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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
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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
ci
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)
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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:
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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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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 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
n v tnh Ci g ci ci
S lng 15 200 15 1
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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- 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 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 <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 1015 pht. - Ra sch x phng bng nc my. - Lc vi cn 70o trong 30 giy. - Ra sch cn bng nc ct v trng 56 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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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
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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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. - S phn chia ca cc t bo tng tng: cc t bo bo tng tng ca phn ln
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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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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 2,4-D 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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Phoi soma
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[C5H7O-4COONa]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
n v tnh ml ml gi
Ghi ch
2.2 - Dng c, thit b cn cho mt nhm (30 sinh vin) thc hnh STT 1 2 3 4 5 6 7 8 9 10 11 Tn dng c, thit b 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
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
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 - Dng pipette ht mt phi trong dung dch MS chuyn vo trong git nh ang dng u bnh chit.
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- 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.
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- So snh gia v ht ging t nhin v ht ging nhn to - Nu ngha v cch bo qun ht ging nhn to
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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
Ghi ch
C n ri t trn xung
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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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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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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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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