TRNG I HC NNG LM HU D N HP TC VIT NAM H LAN

BI GING

NG DNG CNG NGH SINH HC TRONG CHN GING

Ngi bin son: TS. L Tin Dng

Hu, 08/2009

Chng I CNG NGH SINH HC V CY TRNG I. KHI NIM CHUNG CNG NGH SINH HC Khong t nhng nm 1970 tr li y, ngi ta hay bt gp thut ng "Biotechnologie". Thut ng ny c hnh thnh t ting La tinh - "Bios", sinh hc v "Technology"- cng ngh. S ra i ca cng ngh sinh hc (CNSH) th hin s pht trin nhanh chng v tri thc khoa hc, sinh hc v cc ngnh lin quan n sinh hc nh ho hc, vt l hc, ng thi ni ln ngha thc tin to ln ca ngnh sinh hc. Nhiu nguyn l, qu trnh sinh hc mi c khm ph v ng dng trong sn xut, gii quyt nhiu kh khn trong cuc sng ca loi ngi trong nhiu lnh vc. Cc nh khoa hc c nhng kin khc nhau v CNSH. C ngi cho rng n l mt s pht trin tip theo ca cng ngh vi sinh, hoc ca sinh hc phn t v k thut gene. Nhng khi nim u cha hon chnh, khng i din c mt cch tng th v ni dung khoa hc v thc tin ca CNSH. Gn y c ngi a ra mt nh ngha bao qut, khoa hc hn: CNSH l qu trnh nghin cu, khai thc, s dng cc nguyn l v qu trnh sinh hc gii quyt cc vn thc tin sn xut phc v i sng con ngi quy m cng nghip. Xut pht t quan im trn, ngi ta phn bit CNSH lm hai loi: - CNSH c truyn: Khai thc cc nguyn l v qu trnh sinh hc da trn c s nhng hiu bit s ng v kinh nghim sng ca con ngi. Chng ta c th lit k mt s hot ng nh: thun ho, tuyn chn vt nui v cy trng, ln men nu ru, lm bnh m, sa chua, lm tng, dm, mui da v cha, chn on bnh bng phng php y hc c truyn. - CNSH hin i: l qu trnh nghin cu, khai thc cc nguyn l sinh hc trn c s nhng kin thc khoa hc tin tin v bng nhng phng php nghin cu hin i. Kt qu v sn phm ca CNSH hin i c th mang li nhng cuc cch mng, thc y pht trin sn xut, p ng nhu cu cuc sng ca loi ngi. CNSH pht trin qua ba giai on: + Trc ht n c hnh thnh t khi con ngi cha hiu bit nhiu v sinh hc. Bng nhng kinh nghim, quy nh sn xut th cng, h to nn nhng sn phm t vi sinh, ng vt v thc vt. V d: lm a, lm dm, nu ru, lai to ging mi, nhn ging... + Giai on hai: Bt u t cui th k 19 khi con ngi hiu bit v cc qu trnh sinh l, sinh ho, di truyn ca sinh vt v c nhiu kt qu s dng chng phc v nhu cu con ngi. Trc ht s dng vi sinh vt trong sn xut sinh khi, chit xut mt s ho cht nh butanol, aceton v cc loi nm men c li khc, nh lm sch nc cho thnh ph. Cng tc nui cy phn lp cc chng vi sinh mi pht trin. Nhiu quy trnh cng ngh s dng nhng ni ln men vi cng sut ln ra i v to ra nhng sn phm mi nh thuc khng sinh, phn bn, ch phm vi sinh...

+ Giai on ba: T nhng nm 60, 70 tr li y, CNSH pht trin mnh m nh nhng tin b vt bc ca sinh hc phn t, iu ch enzym, k thut gene, cng ngh nui cy m v t bo thc vt.... Ngy nay ngi ta c th khai qut tng di y ni dung cng nh nh phm tr hot ng ca CNSH. N c xy dng da trn nn tng ca s hp cc cng ngh c bn: - Cng ngh vi sinh. - Cng ngh enzyme. - Cng ngh ln men (Fennenlation) - Cng ngh nui cy t bo ng vt v min dch hc - Cng ngh nui cy t bo thc vt. - K thut gene di truyn. CNSH ngy cng pht trin v hon thin, i su thm nhp vo mi lnh vc, ngnh khoa hc khc nhau nh cng nghip m, a cht, ch bin thc phm, y t, mi trng v cc ngnh sn xut nng, lm, ng nghip... Thc s, CNSH ng vai tr ht sc quan trng, to ra nhiu sn phm, p ng c nhu cu pht trin ngy mt a dng v phong ph ca con ngi: Lng thc thc phm: sn phm c, tht, tinh bt, ng ; thc phm b tr, cht mu, hng v; cc loi vitamin v acid amin quan trng. Nng nghip: thc n gia sc, thuc tr su, vi khun, vi rt, ch phm vi sinh, nhn ging v tnh, cy hp t, sn xut phi, vaccin. Ho hc: acid hu c, ru, men, cht cao phn t, metal extraction, bioaccumulation. Dc hc: cc cht khng sinh, cc cht chn on bnh, men c ch, vaccin, steroids, alcaloids. Ln men: bia ru, cht kch thch, lng thc, bnh m, b, protein t bo, cn cng nghip, men, cht khng sinh, thuc, vitamin, vaccin. Nng lng: sinh cht, ethanol, methane. Mi trng: x l cht thi, lm sch nc, tinh ch, phn gii du gy nhim. II. VAI TR CA THC VT Thc vt l cha kho ca cuc sng trn tri t. Thc vt l sinh vt sn xut u tin trong tt c cc chui thc n trong h thng t nhin v l ngun cung cp nng lng v chng c kh nng ti sinh. Thc vt l c th t dng, c nhu cu sinh dng rt n gin, s dng nc, mui khong, CO2 v nng lng nh sng mt tri tng hp cc ng n. y l sn phm sinh tng hp u tin t chng c chuyn ho tng hp cc phc cht khc. ng c s dng ngay hoc d tr a vo cng vi mui v c v nc, tng hp nn nhng i phn t cn thit cho s sinh trng, pht trin v tn ti ca thc vt. Nh vy thc vt c kh nng duy nht chuyn ho nng lng mt tri thnh nng lng sinh hc. Trong s ton b nng lng thc vt chuyn ho

c chng ch li 10% cho mnh, cn 90% c s dng bi ng vt v con ngi, ng thi chng cn cung cp 80% lng protein c trong t nhin (20% c sn xut t ngun sinh vt khc). Theo Simmonds (1976), chng ta c khong 120-130 cy trng chnh, thuc 64 h v 180 ging (genera). R rng ngun cy trng ca chng ta rt a dng v phong ph. Tuy vy chng ch chim mt phn rt nh trong s 300 h v 3000 ging (genera) thc vt tn ti hin nay. S a dng ny phn nh s a dng v nhu cu sn phm ca con ngi. ng thi chng cn c s a dng v v tr a l m chng c thun ho (Simmonds 1989). III. CNG NGH SINH HC V THC TIN CI TO CY TRNG NNG NGHIP Giai on u, con ngi tn ti v pht trin c nh sn bn v hi lm, sau chuyn sang mt dng vn ng cao hn: Trng trt v thun ho vt nui, cy trng. Trong qu trnh , cy trng thay i rt ln. S thay i ny xy ra qua s chn lc c thc theo nhng c tnh c li cho con ngi. Qu trnh thc tin lm cho con ngi v cy trng lin kt cht ch vi nhau, to nn mi quan h mt thit ph thuc ln nhau. T ngy u trng trt cho ti cui th k 19, tt c nhng ci to cy trng l do chnh ngi nng dn thc hin. Vo cui th k 19 n gia th k 20, tc ci tin cy trng din ra cng nhanh. Di y l mt s khm ph sinh hc v nh hng ca n i vi lnh vc ny: S khm ph li nh lut ca Mendel cung cp cho cc nh khoa hc nhng c s khoa hc trong cng tc lai to v lm sng t c ch ca s phn ly v quy lut di truyn cc tnh trng. - Nhng nh lut di truyn ca Mendel, quy lut bin d ca Darwin v nhiu khm ph sinh hc khc gip ngi ta tin on c nhanh v chnh xc cc tnh trng, lm cho cng tc lai to ging tin nhanh v hiu qu hn. - Morgan v cng s xy dng c bn gene rui dm. N gii thch c s lin kt v ti t hp ca cc c tnh. - S thu thp ngun gene ging cy trng, cy thun ho v cy di thnh lp ngn hng gene, cung cp thc liu cho chng trnh lai to. - S pht trin di truyn t bo gip nhng ngi to ging hiu bit v cu trc chc nng v c ch ti t hp. - Khm ph ra colchicine, nh to nhng ging mi bng s a bi ho. - Vai tr ca tia X trong vic to ra nhng bin d mi, ra i cc k thut gy t bin khc l cng c tch cc cho nhng ngi lm cng tc ging. - Nhng pht trin trong di truyn s lng gip cc nh to ging hnh thnh c chin lc chn ging v phn tch tnh n nh ca tnh trng. Nh nhng thnh tu trn, ngi ta c tnh ch trong th k 20 nng sut sn lng cy trng tng 50%.

V d ni bt v s thnh cng ca nhng ngi lm cng tc ging v nh hng ca n n vic cung cp lng thc th gii l cuc cch mng Xanh Bc M 1950-1960. Cng tc nghin cu khoa hc dn n cch mng Xanh c bt u vo nhng nm 1940, nhng thc s cch mng Xanh c nh hng v c ngi ta t tn cho n vo nm 1960, do William S.Gang - ngi M xng. Ni dung nghin cu ca cch mng Xanh l s chuyn giao cng ngh tng hp nhiu mt: Lai to ging, canh tc, ti tiu., phn bn, bo v thc vt t cc nc pht trin sang cc nc ngho - i tng p dng cho cy khoai ty v la m. Trong hai cy sau ny vic chuyn giao sn xut cy la m thu c nhiu thnh qu, c tc dng ln to ra cuc cch mng. Cn cy khoai ty kh p dng trin khai hn. Nm 1947 ging la m ln (Norin dwarf) t Nht Bn c chuyn sang nghin cu M v nm 1954 Borlaug mang n sang Mexico v ng chn to c nhng dng ging la m ln c thi gian sinh trng ngn, thch nghi rng, nng sut cao, trng c hai v/nm, chu nng, c th pht trin c vng nhit i. Nhng nm 1960- 1970, cc ging la m ny pht trin nhanh chng n , Pakistan v sn lng la m y tng gp 2 ln, din tch trng la m ln ti 10 triu hecta. t nm sau (1956) Vin IRRI bt u thc hin chng trnh nghin cu theo m hnh cy la m, p dng cho cy la nc v nm 1962 ngi ta to ra nhng ging la nc thp cy, thi gian sinh trng ngn, c b l ng, kho, cho nng sut cao. Vo nm 1970, nhng ging la ny cng pht trin nhanh, t din tch 10 triu ha Pakistan, n , lndonesia v Trung Quc (ging u tin l IR8). Tip theo, cc ging la c trng v kho st Vit Nam nm 1975, 1976 v ging la lR8 nhanh chng pht huy tc dng hnh thnh thm 1 v la xun min Bc nc ta, cho nng sut ti 3 tn/ha, sau ln 4-5 tn/ha. Hin nay chng ta trng ch yu l cc ging mi thp cy, ngn ngy, nng sut, cht lng ca chng lun lun c ci thin. Vin Nghin cu la Quc t ang chun b a ra nhng ging la mi c kiu hnh c bit gi l "Superrice". Superrice c nhng c im u vit sau (bng 1.1): Bng 1.1. Mt s ging la u vit ni bt Sinh khi Ch s thu Nng sut Thi gian sinh (tn/ha) hoch (tn/ha) trng Modernrice 20 0,5 8 100 120 Supenrice 22 0,6 12 100 120 Localrice 12 0,3 3 160 Ging ny c ngun gc lai to t Indica v Tropical Javania (Kush 1966 cha cng b). Tn ging

Chng II NUI CY M V T BO THC VT I. LCH S PHT TRIN K thut nui cy m v t bo thc vt tri qua gn mt trm nm pht trin. C th tm thi phn chia qu trnh pht trin thnh 4 giai on: 1. Giai on khi xng (1898 - 1930) Bt u t nhng th nghim u tin ca Haberlandt (1898) khi ng xng ra tnh ton th ca t bo v tm cch nui cy t bo phn lp t tng t bo lc, t bo tng nhu m, tng biu b v lng ht ca thc vt chng minh cho lun im ca ng nhng khng thnh cng. Tip theo Haberlandt, cn c mt s phng th nghim khc nh Winkler ( 1902), Thielmann (1924) v Kuster (1928) cng tin hnh th nghim tng t nhng khng nhn c t bo phn chia no. Winkler lc by gi c nhng quan st rt tinh t. ng nhn thy: t bo non trng ln khi ht phn ny mm v ngh nui t bo dinh dng phn lp cng vi ng phn trong mt git treo bt ng phn kch thch t bo dinh dng phn chia. Ngoi ra ng cn ngh b sung vo mi trng dinh dng dch chit t cc phn m dinh dng khc, v d nh dch ly t ti phi v ng tin rng bng con ng c th nui thnh cng mt t bo dinh dng phn lp thnh phi m ng nh gi l phi nhn to (artificial embryo). Phi n nhng nm 30 ca th k 20 ngi ta mi t c nhng tin b thc s: Schmucker (1929), Scheitterer (1931), Pfeiffer (1931, 1933), Larue (1933) thng bo v nui cy thnh cng on u r ring r. Trong mi trng nhn to cc on r ny pht trin thnh nhng chic r hon chnh. y l tin b nh du mt giai on pht trin mi. 2. Giai on nghin cu sinh l (1930 - 1950) Bt u bng thnh cng ca White (1934) nui cy c mt dng r c chua sinh trng mnh v lin tc. Cng nm, Gautheret thng bo thnh cng trong vic nui cy m tch t tng tng (cambium) ca cy Salix apraea v cy Populus nigra. M nui cy lin tc phn chia trong nhiu thng trn mi trng Knop b sung glucose v cysteinhyochloride. Trong thi k ny Went v Thimann (1937) pht hin ra IAA l mt auxin tn ti t nhin trong c th thc vt. IAA c cng nhn l mt loi hormon thc vt, c chc nng nh mt cht iu khin sinh trng tc ng ln qu trnh phn chia t bo v hnh thnh r. IAA lp tc c Gautheret s dng vo mi trng nui cy, kt qu thu c mi ch hn ch m tng tng ca cy Salix.

Tip nm 1938 Nobecourt nhn c phn bo m c c rt Daucus carota. Cng nm 1938, White nui cy c m tng tng ca cy thuc l lai Nicotiana glauca x N.langsdorffi. Vo cui thi k ny c nhng quan st v s phn ho c quan r, l trong m nui cy ca cy c rt hoc cy thuc l lai. Thnh cng quan trng ca thi k ny l xy dng v s dng c kt qu mt s loi mi trng bn nhn to, ng thi pht hin c vai tr ca mt s vitamin m bo s thnh cng i vi vic nui cy c quan (r) v m (tng tng) thc vt. 3. Giai on nghin cu pht sinh hnh thi (1950 - 1960) i din cho giai on ny l Miller, Skoog, Steward, Reinert. Giai on pht sinh hnh thi bt u bng cng trnh ca Camus (1949) ghp chi ln khi m nui cy v thy qu trnh phn ho ng mch xy ra trong khi m. y l tin nghin cu v s iu khin qu trnh phn ho t bo trong khi m nui cy. Tip theo l cng trnh ca Miller v Skoog (1956) to chi thnh cng t m thuc l nui cy. Trong giai on ny, Skoog pht hin ra kinetin l mt cht iu khin qu trnh phn bo (thuc nhm cytokinin) v phn ho mm chi. 1958-1959 Steward Reinert s dng nc da (c cha cc cht nhm cytokinin) vo nui cy t bo c rt (Daucus carota) v thu c phi t nui cy t bo c rt. K thut nui cy t bo n trong dch lng c Muir (1953) xy dng i vi t bo ca cy Tageter erecta v Nicotiana tabacum v Nickell (1956) nui c t bo n ca Phaseolus vulgaris trong dch lng thng qua cy truyn 4 nm lin tc. Nh vy, sau 50 nm gi thit ca Haberlandt c Muir chng minh l ng trong lun vn tin s ca ng, Muir s dng t bo Crown-gall mt loi t bo khi u thc vt v nui cy thnh cng t bo n. 1960 Bergmann pht trin k thut t bo n ln mt bc mi to c khi m so t mt t bo n bng k thut gieo tri t bo thc vt trn a thch (cell platting) nh tri t bo vi sinh vt. 4. Giai on trin khai nui cy m vo cng ngh sinh hc thc vt 1959 Melchers s dng m n bi ca Antirrinum majus nghin cu tnh bin ng mc bi th trong nui cy v gy t bin. 1967 Nitsch, 1968 Nakata v Tanaka to c cy n bi t bao phn thuc l, m ra mt trin vng ng dng n bi vo cng tc ging v nghin cu di truyn. 1960 Cocking tch c t bo trn protoplast.

1964 Guha v Maheswari to c cy c c dc (Datura innoxia) c b nhim sc th n bi t nui cy bao phn. 1968 Niieki v Ono nui cy thnh cng bao phn v to c cy n bi la (Oryza sativa). 1971 Takebe ti sinh c cy thuc l hon chnh t protoplast thuc l ging Xan thi. 1977 Melchers lai sma thnh cng cy c chua v cy khoai ty. 1985 cy thuc l mang gene bin np u tin c cng b. Khi nim transgenic plant tr thnh ph cp trong thut ng cng ngh sinh hc thc vt. 1994 ging c ci ng mang gene khng bnh virus bin np c a vo sn xut i tr ti Nauy. M c hng trm ging cy mang gene bin np c sn xut chp nhn. Th nhng cho n nay nhng quan nim v qui nh v an ton sinh hc ca cc nc ang cn rt khc nhau, v th vic a cy trng mang gene bin np vo sn xut i tr cn ph thuc vo trnh khoa hc v chnh sch ca tng nc. II. YU CU C BN CA K THUT NUI CY M V T BO THC VT 1. Phng th nghim Cn c b tr theo s sau: a) Bung chun b: c b tr lin hon cho cc khu pha ch v bo qun lnh cc dung dch cht khong, vitamin...; pha ch v kh trng (hp nhit v p sut cao) mi trng nui cy; chun b s b mu vt trc khi cy. Cc thit b ti thiu cn thit l cn phn tch (10 -3g), cn k thut (l kg), t ho cht, t lnh c ngn lnh su, my o pH, ni kh trng. b) Bung cy: C th la chn cc phng n sau: Bung v trng iu kin ti thiu l phi kn gi, c ph knh hoc p gch men knh. Hng ngy sau gi lm vic tin hnh tit trng bng: - Dung dch formol 5-10% phun thnh sng m. - Chiu UV (254nm) trong 2-3 gi. Nh th cng ch m bo cho thao tc 2-3 gi, sau li phi kh trng t u. T cy v trng C th t thit k v ng ly theo dng t cy vi sinh, c 2 l lun tay vo thao tc. T cng c kh trng nh bung v trng bng formol v n cc tm. Bn cy v trng Thit b l tng l bn cy v trng lm vic theo nguyn tc lc khng kh v trng qua mng v thi khng kh v trng v pha ngi ngi thao tc. Hin nay

trn th gii c rt nhiu hng sn xut loi bn cy v trng ny dng trong nhiu lnh vc nghin cu , dch v v sn xut nh y dc hc, sinh hc, in t... Gi tu theo cht lng tng loi, nm trong phm vi 1.000 10.000 USD. c) Bung nui cy Nhit Yu cu chnh i vi bung t cc bnh nui cy l phi m bo nhit n nh trong khong 25 oC. C nhng loi cy yu cu nhit cao hn, tu theo m b tr thm nhng t nui c ch nhit khc nhau cho thch hp. nc ta, gi c nhit 25oC nht thit phi s dng my iu ho nhit . Thng thng 1 my cng sut 1,5 KW gi mt cho 15m2 hoc 50m3 bung nui. nh sng Theo ch chiu sng m chia ra thnh: 1. Bung ti lin tc (nui to m so, gi m so). 2. Bung sng theo chu k quang 12-16 gilngy. 3. Bung sng lin tc. Tu theo yu cu c th ca nui cy m b tr vic chiu sng cho thch hp nh sng trn dn nui phi l loi nh sng c ph gn nh nh sng t nhin (230780 nm). Cc loi n hunh quang nh sng trng ni chung u p ng c yu cu trn. Cng c nhng pht hin cho thy nh sng vng cn tm (350-400 nm) c tc dng kch thch sinh trng ca thc vt ni chung, trong c cy nui cy invitro. Cng nh sng trn dn nui cy ti thiu phi t 2000 lux (o c ly 25 - 30cm, tng ng mt y ca bnh nui cy). C hai cch b tr ngun sng: 1. Trc tip trn mi tng ca gi hoc t nui cy. n mc song song vi mt phng cn chiu sng. 2. Bn cnh, n mc vung gc vi mt phng cn chiu sng. Cch ny trnh c hin tng dn nui b t nng do n v nhit lng do n sinh ra c li thot ln trn theo lung kh i lu, nhng cch mc n ny c nhc im l nh sng phn b khng u, cc bnh nui pha ngoi nhn c cng chiu sng yu hn. Gi nui cy c ng bng g hay hn bng kim loi (st gc) thnh khung. Kch thc thng dng ca khung cao 2000 mm, di 1500-2000 mm, rng 450 mm, chia thnh 4 tng, mi tng cao 500 mm. c lt bng knh 4-5mm cho tin lau chi v khng cn nh sng. 2. Mt s thit b chnh dng cho nui cy m v t bo thc vt a) Bnh nui cy Phi c sn xut bng thu tinh trung tnh, c kh nng chu nhit tt o 200 C v p sut 1,5 at.

