1

Chng 2. NHNG KHI NIM C BN 2.1. Hoc thuyt t bao

Nm 1662, Robert Hooke a thit k kinh hin vi n gian u tin va quan sat c cu truc cua ming bc bn bao gm nhiu hat nho, ng goi cac hat nho o la t bao (cells). Nm 1675, Anton Van Leeuwenhoek xac nhn c th ng vt cung bao gm cac t bao. ng quan sat di kinh hin vi thy mau ng vt co cha cac hng cu va ng goi o la cac t bao mau. Nhng mai n nm 1838, Matthias Jacob Schleiden (nha thc vt hoc) va 1839, Theodor Schwann (nha ng vt hoc) mi chinh thc xy dng hoc thuyt t bao. Schleiden va Schwann khng inh rng: Mi c th ng thc vt u bao gm nhng th tn tai hoan toan c lp, ring re va tach bit, o chinh la t bao. Co th noi Schleiden va Schwann la hai ng t cua hoc thuyt t bao. Tuy nhin, ca hai ng khng phai la cac tac gia u tin phat biu mt nguyn tc nao o, ma chi la din at nguyn tc y ro rang va hin nhin ti mc no c ph bin rng rai va cui cung a c a s cac nha sinh hoc thi y tha nhn. 2.1.1. Tinh toan th cua t bao (cell totipotency). Haberlandt (1902) la ngi u tin xng ra phng phap nui cy t bao thc vt chng minh cho tinh toan th cua t bao. Theo ng mi mt t bao bt ky cua mt c th sinh vt a bao u co kha nng tim tang phat trin thanh mt ca th hoan chinh.Nh vy mi t bao ring re cua mt c th a bao u cha y u toan b lng thng tin di truyn cn thit cua ca sinh vt o va nu gp iu kin thich hp thi mi t bao co th phat trin thanh mt c th sinh vt hoan chinh. Hn 50 nm sau, cac nha thc nghim v nui cy m va t bao thc vt mi at c thanh tu chng minh cho kha nng tn tai va phat trin c lp cua t bao. Tinh toan th cua t bao thc vt a c tng bc chng minh. Ni bt la cac cng trinh: Miller va Skoog (1953) tao c r t manh m ct t thn cy thuc la, Reinert va Steward (1958) a tao c phi va cy ca rt hoan chinh t t bao n nui cy trong dung dich, Cocking (1960) tach c t bao trn va Takebe (1971) tai sinh c cy hoan chinh t nui cy t bao trn cua la cy thuc la. Ky thut tao dong (cloning) cac t bao n c phn lp trong iu kin in vitro a chng minh mt thc t rng cac t bao soma, di cac iu kin thich hp, co th phn hoa phat trin thanh mt c th thc vt hoan chinh. S phat trin cua mt c th trng thanh t t bao n (hp t) la kt qua cua s hp nht s phn chia va phn hoa t bao. biu hin tinh toan th, cac t bao phn hoa u tin trai qua giai oan

Cng ngh nui cy m t bo thc vt

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2 phan phn hoa (dedifferentiation) va sau o la giai oan tai phn hoa (redifferentiation). Hin tng t bao trng thanh tr lai trang thai phn sinh va tao ra m callus khng phn hoa (undifferentiation) c goi la phan phn hoa, trong khi kha nng cac t bao phan phn hoa tao thanh cy hoan chinh (whole plant) hoc cac c quan thc vt c goi la tai phn hoa. ng vt, s phn hoa la khng th ao ngc tr lai. Nh vy, s phn hoa t bao la kt qua c ban cua s phat trin nhng c th bc cao, no thng c goi la cytodifferentiation. 2.1.2. Th bi v gen Gen quyt nh cc tnh trng thc vt. C tnh trng tng ng vi mt gen nhng cng c nhiu tnh trng lin quan n nhiu gen, cc tnh trng gi l tnh trng n gen v tnh trng a gen. Hai gen nm trn mt v tr nht nh trn nhim sc th tng ng gi l allen. Tuy cng tham gia quyt nh mt tnh trng nhng mi allen qui nh mt c im ring. V d mu hoa, mt allen c th mang thng tin di truyn cho hoa mu , allen kia cho hoa mu trng.Trng hp ny ta c c th d hp t v tnh trng mu hoa, nu c 2 allen u mang thng tin di truyn cho mu th ta c c th ng hp t. i vi c th d hp t, mt allen c th l allen tri, allen cn li l allen ln. Allen tri quyt nh tnh trng. C trng hp tri hon ton v tri khng hon ton. Tri khng hon ton khi t hp 2 allen s cho tnh trng trung gian. Th bi l danh t ch s b nhim sc th c trong t bo, m, c th thc vt vi qui nh chung l cc t bo sinh sn c 1 b nhim sc th c gi l th n bi. Hp t, sn phm dung hp ca 2 giao t n bi, c th l nh bi vi s nhim sc th 2n. Tt c cc t bo soma hnh thnh do s phn chia hp t u l nh bi. Trn thc t c th tm thy cng lc nhiu mc bi th khc nhau cc m khc nhau ca c th thc vt.(4n, 8n) l hin tng a bi ha do ni gim phn. Khong mt na thc vt bt cao mc a bi th. S nhim sc th c bn ca loi l X ( l s n bi nh nht trong dy a bi), cc c th c X nhim sc th c gi l th nht bi phn bit vi th n bi. V d : cy la m c 2n=42 . Trn thc t n l th lc bi 6X, trong s nhim sc th c bn ca loi l X=7. Th n bi ca cy la c n=3X=21 nhim sc th. 2.1.3.Th bo t v th giao t Th bo t gm c hp t v tt c cc t bo sn sinh t hp t k c ht phn trong ti phn v non.

Cng ngh nui cy m t bo thc vt

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3 Th giao t gm c ht phn ny mm v tt c cc t bo do n sn sinh ra, bao gm cc giao t. Khi 2 giao t khc ging dung hp, th bo t 2n c ti lp. thc vt bc cao, th giao t thng khng qu 3 t bo trong 2 t bo l cc giao t. cc loi thc vt mc th bi dao ng theo chu trnh sau:

Th bo t (2n)

Gim phn

Bo t (n)

Th tinh Th bo t n bi (n) giao t (n) Th giao t (n)

S 2.1. Chu trnh dao ng mc bi th thc vt bc cao, th giao t ( trong cc trng hp c bit, c th pht trin thnh bo t n bi) cha n nhim sc th. Th bo t n bi c th ra hoa nhng cc bo t hnh thnh khng c sc sng. To th bo t n bi v nhng cy n bi kp l mc ch ca nui cy ti phn v ht phn. 2.1.4. Sinh sn hu tnh v sinh sn v tnh Sinh sn v tnh l hin tng 1 c th to ra cc c th mi t mt phn c quan sinh dng ca mnh, khng h c s tham ca cc yu t quy nh gii tnh, c th con sinh ra hon ton ging ht c th m. Sinh sn v tnh c rt nhiu hnh thc. sinh vt n bo c phn i t bo. Mt s c th a bo bc thp th mt t bo sinh dng phn chia to ra mt nhnh mi v sau tch ra khi c th chnh nh thy tc chng hn, cng c th mt mu ca c th m t ra ri n mc ra mt c th khc kiu nh to lam. Mt s khc th c hn mt loi t bo sinh sn ring nhng m vn hon ton khng c tnh cht gii tnh g c m ch l t c th m to ra m thi. chnh l hin tng sinh sn v tnh bng bo t. Bo t cc c th n bo c th l khi mi trng bt li th chng t rt nc ra khi t bo, tr thnh dng tim sinh i thi c sng li. sinh vt a bo th ti ng cc t bo gi l bo t v tnh. n ma sinh sn chng s pht tn cc t bo ra mi trng xung quanh. Khi gp iu kin thun li th mi bo t to ra mt c th mi. thc vt th khc, n tn ti c hai kiu sinh sn v

Cng ngh nui cy m t bo thc vt

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4 tnh v hu tnh. Sinh sn v tnh y cng l t mt phn ca c th m tch ra v to ra mt c th mi. Sinh sn hu tnh phi c s tham gia ca cc yu t quy nh gii tnh, bao gm c v ci. Cc yu t ny c th trn cng mt c th hay khc c th, bn cht ca cc yu t l do cc nhim sc th gii tnh quy nh. Sinh sn hu tnh cng c nhiu kiu. Kiu s khai nht l tip hp, l hin tng hai t bo c, ci trao i nhn cho nhau. Sau l sinh sn hu tnh bng bo t nh ru, dng x,... Ln ti nhng lp trn th l th tinh vi s tham gia ca cc giao t c v ci, mi loi giao t nm cc t bo khc nhau.

2.2. T bo thc vt.

C th sng cu to t mt t bo n c hoc mt phc hp cc t bo. T bo rt a dng, khc nhau v hnh thi, kch thc, cu trc v chc nng. T bo ng vt v t bo thc vt l nhng bin i ca cng mt kiu c s ca n v cu trc. Trn c s hc thuyt t bo c hnh thnh do Mathias Schleiden v Theodor Schawn vo na u th k XIX. Thut ng t bo ln u tin c Robert Hooke t ra vo nm 1665 da trn nhng quan st cc khoang nh c vch bao quanh ca nt bn v v sau ng cn quan st thy trn m ca nhiu cy khc. Ni cht ca t bo v sau mi c pht hin v c gi l cht nguyn sinh, cn thut ng th nguyn sinh l do Hanstein xng nm 1880 ch cht nguyn sinh c trong 1 t bo n c. Nhn c Robert Brown pht hin nm 1831. Mi t bo l mt h thng m, t duy tr v t sn xut: t bo c th thu nhn cht dinh dng, chuyn ha cc cht ny thnh nng lng, tin hnh cc chc nng chuyn bit v sn sinh th h t bo mi nu cn thit. Mi t bo cha mt bn mt m ring hng dn cc hot ng trn. Mi t bo u c mt s kh nng sau: - Sinh sn thng qua phn bo - Trao i cht t bo bao gm thu nhn cc vt liu th, ch bin thnh cc thnh phn cn thit cho t bo, sn xut cc phn t sinh nng lng v cc sn phm ph. thc hin c cc chc nng ca mnh, t bo cn phi hp thu v s dng c ngun nng lng ha hc d tr trong cc phn t hu c. Nng lng ny c gii phng trong cc con ng trao i cht - Tng hp cc protein, y l nhng phn t m nhim nhng chc nng c bn ca t bo, v d nh enzyme. Mt t bo ng vt thng thng cha khong 10,000 loi protein khc nhau.

Cng ngh nui cy m t bo thc vt

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5 p ng vi cc kch thch, hoc thay i ca mi trng bn trong v bn ngoi nh nhng thay i v nhit , pH hoc ngun dinh dng. Di chuyn cc ti tit. 2.2.1. Cu trc ca t bo thc vt Cc t bo thc vt cc c th khc nhau, hoc cc m, cc c quan khc nhau ca cng mt c th s khng ging nhau v hnh dng, kch thc v cu trc nhng v bn cht c bn cc t bo u c mt s c im chung. T bo thc vt chia lm 2 phn chnh: Thnh t bo v phn nguyn sinh cht, y l phn quyt nh nhng c tnh sng ch yu ca t bo thc vt.

Hnh 2.1. M hnh cu trc t bo thc vt in hnh Mi t bo u c mng t bo, dng bao bc t bo, cch bit thnh phn ni bo vi mi trng xung quanh, iu khin nghim ngt s vn chuyn vo v ra ca cc cht, duy tr in th mng v nng cc cht bn trong v bn ngoi mng. Bn trong mng l mt khi t bo cht c (dng vt cht chim ton b th tch t bo). Mi t bo u c cc phn t DNA, vt liu di truyn quan trng v cc phn t RNA tham gia trc tip qu trnh tng hp nn cc loi protein khc nhau, trong c cc enzyme. Bn

Cng ngh nui cy m t bo thc vt

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6 trong t bo, vo mi thi im nht nh t bo tng hp nhiu loi phn t sinh hc khc nhau. 2.2.1.1. Thnh t bo Thnh t bo l cu trc thit yu i vi nhiu qu trnh sinh l v pht trin ca thc vt. L lp v bao bc, thnh t bo c vai tr nh b khung xng qui nh hnh dng t bo. Thnh t bo c mi quan h mt thit n th tch v p sut ca t bo do rt cn thit cho qu trnh trao i nc bnh thng thc vt. Thnh t bo thc vt tham gia xc nh di c hc ca cu trc thc vt, cho php chng sinh trng n mt cao kh ln. S a dng v chc nng ca thnh t bo bt ngun t s a dng v phc tp trong cu trc ca chng. Nhn chung cc thnh t bo c chia thnh hai nhm chnh: thnh s cp v thnh th cp. Thnh s cp hnh thnh bi cc t bo ang tng trng v thng c coi l tng i cha bit ha. Thnh th cp c hnh thnh sau khi t bo ngng tng trng, c mc chuyn ha cao c v thnh phn v cu trc. Trong thnh t bo s cp cc vi si xeluloza c gn cht trong mt mng li hydrat ha cao. Mng li ny bao gm s cc nhm polisaccarit thng l hemixenluloza v pectin cng 1 lng nh protein cu trc. B khung t bo l mt thnh phn quan trng, phc tp v linh ng ca t bo. N cu thnh v duy tr hnh dng t bo; l cc im bm cho cc bo quan; h tr qu trnh thc bo (t bo thu nhn cc cht bn ngoi); v c ng cc phn t bo trong qu trnh sinh trng v vn ng. cc protein tham gia cu thnh b khung t bo gm nhiu loi v c chc nng a dng nh nh hng, neo bm, pht sinh cc tm mng. 2.2.1.2. Cc bo quan Khng bo Khng bo l mt khoang ln nm trong trung tm cht nguyn sinh ca t bo. Nhng t bo thc vt trng thnh thng c mt khng bo ln cha y nc v chim t 80-90% th tch t bo. Khng bo c bc trong mt lp mng gi l mng khng bo (tonoplast). Trong khng bo cha nc, cc mui v c, ng, cc enzim v nhiu cht trao i th cp. Mng sinh cht Ranh gii gia thnh t bo vi cht nguyn sinh cng nh gia cht nguyn sinh vi khng bo c hnh thnh bi cc mng. Mng sinh cht ngn cch cht nguyn sinh vi mi trng xung quanh nhng cng cho php cht nguyn sinh c th hp th hay o thi cc cht khc ra khi t bo.

