c biu hin m so ho mnh, nhng thng l loi m so xp, mng nc, km ti sinh.

Ngun cacbon
Cy in vitro sng ch yu theo phng thc d dng, mc d di nh sng nhn to chng c kh nng hnh thnh dip lc v quang hp nhng b hn ch cho nn vic a vo mi trng nui cy ngun cc bon di dng hu c l bt buc, gip cho t bo phn chia v tng sinh khi. Tu thuc mc ch v s i hi ca tng h m c th dng cc loi ng khc nhau: Sucrose, Maltose, Glucose, Galactose v Lactose. Hm lng ng b sung vo mi trng nui cy thng l 20 - 40 g/l.

Cc vitamin.
Cc loi m v t bo thc vt nui cy in vitro u c kh nng t tng hp c hu ht cc loi vitamin, nhng thng khng v lng nn phi b sung thm t bn ngoi vo, c bit l cc vitamin nhm B. - Vitamin B1 (thiamin) ng vai tr quan trng trong qu trnh bin i cacbon v tham gia vo thnh phn t hp enzym xc tc qu trnh oxy ho kh cacbon axit hu c. Nng thng dng t 0,1- 10 mg/l. - Vitamin B6 (piridoxin) tham gia vo thnh phn cc enzym kh cacbon v thay i v tr nhm amin trong cc axitamin. Nng dng t 0,1 - 1 mg/l. -Myo - inositol cn c b sung mt lng kh ln t 50 - 500 mg/l v t ra c tc dng rt r n s phn chia ca m.

-Cc cht b sung:

Bao gm nc da,dch chit nm men,cht n

Cc cht iu ho sinh trng

Cht iu ho sinh trng gm c cc phytohormone v cc cht iu chnh sinh trng tng hp nhn to. Phytohormone l cc cht ho hc c thc vt tng hp vi mt lng rt nh cc b phn, c quan nht nh v c vn chuyn n cc c quan, b phn khc iu ho cc qu trnh sinh l trong cy, m bo mi quan h hi ho gia cc c quan, b phn.

Auxin

Auxin - hormone kch thch thc vt sinh trng

Auxin c nh ngha l cht c hot tnh sinh hc ging vi IAA:
kch thch ko di cc t bo l bao mm v lt ct thn cy tch ri. cng vi cytokinin kch thch s phn chia ca cc t bo m so (callus culture). kch thch hnh thnh r t l v thn cy tch ri. kch thch mt s hin tng sinh trng v pht trin khc tng t nh hot tnh ca IAA.

Kch thch s hnh thnh,s sinh trng ca qu v to qa khng ht Gy ra tnh hng ng ca cy,km hm s rng l,hoa,qu

Trong cy c tng hp m non, c bit l ang pht trin v vng nh sinh trng

Gibberellin - cht iu ho phn chia t bo thc vt

Hin xc nh c 136 GA, chng c cu trc ho hc khc nhau song c u c b khung gibberellase GA c t tn theo th t pht hin: GA1, GA2,..., GAn, trong GA3 l loi c phn tch cu trc u tin, y cng l loi GA c hot tnh mnh v c ng dng rng ri.

Trao i gibberellin
Tng hp : Cc GA bn cht l cc acid diterpene c tng hp bng con ng terpenoid trong lp bo, sau c bin i ho hc trong li ni cht v t bo cht cho ti khi hon thin cu trc tr thnh dng hot ng GA c tng hp
Trong phi ang sinh trng, l non, r non, qu non; Ch yu trong lc lp

Vn chuyn khng phn cc trong xylem v phloem hay gia cc t bo cnh nhau C th lin kt vi ng (thng l glucose) tr thnh dng khng hot ng GA kh bn vng trong cy, t b phn hu

C ch
S sinh trng ko di ca thn:
GA kch thch s ko di ca t bo (khng bng c ch sinh trng acid nh auxin), tuy nhin trong thc t GA lun xut hin cng auxin c th tc dng ko di ca GA ph thuc vo auxin.
GA kch thch s phn chia t bo bng cch khi ng mt s gene CDK (cyclin-dependent protein kinase) c vai tr trong iu ho chu trnh t bo (chuyn t bo t pha G1 sang pha S).