 ng nghim nui cy: y trn hay phng, kch thc ti u l 24 x 160mm hoc 32 x 160mm. Ming khng c g. a petri: ng knh 70mm, cao 15mm hay ng knh 95mm, cao 15mm. Hin nay a petri cht liu plastic cc chng loi ng knh t 30mm u 150mm ang c s dng thay th cht liu thu tinh. Cc loi a ny thng khng chu nhit, c tit trng bng chiu x v ch dng mt ln. Bnh tam gic: Loi 50, 100 v 250ml, ming rng hoc ming hp. Cc loi bnh nui dch lng: - Bnh cu - Bnh cu chuyn dng c nhiu nhnh ph Hp nui cy: Mt s hng sn xut loi hp (kch thc 75x75xl00 mm) bng nha trong chu nhit c np y chuyn dng nui cy invitro. Dng loi hp ny rt tin li trong khi thao tc cy v chuyn cy ra ngoi t. Hin nay ang lu hnh loi hp nha hnh khi lp phng 100x100x100 mm, ming trn, rng bn hng. Khi nui c th xp chng ln nhau, khng gian v che chn nhau. Trong nhn ging cng nghip cc loi cy rt tin li. Chai nui cy: Mt s loi chai ming rng thng dng trong cng nghip thc phm, c np y xoy bng hp kim nhm khng r hoc gn y ngi ta ci tin dng np nha trong sut v chu nhit c th kh trng. Loi chai ny ch yu c a vo lm bnh nhn ging cy invitro theo qui m cng nghip. b) Nt y Bng khng thm nc l loi nguyn liu lm nt tt nht. nc ta iu kin v sinh phng nui cha cao nn dng giy qun thnh np y ngoi chng bi v chng m. Giy nhm (hay gi nhm l giy bc) l vt liu lm np y ph bin nht hin nay. u im ca loi vt liu ny l khng bt bi, trnh c hin tng nhim trng do nt bng gy ra i vi nhng nui cy dch lng. Vt liu nhm c th h trn la trc tip kh trng trong khi cy. Gn y loi vt liu nha trong sut, chu c nhit cao, c th kh trng ang c s dng nhiu. c) Phng tin v ho cht kh trng - T sy cho dng c thu tinh v dng c sy bo m 160-200 oC. - Ni hp tit trng c kh nng chu 1,2-1,5 at v nhit 120-130oC, s dng cho vic kh trng mi trng nui cy bng hi nc p sut v nhit cao. - Dung dch kh trng ho hc kh trng b mt mu vt s c nui cy, thng dng: Ca-hypochloride, Na-hypochloride, clorua thu ngn (HgCl), nc brm, oxy gi (H2O2), cn kh trng s b v t dng c khi nui cy. - Phu lc v trng loi microspore (0,2 m) dng cho nhng trng hp bng c kh trng bng nhit cao. V d: mi trng nui cy protoplast, dung dch enzym hay dung dch GA3

III. THNH PHN MI TRNG DINH DNG Vo thi k Haberlandt tin hnh cc th nghim nui cy t bo phn lp, nhng hiu bit v nhu cu dinh dng khong ca m v t bo thc vt cn rt hn ch, c bit l vai tr ca cc cht iu ha sinh trng hu nh cha c khm ph. Chnh v vy m Haberlandt khng thnh cng. n nay c hng trm loi mi trng dinh dng nhn to c xy dng v th nghim c kt qu. Hu ht cc loi mi trng u bao gm nhng nhm cht chnh sau y: 1. Cc loi mui khong 2. Ngun carbon 3. Vitamin 4. Cc cht iu ha sinh trng 5. Cc nhm cht b sung 6. Cht n 1. Cc loi mui khong Cc nguyn t khong dng trong mi trng dinh dng nui cy m v t bo thc vt c phn chia thnh 2 nhm theo hm lng s dng: nhm a lng v nhm vi lng. a) Cc nguyn t khong a lng Bao gm cc nguyn t khong c s dng nng trn 30 ppm (phn triu = part per million), tc l trn 30 mg/l. Nhng nguyn t l: N, S, P, K, Mo v Ca. Ring Na v Cl cng c s dng trong mt vi loi mi trng, nhng cha r vai tr ca chng. Nit (N): c s dng hai dng NO3- v NH4+ ring r hoc phi hp vi nhau. Hu ht cc thc vt u c kh nng kh nitrat thnh ammonium thng qua h thng nitrat reductase (NR). Ammonium c t bo thc vt ng ho trc tip sinh tng hp nn cc cht m hu c nh amino acid. iu ng lu l nu ch dng ammonium ( khng c nitrat) th sinh trng ca t bo gim, thm ch ngng hon ton. Nguyn nhn chnh l do qu trnh trao i ln ca t bo xy ra lch dn n tnh trng thay i pH ca mi trng. C th: Khi ch dng nitrat, pH ca mi trng tng dn v khi ch dng ring ammonium, pH ca mi trng gim dn do t bo hp thu NO3- hoc NH4+ v thi ra mi trng loi ln c ho tr tng ng. Khi pH gim th qu trnh trao i Fe ca t bo km i, kt qu l t bo sinh trng chm li. V vy hu ht cc loi mi trng u dng nitrat v ammonium dng phi hp, nhng tu theo c tnh hp thu nh ca loi cy m phi hp theo t l thch hp. Lu hunh (S): Ch yu v tt nht l mui SO42-. Cc dng ion khc nh SO3 hoc SO2 thng km tc dng, thm ch cn c. Phospho (P): M v t bo thc vt nui cy c nhu cu v phospho rt cao. Phospho l mt trong nhng thnh phn cu trc ca phn t acid nucleic. Ngoi ra

10

khi phospho dng H2PO4- v HPO42- cn c tc dng nh mt h thng m (buffer) lm n nh pH ca mi trng trong qu trnh nui cy. b) Cc nguyn t vi lng L nhng nguyn t c s dng nng thp hn 30 ppm. l Fe, B, Mo, Cu, Zn, Ni, Co. St (Fe): Thiu st, t bo mt kh nng phn chia. Th nghim vi st nh du bng ng v phng x 59Fe cho thy Fe c d tr trong nhn rt nhiu. Thiu Fe lm gim lng RNA v gim sinh tng hp protein nhng lm tng lng DNA v amino acid t do. Kt qu l gim phn bo Fe thng to phc hp vi cc thnh phn khc v khi pH mi trng thay i, phc hp ny thng mt kh nng gii phng Fe cho cc nhu cu trao i cht trong t bo. Tt nht l nn s dng Fe dng phc chelat vi citrat hoc vi EDTA (Ethylen Diamin Tetraacetic Acid). T cc phc cht ny Fe c gii phng trong mt phm vi pH kh rng. Mangan (Mn): Thiu Mn cng lm cho hm lng cc amino acid t do v DNA tng ln, nhng lng RNA v sinh tng hp protein gim dn n km phn bo. Bo (B): Thiu B trong mi trng gy nn biu hin nh tha auxin v thc t B 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. Molypden (Mo): L ion ng vai tr co-factor trong h thng nitrat reductase, nh vy Mo tc ng trc tip ln qu trnh trao i m trong t bo thc vt. 2. Ngun cacbon M v t bo thc vt nui cy invitro sng ch yu theo phng thc d dng mc d nhiu trng hp chng c th sng bn d dng nh iu kin nh sng nhn to v lc lp c kh nng quang hp. V vy vic a vo mi trng nui cy ngun cacbon hu c l iu bt buc. Ngun cacbon thng dng nht c kim chng l saccharose. Nng thch hp ph bin l 2-3 %, cng cn ph thuc vo mc ch nui cy m thay i, c khi xung ti 0,2% (chn dng) v tng ln n 12% (nhm gy cm ng stress nc). Tip n l glucose v maltose cng hay c a vo mi trng nui cy (glucose cho nui cy protoplast v maltose cho nui cy bao phn la). Cc loi ng khc nh fructose, raffinose, lactose, galactose cng c th nghim nhng t ra km hiu qu v ch c dng trong nhng trng hp c bit. Cc dng polysaccharide nh tinh bt, pectine, dextrine cng c th dng nui cy. Tuy nhin, nhng loi t bo c nui trn mi trng ch c cha mt trong cc polysaccharide trn nht nh phi th hin kh nng thu phn thng qua cc enzyme nh amylase chng hn. C nhng chng t bo nui cy gii phng ra mi trng cha tinh bt kh nhiu amylase. Chuyn chng ln mi trng ch cha saccharose, lng amylase thi ra gim ngay, nguyn nhn chnh do cc promotor ca gen amylase chu tc ng khng ch ca saccharose.

11

Cc loi ru nh glycerin cng c th c t bo s dng. Manitol hoc sorbitol hon ton trung tnh v khng thm nhp vo bn trong t bo, nhng chng c s dng rng ri trong nui cy huyn ph v nui cy protoplast v chc nng l cht n nh p sut thm thu. Cc loi ru mt ln ri ethanol, methanol t hiu qu, cn propanol v butanol th rt c. Axit hu c thng khng phi l ngun cacbon thch hp cho t bo thc vt nui cy. Th nghim vi folic acid, succinic acid, pyruvic acid v keto glutarc acid ch t 15% sinh trng so vi saccharose. 3. Vitamin Mc d tt c cc loi m v t bo thc vt nui cy invitro c kh nng t tng hp c hu ht cc loi vitamin, nhng thng khng v lng, do phi b sung thm t bn ngoi vo, c bit l cc vitamin thuc nhm B. a) Vitamin B1 (Thiamin): L mt cht b sung rt cn cho mi trng nui cy. Khi kh trng bng cch hp nhit cao, B1 b nhit phn thnh pyrimidin v thiazol l hai cu t ca B1, nhng t bo nui cy c kh nng tng hp chng li thnh phn t B1. V vy khng nht thit phi kh trng bng phng thc khc nh lc. b) Vitamin B2 (Riboflavin): C th tit trng bng phng php nhit, nhng li d b nh sng phn hu. i vi nui cy sng ch dng nng 0,01 ppm, nhng i vi nui cy trong ti c th tng ln 10-50 ppm. c) Vitamin B6 (Pyridoxin, Adernin): L tin cht ca pyridoxalphosphat cofactor ca cc nhm enzym nh carboxylase v transaminase. Khi hp nhit cao, phn ng xy ra: Pyridoxin + Phosphat Pyridoxalphosphat. d) Myo Inositol (Bios I): C vai tr trong sinh tng hp thnh t bo, c th l sinh tng hp polygalacturonic acid v pectine. Inosit l cht bn vng khi kh trng. Thng c s dng nng cao 100 ppm. Khi phn tch thnh phn ca nc da, ngi ta thu c inosit trong mt phn on trung tnh. e) Biotin (Bios II): Cn thit cho phn bo ca mt s loi m. Ch s dng nng rt thp 0,001-0,01. f) Pantothenic acid (Bios III, Vit. B5): c s dng lm thnh phn ca coenzym A. 4. Cc hn hp cht t nhin Cc nh sng lp ca ngnh nui cy m thng s dng mi trng dinh dng rt n gin gm mui khong v ng. Ngy nay ngi ta khng nh rng li mi trng n gin nh vy khng cho t bo sinh trng bnh thng. V vy thnh phn mi trng ngy cng phong ph, y v phc tp hn. Ngi ta s dng mt s hn hp dinh dng t nhin nh: a) Nc da: T 1941 c s dng nui phi ca Datura v 1949 nui ca Daucus. Kt qu phn tch thnh phn ca nc da t non n gi ca Tulecke v ctv (1961) cho thy, trong nc da c:

12

- Amino acid t do: t nng t 190,5 ppm n 685 ppm trong nc da tu theo tui ca qu tnh t non n gi. Khi hp nhit cao ch cn 70 ppm. - Amino acid dng lin kt c trong protein v peptid - Axit hu c - ng - RNA v DNA Ngoi ra nc da cn cha cc hp cht quan trng i vi t bo nui phn lp nh: - Myo Inositol - Cc hp cht c hot tnh auxin - Cc cytokinin dng glycoside b) Dch chit mm la m (mch nha): Thnh phn ho hc cha c phn tch k, ch yu cha mt s ng, vitamin v mt s cht c hot tnh iu khin sinh trng. c) Dch chit nm men (Yeast Extract: YE): Vi dch nm men, White (1934) ln u tin nui thnh cng r c chua trong ng nghim ko di v thi hn. Thnh phn ho hc ca dch nm men t c ch phn tch. Ch yu cha: ng, nucleic acid, amino acid, vitamin, auxin, khong. Tc dng ca YE vi r tt, nhng vi m so khng r rng. d) Dch thy phn casein [Casein Hydrolysae (CH)]: c s dng rng ri trong k thut vi sinh vt, nui cy m t bo thc vt, ch yu c s dng lm ngun b sung acid amin. e) Hn hp amino acid nhn to: Da trn nhng kt qu phn tch cc hn hp cht t nhin trn, nhiu tc gi ra nhng cng thc pha ch hn hp acid nhn to b sung vo mi trng dinh dng. Kt qu s dng cc hn hp ny cn rt khc nhau: C th yu cu acid amin ca tng loi t bo rt khc nhau. Trong mi trng lng nui m so la v mi trng ti sinh cy la t m so prolin l mt thnh phn quan trng. 5. Cc cht iu ha sinh trng Trong mi trng nui cy m v t bo thc vt, thnh phn ph gia quan trng nht quyt nh kt qu nui cy l cc cht iu ha sinh trng. Nhng cht iu ha sinh trng thuc cc nhm sau: a) Auxin c gi l hoc mn sinh trng do Went v Thimann (1937) pht hin, ch yu kch thch sinh trng ca t bo, nhng cng lm tng phn bo. C 4 loi auxin thng c s dng trong nui cy m l: 1. Indolylacetic acid (IAA) tn ti trong t nhin 2. Naphthylacetic acid (NAA) 3. 2,4-Dichlorphenoxyacetic acid (2,4-D) 4. Indolylbutyric acid (IBA)

13

Ring IAA l auxin t nhin, cn li NAA, IBA v 2,4-D l cc auxin nhn to. Thng th cc auxin nhn to c hot tnh mnh hn do c im phn t ca chng nn cc enzym oxy ho auxin (auxinoxidase) khng c tc dng. Kinh nghim s dng auxin trong nui cy m: lc u s dng nng cao, sau thp hn trnh tnh trng m b "say" v nhim c.

Hnh 1: Cu trc phn t cc loi auxin t nhin (IAA, H.5.la) v tng hp (IBA, H.5.1b ; IBA, H.5.1d v 2,4-D, H.5.1c); cc loi cytokinin (KIN,H.5.1e; BAP, H.5.1g; Zeatin, H.5.1f; giberellic acid (GA3, H.5.1h) v absicic acid (H.5.1i) dng trong nui cy m t bo thc vt

14

b, Cytokinin (hormone phn bo): Ln u tin c Skoog (khong 1950) pht hin trong mt th nghim chit xut acid nucleic b s sut. l nhng cu t ca nucleic acid b phn hu thnh. 3 loi cytokinin chnh thng c dng trong nui cy m l: Kinetin l sn phm c pht hin u tin, c cu trc phn t l: 6-(2furfuryl)-aminopurin (H.5.1e). Kinetin c phn lp t ch phm DNA c hoc lnucleic acid mi sau khi hp nhit cao hay un si. Trong c th sng c th khng c kinetin tn ti. Sn phm ny kch thch s pht sinh chi ca cy thuc l nui cy, nhng nu phi hp x l cng auxin t l nng thch hp th s kch thch qu trnh phn chia t bo (do c tn l kinetin). Kinetin thc cht l mt dn xut ca base hu c adenin, nh vy c th coi y l mt cht "nhn to". Trong t nhin c tn ti mt hoc mn phn bo khng? Letham l ngi u tin phn lp tinh ch v cho kt tinh thnh cng hoc mn phn bo t nhin t ni nh ang dng sa ca ht ng. Hp cht cytokinin t nhin c gi l zeatin (zea = ng). Zeatin cng l mt dn xut ca adenin. Cng thc ho hc ca zeatin l: 6(4-hydroxy-3-methylbut-2-enyl) aminopurin (H.5.1f). Trong thc tin nui cy, ngi ta ch dng zeatin trong nhng trng hp c bit v qu t m thng s dng kinetin hoc mt sn phm tng hp nhn to. l: 6-Benzylaminopurin (BAP) Hot lc ca BAP (H.5.1g) cao hn nhiu so vi kinetin v bn thn BAP bn vng hn zeatin di tc ng ca nhit cao. c) Gibberellic acid: c pht hin vo nhng nm 1930. Lch s pht hin nhm hoc mn ny bt u t 1895 khi ngi Nht ni v bnh la von. 1926 th pht hin c bnh l do loi nm Gibberlla fujikuroi gy ra. n nhng nm 30 th mi phn lp v tinh ch c hot cht, c gi l gibberellin. Mi sau Chin tranh th gii th hai, nm 1950 ngi Anh v ngi M mi bit n cng trnh ny ca ngi Nht. Ti nay pht hin c trn 60 loi thuc nhm gibberellic acid. Loi gibberellic acid thng dng nht trong nui cy m l GA3 (H.5.1h). Trong i sng thc vt, gibberellin ng vai tr quan trng i vi nhiu qu trnh sinh l nh: - Sinh l ng ngh ca ht v chi - Pht trin ca hoa - Lm tng sinh trng chiu di ca thc vt Nhng trong nui cy m v t bo thc vt, tc ng ca gibberellic acid cha tht r rng. Nhiu tc gi c s dng v coi l thnh phn khng th thiu ca mt loi mi trng chuyn dng no . d, Absicic acid (ABA)

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ABA thuc nhm cc cht c ch sinh trng c cng thc ho hc nu trong hnh 5.1i. Abscisic acid c tc dng tng cng kh nng chng chu ca t bo thc vt i vi iu kin ngoi cnh bt li, v vy ABA c a vo mi trng ti sinh cy v mang li hiu qu nht nh. 6. Cht n - Thch (Agar) L mt loi polysaccharid thu c t mt s loi to (ch yu l to hng Rhodophyta), trong c rau cu mc vng m ph Vit Nam (Glacia sp.). c s dng lm cht m cho mi trng dinh dng rn li. 80 oc thch ngm nc chuyn sang trng thi sol v 40 oc tr v trng thi gel. Kh nng ngm nc ca thch l 6- 12g thch/l lt nc. 7. Nc Nc pha mi trng cy phi l loi nc hon ton sch ion. Thng thng ngi ta s dng nc ct 2 ln v tt nht l s dng h thng ct nc thu tinh. 8. pH ca mi trng pH ca mi trng dinh dng nh hng trc tip ti qu trnh thu nhn cc cht dinh dng t mi trng vo t bo. V vy i vi mi loi mi trng nht nh v i vi tng trng hp c th ca cc loi cy phi chnh pH ca mi trng v mc n nh ban u. i vi m so ca nhiu loi cy, pH ban u thng l 5,5-6,0. Sau 4 tun nui cy t c 6,0-6,5. c bit khi s dng cc loi ph gia c tnh kim hoc tnh axit cao nh axit amin, vitamin th nht nh phi dng NaOH (dng 1 M Tris cng an ton lm tng hoc HCI long lm gim pH mi trng v 5,5-6,5. Nhng th nghim nui cy t bo n hay t bo trn trng lng mi trng nh th vic chnh pH l bt buc. IV. MT S LOI MI TRNG C BN Thnh phn c bn ca mt s loi mi trng nui cy m v t bo thc vt c trnh by trong bng 5.1. Sau y l mt vi gi mang tnh nguyn tc trong khi s dng cc loi mi trng khc nhau - Mi trng MURASHIGE-SKOOG (MS): L mt trong nhng loi mi trng c s dng rng ri nht trong nui cy m v t bo thc vt. Mi trng MS thch hp cho c thc vt hai l mm v mt l mm. Ti nay c rt nhiu cng thc ci tin mi trng MS trn c s cng thc gc do Murashige v Skoog cng b nm 1962 (bng 5.1, ct MS). - Mi trng ANDERSON: L loi mi trng chuyn dng cho vic nhn ging v tnh cc cy thuc h Rhododendron (Mu n). Nng cc loi mui

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khong dng trong mi trng ny ch thp bng na so vi trong mi trng MS (bng 5.1, ct A). Loi mi trng ny cng c dng cho mt s loi cy thn g nh. - Mi trng GAMBORG: L loi mi trng c th nghim u tin cho cy u tng, nhng cng c dng nhiu trong nhn ging v tnh v c bit l trong tch v nui t bo trn (bng 5.1, ct G). - Mi trng nui cy phong lan: Trn c s cc cng thc mi trng VACIN v WENT, mi trng KNUDSON hoc LINDEMANN, hng Sigma a ra mt s cng thc mi trng nui cy phong lan, v d mi trng V (bng 5.1 ct V) cho cc loi Cymbidium hoc mi trng Phytamax cho phong lan ni chung (bng 5.1, ct P). - Mi trng CHU (N6): L loi mi trng rt hiu qu trong nui cy bao phn ca la v cy ho tho (bng 2.1, ct N6).

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Bng 2.1: Thnh phn mt s loi mi trng thng dng dng trong nui v t bo thc vt T Thnh phn (mg/l) MS A G V P T Mui khong 1 Ammonium nitrate 1650, 400, 134,0 825,0 2 Ammonium sulfate 0 0 500, 3 Boric acid 3,0 0 3,1 4 Calcium chloride 6,2 6,2 113,2 166,0 5 Calcium phosphate tribasic 332,2 332, 6 Cobalt Cloride.6H2O 2 0,025 200, 0,012 7 Cupric sulfate.5H2O 0,025 0,025 0 5 8 Ethylene diaminetetraacetic 0,025 0,02 0,012 acid, 2Na.2H2O 5 37,26 5 9 Ferric tatrate.2H2O 37,26 0,02 10 Ferrous sulfate.7H2O 5 27,8 37,26 11 Magnesium sulfate 27,8 122,1 28,0 12 Manganese sulfate 180,7 74,5 10,0 27,8 13 Molypdic acid (Na2).H2O 16,9 0,25 122, 90,35 14 Potasium iodide 0,25 55,7 0,75 1 8,45 15 Potasium nitrate 0,83 180, 2500, 5,07 0,125 16 Potasium phosphate 1900, 7 0 8 0,415 17 monobasic 0 16,9 130,5 950,0 18 Potasium chloride 0,25 85,5 Zine sulfate.7H2O 0,3 2,0 525, 8,6 480, 0 5,5 0 250, 330, 0 6 8,6 Cc cht hu c v vitamin 19 Glycine 2,0 20 Thiamine.HCI 0,4 0,4 10,0 0,4 1,0 21 myo Inositol 100,0 100, 100,0 100,0 22 Pyridoxine HCI 0,5 0 1,0 0,5 23 Nicotinic acide 0,5 1,0 0,5 24 MES 1000 25 Peptone 2000 Cc cht iu khin sinh 26 trng

cy m N6 K

463,0 1,6 125,3 37,26 27,8 90,35 3,33 0,8 2830, 0 400,0 1,5

600,0 3,0 453,0 0,025 0,025 37,26 27,8 146,5 10,0 0,25 0,75 1900, 0 170,0 300,0 2,0

2,0 1,0 100,0 0,5 0,5 -

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27 Adenine Hemisulfate 28 6-Dimethylaminopurine 29 2,4-Dichlorphenoxyacetic acid 30 Indole-3-acetic acid alpha-Naphthaleneacetic acide 6-Benzylaminopurine 32 Saccharose (g/l) 33 Agar (g/l) pH 30,0 8,0 5,6

80,0 2,0 2,0 0,5 0,5 2,0 30,0 8,0 5,8 30,0 8,0 5,8 20,0 5,8 20,0 5,8 20,0 8,0 5,8 20,0 5,8

19

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V. KH NNG NUI CY CA CC LOI M V T BO V nguyn tc, mi loi t bo ca mt c th thc vt u cn lu gi c tnh ton nng, c ngha l vn cn kh nng nui cy thnh cng. Tuy nhin trong thc tin cc loi t bo v loi m khc nhau th c biu hin rt khc nhau v trin vng, nui cy thnh cng. Mt nguyn tc c bn c tng kt l cng gn trng thi t bo phi bao nhiu, kh nng nui cy thnh cng cng cao by nhiu. Nh vy, t bo v m ca phi non l c trin vng nht, sau n l t bo ca cc nh sinh trng trng thi hot ng (chi nh ngn, u r) hay trng thi ng ngh (chi nch). Cng rt gn phi l cc t bo sinh dc nh non bo v t bo ht phn giai on non. Cc t bo ca m tng tng (cambium) cng l ngun mu vt nui cy rt l tng. Hnh 5.2. Trnh by s s dng cc loi m v t bo vo cc k thut nui cy khc nhau v trin vng ng dng thc tin ca chng.

