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Bi 1. Phng php pha ch mi trng nui cy m thc vt Bi 2. Phng php kh trng m thc vt Bi 3. Kh trng nui cy chi ng cc v ht cam Bi 4. Vi nhn ging da bng nui cy chi ng Bi 5. Phng php nui cy to m so thc vt Bi 6. K thut to o bao ht ging nhn to Bi 7. Phng php tch v nui cy nh sinh trng Bi 8. Phng php vi ghp thc vt Bi 9. Phng php thun ho cy ra vn m TI LIU THAM KHO 4 2

2 8 12 15 20 25 30 34 38

THC HANH NUOI CAY MO THC VAT

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BI I PHNG PHP PHA CH MI TRNG NUI CY M THC VT1 - NGUYN TC 1.1 - Nguyn tc pha ch mi trng Mi trng nui cy m thc vt khc vi cc loi mi trng nui cy vi sinh l c thnh phn rt phc tp, s hin din ca rt nhiu loi ho cht vi hm lng rt nh. Do vy, thun tin cho vic pha cc mi trng nui cy (mi trng lm vic), ngi ta khng cn ho cht mi ln pha mi trng m chun b trc di dng nhiu loi dung dch vi m c t X10 X100, ch cn pha vi nc ct hai ln hay nc v khong khi s dng. Cc dung dch ny gi l dung dch m (stock). Dung dch m c bo qun di ngy trong t lnh thng hoc t lnh su. Thng thng pha cc dung dch m: stock a lng, stock vi lng, Fe EDTA, stock vitamin, stock cht sinh trng, v cc stock cn thit khc. 1.2 - Phn loi mi trng nui cy m thc vt Ty theo thnh phn cc cht c trong mi trng, c th phn thnh 3 loi mi trng: - Mi trng ngho cht dinh dng: nh mi trng White, Knop. Mi trng ny thch hp vo cc giai on cui ca quy trnh nui cy m thc vt khi chun b chuyn cy t ng nghim ra vn m. Mi trng ngho cht dinh dng cng c th s dng trong cc sinh trc nghim kim tra c tnh bin dng ca m thc vt. - Mi trng c hm lng cht dinh dng trung bnh: mi trng B5, Gamborg. Loi mi trng ny c s dng i vi mt s loi cy c nhu cu cc cht dinh dng trong mi trng va phi hoc cng c th s dng khi nui cy m thc vt di ngy. - Mi trng giu cht dinh dng: mi trng MS (Murashige Skoog). y l mi trng khi u cho mi qu trnh nui cy m i vi mi i tng nui cy. Mi trng MS l mt mi trng thch hp vi nhiu loi cy, do giu v cn bng v cht dinh dng. 2 - NGUYN LIU - HO CHT - DNG C 2.1 - Ho cht - Saccharose (sucrose) - Agar - NH4NO3 - KNO3 - MgSO4.7H2O - KH2PO4 - CaCl2.5H2OTHC HANH NUOI CAY MO THC VAT

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- KI - Na2MoO4.2H2O - CoCl2.6H2O - H3PO3 - MnSO4.4H2O - ZnSO4.7H2O - CuSO4.5H2O - Thiamine HCl - Myo-inositol - Glycine - NAA - BA (6-Benzyl aminopurin) - Na2-Ethylen diamin tetraacetat (Na2 EDTA -Fe2(SO4)3 2.2 - Dng c Thit b cn cho 1 nhm (30 sinh vin) thc hnh STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tn dng c, thit b - Bnh nh mc: 100 ml - Bnh nh mc: 500 ml - ng ong: 100 ml - ng ong 500 ml - a khuy - Bnh mu 200 ml - Qu bp cao su - B n nhit - Cc 100 ml - Cc 200 ml - Pipett: 1 ml - Pipett: 5 ml - Pipett: 10 ml - Bnh nh mc 250 ml - ng nghim 25 - Bng m n v tnh ci ci ci ci ci ci ci ci ci ci ci ci ci ci ng g S lng 10 3 12 3 15 5 15 1 30 15 15 15 15 15 100 200 Dng pha Fe-EDTA Ghi ch


