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Bai 1

M uI. Cac thit bi cua phong thi nghim nui cy m va t bao thc vt(ghi chu: + rt cn, - tuy theo nhu cu) 1. Phong ra va ct nc + May ct nc 1 ln (8 L/gi) + May ct nc 2 ln (4 L/gi) - May san xut nc kh ion 2. Phong sy hp + Tu sy 60-300oC + Ni kh trung (autoclave) loai nho (25 L) - Ni kh trung loai ln (50-100 L) 3. Phong chun bi mi trng + pHmeter + May khuy t + Cn phn tich 10-4g + Cn ky thut 10-2g + Bp in - Microwave + Tu lanh 100-200 L - Tu lanh su (-20 -80oC) 4. Phong cy v trung + T cy v trng (Laminar, Clean Bench) + Quat thng gio + en t ngoai treo trn hoc treo tng (1,2 m) - Thit bi loc khng khi

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5. Phong anh - May anh k thut s - H thng n chiu 6. Phong kinh hin vi + Kinh hin vi 2 mt ( phong ai 1000 ln). + Kinh hin vi ao ngc ( phong ai 1000 ln) co gn may chup anh k thut s. + Kinh lup 2 mt ( phong ai 75 ln). - Microtome va cac dung cu quan sat t bao hoc. - Knh hin vi hunh quang 7. Phong nui + Cac gian en huynh quang nhiu ngn, co chiu sang ch binh nui cy t 2000-3000 lux. + May iu hoa nhit - May lc nm ngang (100-200 vong/phut) + Tu m - Ni phn ng sinh hc (bioreactor) 8. Phong sinh hoa + May sc ky (HPLC, sc ky ct, sc ky lp mong...) + Thit bi in di gel (agarose va polyacrylamide) + May chup anh, phn tch v lu tr hnh nh ca DNA, RNA v protein (Gene documentation system) + May quang ph co dai o t 190-1100 nm - Mt s thit bi khac phn tich thanh phn sinh hoa cua cac t bao va m nui cy.

II. Cac nhn t am bao thanh cng trong nui cy m-t bao thc vtCo 3 nhn t chinh: - Bao am iu kin v trung. - Chon ung mi trng va chun bi mi trng ung cach.

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- Chon m cy, x ly m cy thich hp trc va sau khi cy. 1. Bao am iu kin v trung 1.1. Y nghia cua v trung trong nui cy m va t bao thc vt Mi trng nui cy m va t bao thc vt co cha ng, mui khoang, vitamin... rt thich hp cho cac loai nm va vi khun phat trin. Do tc phn bao cua nm va vi khun ln hn rt nhiu so vi cac t bao thc vt, nu trong mi trng nui cy chi nhim mt vai bao t nm hoc vi khun thi sau vai ngay n mt tun, toan b b mt mi trng va m nui cy se phu y mt hoc nhiu loai nm va vi khun. Thi nghim phai bo i vi trong iu kin nay m nui cy se khng phat trin va cht dn. Thng thng, mt chu ky nui cy m va t bao thc dai t 1-5 thang (tuy i tng va muc ich nui cy), trong khi thi nghim vi sinh vt co th kt thuc trong mt vai ngay. Noi cach khac, mc v trung trong thi nghim nui cy m va t bao thc vt oi hoi rt nghim khc, iu kin nay c bit quan trong khi nui cy cac t bao n thc vt trong cac ni phan ng sinh hoc (bioreactor), iu kin v trung phai rt cao mi co hy vong thanh cng c. 1.2. Ngun nhim tap Co 3 ngun nhim tap chinh: - Dung cu thuy tinh, mi trng va nut y khng c v trung tuyt i. - Trn b mt hoc bn trong m nui cy tn tai cac si nm, bao t nm hoc vi khun. - Trong qua trinh thao tac lam ri nm hoc vi khun theo bui ln b mt mi trng. 1.3. V trung dung cu thuy tinh, nut y va mi trng a. Dung cu thuy tinh Dung cu thuy tinh dung cho nui cy m va t bao thc vt phai la loai thuy tinh trong sut anh sang qua c mc ti a va trung tinh tranh kim t thuy tinh gy anh hng n s phat trin cua m nui cy.

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Cn ra sach dung cu thuy tinh trc khi a vao s dung. Thng thng, chi cn x ly dung cu thuy tinh bng sulfochromate mt ln u khi a vao s dung, v sau chi cn ra sach bng xa phong, trang sach bng nc may nhiu ln va cui cung trang bng nc ct. Sau khi rao nc, dung cu thuy tinh (tr cac loai dung do th tich) cn c v trung kh bng cach sy 60-70oC/2 gi. Sau khi ngui c ly ra ct vao ch it bui. b. Nut y Thng dung nht la cac nut y lam bng bng khng thm nc. Nut phai tng i cht am bao bui khng i qua c, ng thi nc t mi trng khng bi bc hi qua d dang trong qua trinh nui cy. Bng khng thm nc la loai nut n gian nht, nhng co cac nhc im sau: + Nu khi hp nut bng bi t hoc dinh mi trng thi v sau se bi nhim nm, nht la cac thi nghim nui cy trong thi gian dai. + Thao tac lam nut bng chm, khng thun tin khi nui cy trn quy m ln. + Chi dung c mt vai ln, sau phai bo. Hin nay, ngi ta s dung nhiu loai np y khac thay th nut bng. Cac hang san xut dung cu nui cy m cung cp loai np ng nghim va binh tam giac bng nha chiu nhit co th hp v trung nhit 121oC (khoang 1 atm) ma khng bi bin dang. Mt s phong thi nghim dung np ng nghim inox hoc cao su rt thun tin cho vic v trung kh hoc t. Cung co th s dung giy nhm lam np y. Trong giao trinh nay, chung ti s dung giy nhm lam nut y. c. Mi trng Noi chung, mi trng c pha ch va d tr trong iu kin khng v trung va em hp v trung khi a phn phi vao cac dung cu thuy tinh a y nut hoc np. Thi gian hp t 15-20 phut ap sut khoang 1 atm (121oC). Sau khi v trung cn phai lam kh np ng nghim hoc nut bng. Cac dung dich me (stock solutions) dung pha ch mi trng (dung dich mui khoang, vitamine, cht kch thch sinh trng...) cn c bao quan trong tu lanh. Dung dich me cua hn hp vitamin nn chia thanh nhiu lo nho va bao quan trong ngn a cua tu lanh. Khng nn pha

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mt lng qua ln dung dich me cac cht sinh trng, thng chi nn dung cac lo co dung tich t 100 n 200 mL. ap sut khoang 1 atm hu ht cac vi sinh vt co trong mi trng u bi dit, k ca cac vi sinh vt dang bao t. Thi gian hp thng t 15 n 20 phut 1 atm. Trong nui cy m va t bao thc vt phai dung nc ct hoc nc kh ion, tt nht la dung nc ct 2 ln t cac may ct hoan toan bng thuy tinh. i khi ta cn cho vao mi trng nui cy cac ch phm mn cam vi nhit, co th bi phn huy khi ta hp 121 oC. Trng hp nay cn tin hanh loc v trung ring cac ch phm o va sau a vao mi trng c kh trung. d. Loc v trung Phng phap n gian nht la dung cac mang loc Millipore (Hnh 1.1) do hang Millipore san xut hoc dung cac phu loc thuy tinh xp s 5.

Hinh 1.1. Mt s loai mang loc v trung cua hang Millipore (disposable). y l loi mng lc c kh trng bng chiu x v ch dng mt ln.