Chng III PHC TRNG V TO CY SCH BNH I. THIT HI KINH T DO BNH VIRUS THC VT Trong sn xut nng nghip, thit hi kinh t hng nm do cc ngun bnh ni chung (nm, vi khun, tuyn trng...) v virus ni ring rt ln. V d, bnh virus Tungro pht trin trn cy la, nm 1971 cp i na triu tn thc ca nhn dn Philipine. Bnh virus khm lm cho nng sut la m, i mch cc nc chu u (Anh, Php) v M gim ti 20-30%. Nhn chung mc gy hi v tnh nghim trng ca bnh virus th hin cc nhm cy khc nhau. i vi cy hng nm, s thit hi th hin qua vic gim nng sut hoc gy mt ma ton b trong mt v. i vi cc cy lu nm, thn g (cam, chanh, mn, l, to), bnh virus khng nhng lm gim cht lng v nng sut ngay i vi cy b nhim bnh, m cn l nguy c cho cc cy kho nhng nm sau. c bit, i vi cc cy nhn ging v tnh, bnh virus ly lan nhanh, d pht trin thnh dch gy hi nghim trng hn c. Virus khng th phng chng, tiu dit bng x l cc cht ho hc nh nhng bnh vi khun, nm v su b. Cch duy nht loi b virus l phi tch chng ra khi cy b bnh, tr li cho cy trng mt cuc sng bnh thng kho mnh.

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II. NGUYN L LM SCH VIRUS V DUY TR TNH SCH BNH Danh t lm sch virus ch ng v ni dung ca cng vic. l vic phi gii phng cc thc vt b nhim virus khi virus. y ch cp ti cc cy trng nhn ging v tnh v phng thc nhn ging ny l nguyn nhn truyn bnh t th h ny sang th h khc. V vy bin php lm sch bnh virus lun phi kt hp vi bin php duy tr tnh sch bnh. C hai bin php nm trong phm vi phc trng ging, ngi ta gi l bin php gi sch bnh. Bn cnh hai nhim v l duy tr c tnh ging v tnh ng u ca ging, nhim v ch yu ca cng tc phc trng ging l cung cp c tp on cy b m v ht ging sch virus. Kt qu vic lm sch virus thu c l sch hay t virus ph thuc rt nhiu vo thc trch nhim ca ngi nghin cu. Kinh nghim thc t cho hay, nhng bin php phc trng ging c hiu qu l bin php c thc hin mt cch trit v c trch nhim. Do vy lm virus c coi l mc tiu ca cng tc phc trng ging. Bn cnh x l nhit v xc nh tnh sch bnh, cc phng php thu c cy sch virus bao gm ch yu vn l nui cy nh sinh trng. ng nhin k thut nui cy nh sinh trng y c thc hin theo mt mc ch khc nn phc tp v tn km hn l trong nhn ging v tnh. V th ngi ta phn bit r gia nui cy nh sinh trng trong cng tc phc trng ging ni chung v lm sch virus ni ring. Mc ch ca ngi bo v thc vt trong nui cy nh sinh trng v nui cy m, phn bit r vi nhng ngi lm cng tc duy tr ging. i vi ngi lm cng tc bo v thc vt, yu cu ln nht l lm sch virus. Trong thc t iu hu nh khng th t c. V vy phi kt hp nhiu bin php m bo kt qu. X l nhit nui cy nh sinh trng v xc nh (th) virus phi c thc hin theo mt chu trnh kn. Cc nh duy tr ging i hi phi c nhng c th thc s sch virus, ri thng qua phng php nhn invitro c th nhn thnh s lng cy bt k m khng b ti nhim. Cc nh sinh l thc vt rt quan tm n phng php nhn ging invitro (Murashige, 1974. Pierik, 1975). y l do chnh khin cc nh sinh l thc vt quan tm l vic lm sch virus i vi cy trng. V l do kinh t ngi ta ch gii hn vic nhn ging invitro nhng cy trng m i vi chng cc phng php c in nhn nhanh nhng ging mi hoc lm sch virus khng thc hin c. Phng php nhn ging invitro loi tr c nguy c ti nhim v v th n t ra u vit hn cc phng php c in. Tuy vy theo kinh nghim thc tin, cc cy trng c nhn ging invitro vn cn mang t nhiu tc nhn gy bnh. Gn y c nhiu cng trnh nghin cu x l ho cht gp phn nng cao hiu qu sn xut cy sch bnh (Tomlinson, 1982). Nh vy loi b virus, ngi ta kt hp cc gii php: - X l nhit v ho cht - Nui cy nh sinh trng v chn lc bng phng php th virus.

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III. K THUT LM SCH VIRUS 1 X l nhit y l bin php lm sch bnh c c s thc tin. Nhng cy ma b bnh cho nng sut cao hn khi ngm trong nc nng. Nhiu nghin cu cho thy dng kh nng thun li hn i vi a s cy trng v chng chu ng tt hn v virus b loi dn dn. Nhng hiu bit v c ch lm sch bnh bng x l nhit cn cha y . Gi thit chung l virus b c ch sinh sn 39 40 oc. Qu trnh sinh trng ca thc vt nhit cao cng b c ch nhng mc thp hn v v vy nhng b phn sinh trng nhanh thng l sch bnh hoc ngho virus. Nhiu nghin cu cho thy, c th thc vt c bo tn trng thi ti thch trong thi gian di nhit cao. Chu k quang thch hp cho x l nhit cao l 16 h /ngy. Tuy vy cn thn trng v chn thi gian thch hp cho tng loi cy, nhit cn c kim tra lin tc, m cn gi 50%. C ch ca qu trnh ny (theo Kassamis, 1957) l khi nhit cao, qu trnh tng hp b ngng nhng vn din ra s phn gii cht trong virus. Nh vy, khi x l nhit 40 oc, m phn sinh cy vn pht trin trong khi virus ngng sinh sn do s sao chp nhn RNA v DNA ca chng b ph v. Cc nh meristem do vy c kh nng khng cha virus. Do vic x l nhit cao c nh hng n cy v mu cy, thi gian v nhit x l phi c chn lc thch hp cho tng loi cy v tng loi virus. V d: loi virus cun l khoai ty, c khoai ty c x l 40 oc, 4 gi. i khi kt hp 2 loi x l mang li hiu qu cao hn. Chng hn vi virus khm thuc l, u tin x l nhit 40 oc, 16 ting, sau 22 oc, 8 ting. T l cy sch bnh thu c trong trng hp ny cao hn so vi khi x l 40 oc vi thi gian 20h (Walkey v Freeman, 1977). Ngoi ra nhit thp cng c tc dng c ch loi b mt s virus. Moskovers (1973) thu c cy khoai ty sch virus A v Y bng cch nui cy nh sinh trng c x l nhit 5 - 15oc. iu kin thch hp hoa cc c kh nng chu nhit 38 oc trong thi gian na nm; tip theo l hoa anh tc cng chu c thi gian kh di, hoa thu tin chu c nhit 34 oc trong thi gian 4 - 6 tun. 2. Nui nh sinh trng Limasset v Cornnel (1949) chng minh c rng, nng virus trong thc vt gim dn b phn gn nh sinh trng, ring nh sinh trng th hon ton sch virus (Morel v Martin, 1952). Thc t ny c ng dng lm sch virus bng cch tch nh sinh trng iu kin v trng ri nui cy chng thnh cy hon chnh. Vic phn lp nh sinh trng c kch thc 0,01 0,1mm rt kh khn (Hollings v Storie, 1964). Qua tnh sch bnh ca mu vt nui cy b

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gim xung nhng tc ti sinh cy tng ln v chnh l phng php c ng dng trong thc tin. thu c cy sch bnh khng nht thit phi c nh sinh trng hon ton sch cc phn t virus m n c hon thin trong qu trnh phn ho ca cc t bo cha phn ho. V vy trong thc tin phi gii hn nng virus v khi lng m phn ho mc nht nh nu cn c cy sch virus. Vic phi hp x l nhit vi nui cy nh sinh trng l phng php thun li bi v thng qua x l nhit, qu trnh sinh sn ca virs trong chi ngn b c ch mnh v thng qua qu trnh phn ho nh sinh trng, tnh sch virus s c m bo vi xc sut cao. y khng cp ti vn chn cc mi trng thch hp. Thng thng ngi ta s dng mi trng ca Murashige v Skoog, Buys, White v Heller. Theo quan im l thuyt v kinh nghim thc tin, ngi ta thu c nhng kt qu khc nhau. Trong tng phng th nghim nu vic nui cy nh sinh trng c thc hin trn quan im sn xut ln th cn ch nhng im sau y: m bo ng nht ca ging trong tt c cc khu nui cy, m bo tc sinh trng nhanh v u i vi s lng nh sinh trng ln, ng thi kt qu a cy ra t cng cn phi c m bo. Cc nh sinh trng cn c nui sau khi phn lp cc bung nui cy hon ton khng ch t mt kh hu nhit 22oc v 6h Chiu sng 1 .000 - 3.000 lux. Ngoi m phn sinh nh, mt s m v t bo khc cng c nui cy to cy sch bnh, bao gm m so, protoplast v m c quan sinh sn. Mt s cng trnh nghin cu thu c nhng cy kho mnh t m so thuc l b nhim bnh khm virus (Mori, 1977). Nu em khi m so t bo thuc l kim tra xt nghim th thy c khong 30 - 40% t bo b nhim bnh. Nh vy trong khi m so nhim bnh xut hin nhng vng m sch bnh. iu ny c th gii thch l s nhn, sao chp virus chm hn s phn chia t bo (Svobodva, 1965). Nui cy t bo trn thu cy sch bnh c Shepard (1975) thc hin trn cy khoai ty. T protoplast ca l b nhim virus X, ng ti sinh c cc cy khoai ty sch bnh virus X; trong tng s 4140 cy, c 7,5% l cy sch bnh. Vic nui cy m t hoa to cy sch bnh c p dng c kt qu i vi cy c mi, v c mt s virus khng lan truyn qua ht. Phi tm v ti phi c s dng c kt qu trong vic to ra nhng cy cam sch bnh (Navarro v Juarcz, 1977). 3. X l ho cht . Ni chung x l ho cht khng th coi nh mt phng tin loi b virus khi cy trng. Cc cht ho hc thng dng c ch pht trin cc triu chng v s nhn nhanh, ly lan ca virus (Tomlinson, 1982). Chng khng trc tip km hm hot ng ca virus m lm tng kh nng khng ca c th thc vt (Antonin v ctv, 1981). Cc cht riboside (virazole hoc ribavirin) c cng thc 2,4

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Triazole - 3 - carboxamide lm tng t l cy sch bnh ln ng k, gip ch thc s cho cc k thut khc loi b virus c hiu qu hn. Cht ribavirin l ng phn ca guanosin c ph hot ng khng virus rng i vi c hai loi virus (virus cha RNA v DNA) (Sidwell v ctv, 1972). Ngoi ra, n cn c nhng hot ng c ch qu trnh sao chp, nhn nhanh ca virus trong ton b c th ca cy trng. Tt c cc cht ho hc c tc dng km hm s hot ng ca virus c Kartha (1986) tp hp trong bi tng quan v ng ch ra rng chng l ng phn ca purine v pirimidine, amino acid, hoc mn sinh trng thc vt v cc khng sinh. Trong qu trnh nui cy m, t bo, s c mt ca cc cht ny (virazole) c th duy tr trng thi v trng lu hn, gip cho vic loi b virus c hiu qu hn (Hansen v Lane, 1 985). Gn y ngi ta cn s dng mt nhm cc cht khc, nh arabinoside hoc vidarabine lm sch virus trong qu trnh nui cy m phn sinh khoai lang v cho kt qu tt (Stone v ctv, 1978). IV. KIM TRA, XT NGIIIM VIRUS Khng phi tt c cc loi cy sau qu trnh x l phi hp nhit v nui cy nh sinh trng u sch virus, v vy cn phi tin hnh xt nghim. Khong thi gian t lc ti sinh cy cho n khi xt nghim cn t c 4 n 6 thng cc th virus cn tn li trong thc vt t c nng cn thit cho vic xt nghim m bo chnh xc. Xt nghim virus trong khun kh ca qui trnh lm sch virus hon ton khc qu trnh phn tch virus mt cy trng. i vi vic lm sch virus th chnh xc ca phng php th virus trong mi loi xt nghim mang ngha quyt nh, cho nn mi mt cy cn c xt nghim theo nhiu phng php khc nhau, trong cn ch ti cc phng php xt nghim nhng loi virus ph bin v c ngha kinh t. i vi vic phn tch virus mt loi cy trng, ngi ta ch ch ti s lng cng nh s phn loi ca chng. nhy cm ca mi phng php xt nghim c vai tr th yu. Sau y l mt s phng php xt nghim virus c ng dng cho cc loi cy hoa: 1. Xt nghim bng cy ch th Dng dch p ca thc vt cn c xt nghim gy nhim trn mt cy ch th thch hp hoc dng phng php ghp c th chng minh c s c mt ca virus. Ch sau khi thc hin phng php th ny, kt lun v bnh virus mi thc s m bo tnh chnh xc ca n. 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 thuc cc yu t khc na. Trong trng hp xt nghim hng lot, cng vic gy bnh nhn to i vi s lng cy ch th l 10.000 n 100.000 trong thi gian nhiu thng th chnh

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xc ca phng php gim i v khng th tin hnh cc th nghim lp li. Tui ca cy trng cng nh trng thi sinh l ca chng trong cc ma khc nhau ca 1 nm nh hng rt nhiu n tnh chnh xc ca phng php xt nghim. V l do trong trng hp phi xt nghim hng lot, phng php dng cy ch th khng m bo bng phng php min dch. Nu ch xt nghim mt s lng cy va phi, v d mt nghn c th th phng php dng cy ch th khng cn thay bng phng php khc v cng vic c th tin hnh trong mt thi v thch hp. Triu chng bnh l c th quan st c sau 3 n 5 ngy song thng thng l sau hai thng, v vy cn mt din tch nh knh kh rng trong mt thi gian tng i di, chi ph cho xt nghim bng phng php cy ch th thng t gp 3 ln so vi phng php huyt thanh. 2. Phng php th huyt thanh Phng php xt nghim ny da trn c s kh nng lin kt gia cc khng th vi khng nguyn c trng ring ca n. Khng nguyn l mt cht protein c kh nng to ra mt phn ng min dch khi chng c a vo c th ng vt. Hu ht virus thc vt u c tc dng nh nhng khng nguyn. Khi chng c a vo mt c th ng vt thch hp (nh th), chng s kch thch sn xut ra nhng khng th. Nhng khng th ny c th c dng trong cc xt nghim huyt thanh (Van Regenmontel, 1982). Phn ng kt ta xy ra khi c s kt hp gia mt khng nguyn v mt khng th thch hp. Trong cc phn ng nh vy, khng nguyn v khng th lin kt vi nhau to thnh nhng th huyn ph khng tan (latex). Phn ng min dch ny chnh l nguyn l ca nhng phng php xt nghim huyt thanh. Da vo hnh dng, kch thc, s phn b ca sn phm phn ng, ngi ta c th phn bit c cc loi xt nghim sau: - Xt nghim cn c vo s kt ta ca cc thnh phn khng nguyn v khng th xy ra trong mi trng lng. Phng php th ny thng c dng so snh s khc nhau ca cc virus. Mi quan h gia cc virus c xc nh thng qua mc pha long ca khng huyt tng trong phn ng kt ta. Chun cho mt khng huyt tng l mc pha long cao nht ca khng huyt tng thc hin phn ng min dch vi mt loi virus ng dng. a) Xt nghim min dch khuch tn, c thc hin trong thch. Sn phm phn ng lin kt gia khng nguyn v khng th s khuch tn u qua thch (Akers v Steere, 1967). Min dch khuch tn dng nhn bit mt loi virus gi l Radial diffusion (Mancini v ctv, 1965). Trong min dch khuch tn kp (gel double-difusion), khng nguyn v khng th c cha trong nhng h thch v chng s khuch tn ln vo nhau (Ouerlory, 1968), hnh6.1.

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b, Phn ng ngng kt (Agglutination test) Trong phn ng ny, khng th hoc khng nguyn c bm vo nhng tiu phn ln hn. Do vy khi phn ng xy ra, phc cht gia Ab- Ag c kch thc ln hn, d quan st. Phng php ny s dng pht hin nhanh virus ly nhim khoai ty trn ng rung (Van Slogteren, 1955). Gi th khng nguyn hoc khng th bm c th l Polystyrene hoc vi khun (Staphylococas aureus). c) Min dch lin kt vi enzyme [Enzyme linked immuno sorbent assay (ELISA)] Trong phng php ny nhy cm ca phn ng min dch pht hin c virus tng ln do s lin kt ca phc cht Ab - Ag vi enzyme nn. Do s b sung thm cht enzyme nn, mu ca phn ng th hin r hn, cho php ngi ta nh lng c phn ng min dch v pht hin virus nng rt thp (Voller v ctv 1976). Ngy nay ngi ta s dng ELISA nhn bit nhiu loi virus thc vt khc nhau (Koening v Paul, 1983). Mt s trung tm nghin cu nng nghip quc t iu ch nhng b xt nghim ELISA khc nhau: CIP sn xut b ELISA nhn bit cc virus X, Y, M khoai ty, virus chn chim cy khoai lang v virus da cc cy sn. T chc nghin cu chui th gii (INBAP) nghin cu sn xut ch phm ELISA pht hin virus chm ngn cy chui. R rng phng php xt nghim huyt thanh ngy cng c ci tin v s dng phn bit nhiu loi virus. Nhng u im chnh ca phng php l: chnh xc, nhanh (kt qu thu c chm nht sau 48 gi), chi ph cho xt nghim thp nhu cu v ho cht, iu kin thc nghim n gin, d kim. 3. Xt nghim bng lai phn t Lai phn t acid nucleic l mt k thut mi pht trin gn y, c p dng nhn bit, pht hin vt liu di truyn ca virus. y l phng php phn tch c nhy, tnh c trng cao nhn bit RNA hoc DNA ca virus (Abu, Samah v Raudles. 1983). K thut bao gm nhng ni dung sau: sn xut DNA tng ng da trn cc ch phm acid nucleic ca virus. DNA tng ng ny s c gn vi cc

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ng v phng x 3H hoc 32p, v sau cho kt hp vi dch chit t cy b nhim bnh. Trc , acid nucleic trong dch chit c tch ra v phn gii qua sc k trn thch v c nh trn mng nitrocellulose. Biu cht ng v phng x c s dng pht hin cc cp lai dng tnh. DNA tng ng c sn xut c trng cho tng loi virus v k thut ny cho php sng lc mt s lng ln mu bnh. 4. Xt nghim bng knh hin vi in t Knh hin vi in t vi s hon chnh v k thut v sau khi ng dng phng php nhng (Brandes, 1957) c th a vo xt nghim hng lot vi s lng mu va phi. Khi chng minh virus hnh a v hnh si hoa phong lan (Corbert, 1 974), hoa hu (Asges v ctv, 1974) knh hin vi in t mang li nhng kt qu ng tin cy i vi xt nghim hng lot. Kh khn ch yu hin nay l chi ph cho thit b v s lng mu c xt nghim b hn ch, ng thi loi virus c chng minh cng ch l loi hnh a v hnh si. Nu virus tn ti dng cu th rt kh pht hin v n kh ging cc c quan t ca t bo thc vt bnh thng. 5. Kim nh l phng php duy nht duy tr tnh sch bnh. Trng vt liu sch bnh thu c bng phng php chn lc qua kim nh c th coi l gii php ti thiu chng mang li kt qu nu khng tin hnh cc phng php khc c. Virus stunt ca hoa cc khng mt i hoc hn ch dn khi x l nhit. V vy ch dng phng php chn lc bng xt nghim ghp ln cy ch th. bo m s lng ging cn thit, hng nm c phi xt nghim 100000 cy ghp. s dng mt cch ti u chi ph ln ny, ngi ta a xt nghim bng phng php ghp vo mt qui trnh khp kn. Vi vic thc hin mt cch lin tc bin php ny trong vng 6 nm ngi ta h t l nhim virus stunt ca hoa cc t 30% xung 0% (Oertel, 1976). 6. Kim nh l khu cui cng trong qui trnh lm sch bnh Nu trong qui trnh lm sch virus c th p dng c k thut x l nhit v nui cy nh sinh trng th vic xt nghim virus ch cn l bin php kim tra cui cng ca qu trnh lm sch virus. Nh vy s c mu thun vi mc ch lm sch virus nu ngi ta s dng 50% cy trong tp on cy trng b bnh lm vt liu ban u v coi chng l nhng cy c phm cht tt. Mt mt thng qua ci tin phng php xt nghim a chnh xc ca phng php ln cao, ngi ta phi lun tnh n s lng virus bo tn nng ti thiu m phng php xt