Hiu qu sinh l
Kch thch s sinh trng ko di ca thn (c xc nh do vai tr ca GA1)

Sinh trng cc t bin ln (thiu gene chu trch nhim tng hp enzyme trong con ng tng hp GA)

Hiu qu sinh l
Kch thch s vn di ca cc ging cy h la

Kch thch s ny mm ca ht, c

Hiu qu sinh l

Kch thch s ra hoa c ch pht trin hoa ci, kch thch pht trin hoa c

Cytokinin - cht iu ho phn chia t bo thc vt

Cytokinin c nh ngha l nhng cht ho hc c hot tnh ging vi trans-zeatin

kch thch phn chia t bo m so kch thch hnh thnh r/chi trong m so lm chm qu trnh gi ha ca l kch thch pht trin ca l mm

 V cu trc, a s cytokinin c dng purine c thay th N ti v tr C6

Zeatin

cytokinin l nhng dn xut t tARN thc vt, chng c tng hp trong h r, ri c vn chuyn khng phn cc trong xylem ln ngn. Ngoi ra chng cn c tng hp chi, l non, qu non, tng pht sinh
cytokinin trong thc vt c th dng t do hoc lin kt, chng nhanh chng b chuyn ho nh cc enzyme oxidase thnh adenine ri theo con ng chuyn ha adenin ti s dng trong c th hoc phn gii thnh sn phm cui cng l urea.

Cytokinin c b sung vo mi trng ch yu kch thch s phn chia t bo v quyt nh s phn ho chi bt nh t m so v c quan Tc ng phi hp ca Auxin v Cytokinin c tc dng quyt nh s phn ho ca m theo hng to r

iu ho s phn ha c quan

auxin/cytokinin cao kch thch ra r auxin/cytokinin thp kch thch ny chi

Quy trnh tng qut cng ngh nui cy m t bo thc vt in-vitro

1. Quy trnh
Bc 1
Bc 2

Chn vt liu nui cy

Kh trng

Bc 3
Bc 4 Bc 5 Bc 6

To chi
To r Cy cy vo mi trng thch ng Trng cy trong vn m

S : quy trnh nhn ging bng nui cy m TB

Bc 1: Chn vt liu nui cy - nh chi, nh r : l b phn non, d

tham gia vo qu trnh phn ho v phn phn ho to nn c th mi.Thng t nhim bnh.

Bc 2 : Kh trng

Bung kh trng

Bc 2 : Kh trng
- Mu v dng c c ty ra kh trng.
- Ct nh sinh trng thnh phn t nh, ty ra, kh trng bung v trng. VD: - Vt liu nui cy tin kh trng vi HgCl 0,1% v nc ct. - Que cy, ng nghim v gi thc kh trng trong ni hp.

Bc 3 : To chi

Khi callus chun b to cy con Cy con mi ang hnh thnh

Bc 3 : To chi
- cy c th pht trin thn cnh - Cm vt liu nui cy trong mi trng dinh dng c b sung thm xytokinin hot ho to chi.

Bc 4 : To r
- Khi cy t tiu chun v chiu cao, s l th chuyn sang MT to r. l MT dinh dng thch hp b sung cht KT auxin, IBA

To r

Bc 5 :Cy cy vo mi trng thch ng

Bc 5 :Cy cy vo mi trng thch ng

- Chuyn cy sang MT thch ng gn ging vi
MT t nhin v: nhit , m, nh sng... - Khng th b qua bc ny c v cy mi to ra rt yu. Nu trng trc tip vo MT t nhin cy s d nhim bnh v cht.

Bc 6: Trng cy trong vn m
- Cho cy thch ng vi MTSX.
- Khi cy chiu cao, thn l th chuyn cy ra vn m nhn nhanh SX.

Quy trnh nhn ging Lan bng nui cy m in-vitro

Cc phng thc nui cy m t bo thc vt

Nui cy m thc vt trn mi trng cng Nui cy huyn ph t bo

Nui cy m t bo thc vt trn mi trng cng

Ti sinh chi trc tip t mu m nui cy
Vt liu nui cy: Ch yu l nhng khi m v c quan tch ri:m phn sinh, nh sinh trng,mnh l,.. Cc bc tin hnh: + La chn vt liu + Kh trng vt liu nui cy + Cy vo mi trng mu + Nhn nhanh chi + Chuyn sang mi trng to r,to cy hon chnh

Ti sinh chi gin tip qua giai on m so(callus)

Bao gm cc bc: +La chn vt liu +Kh trng vt liu nui cy +Vo mu mi trng: Khng bt chi trc tip m to m so hoc protocom Nhn nhanh m so hoc protocom Cho m so hoc protocom ti sinh to chi +Nhn nhanh chi +To r to cy hon chnh +a cy ra mi trng ngoi

