Trang 1 /8 Gio trnh nui cy m. Tp hp 8 bi thc hnh v nui cy m. c su tm, xin gii thiu n cc bn ng nghip v cc bn hc sinh, sinh vin.

BI 1: M U

1.CC THIT B C BN CA MT PHNG TH NGHIM NUI CY M V T BO THC VT a. Phng ra v ct nc - My ct nc 1 ln - My ct nc 2 ln b. Phng hp sy - Autoclave - T sy 60 200oC c. Phng chun b mi trng - Cn phn tch (chnh xc n 0,0001 g) - Cn k thut (chnh xc n 0,01 g) - pH k - My khuy t - T lnh - L vi sng (microwave) d. Phng thao tc nui cy - T cy v trng (laminar) - Qut thng gi - n t ngoi treo tng e. Phng nui cy - Cc gin k c gn n hunh quang - My iu ha nhit - My lc nm ngang - T m f. Phng th nghim: (phng ny dng tin hnh cc phn tch sinh ha, phn t v di truyn) - Knh hin vi 2 mt ( phng i 1000 ln) - Knh lp 2 mt ( phng i 75 ln) - Microtome - My nh k thut s - H thng n chiu - Quang ph k

2. CC NHN T M BO THNH CNG TRONG NUI CY M T BO THC VT C 3 nhn t chnh: - Bo m iu kin v trng - Chn ng mi trng v chun b mi trng ng cch - Chn m cy v x l m cy thch hp trc v sau khi cy. 2.1. ngha ca v trng trong nui cy m v t bo thc vt Mi trng nui cy m v t bo thc vt c cha ng, vitamin, mui khong rt thch hp cho cc loi nm v vi khun pht trin. Do tc phn bo ca nm v vi khun ln hn rt nhiu so vi t bo thc vt, nu trong mi trng nui cy ch nhim mt vi bo t nm hoc vi khun th ch sau vi ngy n mt tun, ton b b mt mi trng v m nui cy s ph y mt hoc nhiu loi nm v vi khun. Th nghim phi b i v trong iu kin ny m nui cy s khng pht trin v cht dn. 2.2 Ngun tp nhim C 3 ngun tp nhim chnh: - Dng c thu tinh, mi trng nui cy v nt y khng c v trng tuyt i - Trn b mt hoc bn trong m cy tn ti cc si nm, bo t nm hoc vi khun - Trong qu trnh thao tc lm ri nm hoc vi khun theo bi ln b mt mi trng 2.3 K thut v trng 2.3.1 V trng dng c thu tinh, nt y v mi trng a. Dng c thu tinh Thng thng cc dng c thu tinh dng trong cc th nghim thng c x l bng dung dch sulfocromate mt ln u trc khi a vo s dng; v sau ch cn ra sch bng x phng, trng sch bng nc ct v tht ro trc khi s dng. Trong trng hp cc dng c thu tinh dng trong cc th nghim nui cy m v t bo thc vt i hi v trng, c th kh trng trong t sy nhit cao trong nhiu pht hoc nhiu gi. Cc dng c ny lun c gi trong giy nhm hoc hp kim loi trnh b nhim tr li sau khi kh trng. Bng 1.1: Thi gian kh trng dng c thu tinh bng nhit v nhit kh trng Nhit (oC) 160 170 180 190 b. Nt y Thng dng nht l cc nt y lm bng bng gn khng thm nc.Nt phi tng i cht m bo bi khng i qua c, ng thi nc t mi trng khng b bc hi qu d Thi gian kh trng(pht) 45 18 7,5 1,5

dng trong qu trnh nui cy. Bng khng thm nc l loi nt n gin nht nhng c cc nhc im sau: - Nu khi hp nt bng b t hoc dnh mi trng th v sau s rt d b nhim nm, nht l vi nhng th nghim tin hnh trong mt thi gian di - Thao tc lm nt bng chm, khng thun tin khi nui cy trn qui m ln - Ch dng c vi ln l phi b Hin nay ngi ta s dng nhiu loi np y khc thay th nt bng. Cc hng sn xut dng c nui cy m cung cp loi np ng nghim v bnh tam gic bng nha chu nhit c th hp v trng nhit 1210C m khng b bin dng. Mt s phng th nghim dng np ng nghim bng inox hoc cao su rt thun tin cho vic v trng kh hoc t. Cng c th s dng giy nhm lm np y c. Mi trng Mi trng nui cy thng c hp kh trng trong ni hp (autoclave), kh trng bng p sut hi nc bo ha. Thi gian hp t 15-30 pht p sut hi nc bo ha l 103,4 kPa (1atm) tng ng vi nhit 1210C. nhit 1210C, hu ht cc sinh vt c trong mi trng u b tiu dit, k c dng bo t. Sau khi v trng cn phi lm kh np ng nghim hoc nt bng trnh b nhim tr li. Bng 2: Thi gian kh trng dung dch v cc mi trng lng bng ni hp (autoclave) 121oC ti 103,4 kPa Th tch mi trng (mL) <50 75 250-500 1000 Thi gian hp kh trng(pht) 15 20 25 30

Vic hp kh trng bng ni hp th khng thch hp vi nhiu ho cht nhy cm vi nhit nh: cc acid amin, cc vitamin, cc hormon tng trng, v cc cht khng sinh. Cc cht nh vy thng phi c kh trng bng cch lc v trng. Mng lc c lm bng mng polyethylen hoc sI cellulose. Cc l trn mng siu lc ny c thit k hiu qu cho vic gi li cc vi sinh vt gy nhim. d. Lc v trng Phng php n gin nht l dng cc mng lc Millipore hoc dng cc phu lc thy tinh xp s 5. Mt s loi mng lc v trng ca hng Millipore. y l loi mng lc c kh trng bng chiu x v ch dng 1 ln Phng php s dng mng lc Millipore: hng Millipore cung cp mng lc v gi bng nha hu nhit. Di y m t mng lc loi Millipore Swinex c ng knh 25mm. B lc gm c gi bng loi nha chu nhit gm np v , vng cao su v mng lc. t mng lc (c kch thc l 0,25m) trn , t vng cao su ln v vn cht np vo . Gi ton b b lc vo trong mt t giy nhm v kh trng trong autoclave 121oC trong 15-20 pht.

ng thi cng hp v trng mt bnh hu tinh hng dch lc. Dng ng tim ht dch lc v bm qua b lc. 2.3.2 Kh trng m thc vt M cy c th l hu ht cc b phn khc nhau ca thc vt nh ht ging, phi, non, hoa, l, u r, thn ctu theo s tip xc vi mi trng bn ngoi, cc b phn ny cha nhiu hay t vi khun v nm. ng la non khi cn trong b, m tht bn trong qu thng t b nhim vi sinh vt; ngc li, l, thn c bit cc b phn nm su trong t nh r, c c lng nm, khun tp rt cao. Hu nh khng th v trng m cy c nu nm, khun nm su cc t bo bn trong m ch khng hn ch b mt. L khoai lang c th v trng d dng trong ma kh nhng khng th lm c trong ma ma. Phng php v trng m cy thng dng nht hin nay l dng cc cht ho hc c hot tnh dit nm khun. Hiu lc dit nm khun ca cc cht ny ph thuc vo thi gian x l, nng v kh nng xm nhp ca chng vo cc k ngch li lm trn b mt m cy, kh nng y ht cc bt kh bm trn b mt m cy. tng tnh linh ng v kh nng xm nhp ca cht dit khun, thng thng ngi ta x l m cy trong vng 30s trong ru ethylic 70% sau mi x l dung dch dit khun. ng thi ngi ta thm cc cht gim sc cng b mt nh Tween 80, Fotoflo, Teepol vo dung dch dit nm khun. c khi nim v nng v thi gian s dng cc cht dit nm khun x l m cy, xin dn ti liu nghin cu ca Street (1974) bng sau: Tc nhn v trng Calci hypochlorit Natri hypochlorit Hydro peroxid Nc Brom HgCl2 0.1 1 Cht khng sinh Nng % 9 10 2 10 12 1-2 2 10 4 50 mg/l Thi gian x l 5 30 5 30 5 15 2 10 Trung bnh 30 60 Kh tt Hiu qu ( pht) Rt tt Rt tt Tt Rt tt