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7 Mng t bo - Tm o ngoi

V bc bn ngoi ca mt t bo eukaryote gi l mng sinh cht (plasma membrane). Mng ny cng c cc t bo prokaryote nhng c gi l mng t bo (cell membrane). Mng c chc nng bao bc v phn tch t bo vi mi trng xung quanh. Mng c cu thnh bi mt lp lipid kp v cc protein. Cc phn t protein hot ng nh cc knh vn chuyn v bm c nm khm vo lp lipid mt cch linh ng (c th di chuyn tng i). V bc bn ngoi ca mt t bo eukaryote gi l mng sinh cht (plasma membrane). Mng li ni cht Mng ni cht l mt h thng mng phc to,th hin trn bn ct ngang l h thng cc ti dp hoc cc ng nh gm hai lp mng v gia l mt khong hp T bo cht Bn trong cc t bo l mt khng gian cha y dch th gi l t bo cht (cytoplasm). N bao hm c hn hp cc ion, cht dch bn trong t bo v c cc bo quan. Cc bo quan bn trong t bo cht u c h thng mng sinh hc phn tch vi khi dung dch ny. Cht nguyn sinh (cytosol) l ch ring phn dch th, ch khng c cc bo quan.i vi cc sinh vt prokaryote, t bo cht l mt thnh phn tng i t do. Tuy nhin, t bo cht trong t bo eukaryote thng cha rt nhiu bo quan v b khung t bo. Cht nguyn sinh thng cha cc cht dinh dng ha tan, phn ct cc sn phm ph liu, v dch chuyn vt cht trong t bo to nn hin tng dng cht nguyn sinh. Nhn t bo thng nm bn trong t bo cht v c hnh dng thay i khi t bo di chuyn. T bo cht cng cha nhiu loi mui khc nhau, y l dng cht dn in tuyt vi to mi trng thch hp cho cc hot ng ca t bo. Mi trng t bo cht v cc bo quan trong n l yu t sng cn ca mt t bo. Nhn t bo - trung tm t bo: Nhn t bo l bo quan ti quan trng trong t bo eukaryote. N cha cc nhim sc th ca t bo, l ni din ra qu trnh nhn i DNA v tng hp RNA. Nhn t bo c dng hnh cu v c bao bc bi mt lp mng kp gi l mng nhn. Mng nhn dng bao ngoi v bo v DNA ca t bo trc nhng phn t c th gy tn thng n cu trc hoc nh hng n hot ng ca DNA. Trong qu trnh hot ng, phn t DNA c phin m tng hp cc phn t RNA chuyn bit, gi l RNA thng tin (mRNA). Cc mRNA c vn chuyn ra ngoi nhn, trc tip tham gia qu trnh tng hp cc protein c th. cc loi prokaryote, cc hot ng ca DNA tin hnh ngay ti t bo cht (chnh xc hn l ti vng nhn).

Cng ngh nui cy m t bo thc vt

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8 Ribosome - b my sn xut protein : Ribosome c c trong t bo eukaryote v prokaryote. Ribosome c cu to t cc phn t protein v RNA ribosome (rRNA). y l ni thc hin qu trnh sinh tng hp protein t cc phn t mRNA. Qu trnh ny cn c gi l dch m v thng tin di truyn m ha trong trnh t phn t DNA truyn qua trnh t RNA quyt nh trnh t amino acid ca phn t protein. Qu trnh ny cc k quan trng i vi tt c mi t bo, do mt t bo thng cha rt nhiu phn t ribosomethng hng trm thm ch hng nghn phn t. Ty th v lc lp - cc trung tm nng lng : Ty th l bo quan trong t bo eukaryote c hnh dng, kch thc v s lng a dng v c kh nng t nhn i. Ty th c genome ring, c lp vi genome trong nhn t bo. Ty th c vai tr cung cp nng lng cho mi qu trnh trao i cht ca t bo. Lc lp cng tng t nh ty th nhng kch thc ln hn, chng tham gia chuyn ha nng lng mt tri thnh cc cht hu c (trong qu trnh quang hp). Lc lp ch c cc t bo thc vt.

Mng li ni cht v b my Golgi - nh phn phi v x l cc i phn t:

Mng li ni cht (ER) l h thng mng vn chuyn cc phn t nht nh n cc a ch cn thit ci bin hoc thc hin chc nng, trong khi cc phn t khc th tri ni t do trong t bo cht. ER c chia lm 2 loi: ER ht (rm) v ER trn (nhn). ER ht l do cc ribosome bm ln b mt ngoi ca n, trong khi ER trn th khng c ribosome. Qu trnh dch m trn cc ribosome ca ER ht thng tng hp cc protein tit (protein xut khu). Cc protein tit thng c vn chuyn n phc h Golgi thc hin mt s ci bin, ng gi v vn chuyn n cc v tr khc nhau trong t bo. ER trn l ni tng hp lipid, gii c v b cha calcium.

Lysosome v peroxisome - h tiu ha ca t bo : Lysosome v peroxisome

thng c v nh h thng x l rc thi ca t bo. Hai bo quan ny u dng cu, mng n v cha nhiu enzyme tiu ha. V d, lysosome c th cha vi chc enzyme phn hu protein, nucleic acid v polysacharide m khng gy hi cho cc qu trnh khc ca t bo khi c bao bc bi lp mng t bo. Vt liu di truyn - Yu t duy tr thng tin gia cc th h : Vt liu di truyn l cc phn t nucleic acid (DNA v RNA). Hu ht cc sinh vt s dng DNA lu tr di hn thng tin di truyn trong khi ch mt vi virus dng RNA cho mc ch ny. Thng tin di truyn ca sinh vt chnh l m di truyn quy nh tt c protein cn thit cho mi t bo ca c th. Tuy nhin, mt nghin cu mi y cho thy c th mt s RNA cng c s dng nh l mt bn lu i vi mt s gene phng sai hng.

Cng ngh nui cy m t bo thc vt

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9 cc sinh vt prokaryote, vt liu di truyn l mt phn t DNA dng vng n gin. Phn t ny nm mt vng t bo cht chuyn bit gi l vng nhn. Tuy nhin, i vi cc sinh vt eukaryote, phn t DNA c bao bc bi cc phn t protein to thnh cu trc nhim sc th, c lu gi trong nhn t bo (vi mng nhn bao bn ngoi). Mi t bo thng cha nhiu nhim sc th (s lng nhim sc th trong mi t bo l c trung cho loi). Ngoi ra, cc bo quan nh ty th v lc lp u c vt liu di truyn ring ca mnh (xem thm thuyt ni cng sinh). V d, mt t bo ngi gm hai genome ring bit l genome nhn v genome ty th. Genome nhn (l th lng bi) bao gm 46 phn t DNA mch thng to thnh cc nhim sc th ring bit. Genome ty th l phn t DNA mch vng, kh nh v ch m ha cho mt vi protein quan trng. 2.2.2. Cc qu trnh chc nng ca t bo 2.2.2.1. Sinh trng v trao i cht ca t bo Gia nhng ln phn bo, cc t bo thc hin hng lot qu trnh trao i cht ni bo nhm duy tr s tn ti cng nh sinh trng ca mnh. Trao i cht l cc qu trnh m t bo x l hay ch bin cc phn t dinh dng theo cch ring ca n. Cc qu trnh trao i cht c chia lm 2 nhm ln:

Qu trnh d ha (catabolism) nhm phn hu cc phn t hu c phc tp thu nhn nng lng (di dng ATP) v lc kh; Qu trnh ng ha (anabolism) s dng nng lng v lc kh xy dng cc phn t hu c phc tp, c th v cn thit.

Mt trong cc con ng trao i cht quan trng l ng phn (glycolysis), con ng ny khng cn oxy. Mi mt phn t glucose tri qua con ng ny s to thnh 4 phn t ATP v y l phng thc thu nhn nng lng chnh ca cc vi khun k kh. i vi cc sinh vt hiu kh, cc phn t pyruvat, sn phm ca ng phn, s tham gia vo chu trnh Kreb (hay cn gi l chu trnh TCA) phn hu hon ton thnh CO2, ng thi thu nhn thm nhiu ATP. sinh vt eukaryote, chu trnh TCA tin hnh trong ty th trong khi sinh vt prokaryote li tin hnh ngay t bo cht. 2.2.2.2. Sinh tng hp protein

S qu trnh sinh tng hp protein:

Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

10 Trong vng cht nhn, cc gene c phin m thnh nhng phn t RNA. Sau khi thc hin cc sa i sau phin m, phn t mRNA trng thnh c vn chuyn ra t bo cht tin hnh tng hp protein ti y. Cc ribosome tin hnh dch m ca mRNA nh mi lin kt hydro theo nguyn tc b sung gia b ba m sao trn mRNA vi b ba i m trn tRNA tng ng. Nhng phn t protein sau khi c tng hp thng c tin hnh mt s sa i cho ph hp vi chc nng, v d gn thm cc gc ng

Sinh tng hp protein l qu trnh t bo tng hp nhng phn t protein c trng v cn thit cho hot ng sng ca mnh. Qu trnh phin m l qu trnh tng hp nhng phn t RNA thng tin da trn trnh t khun ca DNA. Trn khun mRNA mi c to ra, mt phn t protein s c to thnh nh qu trnh dch m. B my t bo chu trch nhim thc hin qu trnh tng hp protein l nhng ribosome. Ribosome c cu to t nhng phn t RNA ribosome v khong 80 loi protein khc nhau. Khi cc tiu n v ribosome lin kt vi phn t mRNA th qu trnh dch m c tin hnh. Khi , ribosome s cho php mt phn t RNA Hnh 2.2. Qu trnh sinh vn chuyn (tRNA) mang mt loi amino acid c trng i tng hp protein vo. tRNA ny bt buc phi c b ba i m c trnh t b sung vi b ba m sao trn mRNA. Cc amino acid ln lt tng ng vi trnh t cc b ba nucleotide trn mRNA s lin kt vi nhau to thnh mt chui polypeptide. 2.2.2.3. Hnh thnh cc t bo mi

Phn bo l qu trnh sinh sn t mt t bo (gi l t bo m) phn chia thnh hai t bo non. y l c ch chnh ca qu trnh sinh trng ca sinh vt a bo v l hnh thc sinh sn ca sinh vt n bo. Nhng t bo prokaryote phn chia bng hnh thc phn ct (binary fission) hoc ny chi (budding). T bo eukaryote th s dng hnh thc phn bo l nguyn phn (mitosis) (mt hnh thc phn bo c t). Nhng t bo lng bi th c th tin hnh gim phn to ra t bo n bi. Nhng t bo n bi ng vai tr giao t trong qu trnh th tinh hnh thnh hp t (lng bi). Trong phn bo,
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