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Phng phap s dung mang loc Millipore: Hang Millipore cung cp mang loc va gia bng nha chiu nhit. Di y m ta mang loc loai Millipore Swinex co ng kinh 25 mm. B loc gm co gia bng loai nha chiu nhit gm np va , vong cao su va mang loc. t mang loc (c kich thc l 0,25 m) trn , t vong cao su ln va vn cht np vao . Goi toan b b loc vao trong mt t giy nhm va kh trung trong ni ap sut 121oC trong 15-20 phut. ng thi v trung mt binh thuy tinh hng dich loc. Dung ng tim hut dich loc v bm qua b loc. 1.4. V trung m nui cy M nui cy co th la hu ht cac b phn khac nhau cua thc vt nh: hat ging, phi, noan sao, hoa, la, inh sinh trng, u r, thn cu... Tuy theo s tip xuc vi iu kin mi trng bn ngoai, cac b phn nay cha it hay nhiu vi khun va nm. Hu nh khng th v trung m nui cy c nu nm khun nm su cac t bao bn trong ch khng han ch b mt. Phng thc v trung m nui cy thng dung nht hin nay la dung cac hoa cht co hoat tinh dit nm khun. Hiu lc dit nm khun cua cac cht nay phu thuc vao thi gian x ly, nng va kha nng xm nhp cua chung vao cac ke ngach li lom trn b mt m nui cy, kha nng y ht cac bot khi bam trn b mt m nui cy. tng tinh linh ng va kha nng xm nhp cua cht dit khun, thng thng ngi ta x ly m nui cy trong vong 30 giy trong cn 70% sau o mi x ly trong dung dich dit khun. Cac cht khang sinh trn thc t it c s dung vi tac dung khng trit va co anh hng xu ngay ln s sinh trng cua m cy. Trong thi gian x ly, m cy phai ngp hoan toan trong dung dich dit khun. i vi cac b phn thc vt co nhiu bui t, trc khi x ly nn ra ky bng xa phong di dong nc chay. Khi x ly xong, m cy c ra nhiu ln bng nc ct v trung (3-5 ln). Nhng phn trn m cy bi tac nhn v trung lam cho trng ra cn phai ct bo trc khi t m cy ln mi trng. tranh anh hng trc tip cua cac tac nhn v trung ln m cy, nn chu y lai mt lp boc ngoai khi ngm m vao dung dich dit khun. Lp cui cung nay se c ct bo hoc boc i trc khi t m cy ln mi trng. V trung m cy la mt thao tac kho, it khi thanh cng ngay ln u tin. Tuy vy, nu kin tri tim c nng va thi gian v trung thich hp thi sau vai ln th, chc chn se at kt qua.

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Bang 1.1. Nng va thi gian s dung mt s cht dit khun x ly m cy thc vt Thi gian x ly (phut) 5-30 5-30 2-10 5-15 2-10 30-60

Stt 1 2 3 4 5 6

Tac nhn v trung Calcium hypochlorite Sodium hypochlorite Nc Bromine H2O2 HgCl2 Khang sinh

Nng 9-10% 2% 1-2% 10-12% 0,1-1% 4-50 mg/L

Hiu qua Rt tt Rt tt Rt tt Tt Kha Kha

1.5. V trung ni thao tac cy va tu cy v trung Ngun nhim tap quan trong va thng xuyn nht la bui ri vao dung cu thuy tinh cha mi trng trong khi m np hoc nut bng thao tac cy. Ngi ta ap dung nhiu bin phap khac nhau chng lai ngun nhim tap nay. Bung cy thng la bung co din tich hep, rng t 10-15 m2, co hai lp ca tranh khng khi chuyn ng t bn ngoai trc tip a bui vao. San va tng lat gach men co th lau chui thng xuyn. Trc khi a vao s dung, bung cy cn c x ly hi formol bng cach rot formaldehyde (formalin) 4% ra mt s np ia petri rai rac vai ni trong phong cho bc hi t do. ong kin ca phong cy trong 24 gi, sau o bo formaldehyde i va kh hi formaldehyde tha bng dung dich NH3 25% cung trong 24 gi. Mt ban cy, trc khi lam vic phai lau mt ban bng cn 90%. Cac dung cu mang vao bung cy u v trung trc: t ao choang, mu vai, khu trang cua ngi cy, n dao, keo, forceps, giy loc, binh ng nc ct... Trn ban cy thng xuyn co mt en cn (hoc en gas) s dung trong khi cy va mt cc ng cn 90% nhung cac dung cu lam vic. Trc khi cy, k thut vin cn ra tay bng xa phong va lau ky n khuyu tay bng cn 90%. am bao mc v trung cao trong phong cy cn co mt en t ngoai 40W treo trn trn. Chi cho en nay lam vic khi khng co ngi trong phong cy. Nn bt en t ngoai 30 phut trc khi cy. Cn giam s chuyn ng cua khng khi trong bung

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cy n mc ti thiu, vi vy tt ca cac dung cu phuc vu vic cy u phai chun bi y u trong khi cy tranh i lai, ra vao bung cy nhiu ln.

Hnh 1.2. T cy v trng

2. Chon mi trng dinh dng Xem bai 2 3. Chon m cy va x ly m cy Khng co nhng hng dn cu th trong vic chon m cy. V nguyn tc, tr nhng m cy a hoa g, cac m khac trong c th thc vt u co th dung lam m cy. Tuy vy, co th nhn xet chung la cac m ang phat trin, thit qua non, la non, cung hoa, hoa, m phn sinh... khi t vao mi trng co cha mt lng cht sinh trng thich hp u co kha nng phn chia va phn hoa (Bng 1.2). bt u nghin cu nhn ging v tinh mt cy nht inh, trc tin ngi ta chu y n cac chi nach va m phn sinh ngon. Cn bit rng tuy mang mt lng thng tin di truyn nh nhau, cac m khac nhau trn cung mt cy co th sinh trng va phat trin vi kha nng tai sinh chi, r hay cy hoan chinh rt khac nhau. Vi vy, khi khi s chon ging, nhn ging mt cy cu th bng phng phap nui cy m va t bao thc vt, trc ht cn thi nghim tim hiu phan ng cac b phn khac nhau cua cy trong nui cy cac nng cht sinh trng khac nhau. Sau khi cy, m cy cn c t trong iu kin nhit va anh sang n inh. Tuy vao cac muc ich nghin cu ma co cac ch chiu sang khac nhau, chng han qua trinh tao callus co th cn bong ti hoc

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chiu sang nhng qua trinh tai sinh va nhn ging v tinh nht thit cn anh sang. Nhit phong nui nn gi n inh t 25 2oC bng may iu hoa nhit . Cng chiu sang khoang t 2000-3000 lux. 4. Mt s im cn lu y - Cac dung cu dung cho thi nghim nui cy m sau khi tit trung ni kh trng u phai c tit trung sau mi ln dung n bng cach nhung vao cn 90% ri h ln ngon la en cn. - Trc khi cy phai v sinh toan b khu vc cy bng cn 90%. - Nn s mu cy trong ia petri t 4-5 mu, tranh trng hp nhiu khng cy kip mu se bi kh. - Cn dung t dung cu phai c thay sau mi t cy.Bang 1.2. Cac c quan cua thc vt s dung trong nui cy m va t bao Stt 1 2 3 4 5 6 7 8 9 Ngun gc mu vt nui cy M phn sinh inh (meristem) Chi inh (shoot tip) Chi nach (axillary bud) Cung la (leaf petiole) Phin la (leaf blade) R (root) Dang hanh (bulds, scale) Cy mm (seedling) Hat phn (pollen, microspore) Kich thc 0,5-1 mm 0,5-1 cm 0,5-1 cm 0,2-0,3 cm 0,2-1 cm 0,5-1 cm 1-2 cm 2-3 mm 0,1-0,5 mm Mu nui cy inh sinh trng Chop inh co cha mt phn thn Chi bn co cha mt phn thn, la va chi nach Cung la c ct nho, phn na c cy chim vao mi trng Phin la non t trn mi trng, mt di t trn mt thach Mu r c t trn mt thach Mu c t trn b mt hay cy chim phn na vao mi trng Chi non Hat phn trong bao phn

