Bai Giang Thuc Hanh - Nuoi Cay Mo TBTV

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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
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BI I PHNG PHP PHA CH MI TRNG NUI CY M THC VT
1 - 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 - CaCl2.5H2O
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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 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
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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 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)

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; 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:
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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 VT
1 - 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.
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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 S lng 15 15 12 3 15 15 15 5 30 15 5 5 5 60 Dng li dao m nhn Ghi ch
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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 T cy c qut thi v trng Ghi ch
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 mu
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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 CAM
1 - 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, 90o
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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 t
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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 10 15 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 10 15 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 5 6 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 <1. - Cm thn cc vo mi trng sao cho chi ng vn nm trn b mt mi trng . 4.2 - Kh trng v nui cy ht cam a. Kh trng ht - Ra ht cam di vi nc my. - Lc ht vi nc x phng 10 15 pht. - Ra sch x phng bng nc my. - Lc vi cn 70o trong 30 giy. - Ra sch cn bng nc ct v trng 5 6 ln. b. Tch v ht - t ht cam ln trn giy cy. Dng kp gi ht. - Dng dao m ct hai ng dc theo chiu di ht. Hai ng ny i din nhau. Li dao ch va ct rch lp v, khng chm vo phn tht ht. - Dng dao v kp tch b lp v cng (v tru) v v la bn ngoi. - Ht cam l ht a phi, khi tch cc phi c th tch ri nhau. Ta vn c th dng nhng mnh phi nui cy, mi mnh phi s pht trin thnh mt cy cam. c. Nui cy - t ngang ht cam trn mi trng MS. - Dng kp nh ht ln b mt mi trng sao cho ht nhp vo mi trng - t bnh nui cy trong iu kin nh sng 2000 lux, nhit 25oC. 5 - BI BO CO THC HNH 1. Phng php la chn cng thc kh trng. 2. Ti sao phi tch v ngoi (v tru), v la ht cam trc khi nui cy?
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3. Nu bnh nui cy ht cam trong iu kin khng c nh sng, phi c ny mm pht trin c khng? Gii thch?
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BI 4 VI NHN GING DA BNG NUI CY CHI NG
1 - NGUYN TC i vi mt s cy nh tre, ma, da, phng php nhn ging vn thng c s dng hin nay trong nng nghip l nhn ging truyn thng bng chi nch, chi ngm, thn ngm, gim hom Tuy nhin c im chung ca cc phng php ny l h s nhn ging thp, khng p ng c nhu cu v cy ging m rng sn xut. Trc nhu cu ca vic m rng sn xut, trong mt s nm gn nay nhn ging da thng tin hnh bng nui cy chi ng (phng php nhn cm chi). u im ca phng php ny l tc nhn ging cao, h s nhn ging ln. c im ca phng php ny l chi ng c tch t cm chi ng ngn da, sau c nui cy to cm chi trn mi trng in vitro. Cc chi c hnh dng ging nh vy c hoc ht go nm p st vo thn (li), c mu trng. Mi chi c tch ring nui cy to thnh cm chi. 2 - VT LIU HO CHT DNG C 2.1 - Vt liu Chi ng c tch t ngn da dng lm vt liu nui cy. 2.2 - Ho cht - Cn 70o, 90o - Mi trng MS b sung NAA, BA - Javel - X phng bt 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 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 n v tnh ci ci ci ci ci ci ci ci ci S lng 15 15 12 3 15 15 15 5 30 Dng li dao m nhn Ghi ch
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STT 10 11 12 13 14 15 16 17 18 19 20 Tn dng c, thit b - 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 g ci ci ci ci S lng 15 5 5 5 60 15 200 15 15 15 1 T cy c qut thi kh v trng Ghi ch
3 - NI DUNG THC HNH - Hc sinh tin hnh tch b l cc ngn da thu chi ng. - Cc chi ng c tch ri, nui cy trn mi trng MS b sung NAA v BA. 4 - THC HNH 4.1 - Phng php kh trng chi ng da - Tch b ton b cc l phn ngn da. Tch ln lt cc l t di ln trn, t ngoi vo trong trch lm tn thng cc chi ng. - Tch ri phn ci (li) c mang chi ng. - Ra nh nhng ci da di vi nc my, trnh lm t thng cc chi ng. - Dng dao ct ly bn mt ca ci da c mang chi ng. - Lc nh nhng cc mnh c mang chi ng ny trong nc x phng trong 10 pht. - Ra sch nc x phng di vi nc my. - Lc cc mnh m da trong cn 700 trong 2 pht. - Kh trng vi nc javel (t l 3:7) trong 7 pht. - Ra sch li bng nc ct v trng 56 ln.