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17

- Bnh mu: 1000 ml

ci

2

Bo qun cc stock

3 - NI DUNG THC HNH Hc sinh chia nhm thc hnh pha ch cc dung dch m (stock) ca mi trng MS (Murashige Skoog) nh sau: dung dch m khong a lng (Skoog I), dung dch m Fe EDTA (Skoog II), dung dch m khong vi lng (Skoog III), dung dch m cht iu ho sinh trng 4 - THC HNH PHA CH CC DUNG DCH M (Stock) MI TRNG MS (Murashige Skoog) 4.1. Pha stock khong a lng mi trng MS nng m c 20 ln (x20) Cn chnh xc bng cn phn tch cc mui khong a lng. Dng ng ong, ong khong 400 ml nc ct hai ln vo becher 1000 ml. Cho ln lt cc mui a lng vo theo trnh t nht nh sau: Tn ho cht - MgSO4.7H2O - KH2PO4 Nng mi trng nui cy (mg/lt) 370 170 Nng dung dch stock (x20) (mg/lt) 7.400 3.400

- KNO3 1.900 38.000 - NH4NO3 1.650 33.000 - CaCl2.2H2O 440 8.800 Mi ln cho mt loi khong vo phi khuy tan hon ton v b sung thm 100 ml nc ct hai ln trc khi b sung cc khong khc vo (phng php pha th tch tng dn). Do CaCl2.2H2O c th phn ng to kt ta vi MgSO4.7H2O nn hai cht ny phi pha tch ri nhau theo ng trnh t trn, cho CaCl2.2H2O vo sau cng. Cho tt c vo bnh nh, dng nc ct hai ln chun li cho ng 1000 ml. Nh vy ta c 1000 ml dung dch m khong a lng m c 20 ln. Dung dch ny c bo qu dng dn trong chai mu nhit 4 10oC, dng 50 ml cho mt 1000 ml mi trng nui cy. 4.2. Pha stock khong vi lng nng m c 1000 ln (x1000) Do hm lng mt s loi khong vi lng trong stock khong vi lng rt nh, kh c th cn ong chnh xc nh mt s cc khong vi lng khc. V vy y, ta phi tin hnh pha ring dung dch cc khong ny (CuSO4.5H2O, CoCl2.6H2O) c nng m c hn 100 ln so vi dung dch m v gi l dung dch b ngoi. Sau khi pha song cc dung dch b ngoi, tin hnh pha 100 ml stock khong vi lng X1000 tng t nh phn trn. Thnh phn cc khong nh sau: Nng mi trng nui cy (mg/lt) 6,2 22,3 Nng dung dch stock (x1000) (mg/ lt) 6200 22300 Nng dung dch b ngoi (x100) (mg/lt)

Tn ho cht H3PO3 MnSO4.4H2O


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ZnSO4.4H2O Na2MoO4.2H2O KI CuSO4.5H2O CoCl2.6H2O

8,6 0,25 0,83 0,025 0,025

8600 250 830 25 25

2500 2500

4.3. Pha dung dch m Fe-EDTA nng m c 200 ln (x200) St l yu t rt cn thit cho thc vt. Tuy nhin, nhu cu ca thc vt i vi st rt nh v lng lin tc. V vy, cung cp lng st cho thc vt ta thng dng st di dng hp cht Fe-DETA. Hp cht Fe-EDTA c hnh thnh do s ngm Fe ca EDTA di tc dng ca nhit . di dng hp cht ny, st s c phng thch ra mi trng tu theo nhu cu ca thc vt. Phng php pha nh sau: Tn ho cht FeSO4.7H2O Na2 EDTA Nng mi trng nui cy (mg/lt) 27,8 37,3 Nng dung dch stock (x200) (mg/lt) 5560 7460