11 qu trnh nhn i DNA (dn n nhn i nhim sc th) ng vai tr cc k quan trng v thng din ra ti k trung gian gia cc ln phn chia. Cc pha trong chu k t bo: Pha G0 l mt giai on ca chu k t bo cell cycle m t bo trng thi lng yn. Pha G1 l pha pht trin u tin ca chu k. Pha S, trong pha ny DNA c sao chp, ch S xut pht t synthesis of DNA c ngha l tng hp DNA (cn gi l axt nhn ADN: Axt Dezoxy riboNucleic). Pha G2 l pha pht trin th hai, cng l pha chun b cho t bo phn chia. Pha M, hay pha phn bo mitosis, v trng thi hot ng ca t bo ( cytokinesis), s phn chia t bo thc s din ra to thnh hai t bo mi ging nhau. H thng kim sot, cn gi l cc im kim sot, kim tra cc tn thng ca DNA v cc sai st trong cc qu trnh quan trng ca chu k t bo. Trong trng hp c s khng tng thch no , cc im kim sot c th chn qu trnh lun chuyn qua cc pha ca chu k t bo. Chng hn nh, im kim sot iu khin sao chp DNA v gi cho t bo sao chp hon ton DNA trc khi bc vo qu trnh phn bo ( mitosis). Cng vy, im kim sot con thoi (spindle checkpoint) s ngn cn qu trnh chuyn dch t pha bin k (metaphase) sang pha hu k trong (anaphase) trong qu trnh phn bo nu nh khng c tt c cc nhim sc th ( chromosomes) tp trung nh vo thoi phn bo. Nu h thng ny pht hin c iu g bt thng, th mt mng li cc phn t dn truyn thng tin (signal transduction) s hng dn t bo ngng phn chia ngay. Chng cn cn th gip cho t bo bit c c th sa cha tn thng hay khng hay l khi ng qu trnh t bo cht c lp trnh, mt dng ca n c gi l apoptosis. Qu trnh t bo cht c lp trnh gip hn ch cc t bo tn thng pht trin. V d nh, mt protein, c gi l p53, nhn cm cc tnh hiu xut pht t cc DNA tn thng. N p ng bng cch kch thch sn xut ra cc protein c ch dng qu trnh sao chp DNA li. Nu chc nng ca p53 khng hot ng ng th dn n vic ng cc DNA tn thng khng c kim tra. Hu qu trc tip ca iu l cc gene tn thng s pht trin sang cc dng ung th. Ngy nay, nhng thm d cho thy p53 c

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

12 phi hp vi nhiu loi ung th khc nhau nh l mt vi dng ung th v v ung th i trng. Mt vi t bo nh l t bo thn kinh, khng bao gi phn chia khi n lun dng li pha G0. Tuy nhin, cc nghin cu gn y cho thy nhng trng hp tn thng cht t bo th t bo thn kinh vn c th i vo li chu k t bo. Ngoi ra cc cht c ch chu k t bo ngn cn t bo khi ci cht c lp trnh c bit nh l apoptosis.

2.3.

Thc vt

C th thc vt c cu to t nhng n v hnh thi c gi l t bo, mi t bo c lin kt vi nhng t bo khc bi cht kt dnh gian bo bao quanh. Trong khi lin kt c nhng nhm t bo khc bit v hnh thi hoc v chc nng hoc c hai vi nhng nhm khc. Nhng nhm nh th c gi l m. Mt s m cu to n gin, ch gm mt loi t bo, nhng m khc phc tp hn gm nhiu hn mt kiu t bo Cc m t bo trong c th thc vt u c ngun gc t hp t tc l t t bo trng th tinh qua cc giai on pht trin ca phi v sau pht trin thnh c th trng thnh. C th thc vt sinh trng nh c m phn sinh. M phn sinh ngn phn chia v phn ha thnh cc phn mi ca chi v r. l s sinh trng s cp. S sinh trng th cp thc vt hai l mm v ht trn l do hot ng ca m phn sinh th cp c gi l tng pht sinh. Trong s sinh trng th cp cn c tng phn sinh bn l m phn sinh th cp hnh thnh nn chu b. Tng pht sinh v tng sinh bn c gi l m phn sinh bn v n v tr bn ca than v r phn bit vi m phn sinh s cp l m phn sinh ngn. C th thc vt c phi pht trin k t khi ht ny mm gm r pht trin xung t v chi gm thn mang l pht trin trong kh quyn. S pht trin ca chi v r l t cc t bo ca m phn sinh ngn. Thn l v r c gi l c quan dinh dng. Khi trng thnh th hoa c hnh thnh. Sau s th phn l s th tinh v s hnh thnh phi, ht v qu. Nhng c quan c gi l c quan sinh sn. Chu trnh pht trin ca c th thc vt c th k t khi hp t hnh thnh v kt thc trc khi xy ra s th tinh ca cc giao t to nn th h sau.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

13 2.3.1. S ny mm ca ht v s pht trin ca cy con a. S ny mm ca ht S ny mm ca ht bt u khi ht hp thu rt nhiu nc v tng th tch ln mt cch ng k, c khi n 200%. Kt qu ca s hp thu nc ny lm cho phi gii phng gibberellin, v y l yu t cm ng tng hp mt s cc enzim thy gii trong c c amylaza. Nhng enzim ny thy phn nhng cht d tr trong phi nh, cung cp nng lng cho s tng trng ca phi. T bo bt u phn ct, tng hp thm t bo cht mi, gia tng kch thc nh s hp thu nc. Phi tng trng lm bung v ht ra v nhanh chng hnh thnh mt cy con, c r v thn phn bit.

Hnh 2.3. S ny mm ca ht Khi ht ny mm, trc h dip c mc ra trc tin. Trc h dip mc xung theo chiu trng lc, d ht nm theo hng no. Cng lc trc thng dip bt u pht trin nhanh chng, r mm phn cui ca trc h dip, to ra mt h thng r con gn vo trong t v hp thu nc v mui khong. mt s cy song t dip, phn trn ca trc h dip mc di ra thnh dng hnh vm, mc ngc ln v chui ra khi mt t. Khi trc h dip l ra ngoi khng kh, n mc thng ln, t dip v trc thng dip c a ra khi mt t. Sau trc thng dip bt u mc di ra. y l kiu ny mm thng a. Nhng cy Song t dip khc, th d nh u H lan, Nhn c mt kiu ny mm hi khc, nhng cy ny, trc h dip khng mc thnh hnh vm v t dip khng

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

14 c a ln khi mt t. Thay vo l trc thng dip bt u mc di ra ngay sau khi h thng r con bt u c hnh thnh; n lun lun mc thng ng v chng bao lu nh ra khi mt t. Kiu ny mm ny tng t nh ht La, Bp... thuc cc cy n t dip, ch c mt t dip, nhng giu phi nh . y l kiu ny mm h a. Bp trc thng dip bt u di ra ngay sau khi h thng r c thnh lp. Thn non c dip tiu (l u tin hnh ng) bao bc. b. S pht trin ca cy con u tin cy con tng trng hi chm, nhng sau tng trng vi mt tc nhanh hn trong mt thi gian di hn v cui cng chm li v c th dng tng trng khi cy sp trng thnh. nhng cy a nin, s tng trng tip tc xy ra trong sut i sng ca cy, trong khi nhng cy nht nin nh cc cy u, cy C ci... tng trng ngng li khi cy trng thnh v cy cht i sau mt ma sinh trng. S tng trng ca r v thn ca cy con c c l nh s phn ct v s tng di ca t bo. Hai hot ng ny chu nh hng ca nhiu hormon sinh trng khc nhau, c bit l auxin, gibberellin v cytokinin. nhng cy ch c m s cp th s phn ct t bo v s tng di ca t bo ty thuc vo s hot ng ca hai m phn sinh ngn r v ngn thn. 2.3.2. S tng trng ca r v thn a. S tng trng ca r S hot ng ca m phn sinh ngn r lm cho r tng trng. M phn sinh r c bo v bi mt chp r hnh nn, gm mt khi t bo khng phn ct c. Khi r mc di ra v u r mc su vo trong t th mt s t bo mt ngoi ca chp r c th b tn thng v sau c thay th bng nhng t bo mi do s phn ct t bo ca m phn sinh ngn. Ngay sau ca chp r l vng m phn sinh ngn r, vng ny ngn v gm nhng t bo nh c kh nng phn chia tch cc. Phn ln cc t bo mi c to ra nm xa chp r. M phn sinh tip tc phn ct cho t bo mi v u r tip tc mc su vo trong t. Chnh cc t bo c to ra t m phn sinh ny s thnh lp m s cp cho r.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

15 Khi cc t bo c mi c y ra khi vng m phn sinh ngn, do s lng t bo tng ln s phn ct chm li th s gia tng kch thc t bo l qu trnh chnh. Phn ln s tng kch thc lm r tng trng chiu di nhiu hn l chiu rng. S tng di ca t bo chu tc ng ca cc hormon m c bit l auxin v gibberellin. Vng t bo tng di nhiu nht l vng ngay sau vng m phn sinh v thng di ch vi milimet. K tip l vng t bo trng thnh, ni y t bo bt u trng thnh v c hnh thnh dng c trng. Vng ny d nhn bit nh cc lng ht c mc di ra t nhng t bo biu b.

Hnh 2.4 Cu to ca r b. S tng trng ca thn S phn ct t bo m phn sinh

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

16

Hnh 2.5. nh ct dc ca chi ngn ngn thn to ra m s cp ca thn v khi s khi ca l Cc t bo mi c to ra t m phn sinh ngn thn gn nh ngn, mc di y ngn thn thng ng ln. S tng trng ca thn khc vi s tng trng ca r l c s to ra l pha bn ca nh ngn thn. Cch khong u n m phn sinh ngn thn to ra nhng khi s khi ca l (leaf primordia), sau ny s to ra nhng l mi. Ni l mc ra t thn gi l mt (node) v khong gia hai mt l lng (internode). Phn ln thn di ra l do s tng di ca t bo nhng lng cn non. nh ca thn l mt chui nhng lng cha c mc di ra. Nhng khi s khi ca l rt nh ngn cch cc lng un cong; cc khi s khi gi hn, to hn ca l bao ly cc khi s khi tr hn, nh hn bn trong. Cu trc gm m phn sinh ngn v cc lng cha c tng di c bao bc trong cc khi s khi ca l c gi l chi (bud). nhng cy tng trng theo ma th chi c bo v bi nhng vy, l nhng l bin i mc t di y ca chi. Vo ma xun, khi cc chi ng ny n ra, th cc vy che ch rng i v nhng lng cha bn trong cc chi bt u tng di mt cch nhanh chng. Do cc lng s dn dn c tch xa nhau ra, s phn ct t bo xy ra khi s khi ca l v to ra l non. Trc khi l c hnh thnh mt cch hon chnh, mt u nh ca m phn sinh thng mc ra gia y l v lng. Mi mt vng m phn sinh mi ny s to ra mt chi bn (lateral, axillary bud) c c im tng t nh chi ngn . S tng di ca cc lng ca chi bn trong ma sinh trng k tip s to ra nhnh.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

17 2.3.3. S chuyn ha ca t bo Tt c nhng t bo mi c sinh ra t m phn sinh th c bn ging nhau, chng s tr thnh cc loi m khc nhau. Qu trnh t bo thay i t nhng hnh dng cha trng thnh n trng thnh gi l s chuyn ha (differentiation). Trong s tng trng ca r v thn, t bo bt u chuyn ha thnh cc loi m khc nhau khi chng vn cn trong vng m phn sinh. Sau khi s phn ct t bo v s tng di ca t bo hon tt, t bo bt u trng thnh c hnh dng nht nh. lt ct ngang c th phn bit c ba vng ng tm ngay sau m phn sinh ca r l lp tin b (protoderm), k tip l mt vng m cn bn dy nm ngay di tin b v trong cng l m tin dn truyn (provascular tissue) gm nhng t bo. Ngay trong phi, tin b ngoi tr thnh biu b, m cn bn tr thnh v v ni b, phn trong cng to ra m dn truyn s cp, chu lun v tng tng libe g. S chuyn ha trong thn ang tng trng cng theo cch tng t ngoi tr c hai vng m cn bn, mt vng nm gia tin b v tr tin dn truyn s to ra v v ni b, v mt vng th hai nm trong tr tin dn truyn s tr thnh li. S tng trng theo ng knh ca r v thn ty thuc vo s thnh lp m th cp do s hot ng ca nhng m phn sinh bn, c bit l tng tng libe g. Di nh hng ca auxin, nhng t bo mi c to ra pha ngoi ca tng tng s chuyn ha thnh m libe th cp, trong khi nhng t bo mi c to ra pha trong ca tng tng s to nn m g th cp.

Hnh 2.6. S chuyn ho ca r non

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

18 Thc vt c xu hng mc v hng c nh sng. t mt chu cy trong phng, cy s mc cong hng v pha ca s, nu xoay cy hng vo trong, sau mt thi gian ngn cy li mc hng v pha ca s. Hin tng cy p ng li vi nh sng bi s xoay ny c gi l quang hng ng (phototropism) ca thc vt. Thc vt cn c cc tnh hng ng khc nh a hng ng (gravitropism) l p ng ca cy hng theo chiu ca trng lc, thy hng ng (hydrotropism) p ng vi nc. p ng ny l do s sinh trng chuyn ha; mt pha ca thn cy hay r mc nhanh hn pha bn kia, lm cho cy cong i. Thn c quang hng ng dng, xoay v hng c nh sng; r th ngc li, c quang hng ng m, xoay trnh nh sng. ngha thch nghi ca quang hng ng thn l xoay thn l nhn c nh sng ti a cn thit cho s quang hp.