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Bai 2

Mi trng dinh dngThanh cng chinh trong cac thi nghim nui cy m va t bao thc vt la tim ra thanh phn vt cht cua mi trng dinh dng cn thit t bao co th sinh trng va phat trin c. Thanh phn cua mi trng dinh dng thay i tuy theo loai va b phn nui cy, tuy theo s phat trin va phn hoa cua m cy, tuy theo vic mun duy tri m trang thai callus, mun tao r, tao mm hay mun tai sinh cy hoan chinh. Ngi ta a a ra t nhiu loai mi trng khac nhau cho cac thi nghim nui cy m. a s chung co tinh c hiu cao, co nghia la chung c nghin cu ra nui cy nhng m c bit nao o. Mt s mi trng khac co ng dung rng hn va am bao sinh trng tt cho nhiu loai cy, tuy nhin khng co nhng chi dn chung nao cho rng mi trng nao trong chung bao am sinh trng tt hn. bt u, cn phai th trong nhng mi trng thng dng nao o, chng han mi trng Murashige-Skoog (1962) nu khng thanh cng thi sau o th trn cac mi trng khac. Tuy vy, tt ca nhng mi trng nui cy bao gi cung gm nm thanh phn chinh: - ng cung cp ngun carbon. - Cac mui khoang a lng. - Cac mui khoang vi lng. - Cac vitamin. - Cac cht iu khin sinh trng. Ngoai ra, tuy tng tac gia co th b sung thm mt s cht hu c co thanh phn hoa hoc xac inh (cac amino acid, EDTA...) hoc khng xac inh (nc da, dch chit nm men, dch chit ca chua ...).

I. Thanh phn chinh cua mi trng1. ng Trong nui cy nhn tao, ngun carbon m va t bao thc vt tng hp nn cac cht hu c giup t bao phn chia tng sinh khi cua m khng phai do qua trinh quang hp cung cp ma do ng co trong mi trng dinh dng.

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Hai dang ng thng c s dung la saccharose va glucose. Nhng saccharose c s dung ph bin hn, tuy theo muc ich nui cy ma nng saccharose bin i t 1-12%, thng dung la 2-3%. 2. Cac mui khoang a lng Nhu cu mui khoang cua m va t bao thc vt tach ri khng khac nhiu so vi cy trng trong iu kin t nhin. Cac nguyn t a lng cn phai cung cp la: N, P, K, Ca, Mg va S (Bang 2.1).Bang 2.1. Cac mui khoang a lng dung trong nui cy mNguyn t a lng N (NO3-, NH4+) 2 3 4 5 6 P K Ca Mg S Nng (mM) [NO3-, NH4+] khoang 20 khoang 1 khoang 10 khoang 2 0,5-3 khoang 1

Stt 1

Dang s dung Ca(NO3)2.4H2O, KNO3, NaNO3, NH4NO3, (NH4)2SO4 NaH2PO4.7H2O, KH2PO4 KNO3, KCl.6H2O, KH2PO4 Ca(NO3)2.4H2O, CaCl2.2H2O hoc CaCl2.6H2O MgSO4.7H2O (NH4)2SO4

3. Cac mui khoang vi lng Nhu cu mui khoang cua m thc vt trong nui cy la linh vc it c nghin cu. Rt it cac nguyn t vi lng a c chng minh la khng th thiu c i vi s phat trin cua m va t bao nui cy. Tuy nhin, no a c sinh ly hoc thc vt chng minh i vi cy hoan chinh do o co th s dung c hu ht cac nguyn t vi lng cn thit i vi cy cho m va t bao nui cy trong mi trng nhn tao. Vi vy, s cung cp nay co tinh kinh nghim trong nhng trng hp cu th co th la khng cn thit (Bang 2.2). 4. Cac vitamin

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Bang 2.3 trinh bay cac vitamin thng c dung trong cac mi trng nui cy (chu yu la bn loai u). Cac dung dich stock vitamin d hong do nm khun nhim tap, vi vy cn gi trong iu kin lanh di 0oC (trong ngn a tu lanh).Bang 2.2. Cac mui khoang vi lng dung trong nui cy m Stt 1 2 3 4 5 6 7 8 Nguyn t vi lng Mn B Zn Cu Mo Co I Fe Dang s dung MnSO4.4H2O H3BO3 ZnSO4.7H2O CuSO4.5H2O Na2MoO4.2H2O CoCl2.6H2O KI FeSO4.7H2O Nng (mg/L) 15-100 6-100 15-30 0,01-0,08 0,007-1 0,1-0,4 2,5-20 15-27,9

Bang 2.3. Cac loai vitamin thng dung trong nui cy m Nng (mg/L) 100 0,5-1 0,05-0,5 10-50 1-5 1-5 0,1-1 0,1-1

Stt 1 2 3 4 5 6 7 8

Tn vitamin myo-inositol Nicotinic acid Pyridoxine.HCl (Vit B6) Thiamine.HCl (Vit B1) Panthotenate calcium (Vit B5) Riboflavin (Vit B2) Biotin Folic acid

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5. Cac cht iu khin sinh trng Mt s cht sinh trng khng tan trong nc, do o khi pha dung dich me cht sinh trng cn chu y: - i vi 2,4-D, NAA, IAA, IBA v GA3: cn mt lng cht sinh trng u pha trong 50 mL dung dich me vao mt ly kh, thm 2-3 mL cn 90% ri lc n khi tan ht, sau o mi thm nc ct n 50 mL. - i vi BAP (hay BA): trc ht thm 2-3 giot nc ct va vai giot HCl 1 N, lc cho tan sau o thm nc ct n th tich cn pha. - i vi KIN: thm 2-3 giot NaOH 1 N trc khi pha n th tich cn thit. Bao quan dung dich me cht sinh trng trong lo kin (ring IAA bao quan trong lo mau nu), ct gi tu lanh. 2,4-D, NAA tng i bn co th bao quan nh vy trong mt nm. BAP, IBA, KIN, va GA3 bao quan c t 2 n 3 thang. IAA cn pha lai hang thang am bao hoat tinh.Bang 2.4. Ch vit tt cua mt s cht kch thch sinh trng Ch vit tt BA BAP GA3 IAA IBA 2-iP NOA Ch vit tt KIN NAA 2hZ TDZ Zea 2,4-D Pic

Cht kch thch sinh trng Benzyladenin Benzyladeninpurine Gibberellic acid Indoleacetic acid Indolebutyric acid 2-Isopentenyl adenin Naphthoxyacetic acid

Cht kch thch sinh trng Kinetin Naphthaleneacetic acid Dihydrozeatin Thidiazuron Zeatin 2,4-Dichlorophenoxyacetic acid Picloram

Chu y Cac cht sinh trng co th tac ng ln m nui cy nng rt thp (10-8). Cn dung pipette ring cho tng loai cht sinh trng mt. Va chu y ra cn thn cac ly, cc, chai loc a dung ng va pha cac cht sinh trng nng cao. Ngoai tr IAA va GA3, cac cht sinh trng con lai c coi la bn vng trong qua trinh hp v trung. IAA sau khi pha dung dch stock, c lc qua mang loc millipore (xem bai trc) sau o cha trong cac tube eppendof c boc giy nhm bn ngoai, bao quan lanh su. Mi trng sau khi hp kh trung nhit giam xung con

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khoang 50-60oC khi o mi cho IAA a loc vao (cac thao tac thc hin trong tu cy v trung).