Trong thi gian x l, m cy phi ngp hon ton trong dung dch dit khun. i vi cc b phn c nhiu bi ct, trc khi x l nn ra cn thn bng nc x phng bt v ra sch li bng nc my. Khi x l xong, m cy c ra nhiu ln bng nc ct v trng (ti thiu l 3 ln). Nhng phn trn m cy b tc nhn v trng lm cho trng ra cn phi c ct b trc khi t m cy ln mi trng. trnh nh hng ca tc nhn v trng ln m cy, nn ch li mt lp bc ngoi khi ngm m vo dung dch dit khun. Lp ngoi cng ny s c lt b i trc khi t m cy ln mi trng. V trng m cy l mt thao tc kh, t khi thnh cng ngay t ln u tin. Tuy vy, nu kin tr tm c nng v thi gian v trng thch hp th sau vi ln th chc chn s t kt qu. C th dng khng sinh kim sot hoc loi b s nhim nm trn m cy. Hu ht cc khng sinh nhy cm vi nhit do khng th hp v trng. Chng ho tan c trong nc hoc dung mi thch hp khc, lc v trng v thm vo mi trng v trng khi mi trng ny c lm ngui cn 45-50oC. Khng phi tt c cc khng sinh u thch hp cho s dng trong nui cy m thc vt. Cc khng sinh nh Streptomycin, Kanamycin v Neomycin thng c cho m thc vt v khng hot ng tt trong mt phm vi pH nht nh. Tetracylin cng l c t thc vt, c khuynh hng c ch s tng trng ca m thc vt sau khi b x l trong mt thi gian di. Chloramphenicol c ph hot ng rng nhng c cho thc vt (v ngi) nng thp.

Cc khng sinh thng c dng trong nui cy m thc vt gm Rifampicin (thng c dng kt hp vi cc khng sinh khc), cc polymicin v vancomycin. Cc loi khng sinh Kh nng hot ng Aminoglycoside :c ch s sinh tng hp protein bng cch tc ng ln cc ribosome 30S hoc 50S -Streptomycin - Kanamycin - Neomycin Quinolone Gy tr ngi cho qu trnh sao chp DNA bng cch c ch enzym DNA gyrase - Nalidixic acid - Ofloxacin - Norfloxacin -Lactam - Penicillin - Ampicillin - Carbenicillin c ch s tng hp vch t bo vi khun

Tetracyclin c ch sinh tng hp protein bng cch tc ng ln ribosome 30S Trimehtoprim v sulphonamide c ch s sinh tng hptetrahydrofolate Chloramphenicol c ch s sinh tng hp protein bng cch tc ng ln Rbx 50S Macrolide v lincosamide c ch s sinh tng hp protein bng cch tc ng ln Rbx 50S - Erythromycin -Lincomycin Glycopeptide Tc ng ln s sinh tng hp vch t bo vi khun. - Vancomycin Polymixin Gn ln mng t bo lm thay i dng ion dn n s ph v t bo - Polymixin B - PolymicinE Rifampicin Tc ng ln RNA bng cch gn vo RNA polymerase 2.3.3 K thut cy v trng trnh b nhim trong sut thao tc cy m thc vt, cc nh khoa hc lm vic trong t cy v trng (laminar). l cc t cy c thit b thi khng kh lc v trng vo ch thao tc cy. T cy v trng loi tr mt cch hiu qu ngun tp nhim t bn ngoi v to iu kin thoi mi chongi cy, nn hin nay c s dng rt ph bin trong cc phng th nghim. Khng kh t bn ngoi c qut ht vo qua mt mng lc th. 99% bi trong khng kh c gi li mng lc th. Sau khng kh c thi qua mng lc tinh v phn phi u ra khp b mt ca t cy, khng to nhng xoy khng kh a bi vo ch cy. Mng lc tinh ngn cn cc phn t ln hn 0.3micron vi hiu qu 99,99% v do cc hng chuyn sn xut mng lc cung cp. Tui th ca mng lc th tu theo s lng bi ni lm vic, thng t 6 thng n 1 nm v ca mng lc tinh t 2 n 3 nm. Khi thy hiu qu v trng ca t cy laminar km i th cn phi thay mng lc mi. ko di tui th ca b mng lc trong

t cy laminar- thng rt t tin - nn b tr t cy trong cc phng ring, cng t bi cng tt. 2.3.4 Kh trng ni thao tc cy v dng c cy Ngun nhim tp quan trng v thng xuyn nht l bi ri vo dng c thu tinh cha mi trng trong khi m np hoc nt bng thao tc cy. Ngi ta p dng nhiu bin php khc nhau chng li ngun nhim tp ny. Phng cy thng l bung c din tch hp, rng t 10-15m2, c hai lp ca trnh khng kh chuyn ng t bn ngoi trc tip a bi vo. Sn v tng lt gch men c th lau chi thng xuyn. Trc khi a vo s dng, phng cy cn c x l hi formol bng cch rt formaldehyde (formalin) 4% ra mt s np a petri ri rc vi ni trong phng cho bc hi t do. ng kn ca phng cy trong 24h, sau b formaldehyde i v kh hi formaldehyde cn tha bng dung dch NH3 25% cng trong 24h. B mt ni chun b cy, b mt bn trong v ngoi t cy phi c kh trng trc khi cy bng cch lau sch cc b mt ny bng cn 90%. Tia UV cng c th c s dng kh trng b mt phng cy v t cy nhng n t hiu qu hn v nguy him hn l s dng cn. Tia UV ch tiu dit c cc mm vi sinh nm trc tip ngay trn cc b mt m tia ny chiu vo; nhng n khng th thm qua cc lp bi tiu dit cc vi sinh vt nm bn di cc lp bi ny. Tia UV cn gy hi cho mt v gy ung th da. Cc dng c mang vo phng cy u v trng trc: t o chong, m vi, khu trang ca ngi cy n dao, ko, kp (forceps), giy lc, bnh ng nc ct... Trn bn cy thng xuyn c mt n cn s dng trong khi cy v mt cc ng cn 90% nhng cc dng c lm vic. Trc khi cy, ngi cy cn ra tay bng x phng v lau k n khuu tay bng cn 90%. m bo mc v trng cao trong phng cy n c mt n t ngoi 40W treo trn. Ch cho n ny lm vic khi khng c ngi trong phng cy. Nn bt n t ngoi 30 pht trc khi cy. Cn gim s chuyn ng ca khng kh trong phng cy n mc ti thiu, v vy tt c cc dng c phc v cho vic cy u phi chun b y trong thi gian cy trnh i li ra vo phng cy qu nhiu. Cc dng c bng kim loi nh kp cy, dao m, que cy vng, kim mi nhn c th c kh trng bng cch t di ngn la n cn. Nhng dng c ny trc ht phi c nhng vo cn tuyt i ri mi t. Nh ro i cc git cn tha ri mi a vo ngn la n cn. Khi m np chai hoc np ng nghim mi trng nui cy, th dng ngn tay k t v ngn tay t cm ly np gn, v khng chm tay vo b mt bn trong ca np gn cng nh khng th np gn xung bt c b mt no cho n khi gn n tr li chai mi trng. BI 2: K THUT PHA CH MI TRNG 1.VN LA CHN MI TRNG Khi khi s nui cy m v t bo i vi mt s i tng nht nh, vn t ra l chn mi trng no v trn c s no phi hp t l cc cht dinh dng. Cch thng lm l qua cc ti liu xut bn, cc cng trnh nghin cu v i tng hoc cng h tng ng xem cc tc gi s dng mi trng loi no. Bc u c th gi nguyn mi trng ca tc gi hoc trn c s m ci tin cho ph hp qua cc th nghim thm d.