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nghim khng chng minh c. V vy theo kinh nghim thc t cn tin hnh xt nghim theo phng thc sau: a vt liu ban u vo x l nhit trong mt thi gian di lm sch nhng virus mn cm nhit , sau dng phng php nui cy nh sinh trng. Sau t 4 n 6 thng k t ngy nui cy mi bt u xt nghim. Thi gian ny to virus bo tn trong cy nng cho php chng minh c. Ngi ta hn ch xt nghim s lng 20000 - 30000 cy, hoa cc l loi virus stunt, virus khm, virus asperenie, virus B. Nhng c th c xc nh l sch bnh l ngun vt liu ban u cung cp cy m cho sn xut. Khong 5 10% s c th ny c tch ring ra thnh tp on nhn cc nh to ging ci tin tnh cht theo mun v thun chng (ng nht) ca ging. Ngi ta kim tra nhng c th ny bng tt c cc phng php xt nghim virus hin c. Vi hoa cm chng ngi ta kim tra bng phng php huyt thanh cc loi virus carnation woltle, carnation latent v bng cy ch th nhm loi tr nhng virus him sinh sn khng c biu hin nhn bit c. Nhng cy xc minh c l kho, ngi ta tin hnh m cnh ri sau a vo x l nhit, tip theo ngi ta nui cy nh sinh trng v cui cng kim tra bng xt nghim virus. l ton b qui trnh lm sch virus khp kn. Phng php lm sch virus trong qui trnh khp kn c thc hin nhiu nm x nghip cy con thuc tnh Dresden ca c i vi hoa cc, cm chng (Oertel, 1978). V. DUY TR TNH SCH VIRUS Vn c tm quan trng ng k v cng l vn quyt nh cui cng i vi thc tin nng nghip lin quan ti thi gian duy tr c cy trng sch virus i vi thc tin sn xut th cy coi l b bnh ch khi no nng sut gim xung. Hin nay trong sn xut nng nghip v trng cy n qu, vn ny cn cha c gii quyt tho ng. Vic sn xut dng elite trong qui trnh sn xut khoai ty ging ko di nhiu nm, trong khi nguy c ti nhim thng qua yu t truyn bnh lun tn ti v ph thuc vo iu kin kh hu. Trong ngnh trng hoa tnh hnh thun li hn nhiu. Hin nay CHLB c, vi tp on nhn ca hoa cc v cm chng ngi ta duy tr c tnh sch bnh trong mt nm ri, trong khi ch cn mt nm l c th thay c hon ton tp on ging. V vy vn nu ra trn c th c tr li tm tt nh sau: Khi lng v cht lng vt liu c sn ban u xc nh kh nng sn xut mt v khng b gim nng sut do bnh virus. Gim nng sut c th xut hin nu ngun ging sch virus b nhim sm. i vi khoai ty th nhim ch yu do cc yu t truyn bnh sng iu kin t nhin. i vi cy hoa th ti nhim xy ra khi a cy ging sch bnh vo cc x nghip sn xut b nhim sn. C th ni rng, trong ngnh trng hoa qui trnh lm

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sch virus c coi nh m hnh phng php. Cng qua chng ta nhn thy phng php lm sch virus khng phi l bin php cha bnh mt ln m l mt qu trnh phc tp i vi cy trng b bnh t trc. Ngi ta c th so snh bnh virus ca thc vt nhn ging v tnh nh bnh x hi ca con ngi, khng th cha bng thuc men m phi thay i c thi quen sinh hot. cc x nghip cng nghip sn xut ging cy trng c th gi cc tin b khoa hc k thut l mt loi stress, khi m t mt cy cc m mt nm cho 100 cy m, trc kia ch thu c 15 v mt cy m ch cn 11 ngy ra r, trc y cn 21 ngy. to iu kin cho cc x nghip sn xut cng nghip cy ging thu c nhng thnh tch to ln hn na th vic u t hng nm cho cng tc chng bnh virus tr nn cn thit. Trong trng hp nhn ging v tnh invitro th vic lm sch virus cng phi c coi l iu kin trc tin. VI. QUY TRNH PHC TRNG Phc trng, sn xut cy sch bnh bao gm nhiu giai on, s dng kt hp nhiu cng ngh. vt liu sch bnh phn b n ngi nng dn phi qua nhiu cng on, chng cn c thc hin mt cch nghim ngt (hnh 6.1). VII. MT S KT QU PHC TRNG, TO CY SCH BNH 1. Cy lng thc - Khoai ty: Khoai ty l cy trng chu u, c nhn ging v tnh v b virus ph hoi nhiu nht. Trong thi gian qua vic lm sch virus khoai ty mi c ng dng mt cch chm chp trong qu trnh duy tr ging. Ngi ta ch nhiu nht ti vic to ra cc cy ging sch bnh bng qui trnh th virus v trng cc khu vc sch bnh trnh ti nhim thng qua cc loi rp l. Qui trnh c s dng ch yu l git cc cy c c th truyn bnh vo c khoai ty(klinkowski v schmelzer, 1974). Ti liu cho bit qui trnh ny c th nng cao hiu sut thng qua x l nhit v nui cy nh sinh trng. Qu trnh x l nhit nhit gia 32 v 38 oc trong thi gian 7 ngy n 7 tun v sau nui cy nh sinh trng c th loi tr c virus A, xon l, x v y trong khi virus M v S cng c gim i mt cch ng k (Kassanis, 1950; Thomson, 1956-1958; Quak, 1961; Kassanis v Varna, 1967; Stace-smith v Mellor, 1968; Mc Donald, 1973; Pett, 1974a; Wang v Huang, 1975).

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Hnh 3.1 Cc nh hin vi in t ca nh sinh trng khoai ty c ln t 80100mm cho thy chng vn cn cha trong tiu bn ti 12 th virus x (Krylova v ctv, 1973). Tuy vy sau qu trnh phn loi t cc nh sinh trng vn thu c mt t l phn trm nht nh cc cy sch virus. 2. Cy thc n gia sc sn xut ht ging cy trng lm thc n gia sc, v d c Tam ip, cn phi c cy b m sch bnh virus trnh s ly bnh thng qua ht ging v m bo thu c nng sut ht cao. Ngi ta nghin cu nhiu phng php lm sch virus khc nhau. X l lnh v nui chi ngn mang li tc sinh trng cao, nhng ch sch virus tng phn. Nu x l nhit kt hp vi nui cy nh sinh trng th thu c phn ln cc cy sch virus (Frosheiser, 1969. Barnett v ctv, 1975). 3. Hoa huplng

31

Cc loi bnh virus hoa huplng thng lm gim nng sut mt cch ng k (Schmidt, 1966). Tin hnh chn lc bng mt thng (Schmidt v ctv, 1972) x l nhit v nui cy nh sinh trng (Gipperp v ctv, 1974) ci tin dn tnh trng sch bnh. Adams (1975) gii phng 66% cy khi virus hop - mosaic v latent bng nui cy nh sinh trng, sau lm sch virus prumis necrotic ringspot bng x l nhit trong thi gian 10 ngy. 4. Cy thc phm - Cy rau: Hu ht cc loi rau, tr mt vi trng hp ngoi l, u c nhn ging bng ht. Truyn bnh virus qua ht va mi c chng minh loi virus gy bnh khm x lch v u (u n qu trng hoc xanh), v vy nhng cy trng ny cn phi chn lc nhng cy lm ging v thng qua bin php trng trt cch ly to ra ht ging sch virus. i vi cc loi virus gy bnh cy rau khc th qu trnh ly lan thng xy ra do c hc hoc do rp l, v vy cn c bin php v sinh ng rung v phng tr tc nhn truyn bnh. mt s cy rau nhn ging v tnh (nm rm...), cn s dng phng php nui cy nh sinh trng hoc x l nhit gii phng virus (Holmes, 1965. Paludan, 1971. Mori, 1971. Walkey, 1968). i vi nm rm, c th lm sch bnh bng phng php x l nhit (Gandy v Hollings, 1962) v trong thi gian gn y ngi ta to c phng php min dch trong agar gel (Meyer v Smith, 1976). Ngoi ra nhng cy trng dng sn xut ht ca chng, c th nhn ging v tnh qua nhiu nm, v d nh sp l ngi ta cng cn phi c vt liu sch bnh virus ban u. Thng qua nui cy m, ngi ta to c mt vi trm cy v thng qua bin php th virus thu c 3220 cy sch bnh (Walkey v ctv, 1974). 5. Cy n qu Cy n qu thng b virus ph hoi mt cch mnh nht. Cc th virus gy bnh khng nhng lan truyn khi nhn ging v tnh m c khi nhn ging bng ht. Ngoi ra cy n qu thng l cy lu nm, lun lun chu tc ng ca cc tc nhn truyn bnh, v th chng rt d b nhim bnh. Vic chng minh virus nhim cy n qu gp nhiu kh khn. Hn na thi gian bnh di v kh nng chng chu cao gy nhiu kh khn cho vic lm sch virus cy n qu, v vy cn tin hnh cng tc chng virus gy bnh cy n qu mt cch lin tc. V vic x l nhit i vi cc cy thn g v vic nui cy nh sinh trng ca chng kh khn hn nhiu so vi cc loi cy thn c cho nn t lu ngi ta s dng phng php th tm ra cc vt liu sch bnh ban u (Kegler, 1964a).

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Qu trnh x l nhit i vi cy n qu n nay thng c tin hnh ch yu nhng on cnh m cc mt ca chng s c s dng ghp sau ny. Theo ti liu tng hp ca Nyland v Coheen (1969) v vn x l nhit cc cy thn g c th loi tr c 4 loi virus cy anh o, 1 loi virus cy mn, 7 loi virus cy to, 2 virus cy nho t (nho ty), 6 virus cy o v 2 virus phc bn t. Thnh cng trong x l nhit cy n qu khng bao gi t c 100% mi i tng t nht cn li mt, thm ch mt s loi cn ti 4 virus khng b mt hot tnh khi x l nhit. Nui cy nh sinh trng cy n qu ti nay mi ch c s ng nhng i tng sau: Du chua (Putz, 1974), du chua qu (Yones v Vine, 1968), du chua qu en (Zatyko v ctv, 1974-1975) v cy to (Walkey, 1972). Thc tin cho thy, i vi cy n qu (cy thn g) vic nui cy nh sinh trng cn gp kh khn hn bi v kh nng ti sinh ca chng yu hn so vi cy thn c. Cc th nghim trong nhng nm sp ti chc chn s nu ra nhng kt qu mi, v d: ngi ta ang nghin cu i tng Promis amygdalor (Mehra, 1974) v Promis serrulata (Boxus v Quoirin, 1974). 6. Cy hoa i tng cy hoa ch gp nhng cy nhn ging v tnh thng b bnh virus Oertel, 1962) trong khi bc u ngi ta ch tp trung lm sch nhng cy hoa c ngha kinh t quan trng (v d: hoa cc, hoa anh tc, hoa thu tin....). Hin nay ngi ta bt u nui cy cc loi hoa khc, v d nh hi v n, hoa hu, hoa layn... X l nhit kt hp vi nui cy nh sinh trng c s dng lm sch virus i vi hoa anh tc (Quak, 1957) v hoa cc (Houings v Kassanis, 1957). Vic to cy sch bnh trong cc x nghip chuyn sn xut hoa tr thnh quen thuc, trong nhng nm gn y H Lan c nhng c s ca t chc trng hoa chuyn mn nhn cc loi vt liu lm sch virus. Anh cng mi t chc mt c quan nh vy gi tt l NTD (Lane, 1968). c, x nghip m cy con thnh ph Dresden cng nhn cc loi hoa nh cc, hoa anh tc, hoa thu tin x l nhit v lm sch virus. Cc iu kin c nu chng V lm sch virus i vi cy hoa thng c thc hin d dng, v th nhng loi cy trng ny vic lm sch virus thng c thc hin c kt qu nht.

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Chng IV NHN G ING INVITRO I. MC CH CHUNG Nhn ging invitro hay vi nhn ging (micropropagation) l mt trong 4 lnh vc ng dng chnh ca cng ngh t bo thc vt (bao gm: Lm sch virus; Nhn nhanh cc ging cy trng qu, Sn xut v chuyn ho sinh hc cc hp cht t nhin v Ci lng v mt di truyn cc ging cy trng) v mang li hiu qu kinh t ln nht. Trong chng ny ch yu ni v lnh vc th 3 l nhn ging v tnh cc cy trng trong nng nghip, lm nghip v y t, trong k thut nhn nhanh c ng dng nhm phc v cc mc ch sau: - Duy tr v nhn nhanh cc kiu gene qu him lm vt liu cho cng tc to ging. - Nhn nhanh v duy tr cc c th u dng tt cung cp ht ging cc loi cy trng khc nhau nh cy lng thc c c, cc loi cy rau, cy hoa, cy cnh, cy dc liu thuc nhm cy thn tho. - Nhn nhanh v kinh t cc kiu gene qu ca ging cy lm nghip v gc ghp trong ngh trng cy n qu, cy cnh thuc nhm cy thn g. - Nhn nhanh iu kin v trng cch ly ti nhim kt hp vi lm sch bnh virus. - Bo qun cc tp on ging nhn ging v tnh v cc loi cy giao phn trong ngn hng gene. II. DNG V TNH Khi nim dng v tnh (Clone) c Weber (1903) cp ti nh l mt phn m, c quan sinh dng c tch ra t mt c th thc vt c kh nng ti sinh thnh cng. Shull (1912) cho rng "Clone" l mt nhm c th c kiu gene tng t nhau, chng c nhn bng sinh sn v tnh. Stout (1940) coi Clone l mt n v nhn to, bao gm cc c th ng nht v mt di truyn, chng c nhn v tnh t mt c th. Ngy nay k thut nui cy invitro cho php t m, t bo c th ti sinh mt s lng ln cy, chng c xem nh mt Clone. Cc phng php nhn ging v tnh bng gim cnh, chit ghp, tch cy con, c t cy m u l nhng k thut to ra cc dng v tnh. III.CC PHNG THC NHN G ING V TNH INVITRO

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Phng php nhn ging invitro b sung cho cc k thut nhn ging v tnh c in nh dm cnh, dm chi, chit, ghp, tch dng... mt k thut tin b vi nhng u th chnh sau y: - Tnh kh thi rng V d: K thut dm cnh ch c th ng dng thnh cng mt s cy trng nht nh v vi kch thc 5-20cm kh nng to r ph ca vng m tng tng gn vt ct hoc kh nng nh thc chi ph vn b cc vng t bo ln cn v ton b phn cn li ca on dm khng ch. Nu tin hnh nui cy m vi kch thc 5-20mm, tc l lm gim th tch khi m xung 10 3 ln th r rng mi tng tc gia cc t bo v cc loi m n gin i rt nhiu, hiu qu tc ng ca cc bin php nui cy s phi cao hn. - Tc nhn ging cc k cao. H s nhn ging invitro thng t cc loi cy khc nhau nm trong phm vi t 3 6 10 12/nm, nh vy khng c mt k thut nhn ging v tnh no khc li c h s nhn ging cao hn. - C tim nng cng nghip ho cao. Nui cy trong iu kin n nh mi trng dinh dng, v ch chiu sng nhit l tin hon ton thot khi s l thuc ma v vn xy ra trong sn xut nng nghip v c th cng nghip ho hon ton cng vic sn xut cy ging trong mt dy chuyn sn xut lin tc. Sau y l mt s phng thc nhn ging v tnh invitro: 1. Nui cy m phn sinh hoc nh sinh trng Khi nim m phn sinh ch ng khi mu vt nui cy c tch t nh sinh trng c kch thc trong vng 0,1mm tnh t chp ca thp sinh trng. Trong thc t, mu nui cy c tch vi kch thc nh vy ch khi no ngi ta tin hnh nui cy vi mc ch lm sch virus cho cy trng. Thng s gp kh khn ln trong vic nui thnh cng cc nh sinh trng ring r nh vy. Ph bin nht cc i tng nh phong lan, da, ma. nh sinh trng c tch vi kch thc t 5-10mm, ngha l ton b m phn sinh v mt phn m xung quanh.

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Hnh IV.2. S nhn ging invitro cy da si Agave Tng quan gia ln chi tch t l sng v mc n nh v mt di truyn ca chi c biu hin nh sau: ln , t l sng v tnh n nh ln , t l sng v tnh n nh Nhng xt hiu qu kinh t nui cy (th tch bnh nui, lng dung dch mi trng dinh dng) th: ln , hiu qu kinh t ln , hiu qu kinh t (Du biu hin tng v du biu hin gim) Do phi kt hp c cc yu t tm ra phng thc ly mu ti u. Mt nh sinh trng nui cy iu kin thch hp s pht trin thnh mt hay nhiu chi v cc chi s pht trin thnh cy hon chnh c r y . Xt v ngun gc ca cc cy c 3 kh nng: - Cy pht trin t chi nh (chi ngn). - Cy pht trin t chi nch ph ng. - Cy pht trin t chi mi pht sinh. Tuy nhin rt kh phn bit c chi ph ng v chi pht sinh mi. C hai phng thc pht trin cy hon chnh t nh sinh trng nui cy

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a) Pht trin cy trc tip: Ch yu cc i tng hai l mm nh khoai ty, thuc l, cam chanh, hoa cc nhng c c cy mt l mm nh da si (hnh 7.2), ma. V d: Khoai ty (hnh 7.1) Mm (nh sinh trng) Chi nch Cy. b, Pht trin cy qua giai on d hnh (protocorn) Ch yu gp cc i tng n t ip (mt l mm) nh phong lan, da, hu. Cng mt lc nh sinh trng to hng lot protocorm v cc protocorm c th tip tc phn chia thnh cc protocorm mi hoc pht trin thnh cy hon chnh. Bng phng thc ny, trong mt thi gian ngn ngi ta c th thu c hng triu c th. V d: Lan "Hi v n" Cymbidium. Cc i tng hoa lan Orchidaceae mang li hiu qu kinh t c bit cao. Sau nhng kt qu u tin Cymbidium ca Morel (1966), ngi ta thu c kt qu rt tt 22 chi khc nhau ca h ny. S d nhn ging v tnh hoa lan t c thnh cng ln v c ng dng rng ri nh vy l v phong lan c phng thc sinh sn qua dng d hnh (protocorm). Nh c phng thc nhn ging nhanh v r tin m hoa Cymbidium vn t tr nn c gi phi chng v c nhiu ngi a chung. Nhng thnh cng i vi h Orchidaceae khng nhng ch l bng chng m cn m ng cho vic ng dng k thut ny i vi cc loi cy khc. Lnh vc ng dng mi y nht cng bt u c kt qu l cc cy n qu v cy lm nghip, trong c cy qu nh c ph, to, l, cy thng, b .... Tng s c trn 30 chi khc nhau c nui cy thnh cng. V rng cc cy trng rng v cy n qu l nhng cy trng lu nm nn mi chi ph trong nhn ging invitro u c th chp nhn c. 2. Ti sinh cy hon chnh t cc b phn khc ca cy Ngoi m phn sinh v nh sinh trng l b phn d nui cy thnh cng cc b phn cn li ca mt c th thc vt u c th s dng cho vic nhn ging invitro c. Cc b phn l: - on thn: thuc l, cam, chanh.... - Mnh l: thuc l, c chua, bp ci.... - Cung l: Narcissus. . - Cc b phn ca hoa: xp l, la m.... - Nhnh c: hnh ti, h hoa hu Liliaceae, Iridaceae, Amaryllidaceae. mt s ging rau, ngi ta thnh cng v nhn ging bng cc b phn khc: - Hnh: on b c ( Debrgh v ctv, l976). - Bp c: on r, on thn, mnh l, l mm ( Bajaj v Nitsch, l975). - Mng t: on mm ( Kohler, l975).

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- Xp l: hoa t ( Kohler, 1978). 3. Nhn ging qua giai on m so Trong khun kh ca mc ch nhn ging v tnh, nu ti sinh c cy hon chnh trc tip t mu vt cy ban u th khng nhng nhanh chng thu c cy m cc cy cng kh ng nht v mt di truyn. Tuy nhin trong nhiu trng hp m nui cy khng ti sinh cy ngay m pht trin thnh khi m so. T bo m so khi cy chuyn nhiu ln s khng n nh v mt di truyn. trnh tnh trng nht thit phi s dng loi m so va pht sinh, tc l m so s cp, hy vng s thu c cy ti sinh ng nht. Thng qua giai on m so c th thu c nhng c th sch virus nh trng hp ca Kehr v Sehaffer (1976) thu c ti. Mt phng hng nhn ging v tnh na l to phi soma t t bo m so. Cho ti nay mi thnh cng hai i tng l: - C rt (Daucus carota) ca Reinert (1959). - Thuc l (Nicotiana tabacum) ca Lorz (1977). Tuy nhin vn cha c bng chng c th v s ng u ca nhng c th ny. Nh vy c 3 con dng to cy: - Mu m trc tip to chi v cy hon chnh. - Mu m pht sinh m so v m so to chi. - Mu m to m so t t bo n, m so pht trin phi v t phi thu c cy hon chnh. IV. QUY TRNH NHN G ING INVITRO Cho ti nay vic s dng nhn ging invitro dc p dng cho rt nhiu loi cy (350 loi). Murashighe (1974) chia quy trnh nhn ging thnh 4 giai on: 1. Nui cy khi u, ti sinh chi, cm chi. 2. Nhn nhanh chi, cm chi trong iu kin invitro. 3. To cy con hon chnh, hun luyn cy con. 4. Phc hi, chuyn cy ra trng trong iu kin t nhin Hoa. l, thn, c, cung l, m phn sinh. Hnh 7.3. Quy trnh nhn ging invitro 1. Giai on I Cy gy a mu vt t bn ngoi vo nui cy v trng phi m bo nhng yu cu sau: - T l nhim thp.

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- T l sng cao. - Tc sinh trng nhanh. Kt qu bc cy gy ny rt ph thuc vo cch ly mu. Quan trng nht vn l nh sinh trng, chi nch, sau l hoa t, hoa, on thn, mnh l, r.... V d: Mng ty chi ngn Kohler.1975. Khoai ty mm Morel.1952. Da chi nch Paunethier. 1 976. Bp ci mnh l Bimomillo.1975. Xp l hoa t Kohler.1978. Hoa Narcissus cung hoa Seabrock.1976. Da (Cucurbita pepo) l mm Jelaska. 1 972. Geranium bao phn Abo El-Nil. 1971 . Chn ng phng php kh trng s a li t l sng cao v mi trng dinh dng thch hp s t c tc sinh trng nhanh. Mt s dng mi trng dinh dng ph bin: Mui khong: Theo White (1943), Heller (1953) hoc Murashige v Skoog (1962). Cht hu c: ng saccharoza 1 - 6% Vitamin: B1, B6, inositol, nicotinic acid. Amino acid: Arg, Asp, Asp-NH2 Glu, Glu-NH2 Tyr. Hoc mn: Auxin: IAA, IBA, NAA, 2,4-D. Cytokinin: BAP, Kin, 2P. Gibberellin: GA3. 2. Giai on II - Nhn nhanh giai on ny ngi ta mi kch thch to c quan ph hoc c cu trc khc m t cy hon chnh c th pht sinh. Nhng kh nng to cy l: - Pht trin chi nch. - To phi v tnh. - To nh sinh trng mi. Trong giai on ny cn nghin cu cc tc nhn kch thch phn ho c quan, c bit l chi nh: - B sung t hp hoc mn sinh trng mi (tng kinetin gim auxin). - Tng cng nh sng 16 gi/ngy ti thiu 1000lux, nh sng tm l thnh phn quan trng kch thch phn ho mnh (Weiss v Jaffe,1969). - Bo m ch nhit 20-300C Mc tiu quan trng nht ca giai on ny l xc nh c phng thc nhn nhanh nht bng mi trng dinh dng v iu kin kh hu ti thch.