6. Cac cht hu c khac 6.1. Nc da Cht co hoat tinh trong nc da hin a c chng minh la myo-inositol va mt s amino acid khac. Lng nc da dung trong mi trng nui cy thng kha cao, t 10-20% th tich mi trng. Ly nc da gia, loc trong, cho vao cac tui nilon va bao quan trong lanh su cho n khi dung. Thi gian bao quan khng qua vai thang. Tt nht la nn s dung ti. 6.2. Dch chit nm men va dich thuy phn casein y la cac ch phm thng dung trong nui cy vi sinh vt, m va t bao ng vt a c tiu chun hoa va ban di dan thng phm, thanh phn hoa hoc khng ro. Dung dich thuy phn casein cung cp mt s amino acid, lng thng dung la 1g/1 L mi trng.

II. Vn la chon mi trngKhi khi s nui cy m va t bao mt s i tng nht inh, vn t ra la chon mi trng nao va trn c s nao phi hp ty l cac cht dinh dng. Cach thng lam la qua cac tai liu a xut ban, xem cac tac gia nui cy m trn cung i tng y hoc cac i tng gn gui v mt phn loai a dung loai mi trng gi. Bc u co th gi nguyn mi trng cua cac tac gia o hoc trn c s o ma cai tin cho phu hp qua mt s thi nghim thm do. Trong hang trm mi trng do rt nhiu tac gia nghi cho nhiu loai cy khac nhau, nhiu muc ich nui cy khac nhau, co th chia ra lam ba loai: - Mi trng ngheo cht dinh dng: in hinh la mi trng White, Knop v Knudson C. - Mi trng trung binh: in hinh la mi trng B5 cua Gamborg. - Mi trng giau cht dinh dng: in hinh la mi trng Murashige-Skoog v Linsmaier-Skoog. Vi vy, khi bt u nghin cu nui cy m mt s i tng mi, cha co tai liu trc thi nn thm do so sanh ba loai mi trng trn xem i tng nghin cu thich hp vi loai mi trng nao nht. Sau o, cn

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tim ty l NO3-/NH4+ thich hp. Vic s dung mang tinh kinh nghim i vi mt s mi trng a can tr kha nhiu s tin b trong cac nghin cu v nui cy m va t bao thc vt. Hin nay, mi trng Murashige-Skoog c coi nh la mt mi trng thich hp vi nhiu loai cy do giau va cn bng v cht dinh dng. Vi vy, nhng ngi mi tp s nui cy m thng bt u vi mi trng nay trc khi tim ra c mi trng ring cua minh.

III. Chun bi cac dung dich lam vic thun tin cho vic pha cac mi trng nui cy (mi trng lam vic), ngi ta khng cn hoa cht cho mi ln pha mi trng ma chun bi trc di dang m c (stock), sau o chi cn pha loang khi s dung. Cac dung dich m c c bao quan dai ngay trong tu lanh thng hoc tu lanh su. Nu chun bi mi trng tt thi se giam mt s thi gian ang k cho cng tac nui cy. 1. Chun bi mi trng Murashige-Skoog (MS, 1962). Chia lam 5 phn:Bang 2.5. Thanh phn mi trng MS Nng (mg/L) 1900 170 1650 370 440 6,2 22,3 0,025 0,025 8,6 0,25 0,83 27,8 ( 20) ( 20) ( 20) ( 10) Nng trong dung dich me (g/200 mL) 19 1,7 16,5 3,7 8,8 0,124 0,446 0,5 mg 0,5 mg 0,172 5 mg 16,6 mg 0,556 10 mL 10 mL 10 mL 20 mL Dung tich dung cho 1 L mi trng

Dung dich stock MS1: KNO3 KH2PO4 NH4NO3 MgSO4.7H20 MS2: MS3: CaCl2.2H2O H3BO3 MnSO4.4H2O CoCl2.6H2O CuSO4.5H2O ZnSO4.4H2O Na2MoO4.2H2O KI MS4: FeSO4.7H2O

165 Na2-EDTA MS5: myo-inositol Thiamine.HCl Pyridoxine.HCl Nicotinic acid Glycine 37,3 100 0,1 0,5 0,5 2 ( 20) 0,746 2 2 mg 10 mg 10 mg 40 mg 10 mL

2. Chun bi mi trng Nitsch (Nt, 1956). Chia lam 5 phn:Bang 2.6. Thanh phn mi trng Nitsch Nng (mg/L) 950 68 720 185 166 10 25 0,0025 10 0,25 27,8 37,3 100 0,5 0,5 5 0,05 2 ( 20) ( 20) ( 20) ( 10) Nng trong dung dich me (g/200 mL) 9,5 0,68 7,2 1,85 ( 20) 1,66 0,2 0,5 0,05 mg 0,2 0,5 mg 0,556 0,746 2 10 mg 10 mg 100 mg 40 mg 1 mg 10 mL 10 mL 10 mL 10 mL 20 mL Dung tich dung cho 1 L mi trng

Dung dich stock Nt1: KNO3 KH2PO4 NH4NO3 MgSO4.7H20 Nt2: Nt3: CaCl2.2H2O H3BO3 MnSO4.4H2O CuSO4.5H2O ZnSO4.4H2O Na2MoO4.2H2O Nt4: FeSO4.7H2O Na2-EDTA Nt5: myo-inositol Thiamine.HCl Pyridoxine.HCl Nicotinic acid Glycine Biotin

166 Acid folic 0,5 10 mg

3. Chun bi mi trng nui cy protoplast (theo Trigiano & Gray, 2000) - Mi trng phn lp (Protoplast Isolation medium PI)Bng 2.7. Thnh phn mi trng phn lp PI Nng (mg/L) 1480 27,2 101 246 0,025 0,16 5,8

Stt 1 2 3 4 5 6

Thnh phn CaCl2.H2O KH2PO4 KNO3 MgSO4.7H2O CuSO4.5H2O KI pH mi trng

- Mi trng nui cy (Protoplast Culture medium PC)Bang 2.8. Thanh phn mi trng PC Nng trong dung dich me (g/200 mL) ( 20) 2 9 50 ( 20) 3,4 1,68 5 60 mg 132 mg 0,5 mg ( 20) 0,5 mg 40 mg 10 mL 10 mL Dung tich dung cho 1 L mi trng 10 mL

Dung dich stock PC1: Ca(H2PO4)2.2H2O CaCl2.2H2O PC2: KNO3 NaH2PO4.2H2O (NH4)2SO4 MgSO4.7H20 PC3: H3BO3 MnSO4.4H2O CoCl2.6H2O CuSO4.5H2O ZnSO4.7H2O

Nng (mg/L) 100 450 2500 170 134 250 3 13,2 0,025 0,025 2

167 Na2MoO4.2H2O KI PC4: myo-inositol 0,25 0,75 100 1 ( 20) 5 mg 15 mg 2 20 mg 10 mL

Nicotinic acid

Ly mi loai stock PC (PC1, PC2, PC3 va PC4) 10 mL. B sung thm: + Sequestrene 330 + Sucrose + Glucose + Mannitol 28 mg/L 10 g/L 18 g/L 100 g/L

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Bai 3

Nui cy nh sinh trngI. Muc ich va yu cuMt phng thc d dang nht at c muc tiu trong nui cy m va t bao thc vt la nui cy inh sinh trng (bao gm nui cy chi inh va chi bn). Mi trng thich hp thay i ty theo tng loai cy trng c a vao nui cy nhng c ban la mi trng cha y u cht dinh dng khoang v c va hu c, c b sung cht kich thich sinh trng thich hp. T mt inh sinh trng sau mt thi gian nui cy nht inh se phat trin thanh mt chi hay nhiu chi. Sau o chi tip tuc phat trin vn thn, ra la va r tr thanh mt cy hoan chinh. Cy con c chuyn ra t co iu kin sinh trng phat trin binh thng. y la mt chu trinh ngn nht va tin li hn cac phng thc nhn ging thng thng, c thc hin bng ky thut nhn ging v tinh in vitro thng qua nui cy inh sinh trng. Khi chon inh sinh trng nhn ging cn lu , nu dung inh sinh trng nho qua thi nui cy kho thanh cng, nu dung inh sinh trng ln qua thi nui cy d thanh cng nhng cung d bi nhim trung. Kich thc cua inh sinh trng thay i tuy theo tng loai cy, tuy nhin mt inh sinh trng co kich thc 5-10 mm la thich hp hn ca. Con ng phat trin cua inh sinh trng thanh cy co th trc tip hoc thng qua giai oan protocorm (de hanh). II. Dung cu, thit bi va hoa cht 1. Dung cu - Binh tam giac (100 mL, 250 mL) - Giy nhm - Giy thm v trung - Lo thy tinh kh trung mu vt nui cy - Forceps, keo, dao m - ia petri v trung - Cc chiu nhit, ng ong, micropipette cac loai