Chun b hai becher 500 v 1000 ml. Cho vo mi becher 400 ml nc ct hai ln. un nng hai becher trong b n nhit khong 80oC. Cn chnh xc 5,56 g FeSO4.7H2O cho vo becher 500 ml c 400 ml nc trn, khuy tan hon ton. Cn chnh xc 7,46 g Na2 EDTA cho vo becher 1000 ml trn, khuy tan hon ton. Cho t t dung dch FeSO4.7H2O vo dung dch Na2 EDTA, va va khuy u. Cui cng b sung nc cho 1000 ml. Nh vy, ta c 1000 ml dung dch stock Fe-EDTA m c 200 ln c mu vng nht, trong sut. Dung dch ny c bo qu dng dn trong chai mu nhit 4 10oC, dng 5ml cho mt 1000 ml mi trng nui cy. 4.4. Pha stock hormon Cc hormon khc nhau s tan trong cc dung mi khc nhau. IAA, NAA, BA pha trong NaOH 1N (thm 2-3 ml NaOH 1N v lc cho n khi tan ht, sau thm nc n th tch cn). GA3, 2,4-D pha trong cn 50o cho th tch. pha cc cht sinh trng, c hai cch khc nhau: + Cch 1: pha theo h mol V d: Pha 100 ml dung dch stock IAA (M=176,19) c nng 1 mmol (milimol) t IAA tinh khit. 176,19 g IAA pha trong 1000 ml nc 1000 ml dung dch c nng 1 mol/lt. pha dung dch c nng 1 mmol/lt cn 0,17619 g IAA. Nh vy, mun c 100 ml dung dch IAA nng 1 mmol/lt cn 0,017619 g IAA. (MNAA: 186,2; M2,4-D: 221,04; MBA: 225,2) + Cch 2: pha theo h mg/lt: lm tng t nh cch pha khong. Cc dung dch stock hormon sau khi pha song c bo qun trong cc chai mu nhit lnh 4-10oC. 4.5. Pha stock vitamin mi trng MS nng m c 500 ln (x500) Dng ng ong, ong 50 ml nc ct hai ln vo mt becher 100 ml. B sung cc vitamin vo theo trnh t sau:THC HANH NUOI CAY MO THC VAT

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Tn ho cht Acid nicotinic Thiamin HCl

Nng mi trng nui cy (mg/lt) 0,5 0,1

Nng dung dch stock (x500) (mg/lt) 250 50

Pyridoxin HCl 0,5 250 Myo-inositol 100 50.000 Glycine 2 1.000 Cho tt c dung dch vo bnh nh mc, dng nc ct b sung cho 100 ml. Nh vy, ta c 100 ml stock vitamin MS x500. Dung dch ny c bo qu dng dn trong chai mu nhit di 0oC, dng 2ml cho mt 1000 ml mi trng nui cy. 4.6. Pha mi trng lm vic (mi trng nui cy) Pha 1 lt mi trng MS c bn: - Dng ng ong, ong 500 ml nc ct hai ln vo becher. - B sung 30 g ng sucrose vo, khuy tan hon ton - B sung 50 ml dung dch stock a lng x20, khuy u. - B sung 1 ml dung dch stock vi lng x1000, khuy u. - B sung 5 ml dung dch stock Fe-EDTA x200, khuy u. - B sung 2 ml dung dch stock vitamin x500. - B sung stock hormon vi lng ty mc ch nui cy. - Chuyn tt c dung dch vo bnh nh mc, dng nc ct chun li cho 1000 ml - Chnh pH ca dung dch bng NaOH 1N hay HCl 1N v pH=5,8. - B sung 6-8 g agar. - un si cho tan agar, quy u. Chia hn hp mi trng vo cc bnh nui cy, cn chia xong trc khi dung dch mi trng MS ngui xung 50oC. Hp kh trng (i vi cc thnh phn b bin tnh nhit cao s c lc v trng v b sung vo mi trng sau khi hp). Mi trng sau khi hp c bo qun 25oC. 5 - BO CO THC HNH 1. Trnh by cch pha Skoog I, Skoog II v Skoog III. 2. Quan st hin tng phn ng khi pha stock Fe-EDTA. Gii thch. 3. T BA tinh khit, trnh by cch pha 100 ml stock BA c nng 100 mol/lt. Bit MBA =225,2. 4. Cn bao nhiu ml dung dch stock IAA c nng 150 mol/lt pha mi trng lm vic c nng IAA l 0,1 mol/lt.


