Bai Giang Thuc Hanh _ Nuoi Cay Mo TBTV

Embed Size (px)

Text of Bai Giang Thuc Hanh _ Nuoi Cay Mo TBTV

Ti liu chia s ti: wWw.SinhHoc.edu.vnMC LC

Bi 1. Phng php pha ch mi trng nui cy m thc vt.............................................2 Bi 2. Phng php kh trng m thc vt...........................................................................8 Bi 3. Kh trng nui cy chi ng cc v ht cam............................................................12 Bi 4. Vi nhn ging da bng nui cy chi ng...............................................................15 Bi 5. Phng php nui cy to m so thc vt............................................................20 Bi 6. K thut to o bao ht ging nhn to.....................................................................25 Bi 7. Phng php tch v nui cy nh sinh trng.......................................................30 Bi 8. Phng php vi ghp thc vt................................................................................34 Bi 9. Phng php thun ho cy ra vn m.................................................................38 TI LIU THAM KHO .................................................................................................................................................. 42

THC HANH NUOI CAY MO THC VAT

Trang 1

Ti liu chia s ti: wWw.SinhHoc.edu.vnBI 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

Trang 2

Ti liu chia s ti: wWw.SinhHoc.edu.vn- 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 17 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 - Bnh mu: 1000 ml n v tnh ci ci ci ci ci ci ci ci ci ci ci ci ci ci ng g ci S lng 10 3 12 3 15 5 15 1 30 15 15 15 15 15 100 200 2 Bo qun cc stock Dng pha Fe-EDTA Ghi ch

THC HANH NUOI CAY MO THC VAT

Trang 3

Ti liu chia s ti: wWw.SinhHoc.edu.vn3 - 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 10 oC, 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: Tn ho cht H3PO3 MnSO4.4H2O Trang 4 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)

THC HANH NUOI CAY MO THC VAT

Ti liu chia s ti: wWw.SinhHoc.edu.vnZnSO4.4H2O 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; M 2,4-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

Trang 5

Ti liu chia s ti: wWw.SinhHoc.edu.vnTn 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.

THC HANH NUOI CAY MO THC VAT

Trang 6

Ti liu chia s ti: wWw.SinhHoc.edu.vnBI 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.