169

2. Thit bi - Ni kh trung - Tu sy - Laminar - Tu lanh - Microwave - Cn phn tich 10-4g - Cn ky thut 10-2g - May ct nc 2 ln 3. Hoa cht - Dung dich stock mi trng MS (MS1, MS2, MS3, MS4 v MS5) - Dung dich HgCl2 0,1% - Cn 90% - Agar - Saccharose - Nc ct v trung - Dung dich stock cac cht kich thich sinh trng: BAP v NAA. - Nc da

III. Phng phap tin hanh1. Nui cy phat trin thanh cy trc tip a. Nguyn liu thc vt - inh sinh trng cua cy hng (Paulownia fortunei) - nh sinh trng cy keo lai (Acasia hybrid) b. Mi trng nui cy - MS y u - Saccharose 3% - Agar 0,8% - BAP 2 mg/L - NAA 1 mg/L

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- pHmi trng 5,8 c. Tin hanh Pha mi trng dinh dng (1 L). Chia mi trng vao trong 20 hoc 40 binh tam giac loai 250 hoc 100 mL, tng ng. Nut ming binh bng giy nhm, sau o em kh trung 121oC (1 atm)/15-30 phut (mi trng chun bi trc t 2 ngay tr ln). Ra sach inh sinh trng bng xa phong di dong nc chay, ct bo nhng la chung quanh chi lai mt vai la, cho vao lo thuy tinh v trung chun b kh trng mu vt. Trc khi cy, laminar phai c bt en kh trung 30 phut. Cac dung cu cn thit cho qua trinh cy (forceps, keo, dao, binh kh trung, cn, giy thm...) c t trong laminar trc khi bt en. Kh trung s b bng cn 90% t 30 giy n 1 phut, sau o bng HgCl2 0,1 % trong 5 phut, cui cung ra sach HgCl 2 bng nc ct v trung t 4-5 ln (cac thao tac nay thc hin trong laminar). Ly mu vt ra thm kh trn giy thm v trung boc bo nhng la chung quanh, chi gi lai inh sinh trng (nhng phn m thm dung dich kh trung cung cn phai ct bo). Sau o, cy mu vao cac binh tam giac cha mi trng dinh dng a c chun bi sn. Cc bnh mi trng cy mu c t trong phng nui vi nhit 25 2oC, thi gian chiu sng 8-10 gi/ngy vi cng 2000-3000 lux. Sau 3-4 tun, inh sinh trng se phat trin thanh cy nh qua trinh keo dai chi (shoot elongation). 2. Nui cy phat trin cy thng qua giai oan protocorm a. Nguyn liu thc vt - inh sinh trng hoc chi nach cua hoa lan Dendrobium - nh sinh hoc da (Ananas comusus) b. Mi trng nui cy - Lan: MS y u Saccharose 2% Agar 0,8% Nc da 15%

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NAA 0,1 mg/L BAP 1,0 mg/L pHmi trng 5,8 - Da: MS y u Saccharose 2% Agar 0,8% NAA 0,5 mg/L BAP 0,2 mg/L pHmi trng 5,8 c. Tin hanh Cac bc tin hanh tng t nh trng hp nui cy phat trin thanh cy trc tip. Nhng lu y thm: - inh sinh trng cua cac loai lan hoc da thng rt bn nn phai ra tht ky bng xa phong di dong nc chay. - Lan va da la nhng cy c kh nng sinh trng kh manh nn thi gian kh trung inh sinh trng cua chung co th lu hn cac i tng khac (khoang 7-10 phut) am bao ty l nhim bn thp. Cac binh mi trng a cy mu c t trong phong nui vi thi gian chiu sang 8-10 gi/ngay, cng anh sang 2000-3000 lux, nhit phong 25 2oC.

172

Bai 4

Nui cy n bi in vitroI. Muc ich va yu cuTrong cng tac ging cy trng, ky thut tao dong thun chung lun lun gi vai tro quan trng. Co nhiu phng phap tao dong thun chung khac nhau, nhng thng thng nht la tin hanh chon loc va kim tra qua nhiu th h t phi. Cach lam nay mang lai kt qua chc chn, nhng oi hoi chi phi ln v trng trot va mt nhiu thi gian. Bng con ng n bi, nghia la chn ra nhng ca th 1n ri sau o lng bi hoa chung, trong mt thi gian ngn se thu c nhng ca th ng hp t tuyt i, o la nhng dong thun rt ly tng. Hai bin phap truyn thng thu c cy n bi la: - Chon lc n bi xut hin ngu nhin. - Lai xa va chon lc sau khi b nhim sc th cua mt trong hai giao t mt i (thoai bin) se thu c th n bi, hin tng nay chi mi phat hin c mt s cp lai khac loai cua ai mach. Tuy nhin, vn t ra la lam sao co c mt s lng cy n bi ln. Phng phap nay hu nh khng th giai quyt vn noi trn. Nm 1964, Guha va Maheshawari a a ra mt phng phap tao cy n bi mi, o la tao cy n bi t hat phn thng qua nui cy in vitro. Trn nhiu i tng cy trng, phng phap nay a tao c hang loat ca th n bi trong mt thi gian ngn (chi mt vai thang). Chinh vi vy, a co nhiu chng trinh tao ging cy trng ra i trn c s ap dung ky thut n bi in vitro va a thu c kt qua tt. Co hai trng hp tao cy n bi t hat phn: hat phn tao trc tip cy n bi (thng gp thuc la) hay hat phn to gin tip cy n bi thng qua giai oan phat trin callus ( lua). to cy n bi kp c th s dng hai phng thc: cy n bi kp to t cy n bi c x l colchicine hay cy n bi kp ti sinh t callus ca cy n bi (trng hp ny thng ch t hiu sut khong 60 %).

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II. Dung cu, thit bi, hoa cht1. Dung cu - ng nghim (1,5 cm 20 cm) - Giy thm v trung - Giy nhm - Lo kh trung mu vt nui cy - Forceps, keo, dao m, kim nhon - ia petri v trung - Cc chiu nhit, ng ong, micropipette 2. Thit bi - Ni kh trung - Tu lanh - Laminar - Tu sy - Microwave - Cn phn tich 10-4g - Cn ky thut 10-2g - May ct nc 2 ln - Kinh hin vi 3. Hoa cht - Dung dich stock mi trng Nt (Nt1, Nt2, Nt3, Nt4 va Nt5) - Dung dich HgCl2 0,1% - Cn 90% - Agar - Saccharose - Nc ct v trung - Dung dich stock cc cht kich thich sinh trng: IAA, BAP, KIN v 2,4-D

III. Phng phap tin hanh1. Nguyn liu thc vt

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S dng bao phn ca cy thuc la (Nicotiana tabacum) nui cy n bi. 2. Mi trng nui cyBang 4.1. Thanh phn cac mi trng nui cy bao phn thuc laThanh phn mi trng (1) Nitsch y u (Nt1, Nt2, Nt3, Nt4 va Nt5) Saccharose (%) Agar (%) IAA (mg/L) 2,4-D (mg/L) KIN (mg/L) BAP (mg/L) Tao cy 1n (2) + 2 0,8 0,1 0,1 Tao callus 1n (3) + 2 0,8 0,1-0,5 0,1 To chi 1n (4) + 2 0,8 0,1-1 0,1-1 Tao r 1n (5) + 2 0,8 0,1 -

Ch Mi trng c chun b trong ng nghim (lm thch nghing).