Trong hng trm mi trng do rt nhiu tc gi ngh cho nhiu loi cy khc nhau, nhiu mc ch nui cy khc nhau. V c bn c th chia ra lm 3 loi: - Mi trng ngho cht dinh dng: in hnh l mi trung White,Knop v Knudson C - Mi trng c hm lng cht dinh dng trung bnh: in hnh lmi trng B5 ca Gamborg - Mi trng giu dinh dng: in hnh l mi trng MS (Murashige-Skoog) V vy khi bt u nghin cu nui cy m mt s i tng mi, cha c ti liu trc th nn thm d so snh 3 loi mi trng trn xem i tng nghin cu ph hp vi loi mi trng no nht. Sau cn th tm t l NO3/ NH4+ thch hp. Cc tc gi phng Ty lm vic vi cy trng cn thng khng a NH4+ vo mi trng. Nhng i vi nhng cy dinh dng NH4+ mnh nh cy la, vic thm vo mi trng nui cy mt t l NH4+ thch hp chc chn s c li. Vic s dng mang tnh kinh nghim ch ngha i vi mt s mi trng cn tr kh nhiu s tin b ca cng tc mt s phng th nghim nui cy m thc vt. Thuc l v cart l 2 loi cy kinh in ca nui cy m thc vt. Mi trng nui cy 2 loi cy ny c xy dng kh hon chnh. Tuy vy, khng th dng nguyn cc mi trng nghin cu cc cy ho tho hoc cc cy h u m khng c s ci tin, sa i. iu ny gii thch s tin b chm chp ca nui cy m mt s cy ho tho so vi cy 2 l mm. Hin nay, mi trng MS c coi nh l mi trng thch hp vi nhiu loi cy do giu v cn bng v mt dinh dng. V vy, nhng ngi tp s lm nui cy m thng bt u vi mi trng ny trc khi tm c mi trng ca ring mnh. 2.CHUN B MI TRNG thun tin cho vic pha cc mi trng nui cy ngi ta khng cn ho cht mi ln pha m thng chun b trc di dng cc dung dch m c (cn gi l cc stock), sau ch cn pha long khi s dng. Cc stock ny thng c bo qun di ngy trong t lnh thng hoc t lnh su. 2.1 Thnh phn mi trng dinh dng Nc ct Cht hu c - ng- Acid amin- Vitamin (B1, B6, H, PP)- Cc cht iu ha sinh trng thc vt (Auxin, Cytokinin,Gibberellin) Cht v c a lng N P K Ca Mg S Vi l ng Fe Zn B Co N Mn Cu Al Mo I Cc hp cht khng bit r thnh phn Nc da, nc khoai ty, nc chui, casein,hydrolysalt, trypton, pepton THNH PHN MUI KHONG C BN CA MI TRNG MS (Murashige v Skoog, 1962)

SKOOG I SKOOG II SKOOG III NH4NO3 :1650 mg/L FeSO4.7H2O : 27,8 mg/L KNO3 : 1900 mg/L MnSO4.4H2O : 22,3 mg/L KH2PO4 : 170 mg/L H3BO3 : 6,2 mg/L MgSO4.7H2O : 370 mg/L ZnSO4.7H2O : 8,6 mg/L CaCl2.2H2 : 440 mg/L Na2EDTA : 37,3 mg/L

KI : 0,83 mg/L Na2MoO4.2H2O : 0,25 mg/L CuSO4.5H2O : 0,025 mg/L CoCl2.6H2O : 0,025 mg/L

THNHPHN VITAMIN CA MOREL (MorelsVitamin) Pirydoxine (B6) : 1 mg/L Biotin (H) : 0,01 mg/L Meso-inosito : 100 mg/L Nicotinic acid (P.P) : 1 mg/L Thiamin HCl (B1) : 1 mg/L Pantotate Calci :1 mg/L 2.2 Cch pha cc dung dch m (stock) * Stock a lng MS : SKOOG I (x10) (Pha thnh 1Lvi nng s dng khi pha 1Lmi trng MS l 100mL/L) NH4NO3 : 16500 mg KNO3 : 19000 mg KH2PO4 : 1700 mg MgSO4.7H2O : 3700 mg CaCl2.2H2O : 4400 mg Cn v dng nc ct ho tan ln lt tng cht trong bcher cho tan hon ton. Dng ng ong 1L iu chnh cho 1 lt. * Stock st MS: SKOOG II (x100) (Pha thnh 200mL vi nng s dng khi pha 1L mi trng MS l 2mL/L) Na2EDTA : 3730 mg FeSO4.7H2O : 2780 mg Cn v ho tan tng cht bng 100mL nc ct trong mi bcher ring. t 2 bcher dung dch ln bp v gia nhit cho dung dch m ln va c hi nng bc ln l c. Khuy u v dung dch Na2EDTA vo ng ong 200 mL, ri cho t t dung dch FeSO4vo, va cho va khuy u. nguI ri cho vo bnh ti bo qun trong t lnh. * Stock vi lng MS: SKOOG III (x100) Trc tin cn chun b cc stock con: Dung dch KI: (83mg/mL) Cn 8300 mg KI ho tan vi nc ct cho 100mL Dung dch Na2MoO4.2H2O: (25mg/mL) Cn 2500mg Na2MoO4.2H2O ho tan vi nc ct cho 100mL