Nui cy huyn ph t bo
Nui cy dch huyn ph t bo cha cc t bo v cc khi t bo, sinh trng phn tn trong mi trng lng Thng c khi u bng cch t cc khi m callus d v vn trong mi trng lng chuyn ng (lc hoc khuy). Nui cy dch huyn ph v th l s tin trin t thc vt n mu vt, ti callus, v cui cng ti dch huyn ph. Nui cy dch huyn ph thch hp hn cho vic sn xut sinh khi ca t bo thc vt so vi nui cy callus, do nui cy dch huyn ph c th duy tr v c thao tc tng t vi cc h thng ln men vi sinh vt c ngp chm trong mi trng lng.

Cc phng php nui cy huyn ph t bo

+Nui cy dch th tnh: Ch p dng vi 1 s trng hp nghin cu v 1 s th nghim + Nui cy dch th ng: Mu nui cy c th trc tip vo mi trng dch th,song phi c bin php sc kh vo mi trng.Phng php ny ch yu to m so v phi soma C 2 phng php: * Chm lin tc ( tun hon ): Mu nui cy lc no cng chm trong dch th,phi dng thit b khuy t * Chm khng lin tc ( khng tun hon ) : S dng h thng bp bnh

Xc nh tc sinh trng t bo
+Xc nh s lng t bo: m di knh hin vi
*Ly 1 th tch dch th nht nh c cha t bo * Tch ring r cc t bo bng dd cloromium trioxide ( 5-10% W/v ), ng thi un nng 700C trong 5-10 gy ra s phn tch cc t bo nhng cha lm phn hu t bo,sau lm ngui v lc mnh * Pha long huyn ph t bo n nng ph hp,nhum v m trong bung m t bo T tnh c s t bo trong 1ml dd cy + Xc nh th tch t bo: Ly ngu nhin 1 lng th tch nui cy dd huyn ph sau em ly tm (thng 200 vng/p ) trong 5,thu ly t bo ri o th tch ca tng khi t bo +Xc nh khi lng t bo kh,ti : ng dng thu cht th cp

 ngha khoa hc
- To ra 1 qun th cy con ng u gi nguyn c tnh ca cy m

- H s nhn nhanh cao

- Rt ngn thi gian a ging vo sn xut v pht huy c hiu qu kinh t

- Nhn c s lng cy ln trong 1 din tch nh

- m bo cc cy ging sch bnh - Cy con c to ra nui cy m c tr ho cao - Thun tin v h gi thnh vn chuyn - Bo qun cy ging thun li

ng dng trong thc tin

- Nhn cy ging,to cy ging - Sn xut cht th cp + Cht th cp ng dng trong cng nghip thc phm: cht to mi,mu,cht ngt + Cht th cp ng dng trong dc phm: ankanoid,steroid,cc cht ring l khc ( ubiquinon,saponin ),cc protein ti t hp

Mt s ng dng trong thc tin

Lm sch virut qua nui cy m phn sinh nh
*Nguyn l: - Nng virut gim dn b phn gn nh sinh trng do: + virut khng vn chuyn c trong m phn sinh nh + Cc t bo m phn sinh nh khng cho php s sao chp thng tin di truyn cua virut

+ H thng v hiu ho virut m phn sinh nh mnh hn cc vng khc

+ Nng auxin cao ngn cn qu trnh sao chp ca virut

*Cc k thut lm sch virut in vitro

+ Nui cy m phn sinh nh : Bao gm cc phng php: - Nui cy m phn snh nh kt hp vi x l nhit cao

- Nui cy m phn snh nh kt hp vi x l ho cht

- K thut vi ghp

Chn dng bin d soma

C s chn dng bin d soma: l ch tt c cc bin d th
Gm 2 nhm chnh: Bin d kiu gen v bin d kiu hnh hin cc t bo,m nui cy,cy c ngun gc t nui cy m t bo

Cc nguyn nhn gy bin d dng soma

+ S a dng di truyn t nhin ca cc t bo nui cy

+ Phng thc nhn ging in vitro

+ Loi v nng cht iu ho sinh trng s dng + Thi gian nui cy v s ln cy chuyn

Phng php chn dng bin d soma

+ Chn trc tip + Chn gin tip

+ Chn tng th