3. Nui cy bao phn Nu thuc la hai giai oan canh hoa sp lo ra khoi la ai (nu dai khoang 10-15 mm) c kh trung theo th t: 2 phut trong cn 70%, 5 phut trong dung dich HgCl2 0,05% va ra bng nc ct v trung t 4-5 ln. Trong iu kin v trung, dung forceps va dao m tach nu ly cac bao phn cy vao ng nghim cha mi trng dinh dng a chun bi sn (Bang 4.1, ct 2). Mi ng nghim cy 5-10 bao phn v t nhit 25-27oC, chiu sang t 10-12 gi/ngay vi cng chiu sang 20003000 lux. Sau 6-8 tun nui, vo bao phn se nt ra va xut hin cac cy thuc la n bi, cy chuyn nhng cy thuc la nay sang nhng binh tam gic loi 250 mL cha 50 mL ca cung mt loai mi trng cy n bi pht trin.

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Chu y: Cac thi nghim nui cy n bi chi thanh cng vi iu kin hat phn phai giai oan t t hoc n nhn, do o thng ngi ta phai lam tiu ban hin vi quan sat s phat trin cua hat phn, chon giai oan thich hp ri mi tin hanh nui cy.

4. Nhi bi hoa thng qua giai oan callus Thn ca cy thuc la n bi nui cy trong ng nghim c ct thanh tng oan dai 5 mm va cy ln mi trng tao callus (Bang 4.1, ct 3). Sau 7-10 ngay, t oan thn cy thuc la 1n se hinh thanh mt khi callus nho c goi la callus s cp. T bao callus s cp thng d tai sinh thanh chi khi gp iu kin thun li. Cac cy tai sinh t t bao callus nui cy trn mi trng bang 4.1, ct 4 co bin ng v s lng nhim sc th rt ln. - Phng phap kim tra nhim sc th Mnh la non (1-2 mm) hoc u r (2 mm) c tach ra t cy thuc la n bi nui cy trong ng nghim, c inh bng hn hp cn: acetic (3:1) trong 24 gi. Mu c bao quan cn 70%, nhum nhim sc th bng acetocarmin. m s lng nhim sc th di kinh hin vi. Kt qua phn tich s lng nhim sc th cua cac cy tai sinh t qun th t bao callus n bi la khng ng nht, cho thy: cy 1n chim ty l khong 21,9 %, cy 2n khong 61,5 % va khoang 11,5 % la nhng cy co mc bi th cao hn nhi bi.

176

Bai 5

Nui cy t bao dich huyn phI. Muc ich va yu cuTrong nui cy t bao dich huyn phu (dch lng), dich nui cy cha cac t bao va cac khi t bao, sinh trng phn tan trong mi trng long. Nui cy t bao dich long thng c khi u bng cach t cac khi m callus d v vun trong mi trng long chuyn ng (lc hoc khuy). Vi th qua trinh nui cy nay la s tin trin t thc vt n mu vt, ti callus, va cui cung ti t bao dich huyn ph. Nui cy t bao dich huyn ph thich hp hn cho vic san xut sinh khi cua t bao thc vt so vi nui cy callus do qua trinh nui cy nay c thao tac tng t nh nui cy vi sinh vt trong ni ln men. K thut nui cy t bo dch huyn ph ch yu c s dng sn xut cc hp cht th cp (cc hp cht t nhin) vi hiu sut cao. Cc sn phm th cp nh: cc cht dng lm dc liu, cc cht to mi, cc cht dng lm gia v, cc sc t v cc ha cht dng trong nng nghip, ngy nay c sn xut thnh cng trong nhiu nui cy in vitro t bo thc vt. Sn xut cc hp cht th cp t nui cy t bao thc vt da trn c s lp lun l sn phm co cung gia tri c san xut t nhin trong cac c quan, qua, hoc cac m khac nhau cua cy co th c kich thich tich luy trong cac t bao cha phn hoa r. Nhng t pha trong ky thut nui cy t bao (vi du nui cy r t) cho thy trong tng lai gn phn ln nhng hoa cht cng nghip nao co ngun gc thc vt cho du o la dc liu, thuc nhum hay cht mau thc vt u co th san xut trong cac ni phan ng sinh hoc (bioreator) hay cn gi l h ln men (fermenter).

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Hnh 5.1. Nui cy dch huyn ph t bo thc vt trong bioreactor

II. Dung cu, thit bi, hoa cht1. Dung cu - Binh tam giac (250 mL) - Giy nhm - Giy loc v trung - Lo kh trung mu vt nui cy - Forceps, keo, dao m

178

- ia petri v trung - Cc chiu nhit, ng ong, micropipette 2. Thit bi - Ni kh trng - Tu lanh - Laminar - Tu sy - Microwave - Cn phn tich 10-4g - Cn ky thut 10-2g - May ct nc - May lc - May hut chn khng - Phu s Buchner 3. Hoa cht - Dung dich stock: mi trng MS (MS1, MS2, MS3, MS4 v MS5) - Dung dich HgCl2 0,1% - Cn 90% - Agar - Saccharose - Nc da - Dung dch stock cc cht kich thich sinh trng: KIN, BAP, NAA v 2,4-D. - Nc ct v trung

III Phng phap tin hanh1. Nguyn liu thc vt - Hat lua (Oryza satica): nui cy tao callus. - Cy ngh en (Curcuma zedoaria) in vitro: nui cy tao callus. 2. Mi trng nui cy tao callus

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a. Lua - MS y - Saccharose 3 % - Agar 0,8 % - 2,4-D 5 mg/L - KIN 0,1 mg/L - pHmi trng ~ 5,8 b. Ngh en - MS y u - Saccharose 2 % - Agar 0,8 % - BAP 3 mg/L - 2,4-D 3 mg/L - pHmi trng ~ 5,8 3. Mi trng nui cy t bo dich long Thanh phn mi trng tng t mi trng tao callus nhng khng b sung agar. 4. Tin hanh a. LA: chun bi mi trng theo cac bc tng t cac bai trc. - Tao callus Hat lua boc vo, ra sach bng xa phong di dong nc chay. Cho hat lua vao binh kh trung, kh trung s b bng cn 70% trong 1 phut. Sau o kh trung bng HgCl2 0,1 % trong 6 phut. Ra lai bng nc ct v trung 4-5 ln trc khi cy. Cy hat la kh trng vao mi trng a chun bi v nui nhit 25 2oC, thi gian chiu sng 8-10 gi/ngay, cng chiu sang 2000-3000 lux . Sau 4-5 tun cac callus mau trng sa xut hin trn cac hat lua. - Nhn callus Dung dao m tch ly cac khi callus nh c ng knh 1-2 mm cy chuyn ln mi trng nhn callus (thanh phn mi trng tng t

180

nh mi trng tao callus). Sau 3-4 tun, cac callus se phat trin nhanh chong thanh cac khi callus ln (ng knh khong 3-5 mm). - Nui cy t bao dich huyn ph Chun bi mi trng nui cy dich huyn ph. Cac bc tin hanh va kh trung tng t mi trng co agar. Mi bnh tam giac loai 250 mL cha 50 mL mi trng. Chon cac khi callus co mau trng nga, rn t mi trng nhn chuyn vo mi trng lng. Mt gam callus cho vao 1 bnh tam gic loi 250 mL cha 50 mL mi trng (cac thao tac tin hanh trong iu kin v trung). t cac binh mi trng cha callus trn may lc trong phng nui cy (nhit 25 2oC) vi tc khoang 100-150 vong/phut C 2 ngay ly ra mt binh nui cy, loc sinh khi t bao bng may ht chn khng, cn lng mu ti thu c. Sau , em sy mu 65oC trong 2 gi cn trng lng kh. So sanh vi kt qua ban u theo doi tc sinh trng cua callus Xy dng ng cong sinh trng (tng 2 ngay) cho trng lng ti v trng lng kh ca t bo. b. NGH EN S dung cac cy ngh en in vitro, ct cac oan gc la cy vao mi trng tao callus. Sau 4-5 tun cac callus mau vang bt u xut hin. Tach cac callus nay cy ln mi trng nhn callus (thanh phn mi trng nhn callus tng t mi trng tao callus). Cac bc tin hanh nui cy t bo dch huyn ph tng t nh i vi lua.