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Hnh 1. Ct cc mt c mang chi ng t ci da 4.2 - Phng php nui cy - Dng dao m ct nhng ng dc, ngang tch ri tng chi ng mun ly. - Ct vt 4 cnh di ca ming m da c mang chi ng to thnh hnh thp. - Cm phn u hnh thp ngp vo mi trng nui cy. - t cc bnh nui cy trong iu kin nh sng 3000 lux, nhit 25oC. 4.3 - Phng php to cm chi Sau thi gian nui cy t 20-30 ngy cc chi c cy chuyn sang mi trng nui cy to cm chi. Mi trng nui cy c nng cht iu ho sinh trng NAA 1mg/lt, BA 0,1mg/lt. Sau 30-40 ngy, cc chi nh xut hin pha gc chi. S lng cc chi nh tng dn ln, sp xp chng cht ln nhau thnh mt cm chi (thng l sau 3-4 thng nui cy). Cc chi nh c tch v chuyn sang mi trng ti sinh cy hon chnh. iu kin nui cy to cm chi da: cng nh sng 4000 lux, thi gian chiu sng 10-14 gi/ngy, nhit 25-270C.
Hnh 2. Cch ct ri chi ng da 5 - BI TNG TRNH 1. Trnh by nhng im cn lu khi tch chi ng da 2. Trnh by quy trnh nhn ging da bng chi ng. 3. Ti sao phi ct vt 4 cnh di ca ming m da c mang chi ng?
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BI 5 PHNG PHP NUI CY TO M SO THC VT
1 - NGUYN TC Trong c tnh sinh l ca c th thc vt, khi b nhng tn thng v mt vt l (nhng vt ct trn c th, nhng tn thng do cn trng tn cng) thc vt c kh nng hnh thnh nhng t bo mi hn kn nhng ch tn thng . Nhng t bo mi c hnh thnh l t bo m so. M so l mt khi t bo nhu m pht trin v t chc, hin din trong cc giai on ho lignin khc nhau ca thc vt, thng do cc t bo trong vng tng tng (vng phn sinh) nh tng tng liber mc, tng tng v gc ca on ct to thnh. Nhng t bo m so thng c hnh cu, mu trng hoc nu nht. Khi m so c kh nng ti sinh thnh cy hon chnh trong iu kin mi trng khng c cht kch thch sinh trng to m so. Nui cy to m so c thc hin i vi cc loi thc vt khng c kh nng nhn ging thng qua nui cy nh sinh trng. Nhng m ca thc vt c th dng nui cy to m so l: tng tng libe mc, tng tng v, phi nh, t bo dip nhc, l, tr b r, t dip Cy ti sinh t m so c c tnh ging nh cy m. T mt cm t bo m so c th ti sinh cho cng mt lc cho nhiu chi hn l nui cy nh sinh trng.
Hnh 3. S ti sinh chi t m so. A: M so, B: Chi ti sinh. M so thng c to ra do nhng xo trn trong qu trnh to c quan, nht l trong s to r. Do , cy non hay nhng mnh thn non ca cy trng thnh d to m so. Ngc li, nhng mnh c quan trng thnh khng c kh nng to m so. S to m so thc vt xy ra khi mi trng nui cy c b sung mt lng auxin (2,4-D) thch hp. S to m so do tc dng ca auxin do 3 qu trnh: - S phn phn ho ca t bo nhu m: xy ra cc t bo nhu m mc v libe, nhu m v hay li.