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3. Giai on III - Chun b v a ra ngoi t y l giai on quan trng bao gm vic to r, hun luyn thch nghi thay i nhit , m, s mt nc, su bnh v chuyn t trng thi d dng sang t dng hon ton. Giai on ny thng b b qua mt cch thiu cn c. Khi nghin cu v cu trc ca l cy khoai ty nui cy invitro v so snh vi l cy khoai ty trng bn ngoi chng ti thy chng rt khc nhau. C th s lng kh khng cao hn nhiu ln so vi l cy sng ngoi t nhin. iu chng t phi tin hnh thch nghi dn dn cy nhn ging invitro vi iu kin t nhin. Qu trnh thch nghi y phi c hiu l qu trnh thay i nhng c im sinh l v gii phu ca bn thn cy non . Thi gian ti th cho s thch nghi l 2-3 tun, trong thi gian ny cy phi c chm sc v bo v trc nhng yu t bt li sau: - Mt nc nhanh lm cho cy b ho kh. - Nhim vi khun v nm gy nn hin tng b thi nhn. - Chy l do nng. V. NHN GING INVITRO V VIC S DNG GING U TH LAI Trong chn nui, vic s dng ging vt nui mang u th lai th h Fl tr thnh bin php tng nng sut quan trng bc nht. ngnh trng trt, ging u th lai mi ch c ng dng mt s i tng nh ng, c chua, la, ci du, bp ci, hnh ty, mng ty, c bit l cc ging hoa S dng u th lai khng nhng lm tng nng sut t 20-40%, m ging lai cn c c im l rt ng u so vi ging b m. Tnh ng u ca ging l tin quan trng cho sn xut theo phng thc cng nghip. xp l chng hn, phng thc sn xut cng nghip i hi phi thu hoch ton b din tch bng c gii vo mt thi im. iu ny ch thc hin c kh s dng ging u th lai F1. Nu dng ging thun chng loi tt th vn phi thu hoch lm 4-5 ln. Mun s dng ging u th lai F1, nht nh phi c ging b m thun chng. Nu bo qun ging thun chng theo phng thc t phi th khng m bo v cc ging rau h Brassicaceae thng xut hin hin tng bt t th. V vy phng php nhn ging v bo qun ging trong ng nghim i vi mt s ging rau v ging hoa c mt gi tr kinh t cao. Vn t ra hin nay l phi nghin cu cc qui trnh nhn ging invitro ti u cho tng loi cy trng v ci tin qui trnh gim ti mc ti a cc tn km v nhn cng lao ng trong cc cng on nui cy v a cy con ra ngoi t. nh gi u th lai: Ng: l di bp th hin u th lai r nht.

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K (mile): l th nm th hin u th lai r nht. Petunia (mo g): l mm th hin u th lai tt nht. Nui cy invitro cho php nh gi sm u th lai. V d: Trong chn ging cy lu nm nh cao su, nui cy invitro cho php nh gi sc sng ca cy trc 5 nm. Cy cao su pht trin theo mt chu k nht nh khng ph thuc vo ma. Nhn ging cao su bng phng php ghp So snh sc sng ca N. synvestris ng hp t 1. Cy ng hp t do t th 9 th h (T). 2. Cy lai gia 2 dng ng hp t nhn bng phng php n bi (Hi x Hj) 3. Cy ng hp t th h 1 bng phng php n bi (Ho ). iu kin t nhin T (Hi x Hj) Ho nui cy invitro T (Hi x Hj) Ho VI. NHN GING INVITRO V CC C IM KHNG DI TRUYN (EPIGENETIC) Bn cnh nhng c im di truyn thc vt, ngi ta cn pht hin c hng lot c im hnh thi v sinh l khng di truyn, l cc c im pigenetic. Qu trnh nhn ging invitro c nh hng g ti cc c im pigenetic ny. 1. Hin tng cc c im epigenetic c lu li Nghin cu nhn ging v tnh v cy Phyllanthus amarys (cy ch ) cho thy: Phyllanthus l mt cy c c thn thng ng v cnh ngang ging l kp nhng nch l c hoa cho nn vn tm coi nh cnh ngang. Cng thc dip t thn ng l xon c, nhng cnh ngang l tng i so le: nch c cnh ngang chi nch pht trin mnh thnh cnh thng ng nh thn. Nu ct cnh ngang m th s thu c cnh ngang di hng mt. Cnh ngang pht trin v hn theo mt chng trnh "ngang" nh trc. Nu m cnh thng th s thu c cy thng ng. Nh vy m phn sinh ca cnh khc m phn sinh ca thn v khi nhn v tnh cc b phn khc nhau s thu c cc dng cy khc nhau. y m phn sinh nh iu khin m phn sinh cnh pht trin theo hng ngang. Nu ct b phn sinh nh sm th cnh ngang s pht trin thnh cnh ng.

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T bo sinh trng nh (organisator) iu khin s hot ng ca cc t bo khc (tng t nh ng vt). Hin tng iu khin hng pht trin ny c mt ngha thc tin ln lao. Cc cy c ph v ca cao cng c c im sp xp cnh tng t nh vy, v vy khi tin hnh nhn ging v tnh cc loi cy ny phi ch n hin tng iu khin hng pht trin nh Phyllanthus. 2. Hin tng cc c im epignetic khng lu li Nhn ging v tnh invitro ging da Cayen khng gai thng qua phn chia protocorm ngi ta thu c mt t l ng k cc c th c gai. Bnh thng trong k thut trng da ngi ta s dng hom da c kch thc kh ln 15-30cm. c im khng gai c chng trnh ho trong cc t c kch thc ln v vn da trng nh vy u khng c gai. V sao khi tch nh sinh trng v nui cy thnh protocorm v cy da non th gai li xut hin? C th giai on sinh l tr ca protocorm v qu trnh nui cy nh sinh trng phn lp m phn sinh ca nhng cy da non tng lai c gii phng mt phn khi nh hng ca t chc iu khin v v th chng pht trin t do theo c im c gai ca th h xa xa. Hin tng tng t chng ta cng bt gp trng hp cy cam khng gai: Khi gieo ht hoc nui cy phi soma tr m phi tm s thu c nhng c th c gai. Vn ny hin nay ang c quan tm v mt s c tnh chng chu nh chu mn, chu bnh, chu lnh vn thng l nhng c tnh epigenetic v liu thng qua nhn ging invitro cc c tnh c cn c gi li hay khng?

Chng V TH PHN V NUI CY PHI INVITRO 1. Tnh bt hp ca giao t trc khi th tinh Trong t nhin, qu trnh th phn ca thc vt thng xy ra theo trnh t sau: Ht phn chn ri ln nm nhy ny mm v to ra ng phn. ng phn mc di xuyn dc theo nhy v tip cn ti phi. Lc ny 2 nhn tinh t n bi ca ht phn thm nhp vo ti phi v thc hin qu trnh th tinh kp. Mt tinh t kt hp vi t bo non n bi to thnh hp t nh bi sau pht trin thnh phi, tinh t cn li kt hp vi t bo ni nh nh bi to ra hp bo ni nh tam bi.

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Nu mt ht phn l ri ln nm nhy th lp tc nhy s to ra mt cht c ch s pht trin ca ng phn hoc lm bin dng ng phn ngn cn s th tinh. chnh l tnh bt hp giao t trc khi th tinh. 2. Tnh bt hp giao t sau khi th tinh Trong mt s trng hp khc khi ht phn ca loi l ri ln nm nhy, ng phn vn mc bnh thng v qu trnh th tinh xy ra, nhng ht khng pht trin c. Nguyn nhn ch yu l gia ni nh v phi hnh thnh mt c ch c ch s pht trin ca phi. y l trng hp hay gp khi tin hnh lai xa (lai khc loi v khc chi). 3. Th phn invitro Tnh bt hp ca giao t c th vt qua c bng k thut th phn trong ng nghim. Cng vic ny bao gm tch bu qu cng vi non ra v nui cy trong iu kin v trng. Ht phn c ly v t ln u nhy mt cch kho lo. Khi th phn thnh cng, phi s sinh trng v pht trin trn mi trng nui cy. Bin php ny p dng cho nhng trng hp phi khng pht trin bnh thng hoc cho nhng cy c ht phn t v ng phn pht trin chm, t l ht phn ny mm thp. iu kin c bn l phi nui cy thnh cng bu qu hay non phn lp v ch ng iu khin qu trnh ny mm ca ht phn trong mi trng nui cy v trng. Nh vy sau khi th phn thnh cng, phi s c nui ngay trn mi rng v trng. 1942: La Rue l ngi u tin nui thnh cng hoa ca nhiu loi nhng qu li khng c ht. 1951 Nitsch nui n c chua v da chut v ln u tin thu c ht c kh nng ny mm. 1958: Maheshwari tch non v th tinh thnh cng cy thuc phin Papaver soniferum. 1966: Guha v Johri nui c qu hnh ly ht. 1975: Inomata thu c ht tam bi Brassica pakonenusis x Br.sinensis bng nui n qu. 1974: Beasley thnh cng trong vic phn lp non ca nhiu loi cam chanh. Trn c s nhng kt qu t c, ngi ta tin hnh th nghim th phn trong ng nghim, ngha l thc hin qu trnh to hp t khng ph thuc vo c th m. Cng vic gm cc bc. - Kch thch ht phn ny mm.

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- Kch thch sinh trng ng phn. - Nui non v th tinh non. - Nui hp t thnh ht. Mt s tc gi thnh cng: - Kanta v ctv (1962) loi anh tc Papaver sonniferum. - Dulieu (1963) thuc l Nicotiana tabacum. - Zenktenler (1965) h Caryophyllaceae. - Rangaswamy (1967) hoa mo g Pentunia. Mt thi gian di phng php th phn invitro ch thnh cng mt s i tng thuc h Paparaceae, h Caryophyllaceae v h Solanaceae. cc h ny bu qu cha nhiu non nn tng i d nui cy. h ho tho Graminaceae bu qu ch c mt non rt kh nui cy, ng thi ht phn cng kh kch thch ny mm. Sau gn 10 nm tm ti nghin cu ngi ta bt u thu c kt qu mi trn i tng ho tho: Sladky v Havel (1976) bc u nghin cu trn cy ng. Nitzsche v Hennig (1976) nui thnh cng bu qu ca Lobium th phn Festuca. Glunewald (1976) th phn invitro thnh cng non i mch (Hordeum) bng ht phn ca mch en (Secale) v la m (Triticum). 4. Cu phi Trong mt s trng hp, sau th phn phi tiu bin, khng pht trin c. Nguyn nhn ca tnh trng ny c nhiu, nhng ch yu l do ni nh khng pht trin bnh thng v khng cung cp thc n cho phi pht trin. iu ny c th xy ra trong lai cng loi, nhng ch yu l khi lai cc loi khc nhau. V vy, thu c ht lai trong nhng trng hp ny, ngi ta phi nui cy nhm cu phi sau khi th phn c hon thnh. Phi non c tch ra v nui cy trn mi trng thch hp. Thnh cng thu c trong nhiu trng hp lai cng loi: Bng, i mch, c chua, la v khc loi: i mch x La m, Tripsacum x Zea (Raghavan, 1977). Nui cy phi cn c p dng trong cc trng hp t bt th. Mt s loi cy nhit i nh chui di, khoai nc c kh nng sinh sn hu tnh nhng ht ca chng khng ny mm. Do vy, tch phi v nui cy chng trong mi trng thch hp d dng thu c nhng cy con. Vic ny c ngha trong vic chn to ging. Laibach ( 1925) ln u tin nui c phi non ca cy lai bt dc thnh cy non kho mnh theo s di y: Linum perenna x L.austriacum.

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 Fl bt dc (to c phi nhng khng sng) Phn lp phi non, nui cy invitro. Cy non kho mnh. Mt s cp lai khc loi v khc chi thnh cng nh phng php nui cy phi: - Triticum x Secale Tritical. - Hordeum x Secale (Brink v ctv, 1944). - Elyms x Triticum (Ivanovskaja, 1946). - Trisacum x Zea (Farquarharson, 1957). Trong c cy lai hu th ni ting Hordecale: Hordeum maritinum x Secale cereale (Dehue v ctv, 1977). 5. Mt s kh khn trong nui cy phi a) Mi trng dinh dng: Cc mi trng dinh dng ang s dng hin nay thng c p sut thm thu thp hn dch non c nui cy trn mi trng . Rt c th mi trng khng hon ton thch hp. V vy ngi ta ang tm cch s dng dch chit sut t ni nh. Kruse (l974) a ra cng thc "v em" Mi trng dinh dng + Ni nhu khe + Phi lai Vai tr ca ni nh khe cung cp cho phi lai nhng cht cn thit. Cy cho ni nh c th l nhng loi khc nhau ca cng mt chi. b, Pht sinh m so: Khi nui phi non ca t ht lai: Hordedum vulgare x Secale cerale, cc tc gi (Clauss v Kunert, l977) thy phi pht trin thnh khi m so. iu ny ch c th gii thch c rng mi tng tc gia phi v mi trng dinh dng khng bnh thng nh gia phi v ni nh. D sau c th ti sinh c cy hon chnh t khi m so th cy ti sinh cng s mang nhng thay i v m so thng khng n nh v mt di truyn. Ternovsky v ctv (1976) t c mt kt qu l th l thu c cy thuc l c tnh chng chu mi khi nui phi t ht lai khng ny mm.

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Chng VI NUI CY BAO PHN V TO DNG THUN 1. Hin tng n bi ca thc vt Hu ht cc loi cy trng ca chng ta u c mc bi th ln hn 1, ph bin l nh bi 2n v t bi 4n. Nh vy mi c im di truyn nhng c th ny u b hai hay nhiu allen ca mt gene chi phi. Nu l nhng c th d hp t, tc l cc gene trong mi b gene nh bi hay t bi khc nhau th biu hin tnh trng (phenotyp) ca gene hon ton tu thuc vo tnh trng ln hay tri ca chng quyt nh. V vy mc bi th l tng tin hnh nghin cu di truyn cc tnh trng phi l mc n bi hoc cc mc a bi khc nhng chng phi ng nht tuyt i. Haploidy l b nhim sc th c trng ca trng thi bo t ca chu k pht trin ca cy kho t. N th hin s sinh sn hu tnh bnh thng. Thnh thong cc t bo n bi c th phn chia v sinh trng to thnh cy hon chnh, c s nhim sc th bng 1/2 cy m. Cy n bi thu c t cy nh bi gi l monohaploid, t cy a bi c gi n gin l haploid. Thc t c s khc bit v bn cht, v d nu xut pht t tetraploid th c gi l dihaploid, mc bi th cao hn gi l polyhaploid (khc vi polyplid). thc vt, cy n bi c hnh thnh t nhiu cch khc nhau. Hin tng parthenogenesis hoa ci, khi t bo ci khng c th phn vn pht trin thnh cy. Tng t, hoa c hin tng nh vy cng xy ra. Cy n bi cn xut hin khi phn ho phi. Trng hp ny c tn s qu thp trong t nhin. T lu cc nh di truyn v chn ging cy trng bit s dng trng thi n bi ca cy trng tin hnh nghin cu v thng qua a bi ho th n bi thu c cc dng ng hp t tuyt i. Thng thng ngi ta c th thu c n bi bng hai cch: - Chn lc n bi xut hin ngu nhin vi t l rt thp. - Lai xa v sau khi mt b nhim sc th thoi bin s cn li b nhim sc th n bi. C hai cch ny mi ch thnh cng nhiu cc cy trng thuc h ho tho (Gramineae). K thut to cy n bi invitro thng qua kch thch tiu bo t pht trin thnh cy trong nui cy bao phn v ht phn cho php nhanh chng to ra hng lot cy n bi l mt li thot k diu i vi lnh vc ng dng n bi vo nghin cu di truyn v to ging cy trng. Vi cc th bi ca thc vt bc cao ngi ta c th s dng vo cc nghin cu: - Nghin cu di truyn v mi tng tc ca cc gene.

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- To t bin mc n bi. - To dng ng hp t tuyt i. 2. Phng php to cy n bi invitro a) K thut nui cy Nui cy ht phn: Nui cy ht phn thnh cng u tin vo nm 1950 i vi ht phn cy kho t y ng phn v t bo sinh sn to thnh m so. Guha Maheshawari ( 1964, n ) nui cy thnh cng ht phn cy b t to phi v t cc phi ny cho cy con hon chnh. c bit nm 1968, Nizecki v Ocno nui cy thnh cng ht phn la, thu c cy n bi, to ra mt kh nng ng dng rt ln cho cng tc chn to ging la. C 3 phng thc nui cy ht phn sau y: 1. Nui cy ht phn trng thnh trn mi trng na cng. Callus v cy con c pht sinh trc tip t ti phn. 2. Nui cy ti phn trong mi trng nc, ht phn gii phng ra ngoi, phi cy con ti sinh t ht phn. 3. Nui cy ht phn non. Kt qu thu c qua nui cy ti phn c th l: m so, phi, cy con. Nhiu khi trn cng mt th nghim nui cy mt loi ht phn c th thu c c ba loi. Kt qu ph thuc vo giai on pht trin ca ht phn v trng thi sinh l ca cy cho phn. Nhn chung t l ht phn, ti phn cho kt qu rt thp, khong 1-2% s ht phn, ti phn c cy. D nhin l t l ny ph thuc vo bn cht kiu gene v cc cht iu ho sinh trng c b sung vo mi trng, nht l nhm cytokinin BA, zeatin, kinetin, hoc ngun carbon cung cp, glucoza, maltosa v.v... Nhng mi trng chnh s dng cho nui cy ti phn v phn c tp hp trong hai nhm (xem phn ph lc) Nhm 1: Bao gm mui khong, vitamin v ng. Thnh phn mi trng ny dng chung cho c hai giai on: to m so v ti sinh cy con. Nhm 2: Ngoi nhng thnh phn trn, mi trng phi c b sung thm cc cht iu ho sinh trng auxin, cytokinin vi mt t l thch hp, hoc than hot tnh v cc axit amin khc nh: glutamin, serin v asparagin. Nhng loi thc vt s dng mi trng nhm 1 l: thuc l v cy h c c dc. Nhng thc vt s dng mi trng nhm hai l: la, la m. Cc phn v ti phn cy ny to m so sau mi ti sinh cy n bi, hoc 2n. b, Phng thc pht sinh cy n bi Hin tng pht sinh cy n bi t cc t bo giao t c ca thc vt c gi l sinh sn n tnh c (Androgenesis).

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Ngi ta phn bit 3 phng thc sinh sn n tnh c: Sinh sn n tnh trc tip t tiu bo t: Tiu bo t trong bao phn hay phn lp Phi Cy n bi. Cu trc dng phi (embryoid) pht trin trc tip t ht phn. Qu trnh ny thng xy ra trong bao phn. V d in hnh l: C c dc Datura, Thuc l Nicotiana Sinh sn v tnh qua m so: Tiu bo t trong bao phn hay t do M so Chi Cy hon chnh (n=l) Cy hon chnh pht trin t khi m so, khi m ny thng pht trin ra ngoi bao phn. V d: La (Oryza, Ci (Brassica), Hoa hu (Lolium), La mch (Hordeum). Sinh sn n tnh hn h: Giai on pht trin m so xy ra rt ngn v kh nhn bit. V d: C c dc (Datura); C chua (Lycopersicon). c) Cc bc pht trin bnh thng v pht trin phi ca ht phn Bnh thng sau khi c gii phng t t t ht phn cha mt nhn, giai on ny c gi l giai on ht phn n nhn. Sau nhn chia i to thnh 1 nhn dinh dng v 1 nhn sinh dc. Nhn sinh dc li chia i v 2 nhn ny s lm nhim v th tinh kp cho non v ni nh ca ti phi. Sunderland (1970) cho rng: Trong nui cy invitro, bc pht trin u tin ca ht phn vn xy ra nh bnh thng cho ti giai on 2 nhn. Qu trnh to phi thng bt u t nhn sinh dc, nhn dinh dng hay c hai nhn, song ch yu l nhn dinh dng, trong khi nhn sinh dc ch phn chia mt vi ln ri ngng hn. C trng hp ngoi l cy n bi pht trin t nhn sinh dc nhng thng rt yu. Cng c kin ngc li: Raghavan (1977) cho rng phn ln phi Hyocyanus niger thu c bng nui cy bao phn c ngun gc nhn sinh dc. V nguyn tc k thut, nui cy invitro cho php to c n bi bng nhiu con ng khc nhau, nhng con ng bt u t tiu bo t vn l tin li nht.

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Hnh 6.1: Cc con ng pht trin ca tiu bo t trong nui cy bao phn v ht phn dn n s hnh thnh phi n bi d, Nhn bit v cc giai on pht trin ca tiu bo t Mun nui cy thnh cng trc ht phi nm vng 3 giai on pht trin ca cc giao t c trong ti phn. cy thuc l v cc cy hai l mm c th s dng cc ch tiu nh chiu di n, chiu di l i, chiu di cnh hoa v chiu di ch nh (hnh 9.2).

cy mt l mm nh la m hay la nc c th s dng cc ch tiu nh nu trong hnh di y. Mc pht trin ca bao phn rt ph thuc vo v tr hoa trn bng. Do khi tin hnh ly hoa tch bao phn phi ch n v tr hoa trn bng (hnh 6.3)

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e) X l trc khi nui cy N hoc ng trc khi nui cy c x l gy shock bng cch ct khi cy v 2- 5oC trong thi gian 24-72 gi s c hiu qu. Bn cht ca hiu qu kch thch l do tc ng ca lnh: a) Kch thch s pht trin khng bnh thng ca giao t c. b, Tch lu ht phn n nhn (c ch s pht trin tip cc giai on sau). 3. Tnh ton hiu qu nui cy n bi Kt qu nui cy c tnh theo mt s t s sau: a) T l bao phn to callus v phi: (NAC + NAE) CR = 100 NA CR : T l to m so tnh theo % NAC : S bao phn c callus. NAE : S bao phn c phi NA : Tng s bao phn c cy. b, T l bao phn c phi (CE tnh theo %): NAE CE = 100 NA c) T l callus v phi trn s ht phn nui cy (SE tnh theo %): (NC + NE) SE = 100 Na.f

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NC : S callus. NE : S phi. f : S ht phn/Bao phn. d, Nng sut to callus hay to phi (PE): NC + NE PE = NA Quan st ca nhiu tc gi cho thy, dng nhng cy m c ngun gc bao phn (v d nhng cy c ng hp t ho bng nui cy bao phn invitro) thu bao phn cho nui cy tip theo th nng sut to callus v to phi tng ln ng k. La m: - Ging ln Cesar (2n=42) cho CR = 1% - Dng lng n bi (2n=42) cho CR = 1 0% Thuc l: - Ging thun cho CR = 0,27% - Ging lai CR = 1,45% - Ging hn hp CR = 0,55% - Dng lng n bi CR: 4-20 % 4. Hin tng bch tng trong nui cy n bi cc i tng cy hai l mm nh Datura, Atropa, Nicotiana, Brassica... khi nui cy bao phn cy n bi thng pht trin trc tip t tiu bo t v t khi xut hin cy bch tng. Nhng nhng i tng cy mt l mm nh la nc (Oryza), la m (Triticum)... cy hon chnh pht sinh thng qua giai on m so th t l cy b bch tng chim kh cao (20-30 % hoc cao hn na). T l cy bch tng ph thuc vo: a) Tui m so cy chuyn t mi trng to m so sang mi trng ti sinh cy. Cng cy chuyn mun t l bch tng cng cao. b, Nhit nui cy: Nhit cao thng lm tng s lng cy bch tng. Nghin cu v siu cu trc t bo l cy bch tng cho thy trong tin lp th ca cy bch tng khng c ribosom. Nh vy qu trnh sinh tng hp cc protein hoc cc tiu phn protein ca lp th ny khng hon chnh, dn n tnh trng lp v sc khng pht trin thnh lc lp c. Cng c gi thuyt gii thch hin tng bch tng l kt qu ca hiu ng m: Hiu ng m biu hin r mt s c im di truyn t bo cht: Khi th phn ch c nhn ca t bo sinh dc c c chuyn sang t bo non. V vy cc tnh trng di truyn t bo cht ch di truyn theo ng m. Ht phn l t bo cha rt t nguyn sinh cht, tc l s lng ty th v tin lc lp cng rt t. Khi nui nhng t bo ny thnh nhng c th thc vt hon chnh c th xy ra hin tng mt cn i trong tng tc di truyn gia nhn v c quan t, dn n sai lch trong qu trnh pht sinh c quan t, c bit l lc lp.