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BI 2 PHNG PHP KH TRNG M THC VT1 - NGUYN TC Mt th nghim nui cy m thc vt khc vi mt th nghim nui cy vi sinh thng tin hnh trong mt thi gian rt lu di. Vi mt th nghim trng din nh th, ch cn mt t bo vi khun, mt bo t nm mc ri vo mi trng nui cy, sau mt thi gian ngn s sinh si lm hng mi trng, lm hng th nghim v phi tin hnh lm li t u. Do vic v trng tc loi b ht nm khun trong nui cy m thc vt v cng quan trng. 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 c ty theo s tip xc vi mi trng bn ngoi, cc b phn ny cha nhiu hay t vi khun v nm. chuyn m thc vt t mi trng t nhin vo mi trng nui cy in vitro, m thc vt phi tri qua giai on kh trng loi b ht cc mm vi sinh vt. Tuy nhin qu trnh ny phi m bo l m thc vt cn sng, c kh nng tng trng, pht trin tt. Phng php v trng m cy thng dng nht hin nay l dng cc cht ha 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 lc m cy vi cn 70%, sau mi x l dung dch dit khun. Bng 1. Mt s tc nhn kh trng dng cho thc vt Tc nhn v trng Canxi hypoclorid Natri hypoclorid Hydro peroxid Nc Brom HgCl2 Cht khng sinh Nng (%) 9 10 2 10 12 12 0,1 1 4 50 Thi gian x l (pht) 5 30 5 30 5 15 2 10 2 10 30 60 Hiu qu Rt tt Rt tt Tt Rt tt Trung bnh Kh 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 k bng x phng bt v ra sch li bng nc my. Khi x l song, 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 ct b trc khi t m cy ln m trng. trnh nh hng trc tip ca cc tc nhn v trng ln m cy, nn ch li mt lp bc ngoi khi ngm m vo dung dch dit khun. Lp cui cng ny s c ct b hoc bc i trc khi t m cy ln mi trng.THC HANH NUOI CAY MO THC VAT

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Nh vy, c th t c mc ch kh trng (loi b ht mm vi sinh vt, m thc vt c th tng trng, pht trin), th vic kh trng l phi tm ra c ba yu t lin quan cht ch vi nhau v lin quan ti m thc vt em kh trng l: - Cht kh trng - Nng cht kh trng - Thi gian kh trng. V trng mu l mt thao tc kh, t khi thnh cng ngay ln u tin. Tuy vy, nu kin tr tm nng v thi gian v trng thch hp th sau vi ln th chc chn s t kt qu. 2 - VT LIU - HO CHT DNG C 2.1 - Vt liu - Cy Tru b - Ht thuc l 2.2 - Ho cht - X phng bt - Cn 70o, 90o - Javel - Ca(OCl)2 (Caxi hypoclorid) 2.3 - Dng c Thit b cn cho 1 nhm (30 sinh vin) thc hnh STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Tn dng c, thit b - Dao m - Li dao m - ng ong: 100 ml - ng ong 500 ml - a khuy - Cc 500 ml - Qu bp cao su - Cc 1000 ml - Cc 100 ml - Cc 200 ml - Pipett: 1 ml - Pipett: 5 ml - Pipett: 10 ml - Erlen 250ml n v tnh ci ci ci ci ci ci ci ci ci ci ci ci ci ci Trang 8 S lng 15 15 12 3 15 15 15 5 30 15 5 5 5 60 Dng li dao m nhn Ghi ch