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- S phn chia ca cc t bo tng tng: cc t bo bo tng tng ca phn ln cy hai l mm d dng phn chia di tc ng ca auxin. - S xo trn ca cc m phn sinh s khi (chi hay r). 2 - VT LIU HO CHT DNG C 2.1 - Vt liu Ht thuc l (Nicotiana tabacum) c gieo trn mi trng MS. S dng cc cy con lm vt liu th nghim. 2.2 - Ho cht - Cn 70o, 90o - Mi trng MS b sung 2,4 - D 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
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3 - NI DUNG THC HNH - Hc sinh tin hnh ct t thn, mnh l, on r cy thuc l nui cy to m so, nhng on c mang chi ng c nui cy ti sinh cy. - Cc thnh phn ny c nui cy trn mi trng MS b sung 2,4 D. 4 - THC HNH 4.1 - Vt liu nui cy a. M cy t cy thuc l to ra qua nui cy ht - Tin hnh kh trng ht thuc l. - Nui cy ht thuc l trong mi trng MS. - Bnh nui cy c c trong iu kin ti, nhit 250C cho ny mm. - Cy con c nui cy trong iu kin c nh sng. - Sau 15-20 ngy nui cy, khi cy c t 6-8 l, cao khong 10-15cm, c th s dng l nguyn liu cho nui cy m so b. M cy t cy thuc l mc ngoi t nhin Cy thuc l mc ngoi t nhin c sc tng trng mnh, kh nng hnh thnh m so rt ln. Nhng m t cy ly t cy thuc l mc ngoi t nhin phi tri qua giai on v trng mu loi b ht nm, khun tn ti trong v trn mu cy. 4.2 - Chun b mu cy a. Ct t thn - Dng kp gp mt cy thuc l trong bnh nui cy t ln giy cy. - Dng dao m ct ri tt c l, r ra khi thn. - Ct thn thuc l thnh nhng on nh, mi on l mt t thn. T cc t ny, tip tc chia nh thnh cc on, mi on c kch thc khong 0,2 0,4 cm. Cc on nh ny c nui cy to m so. - iu lu khi ct cc on thn l phi b tt c cc phn mt ca t. Cc phn ny khng c kh nng to so, ch c th c s dng nui cy to chi. b. Ct mnh l - T cc l phn trn, s dng dao m ct b gn chnh v ra l. - Cc phn l ny c ct thnh cc mnh nh c kch thc 0,2 x 1 cm. - Cc mnh l ny c nui cy to m so. c. Ct on r - Sau khi ct cc on thn, l, phn r cn li c ra sch agar trong nc ct v trng. - Dng dao ct r thnh cc on nh c kch thc 0,2 - 0,4 cm. Cc on ny c nui cy to m so.
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A
B C
Hnh 4. Phng php ct mnh l (A), t thn (B), on r (C) 4.3 - Nui cy to m so a. Nui cy thn, l - Dng kp gp cc on thn mnh l cy trn mi trng MS b sung 2,4-D. - Khong cch gia cc on thn, mnh l l 1cm. Trnh cy qu dy. - S dng mi trng MS b sung 2,4-D c nng l 1mol/lt mi trng. b. Nui cy on r - Cc on r c nui cy trn mi trng MS b sung 2,4-D c nng 0,1 mol/lt mi trng. Nhng khi m so hnh thnh qua qu trnh nui cy c th c tch ra v chuyn sang nui cy c nng 2,4-D gim (0,05mol/lt mi trng) tip tc phn chia to thnh nhng khi m so mi. Thi gian cy chuyn gia cc ln nui cy l 4-6 tun. Khi m so cng c th c cho ti sinh thnh cy nu mi trng nui cy loi b 2,4D v b sung BA vo. 5. BI BO CO THC HNH 1. Khi nim m so? Nguyn nhn ca vic hnh thnh m so thc vt? 2. Vai tr ca auxin i vi vic hnh thnh m so thc vt. Nng thch hp nht kch thch to m so t thn, l, r cy thuc l. 3. Phng php ct t thn, on r, mnh l to m so cy thuc l.
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BI 6 K THUT TO O BAO HTGING NHN TO
1 - NGHA mt s loi thc vt nh ma, khoai ty, khoai mm du ty... u khng c kh nng nhn ging bng ht. Bn cnh c nhiu loi thc vt to c ht ging nhng ht ging km cht lng hay ht ging rt t. gii quyt tt c cc vn trn, mt phng php nhn ging mi c nghin cu v ng dng l phng php to ht nhn to. Ht ging nhn to v c bn ging nh ht ging t nhin. Ch c s khc nhau l ht ging nhn to c cu to l phi c bao bc bi lp o bn ngoi thay cho ni nh, v v phi ht nhn to l phi v tnh (phi soma) (hnh 1). Ht ging nhn to gm 3 phn (hnh 2): - Phi v tnh - V bc polymer nh alginat - Mng ngoi: c cu to t alginat canxi Phi soma ht nhn to ging nh phi hp t ht t nhin l c hai cc (nh chi v nh r), nh th c th pht trin thnh mt cy hon chnh tng t nh ht ny mm. Cu trc phi soma ging nh phi hp t nhng phi soma ca ht ging nhn to khng c v bao ht cng nh m d tr.