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5. ng dng n bi Th n bi thc vt c s dng trong cc lnh vc sau: a) Nghin cu v phi hc thc nghim: ch yu trn cc i tng m phi pht trin trc tip t tiu bo t trong nui cy bao phn thng qua qu trnh sinh phi n tnh (androgensis). b, Nghin cu v t bo hc: Cy n bi c th sinh trng v pht trin ti giai on ra hoa, nhng bt dc. Khi nghin cu qu trnh phn bo gim nhim u tin ca t bo m ht phn cy n bi, c th pht hin c nhiu iu l th v quan h tng tc gia cc nhim sc th, bi v b nhim sc th n bi khng th gim nhim bnh thng c. V d: Nghin cu b nhim sc th cy thuc l (Nicotiana tabaccum) nh bi ngi ta thy c 48 NST, lc phn chia gim nhim chng sp thnh 2 dy, gi l dy S v dy T. Mi dy gm 24 NST. Nui cy n bi ca n c s lng NST n=24 v theo di phn chia gim nhim ca t bo m ht phn cng thy cc NST xp thnh cp: 12S + 12T. iu ny chng t b NST n bi ca cy thuc l c nhng biu hin nh mt b NST lng bi, v cc NST trong dy S u tm thy NST tng ng trong dy T. y l kt qu rt ph hp vi lch s pht sinh chng loi ca cy thuc l trng: Nicotiana tabaccum = N. Sylvestris x N. tomentomis (2n = 48) (2n = 24) (2n: 24) (n = 12S) (n = 12T) Hp bo (2n = 12S + 1 2T) Lng bi ho (4x = 24S + 24T) (=b NST ca cy thuc l trng) Nh vy cy thuc l trng l mt dng t bi, nhng khng hon chnh (allotetraploid), biu hin l cy n bi n = 12S + 12T bt th do khng phi tt c NST ca b S u c NST tng ng b T. Cy khoai ty (Solanum tuberosum) cng l mt th t bi khng hon chnh. c) Nghin cu t bin v di truyn Trong b gene n bi khng c quan h tnh tri ln m ch cn quan h b sung gia cc gene, v vy vic nghin cu chc nng v mi tng tc gene c n gin ho ng k. d,To ging thun *To dng ng hp t:

53

Thng thng bng con ng t phi, nu mun t c dng ng hp t ca b gene 2x th phi qua 10 th h v b gene 4x th phi qua 30 th h. Bng nui cy n bi v a bi ch cn 1 th h. so snh hiu qu chn ging bng cc phng php khc nhau: + Cy t th Fl Aa F2 1/4 AA ; 1/2 Aa ; 1/4 aa + Nui cy n bi Fl Aa s cho 1/2 A v 112 a giao t c F H 1/2 AA v 1/2 aa Nu s locus l 10 th t phi cn 410 c th mi c mt c th ng hp F2. Trong khi n bi ch cn 210 cy H c mt c th ng hp. Nu s locus l n th n bi cho php dng ng hp xut hin theo tn s n (1/2) v phng php c in s l (1/4)n . a thm cc h s: NE + EC Pl: tn s to n bi = NA.f P2: t l nh bi ho thnh cng Ta c t s: P1 . P2 . (1/2)n E= (1/4)n Biu th hiu qu so snh gia phng php n bi v phng php c in. Nu E = 1 phng php n bi hiu lc hn c nh vy th: Pl . P2 > (1/2)n, nu n cng ln th Pl . P2 cng nh v nh vy hiu lc ca phng php cng ln. Nhng v gia cc gene c s lin kt vi nhau cho nn phi tnh: Pl.P2 = (1/2)n + n n: biu th s lin kt + C th thng qua tnh ton chn phng php thch hp V d: C 20 gene c lp hoc lin kt theo phng thc tri, siu tri, bng tnh ton cho thy phng php n bi c u th khi: - Gene c tng tc di truyn - B nhim sc rt di hp nhng trng hp tnh i truyn n nh th hai phng php nh nhau. nhng trng hp tnh di truyn khng n nh th phng php n bi c hiu qu hn. * Phn tch so snh hiu qu chn lc trn ng rung. Trong qu trnh nh gi v chn lc ging, hiu qu ca chng thng b nhng yu t sau y cn tr: - Kh nng xut hin cc bin d do gene c hiu ng cng thp. - Cc c tnh gene tri th hin mnh ln t cc c tnh c li khc.

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- Phn ly trong dng cao. Qua thc t lai to, so snh chn lc gia hai phng php cho thy, qua nui cy, to cy nh bi t cy n bi c nhng u im, khc phc c cc thiu st trn. Bng 6.1: Nhng dng kiu hnh (phenotype) cc th h khc nhau Th h F2 (gia cc c th) F3 (gia cc dng) F1 (nhng cy nh bi t cy n bi) Cc bin d VA + VD + VE1 VA + 1/2VD + VEP 2A + VEP

Nh vy chn lc t qun th cc cy nh bi t cy n bi loi tr c s nh hng ca gene tri, ng thi lm tng c kh nng tch lu cc gene c hiu ng cng ln gp i. . - Khi ta xt cc bin d con lai ca cc th h trong phm vi cc l, dng, chng ta cng thy cc u im ca cc phng php to cy n bi. Nhng bin i xut hin trong l: Th h Bin d Trong cc l F3 1/2 VA + 1/2VD + VE1 Trong cc l F4 1/4 VA + 1/4 VD + VE1 Trong qun th cy nh bi t n bi VE1 R rng trong qun th con lai F3 v F4 qua t phi, tn s xut hin cc bin d do gene tri v gene tnh cng vn cn. Nhng qun th cy nh bi ho cy n bi ch chu mt yu t tc ng ln qu trnh chn lc l mi trng. Kasha, Reinberg v Fedak (Canada) i mch x Kiu mch Avena Hordeum Chn lc theo 3 phng php (bng 9.2): - Chn ng Generalogic G - Chn c th Single Seed Desent (SSD) - n bi (DH) Bng 6 .2. Phng php SSD(FG) G(FG) DH Ch tiu Cp lai: Paragon x Zephyr Ngy tr 53,6 54,6 55,8 Nng sut khm 18,8 18,1 17,2 Cp lai OB73 x Champlain Ngy tr 53,0 55,1 53,1 Nng sut khm 21,5 20,4 22,1

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So snh 60 dng DH vi cc dng t phi canada c: 17 dng DH > dng t phi v sn lng 16 dng DH > dng t phi v kch thc ht 14 dng DH chn sm hn 6. Sc sng ca cc c th lng nh bi Nhn chung sc sng ca cc c th DH km hn cc c th d hp. Tnh d hp bo lu tng dn ln v nhng thay i v t khc NST (chromosome) khng sa cha c. Trong trng hp cy DH phi tin hnh t th phn tch lu tr s di hp bo lu - tng sc sng. Hin tng tng gim sc sng lin quan ti tnh d hp c gii thch nh sau: Crick (1974) cho rng nhng on ADN khng to si kp m to cc dng si n. on phn t n ny mn cm vi cc phn t ca mi trng. Gi thit v s sp xp gene trn phn t ADN c vng khuyn S i: gene cm ng R i: gene iu khin S ij: gene cu trc Wallace (1975-1976) ng dng m hnh ny gii thch hin tng siu tri Drosophilla - t bin lm tng sc sng: A1 A 1 A 2 A2 A 1 A2 Hin tng siu tri khng th do mt t bin gy ra c. I th t cc gene mn cm S c sp xp theo th t S 1, S 2, S 3, S 4, nhng do 2 t bin m th t thay i thnh I . vi trt t S 1 , S 3 , S 4 , S 2 . Nu xt theo thi gian th sau khi o trt t cc gene cm ng sn phm gene s xut hin nhanh hn, sc sng ca c th cao hn. 7. Nhng tn ti trong nghin cu n hi Mc d vic kch thch ht phn pht trin thnh cy n bi l nim hy vng to ln cho cng tc ci lng ging cy trng, Melchers (1977) ngh ng dng phng php ny kt hp vi cc phng php c in khc, nui cy bao phn hin nay vn cha p ng c yu cu to ln ca cng tc ging cy trng, v k thut ny mi thnh cng khong trn 30 loi ca trn 20 chi. iu ni bt l thnh cng ny mi ch yu cc chi v loi thuc h c (Solanaceae), h ci (Brasicaceae) v ho tho (Graminaceae). Thi gian qua ngi ta tin hnh cc th nghim vi qui m ln trn i tng cy trng ng cc thuc h ho tho, nhng kt qu mi hn ch la m (Piquard, 1978) v la nc (Niiseki v Oono, 1971, cc tc gi Trung Quc, n , Vit Nam ...).

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Mun ng dng phng php n bi c hiu qu i hi phi c s lng n bi ln. Nhng n nay c th ni chng ta cha nm c chnh xc yu cu dinh dng cn thit. i vi thuc l, mi trng dinh dng nui bao phn rt n gin gm mui khong v ng saccharose, khng cn cc cht hu c khc v hoc mn sinh trng (Vasil v Nitsch, 1975). Th nhng nui cy bao phn la nc v la m thnh cng, cc tc gi Trung Quc phi dng thm dch chit khoai ty m thnh phn cha c bit ti. Mc d vy bao phn cc loi ng cc c nui cy cng ch to m so, c cy hon chnh phi tin hnh to chi t m so . Thng thng th t l bch tng rt cao. V d Mix v ctv (1977) nhn c 3400 cy bch tng trong s 4000 cy yn mch ti sinh t m so bao phn. Ngoi ra trong s c th thu c thng qua bc ti sinh t m so mt t l ng k l cy nh bi (60%). Trong nui cy bao phn, vic xut hin nhng phi lng bi t t bo lng bi ca v bao phn cha th loi tr c. V vy ngi ta ang th nghim to cy n bi t ht phn phn lp. ng nhin mi trng nui cy ht phn phn lp i hi phc tp hn mi trng dinh dng nui cy bao phn. Mi trng nui ht phn Petunia c cha auxin, cytokinin v acid boric. Thng l ngi ta phi nui c bao phn 4- 16 ngy trn mt mi trng dinh dng ri sau mi tch ring ht phn nui tip tc trn mi trng c . Hiu qu ca phng php ny rt cao, t ti 1000 phi/a petri. Thng qua qu trnh nui trc mi trng dinh dng c b sung thm nhng cht cn thit do bao phn tit ra. Glutamin l mt thnh phn quan trng, nhng cn nhiu cht khc vn cha c bit ti. Sau y trnh by s lc qui trnh to cy n bi thuc l t ht phn phn lp (theo Nitsch, 1976). a) Cm ng + To cy n bi: N hoa c x l bng ly tm 2000 vng pht trong thi gian 30 pht nhit 5- 10oC, sau khi ct 48 gi 2-5oC + To cy nh bi: N hoa c ngm trong dung dch 0.04% colsicin v 2% dimethyl sulfoxide (cht dn np) trong thi gian 24 gi 2-5oC v ht chn khng. Sau ra sch dung dch colchicin v x l lnh tip 24 gi. b, Nui bao phn Bao phn c tch t n, nui 3 ngy trong mi trng khong Halperin cha 2% ng saccharoza v Fe - EDTA (5ml/l; Na2 EDTA 754 mg + FeSO4.7H2O 557mg pha trong 100ml nc si), pH 5,8. Nui 50 bao phn trong 5ml mi trng lng. c) Tch v nui ht phn p bao phn bng a thu tinh, lc ht phn bng li c mt 48 m v a vo ng ly tm v trng c bng y. Ly tm 850 vng/pht v ra hai ln bng mi trng mi. Nui ht phn vi nng 104 ht phn/ml, mi a petri ng

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knh 5 cm nui 2,5 ml dung dch, dn parafilm v ni c nh sng nht. Sau 30 ngy s xut hin phi non. Bng 6.3: Mt s i tng nui ht phn phn lp thnh cng S TT 1 2 3 4 5 i tng Ci (Brassica) C chua (Lycopersicon) C c dc (Datura) Thuc l (Nicotiana) Hyoscyanus Tc gi, nm Kameya v Hinata (1970) Sharp v ctv (1972) Nitsch v Noreel (1973) Nitsch (1976) Wernicke v Kohlenbach (1977)

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Chng VII NUI CY T BO TRN V NG DNG Da trn nhng thnh cng trong dung hp t bo ng vt v ngi (Honor Fell m t nhng th nghim u tin vo nm 1959) m cc nh sinh l thc vt ny sinh ra tng ghp t bo thc vt vi nhau. Thng thng t bo ng vt khng c v nn d dng ghp vi nhau, trong khi t bo thc vt c v cellulose bao bc khng th ghp vi nhau c. Cc nh thc nghim phi mt nhiu nm mi ph b c thnh cellulose ca t bo thc vt. Hai bin php ph thnh t bo l bin php c hc v bin php enzyme. Ngi c cng ln trong vn ny l Cocking (1960-1973). Tuy vy k thut protoplast ch pht trin mnh t sau cng trnh ca Takebe (1971) ti sinh cy hon chnh t protoplast tch t l thuc l. I. PHNG PHP TCH V NUI CY T BO TRN Mc d t liu khoa hc tng kt rng c hai cch tch protoplast. l bin php c hc v bin php men hc. Nhng trong thc t, phng php men hc c hiu qu hn rt nhiu phng php c hc, phng php men hc c th tch c hng gram protoplast. Cc enzym c dng l cellulase, pectinase hay hemicellulase hon ton khng c hi i vi t bo. Bi v thnh t bo c thnh phn gm pectin, cellulose, hemicellulose cho nn phi s dng hn hp enzyme bao gm: Pectinase ph hu pectin. Cellulase ph hu cellulose. Hemicellulase ph hu hemicellulose. Ngoi ra, protoplast khng b v sau khi thnh cellulose b ph hu, ngi ta phi s dng nhng cht lm tng p sut thm thu vo dung dch enzyme. Thnh phn dch enzyme gm c (theo Constabel, 1975): - Pectinase - Cellulase - Hemicellulase - Sorbitol - Mannitol - CaCl2.2H2O - CaH4(PO4). 2H2O Tu theo tng i tng v tng loi m m thay i nng cho thch hp. - Sau khi x l enzyme cellulas, protoplast c thu nht li bng my ly tm, sau em ra sch vi mi trng khng cha men v cui cng protoplast chuyn sang dung dch manitol: - To nh dch protolast vi mt l0 4 - l07/ml cht nguyn sinh c cy trn a trn mi trng mm, hoc trong mi trng lng:

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- Sau khi cy 5 - 10 ngy, cht nguyn sinh tng hp thnh t bo mi v bt u phn chia. Trong giai on ny, khi t bo phn chia, mi trng c th c pha long vi mi trng mi duy tr s sinh trng v phn chia ca t bo. V protoplast thc cht l t bo trn khng c thnh cho nn c th tch c t nhiu ngun khc nhau nh cc b phn ca cy (ch yu l mesophyl ca l, m so, t bo n). II. NG DNG T BO TRN Protoplast c th c s dng nghin cu ba vn chnh sau y: - Chn dng t bo. - Dung hp protoplast lai v tnh t bo thc vt. - Bin np di truyn, a cc c quan t virus v DNA ngoi lai vo t bo thc vt. 1. Chn dng t bo Thc vt thng ng thng c kch thc kh ln cho nn cc k thut x l v chn dng kh thc hin vi s lng c th theo tnh ton xc sut thng k, chnh v vy k thut chn dng thng c ng dng i tng vi sinh vt v t c kt qu rt kh quan. Vi bin php b thnh cellulose v a c th thc vt v trng thi tng t bo ring r vi kch thc khng ln hn nhiu so vi c th vi sinh vt cho php tin hnh k thut chn ging vi sinh vt i vi thc vt thng ng v h ng. Mt a petri ng knh 5-7cm cho php nui ti 5.106 protoplast thuc l trong khi mun nui trng 5.10 6 cy thuc l cn c trn 5.106 m2, tc trn l l00ha t canh tc. Ngoi ra c th thc vt thng ng l c th a bo c phn ho thnh cc t chc khc nhau: Nu tin hnh x l t bin ton cy th rt kh t c tn s cn thit. S dng t bo trn ring r cho php loi b mi tng tc vi cc t bo bn cnh v nhng thay i di truyn c iu kin biu hin r rng hn. Nhng kt qu c th ng dng protoplast trong chn ging t bo s c trnh by chng VII. 2. Dung hp t bo trn v lai v tnh a) Bn cht ca lai sinh dng (somatic hybridization) L qu trnh dung hp hai t bo trn sinh dng (soma) to thnh mt t bo lai hn hp. Trong vic lai cc t bo soma xy ra hai hin tng: - Dung hp cht nguyn sinh, cc c quan t. - Dung hp nhn.

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Trng hp lai sinh dng trn vn l trong c hai qu trnh dung hp u xy ra, nhng tn s xy ra thp. Thng th ch xy ra dung hp nhn, cn t bo cht sau khi dung hp mt thi gian ngn c s chn lc o thi, v ch mt trong hai t bo pht trin. C hai hnh thc u cho ra nhng t bo lai thc s gi l hybrids. mt s trng hp khc, dung hp ch xy ra vi t bo cht v c quan t, cn nhn vn gi nguyn, to nn cc t bo cybrids (hnh l0.1). b, Cc phng php dung hp t bo T Vic lai hai t bo trn thc vt c th c thc hin bng hai phng php: 1. Ho hc: Cc cht nguyn sinh c p sut thm thu ging nhau, dung hp chng ch cn s c mt ca mt s cht ho hc nh: - NaNO3 to iu kin t bo dung hp c 25%.

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Hnh 7.2. Chuyn gene CMS bng dung hp t bo trn cy thuc l - Polyvinyl alcohol dextrant v PEG, cng vi Ca++ v pH=8 c th gy mt t l kt hp cc t bo t 1 - 100% . 2. L hc (Electro-fusion): Nh nhng xung in ca dng in trc tip lm thay i din tch b mt ca mng plasmalema, to ra sc ht gia cc t bo trn. Bng cch ny t bo dung hp t t l 100%. Vic nhn bit, chn lc cc t bo lai c quan st bng mt di knh hin vi ngc. Thng qua nhng du hiu ch th nh: tnh pht quang hoc nui cy chng trn mi trng c nhng enzyme, hoc sn phm ho hc c trng: c) Mt s th nghim v kt qu nghin cu Nm 1972, Carlson ti sinh c cy thuc l lai t sn phm dung hp protoplast ca hai loi: Nicotiana glanca + N. langstorff, bt u mt thi k pht trin mi ca k thut protoplast. Th nghim ca Melchers (1974): ng dng protopplast ca hai loi thuc l Nicotiana tabacum: 62

- Ging s (sublethal) khng tng hp c dip lc bnh thng nn rt mn cm vi nh sng cng cao. - Ging v (virescent) cng khng to c lc lp bnh thng l non trng hon ton v mn cm vi nh sng cng cao. Hai ging ny l hai dng t bin c th b sung cho nhau c khi lai to, ngha l nui cy nh sng 800 lux ti giai on ra hoa, sau cho th phn cho s thu c cy lai F1 c l xanh bnh thng v chu nh sng cao (10000 lux). Melchers tin hnh tch protoplast ca cy n bi thu c t nui cy bao phn ca cy s v cy v ri cho dung hp. Sn phm dung hp c chn lc trn mi trng chiu 100001ux cho kt qu ch nhng hp bo sng v pht trin thnh khi m so xanh, trong khi cc loi t bo b m u cht. Cy ti sinh t khi m so xanh c l xanh bnh thng v chu c nh sng cng cao. Thnh cng ca Melchers l s dng hai t bin b sung v chng minh c sn phm dung hp mang gene b sung . Th nghim ca Schieder 1975: Schieder s dng 2 loi t bin dinh dng ca loi ru Sphaerocarpos donollii: - t bin A khng tng hp c axit nicotinic - t bin B khng to c glucose Mun nui A phi a axit nicotinic vo mi trng v mun nui B phi a glucose vo mi trng. Tch protoplast t A v B sau tin hnh dung hp. Nui ton b mi trng thiu glucose v axit nicotinic. Cc loi protoplast b m hoc sn phm dung hp cng loi cht v thiu glucose hoc axit nicotinic. Ch c sn phm dung hp chnh thc gia A v B c th sng st c, sau c ti sinh thnh cy lai c kh nng tng hp axit nicotinic v glucose. Phng thc to cy lai nh va trnh by khng bao gi c trong thin nhin, m ch thc hin c bng con ng lai protolast. Thi gian qua ngi ta tin hnh lai mt s cp rt xa nhau. V d: Maliga (1976) lai t bo trn ca tng v la, nhng sn phm dung hp mt dn kh nng phn chia. Cng Hungari trong Trung tm nghin cu sinh hc Szeged ngi ta tin hnh lai t bo c rt vi t bo ngi v sn phm dung hp sng c mt thi gian c phn bo v tng hp axit nucleic. 1976, Kao nhn c t bo lai gia tng vi thuc l v nui c 4 thng. Qua hng ngn th h phn bo, hai b nhim sc th vn ng thi hot ng v th hin tnh cht ca t bo lai. Kt qu ca Melchers trong lai protoplast gia c chua v khoai ty nh du mt mc quan trng trong lnh vc ny. Cy lai c chua v khoai ty u tin ra i nm 1977 khi lai protoplast ca cy c chua Lycopersicon esculentum (Mill)/var.cerasiforme (Dunal) Alef (t bin xanh vng Rich) vi protoplast t bo m so nui trong dch lng ca dng khoai

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ty lng bi DH s HH 258. Sn phm dung hp lc u ch ti sinh r v sau thu c chi nhng khng phn bit c chi cy lai hay chi khoai ty c mu l bt thng. Phn tch nhim sc th khng kt qu v nhim sc th khoai ty v c chua rt kh phn bit hnh dng. Nh c bin php ca Holdn l gia c chua v khoai ty c s khc nhau v bng protein ca enzim ribulose-l,5- biphosphat carboxylase trn in di SDS t m Melchers phn bit c sn phm dung hp vi t bo b m. Cc dng cy lai c pht hin: - Cy lai c plastome(b gene lc lp) khoai ty - Cy lai c plastome c chua Dng 1: potato + tomato c plastome ca potato c gi l pmatoes (ting c Karmaten ghp ca hai t kartoffel + tomaten). Dng 2: potato + tomato nhng c plastome ca tomato c gi l topatoes (ting c tomoffeln ghp ca hai t tomaten + kartoffeln). Trong hai dng ny, mt s cy c kh nng to b phn ging c nhng hoa vn bt th. Vn ny ang c nghin cu, hy vng s to ra c cy rau c qu c chua v c khoai ty. 3. Bin np gen vo t bo trn Thng tin di truyn desoxyribonucleic acid DNA tn ti 3 dng chnh: - DNA c th thng ng trong c DNA nhn, DNA c quan t - DNA vi sinh vt - DNA plasmid. Bin np thng tin di truyn (transformation) l k thut s dng DNA tinh khit a vo c th hay t bo khc v theo di biu hin ca thng tin di truyn mi ny. Hin tng transformation c pht hin ln u tin vi khun Pneumococcus trong cc th nghim ca Avery, Macleod v Mc Carty (1944). a) Cc phng php a DNA vo c th v t bo khc 1) X l trc tip vi dung dch DNA: Phng php ny c Ledoux v Hep p dng i vi ht hoc mm cy. Hiu qu khng cao v khng chc chn. Tn s 10 -5- 10-7. Ngu nhin 10 -6. Mt s tc gi ci tin bng cch x l DNA + protamin (mt thnh phn ca protein histon trong nhim sc th) a c tn s bin np t 29,4% ng. y l mt kt qu bt ng vt qu sc tng tng. Phi kim tra li. Mt s cng trnh s dng DNA dng plasmid trn vi ht phn th phn nhn to v hy vng ng thi thu c kt qu bin np. Nhn chung k thut x l trc tip vi DNA khng c ph cp v km hiu qu v kh lp li. Tuy nhin n c mt s phng th nghim thc hin trong thi k s khai ca k thut DNA ti t hp vo nhng nm u ca thp nin 70.