2.4.

Phng th nghim
2.4.1. Cc thit b , dng c cn thit ca phng th nghim nui cy m Mt phng th nghim nui cy m t bo thng bao gm: -Phng ra dng c -Phng chun b mi trng, hp tit trng v cha dng c

- Phng cy v trng - Phng nui mu - Phng quan st v thu nhn s liu S tng quan nh sau: 1 2 3 4 5 6 10 9 8 7

1.Phng ra v sn xut nc ct 2. Phng sy hp, kho thy tinh sch

Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

19 3. Phng chun b mi trng 4. Phng chun b mu 5. Phng cy v trng 6. Phng nh 7. Phng knh hin vi 8. Phng nui 9.Phng nui 10. Phng sinh ha Bn cnh phng th nghim cn c h thng nh li v vn m trng cy ly nguyn liu nui cy v trng cy ti sinh trong qu trnh chn lc nvitro. a. Phng ra v ct nc: Phng ra dng c phi c bn ra ln , c ng thot nc ring cho axit, c k cc thit b: - My ct nc mt ln - My ct nc hai ln - My sn xut nc kh ion b.Phng sy hp: - T sy 60-6000C (loi c dung tch ln) - Ni p sut loi nh (20-30 lt) - Ni p sut loi ln (70-100 lt) c.Phng chun b mi trng: - pH meter - My khuy t - Cn phn tch 10-4 g - Cn k thut 100-2g - My rt mi trng - Bp in - Microwave - T lnh 100-200 l - T lnh su (-20 n -800C) d. Phng cy v trng Phng cy v trng nn l mt phng nh, kn, sn v tng cn c lt gch men hoc sn lau chi v kh trng thng xuyn. Ca phng cy nn l ca knh v trong khi thao tc cy rt d b pht n cn do cn phi d lin lc vi bn ngoi trong lc cn thit. Trn tng gn n UV kh trng phng.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

20 C hai loi t cy thng c s dng: t cy tnh v t cy thi kh v trng. Trong t cy phi c n trng d lm vic v c n UV kh trng trc khi lm vic - Laminar -Qut thng gi - n t ngoi treo trn hoc treo tng - Thit b lc khng kh - Gi v bn mi trng - B dng c cy, n cn e. Phng nui mu cy: Tt c cc mu cy u c nui trong iu kin nhit nh sng, m, di chiu sng, thng kh thch hp. Phng nui c nhit 15-300C ty theo mu cy v mc ch ca th nghim. Nhit phi c phn b u trong ton phng nui, phi c y nh sng hunh quang v c th iu khin c cng v thi gian chiu sng. Phng nui phi c thi kh ng nht v bin m c iu chnh t 20-98%. - Phng nui sng: tng nn sn mu trng. Cc gi n c lp n ng chiu sng. Trong phng cn gn cc my mc kim tra chnh xc nhit , m. - Phng nui ti nui m so v cc x l c bit. Phng cn tt c cc iu kin nh phng sng ch khc l khng cn lp n chiu sng cho cy, ca s cn c che kn bng vi en. - Cc gin n hunh quang nhiu ngn, c chiu sng ch bnh nui cy t 2000-3000 lux. - My iu ha nhit - My lc nm ngang 100-200 vng/pht - Cc thit b v dng c nui cy t bo n -T m. f. Phng sinh ha : Phng ny dng tin hnh cc phn tch sinh ha, phn t v di truyn. - T ht, t m - Cn cc loi - My ct tiu bn - My o pH - Ly tm lnh - My in di, my soi AND - My PCR,my sc k,quang ph

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

21 - T lnh thng, t lnh su - L vi sng - Pipet t ng cc loi - My soi v chp nh gel -Cc t ng ha cht, t ht kh c. * Cc nhn t m bo thnh cng trong nui cy m t bo thc vt: c 3 nhn t chnh: - m bo iu kin v trng. -Chn ng mi trng v chun b mi trng ng cch - Chn m cy v x l m cy thch hp trc v sau khi cy. 2.4.2. Cc th tc c bn trong phng th nghim: 2.4.2.1. Cn Vic chun b mi trng i hi thao tc cn phi chnh xc. Trc ht cn phi c t v tr n nh, khng b rung, khng kh khng b dao ng nhiu. Cn v da cn phi c gi gn sch s. Quan trng nht l khng c cn qa trng lng cho php v nn s dng cc vt ng ha cht c trng lng nh hoc bng giy khi cn. Khng c ha cht tip xc trc tip vi mt cn. 2.4.2.2. ong cht lng Cc dng c thy tinh c chia vch (ng ht c chia , cc thy tinh c chia vch, ng ong) cn thit pha mi trng. ng ong c th tch 10,20,100 v 1000ml c s dng ong nhng cht lng c th tch ln cn ng ht c chia , bnh nh mc dung ong nhng th tch cn chnh xc. ong cc dung dch ch chnh xc khi y ca khng kh ngang vi vch nh du. 2.4.2.3. Xc nh pH pH ca mi trng cy hu ht c chnh 5,5 +-0,1 trc khi hp kh trng. pH nh hng n kh nng ha tan ca cc ion trong mi trng khong, kh nng ng t agar v s tng trng ca t bo. V vy xc nh chnh xc pH l cn thit. Murashige v Shoog nhn thy rng pH 5,7-5,8 thch hp duy tr s ha tan cc cht khong trong mi trng MS. Nu mi trng MS c s dng dng lng th c th chnh pH 5, mi trng cy huyn ph c pH thp phn no gim bt tnh trng nhim. pH ca mi trng thng c iu chnh bng NaOH hoc HCl sau khi pha xong mi trng v chun b a v hp kh trng. C th chnh pH bng pH k bn, pH k cm tay hoc giy o pH. Thng th nhit cao s lm tng tnh axit ca

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

22 mi trng. Mann v cng s nhn thy rng nu trc khi hp pH=5,7 th sau khi hp pH =5, Nu mun pH =5,7-5,9 th trc khi hp kh trng cn iu chnh pH n 7. 2.4.2.4. Ra dng c thy tinh v bnh nui cy bng plastic Thng thng bnh nui cy sau khi s dng cn phi c ra k bng x bng bt cho ht cc cht bm trn thnh chai ri trng li nhiu ln bng nc sch cui cng trng li bng nc ct. Cc dng c thy tinh b qu bn cn phi c ngm trong axit HCl hoc sulfuric sau ra sch bng nc my v trng bng nc ct. Cc bnh mi trng b nhim trng trong qu trnh nui cy cn phi c hp tit trng trc khi ra. Cc dng c thy tinh sau khi ra phi c sy kh trong t sy v c ct cn thn.

2.5. m bo iu kin v trng

2.5.1. ngha ca v trng trong nui cy m v t bo thc vt Mi trng nui cy m v t bo thc vt c cha ng, mui khong, vitamin..rt thch hp cho cc loi nm v vi khun pht trin. Do tc phn bo ca nm v vi khun ln hn nhiu so vi cc t bo thc vt, nu trong mi trng nui cy b nhim bo t nm hoc vi khun th sau vi ngy s ph y vi khun hoc nm,khi m nui cy s cht dn th nghim phi b i. Thng thng mt chu k nui cy m v t bo thc vt di t 1-5 thng, trong khi th nghim vi sinh vt c th kt thc trong mt vi ngy. Nh vy mc v trng trong th nghim nui cy m v t bo thc vt i hi rt nghim ngt, iu kin ny c bit quan trng trong nui cy t bo n trong cc bioreactor. C 3 ngun nhim tp chnh: - Dng c thy tinh, mi trng v nt y khng c v trng tuyt i. - Trn b mt hoc bn trong m nui cy tn ti cc si nm, bo t vi khun. - Trong qu trnh thao tc lm ri nm hoc vi khun theo bi ln mi trng. 2.5.2.Kh trng 2.5.2.1. Kh trng phng cy v t cy Phng cy thng l phng c din tch hp, rng t 10-15m 2, c hai lp ca trnh khng kh chuyn ng t bn ngoi trc tip a bi vo. Sn v tng c lt gch men d lau chi. Trc khi a vo s dng bung cy cn c x l bng hi Formol bng cch rt formaldehyde (formalin)4% ra mt s da petri ri rc vi ni trong phng cho bc hi t nhin. ng kn ca phng cy trong 24 gi, sau b formaldehyde i v kh hi formaldehyde d bng dung dch NH3 25% trong 24 gi. Cc

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

23 dng c mang vo bung cy u v trng trc: t qun o chong, m vi, khu trang, dao ko..Trn bn cy thng xuyn c mt n cn s dng khi cy v mt cc ng cn 95% nhng dng c lm vic Trc khi cy, th nghim vin cn ra tay bng x phng v lau k n khuu tay bng cn 900. m bo mc v trng cao cn c mt n t ngoi treo trn trn. Phng cy ln c kh trng tin nht l bng n cc tm.Thi gian kh trng ty theo kch thc ca phng v n cc tm ch c s dng khi khng c ngi. Phng cng c th c kh trng bng cch lau ra hai ln/thng vi cc dung dch chng nm. Phng nh hn v t cy cng c kh trng bng tia cc tm hay cc dung dch kh trng. T cy thi gi c kh trng bng cch m qut gi v lau tt c cc b mt bng cn 95% trong 15 pht trc khi bt u lm vic. Phng nui cng c kh trng trc ht l bng x bng bt. Sau lau bng dung dch hypoclorit sodium 2% hoc bng cn 95%. Tt c sn, trn u c lau nh vy mi tun. Cn thn khng nn khuy ng nhng ni b nhim trnh pht tn bo t.Cn gim s chuyn ng ca khng kh trong bung cy n mc ti thiu v vy tt c cc dng c phc v vic cy u phi chun b y trong khi cy trnh i li ra vo bung cy nhiu ln. 2.5.2.2. Kh trng bnh cy v cc dng c khc a. Dng c: Dng c thy tinh dung cho nui cy m v t bo thc vt phi l bnh thy tinh trong sut nh sng qua c mc ti a v trung tnh trnh kim t bnh thy tinh gy nh hng n s pht trin ca m nui cy. Cn ra sch dng c thy tinh trc khi a vo s dng. Thng thng ch cn x l bng sulfochromate mt ln u khi a vo s dng v sau ch cn ra sch bng x phng, trng nhiu ln bng nc my cui cng trng bng nc ct. Sauk hi ro nc dng c thy tinh cn c v trng bng cch sy 1600C/gi. Sau khi ngui c ly ra ct vo ch t bi. Dng c kim loi, giy nhm cn c kh trng bng khng kh nng (1301700C) trong 2-4 gi trong t sy. Tt c cc vt dng ny phi c gi kn trc khi kh trng nhng khng c gi bng giy v giy b phn r 170 0C. Khng nn hp kh trng dng c kim loi v iu kin nng m s lm cho kim loi b r st v b n mn. Trc khi s dng cc dng c c kh trng bng khng kh nng, cc dng c c ly ra khi giy gi, nhng vo cn 95 0 v t trn ngn la n cn. Sauk hi

Cng ngh nui cy m t bo thc vt

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24 dng xong, dng c ny phi c t li bng cn trc khi s dng tip. Khi s dng cn cn lu y n s an ton ti a v rt d b pht. Autoclave l phng php kh trng bng hi nc di p sut nht nh.Nt gn, vi, cc dng c thy tinh, bnh nui cy bng plastic, nt cao su, pipet, nc, mi trng khong u c th kh trng bng ni hp. Gn nh tt c vi sinh u b cht bi hi nc trong ni hp trong 10-15 pht 1210C. b. Nt y: Thng dng nht l nt y lm bng bng khng thm nc. Nt phi tng i cht m bo bi khng i qua c, ng thi nc t mi trng khng b bc hi qu d dng trong qu trnh nui cy. Bng khng thm nc l loi nt n gin nht nhng c nhc im sau: + Nu khi hp nt bng b t hoc dnh mi trng th v sau s b nhim nm nht l cc th nghim nui cy trong thi gian di. +Thao tc lm nt bng chm, khng thun tin khi nui cy trn qui m ln. + Ch dng c mt vi ln sau phi b. Hin nay ngi ta s dng nhiu loi np y khc nhau thay th nt bong. Cc hng sn xut dng c nui cy m cung cp loi np ng nghim v bnh tam gic bng nha chu nhit c th hp 1200C m khng b bin dng. Mt s phng th nghim s dng np inox hoc cao su rt thun tin cho vic vt trng. Cc dung dch m dng pha ch mi trng (dung dch mui khong, vitamin..) cn c bo qun trong t lnh. Dung dch vitamin nn chia thnh nhiu loi nh v bo qun trong ngn ca t lnh. Khng nn pha mt lng qu ln dung dch m cc cht sinh trng, thng ch nn dng cc l c dung tch 100-200 ml. c. Kh trng mi trng khong: kh trng mi trng khong thng s dng hai phng php hp tit trng v lc bng mng lc v trng. Mi trng nui cy, nc ct v cc ha cht n nh khc c th cha trong bnh thy tinh v y bng nt gn, giy nhm hoc np nha. Tuy nhin mi trng c cc cht khng bn nhit th cn s dng phin lc milipore. Ni chung mi trng khong c hp tit trng 121 0C, 1 atm. Vi nhng th tch nh (100 ml hoc t hn) thi gian kh trng l15-20 pht. Vi lng mi trng ln th phi kh trng trong 30-40 pht. p sut khng nn cao qu 1 atm vi p sut cao s lm phn hy carbohydrate v cc phc hp nhy cm vi nhit Bng 2.1 . Thi gian ti thiu hp kh trng mi trng nui cy m 1210C (Burgerr, 1988) Th tch mi trng (ml) Thi gian kh trng ti thiu (pht)