Hnh 5 - So snh cu trc ht t nhin v ht nhn to
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Phoi soma Noi nhu nha n tao Vo nha n tao
Hnh 6 - Cu to ht nhn to Ht nhn to thmg c v lm bng alginat Na v alginat Ca hay B-hydrogel. Alginat Na l mt cht cao phn t c chit sut t rong bin (rong M Sargassum sp l mt loi rong nu). Trong rong M alginat Na tn ti di dng alginit khng tan l hp cht ca cc axid manuronic. chit tch alginat , thng tin hnh nu rong vi Na2CO3. [(C5H7O4COO)2Ca]n + nNa2CO3 Alginat Ca 2[C5H7O4COONa]n + nCaCO3 Alginat Na
Alginat Na l dng tan trong nc. Khi Alginat Na tip xc vi dung dch CaCl2, xy ra phn ng trao i ion gia hai dung dch ny theo phn ng: 2[C5H7O4COONa]n + nCaCl2 Alginat Na [(C5H7O4COO)2Ca]n + 2nNaCl Alginat Ca
dng ny, alginat Ca kt ta, tr thnh dng khng tan, to thnh mng khng thm nc. Ton b b mt ca got hn hp trn s c bao bc bng mt lp o alginat Ca khng thm nc v to thnh mt ht. Phn alginat Na bn trong vn trng thi tan. V ht nhn to c to ra theo nguyn tc ny. Ht nhn to c th bo qun, tn tr v trng trt bng nhng k thut canh tc truyn thng. V c bit l c th ch ng trong sn xut, trng trt, canh tc khng ph thuc vo cc iu kin thi tit, ma v. Trong bi thc hnh ny, chng ta s tm hiu k thut to o bao cho phi v tnh cu thnh mt ht ging nhn to hon chnh. 2 - NGUYN LIU - HO CHT DNG C 2.1 - Nguyn liu, ho cht STT 1 2 Tn ho cht, ph liu Alginat Na 4% Mi trng MS n v tnh ml ml S lng 100 1000 Ghi ch
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STT 3 4 5 6 Tn ho cht, ph liu CaCl2 2,5% Nc ct Giy lc Phi v tnh 2.2 - Dng c, thit b cn cho mt nhm (30 sinh vin) thc hnh Tn dng c, thit b STT 1 2 3 4 5 6 7 8 9 10 11 Bnh chit 500ml Pipette 10 ml Kp gp 25cm Phu ln Erlen 500ml Erlen 250 Qu bp cao su ng h c nhn a petri Cc 1000 ml Cc 250ml n v tnh ci ci ci ci ci ci ci ci ci ci ci S lng 2 2 2 2 2 2 2 2 2 2 2 Ghi ch To git nh Ht v chuyn phi Gp ht n v tnh ml ml gi S lng 300 500 1 Ghi ch
3. NI DUNG THC HNH - Hc sinh tin hnh nh git v chuyn phi to ht - Hc sinh thc hin ngm ht trong dung dch CaCl2 v nc to ht hon chnh 4 THC HNH 4.1 To git nh - Ho tan 100 ml Alginat Na 4% vo 1 lt mi trng MS c cha cc mui khong, ng, vitamin, cc cht sinh trng. Cho hn hp ny vo bnh chit. - M kho nh git ca bnh chit, nh tng git nh nhng hn hp ny vo mt cc dung dch CaCl2 2,5%. Mi ln nh, khi git dung dch va l ra u ng nh git ca bnh chit, vn kho li dng git dung dch u ng nh git 4.2 Chuyn phi v to ht
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- Dng pipette ht mt phi trong dung dch MS chuyn vo trong git nh ang dng u bnh chit. - M kho bnh chit git c cha phi nh vo trong erlen ng CaCl2. - Ngm ht ny trong dung dch CaCl2 20 pht, c th lc nh erlen. 4.3 Ra v lm kh ht - Sau 20 pht, dng kp gp ht nhn to sang mt cc ng nc ct. Ht c ngm nc 5 pht lm cng v ngn chn phn ng. - Sau 5 pht, dng kp chuyn ht ra mt a petri c lt giy lc lm kh ht. - Ht sau khi c lm kh c th c bo qun hay s dng cho gieo trng trc tip. Hon hp alginat Na 4 % va moi trng MS
Chuyen phoi vao giot nhieu Tao mang bao alginat Ca trong 20 phut
Ngam nc va thu hat nhan tao

Hnh 7. K thut to o bao ht ging nhn to 5 - NHNG IM CN LU - Tt c mi cng vic to o bao ht nhn to u phi c thc hin trong t cy v trng - Vic vn kho ca bnh cu nh git phi thc hin cn thn c th gi c git dung dch u ng. - Thao tc chuyn phi vo git nh thc hin nh nhng khng lm ri git dung dch. - Thi gian ngm ht phi chnh xc 20 pht. 6. TNG TRNH TH NGHIM - Lm tng trnh th nghim
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- Gii thch nguyn tc to o bao ht ging nhn to - So snh gia v ht ging t nhin v ht ging nhn to - Nu ngha v cch bo qun ht ging nhn to