Dung dch CuSO4.5H2O (2,5mg/mL) Cn 250mg CuSO4.5H2O ha tan vi nc ct cho 100mL Dung dch CoCl2.6H2O (2,5 mg/mL) Cn 250 mg CoCl2.5H2O ha tan vi nc ct cho 100 mL SKOOG III (x100) : pha thnh 500mL vi nng s dng khi pha 1L mi trng MS l 5mL/L MnSO4.4H2O : 2230 mg H3BO3 : 620 mg ZnSO4.7H2O : 860 mg KI : 83 mg/1 mL Na2MoO4.2H2O : 25 mg/1 mL CuSO4.5H2O : 2,5 mg/1 mL CoCl2.6H2O : 2,5 mg/1 mL Cn 3 cht u tin v ho tan vi nc ct trong tng bcher ring.Cho tng dung dch theo th t vo ng ong 500mL. Sau ht 1mL dungdch trong stock con ca 4 cht tip theo cho vo ng ong, va cho va khuy u v thm nc ct cho 500mL * Thnh phn vitamin Pha cc stock con Dung dch pirydoxine (B6) (10mg/mL) Cn 1000mg B6 ha tan vi nc ct cho 100mL Dung dch Biotin (H) (1mg/mL) Cn 100mg biotin ha tan vi nc ct cho 100 mL Dung dch Nicotinic Acid (P.P) (10mg/mL) Cn 1000 mg nicotinic acid ha tan vi nc ct cho 100mL Dung dch Thiamin-HCl (B1) (10mg/mL) Cn 1000 mg B1 ho tan vi nc ct cho 100 mL Dung dch Pantotheate-Ca (10mg/mL) Cn 1000 mg Pantotheate-Ca ha tan vi nc ct cho 100mL Vitamin Morel (x 100) Pirydoxine (B6)100g/10mL Biotin (H)1 mg/1 mL Meso-inosito l10 g/10g Nicotinic acid(P.P) 100mg/10mL Thiamin HCl(B1)100mg/10mL Pantotate Calci100 mg/10 mL Cn meso-inositol ri ha tan vi nc ct. ong tng cht theo th t cho vo ng ong c cha nc ct , va cho va khuy u v thm nc cho 200mL * Thnh phn cc cht iu ha sinh trng thc vt (tnh theo mg) Dung dch BAP (1mg/mL) Cn 100 mg Benzylaminopurine (BAP) ho tan trong 5mL NaOH 1N.Cho thm nc ct cho 100 mL. Mi mL dung dch c cha 1mg BAP Dung dch IAA (1mg/mL) Cn 100mg Indolacetic acid (IAA) ha tan trong 5 mL NaOH 1N. Cho thm nc ct cho 100mL. Mi mL dung dch c cha 1mg IAA.

Dung dch IBA (1mg/mL) Cn 100mg Indolebutyric acid (IBA) ha tan trong 5mL NaOH 1N. Cho thm nc ct cho 100mL. Mi mL dung dch c cha 1mg IBA Dung dch Kinetin (1mg/mL) Cn 100mg Kinetin (KIN) ha tan trong 5 mL NaOH 1N. Cho thm nc ct cho 100mL. Mi mL dung dch c cha 1mg KIN. Dung dch NAA ( 1mg/mL) Cn 100mg Naphthaleneacetic acid (NAA) ha tan trong 5mL NaOH 1N. Cho thm nc ct cho 100mL. Mi mL dung dch c cha 1mg NAA. Dung dch 2,4-D (1mg/mL)Cn 100mg 2,4-D (2,4-dichlorophenoxyacetic acid) ha tan trong 50mL ethanol 50%. Cho thm nc ct cho 100mL. Mi mL dung dch ccha 1mg 2,4-D. * Cc dung dch cht iu ha sinh trng thc vt (tnh bng mol/l) BAP c trng lng phn t (MW) l 225.26 - Ho tan 225.26gr BAP trong 1l nc ct tc ta c 1l dung dch BAP1M hay 1mol/l - Cn 225.26 mg BAP ho tan trong 1l nc ct tc ta c 1l dung dch BAP 1mM hay 1mmol/l - Cn 225.26x10-3 mg BAP ho tan trong 1l nc ct tc ta c 1l dung dch BAP 1M hay 1mol/l Dung dch BAP ( MW 225.26) (10mM) Cn 225.26 mg BAP ho tan trong 5 mL NaOH 1N. cho thm nc ct cho 100 mL , tc ta c 100 mL dung dch BAP 10mM V d: Cho 1mL dung dch BAP 10mM vo mi trng MS pha c 1L mi trng. Nh vy ta c 1L mi trng MS c cha 10M BAP. Mun pha 1Lmi trng MS c cha 1M BAP, ta s dng 0.1mL dung dch BAP 10 mM. Tng t nh vy pha cho cc loi kch thch t cn li, bit: - IAA (MW 175.19) - IBA (MW 203.24) - Kinetin (MW 215.22) - NAA (MW 186.21) - 2,4-D (MW 221.04) 3. THC HNH - Tin hnh pha Skoog I, II v III ca mi trng MS - Pha stock vitamin Morel, glycin v inosytol - Pha cc stock cht iu ho sinh trng nh hng dn phn trn BI 3: GIEO HT IN-VITRO I.CHN M CY V X L M CY 1.1 Chn la m cy Khng c nhng hng dn c th trong vic chn m cy. V nguyn tc, tr nhng m cy ha g, cc m khc trong c th thc vt u c th dng lm m cy. Tuy vy c th nhn xt chung l cc m ang pht trin, tht qu non, l non, cung hoa, hoa, m phn sinh khi t vo mi trng c cha mt lng cht sinh trng thch hp u c kh nng phn chia v phn ha. bt u nghin cu nhn ging v tnh mt cy nht nh, trc tin ngi ta ch n cc chi nch v m phn sinh ngn. Cn bit rng tuy mang mt lng

thng tin di truyn nh nhau, cc m khc nhau trn cng mt cy c th sinh trng v pht trin vi kh nng ti sinh chi, r hay cy hon chnh rt khc nhau. V vy, khi khi s chn ging, nhn ging mt cy c th bngphng php nui cy mv t bo thc vt, trc ht cn th nghim tm hiu phn ng ca cc b phn khc nhau ca cy trong nui cy cc nng cht sinh trng khc nhau. Sau khi cy, m cy cn c t trong iu kin nhit v nh sng n nh. Tu vo cc mc ch nghin cu m c cc ch chiu sng khc nhau, chng hn qu trnh to m so c th cn bng ti hay nh sng, nhng qu trnh ti sinh v nhn ging v tnh nht thit phi c nh sng. Nhit phng nui nn gi n nh 25+2oC bng my iu ha nhit . Cng chiu sng khong t 2000 3000 lux. 1.2 X l v trng mu cy Mu a vo nui cy invitro c nhiu ngun gc khc nhau. C th l nhng mu cy v trng nh cy con invitro cho ny mm trong iu kin v trng; cng c th l cc mu cy cha v trng, ly trc tip t bn ngoi t nhin nh chi non, l, thn, c, r Thun li nht l s dng cc mu cy v trng bi l cc phng php v trng mu cy trc tip thng t nhiu gy hi cho mu cy do cht kh trng gy ra. C nhiu phng thc v trng mu cy: a. V trng ht: - Ra ht bng x phng, lc u 2-3 pht. Vi cc loi ht nh, thng ng ht trong ti vi mng. - Ra sch x phng bng nc my di vi nc chy mnh - V trng s b bng cn 70%, lc u 1-2 pht - Ra sch cn bng nc v trng - Cho ht vo dung dch kh trng NaOCl 1-15%. Thm vi git bm dnh Tween20. Kh trng trong 15-20 pht tu mu. - Ra sch cht kh trng nhiu ln bng nc v trng (khong 5 ln) cho n khi ht mi javel. - Ht v trng c th nui cy trn mi trng to mu v trng. b. Cc phng thc khc v trng ht - Kh trng ln th nht vi NaOCl 5,25% - Kh trng ln th hai vi HgCl2 0,1 1%, thm vi git HCl 0,5% trong 10 pht. - Ra mu vi H2O2 6% v trng trc khi ra li bng nc v trng - C th kh trng s b ht bng acid sulfuric trong 2-10 pht - Vi ht c v cng, c th dng phng php t kh trng s b. c. V trng chi non, l v thn c: - V trng s b bng cch ngm mu vo cn 70% trong 1-3 pht, thI gian x l ny cng cn tu thuc vo cng hay mm ca mu - V trng mu vi cht kh trng NaOCl 1% hay vi javel thng phm theo t l 1:4 hay 1:5 trong thi gian 5-20 pht tu theo mu - Ra li bng nc v trng cho sch hon ton cht kh trng d. V trng c, r - Mu nui cy phi c ra di vi nc chy mnh cho tht sch t ct bm trn mu, sau ngm trong nc x phng long 10 pht - V trng s b bng cn 70% trong 1-5 pht - V trng vi cht kh trng c nng v thi gian thay i tu mu - Ra li bng nc v trng cho tht sch cht kh trng