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25 20-25 24 50 26 100 28,5 250 31,5 500 35 1000 40 2000 48 3000 55 4000 63 Nhiu loi protein, vitamin, amino axit, hormone..khng bn nhit v vy nn dng phin lc micropore kh trng. Phin lc milipore hoc phin lc seitz u c th s dng vi kch thc l khng ln hn 0,2 m. Cc bnh ng thy tinh cn phi c hp tit trng trc khi lc. Mi trng khong c cht khng bn nhit c th c tin hnh chun b theo cc bc sau: u tin cc cht khong bn vi nhit c hp tit trng, sau lm lnh xung cn 50-600C trong iu kin v trng khi nhng cht khng bn vi nhit s c lc v trng. Dung dch kh trng ny s phi hp vi nhau trong iu kin v trng to ra mt mi trng hon chnh 2.5.2.3.Kh trng mu cy thc vt Cc loi mu cy thng c s dng trong nui cy m V mt nguyn tc, cc t bo cn sng phn ha u c kh nng phn phn ha tr li trng thi tr ha v ti lp kh nng phn chia. Tuy nhin c th nhn xt chung l cc m ang pht trin mnh (m phn sinh ngn, tng tng) khi t vo mi trng c cha mt lng cht sinh trng thch hp u c kh nng to m so cao. bt u nghin cu nhn ging v tnh mt cy nht nh, trc tin ngi ta ch y n chi nch v m phn sinh ngn.Cc m thc vt thng c s dng nui cy l: - nh sinh trng thn, r - Chi bn - Tng tng - Vy c - Chi ngn - Nhu m l, nhu m v thn - Chi ny t c Cn bit l tuy mang mt lng thng tin di truyn nh nhau, cc m khc nhau trn cng mt cy c th cho cc m so pht trin khc nhau vi kh nng ti sinh chi, r hay cy hon chnh rt khc nhau. V vy khi khi u chn ging, nhn ging mt cy

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26 c th trc ht cn tm hiu phn ng ca cc b phn khc nhau ca cy trong nui cy cc nng cht sinh trng khc nhau. Kh trng mu cy l vic lm kh v mu sng khng th kh bng nhit cao m phi gi c bn cht sinh hc ca n. Do mu cy thc vt phi c kh trng bng cc dung dch kh trng. Cc dung dch kh trng thng dng l hypoclorit calcium, hypoclorit sodium, chlorua thy ngn, oxi gi T l v trng thnh cng ph thuc thi gian kh trng v nng cc cht kh trng v kh nng xm nhp ca chng vo cc k lch li lm trn b mt m nui cy, kh nng y ht cc bt kh bm trn b mt m nui cy. Cc dung dch dng kh trng mu phi bo v c m thc vt nhng thi gian kh trng phi tiu dit ngun gy nhim l nm v vi khun. Cc mu cy khi chn la phi c ra trc bng x phng di dng nc chy ri mi cho vo ngm trong dung dch kh trng tng tnh linh ng v kh nng xm nhp ca cht dit khun, thng thng ngi ta x l m nui cy trong cn 70% trong 30 giy, sau mi x l trong dung dch dit khun. Trong thi gian x l, m cy phi c ngp hon ton trong dung dch dit khun. Khi x l xong m cy c ra nhiu ln trong nc ct v trng (3-5 ln). Nhng phn trn m cy b tc nhn v trng lm cho trng phi ct b trc khi t m cy ln mi trng. trnh nh hng trc tip ca tc nhn v trng ln m cy, nn ch y li mt lp bc ngoi khi ngn 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. i vi nhng mu kh kh trng th vic x l mu phi c lp li sau 24-48 h trc khi cy. iu ny cho php nhng vi sinh vt cha cht c thi gian pht trin n giai on nhy cm vi thuc kh trng. Bng 2.2. Nng v thi gian s dng 1 s cht dit khun x l m cy thc vt

Stt 1 2 3 4 5

Cht kh trng Hypochlorite calcium Hypochlorite sodium Nc bromine Oxy gi Chlorua thy

Nng 9-10% 0,5-5% 1-2% 3-12% 0,1-1%

Thi gian kh Hiu qu trng (pht) 5-30 Rt tt 5-50 2-10 5-15 2-10 Rt tt Rt tt Tt Tt

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27 ngn Nitrate bc Khng sinh

6 7

1% 4-50mg/l

5-30 30-60

Tt kh

Cc cht khng sinh trn thc t t c s dng v tc dng khng trit v c nh hng xu ngay ln s sinh trng ca m cy. Vic x l thnh cng ngun gy nhim phn ln ph thuc vo k thut x l trong nui cy v trng. Cc ngun gy nhim l bi tc, tay, qun o v vy trong khi cy phi ra tay,lau bng cn 700 ti khy tay, tay o phi xon cao ln, kp tc gn.. Khng ni chuyn hoc nhy mi trong khi ang cy. Khi cy khng nn chm tay vo mt trong ca bnh cy cng nh cc dng c cy. Khng a vo t cy cc bnh cy b nhim v bo t c th pht tn trong t cy.

2.6. Mi trng
Mt trong nhng yu t quan trng nht trong s tng trng v pht trin hnh thi ca t bo v m thc vt trong nui cy m l thnh phn mi trng nui cy. Thnh phn mi trng nui cy t bo v m thc vt thay i ty theo loi v b phn nui cy. i vi cng mt mu cy nhng ty theo mc ch th nghim th thnh phn mi trng cng s thay i ty theo giai on phn ha ca mu cy. 2.6.1. Thnh phn ho hc ca cc mi trng nui cy m, t bo thc vt Mi trng nui cy m v t bo thc vt tuy rt a dng nhng u gm mt s thnh phn c bn sau: 1- Cc mui khong a lng v vi lng 2- Cc vitamin 3- Cc amino axt 4- Ngun cc- bon: mt s cc loi ng 5- Cc cht iu ho sinh trng 6- Cc cht hu c b sung: nc da, dch chit nm men, dch chit khoai ty, bt chui kh... 7- Cht lm thay i trng thi mi trung: cc loi thch (agar) Tt c cc hp cht ny u tham gia vo mt hoc nhiu chc nng trong s sinh trng v phn ho ca thc vt nui cy in vitro.

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28 Cc nh khoa hc s dng cc mi trng nui cy rt khc nhau. Vic la chn mi trng nui cy vi thnh phn ho hc c trng ph thuc vo mt s yu t: 1- i tng cy trng hoc m nui cy khc nhau c nhu cu khc nhau v thnh phn mi trng. 2- Mc ch nghin cu hoc phng thc nui cy khc nhau (nui cy to m so phi ho hoc phi v tnh, nui cy t bo trn hoc dch lng t bo, vi nhn ging) 3- Trng thi mi trng khc nhau (c, lng, bn lng). 2.6.1.1. Cc cht khong i vi cy trng, cc cht v c ng vai tr rt quan trng. V d, Mg l mt phn ca phn t dip lc, Ca l thnh phn ca mng t bo, N l thnh phn quan trng ca amino axt, vitamin, protein v cc axt nucleic. Tng t, Fe, Zn v Mo cng l thnh phn ca mt s enzym. Cc mi trng khc nhau c hm lng v thnh phn cht khong khc nhau, v d thnh phn v nng khong ca mi trng White hoc Knop kh ngho nn, nhng li rt giu mi trng MS v B5. Mui khong l thnh phn khng th thiu trong cc mi trng nui cy m v t bo thc vt: - Mui khong l cc vt liu (ngun N, S, P....) cho s tng hp cc cht hu c. Nit, lu hunh, pht-pho l cc thnh phn khng th thiu ca cc phn t protein, cc axt nucleic v nhiu cht hu c khc. Canxi v axt boric c tm thy ch yu thnh t bo, c bit l canxi c nhim v quan trng gip n nh mng sinh hc. - ng vai tr nh mt thnh phn khng th thiu ca nhiu enzym (l cc cofactor): Magie, km, st... v nhiu nguyn t vi lng l nhng phn quan trng ca cc enzym. - Cc ion ca cc mui ho tan ng vai tr quan trng n nh p sut thm thu ca mi trng v t bo, duy tr th in ho ca thc vt. V d, K v C rt quan trng trong iu ho tnh thm lc ca t bo, duy tr in th v tham gia hot ho nhiu enzym. Trong mi trng, cc mui khong c chia thnh cc nguyn t vi lng v a lng: - Cac cht dinh dng a lng bao gm su nguyn t: nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) va sulphur (S) tn tai di dang mui khoang, la thanh phn cua cac mi trng dinh dng khac nhau. Tt ca cac nguyn t nay la rt cn thit cho sinh trng cua m va t bao thc vt. Mi trng nui cy phai
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

29 cha it nht 25 mmol/L nitrate va potassium. Tuy nhin, hu ht cac nghin cu u cho thy ngun N cung cp trong mi trng di ca 2 dang nitrate va amonium (2-20 mmol/L) la tt hn ca. Trong trng hp chi dung amonium, thi cn phai b sung thm mt acid dang mach vong (cycle acids), tricarboxylic acid hoc mt s acid khac na (dang mui), nh: citrate, succinate, hoc malate sao cho moi anh hng c do nng cua amonium vt qua 8 mmol/L trong mi trng c giam bt. Khi cac ion nitrate va amonium cung hin din trong mi trng nui cy, thi ion sau c s dung nhanh hn. Cac nguyn t chinh khac, nh: Ca, P, S va Mg, nng thng dung trong khoang 1-3 mmol/L. 2.6.1.2. Cc nguyn t a lng a. Ngun carbon (C) ng sucrose (saccharoza) l ngun cacbon ch yu v c s dng thng xuyn trong hu ht cc mi trng nui cy m, k c khi mu nui cy l cc chi xanh c kh nng quang hp. Khi kh trng, ng sucrose b thu phn mt phn, thun li hn cho cy hp th. Trong mt s trng hp, v d nui cy m cy mt l mm, ng glucose t ra tt hn so vi sucrose. M thc vt c kh nng hp thu mt s ng khc nh maltose, galatose, lactose, mannose, thm ch tinh bt, nhng cc loi ng ny hu nh rt t c s dng trong nui cy t bo v m thc vt. M va t bao thc vt nui cy in vitro sng chu yu theo phng thc di dng, mc du nhiu trng hp chung co th sng ban di dng nh iu kin anh sang nhn tao va luc lap co kha nng quang hp. Vi vy, vic a vao mi trng nui cy ngun carbon hu c la iu bt buc. Ngun carbon thng dung nht a c kim chng la sucrose, nng thich hp ph bin la 2-3%, song cung con phu thuc vao muc ich nui cy ma thay i co khi gim xung ti 0,2% (chon dong t bo) va tng ln n 12% (cam ng stress nc). Tip n la glucose cung thng c a vao mi trng nui cy va cho hiu qua tng ng sucrose (glucose thng dung cho nui cy protoplast), con fructose cho hiu qua kem hn. Sucrose, trong khi kh trung mi trng, bi bin i thanh glucose va fructose. Trong tin trinh nay, u tin glucose se c s dung va sau o la fructose. Cac carbohydrate khac, nh: lactose, galactose, rafinose, maltose, cellobiose, melibiose va trehalose cung a c thi nghim, nhng to ra kem hiu qua va chi c dung trong nhng trng hp c bit. Cac dang polysaccharide nh tinh bt, pectine, dextrine cung co th dung cho nui cy, tuy nhin nhng loai t bao c nui trn mi trng co cha mt trong cac polysaccharide trn nht inh phai th hin kha nng thuy phn thng qua cac enzyme