STT 15 16 17 18

Tn dng c, thit b - Kp di 25 cm - Bng m - ng nghim 25 - T cy v trng

n v tnh Ci g ci ci

S lng 15 200 15 1

Ghi ch

T cy c qut thi v trng

3 - NI DUNG THC HNH Hc sinh thc hnh kh trng t thn cy Tru b, ht thuc l. Sau khi v trng tin hnh gieo ht thuc l, cy t thn cy Tru b ln mi trng MS. 4 - THC HNH 4.1 - Kh trng cy Tru b a. Thc hin trong phng th nghim - Cy Tru b c ct thnh nhng on nh 30cm. Ra cc on ny di vi nc my cho sch bi t. - Ct thnh nhng on Tru b ny thnh nhng t khong 3cm, mi t mang mt chi ng. Ct b l, r. - Ra cc t vi nc c pha mt t x phng trong cc erlen. - Ra li bng nc my nhiu ln cho sch x phng. b. Thc hin trong t cy - Lc cc t thn vi cn 70o trong 1 pht - Chit b cn. Ngm cc mu trong dung dch Caxi hypoclorid 10% trong 10 pht (hay dung dch javel c pha long vi t l 1:9 trong 5 pht). - Ra sch dung dch kh trng bng nc ct v trng 5-6 ln - Gn b nc. 4.2 - Kh trng ht thuc l - Thc hin trong phng th nghim, ra ht thuc l di vi nc my. - Lc ht vi nc c pha mt t x phng. - Ra li nhiu ln bng nc my cho sch x phng - Thc hin trong t cy, lc ht vi cn 70o trong 1 pht. - Chit b cn, ngm ht trong dung dch Caxi hypoclorid 10% trong 5 pht. - Ra sch dung dch kh trng bng nc ct v trng 5-6 ln. - Gn b nc. 4.3 - Cch cy muTHC HANH NUOI CAY MO THC VAT

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a. Mu cy Tru b - Trong t cy v trng, nhng t cy Tru b kh trng, c t trn giy cy v trng. Dng dao m v kp tch cc l. - mi nch l c mang mt mm ng. Dng dao m ct mt on mu c kch thc khong 1x1 cm cy vo mi trng MS hp kh trng trong cc ng nghim. - Khi cy mu cn ch cy ng chiu ca thn. Cc ng nghim c t trong phng lnh nhit 25oC, cng nh sng 2500-3000 lux. b. Mu ht thuc l - Ht thuc l sau khi kh trng, dng kp t u trn b mt mi trng MS trong cc erlen. Khong cch gia cc ht l 1cm. - Cc erlen c t ni ti hon ton. 5 - BI BO CO THC HNH 1. Trnh by phng php kh trng t thn Tru b, ht thuc l. 2. Vai tr v tc dng ca tc nhn kh trng cn, canxi hypoclorid. 3. Ghi nhn kt qu nui cy.