BI 7 PHNG PHP TCH V NUI CY NH SINH TRNG

1 - NGUYN TC M phn sinh nh cha nhng t bo nh sinh trng, c bao bc bi mt lp v b mt c cu to cutin hn ch thp nht qu trnh mt nc v lp cutin ny bao bc c chi nh. thc vt, s hnh thnh mi cc c quan bt u trong cc m phn sinh nh, cc m ny phn ho ngay t nhng giai on pht trin u ca phi v gi li trong sut i sng ca cy. iu ny xy ra l do m phn sinh c s phn ho ca nhng t bo khi sinh. Tt c cc t bo cn li u xut pht t cc t bo khi sinh. Qu trnh sinh trng ca c quan din ra theo 3 giai on: - Giai on th nht: thng c gi l giai on phi sinh. Trong cc im sinh trng (trong cc m phn sinh nh) xy ra s hnh thnh mm c quan v s phn chia u tin ca n thnh cc m ring bit. - Giai on th hai: giai on di ra do s sinh trng nhanh chng, mm c quan t n kch thc ti a v tr nn c hnh dng nht nh. - Giai on th ba: giai on kt thc s phn ha t bo, bt u s ho g cc thnh t bo, xut hin trn nhng ch dy ln c cu to v kt qu l khng cn kh nng tip tc sinh trng. mi nch l u c chi nch. Chi nch thc cht c cu to khng khc nh sinh trng ca thn. Do hin tng u th ngn, chi nch khng pht trin, nhng khi c nh thc v bt u sinh trng, chng c cu to l y nh thn chnh. Qu trnh sinh tng hp DNA ca virus thc vt khng xy ra trong t bo nh sinh trng do mt c ch hin nay khng r. V vy m nh sinh trng l m duy nht sch virus. Do trong k thut nui cy m t bo, m nh sinh trng c s dng l vt liu nui cy m t bo nhm to cc cy khng nhim virus v cc loi vi khun hay nm gy bnh. Nui cy nh sinh trng l phng php nhn ging quan trng va to ra nhng loi cy trng sch vius va c h s nhn ging rt cao. Phng php ny p dng hiu qu i vi cy thn tho nh cc, cm chng, khoai ty, khoai lang, chui...
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Hnh 8. nh sinh trng 2 - VT LIU HO CHT DNG C 2.1 - Vt liu - Cy hoa D Yn Tho (Petunia sp) 2.2 - Ho cht - Cn 70o, 90o - Mi trng MS b sung BA - X phng bt - Cn 70o, 90o - Javel - BA 2.3 - Dng c Thit b cn cho 1 nhm (30 sinh vin) thc hnh STT 1 2 3 4 5 6 7 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 n v tnh ci ci ci ci ci ci ci S lng 30 30 12 3 15 15 15 Dng li dao m nhn Ghi ch
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STT 8 9 10 11 12 13 14 15 16 17 18 Tn dng c, thit b - Cc 1000 ml - Cc 100 ml - Cc 200 ml - Erlen 250ml - Kp di 25 cm - Bng m - ng nghim 25 - Gi ng nghim l ln - n cn - Knh lp hai mt - a petri 100mm n v tnh ci ci ci ci Ci g ci ci ci ci ci S lng 5 30 15 60 15 200 15 15 15 1 1 C n ri t trn xung Ghi ch
3 - NI DUNG THC HNH - Sinh vin tin hnh tch nh sinh trng cy hoa D yn Tho. - Sinh vin tin hnh cy v nui nh sinh trng va tch trn mi trng MS 4 - THC HNH 4.1 - Kh trng mu tch nh sinh trng Chn cy m trng thnh c phm cht tt (l khng b su, thn khng b dp, chi ngn v chi ng pht trin tt) hi bp non di 2 3 cm, cho vo chai hay ti nilon kn, chuyn v phng th nghim. - Dng dao m ct b bt l. - Kh trng mu ln lt vi nc x phng long, cn v NaOCl 0,5% trong 5 pht. Ra mu vi nc ct v trng, lm ro chun b tch nh sinh trng. 4.2 - Tch nh sinh trng Phng php nui cy nh sinh trng s dng phn nh nht chi nh ca thn lm mu nui cy. Phn ny gm m phn sinh v vi phc th l. Vic tch v nui cy nh sinh trng nhm to cc cy sch bnh. Mu cy cng nh kh nng to cy sch bnh cng cao. V d, m phn sinh t 0,1 n 0,15 mm c th c 100% sch virus. Tuy nhin mu cnh nh kh nng sng st cng thp. Do vy ngi ta thng s dng mu cy t 0,25 n 1 mm. - nh sinh trng c tch di knh lp c phng i t 10 n 40 ln c lm v trng bng cch lau cn tht sch thn knh v bn mu tch. Trong qu trnh tch lun chun b mt tm bng tm cn trn bn mang mu tin vic v trng v lm ngui dao. - Dng hai dao m tht sc thao tc tch nh sinh trng.