181

Bai 6

Nui cy protoplastI. Muc ich va yu cuKy thut protoplast phat trin manh t sau cng trinh cua Takebe (1971) khi tai sinh cy hoan chinh t protoplast ca la cy thuc la. Co ba phng thc phn lp protoplast, o la: - C hoc (khng dung cac enzyme) - S dung ezyme tun t (x l qua hai bc) - S dung hn hp enzyme (x ly ng thi) Tuy theo i tng va tng loai m ma thay i nng cua cac enzyme cho thich hp. Vi proplast thc cht la t bao trn khng co thanh cho nn co th tach c t nhiu ngun khac nhau nh cac b phn cua cy (la, r, hat phn), m seo, t bao n... Protoplast thc vt co ba ng dng chinh sau: - Chon dong t bao bin d soma. - Dung hp protoplast lai v tinh t bao thc vt. - Bin nap di truyn: a cac c quan t, virus va DNA ngoai lai vao t bao thc vt.

II. Dung cu, thit bi, hoa cht1. Dung cu - Binh tam giac (100 mL), a petri nha v trng (disposable) - Giy thm v trung - Forceps, keo, dao m - ia petri thuy tinh v trung - Giy parafilm, giy nhm - Tube ly tm loai 15 mL - Li loc co ng kinh l loc 65m - Cc chiu nhit, ng ong, micropipette 2. Thit bi

182

- Ni kh trung - Tu sy - Laminar - Tu lanh - Microwave - Cn phn tich 10-4g - Cn ky thut 10-2g - May ct nc 2 ln - May ly tm (tc 6000-10000 rpm), rotor cho tube 15 mL - May lc - Bung m - Kinh hin vi ao ngc 3. Hoa cht - Dung dich enzyme tach protoplast - Mi trng phn lp (PI), mi trng nui cy protoplast (PC) - Cn 90% - Nc ct v trung

III. Phng phap tin hanh1. Nguyn liu thc vt L ca cy thuc la in vitro c chon dung lam nguyn liu tach protoplast. S dung cac la c tach trong ngay. 2. Chun bi mi trng Dung dich enzyme tach protoplast: + Onozuka cellulase R10 0,5 % + Onozuka macerozyme R10 0,1 % + Mannitol 13,0 % + pHmi trng ~ 5,8 Dung dich nay c kh trung bng mng loc Millipore loi c ng kinh l loc 0,2-0,25 m.

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3. Tin hanh a. Ngay th nht Cac mu la thuc la in vitro c loai bo ht gn la va t ln ia petri thuy tinh v trung. Dung dao cy bm nho cac manh la. Cho cc mnh l bm nh vo cc ong v trng loi 50 mL b sung 10 mL dung dich enzyme tach protoplast. Boc cc ong bng giy parafilm, ghi nhan va t trong ti qua m trn may lc tc thp (khoang 40 vong/phut). b. Ngay th hai Dung micropipette chuyn dich protoplast ln li loc v trng ( = 65m) t trong cc loai 50 mL. B sung thm 3 mL dung dich ra (PI + 10% mannitol) vao cc cha cac manh la vun, lc manh, va lc tip trn li lc ri chuyn vao hn hp protoplast-enzyme ca bc trn. B sung thm 2 mL dung dich ra ra proptoplast ht khoi li loc (dung forceps gi li loc trong luc ra). Chuyn hn hp protoplast-enzyme (tng cng 15 mL) vao tube ly tm loi 15 mL va ly tm 50 g trong 10 phut. Loai bo phn dich ni phia trn, ti huyn ph tiu th bng 10mL dung dich tach (PI + 20% sucrose), thm 1mL dung dich ra ln trn b mt dung dich tach va protoplast sao cho khng lam hoa ln hai dung dich, tt nht la tao thanh hai pha (nho t t dung dich ra bn thnh tube tranh bn tung toe ln dich ra bn di), ly tm 50 g trong 10 phut. Cac protoplast sng sot se nm trn b mt dung dch (th ni). Dung micropipette ht lp protplast mau xanh luc ni trn tube ly tm v chuyn n sang mt tube ly tm sach, thm 10 mL dung dich ra va ly tm lai. Protoplast se lng xung ay tube va co dang vin (cuc tron). Ra protoplast thm mt ln na trong 10 mL dung dich ra. (ln ra nay co th bo qua nu cac hat protoplast qua be hay qua it chi va u dung). Loai bo phn ni trn mt va tai huyn ph protoplast trong khoang chng 1 mL mi trng nui cy protoplast (PC). t mt giot protoplast trn bung m hng cu va c lng mt protoplast (s lng t bao/s m 10.000). Nui cy protoplast bng cch pha loang 50.000 t bao/mL mi trng nui cy protoplast va chuyn sang mt ia petri v trung.

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Boc lai, ghi nhan va nui cy qua m trong ti 28oC. c. Ngay th ba Chuyn sang nui cy anh sang yu (10-20 mol.sec/m2 hoc boc mt lp vai tha di en huynh quanh anh sang trng, vi chu ky chiu sang 16 gi, nui trong 2 ngay. d. Ngay th nm Chuyn sang nui cy cng anh sang cao hn (50-75 mol/sec/m2) trong 2 ngy bng cach bo lp vai tha ra. e. Ngay th bay Ghi lai kt qua cua bai hoc nui cy protoplast, s t bao phn chia t tng s t bao nui cy ban u, trong mt mu co 100-200 protoplast nui cy. Co du hiu cua s nhim bn khng.Ch thch: Nu co th tim thy tp oan t bao, protoplast co th c pha loang vi mi trng nui cy sach c lng nho mannitol quan sat s phat trin cua callus co ngun gc protoplast. Sau khoang 8-10 tun, callus nay co th c chuyn ln mi trng tao chi.

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Bai 7

Chuyn gen thng qua Agrobacterium tumefaciensI. Muc ich va yu cuCac thc vt chuyn gen a c tao ra bng nhiu k thut khac nhau. Co hai phng thc chnh: - Chuyn gen trc tip: bn gen, xung in, vi tim, siu m, silicon carbide, thng qua ng phn, in di ... - Chuyn gen gian tip (nh vi sinh vt): Agrobacterium tumefaciens. Phng phap c s dung thng dung v t tn km hn c la chuyn gen gian tip thng qua Agrobacterium. Agrobacterium tumefaciens l mt loi vi khun Gram m co trong t, thng c s dung tin hanh bin nap di truyn trn i tng thc vt. oan DNA mun chuyn vao mt cy nao o co th c thao tac d dang trn E. coli va sau o chuyn tr lai vao Agrobacterium. Vic chuyn gen vao thc vt qua Agrobacterium a thc hin thanh cng trn nhiu loai cy: thuc la, u tng, cac cy ho u nhit i, bng cai, khoai ty.