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BI 3 KH TRNG, NUI CY CHI NG CC HT CAM1 - NGUYN TC Trong cc phng php nhn ging v tnh in vitro cy trng, bn cnh nhng phng php i hi ngi lm vic phi c k thut cao nh tch v nui cy nh sinh trng, vi ghp, phng php nhn ging bng nui cy chi ng (chi nch) l phng php n gin, d thc hin v mang li hiu qu cao nht. a s cc loi thc vt, mi nch l u mang mt chi ng. Cc chi ng ny c c tnh hon ton ging vi cc nh sinh trng (l mt nh sinh trng trng thi tim sinh), c kh nng tng trng pht trin thnh mt c th thc vt ton vn. Cc chi ng ny a s b c ch s pht trin bi cc chi nh (tnh u th ngn). Nu tch ring mt on thn c mang chi nch ny ra khi c th thc vt, di tc ng bi nhng yu t kch thch sinh trng ngoi sinh nh cytokinin, auxin th t mt chi nch ny c th cho ra mt cm chi vi rt nhiu chi mi (t hng chc ti hng trm chi). Vi nhng chi mi to thnh, tip tc cy chuyn sang nhng mi trng to chi mi th h s nhn ging rt ng k. Vi nhng loi thc vt c nh sinh trng nh (nh sinh trng du hoa th) th vic tch ly nh sinh trng rt kh thc hin. V vy, vi nhng loi thc vt ny, phng php nhn ging v tnh hiu qu nht l nui cy chi nch. Chi nch em nui cy thng gn lin vi mt on thn, do sc sng ca chi nch thng tt hn rt nhiu ln so vi nh sinh trng tch ri, hiu qu to cm chi cng thng tt hn so vi phng php nui cy nh sinh trng Vic tch chi nch thng thc hin vi nhng on nhnh c mang chi ca thc vt. Nhng on nhnh ny c kh trng trc khi ct nh thnh nhng on c mang chi nch. Bn cnh , vic tch chi nch cng c th thc hin t nhng cy v trng trong ng nghim c to ra do vic nui cy ht ging. Trong bi thc hnh ny, s cp ti phng php nhn ging v tnh thc vt qua nui cy chi nch v to ngun cy cho phng php nui cy chi nch t ht ging. 2- VT LIU HO CHT DNG C 2.1 - Vt liu - Cy hoa cc - Ht cam 2.2 - Ho cht - Cn 70o, 90o - Mi trng MS b sung 2,4 D - X phng bt - Cn 70o, 90oTHC HANH NUOI CAY MO THC VAT

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- Javel - NAA - BA 2.3 - Dng c Thit b cn cho 1 nhm (30 sinh vin) thc hnh STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Tn dng c, thit b - Dao m - Li dao m - ng ong: 100 ml - ng ong 500 ml - a khuy - Cc 500 ml - Qu bp cao su - Cc 1000 ml - Cc 100 ml - Cc 200 ml - Pipett: 1 ml - Pipett: 5 ml - Pipett: 10 ml - Erlen 250ml - Kp di 25 cm - Bng m - ng nghim 25 - Gi ng nghim l ln - n cn - T cy v trng n v tnh ci ci ci ci ci ci ci ci ci ci ci ci ci ci Ci g ci ci ci ci S lng 15 15 12 3 15 15 15 5 30 15 5 5 5 60 15 200 15 15 15 1 T cy c qut thi kh v trng Dng li dao m nhn Ghi ch

3 - NI DUNG THC HNH - Sinh vin tin hnh ct t thn cc, thc hin xc nh cng thc kh trng. Nui cy thn cc trn mi trng MS b sung. - Sinh vin tin hnh kh trng, tch v v nui cy ht cam trn mi trng MS. 4 - THC HNH 4.1 - Kh trng v nui cy thn cc ct tTHC HANH NUOI CAY MO THC VAT

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a. Kh trng - La cy cc cn ti, non, l t b dp. Dng dao ct b hoa v gc. Ct ly mt on cch gc 1015 cm. - Ra sch on thn cc di vi nc my. - Ct thnh nhng on nh. Ct gia hai chi ng. Mi on c mt chi ng. - Lc cc on trong nc x phng t 1015 pht. - Ra sch x phng di vi nc my. - Lc vi cn 70o trong 1 pht. - Lc vi dung dch javel/nc cc t l 1/9, 2/8, 3/7, 4/6, 1/1. - Ra sch javel bng nc ct v trng 56 ln. b. Nui cy - Thn cc sau khi kh trng c nui cy trn mi trng MS b sung BA v NAA vi t l NAA/BA

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