e. Trnh hoi mu Trong qu trnh nui cy thng xut hin nhim l do trong qu trnh nui cy bo t trong khng kh ri vo hay mu cy cha c v trng hon ton. Sau mt thi gian th bo t pht trin nhanh chng v c th s lm cht mu hoc cnh tranh cht dinh dng lm mu khng pht trin. Hoi mu thng do nm mc (khun lc c dng si) hoc do khun (khun lc c dng vn nhy). Nu nm hoc khun ch mc tr6en b mt mi trng, thng l nhim do thao tc cy cha tt; nu c khp trong mi trng l nhim do mi trng cha c tit trng hon ton; cn nu khun v nm xut pht t gc mu lan ra th l do mu cha c kh trng trit . Tu theo quan st v da vo kinh nghim ca ngi cy m c nhn nh chnh xc v nguyn nhn gy hoi mu tm cch khc phc tnh trng ny. Trong trng hp cc bnh nui cy b nhim, cn c hp b trc khi em i ra sch trnh ly lan ngun nhim trong khu vc cy. 2.THC HNH 2.1 Mc ch Gip cho sinh vin lm quen vi thao tc v trng mu cy v k thut cy v trng 2.2 Dng c, thit b v ha cht 2.2.1 Dng c: - Bnh tam gic (250mL) - Giy cy v trng - Bcher (cc t thu tinh) 500mL, 1000mL - Forceps (kp), dao cy, n cn - a petri v trng 2.2.2 Thit b: - Autoclave - T sy - T cy (laminar) - T lnh - Cn k thut - My ct nc 2 ln - My khuy t - pH k 2.2.3 Ho cht - Dung dch Skoog I, II v III ca mi trng MS - Stock vitamin Morel - Stock glycin - ng saccharose - Agar - Nc ct v trng - Cn 90%, 70% - Dung dch hypochloride calcium 10% - X phng bt 2.3 Cc bc tin hnh 2.3.1 Chun b mi trng (thnh phn cho 1L mi trng) - Skoog I (MS):100mL - Skoog II (MS): 2mL - Skoog III (MS): 5mL

- Vitamin Morel: 2mL - Glycine: 2mL - Sucrose: 30g - pH :5,8 - Agar:7g 2.3.2 Nguyn liu thc vt: - Cc ht cam chanh cn nguyn vn, khng b h hi 2.3.3. Cc bc thc hin - Cho ht vo bcher c cha nc x phng long, ra ht cho sch cht nht bm xung quanh ht - Ra ht cho sch x phng bng nc ct. Ngm ht 2pht trong cn 70% - Ra ht sch etanol bng nc ct. Sau ngm ht 10pht trong dung dch hypochloride calcium 10% - Trong t cy v trng, ra ht cho sch cht kh trng bng cch lc ht trong nc ct v trng (3 ln) - Tin hnh tch o ht bng kp v dao m v trng. Cho cc ht va tch b v o vo a petri v trng c cha giy thm v trng v lm m giy thm bng mt t nc ct v trng. - Gieo ht: gieo 4-5 ht vo cc bnh tam gic cha mi trng nui cy c hp kh trng. - y np bnh cy v ghi r h tn, ngy cy, s nhm, tn ging v mi trng. 2.4 Yu cu - Thc hin pha mi trng gieo ht - Thc hin cc thao tc v trng trong nui cy v k thut gieo ht - Thu c cc cy con v trng trong ng nghim BI 4: NUI CY NH SINH TRNG 1.GII THIU Trong nui cy invitro, mt phng thc n gin v thng hay c s dng ti sinh chi invitro l nui cy nh sinh trng. Hiu mt cch ng ngha th nui cy nh sinh trng l s dng phn m phn sinh ngn vi 3-4 tin pht khi l, tc l cc nh sinh trng c kch thc t 0,1 0,15mm tnh t chp sinh trng. K thut ny kh phc tp, phi thc hin di knh lp v kh nng sng st ca mu cy c kch thc nh nh th thng khng cao, do ch c tin hnh khi cn nui cy vi mc ch to cc cy con invitro sch virus. Trn thc t ngi ta thng nui c nh chi non vi kch thc khong vi mm. c th l nh chi ngn hoc nh chi nch. Mi nh sinh trng nui cy iu kin thch hp s to ra mt hay nhiu chi v mi chi s pht trin thnh cy hon chnh. Xt v ngun gc ca cc cy , c th c ba kh nng: - Cy pht trin t chi ngn - Cy pht trin t chi nch ph ng - Cy pht trin t chi mi pht sinh, v d: nui cy on tr h dip ca cy mng cu (Annona squamosa) s cho rt nhiu mm (buds) trn m cy v mt s mm sau s pht trin thnh chi v sau s thnh cy invitro hon chnh. Tuy nhin thng thng rt kh phn bit chi ph ng v chi mi pht sinh. Cc phng thc pht trin cy hon chnh t nh sinh trng nui cy nh sau:

* Pht trin cy trc tip: Ch yu cc i tng hai l mm (dicotyledon) nh khoai ty, thuc l, cam chanh, hoa cc V d: Khoai ty (Solanum tuberosum): Mm (nh sinh trng) Chi nch Cy * Pht trin cy thng qua giai on protocorm Ch yu gp cc di tng mt l mm (monocotyledon) nh phong lan, da, hu Cng mt lc nh sinh trng to hng lot protocorm (proembryo) v cc protocorm ny 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:Hoa lan (Orchidaceae): nh sinh trng Protocorm Cy Theo Champagnat (1977) v Fast (1980), cc chi non ang tng trng di 10-15cm, ca mi nh l thng c dng lm vt liu cho vic nui cy nh sinh trng. t bn c di sch i vi nc chy v l c lt sch cho n khi thy r cc chi bn. Chi non lc ny c nhng vo cn 70% v c kh trng trong dung dch kh trng 10%(v/v). Cc chi bn c ly ra v ra trong nc ct v trng. Sau , chng c kh li thm 10 pht na trong dung dch kh trng 3% c cha Tween80 v c ra li trong nc ct v trng. Trong t cy v trng, phn gc ca nhng chi nh nht c ct b v vic cy c tin hnh. Trn cc chi ln hn, trc ht tch b cc l v phn gc b cht do tc ng ca cht kh trng, sau cy vo mi trng. Phi mt mt thi gian tng i di th protocorm mi c thnh lp. Cc protocorm ny c ct ra v cy chuyn vo mi trng mi. Ngy nay, ngi ta thng cy cc nh chi ln hn (mang 3-4 tin pht khi l) v d thnh cng hn l cy cc nh chi ch c 2 tin pht khi l. Nu protocorm khng c ct khi mu cy th n pht trin thnh ci v ra r; nu c tch ra khi mu cy th s c s thnh lp cc protocorm bt nh mi t cc peotocorm ban u. Khi cy nh sinh trng ca Cymbidium ch c vng xung quanh tin pht khi l u ln v cui cng to thnh protocorm. S thnh lp protocorm c th c to ra m khng cn c nh sinh trng ngn. Khi cy nh sinh trng ca Cattleya, cc m thng nhanh chng ho nu. V l do m nh sinh trng c ct trong mi trng lng hoc nc ct v trng v c vy trong mi trng lng, nh cc cht nu d khuych tn vo trong mi trng v t gy nh hng n m cy (Fast, 1980). Cattleya, ngi ta thng tch mt chi (35mm) vi nhiu tin pht khi l. Mi trng cy Cattleya thng phc tp hn mi trng cy Cymbidium (Fast, 1980 v Champanat,1977) i khi c cha auxin, cytokinin, nc da v peptone. S thnh lp protocorm Cattleya mt nhiu thi gian v lun c to ra phn gc ca l gi nht; thc t nh sinh trng ngn khng ng vai tr g c v mt i. Vic cy cc tin pht khi l Cattleya cng c th p ng cho s thnh lp protocorm. Mi trng nui cy nh sinh trng tng i ngin v rt ging mi trng gieo ht cho nhiu ging lan khc nhau. Cymbidium thng c nui cy trn mi trng khong Knudson C hoc Vacin & Went nh sinh trng thng c cy trn mi trng c (ngoi tr Cattleya)nhng protocorm thng c nhn ln trong mi trng lng, v protocorm ch tng trng thnh chi con khi c cy trn mi trng c, thnh phn mi trng thng khc nhau trong tng giai on. Mi trng c pH trong khong t 4,8 5,8. Nng ng saccharose t 1-3% (w/v) hoc i khi l 1,5% glucose + 1,5% fructose. Mt s loi lan n thn th khng cn ng trong giai on nui cy nh sinh trng v s hin din ca ng c th lm nh sinh trng v v cht. Trong giai on to protocorm, thng ng v nc da (!0-15%) c thm vo mi trng kch thch s thnh lp protocorm. Cht iu ho

sinh trng thc vt ni chung khng cn thit, s hin din ca chng trong mi trng c th lm c hi cho t bin ln hn. Nhit ti u cho nhn ging t 22-28oC. nh sng n hunh quang thng c s dng vi 12-16 gi chiu sng/ngy. C th nui cy cng chiu sng thp nhng cn gia tng nh sng trong giai on to chi t protocorm. Ni chung c th thc hin vic nhn ging lan t nh sinh trng theo 2 cch: hoc trn mi trng c hoc trn mi trng lng. Trong mi trng lng cn lc vng vi vn tc rt khc nhau tu theo loi nhng hu ht cc nh nghin cu thng thc hin vi vn tc thp 2-5 vng/pht. S nhn ging trong mi trng lng thng tt hn trn mi trng c bi v khi lc s cung cp O2 v cht dinh dng cho mu cy hiu qu hn. Khi cy con cao khong 5-7cm vi 3-4 l c th mang ra trng trong vn m. Cc i tng hoa lan mang li hiu qu kinh t c bit cao. Sau nhng kt qu u tin chi 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 hoa lan c phng thc sinh sn qua protocorm. Nh c phng thc nhn ging nhanh v r tin m hoa lan vn t tr nn c gi c phi chng v c nhiu ngi a chung. Nhng thnh cng h Lan khng nhng ch l bng chng m cn m ng cho vic ng dng k thut ny i vi cc loi cy khc. 2.THC HNH 2.1 Mc ch - Kh trng mu nh chi - kho st s ti sinh cy trc tip t nui cy nh chi - Kho st s thnh lp protocorm t nui cy nh chi 2.2 Nui cy pht trin thnh cy trc tip 2.2.1 Nguyn vt liu on thn non cy cam hoc chanh 2.2.2 Mi trng nui cy Mi trng MS b sung BA 2ppm v NAA 0,2ppm 2.2.3 Tin hnh: - Cc cnh mu non ly t vn m v c ct b ht l, ct thnh on 2-3cm, cho vo bcher - Ra sch cnh mu bng nc x phng long, sau ra sch x phng bng nc my nhiu ln di vi nc chy mnh. - a cnh mu vo t cy v trng, ngm trong cn 70% trong 2-3 pht - Ra sch cn bng nc ct v trng 1 ln - X l mu bng Ca(OCl)2 6% trong 10 pht sau thay bng dung dch Ca(OCl)2 5% trong 5 pht - Ra sch mu bng nc ct v trng 6-7 ln cho ht mi javel - Cnh mu c ct b cung l v 2 u ca phn thn b cht kh trng ty trng. Chia cnh mu thnh cc t 1cm - Cm cc t vo mi trng nui cy chun b, cho phn cung l hng ln trn, chi ng phi nm trn mt thong ca mi trng. - Nui mu trong iu kin sng 2000lux/16h/ngy 25oC.

2.3 Nui cy pht trin cy thng qua giai on protocorm 2.3.1 Nguyn vt liu Cc on chi con Dendrobium cao 10cm 2.3.2 Mi trng nui cy nh sinh trng (cho 1L mi trng) - Khong a lng Knudson 100mL - Khong vi lng Heller 5mL - Skoog II MS : 2mL - Vitamin Morel: 2mL - Glycin :2mL - Inositol : 5mL - Vitamin B1: 5mL - ng: 30g - Nc da: 150mL - Khoai ty: 60g - Than hot tnh: 0,25g - pH: 5,5 - Agar: 6g * Khong a lng ca KnudsonC (Morel,G.M.,1965) - NH4NO3: 500mg/L - NH4(SO4)2: 500mg/L - Ca(NO3)2: 241,3mg/L - KCl : 250 mg/L - KH2PO4: 250 mg/L - MgSO4: 122,15mg/L * Khong vi lng ca Heller (1953) - AlCl3.6H2O : 0,054 mg/L - CuSO4.5H2O : 0,03 mg/L - H3BO3: 6,2 mg/L - KI : 0,01 mg/L - MnSO4.H2O : 0,08 mg/L - NiCl2.6H2O : 0,03 mg/L - ZnSO4.7H2O :1,0 mg/L 2.3.3 Cc bc tin hnh: - Ra sch chi Dendrobium di vi nc chy. Dng dao m lt ht cc l non bao xung quanh chi cho n khi l ra cc chi bn v chi ngn - Ngn chi trong nc x phng long v dng gn lau nh ln thn chi. Ra cho sch x phng di vi nc chy - Lc chi trong cn 70% trong 1 pht. Sau x l vi dung dch javel thng phm theo t l 1:5 trong vng 25-30 pht - Trong t cy, dng kp v trng cho chi vo cc bcher c cha nc ct v trng. lc nh th 3-5 ln cho ht mi javel. - t cc chi ln giy v trng.dng dao ct ring tng chi bn v chI ngn ra khi chi m ban u. Nh ct b ht cc m cht b cht kh trng lm trng phn gc chi. Cy tng chi vo ng nghim c cha mi trng chun b. - Nui trong phng sng - Ghi r tn nhm, ngy cy, ging cy vo tt c cc ng nghim