64

2) S dng cc vector t nhin: Chng ta bit trong t nhin vn xy ra hin tng bin np gene nhiu dng khc nhau. V d nh: Thc khun (Phage) khi bm vo thnh t bo th ch a DNA vo t bo ch, DNA genom ca thc khun biu hin v tng hp thc khun mi. Siu vi khun - virus (v d SV40). Cu to gm acid nucleic v protein (tng ng vi nhim sc th) thm nhp vo t bo vt ch v s dng b my sinh tng hp acid nucleic v protein to ra virus mi. Plasmid: Trong t bo ca gii ng vt v gii thc vt u tn ti cc th plasmid, l cc vng DNA t sinh sn c lp. vi khun. thc vt v ng vt plasmid lin quan ti yu t gii tnh ca t bo, n kh nng chng chu cc loi khng sinh.... c im quan trng ca plasmid l chng c th lin kt vo nhim sc th nhng cng c th tn ti bn ngoi nhim sc th mt cch c lp. Ngy nay ngi ta ang s dng nhiu loi vector c ngun gc t nhin khc nhau, k c nhn to hon ton a DNA l vo t bo. V d: thc khun virus SV40, plasmid pBR 322, virus khm xupl CMV, plasmid Ti ca vi khun t Agrobacterium tumerfaciens... Cc loi plasmid u c thit k c mang gene chn lc khng khng sinh nh penicillin, tetracyclin, steptomycin, hygocillin, kanamycin... b) Bin np gene vo prooplast thc vt K thut gene l lnh vc khoa hc cng ngh nghin cu v trin khai cc k thut v nucleic acid v gene nhm ch ng trong vic phn lp, thit k, bin np v th hin chng. dy ch nu s lc cc bc chnh. Trc ht nhn bit gen thng qua chc nng sinh hc ca n, c th l tnh trng lin quan n c im hnh thi (gene ln, gene mu sc hoa,...), c im chc nng (sinh tng hp cao, chng chu...), ni chung l cc c im chng ta quan tm. Xc nh iu kin c trng cho qu trnh biu hin v km hm gene. Trn c s tin hnh cc bc phn lp v xc nh gene theo trnh t sau: Phn lp RNA (mRNA) thng tin ca gene A, chn loi t bo v giai on thch hp m qu trnh sao m transcription DNA mRNA xy ra mnh. V d: 90% mRNA ca t bo tu sng to mu l mRNA ca hemoglobin hoc 95% mRNA ca t bo d con g l mRNA ca lng trng trng. Phn lp bng cc k thut: - in di SDS - Sc k ct poliU-cellulose hoc oligo dT-cellulose Tng hp gene invitro: Bng enzym Reverse Transcriptase (do Baltimore, Timin v Dulbecco phn lp c t virus RS kh v nhn gii Nobel,1975): Transferase thng thng xc tc sinh tng hp RNA t DNA cn transcnptase ngc li xc tc sinh tng hp DNA t RNA (trong thin nhin l 65

phng thc tn ti ca cc loi virus cha RNA). Sn phm thu c l cc phn t cDNA (complementary DNA). Thit lp th vin cDNA t chn lc v nhn cc dng cDNA c trng. S dng cDNA c trng lm phn t nh du phn lp ra on gene tng ng trong th vin genom. Xc nh ton b gene cn thit km cc on iu khin. Cn c cch khc l i t protein l sn phm do gene cn tm to ra. Xc nh trnh t acid amin ca protein . T thit lp trnh t nucleotid ca gene. Tng hp mt on oligonucleotid bng my DNA synthcsizer lm phn t nh du cho lai phn t chn lc gene t th vin genom. Gn gene vi vector: Thng thng ngi ta s dng cc loi plasmid mang gene khng sinh tin li cho vic chn sn phm bin np sau ny. V d: Giai on u plasmis pBR 322 cha gene khng pinicillin v tetracyclin thng c s dng. V sau pUC l8 v pUC 19 tr nn thng dng hn i vi t bo thc vt. Khi s dng phng php bin np qua Agrobacterium, nht thit phi dng Ti dng c ci bin. Dng mt loi enzym ct m vng DNA plasmid. loi enzym ct l restriction endonuclease (RE) tn ti trong cc t bo c kh nng khng phage v virus l theo phng thc chn ct si DNA vo nhng v tr gia cc cp baz nht nh. V d: EcoERI ct gia GA v AG bin vng hay si DNA thnh tng (mu) on nh lm mt kh nng sinh sn ca thc khun hoc virus. Pht hin ra RE c nhn gii Nobel, 1978. Cc on DNA ng vai tr gene l s c ghp vo cc v tr cloning thch hp ca plasmid tu theo mc ch chn lc v theo di sau ny (xem phn sau). a vo t bo: X l vi nng t bo v plasmid thch hp sau mt thi gian nht nh t bo ch cha mt plasmid (phn ln t bo s khng c plasmid). C nhiu phng php bin np plasmid khc nhau: - Bin np bng ho cht: ch yu dng PGE - Bin np bng xung in (trn my chuyn dng) - electroporation - Bin np bng sng bn gene - particle gn - Bin np thng qua Agrobaterium Hiu qu s dng ca tng phng php s c tho lun k chng Cng ngh gene thc vt. Chn lc sn phm bin np: Nui trn mi trng chn lc cha khng sinh c hiu cho loi plasmid c bin np. V d: tetracyclin, kanamycin, hygromycin ... Ch nhng t bo c plasmid mang gene khng khng sinh mi c th sng st v pht trin trn mi trng chn lc. nh gi kt qu bin np v theo di s phn c gene bin np: y l cng on cn gp nhiu kh khn, nht l khi t bo nhn l t bo thng ng. nhng t bo vi khun nhn gene l, hot ng ca gene l biu hin tt. Thng thng c nhng cch thc sau: - Theo di biu hin ca gene bin np 66

- Phn lp v nhn gene bin np bng k thut PCR (polymerization chain reacbon). - Phn tch RFLP (restriction fragment lenght polymorphism). IV. TN TI CA K THUT PROTOPLAST 80% thc n ca nhn loi u do cc cy trng ng cc to ra. Nu mt k thut mi ra i phc v c cng tc ci lng ging, nhng cy trng quan trng ny s mang li hiu qu v cng to ln. K thut protoplast thu c thnh cng cc loi thuc h c Solanaceae, h Brassiccaceae v mt s h khc nhng thnh cng h ho tho Gramineae - l h ca cc cy trng ng cc chnh cn hn ch. Cy la nc (Oryza sativa) l mt i tng ng cc thnh cng nht trong nui cy protoplast, nhng vn ch hn ch cc ging thuc loi ph Japonica. Cc ging la thuc di chi Indica vn kh ti sinh thnh cng v trong s cy ti sinh c, t l bt th thng rt cao. Sau y l nhng yu t c nh hng n kt qu nui cy v ti sinh t protoplast trong nui cy invitro: i vi vic tch protoplast: 1. C s i truyn i vi (kh nng tim tng ca t bo trong nui cy invitro). 2. Tng tc t bo trong cy. 3. C ch phn ho trong qu trnh pht trin c th. 4. Ngun gc c quan v cy hon chnh. 5. Trng thi sinh l ca t bo. 6. Tc ng ca qu trnh phn lp. 7. Tc ng trng thi phn lp. i vi iu kin nui cy: 8. Nhu cu dinh dng. 9. Nhu cu hoc mn. 10. iu kin vt l nui cy. 11. Cc yu t c ch c th xut hin. 12. Tc ng mt qun th t bo i vi qu trnh phn bo u tin: 13. Sinh tng hp thnh t bo. 14. iu khin sinh tng hp thnh t bo. 15. Chc nng ca thnh t bo i vi phn bo. 16. iu khin s phn phn bo. 17. iu khin phn chia nhn. 18. iu khin phn bo. i vi qu trnh phn ho: 19. iu khin phn ho t bo. 20. Tng tc t bo trong nui cy.

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21. iu khin v c ch to kiu m. 22. iu khin qu trnh to c quan, phi.

Chng VIII TO T BIN V CHN DNG T BO K thut chn dng t bo ra i rt sm trong nghin cu vi sinh vt. Nhng i vi cc i tng thc vt thng ng, k thut ny mi c ng dng t thp nin 70. C th tin hnh x l v chn lc t bo thc vt theo ba mc : 1. M so 2. T bo n. 3. T bo trn. Mc ch chn lc c th khi qut nhng im sau: 1. Chn dng t bo chng chu iu kin bt li ca ngoi cnh phi sinh hc nh: nng, lnh, phn, mn, hn, ng... 2. Chn dng t bo khng cc c t do cc tc nhn sinh hc gy ra nh: c t do nm bnh tit ra, cc loi khng sinh do vi sinh vt sn sinh ra... 3 . Chn dng sn xut d tha cc loi sn phm, ch yu l acid amin. 4. Chn cc dng mang nhng c im nh du nghin cu di truyn (genetic markers). 1. Phng thc chn dng t bo a) Chn trc tip Thng qua u th v sinh trng hay s khc bit thy c v mu sc, c th chn c dng t bo t qun th t bo. Mt s dng c kh nng chu khng sinh, chu cc cht tng t amino acid hoc mui cng c th chn trc tip t qun th t bo. H thng t bo hay c s dng l cc t bo nui dch lng. iu kin chn lc y l cc dc t vi nng khc nhau gy tc ng trc tip ln sinh trng ca t bo. Nhng t bo c kh nng phn chia trong iu kin cha c t vi nng tng dn t ln cy chuyn ny n ln cy chuyn khc c sng lc dn. Hoc c th a c qun th t bo vo iu kin mi trng c ch sinh trng hon ton chn ra nhng t bo sng st. Thng ngi ta trn t bo vo mi trng thch cha dc t v ch nhng t bo sng st phn chia thnh khun lc m so, cng c th cy trc tip ln mi trng chn lc cha dc t. Dng chng chu thng xut hin t mt phn ca khi t bo nui cy.

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b, Chn gin tip Trong trng hp ny, c im ca dng c chn l kt qu biu hin khuyt tt ca t bo. Th d in hnh l chn dng thiu enzym nitratreductase (NR). Trn mi trng cha CIO3 nhng t bo c NR s dng CIO3 nh NO3 v kh thnh chlorit. Chlorit tc dng nh mt c t cho nn ch c nhng t bo khng c NR mi sng st. c) Chn tng th Cc 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 c khi li chnh l yu t gy t bin, v d t bin ln chu c S-2-aminoethyl cysteine xut hin sau khi x l t bin phi nui cy. 2. t bin v chn lc cc bin d a) Bin d soma Thut ng "bin d sinh dng" c Larkin v Scowcroft (1981) dng ch tt c cc bin d xy ra trong qu trnh nui cy m, t bo. Bn cht v c ch ca hin tng ny lin quan mt thit n nhng thay i trong genom ca t bo nui cy. Do nhiu nguyn nhn (tc ng ca cc hocmon sinh trng trong thi gian di v cc yu t khc) s nhim sc th trong t bo nui cy c th tng ln (bng 8. 1 ) to ra cc dng a bi lch v mc bi th cao. Bng 8.1: Thay i s nhim sc th trong nui cy Nicotiana tabacum (Murashige & Nakano, 1967) Vt liu nui cy S lng t bo quan st 53 44 15 Mc bi th 8x 16x a bi lch 52.8 70.5 15.9 26.7 20 53.3 100

4x 47.2 9.1 -

Cung l 3,5 10, 5 cm Cung l 15,5 22, 5 cm M so cung l sau mt nm M so cung l sau su nm

Nhiu trng hp, cc on ca nhim sc th c chuyn i hoc o ngc. Cu trc ca phn t DNA cng c th b thay i dn n t bin kiu hnh thc s. Nhng kiu hnh thu c trong nui cy c nhng c im sau: a) Bn vng qua nhiu ln nui cy.

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b, Tn s xy ra thp (10 -6 10 -10), khi dng tc nhn gy t bin, tn s c th tng. c) C quan h cht ch vi nhng sn phm ca gene c trng. Bin d sinh dng c khai thc phc v cng tc ci tin ging cy trng. V d: Nui cy m t bo cy ma thu c nhng dng ma c kh nng chng chu bnh Piji (do virus gy ra) v bnh Hexminthosporium Sachari (Heiz v ctv, 1977). i Loan, ging ma c nng sut v hm lng ng cao c to ra bng cch nui cy v chn lc dng bng m so. thuc l, nui cy ht phn v 1 t bo trn thu c nhng cy t bin c nng sut cao, thi gian sinh trng ngn, hm lng ng v alkaloid thp. khoai ty, thu c 1000 dng khc nhau t nui cy t bo trn. Qua chn lc, ng thu c ging Ruset Burbank c thi gian sinh trng ngn, c u, hnh dng p, mu pht hng. Nhng c tnh ny bn vng qua nhiu th h sinh sn v tnh v hu tnh v lm cho Ruset Burbank hn hn ging gc ban u. b, X l t bin Cc dng t bin chn lc c trong nui cy invitro thng xut hin vi tn s 10 -5 - 10-8 trong trng hp khng x l. Nu nhng t bo c x l vi cc tc nhn th tn s c th tng ln 10 ln (bng 8.2). Bng 8.2. X l gy t bin nui cy t bo thc vt (Maliga, 1980) Loi cy X l T l t bo sng (%) 10 1 50 42-60 Kiu hnh chn lc Chng chu Cycloheximide Chlorate Thay i sc t Chng chu valine Tn s xut hin T nhin C x l 5,4 x 10 7,7 x 8 10 -7 5,4 x 10 - 4,4 x 8 10 -6 6,6 x 10 - 2 x 10 8 -7 10 -5 2,2 x 10 - 4 x 10
6 -4

C rt Thuc l Hoa mo g Thuc l

EMS (2,5%, 2h) NG (100prg/ml,14h) NEV (25mM) Tia X (1000R) Tia t ngoi cc tm (1000erdmm2)

1,4 x 10 -5

EMS: Ethyl methare sulphonate sulplte NEU: N-ethylnitrosourea

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Ngoi cc tc nhn trn (EMS, NEU, tia X, tia t ngoi), ngi ta cn dng Al (NO3)3 AlCl3 NaCl v mt s cht khc. t bin c th gy ra nhng s thay i cu trc bc 1 ca DNA, xut hin tnh trng mi, c di truyn cho th h sau thng qua sinh sn hu tnh. Cc tnh trng ny chu s phn ly di truyn theo quy lut ca Mendel, tn s xut hin thp l0 -6 10-10. c) t bin hay thay i hot tnh gene (t bin phi l kt qu r rng ca s thay i cu trc bc 1 ca DNA c di truyn dn n nhng bin i v tnh trng) Thng thng trong nui cy m t bo, t bin c chn thng qua thay i tnh trng ca t bo. Nhng nhng thay i v tnh trng t bo c th l kt qu ca thay i hot tnh gene. V d: Dng t bo thuc l khng Cycloheximid (CH) ca Maliga (1976) nu cy chuyn 1 -2 ln sang mi trng khng cha CH th kh nng khng CH mt i. iu chng t tnh khng CH do 1 gene quyt nh v lc bnh thng gene khng th hin hot tnh. Tng t nh vy ngi ta cng gp dng t bo c rt chu 2,4-D ca Widholm (1 977). Dng t bo c rt khng colchicin l kt qu thay i tnh cht mng t bo. Cc dng bin i nh vy do mt c ch khng di truyn (epigenetic mechanism) iu khin. Thay i tnh trng do t bin phi l nhng tnh trng chuyn qua th h sau bng sinh sn hu tnh c. t bin ca DNA nhn s phn ly theo nh lut Mendel sau khi lai, cn t bin DNA c quan t nh lc lp v ty th th c th di truyn ng m. Cho ti lc lai xong, tt nht ch nn gi cc dng va phn lp c l cc dng t bo "hoc cc dng thay i tnh trng". Khi ti sinh cy t mt dng t bo cng c th xut hin nhng cy khng di truyn tnh trng chn. iu c th gii thch nh sau: lc chn, mt s t bo mn cm c cc t bo chng chu xung quanh bo v bng cch cung cp cc sn phm trao i cht hoc enzym qua cc khe t bo. Nhng t bo mn cm cng c th xut hin thng qua hin tng tch t bo v tnh, hin tng to khm hoc nhng bin i di truyn (bach mutation = thoi bin) v khng di truyn (epigenetic). 3. Mt s dng t bo c chn thnh cng a) Khng amino acid v cc cht ng ng Mt s acid amin, trong c valine v threonin c ch sinh trng t bo khi a mnh chng vo mi trng dng ng nui cy, v chng c ch t bo s dng NO3 hoc ngn cn acid amin cng ngun gc:

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Ngoi ra nu s dng cc cht tng t acid amin, chng c th c s dng nh acid amin vo vic tng hp ra cc phn t protein, kt qu l cc phn t ny b mt hot tnh. Hin tng c ch ny c th khc phc bng cch cung cp cho t bo thm cc acid amin khc hoc cc hp cht l dn xut bnh thng khc ca chng. i vi t bo c th t khc phc bng cch tng cng tng hp cc acid amin thch ng. l nguyn nhn v sao cc dng t bo khng c cc cht tng t acid amin li sn xut d tha acid amin. C ch ca hin tng sn xut d tha (over production) l qu trnh c ch ngc (feed bach inhibition) km mn cm hn. Trong dng t bo khng 5-methyl tryptophan, c ch c ch sn phm ngc ny km mn cm v vy hot tnh ca anthranilat synthetase vn cao v lng tryptophan c to ra cao gp 5 ln so vi bnh thng. cc dng khng p-fluorophenyl alanin, c ch c ch sn phm ngc hot ng ca enzym chorismat mutase km mn cm vi phenylalanin v tyrosin, v vy lng phenyl c to ra rt cao. Trong thc t hin tng kim tra lng lo cc qu trnh sinh tng hp acid amin vn tn ti khng cn s c mt cc cht ng ng acid amin. Cc acid amin t do ny (phe, tyr, met, lys) c tch lu li hay chuyn ho tip (th d thnh cc hp cht phenol). Hin tng thi acid amin t do ra mi trng ch gp i vi prolin (pro). Ngoi ra cc cht tng t acid amin cn c th do nhng c ch khc gy ra, v d nh: - T bo hn ch thu nhn cc cht tng t acid amin. Hot ng thu nhn cc cht rt yu. - T bo c kh nng chn cc acid amin bnh thng tng hp protein trong khi cc cht tng t acid amin b b li: - Trng hp khng theonin th t bo c nhng thay i trong h thng enzym s dng N03. Nghin cu v tnh khng cht tng t acid amin c ngha thc tin rt ln. Ngi ta hy vng s to c cc ging cy c gi tr dinh dng cao... Chng hn Widholm (1977) chn c mt dng t bo c rt c sn xut 27 ln tryptophan t do nhiu hn bnh thng v tng 25% lng tryptophan tng s (t do + trong protein). mt dng t bo c rt khc cng mt lc khng c 4 cht tng t amino acid th hm lng cc amino acid tng ng l Lys, Phe, Met v Try tng t 6 - 34 ln. Tuy nhin biu hin s sn xut d tha ny c cn gi c sau khi ti sinh cy hay khng ? l iu cha c kt lun. Vn ti sinh cy t dng t bo khng 5-methyltryptophan l vn sinh l kh l th. Khng 5-methytryptophan s sn xut d tha tryptophan m tryptophan li l tin cht ca IAA nn t bo s sn xut d tha IAA v nh vy t bo sinh