II. Dung cu, thit bi, hoa cht1. Dung cu - Binh tam giac (250 mL), ng nghim c np vn - Giy thm v trung - Forceps, keo, dao m - Que cy vi sinh - ia petri (plastic va thuy tinh) v trung - Giy parafilm - Giy nhm - Tube eppendorf (1,5-2 mL) - Cc chiu nhit, ng ong, micropipette

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2. Thit bi - Ni kh trung - Tu sy - Laminar - Tu lanh - Microwave - Cn phn tich 10-4g - Cn ky thut 10-2g - May ct nc 2 ln - May ly tm lanh (14000 rpm) cho tube 1,5-2 mL 3. Hoa cht - Mi trng c ban MS - Mi trng nui cy vi khun LB - Cac loai khang sinh: kanamycin, rifampicin va cefotaxim - Agar - MgSO4 10mM - Dung dch stock cc cht kch thch sinh trng: NAA va BAP - Cn 90% - Nc ct v trung

III. Phng phap tin hanh1. Nguyn liu - E. coli chung TOP 10 co mang pRK 2013 (helper plasmid) - E. coli chung TOP 10 co mang vector pBI 121+ insert DNA (gen ngoi lai mang tnh trng mong mun) - Agrobacterium tumenfaciens chung LBA 4404. - Cy thuc la in vitro. 2. Chun bi mi trng 2.1. Mi trng LB - Bacto-tryptone - Yeast-Extract

3g 1,5 g

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- NaCl - Nc ct n u 300mL.

3g

2.2. Mi trng LB c Mi trng LB b sung 1,5 % agar/100 mL. 2.3. Mi trng LB-R Mi trng LB b sung 100 g/mL rifampicin. 2.4. Mi trng LB-K Mi trng LB b sung 50 g/mL kanamycin. 2.5. Mi trng LB-RK Mi trng LB b sung 100 g/mL rifampicin va 50 g/mL kanamycin. 2.6. Mi trng MSO - MS y u - Agar 0,8 % - Saccharose 3 % - pHmi trng ~ 5,8 2.7. Mi trng nui cy (MS 104) - MS y u - Agar 0,8 % - Saccharose 3 % - BAP 2 mg/mL - NAA 0,2 mg/mL - pHmi trng ~ 5,8 2.8. Mi trng chon loc (MS SEL) - MS y u - Agar 0,8 %

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- Saccharose 3 % - BAP 2 mg/mL - NAA 0,2 mg/mL - Kanamycin 1,5 mL - Cefotaxim 2 mL - pHmi trng ~ 5,8 2.9. Mi trng tao r (MSR) - MS y u - Agar 0,8 % - Saccharose 3 % - Kanamyin 1,5 mL - Cefotaxim 2 mL - pHmi trng ~ 5,8 Mi trng co b sung cefotaxim cn c bao quan trong iu kin khng co anh sang. Cht khang sinh c b sung sau khi a kh trung mi trng ( nhit mi trng ha xung khoang 55-60 oC) iu kin v trung cua laminar. 3. Tin hanh 3.1. Triparental mating (giao phi b ba) a. Nui cy vi khun Cac mi trng LB long c chun bi trong cac ng nghim c np vn, kh trung tin hanh nui cy cac vi khun. Cac bc nh sau: Nui A. tumenfaciens trong mi trng LB-R trong ti, 28 oC, thi gian 48 gi (nui trc khi tin hanh triparental mating 2 ngay). Nui E. coli co mang plasmid helper (pRK 2013) va E. coli co mang vector pBI 121 + insert DNA trong mi trng LB-K (nui trc khi tin hanh lai b ba 1 ngay). b. Nui cy chung 3 loai vi khun Cac bc tin hanh:

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Ly mi ng nghim nui cac loai vi khun 1 mL, ly tm tc khoang 10.000 rpm, trong 5 giy. Loai bo th ni, thm 1 mL mi trng LB, lc nhe trn ( loai bo cht khang sinh). Lp lai 2 bc trn 2 ln. Thm 300 l mi trng LB. 3 loai vi khun trn c cy ln mt ia cha mi trng LB c (a c chun bi trc cac ia petri cha mi trng LB b sung 1,5 % agar) khng b sung cht khang sinh, mi loai vi khun ly 100 l. Dan u trn b mt mi trng n khi kh hoan toan. Nui cy t 1-2 ngay tai 26-28oC, n khi cac tham vi khun c hinh thanh. c. Chon loc vi khun nui cy bng cach trn 3 loai vi khun, s dung cht khang sinh Cac bc tin hanh: Ly 5 mL MgSO4 10 mM cho ln ia petri mi, ly mt it vi khun trong thm a nui cy trn cho vao co mt dich long. Chun bi 4 tube eppendorf v trung, cho vao mi tube 900 L MgSO4 10 mM. Tin hanh pha loang vi khun thanh cac nng pha loang: 10-1, 10-2, 10-3, 10-4. Ly 100 l mi loai vi khun a pha loang dan u ln 5 ia petri cha mi trng LB-RK b sung 2 % agar. Nui trong 2-3 ngay tai 26-28oC trong iu kin ti pht trin khun lc. Nui cy 1 khun lac trong 5 mL mi trng LB-RK tai 26-28oC t 1-2 ngay thu c sinh khi E. coli, xac inh Agrobacterium a c chuyn gen (nu cha s dung ngay, thm vao 15% glycerin v bao quan -70oC). 3.2. Chuyn gen vao cy thuc la thng qua Agrobacterium Cac bc tin hanh: Ly 1 khun lac trn cy chuyn vao 5 mL mi trng LB-K long, nui cy lc iu kin ti, nhit t 27-28oC trong 2-3 ngay.

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Thu tt ca cac t bao a nui cy trn bng cach cho mi trng LB-K long cha khun lac vao tube eppendorf, ly tm t 0,5-1 phut, tc 12.000 rpm. Lp lai nhiu ln. Loai bo mi trng LB phia trn, cac t bao thu c hoa tan trong 1 mL mi trng MSO long. Chun bi 20 mL mi trng MSO trn ia petri. La thuc la in vitro ct thanh cac manh la co kich thc c chng khoang 0,5-0,7 cm 0,5-0,7 cm (loai bo cac gn la), chuyn ln ia petri cha mi trng MSO. T bao Agrobacterium hoa trong 1 mL mi trng MSO c cy chuyn sang mi trng MSO co cha cac manh la, lc nh bng tay va u trong 10 phut. Sau khi ng nui cy, lam kh cac manh la bng cach chuyn chung ln giy loc v trng trong 0,5-1 phut. Cac manh la o c nui trn mi trng nui cy (MS 104) trong 2 ngay trong ti. Cac manh la sau c cy chuyn sang mi trng chon loc (MS SEL) va nui cy trong 3 tun tai sinh chi iu kin chiu sang. Cac chi tao thanh c cy chuyn ln mi trng MSR tao r. Cac cy hoan chinh c chuyn sang nui cy trong cac binh. Cac cy nay la cac cy a c tin hnh chuyn gen. bit vic chuyn gen co thanh cng hay khng cn phai tin hanh cac thi nghim kim tra (tach chit DNA cua Agrobacterium, chy PCR vi cp primer c hiu cho insert DNA v in di san phm PCR trn agarose gel 1 %).

TAI LIU C THM - Murashige T and Skoog F. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15:473-497. - Nistch JP and Nistch C. 1969. Haploid plants from pollen grains. Science 163:85-87. - Trigiano RN and Gray DJ. 2000. Plant tissue culture concepts and laboratory exercises. - Street HE. 1977. Plant tissue and cell culture. Blackwell Scientific Publication. Osney, Mead, Oxford, UK.

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- Nguyn Vn Uyn. 1995. Nhng phng php cng ngh sinh hc thc vt. T1 v T2. NXB Nng nghip. TP H Ch Minh.