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30 chng han nh amylase. Co nhng chung t bao nui cy giai phong ra mi trng cha tinh bt kha nhiu amylase. Chuyn chung ln mi trng chi cha sucrose lng amylase thai ra se giam ngay. Glycerin cung co th c t bao s dung. Mannitol hoc sorbitol hoan toan trung tinh vi khng thm nhp vao bn trong t bao, nhng chung c s dung rng rai trong nui cy huyn phu va nui cy protoplast vi chc nng la cht n inh ap sut thm thu, hoc tng t sucrose chung cung c dung cam ng stress nc. Cac loai ru nh ethanol, methanol it hiu qua, con propanol va butanol thi rt c. Acid hu c thng khng phai la ngun carbon thich hp cho t bao nui cy. Thi nghim vi cac acid: folic, succinic, pyruvic va keto-glutaric chi at 15% sinh trng so vi sucrose. Cac m va t bao thc vt trong mi trng nui cy t c kha nng t dng va vi th cn thit phai b sung ngun carbon bn ngoai cung cp nng lng. Thm chi cac m bt u luc hoa hoc hinh thanh dip luc t di cac iu kin c bit trong sut qua trinh nui cy a khng t dng carbon. Vic b sung ngun carbon bn ngoai vao mi trng lam tng phn chia t bao va tai sinh cac chi xanh. S thuy phn tng phn sucrose xut hin khi mi trng c kh trung. Cac t bao va m nui cy a sinh trng trn mi trng co sucrose c kh trung bng autoclave tt hn trn mi trng co sucrose c kh trung bng mng lc (filter). iu nay cho thy cac t bao thich hp vi ngun d tr co sn cua glucose va fructose do thuy phn sucrose khi kh trung bng autoclave. S dung fructose c kh trng bng autoclave khng c cp n vi no co th gy bt li cho sinh trng cua m.

b. Nit (N): Thnh phn chnh ca hu ht cc mi trng l nit v c di dng nitrat (NO3-) hoc amonium (NH4+). Cc mui c dng ph bin l kali nitrat (KNO 3), nitrat amon (NH4NO3) v canxi nitrat (Ca(NO3)2.4H2O). Nhng hp cht ny cung cp nit v c cho thc vt tng hp cc phn t cht hu c phc tp. M , t bo thc vt trong nui cy c th s dng nitrogen khong nh aminonium v nitrate, ng thi cng s dng cc dng nitrogen hu c nh amino acid. T l amonium v nitrate thay i ty theo loi v trng thi pht trin ca m. Nitrate c cung cp di dng mui Ca(NO 3)2.4H2O, KNO3, NaNO3 hoc NH4NO3. Amonium c cung cp di dng (NH 4)2SO4 hoc NH4NO3. Trong mt s t trng hp c th cung cp di dng urea. Tng nng ca NO 3+ v NH4+ trong mi trng nui cy thay i ty theo i tng nui cy v mc ch nghin cu.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

31 Amonium ch yu c d tr r nh ngun nit hu c. Nitrat c th c vn chuyn theo mch xylem n cc b phn ca cy, ti n s tham gia vo qu trnh ng ho nit. Nitrat c th c d tr khng bo v thc hin chc nng quan trng trong vic iu chnh s thm thu v cn bng ion ca cy trng. S bin i nitrat: Nitrat khng th s dng ngay lp tc sinh tng hp cc cht hu c phc tp m trc tin cn phi c kh thnh amoniac. Phn ng din ra nh sau: NO3- + 8H+ + 8e- NH3 + 2 H2O + OHPhn ng ny c thc hin qua 2 bc nh hai enzym: nitrat- v nitrit reductaza. Trc tin, nitrat c bin i thnh nitrit nh nitrat reductaza. Tip theo, nitrit b kh thnh amoniac nh enzym nitrit reductaza. S bin i ca nitrat thnh nitrit din ra trong t bo cht. Trong hu ht cc cy trng, s kh nitrat c th din ra c l v nh chi. S kh nitrat din ra mc no ph thuc rt ln vo cc nhn t nh: loi, tui cy, nng nitrat. C th, cc loi cy thn g c kh nng kh nitrat rt cao. Khi nng nitrat thp, s kh hu nh din ra r. Ngc li, nu nng nitrat cao th qu trnh ny din ra c l. Cc cation kt hp vi nitrat c vai tr quan trng trong vic hp thu nitrat. Nu cation l K+ th hot ng ca enzym nitrat reductaza r thp v nitrat s c vn chuyn ln cc nh chi ca cy. Trong trng hp cation l Ca 2+ th s kh r li din ra mnh hn. S kh nitrit thnh NH3 nh enzym nitrit reductaza din ra trong l cy, trong in t cn thit cho phn ng ny c cung cp t s kh cc hp cht st trong h thng quang hp. S kh nit cha trong cc hp cht: Amonium v amoniac l nhng cht c i vi thc vt ngay nng thp. Do chng cn c chuyn ho tht nhanh thnh cc hp cht phn t lng nh c cha N nh: asparagin, arginin, allantoin v betain. Sinh tng hp glutamin v glutamat din ra c r v nh chi l cc qu trnh c bn trong s chuyn ho amonium. Bn cnh s kh c tnh ca amonium v amoniac, cc hp cht cha nit phn t lng thp cn c mt s chc nng khc. Chc nng quan trng nht l cung cp N trong cc lin kt hu c v -NH 2 c thc vt hp th nh ngun N hu c cho qu trnh sinh tng hp cc amino axt v protein. Cc hp cht phn t lng nh ny cn c s dng lm cht mang cation nh Mn, Cu vn chuyn cc cation qua h thng mch dn trong cy. Ngoi ra, chng cn nh mt kho d tr nit d tha. Ngc li vi con ngi v ng vt, thc vt khng th bi tit cc hp cht nit hu c nh ur nhng nh c ch ny cho php thc vt d tr c nit hu c.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

32 c. Phospho (P): Photpho l nguyn t quan trng trong i sng thc vt . N tham gia vo vic vn chuyn nng lng, sinh tng hp protein, acid nuclic v tham gia cu trc ca mng. Trong mi trng nui cy, Photpho c cung cp di dng mono hay dihydrogenphosphate potasium hay sodium. Ion photphate ha tr 1 v 2 c th chuyn i ln nhau ty theo pH. Ion H 2PO4- chim u th pH nh hn 7, y l c tnh ca hu ht mi trng nui cy m t bo thc vt cng l ion d c thc vt hp th nht. Photpho thng c cung cp di dng photphate ha tan hn ch. Nng photphate ha tan cao trong mi trng s lm gim s tng trng ca m, c th do calcium v mt s nguyn t vi lng b kt ta trong mi trng hoc b gim hp thu vo trong m. Nng ion photphate cho vo mi trng cao nht l 18,9 mM, trung bnh l 1,7 mM, hu ht cc mi trng cha photphate khong 1.3 mM. Phospho dng HPO42- c hp th nh h thng r ca thc vt v ngc li vi nitrat, sunfat, n khng b kh. N c th c mt trong thc vt di dng P v c hoc dng hp cht este (R-O-P). Nng lng thu c khi gii phng mt nguyn t P khi cc lin kt (cao nng lng) l rt quan trng i vi qu trnh trao i cht ca t bo. Axit nucleic : P l mt nguyn t thit yu trong cu to ca DNA v RNA ni cc n phn t axt ribonucleic to thnh i phn t. Phospholipid: Phospholipid ca mng sinh hc cng c cha mt lng ln P. Trong nhng phospholipid ny, P (qua lin kt este) to nn cu ni gia diglyxerit vi mt amin, axit amin hoc mt ru. Phospholipid c mt u ho nc, phn t diglyxerit v mt u k nc c cha PO 43-. C hai u c chc nng quan trng trong vic gi n nh mng t bo. Mng t bo bao gm hai lp phospholipid n ghp li to thnh lp mng kp lipid. u a nc ca lp phospholipid quay ra ngoi hng v pha cc phn t nc trong khi ui k nc li quay vo pha trong gia hai lp ca mng t bo v tng tc ln nhau. Qu trnh chuyn ho nng lng: Phospho dng lin kt este cao nng (C-P) rt quan trng i vi qu trnh chuyn ho nng lng v tng hp sinh hc thc vt. ng vai tr quan trng hn na l cc lin kt cao nng gia hai nguyn t P nh trong phn t ATP (P-P = 30 kJ). Nng lng gii phng trong sut qu trnh thu phn glucoza, qu trnh oxi ho, phosphoryl ho hoc quang hp c s dng tng hp ATP. Ngc li nng lng ny khi cn li c gii phng ra qua phn ng thu phn ATP thnh ADP v P v c. V vy ATP c chuyn ho v tng hp mi lin tc. Mt gram nh r ang trong giai on trao i cht mnh c th tng hp 5g ATP/ ngy vi tc tng hp trung bnh l 30 giy.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

33 Vng d tr P (phosphat): Trong t bo bao gm hai vng d tr phosphat khc nhau. Vng trao i, ch yu phosphat dng este, nm trong t bo cht v ty th. Vng khng trao i, ch yu l dng P v c, nm trong khng bo. Nu ngng cung cp P cho cy, nng P v c trong khng bo ngay lp tc s gim trong khi vng trao i tc gim chm hn nhiu. Khi tng cng cung cp P, nng P cha trong cc c quan ca t bo cng tng theo, tuy nhin khi tng qu mc bnh thng th ch c P v c trong khng bo tng ln. V vy c th ni, P d tha c d tr khng bo di dng P v c. Cc enzym: P v c cng c kh nng iu chnh tt trong nhiu qu trnh trao i cht ca thc vt. Vy nn s phn b P l cn thit iu chnh qu trnh trao i cht ca t bo. c chua, s gim P v c khng bo trong t bo cht kch thch hot tnh ca enzym phosphofructokinaza. Enzym ny l enzym quan trng trong s phn gii c cht ca phn ng thu phn glyco v lm tng s h hp ca t bo trong qu trnh chn. Cng trong thi gian ny, s thiu ht P c th lm chm qu trnh chn ca qu. Trong qu trnh tng hp tinh bt ca lc lp, P cng ng mt vai tr quan trng. Ch vi nng thp, P v c c th gy c ch qu trnh tng hp tinh bt. S d nh vy l do ADP-gluco-pyrophosphorylaza, enzym quan trng nht trong qu trnh tng hp tinh bt, b km hm bi P v c v c kch thch nh cc triossephosphat. V th, s cn bng gia cc hp cht c cha P l rt quan trng trong iu ho tng hp tinh bt lc lp. Ngoi ra, P v c cng tham gia qu trnh ny theo mt con ng khc. Cc phn t vn chuyn phosphat mng t bo s mang P v c vo t bo v cc triosephosphat ra ngoi t bo, lm nng P v c trong lc lp tng, triosephosphat gim. iu ny li tc ng n qu trnh tng hp tinh bt theo c ch trnh by trn. Ribuloza biphosphat (RuBP), vi vai tr nh mt cht nhn CO 2, l hp cht quan trng trong s c nh CO 2. S ti tng hp ny i hi phi c cc triosephosphat. Khi nng P v c cao s kch thch gii phng cc triosephossphat ra khi lc lp, gy thiu ht cc cht ny, do lm km hm qu trnh c nh CO 2. Ngoi ra, phospho cn quan trng trong vic iu khin hot ng ca nhiu enzym khc. Nng phospho ti u cho s sinh trng ca thc vt l 0,3 0,5 g/ kg trng lng kh. S thiu P lm cy chm ln, l cy c mu xanh thm do trong thi gian b thiu P, s pht trin ca l chm hn s tng hp dip lc t nn lm tng nng dip lc t trong l cy. d. Lu hunh (S): Lu hunh nh SO42- c hp th r cy vi tc chm. Ging nh nitrat, lu hunh phi c kh trc khi s dng sinh tng hp cc hp cht c cha lu hunh

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

34 nh amino axt, protein v enzym. Lu hunh dng cha kh c kt hp trong cc sulpholipid v cc polysaccharid. S ng ho lu hunh: Bc u tin trong qu trnh ng ho lu hunh l s hot ho gc SO42- nh enzym ATP sulfurylaza. Phn ng ny to ra adenosine phosphosulfate (APS) v P v c. Tip theo l hai qu trnh ho hc hon ton khc nhau. Mt qu trnh khng din ra s kh lu hunh m to lin kt vi cc polysaccharide trong sulpholipid. Trong qu trnh th hai, lu hunh c kh thnh nhm SH (nhm thiol) v nhm sulfuryl ca APS c vn chuyn ti gluthantione (Glut-SH). Sau nhm SH c vn chuyn ti cho acetylserine v phn tch thnh acetat v cystein. Cystein l sn phm bn u tin trong qu trnh ng ho v l tin cht ca tt c cc hp cht hu c c cha lu hunh trong thc vt, v d nh: protein, co-enzym, cc hp cht trao i cht th cp... Qu trnh ng ho lu hunh ch yu din ra lc lp. Khi thiu lu hunh, sinh tng hp protein b km hm, lng dip lc t trong l cy b gim st. Cc protein: Lu hunh c mt trong cc protein c cha cystein v methionin. C hai axt amin ny u l tin cht ca tt c cc hp cht c cha lu hunh trong thc vt. Lu hunh, cu t hp thnh ca nhiu coenzym v cc nhm prosthetic, c chc nng quan trng trong rt nhiu phn ng oxy ho kh, c biu din nh sau: R-SH + HS-R R-S-S-R. Gc R c th l phn cn li ca phn t cystein nhng cng c th l tripeptit gluthatione. Gluthatione tan c trong nc v do , n ng vai tr nh mt h oxi ho kh lc lp v dch bo. Cu lu hunh gia hai phn t cystein rt quan trng trong cu trc bc ba ca phn t protein v hot ng ca enzym. Nhm SH, nh cp trn c trong thnh phn ca cc coenzym v APS, to thnh mt phn ca nhm chc nng trong phn t enzym. Cc metallothionein: Cc hp cht phn t lng thp c cha lu hunhmetallothionein, thng hay c tm thy trong thc vt. Hu ht cc cht ny u c cha cystein. c bit, cc kim loi nh: ng, cadimi v km thng lin kt trong metallothionein. Gn nh chc chn nhng phn t protein nh ny tham gia vo s bi tit cc ion kim loi trn khi chng d tha, trc khi lin kt vi nhm chc nng SH ca cc enzym. Lu hunh cha b kh: Lu hunh dng cha b kh l thnh phn cu to sulpholipid, l phn t to thnh cu trc ca mng sinh hc. Lu hunh thng c mt dng hp cht este ca lu hunh v ng 6 cacbon, v d nh glucoza. Sulpholipid c