2.4 Yu cu - Thc hin tt thao tc cy v trng v kh trng mu BI 5: NUI CY M SO 1. GII THIU Nui cy m so l khu rt quan trng trong nui cy m t bo. M so l nguyn liu khi u cho cc nghin cu quan trng khc nh: phn ha m v t bo, chn dng t bo, nui cy t bo trn, nui cy t bo n, nui cy phi soma, sn xut cc cht th cp c hot tnh sinh hcM so l mt khi t bo khng c t chc, hnh thnh t cc m v cc c quan phn ha di cc iu kin c bit (c vt thng, x l cc cht iu ho sinh trng thc vt). Cc t bo thuc cc m hoc c quan ny phi chu mt s phn phn ha trc ln phn chia u tin. Nhn chung s to m so invitro (nh auxin tc ng) do 3 qu trnh: - S phn phn ha t bo nhu m (t nhiu su bn trong c quan) bao gm cc t bo nhu m mc v libe, nhu m v hay li. - S phn chia ca cc tng tng: cc t bo tng tng ca phn ln STD d dng phn chia di tc ng ca auxin thm ch khng cn auxin ngoi sinh nh cc loi cy c hay dy leo. - S xo trn ca cc m phn sinh s khi (chi hay r) qu trnh ny c u tin p dng TD, v cc cy ny tng tng thiu v nhu m kh phn phn ho so vi STD Mu sc ca m so khng ging nhau trn cc mi trng nui cy khc nhau hay trn cc b phn khc nhau v chng thng c mu vng, trng, nu hay trng xanh Nng v loi kch thch t s dng trong mi trng nui cy l nhng yu t c nh hng n s hnh thnh v pht trin m so. Thng m so c hnh thnh trn mi trng giu auxin; c th dng auxin ring r hay kt hp vi nhau hoc c th kt hp vi cytokinin tu tng loi cy. Hm lng hormon ni sinh v chiu di chuyn ca cc hormon ny trong mu cy c nh hng n s pht sinh m so. V vy ngun mu cy, vic ly mu cy, cch t mu cy trn mi trng nui cy s nh hng n s pht sinh m so dn n nhng phn ng khc nhau ca mu cy. Vi mt s cy th vn ny khng quan trng nhng cng c mt s cy chu nh hng rt ln. 2. THC HNH 2.1. Mc ch: Kho st s pht sinh m so t cc b phn khc nhau cy thuc l 2. 2 Vt liu 2.2.1 Mi trng nui cy MS (20g/l ng) c b sung 0.1M 2,4-D v 1M 2,4-D 2.2.2 Nguyn liu thc vt Cy con thuc l in- vitro 2.2.3 Ho cht v dng c - Nuc ct v trng - Dao, kp, a cy, giy cy

2. 3. Cc bc thc hin Cn thn gp cy con in-vitro ra khi bnh nui cy. Trnh kp qu mnh lm dp mu cy (hnh A) - Dng dao cy ct on r, lng thn v l chuyn qua mt da cy khc x l mu (hnh B) - L: ct b gn l v ra l. Phn l cn li c ct thnh nhiu mnh nh vi kch thc 0,8 -1mm x 8 10mm. t cc mnh l ny nui trn cc a petri cha mi trng MS + 1M 2,4-D BC - Lng thn: chn cc on lng thn c ng knh 2-2,5mm c ct lt mng 0,05 0,1mm bng li dao tht sc. Cc lt ct c t nm trn cc a petri cha mi trng MS + 1M 2,4-D R: Ra sch agar bng nc ct v trng, ct nh thnh tng on 1-1,5mm t ln cc a petri c cha mi trng MS + 0.1M 2,4-D - Dng nha nylon cun quanh mp a petri m bo s v trng trong thi gian nui cy. - Ghi r s nhm, tn mu cy, tn mi trng v ngy cy. - t nui trong ti 4. Yu cu: - Thao tc x l mu cy tt, lt ct dt khot cng mng cng tt; trnh dp mu - Ghi nhn v so snh thi gian pht sinh so, hnh dng v mu sc khi m so, v tr pht sinh so t cc mu cy khc nhau. BI 6: KHO ST S PHT SINH CHI T CC B PHN KHC NHAU CA CY THUC L 1.GII THIU Nhn ging cy trng bng phng php nui cy m t bo invitro c gi l vi nhn ging. C nhiu phng php vi nhn ging khc nhau to chi t to cy con invitro hon chnh. Tu thuc vo tng i tng khc nhau m s dng phng php ph hp v tt nhin mi phng php s c nhng u v nhc im ring * Vi nhn ging bng phng php ct t gim cnh - H s nhn thp - Thng ng dng cc i tng kh to cm chi (v d nh lan s) * Vi nhn ging bng cch tch chi t cm chi - H s nhn cao - Phc to hn trong vic kch thch chi pht trin cao thnh cy con hon chnh Cm chi c th c hnh thnh t nhiu con ng khc nhau: - Ti sinh t m so (kh nng t bin cao hn nn thng c s dng trong nui cy chn lc ging mi) - Pht sinh chi bt nh t cc c quan khng sinh sn ca cy nh lng thn, l, cung l, r, trc pht hoa, l i, cnh hoa (mu cy trI qua giai on phn phn ha to cc t bo sinh m; tip n l giai on to c quan vi giai on trung gian to m so m ta c th quan st thy hoc khng ri t mi pht sinh chi bt nh) - Ph trng thi tim sinh ca cc chi ng mu cy l 1 t chc nh t thn, nh sinh trng. Cn kim sot hormon tng trng chn ng s pht trin ca chi to nhiu