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- Gp mu ln bn t mu, dng mi dao m tch b ht nhng l ngoi, ch li hai l nguyn thy. Tch cc l ln lt t ngoi vo trong, trch li dao lm tn thng phn chi nh. - Sau khi tch b ht cc l ngoi, dng mi dao m nhn ct ly phn nh sinh trng c hai phc th l c kch thc 0,6mm. Gi nguyn nh sinh trng trn mi dao chuyn vo mi trng nui cy. 4.3 - Nui cy nh sinh trng i vi nhng loi cy tit nhiu phenol thng nn c nui cy trn mi trng c b sung than hot tnh. Mi trng nui cy nh sinh trng D Yn Tho thng b sung BA 2mg/lt, NAA 0,1mg/lt. Mi trng c th l c hoc lng (c cu giy). - nh sinh trng sau khi tch xong nguyn trn li dao m. - a li dao m vo mi trng nui cy, m xuyn li dao m vo b mt mi trng, nh sinh trng s c gi ngay ngn trn b mt mi trng nui cy. - nh sinh trng c nui cy nhit 250C , nh sng 3000 lux. Nu nh sinh trng tit nhiu phenol th nn cy chuyn lin tc trong qu trnh nui cy. 5 - BI TNG TRNH 1. ngha ca phng php nui cy nh sinh trng? 2. Nu t nh sinh trng khng ngay ngn ln mi trng (mt ct khng tip xc c vi b mt mi trng) nh sinh trng c tng trng c khng? Gii thch? 3. loi b virus cy trng, thng c nhng phng php g?
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BI 8 PHNG PHP VI GHP THC VT
1 - NGUYN TC Nui cy nh sinh trng l mt phng php nhn ging quan trng nhm thu c mt s lng ln cy ging v cy sch bnh virus. Tuy nhin i vi mt s loi thc vt, c bit l cy thn g, nh sinh trng rt kh c th sinh trng trn mi trng nhn to. Do vy i vi nhng loi cy ny, vi ghp l mt phng php quan trng c th p dng. Mt khc, vic lm sch bnh virus cho cy trng, c bit cy n qu c mi thuc h cam chanh ang l nhu cu ln i vi nhiu ni trn th gii. Trong khi cha to c nhng ging khng virus th vic to ra nhng khu vc cy m sch virus cung cp mt ghp v cnh chit u dng l cn thit. Bn cnh nhng bin php ty virus nh cy m x l nhit, to cy trong nui cy phi tm phng php nui cy nh sinh trng ang c p dng rng ri v c hiu qu vo vic lm sch virus i vi nhiu cy trng khc nhau. Trong trng hp cy n qu lu nm, k thut ghp ang chim v tr hng u trong nhn ging v kt hp c kh nng chng chu ca gc cy hoang d vi u im nng sut v phm cht ca mt ghp. Tuy nhin, trong lc ghp theo k thut truyn thng do thi gian trng gc ghp ko di v kch thch mt ghp kh ln, nn bnh virus c th ly truyn. khc phc nhng nhc im trn, k thut ghp nh sinh trng gi tt l vi ghp c th nghim v mang li kt qu tt. V nguyn tc, vi ghp l nui cy nh sinh trng, nhng thng qua dinh dng t nhin ca gc ghp. nh sinh trng lm mt ghp c kch thc 0,20,5 mm, c tch t bp non ang sinh trng mnh ca cy m trng thnh, gc ghp l mm gi mi ny t ht ca ging hoang di, ton b cy ghp c nui dng trong iu kin ng nghim v trng. Nhng cy ghp thu c bng phng php ny hon ton sch bnh v mang c im di truyn ca cy m cho mt ghp. 2 - VT LIU HO CHT DNG C 2.1 - Vt liu - Cy cam nui cy t ht trong ng nghim l nguyn liu dng lm gc vi ghp. - Cnh cam ly t cy cam c cht lng qu cao c s dng lm nguyn liu tch nh sinh trng. 2.2 - Ho cht - Cn 70o, 90o - Mi trng MS b sung BA - X phng bt - Javel - BA, NAA
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2.3 - Dng c Thit b cn cho 1 nhm (30 sinh vin) thc hnh STT 1 2 9 10 11 12 13 14 15 16 17 18 Tn dng c, thit b - Dao m - Li dao m - Cc 100 ml - Cc 200 ml - Erlen 250ml - Kp di 25 cm - Bng m - ng nghim 25 - Gi ng nghim l ln - n cn - Knh lp 2 mt - a petri 100mm n v tnh ci ci ci ci ci Ci g ci ci ci ci ci S lng 30 30 30 15 15 15 200 15 15 15 15 1 Knh lp c n ri t trn xung Dng li dao m nhn Ghi ch