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

35 nhiu trn mng thylakoid ca lc lp v tham gia vo qu trnh vn chuyn ion qua mng sinh hc. Hn na, s c mt ca sulpholipid trn mng t bo chc chn lin quan n kh nng chu mui ca thc vt. Mi v c trng ca mt s loi nh: hnh, ti ch yu c lin quan n s c mt ca cc hp cht c cha lu hunh d bin i. e. Kali (K): K+ l mt cation ch yu trong cy, gip cho cy cn bng cc anion v c v hu c. Ion K+ c chuyn qua mng t bo d dng v c vai tr chnh l iu ha pH v p sut thm thu ca mi trng ni bo. S thiu ht K+ trong mi trng nui cy m thc vt s dn n tnh trng thiu nc. K+ c cung cp di dng mui KNO3, KCl. 6H2O, KH2PO4 Trong thc vt, K+ l mt cation c tnh linh ng cao, c mc t bo cng nh trong qu trnh vn chuyn qua cc khong cch di trong mch xylem hoc mch libe. Trong tt c cc nguyn t, kali l nguyn t c mt vi nng cao nht, t bo cht t 100 200 mM, lc lp t 20 200 mM. Mui kali c vai tr quan trng trong vic iu chnh tnh thm ca t bo. i vi s gin t bo cng nh cc qu trnh khc c iu chnh nh sc trng ca t bo, K + c vai tr nh mt ion trung ho cc ion v c v hu c ho tan trong dung dch, ng thi duy tr pH trong khong 7 8 l pH thch hp cho hot ng ca hu ht cc enzym. Vai tr ca K+ i vi cc enzym: K+ cn thit cho hot ng ca nhiu enzym. Trn 50 enzym ca thc vt hot ng vi s tham gia ca K + hoc b kch thch bi K+. S lin kt ca K+ vi enzym gy ra s thay i cu hnh khng gian ca enzym, do lm tng i lc ca enzym vi c cht. Khi thiu K + ngi ta thy c s tng nng ng ho tan v cc hp cht cha nit, km theo s gim nng tinh bt, ch yu do vai tr thit yu ca kali i vi vic iu khin hot ng enzym trong qu trnh trao i cacbon. Enzym ATPaza mng t bo cng chu s tc ng ca K +. cy trng thnh, K+ cn cho qu trnh tng hp protein. K+ cn cn thit trong qu trnh dch m v tng hp tRNA ribosome. S tng hp ribuloza biphosphat cacboxylaza cng ph thuc rt mnh vo nng K+. y cn l ion cn thit cho hot ng v tng hp enzym nitrat reductaza. Bn cnh vai tr trong hot ng ca nhiu enzym, K + cn iu chnh s cn bng ion v pH ca lc lp trong quang hp. K+ l ion trung ho quan trng nht cho s a dng H+ qua mng thylakoid. Ion ny cng c mt trong s to thnh gradien pH mng t bo cn thit cho qu trnh tng hp ATP. S tng nng K+ dn n s tng cng qu trnh quang hp, h hp v hot ng ca enzym ribuloza biphosphat cacboxylaza.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

36 S gin ca t bo: S tng kch thc ca khng bo trung tm trong t bo l mt qu trnh quan trng trong s gin t bo. hnh thnh khng bo, u tin l s tng kch thc ln ca thnh t bo, tip theo kh nng thm thu ca khng bo tng ln. iu ny c th t c nh s tch t K + gy ra s tng mnh v th tch ca khng bo do tnh thm. GA3 v K+ dng nh c tc dng h tr nhau trong vai tr lm tng chiu cao cy. S cn bng ion: K+ c vai tr quan trng cho vic duy tr cn bng ion. N trung ho cc anion km linh ng trong t bo cht v rt nhiu cc anion linh ng trong mch xylem, libe v khng bo. Trong qu trnh trao i nitrat, K + c chc nng ch yu l vn chuyn ion NO3- qua nhng khong cch di trong mch xylem hoc d tr trong khng bo. Sau qu trnh kh nitrat trong l cy, lng K + cn li c s dng tng hp cc axit hu c trung ho ion K+. Cc mui kali ca cc axit hu c nh kali malat c vn chuyn ti r, sau K+ c th nhn ion nitrat t bo r v vn chuyn chng qua mch xylem. f. Canxi (Ca): Calcium cng l mt cation ch yu gip cn bng cc anion trong cy nhng cch thc khng ging nh K+ v Mg+ v Ca2+ khng phi l ion linh ng. Calciun c th lin kt cc phn t sinh hc li vi nhau do n gp phn vo trong cu trc v hot ng sinh l ca mng t bo v phin gia ca thnh t bo . S hot ng ca nhiu enzim khc ca thc vt cng ph thuc vo Ca2+ v calcium l ng yu t vi nhng enzim phn gii ATP. Trong nui cy t bo, Ca2+ c vai tr trong s pht sinh hnh thi ng thi vi s cm ng ca cc cht iu ha sinh trng c bit l auxin v cytokinin. Ca2+ l thnh phn quan trng ca thnh t bo v mng t bo. S lng ln Ca 2+ gn trn thnh t bo ng vai tr ch yu trong cng c vng chc cho thnh t bo v iu ho cu trc mng t bo. Ion Ca2+ t do c mt trong t bo nng rt thp, khong 1M c tc dng ngn chn s kt ta P v c. Do hm lng ion Ca2+ trong t bo thp nn khng c s cnh tranh vi ion Mg2+ v v tr gn cation v trnh lm bt hot enzym. Ion Ca 2+ ch c th di chuyn qua mng t bo theo mt chiu (Ca2+ ch ra ngoi t bo c nhng khng vo c), do m bo c nng ion Ca 2+ ni bo thp. c bit trong cc t bo l, c mt lng ln canxi lin kt vi cc khng bo. Canxi cn thit cho s thit lp cn bng ion nh trung ho cc anion hu c v v c. Hu nh canxi dng lin kt to mui ocxalat. Mc d hp cht ny kh tan nhng n c vai tr duy tr nng ion

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

37 Ca2+ thp trong lc lp v t bo cht. Mui canxiocxalat cn c chc nng iu chnh s thm thu ca t bo. Canxi c vai tr quan trng trong qu trnh nhn ln ca t bo v r. Ngoi ra s pht trin ca ng phn cng ph thuc vo canxi, y l qu trnh c nh hng nh canxi ngoi bo. IAA tham gia vo qu trnh vn chuyn canxi. Cht c ch auxin nh TIBA cng c ch s phn phi Ca2+ trong thc vt lm xut hin s thiu ht canxi. Vai tr ca Ca i vi thnh t bo: Pectin l thnh phn quan trng ca mng lin kt gia cc b bo vi nhau v c phn hu nh enzym polygalacturonase. Tuy nhin, Ca c ch mnh hot ng ca polygalacturonaza. Hot ng mnh ca enzym ny c ghi nhn khi thiu Ca. Nu nng Ca c th hu ht cc pectin s tn ti di dng mui canxipectat. Nh vy, thnh t bo c kh nng chng chu tt i vi hot ng ph hu ca enzym polygalacturonaza. S c mt ca ion Ca 2+ cng c vai tr quan trng trong vic ngn chn s xm nhim nm. Ion Ca2+ c tc ng ln n s n nh ca mng t bo. S thiu ion Ca 2+ s lm tng kh nng thot ra ngoi mng t bo ca cc hp cht phn t lng nh. Mng t bo c th s b phn hu hon ton khi thiu ht nghim trng ion Ca 2+. Ion Ca2+ c kh nng lm n nh mng t bo thng qua s tng tc vi cc nhm phosphat, cacboxyl ca hp cht phospholipid v protein c mt trong mng t bo. Cc enzym: Khc vi magie l nguyn t tham gia vo qu trnh hot ho ca rt nhiu enzym, canxi ch c tc ng ln mt vi enzym nh: amilaza v ATPaza. Ca ch yu kch thch cc enzym mng t bo, m hot ng ca nhng enzyme ny c qui nh nh cu trc mng. Tuy nhin, ion Ca 2+ cng c tc dng km hm mt s enzym ca t bo cht. Calmodulin trong t bo c kh nng hot ho cc enzym nh phospholipaza bng cch to thnh phc ca Ca2+ - calmodulin vi enzym. Ngoi ra, ngi ta cn cho rng calmodulin c vai tr trong vic vn chuyn ion Ca 2+ ti khng bo. g. Magi (Mg): Magnesium l nguyn t cn thit cho s sinh tng hp dip lc t v ng thi n cng tham gia vo cu trc ca mt s enzim vn chuyn photphate. Ion Mg + l mt ion linh ng, c th khuych tn vo trong t bo nh K + v vy c vai tr nh mt cation c th trung ha cc cation v cc acid hu c. Mi trng nui cy m thc vt thng cha Mg vi nng khng thay i nhiu trung bnh l 6,8mM. MgSO 4 l ngun b sung ion Mg+ duy nht cho m cy.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

38 Mg2+ l mt ion rt linh ng c kh nng hnh thnh phc vi cc nhm chc nng khc nhau. Vai tr Mg2+ ca i vi quang hp: Mg2+ l nguyn t trung tm trong phn t chlorophyl ca h quang hp I v II. Trong phn t chlorophyl, cc photon c hp th to ra dng in t, t to ra ATP v NADPH ng vai tr quan trng i vi c nh CO2. Nu Mg2+ c mt vi nng ti u th khong 10 - 20% ion Mg 2+ trong l c c nh lc lp. Nng cao cc ion Mg2+ v K+ l cn thit duy tr pH khong 6,5 7,5 trong lc lp v t bo cht, tri vi khng bo pH ch vo khong 5- 6. Trong mt chng mc no , pH xc nh cu trc ca protein v enzym nn n nh hng n qu trnh sinh tng hp protein v chc nng ca lc lp. Vai tr Mg2+ i vi hot tnh ca cc enzym: Mg2+ l ion cn thit cho cu trc bc ba ca nhiu phc enzym-c cht v n to ra dng cu trc khng gian ph hp gia enzym v c cht. Mg2+ tham gia vo qu trnh tng hp protein vi nhiu cp khc nhau. Mg2+ to thnh cu ni gia cc di n v ca ribosome. Khi thiu Mg2+, cc di n v s b tch ra v qu trnh tng hp protein b ngng li. S hot ng ca cc enzym nh: RNA polymeraza tham gia vo qu trnh sinh tng hp RNA i hi phi c mt Mg2+, do thiu Mg2+ s km hm sinh tng hp RNA. l cy, 25% protein tng s nm trong lc lp, nu thiu Mg 2+ th ngay lp tc cu trc v chc nng ca lc lp b nh hng. Mg2+ cn quan trng trong hot ng ca enzym ribulose biphosphat cacboxylaza. y l mt enzym ph thuc nhiu vo pH v Mg 2+. Lin kt ca Mg2+ vi enzym lm tng i lc vi c cht CO2 v Vmax. Vai tr Mg2+ i vi chuyn ho nng lng: Mg l mt cht khng th thiu trong qu trnh chuyn ho nng lng ca thc vt do vai tr quan trng ca n i vi sinh tng hp ATP (ADP + P v c = ATP), c bit l lc lp. Trong qu trnh ny, Mg2+ to thnh cu ni gia enzym v ADP. Ngoi ra, Mg 2+ cn c kh nng to phc vi ATP. Enzym ATPaza vn chuyn cc nhm phosphoryl cao nng, cung cp cho protein hoc ng. Mc d Mg2+ c nhiu chc nng nh vy nhng hu nh n li tn ti dng d tr trong khng bo. Ti y, n ng vai tr nh mt ion trung ho vi cc anion hu c v v c trong vic cn bng ion. 2.6.1.3. Cc nguyn t vi lng (Fe, B, Cl, Co, Cu, Mn, Mo, Zn...) Cac nguyn t v c cn mt lng nho nhng khng th thiu cho sinh trng cua m va t bao thc vt c goi la cac nguyn t vi lng. o la cac ion: iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu), va molybdenum (Mo). Fe dng nh thich hp hn khi c cung cp di dang chelate Fe, va Zn c dung binh
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