chi (cm chi) V nguyn tc c th kch thch s thnh lp chi bt nh t tt c cc c quan khng sinh sn ca thc vt. Tuy nhin trn thc t thng ch thnh cng trn i tng l l v cung l ngoi tr mt s i tng kinh in, d lm nh thuc l, c chua Hm lng v loi kch thch t b sung vo mi trng c nh hng quyt nh n s thnh lp chi (thng s dng kch thch t nhm cytokinin vi nng cao kt hp vi nhm auxin c nng thp). Kh nng ti to chi cn ph thuc vo kch thc ca mu cy. Mu cy qu nh c th khng p ng c vi cc iu kin nui cy v s ho nu. 2. THC HNH 2.1. Mc ch Chng minh nguyn tc vi nhn ging v kh nng ti to chi bt nh t cc b phn khc nhau ca thc vt 2.2. Vt liu 2.2.1 Mi trng MS (20g/l ng) c b sung 10M BA 2.2.2 Nguyn liu thc vt Cy con thuc l invitro 2.2.3 Ho cht v dng c - Nc ct v trng - Dao cy, kp, a cy v giy cy v trng 2. 3. Cc bc tin hnh X l mu cy tng t bi 5 v cy vo cc a petri c cha mi trng chun b (R, Lng thn, Phin l) 4.Yu cu - Thc hin tt thao tc x l mu cy - Lt ct lng thn cn mng n kch thc yu cu - Quan st v ghi nhn kt qu v s thnh lp chi , thi gian v s lng chi trn mi mu cy. BI 7: KHO ST S TI SINH CHI T M SO 1.GII THIU Qu trnh hnh thnh c quan trong m xy ra qua 2 giai on: - Giai on th nht l ti phn ha. Trong giai on ny xy ra qu trnh chuyn cc t bo bit ha thnh m so - Giai on th hai l hnh thnh cc mm mng c quan. Bng phng php phng x t bo thy rng nhng t bo ca cc mm mng nhu m m y c hnh thnh mm mng c quan, tng hp DNA v protein xy ra rt mnh, hm lng ng cng tng. Trong qu trnh phn ha, cc m so khng c t chc c hnh thnh cc cu trc hnh thi dn n vic to chi, r, cnh, hoa v cy hon chnh. Qu trnh phn ha ny c th thc hin bng cch thay i mt s cht v cc cht iu ha sinh trng trong mi trng nui cy i vi m so, xu th to c quan gim dn khi m cy chuyn nhiu ln v khi cy chuyn nhiu ln nh th thng hnh thnh cc t bo a bi v lch bi, ngoi ra c th mt cc yu t di truyn. Theo V Vn V (1999) m so khi hnh thnh thng c 2 loi:

 Loi xp: cha nhiu t bo xp vi nhn nh, t bo cht lng v khng bo to. Loi cng: Cc t bo cng, chc thnh khi, nhn to, t bo cht m c v khng bo nh. Dng m so cng c nh hng n kh nng ti sinh c quan ca khi m. Kh nng ti sinh chi sm mt i m so xp nhng vn duy tr m so cng. Nguyn nhn c th do cc t bo m so s mt i kh nng tng hp mt s cht thit yu cho s ti sinh ca n khi s ln cy chuyn tng ln ( Gautht, 1962). V vy khi nui cy m so nhm mc ch ti sinh chi, nht thit phi c gng tm iu kin mi trng thch hp cho s hnh thnh cc khi m so cng, chc; cc m so xp cn c loi b trong cc ln cy chuyn v i khi dng m so ny pht trin rt nhanh v ln t c cc m so cng c kh nng ti sinh phi. 2.THC HNH 2.1. Mc ch: Chng minh kh nng ti sinh chi t m so 2.2. Vt liu: 2.2.1 Mi trng MS (30g ng) + BA 1,5M + NAA 0,5 M 2.2.2 Nguyn liu thc vt M so t lng thn, l v r ca bi 3 sau 4 tun nui cy 2.2.3 Ho cht v dng c - Nc ct v trng - Dao cy, kp, a cy v giy cy v trng 2. 3. Cc bc thc hin - Dng kp gp m so t bnh mu cho vo a petri - Dng dao cy ly sch phn agar cn bm vo mu - Nu m so qu ln th ct nh thnh tng mu khong lng tay - Cy vo mi trng ti sinh chi chun b - Ghi r tn nhm, tn mi trng, tn ging, ngy cy - t mu nui trong phng sng 4. Yu cu: - Thc hin tt thao tc cy chuyn - Ghi nhn t l mu to chi, thi gian ti sinh chi, s chi trn mI mu, kch thc chi, s bin i ca mu - So snh s pht trin ca cc loi m so t cc b phn khc nhau. Rt ra nhn xt. R Lng thn Phinl Chi ti sinh M so

BI 8: A CY RA VN M

1.GII THIU Cc k thut chit cnh, gim cnh v ghp l cc phng php nhn ging v tnh thc vt hay cn gi l nhn ging in vivo. Ngoi ra cn c k thut nhn ging in vitro hay nui cy m l mt phng php nhn ging v tnh thc vt cho h s nhn cao hn cc k thut trn Cy con in vitro khi pht trin hon chnh c y thn, l v r s c chuyn ra vn m. y l giai on kh khn nht trong k thut nhn ging v tnh bng nui cy m. Cy in vitro c nui cy trong iu kin n nh v dinh dng, nh sng, nhit , m nn khi chuyn ra t vi cc iu kin t nhin hon ton khc hn nh dinh dng thp, nh sng c cng mnh, nhit cao, m thp cy con d b stress, d mt nc v mau b ho. Mt khc trong mi trng t nhin c rt nhiu vi khun v nm gy bnh lm thi v cht cy. khc phc tnh trng ny, ngi ta c cc bin php nh sau: - Vn m cy cy m phi mt, cng chiu sng thp, nhit khng kh thp, m thp - Cy con c trng trn lung m cy c c cht d thot nc, ti xp, gi c m. C cht v cy con cn c x l vi thuc chng nm hoc thuc tm phng nga nm bnh cho cy - Trc khi a cy ra vn m nn cc bnh mu ngoi vn m trong 1-2 tun cho cy thch nghi dn vi iu kin t nhin - Trong nhng ngy u a cy ra vn m, lung m cy cn c ph nylon gim qu trnh thot hi nc l ( thng 7-10 ngy sau khi ra cy). Mi ngy nn ti nc 2 ln km theo phun sng thng xuyn khi tri nng nhm duy tr m. - Cy con sau khi trng 7-10 ngy cn phi bn phn cy pht trin ng thi phun thuc nga nm bnh. 2. THC HNH 2.1. Mc ch Lm quen cc k thut a cy ra vn m 2.2. Vt liu 2.2.1.i tng cy ging: - Cy con Lan Hong Tho ( Dendrobium sp.) in-vitro - Cy con Thuc l in-vitro 2.2.1 Nguyn liu v dng c - X da - Tro tru - Ct xy dng - Dy thun - Ti m 12 x 17cm - Kp - Thau nha - R nha - Bnh phun 2. 3. Cc bc thc hin - Cho mt t nc vo cc bnh mi trng c cy ging, lc mnh mi trng v ra. - Dng kp gp nh nhng cy ging ra khi bnh trnh lm t,dp thn, l hoc r cy - Ra sch agar bm r cy bng 1 tm nhn; cn thao tc nh nhng khng lm t v dp r ; nu khng khi em trng vm m cy s d b nhim nm bnh v cht.

- Ngm cy trong dung dch thuc tm 10/00 trong 1 pht sau ra li bng nc sch ro. - i vi Thuc l (gi th trng l hn hp x da, tro tru v ct xy dng): Dng 1 cy a xm cc l m trn ti m vI su khong 5cm. t gn r cy con vo l m v dng tay m cht t li gi cy ng vng. m 5 cy cho 1 ti m - i vi Lan hong tho (gi th trng l x da): Gi gn phn r trong ming x da, dng thun buc nh nh hnh cy. Xp vo r nha, ti phun sng nh trc khi a ra vn m. 4. Yu cu: - Thc hnh thao tc a cy con ra vn m, trnh lm dp v t r qu nhiu - Quan st t l cy con sng st sau khi ra vn m.