3 - NI DUNG THC HNH - Sinh vin tin hnh tch nh sinh trng cy cam nui cy trong ng nghim. - Sinh vin tin hnh ghp nh sinh trng va tch vo gc ghp. 4 - THC HNH 4.1 - Chun b gc ghp Tch ht cam t qu chn, ra sch v kh trong vng 24 gi, ct gi trong t lnh 4oC, dng dn. - Ht c kh trng 510 pht bng dung dch HgCl 0,1%. - Tin hnh tch b v tru, v la. - Gieo ht trn mi trng MS cha 2% ng v 0,8% agar 25oC ni ti hon ton, sau 10 ngy cy mm di 58 cm c chn lm gc ghp. 4.2 - Tch nh sinh trng Chn cy m trng thnh c nng sut v phm cht tt, hi bp non di1,5 3 cm, cho vo chai hay ti nilon kn, chuyn v phng th nghim. - Ct b bt l, kh trng bng NaOCl 0,5% trong 5 pht. - Dng kim nhn gt b nhng l non, sau dng li dao m nhn ct nh sinh trng 0,5 mm nguyn trn li dao chuyn vo vi tr ghp. 4.3 -. Tin hnh ghp
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i vi gc ghp, dng dao m ct b u r, tr trn l mm v hai l mm, li phn r chng 23 cm v phn thn chng 2 cm. Ty theo cch ghp s tin hnh x l gc ghp khc nhau. a. Ghp ln mt ct - Dng dao m ct ngang thn gc ghp to thnh mt mt phng vung gc vi trc thn gc ghp. - Di knh lp, xc nh vng tng tng libe-mc. t mt ghp trc tip ln b mt nht ghp, trn vng tng tng. b. Ghp ch T ngc - Dng u nhn ca li dao m, ct to thnh hnh ch T ngc trn gc ghp cch mt ct bn trn khong 0,1-0,3cm. Vt ct i ht vng v ca gc ghp. Chn ca ch T l mt ct d tip bc lc vng tng tng libe-mc. - Dng mi kim hay u dao m t nh sinh trng va tch c trn vo mt ct ca ch T (chn ca ch T ngc) sao cho mt ct ca nh sinh trng tip xc vi vng tng tng. c. Ghp hm ch - Khot trn thn mm cch mt ct 5 mm mt vt lm hnh hm ch, chiu su vt lm bng chiu dy lp v. - Dng kim nhn hay u dao m nhn t nh sinh trng va tch vo y hm ch.
Hnh 9. Ba kiu vi ghp 4.4 - Nui cy ghp Cy ghp c t vo ng nghim ln (200 x 25 mm) cha 6 ml mi trng MS lng c hm lng ng cao l 7,5%. t ng nghim c cha cy ghp nhit 25oC, chiu sng 12 gi/ ngy, cng chiu sng t 3000-5000 lux. 5 - BI TNG TRNH 1. Gii thch c ch lin kt gia gc ghp v mt ghp (nh sinh trng)? 2. Trnh by nhng phng php vi ghp v nhng im cn lu cho tng phng php?
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3. C th s dng phng php vi ghp cho nhng i tng cy trng no (cy thn g, thn tho), gii thch?
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BI 9 PHNG PHP THUN HO CY RA VN M
1 - NGUYN TC S thnh cng ca mt k thut nhn ging thc vt th hin qua s cy con sng c ngoi vn m. T l cy nui cy sng c ngoi vn m ph thuc nhiu vo k thut thun ho. Do , vic thun ho cy nhm mc ch cho cy nui cy thch nghi c vi nhng iu kin ca mi trng t nhin: + Nhit + m + nh sng + Ch dinh dng + Dch bnh Gia iu kin mi trng bnh nui cy, phng tng trng v mi trng t nhin (mi trng vn m) c s khc bit cn bn v tt c cc yu t, do im cu trc v sinh l gia cy nui cy c s khc bit vi cy t nhin. Nh vy, mc ch ca vic thun ho l chun b cho cy nui cy c c nhng tnh cht nh cy t nhin c th thch nghi v sng c ngoi mi trng t nhin. Nhng tnh cht ny c hnh thnh da trn nguyn tc c s thay i dn dn cc iu kin mi trng ngoi bnh nui cy bng c vi cc iu kin mi trng t nhin: - Nhit : thay i t 26oC n 32-34oC - m: thay i t 80-90% n 50-60% - nh sng: thay i t 3.000-5.000 lux n 12.000 n 15.000 lux - Ch dinh dng: thay i t phng thc d dng thnh phng thc t dng. 2 - VT LIU HO CHT DNG C 2.1 - Vt liu - Cy lan H ip ti sinh hon chnh - Cy Bch n ti sinh hon chnh 2.2 - Ho cht, nguyn liu - Dung dch KMnO4: 0,5% - Gi th hn hp m cy Bch n - Gi th dn, ru bin m cy lan H ip 2.3 - Dng c Thit b cn cho 1 nhm (3 sinh vin) thc hnh