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trng t to auxin ngha l khng cn auxin ngoi a vo mi trng. Lng IAA tng cng lm cho t bo mt kh nng to phi v to chi. Ch c 10 -6 t bo c th to cy, c th do mt t bin th hai xy ra trong trao i cht tryp hay IAA, cho ti nay mi c: 2 trng hp cy ti sinh t m so - Nicotinana khng MS v Val 1 trng hp chn t nui phi v - Hordeun khng AEC 1 trng hp chn c cy - Triticum khng 5MT C kh nng di truyn tnh trng ny cho th h sau qua sinh sn hu tnh. b, Khng chlorat ClO3 Muller v Grafe (1978) chn c 2 loi dng khng ClO3 do thiu hot tnh NR t m so thuc l. - Loi cnx: to cofactor molypden cho NR rt yu. - Loi nia: khng to phn protein ca enzym T dng nia vi hot tnh NR gim (20%) to c cy v gi c tnh trng . ang nghin cu dung hp protoplast b sung di truyn cho cc dng. c) Khng khng sinh Maliga (1973) chn thnh cng dng t bo thuc l khng streptomycin, di truyn ng m Streptomycin c ch sinh tng hp protein lc lp, ty th nhng khng nh hng n sinh tng hp protein trong ribosom t bo cht. Chng khng streptomycin xanh chn bng cch tch nhng cm t bo xanh trn mi trng cha streptomycin. Chng trng chn thng qua kh nng sinh trng trn mi trng cha streptomycin. Kh nng khng ny c th do c lc lp v ty th nhng cng c th ch do mt c quan t quyt nh. Ngoi ra ngi ta cn pht hin c tnh khng t hp, ngha l chn khng mt loi khng sinh ny cng khng c mt s khng sinh khc. V d dng sylvertris khng kanamycin c th khng c streptomycin v neomycin (bng 2. Maliga,1978). d, Khng cc cht ng ng base ca DNA Trong nui cy m t bo ng vt ngi ta dng: 5-bromodeoxy uridin (BUdR) v 8-azaguanidin (AG) chn nhng dng t bo t bin thiu thymidinkinase (TK) v hypoxanthin guanidin photphoribosyl transferase (HGPRT) dng trong cc nghin cu l t bo soma ng vt. C ch chn lc nh sau:

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- Cc t bo c TK v HGPRT bnh thng s s dng BUdR v AG nh cc nucleotide cho sinh tng hp DNA. DNA mang BUdR v AG s khng bnh thng v t bo s cht. Nhng t bo thiu TK v HGPRT khng s dng BUdR v AG chng s sng c. Cc nh sinh l thc vt cng s dng m hnh ny i vi t bo thc vt v bc u thu c mt s kt qu. Brigh v ctv ( 1974- 1975) chn c dng t bo thuc l c hot tnh HGPRT gim 50% song cc dng khng BUdR vn c hot tnh enzym loi ph thuc ATP TK kh ln. iu c gii thch nh sau: trong t bo thc vt c 2 loi TK, do vy phi chn c 2 t bo t bin ng thi mi mt hon ton hot tnh TK. Mt khc dng t bo tng khng BUdR ca Okyana (1976) li do t bo c kh nng sn xut d tha thymidin. Cng c t bo c c ch sa cha DNA tt cho php chng sa c nhng sai lch do BUdR hay AG gy ra v nh vy chng c kh nng khng BUdR v AG. e,Dng t bo c gi tr kinh t cao Gegenbach v Green (1975-l77) chn c dng ng bt dc c c kh nng khng bnh g st do nm Helminthosporium maydis gy ra bng k thut chn dng t bo nui cy bng con ng nh sau: Ging ng bt dc c mang t bo cht Texas Nui cy m so Chn lc trn mi trng cha c t ca H.maydis Dng khng H.maydis Ti sinh cy Kim tra tnh khng H.maydis (kt qu +) Khng c t ca nm bnh r st v tnh bt dc c di truyn ng m v do t bin trong mt gene gy nn. Cch gii thch khc: trong t bo cht c c mt qun th genom ca cc c quan t Chn lc tnh chu bnh tc l chn gene thch hp trong qun th . Tnh

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khng thng do DNA ty th quyt nh v tnh bt dc c trong trng hp ny cng do DNA ty th quyt nh. thuc l ngi ta chn c t bin khng methionin sulfoximine - mt c t ging ca Pseudomonas tabaci tit ra (P. tabaci gy bnh m la thuc l) Cc dng chu Al, NaCl v amitrol lin quan n tnh chu phn, chu mn ca thc vt cng c chn lc, song vn cn phi nghin cu l kh nng ti sinh cy hon chnh v kim tra di truyn cc tnh chng chu . Cc dng t bo chu lnh, chu cc cht dit c di nh 2,4-D tuy c chn, nhng t ra khng di truyn (epigenetic) (bng 3, Meliga. 1978). h, Dng t bo trong nghin cu k thut di truyn Vi nhng c tnh chn lc, ngi ta c th s dng cc loi dng t bo v cc c tnh ca chng nh nhng c im ch th trong nghin cu iu khin di truyn v k thut gene. C th tin hnh nghin cu: - Hin tng b sung di truyn V d: dng cnx v nia ca Muller v gene NR trong thuc l Dng cnx + Dng nia (khng tng hp cofactor) (khng tng hp apoenzym) Dung hp protoplast Callus - Kim tra hot tnh NR Ti sinh cy Cy - Xc nh cc c im di truyn t bo cht, c bit lin quan ti tnh chng chu dc t.

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CHNG IX BO QUN INVITRO NGUN GEN THC VT 1. Khi nim chung Mc ch c bn ca bo qun ngun ti nguyn di truyn thc vt (TNDTTV) l duy tr s phong ph, a dng di truyn trong loi, gia cc loi v trong h sinh thi ni chung, ng thi c s hiu bit v chng lm c s cho vic khai thc, s dng chng cho hin ti v trong tng lai (Khanna v ctv, 1991). t lu cc phng thc bo qun ngun gene ph thuc vo loi cy trng khc nhau, thng da vo bn nng bo tn ni ging ca tng loi. Trn c s c tnh sinh sn, cc loi cy trng c xp thnh cc nhm sau: a) Cy sinh sn hu tnh, ht ca chng chu c iu kin bo qun nhit v m thp (dessication tolerant seed), bao gm ht la, ng, u , la m, i mch, i, ht rau v.v... b, Cy sinh sn hu tnh, ht ca chng hng, mt sc ny mm khi bo qun iu kin nhit thp, kh (dessication sensitic sed), n bao gm cc cy thuc loi recalcitrant nh: cao su, da, xoi, mng ct, su ring, mt, l, chm chm v.v... c) Nhng cy nhn ging v tnh, mt hon ton kh nng sinh sn hu tnh, bao gm khoai s, da, dong ring, hong tinh v.v... Cy nhn ging v tnh cn gi li mt phn sinh sn hu tnh: khoai lang, sn, ma, chui. hnh. ti v.v... Ngun gene cy trng c bo qun theo hai phng thc chnh: 1. Insitu hoc bo qun ti ch: Loi bo qun ny cho php cy trng sinh trng trong iu kin sinh thi t nhin ca chng. Ngun gene cy trng c bo qun ti ch (ni v) l nhng cy sinh sn v tnh (Hawkes, 1982), cy c ht recalcitrant v cc loi cy hoang di. 2. Ex-situ, cc c quan sinh sn ca cy trng c bo qun trong iu kin nhn to ti u. N c th ti sinh thnh cy con hon chnh khi cn thit. Bo qun Ex-situ ng mt vai tr quan trng trong xy dng cc ngn hng gene v n c s dng rng ri cho nhiu i tng cy trng. Trong bo qun Ex-situ, ngun gene ca bt k cy trng no cng c th c duy tr, lu gi. C nhng hnh thc bo qun Ex-situ khc nhau: - Trn vn m, to thnh field genebank - Bo qun ht trong kho lnh to thnh seed genebank - Bo qun invitro, to thnh invitro genebank - Bo qun DNA, acid nueleic, cht mang thng tin di truyn. Vn la chn gii php k thut no l ph thuc vo bn cht cy trng, iu kin kinh t v pht trin khoa hc, cng ngh ca tng quc gia. Trong thc tin, bo tn c s a dng phong ph ngun gene mt cy trng bt k u phi phi hp mt cch cht ch v hp l gia cc hnh thc bo qun trn. iu quan trng y l chn gii php no l chnh, tng quan t l cc phng php

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 l bao nhiu %, n ph thuc vo bn cht sinh hc ca cy trng . Di y l s tnh trng s dng cc phng php bo qun cho ba loi cy trng c trng ca ba nhm: la, khoai lang, da (hnh 13.1).

Cc gii php h tr ln cho nhau, trong chin lc chung bo qun TNDTTV chng c tm quan trng khc nhau, ph thuc vo i tng cn bo v (bng 10.1).

Hnh 10.1. Bn cht cy trng quyt nh cc gii php bo qun Yu t sinh hc 1. Cy lu nm (cc loi cy g) 2. Cc cy hng nm 3. Ht orthodox 4. Ht recalcitrant Nhng phng php bo qun thch hp Insitu, ngn hng gene ng rung. Bo qun: invitro, ht v ht phn - Bo qun ht, ht phn - Ngn hng gene ng rung v invitro Bo qun ht Ngn hng gene invitro ti ch v trn ng

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rung 5. Cy nhn v tnh vi ht v sinh Ngn hng gene ng rung, invitro v bo qun ng kh 6. Cy nhn v tnh vi ht c - Ngn hng gene ng rung phi bnh thng - Bo qun ht, ht phn - Bo qun invitro, ng kh 7. Cy c ht phn vi s sng cao Bo qun ht phn 8. Cy c iu kin nui cy m Tm cc phng php bo qun thay th 9. Cy mc n nh di truyn Tm cc phng php bo thch hp cao 2. Ngn hng gene invitro Bo qun ngun gene invitro s dng hai phng php: nui cy bo qun v bo qun ng kh (Cryopreservation). Gii php cng ngh bo qun invitro c pht trin trong vng khong 10-15 nm tr li y. N c bt u vi vic nui cy bo qun m so, t bo, phc v mc ch nghin cu sinh l, sinh ho v tng hp cc cht th cp. Da trn nguyn l , chng c chuyn sang nui cy bo qun chi, mm, phi v cy con hon chnh phc v cng tc nhn nhanh sn xut cy ging v xy dng ngun gene cy trng. Bo qun invitro tn ti pht trin ng thi vi vic s dng cc phng php s dng ngun gene c truyn. Trong mt tng lai gn, bo qun invitro s cho ra i cc ngn hng gene invitro. Bi l mt ngn hng gene khng th hiu n gin l mt t mu ging bo qun trong kho lnh, hoc mt s ng nghim nui cy mt vi tp on cy trng m ngn hng gene l mt n v hon chnh vi mc t chc qun l cao, thc hin mt cch m bo v c hiu qu cc nhim v: lu gi, bo qun, nh gi, kho st cc tnh trng, x l s liu, t liu ho kt qu cui cng l cung cp thng tin v vt liu phc v cc chng trnh mc ch nghin cu khc nhau cng nh thc hin vic trao i ngun gene gia cc nc trn th gii. Bo qun invitro xy dng ngun gene thc hin hai nhim v: - Bo qun cc tp on hot ng, nhm cung cp vt liu phc v cng tc nghin cu v trao i ngun gene gia cc ngn hng gene trn th gii. - Bo qun tp on c bn, bo tn ngun gene lu di, b sung vt liu cho cc tp on hot ng. Nh vy bo qun invitro tham gia gii quyt mt s cng on trong h thng ngn hng gene (hnh 10.2).

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Hnh 10.2 3. K thut bo qun bng nui cy invitro Bo qun ngun gene trong iu kin invitro c coi l gii php cng ngh c trin vng, c bn i vi cc cy trng nhn v tnh v cy c ht mt sc ny mm nhit v m thp. - Mu a vo bo qun rt a dng, c th l t bo n, huyn ph t bo, m so, thn, r, phi v cy con hon chnh. - Bo qun trong iu kin invitro da trn hai nguyn l khoa hc: + Sinh trng ti thiu (trong bo qun ngn hn) + Ngng sinh trng tm thi trong bo qun di hn). a) Bo qun sinh trng ti thiu c p dng nhiu cho bo qun cy con hon chnh, i vi nhm cy nng nghip nhn v tnh nh khoai lang, khoai ty, sn, khoai s, khoai mi, chui, da v.v... Mu a vo nui cy l nhng c quan sinh sn, phi, chi, mm, t xy ra nhng bin d sinh dng trong qu trnh to cy con. Cy con a vo bo qun invitro phi c lm sch bnh, kho. Trong thi gian bo qun, cc c tnh di truyn ca cy khng b mt hoc thay i.

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Trong bo qun, cy con c t trong iu kin nh sng, nhit v ch dinh dng c bit. Tc sinh trng, pht trin ca chng b c ch, gim 15-20 ln so vi tc sinh trng trong iu kin bnh thng. Nhng yu t c ch sinh trng s dng trong nui cy bo qun khc nhau, ph thuc vo bn cht cy trng, bao gm: - Nhit thp (6 180C) - Cht c ch sinh trng (acid abcisic) - Nhng cht lm gim p sut thm thu dinh dng v oxy (manitol, du ph). - Lm ngho cht dinh dng trong qu trnh nui cy. b, Bo qun ngng sinh trng tm thi c p dng cho nhiu loi cy, nhng ch yu i vi cy c ht khng bo qun lu di nhit v m thp. Mu a vo bo qun bao gm phi, m, t bo v trc khi a vo bo qun chng phi chu x l qua mt s cng on: - Tin sinh trng: x l vi 5-10% proline v 3-6% manitol. - Cht chng ng gm c: dung dch DMSO nng 1M c thm dung dch glycerol 1M v Sucrose 2M. - Bo qun: nhit - 196oC - Ph bng: nhit 40 oC - Phc hi sinh trng. Trong qu trnh bo qun, mu c nhit - 196oC, cc qu trnh trao i cht, sinh trng, pht trin ngng hot ng. Ht thi hn bo qun (c th 2030 nm), mu c ly ra phc hi sinh trng v nui cy to thnh cy con hon chnh. C hai gii php bo qun bng sinh trng chm cng nh ng lnh u c nhng hn ch kh khn chung cc kha cnh sau: - Cha c nhng nh gi y v mi tng tc gia cc yu t mi trng (thnh phn dinh dng trong mi trng nui cy, cht c ch sinh trng, nhit , cng v cht lng nh sng s dng) do vy cn gp rt nhiu kh khn cho vic p dng cng ngh ny trn quy m ln v i vi nhiu loi cy trng khc nhau. - Cha xc nh c khong thi gian ti a mt cch an ton trong qu trnh bo qun bng sinh trng chm cho tng loi cy trng. Vn quan trng v chung cho c hai hnh thc bo qun l s xut hin cc bin d sinh dng v thiu cc bin php kim tra chng. Trong khi nhng kin thc hiu bit lin quan n s n nh v di truyn ca cc hnh thc bo qun ni chung cha c nghin cu mt cch nghim tc v y . 4. Mt s kt qu v tn ti

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Trong bo qun invitro, hnh thc nui cy bo qun sinh trng ti thiu c p dng nhiu v tr thnh mt gii php khng th thiu c trong cc ngn hng gene cy trng. Hn 10 nm qua theo s liu thng k ca ban ti nguyn cy trng th gii, ngun gene ca khong 20 loi cy trng khc nhau c lu gi bng con ng ny. Danh sch cy a vo bo qun invitro 1. Cy da (Annanas spp) 2. Cy cafe (Coffe spp) 3. Cy cao su (Hevea brasiliensis) 4. Khoai lang (Ipomoea spp) 5. Khoai ty (Solanum tuberosum) 6. Sn (Manihot spp) 7. Cy chui (Musa spp) 8. Cy u (Carica papaya) 9. Cy da (Cocos nucifera) 10. Khoai mi (Dicorea spp) 11. Cy ca cao (Theobroma cacao) 12. Cy nho (Vitis vinifera) 13. Cy cam (Citrus spp) 14. Cy gng (Zingiber officinale), cy ngh (Curcuma domestica) 15. Cy c du (Elaeis guineensis) Thnh cng u tin v c ngha thc tin ln l vic ng dng bo qun ngun gene cc tp on cy ly c trong nng nghip. y cc yu t km hm sinh trng cy con trong qu trnh nui cy (iu kin bo qun bao gm nhit , nh sng v thi gian ti a mi ln cy chuyn tp on) hon ton c xc nh (bng 10.2). Bng 10.2. Cy trng Khoai lang (Ipomoea batatas L.) Sn (Manihot esculenta) Chui (Musa spp) Khoai ty (Solanum spp) iu kin bo qun Nhit 16 18oC nh sng 1000 lux manitol 3% Nhit 23 oC nh sng 100 lux Nhit 15 oC nh sng 1200 lux nh sng 2000 lux manitol 3% Thi gian cy chuyn 12 18 thng

18 24 thng 18 thng 24 thng

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Nh s gip ca T chc lng thc (FAO) v Ban ti nguyn di truyn cy trng th gii (IPGR), nhiu trung tm nghin cu nng nghip quc t v nhiu quc gia v ang xy dng nhng ngn hng invitro v chuyn dn cc tp on cy trng nhn v tnh bo qun trn ng rung vo nui cy bo qun invitro. Di y l mt s kt qu thu c trong cng tc bo qun invitro, nht l vic s dng phng php bo qun bng sinh trng chm mt s trung tm nghin cu nng nghip quc t. CIP (Trung tm nghin cu khoai ty) Pru lu gi ngun gene khoai ty v khoai lang vi mt khi lng ln trong iu kin invitro. IITA bo qun invitro nhiu tp on khoai mi, t, vc v sn. CIAT (Trung tm nghin cu nng nghip nhit i) chu trch nhim duy tr ngun gene cy sn v INIBAP (Mng li ci to ging chui th gii) c ngn hng gene invitro chui ti B (bng 10.3). Ngoi ra cn nhiu tp on cy nhn ging v tnh cng nh cy c ht recalcitrant c bo qun invitro trong h thng ngn hng gene ca nhiu quc gia nh n , Trung Quc. Nht Bn v.v... Bng 10.3. S duy tr v bo qun invitro ca mt s cy trng chnh cc vin nghin cu nng nghip quc t (1992) Loi cy trng Khoai lang Khoai ty Sn Chui Tng Bo Tng Bo Tng Bo Tng Bo s mu qun s qun s qun s qun tp invitro invitro invitro invitro on 1000 600 2000 400 250 150 4500 3481 1500 1000 1500

IITA (Nigenia) CIAT (Colombia) CIP (Pru) INIBAP (B)

- Khoai lang bo qun 16 oC + 3% manitol - Sn bo qun 18-20oC - Chui bo qun 15 oC - Khoai ty bo qun 6- 10oC + 3 % manitol, nh sng 1000- 1200 lux. nc ta, bo qun tt hn ngun gene cc cy trng nhn v tnh, nht l cc cy ly c, r v cy n qu, thi gian qua chng trnh qu gene to iu kin cho b mn cng ngh sinh hc ca Vin khoa hc k thut Nng nghip Vit Nam bc u tin hnh nghin cu nui cy invitro bo qun c kt qu mt s tp

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on cy trng. Cng tc nghin cu ny tin hnh t nhng nm 1989 v cho kt qu tt. B mn a c 107 mu ging t tp on khoai lang trn ng rung v bo qun an ton trong iu kin invitro nhit 18-20oC vi thi gian cy chuyn 69 thng. Sau mt thi gian bo qun di (5 nm) mt s trong nhng ging khoai lang ny nh: Hong Long, B, Chim du , t v Tri Sa c trng v kho st li trn ng rung. Kt qu cho thy mt s c tnh: hnh dng, mu sc, thn l, tht c khng sai khc so vi cc mu ging bo qun trong tp on trn ng rung t nhiu nm nay. Cng trn nguyn tc nghin cu th nghim , nhiu mu ging ca tp on cy trng khc nh khoai s, khoai ty, chui, da, mt v cam chanh v ang c chuyn vo bo qun trong iu kin invitro v kim tra li s n nh di truyn ca chng bn ngoi ng rung. Nhng th nghim ny u cho kt qu kh quan (bng 10.4). Nh vy, cho n nay trong ngn hng gene c khong 200 mu ging ca 7 tp on cy trng khc nhau c lu gi thng xuyn v an ton trong iu kin invitro. Bng 10.4. Tnh trng bo qun invitro mt s tp on cy nhn v tnh Tn cy trng Khoai lang Khoai s Khoai ty Chui Da Cy c mi (cam, chanh) S lng mu cy 107 60 26 20 5 16 Trng li kim tra x x x x Nhit bo qun 18oC 22oC 18oC 18oC 18oC 20oC Thi gian cy chuyn (thng) 6 10 46 4 4 6 3

y cng l ngun vt liu sch bnh an ton sn sng tham gia vo cng tc trao i ngun gene ca cc cy trng nhn v tnh ca nc ta vi h thng mng li ngun gene quc t. K t nm 1990 li y, ngn hng gene cy trng cng l ni tip nhn nhiu ln, vi s lng khong 150 mu ging l vt liu nui cy invitro nh: khoai lang, khoai ty v sn t cc t chc nghin cu nng nghip quc t. Chc chn trong thi gian ti vic hp tc trao i ngun gene bng vt liu nui cy invitro tr thnh nhu cu v pht trin i vi nhiu cy trng khc. 5. Kt lun Cng ngh sinh hc thc s c ng dng mt cch c hiu qu trong nhiu lnh vc nh chn to ging cy trng, bo v thc vt, khai thc s dng v phn phi ngun gene v trong k thut di truyn. Nhng ng dng ca cng ngh

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sinh hc thc vt c coi l quan trng v c ngha hn c l nui cy bo qun invitro ngun ti nguyn di truyn thc vt. Bo qun invitro c bit c gi tr i vi cy trng sinh sn hu tnh, nhng ht ca chng khng th bo qun c m v nhit thp v cy trng nhn ging v tnh, m vic duy tr cc tp on ca chng trn ng rung ang c nhiu nguy c tht thot. Bo qun invitro trong mt ngn hng gene l mt s phi hp ca nhiu hot ng nghin cu, bt u l vic thu thp, phn lp, lm sch bnh, bo qun n phn phi v s dng. Ngun gene c lu tr invitro bng hai gii php: km hm sinh trng qua vic gim nhit nui cy hoc thm vo mi trng nui cy cc cht c ch v bo qun ng lnh trong nit lng. C hai hnh thc bo qun mi c nghin cu v ang c mt s hn ch. Tuy nhin chng c nhiu t chc nghin cu nng nghip quc t v ngn hng gene quc gia s dng nui cy bo qun thnh cng trn 20 loi cy khc nhau. nc ta, cng tc nghin cu nui cy, bo qun invitro bc u tin hnh c kt qu i vi mt s cy trng nhn ging v tnh trong nng nghip. Nhng nhu cu pht trin, s dng, bo qun invitro rt ln. Trong iu kin kh hu nhit i chng ta c ngun gene cy ht recalcitrant (xoi, mt, na, su ring, nhn, vi v cy c mi...) v cy nhn ging v tnh c gi tr gm: - Cy n qu, cy cng nghip (chui, da, ma). - Cy lng thc, cy thuc (khoai s, t, vc, khoai lang, sn v dong ring, hong tinh, gng, ngh, sinh a, tam tht...). Ngun gene ny rt phong ph v thuc nhm vt liu bo qun invitro bt buc. Xut pht t nhu cu thc tin trn, trong nhng nm ti Ban ti nguyn di truyn thc vt nc ta cn phi la chn mt chin lc bo qun thch hp, s dng nhng thnh tu ca cng ngh sinh hc, pht trin nhng gii php mi, nui cy bo qun invitro h tr cc phng php c truyn nh ngn hng gene ht v trn ng rung nhm bo qun tt hn ngun ti nguyn ca chng ta.

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