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STT 1 2 3 4 5 6 Tn dng c, thit b - Khay m - Thau nha - R vung 30x50cm - Cc 200 ml - Erlen 250ml - Kp di 25 cm n v tnh ci ci ci ci ci Ci S lng 1 1 1 1 1 1 Ghi ch
3 - NI DUNG THC HNH - Sinh vin tin hnh gp cy khi bnh mi trng nui cy, x l v trng vo gi th trn khay m. - Sinh vin tin hnh chm sc, thay i cc iu kin mi trng vn m thun ho cy. 4 - THC HNH 4.1 Chun b gi th m cy * Gi th m cy Bch n - T l gi th: t (70%) + Phn chung hoai (10%) + tro tru (20%) - m gi th: 70% - Gi th c x l tit trng bng cch hp hoc bng mt s loi ho cht (24 gi trc khi trng): + Vantox M12: 0,5% + Zinep: 0,4% + Formaline: 40% (phun 2-3 ngy trc khi trng. * Gi th m cy lan H ip - T l gi th: Dn hoc ru (60%) + Ht xp (40%) - m gi th: 70% - Gi th c x l tit trng bng cch hp hoc bng mt s loi ho cht (24 gi trc khi trng): + Vantox M12: 0,5% + Zinep: 0,4% + Formaline: 40% (phun 2-3 ngy trc khi trng. 4.2 Chuyn cy ra khi bnh nui cy
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- Bnh cy nui cy m m nt y c 1 tun - Dng kp inox, gp cy khi gi th agar. Trch lm tn thng l v r cy nui cy - t cy vo thau nha, dng vi mm hoc bng thm nc ra sch agar v dng cht trn b mt cy. Trng li 3 ln bng nc sch. - Ngm cy vo dung dch KMNO4 0,5% trong 20 pht dit vi sinh vt. - Vt cy vo r nha, ro 1-2 gi. 4.3 -. Tin hnh trng cy vo gi th - Gi th sau khi trn u theo t l v kh trng, c phn u vo cc l ca khay m, dng tay nn cht va phi. - i vi cy Bch n cy m, dng 1 thanh tre, soi mt l nh vo gia khi gi th, t 1 cy con Bch n vo l soi v dng tay n nh gi th xung quanh gc gi c nh. - i vi cy lan H ip, dng mt t gi th cun xung quanh phn r ca cy (khng qu c r). t cy vo gia l khay m, chn thm mt t gi th gi c nh. Lu , trnh b qu cht hoc lm tn thng r trong qu trnh trng. 4.4 Chm sc, thun ho cy con cy m Khay m cy con cy m c chuyn vo vn m, iu kin mi trng c iu chnh nh sau: - Nhit 30oC - nh sng: + Mt tun u: nh sng t nhin (7.000 lux) + Tun th 2: 70% nh sng t nhin (10.000 12.000 lux) + T tun th 3: 100% nh sng t nhin - m: + Tun u: phun sng m 90% + Tun 2: 70% + T tun 3: 60% Sau 1 thng, tin hnh nh gi s lng cy con sng c ngoi vn m. M t c im sinh trng ca cy con nh: chiu cao cy, s l, mu sc l 5 BI TNG TRNH 1. Giai thch s khc bit v c im gia cy trong bnh nui cy v cy ngoi t nhin? 2. Trnh by phng php thun ho cy con v chuyn cy con ra vn m? 3. Bo co kt qu v t l cy con sng c ngoi vm m, c im sinh trng ca cy con cy m khi c chuyn ra vn m?
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TI LIU THAM KHO
1. Dng Cng Kin, 2000. Nui cy m thc vt. NXB Trng H Khoa Hc T nhin. 2. Kenneth C. Torres, 1957. Tissue culture techniques for horticultural crops. Chapman & Hall, New York - London 3. Nguyn c Lng, 2002. Cng ngh t bo. NXB H Quc Gia Tp. H Ch Minh. 4. Nguyn Vn Minh, 1998. Cng ngh t bo. NXB H Nng lm Tp. H Ch Minh 5. Nguyn Vn Uyn, 1993. Nui cy m thc vt phc v cng tc ging cy trng. NXB Nng nghip. 6. Bi Trang Vit, 2000. Ti liu hc tp Nui cy m thc vt. NXB H Y dc Tp. H Ch Minh
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