39 thng trong cac mi trng nui cy. Cac dang mui tatrate va citrate Fe kho hoa tan va thng hay kt tua trong mi trng. Vn nay co th khc phuc bng cach dung diaminetetraacetic acid (EDTA)-chelate Fe thay cho citrate Fe, c bit i vi qua trinh tao phi. Tuy nhin, cac dang chelate EDTA khng hoan toan n inh trong mi trng nui cy dang long. Mt s mi trng nui cy c lam giau bng cobalt (Co), iodine (I) va sodium (Na), nhng cac yu cu nghim ngt v cac nguyn t nay cho sinh trng cua t bao a khng c thit lp. Noi chung, nng thng c s dung i vi Cu va Co la 0,1 mol/L, Fe va Mo la 1 mol/L, I la 5 mol/L, Zn la 5-30 mol/L, Mn la 20-90 mol/L va B la 2-5100 mol/L c b sung vao mi trng nui cy tuy thuc vao yu cu cua tng thi nghim. Vic chia thnh cc nguyn t vi lng v a lng ch yu da trn nhu cu ca thc vt i vi cc cht ny. Nhu cu ca thc vt i vi cc nguyn t a lng l ln hn, vi nng > 0.5 mM. Cc nguyn t vi lng c s dng trong mi trng nng < 0.5 mM. Nhu cu ca cy i vi nguyn t vi lng l rt thp. Do vy nhng nguyn t ny cng c mt trong mi trng cc nng tng ng. Hu ht cc nguyn t vi lng s dng lng nmol. Mt s nguyn t vi lng c nhu cu nh hn c th thay th d dng bng s ln tp ngu nhin ca chng trong cc thnh phn ca mi trng nh agar, cc cht b sung nh nc da, dch chit nm men (yeast extract), cc mui v nc. Tm quan trng ca mt s nguyn t vi lng trong thnh phn mi trng cn cha c hiu mt cch r rng. Co, Al, Ni... c th c li i vi thc vt nhng cng c th l khng cn thit. Trong thc t, hu ht cc nguyn t vi lng ch c phn khong ca mui (cation) l quan trng, cn vai tr cc anion c th l khng cn thit. Ion SO42- d tha trong mi trng v ch yu pht sinh t cc mui MgSO4, K2SO4 Nhu cu ca thc vt i vi cc nguyn t a lng ln hn. Nguyn t a lng c nng cao nht trong cc mi trng nui cy m v t bo thc vt. Nhn chung c phn anion v cation ca cc nguyn t a lng u quan trng i vi t bo thc vt. V d nh KNO3, c K+ v NO3- l cn thit. Trong nhm cc nguyn t a lng, cc mui c cha nit ch yu dng kali nitrat, amonium hoc calxi nitrat. a. St (Fe): Trong cy, st ch yu c gn vi cc phc cht. Hm lng Fe 2+, Fe3+ t do rt thp (10-10 mM). Hu ht thc vt ch hp thu Fe2+. Do , Fe3+ cn c kh thnh Fe2+

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40 b mt r trc khi n c chuyn vo trong t bo cht (ch mt s loi c l hp thu st ch yu di dng Fe3+). Trong khi vn chuyn i xa, qua mch xylem ca cy, st ch yu c di chuyn di dng hp cht st-cacbonhydrate ( dng Fe 3+-citrate hay dng phc hp stpeptide). Chc nng chnh ca st trong thc vt l to cc lin kt st. Cc chc nng c bn nh mt h thng oxi ho kh thun nghch c biu din trong phn ng di y: Fe(II) <---> Fe(III) + eCc hemoprotein (cc protein cha st: Cc hemoprotein c bit n nhiu nht l cc cytochrome, c cha mt phc h st-porphyrin. Cc cytocrome to thnh mt phn h thng oxi ho trong chui truyn in t lc lp v ty th ca t bo thc vt. Chc nng ca cc cytocrome nh cht trung gian cho in t, cn cho qu trnh kh nitrat thnh nitrit nh enzym nitrat reductase trong qu trnh ng ho nit. Trong qu trnh c nh nit cy h u, cc cytocrome l trung gian trong chui truyn in t qua cc in t c truyn i cui cng kh N2 thnh NH3. Cc catalase v peroxidase tham gia vo qu trnh quang h hp, thu phn ng v kh c ca hydrrogen peroxid, theo cn bng sau: 2H2O2 ---> catalase ---> 2H2O + O2 Hydrogen peroxit c to ra trong qu trnh kh superoxit nhm trung ho cc gc superoxit. Hydrogen peroxit, n lt n li c trung ho nh enzym catalase. Cc peroxidase c rt nhiu trong t bo thc vt. Thnh t bo gn peroxidase xc tc cho qu trnh trng hp ca phenol vi lignin. R cha lng ln peroxit v c vai tr hp thu st ca cy. Lng phenol d tha khi thiu st s c tit ra ngoi. Cc protein st-lu hunh: Nhm protein cha st th hai l cc protein st- lu hunh. St c gn trong nhm thiol (-SH) ca cystein v lu hunh v c. Ferridoxin l protein cha st lu hunh ph bin nht v l cht mang trong cc phn ng truyn in t c xc tc bi nitrit reductaza, sulphat reductaza, qu trnh tng hp NADP + trong quang hp v kh nit c thc hin nh phc h nitrogenaza. Ba loi protein st lu hunh khc nhau, hot ng lin tip nhau, u nm trong chui truyn in t ca phc h nitrogenaza. Bn cnh hai nhm ny, thc vt cn c nhng enzym khc c cha st. Nguyn t ny cn cho cc phn ng oxi ho kh v s nh v ca cc phc hp enzym - c cht.

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41 St rt quan trng trong sinh tng hp chlorophyl: Trong l xanh 80% st nm trong lc lp. Khi thiu st, ton b st s tp trung l. Trong l non, thiu st s dn n s gim nhanh nng chlorophyl do qu trnh tng hp protein b ngng li. S lng ribosom cng gim mnh. Thiu st r ko theo nhng thay i hnh thi. S di r gim nhng din tch v s lng lng r tng. b. Bo (B): Vai tr ca nguyn t B trong sinh ha hc v sinh l hc thc vt cha c bit nhiu. B cn thit cho s hot ng ca nh sinh trng bi v n c mt trong s sinh tng hpcc base nit c bit l uracil, cng nh cn thit cho s sinh tng hp lignin v acid phenolic. Ngi ta c th s dng nhiu nng B khc nhau trong mi trng nui cy t 50-100 m. Thiu B s lm gim s sinh tng hp cytokinin. S phn chia t bo b km hm do c s gim sinh tng hp ARN trong nhn. Bo c hp thu bi r v c chuyn ti cc b phn ca cy nh cc xylem. cc mng t bo, Bo c mt ch yu dng lin kt este. Khng c bt c mt enzym bit no c cha hoc c hot ho bi Bo. C nhng ch dn cho bit Bo c mt trn hoc bn trong cc mng, c th nh hng ti hot ng ca cc enzym lin kt mng. Cc chc nng ch yu ca Bo l ngoi bo, c pht sinh trong qu trnh ho g ca thnh t bo v s phn ho xylem. Thnh t bo: Nhng lin kt este ng- Bo l mt phn cu trc ca cc hemicellulose trong thnh t bo. Hu ht Bo trong thc vt tn ti dng este trong thnh t bo ca cy. Yu cu v Bo i vi cy hai l mm cao hn so vi cy mt l mm. C th gi nh rng B, cng ging nh calci, c chc nng iu ho qu trnh tng hp thnh t bo cng nh n nh cc thnh phn ca thnh v mng t bo. S thiu ht Bo ngay lp tc c ch qu trnh pht trin chiu di ca cc r s cp v th cp. Hn th, Bo gp phn iu ho qu trnh trao i phenol v tng hp lignin. c. ng (Cu): ng hin din trong h thng enzim cytochrome oxidase ca chui vn chuyn in t h hp. Trong thc vt, ng tn ti di dng ion ha tr 1 v 2. Nng ng cao s gy c cho m. Hu ht cc mi trng nui cy c Cu 2+ vi hm lng 0,1-10

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42 m. Cc ion ng c b sung vo di dng sulfate ng, i khi ngi ta cng c th b sung ng di dng CuCl 2 hoc CuNO3. Thc vt hp thu Mo di dng MoO42-, thng c b sung vo mi trng nui cy vi nng n 1 m. ng l mt cation ho tr 2 v c hp thu vo cy di dng Cu 2+ hay di dng phc cht ca n. Nu nng Cu2+ v phc ng tng ng nhau, cy dng nh a ion ng t do hn. Trong xylem v phloem, ng hu nh tn ti di dng phc, v ch yu l dng mt phc amino axit- ng. Trong t bo, ng ch yu l thnh phn ca phc h enzym v rt quan trng trong cc phn ng oxi ho kh [(Cu 2+)/(Cu+)] c thc hin nh nhng enzym ny. Thiu ng lp tc dn n s gim hot ca cc enzym cha ng. Vai tr ca ng i vi quang hp: Khong 50% ng trong lc lp c gn vi plastocyanin, gia chui truyn in t, gia quang h I v quang h II, c cha 1 nguyn t ng trn 1 phn t. Trong trng hp thiu ng, nng cc plastocyanin s b gim. Cng ging nh plastocyanin, cc plastoquinone ng vai tr quan trng trong truyn in t gia quang h I v quang h II. Khi ng b thiu, mng lc lp s thiu 2 protein iu ho chuyn ng ca cc plastoquinone. tng hp cc plastoquinone cn phi c enzym laccase, y l mt enzym cha ng v hot ng ca n s b gim ngay khi thiu ng. Do , hin tng thiu ng nhanh chng ko theo hin tng gim quang hp. Enzym super oxide dismutase: Cu cng km l mt phn ca enzym super oxide dismutaza (Cu-Zn.SOD), ng vai tr quan trng trong qu trnh trung ho gc anion superoxide O2- hot tnh mnh c to thnh trong qu trnh quang h hp. Bn cnh Cu-Zn.SOD, mt SOD cha mangan cng c trong t bo. SOD ho gii c ca gc O2- hot ng thnh H2O2 v O2, nh bo v t bo trc kh nng ph hu ca gc ny. SOD cng vi catalase phn ng nh sau: O2 + e- ---> O2- (superoxide) O2- + O2- + 2H+ ---> SOD ---> H2O2 2H2O2 ---> catalase ---> 2H2O + O2 Superoxide c gii c nh SOD v ngay sau gii phng H2O2 thnh oxi v nc nh catalase. Cc enzym SOD cha ng - km ch yu c tm thy cht nn stroma ca lc lp. Trong l non, 90% SOD tp trung lc lp v ch c 4-5 % ti th. Thiu ng

Cng ngh nui cy m t bo thc vt

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43 s xy ra nhng thay i trong cu trc lc lp, iu ny th hin r chc nng bo v ca ng. ng cng ng vai tr quan trng trong chui truyn in t ca ti th nh cytochrome oxidase c cha 2 nguyn t ng v 2 nguyn t st. d. Mangan (Mn): Manganese l mt trong nhng nguyn t vi lng quan trng nht, gn nh lun c mt trong mi trng nui cy. Nng ca Mn trong mi trng tng ng vi Fe v B. Mn c tc ng ha hc tng t nh Mg+ nn c th thay th cho Mg+ trong mt s h thng enzim. Mangan tn ti trong thc vt dng ion Mn 2+ khng lin kt, ho tr hai v dng ny n c chuyn t r qua mch xylem n cc phn khc ca cy. Nguyn t ny lin kt cht vi mt s loi protein cha kim loi (metalloprotein), hoc nh mt thnh phn cu trc ca enzym hoc nh mt phn trong h oxi ho kh [Mn(II)/Mn(III)]. Phn ng Hill: Mangan c hai chc nng quan trng trong thc vt. Ion ny lin quan n phn ng ban u c gi l phn ng Hill ca quang h II, trong nc c phn ly thnh oxy v cc photon, theo phng trnh sau: 2H2O ---> O2 + 4H+ + 4eC gi thit cho rng 4 phn t mangan l thnh phn ca mt protein, xc tc cho qu trnh quang phn ly nc. Cc electron c gii phng ra tip tc chuyn ti magie cha trong phc h 680, trung tm ca quang h II. Enzym super oxide dismutase: Cho n nay mi ch c mt vi loi enzym cha mangan c phn lp. Enzym c cha mangan quan trng nht l Mn-SOD. (Xem phn v ng bit thm thng tin v SOD). Cng ging nh ng, nu thiu mangan, s xy ra cc thay i trong cu trc ca lc lp, th hin r nht h thng bo v ca mangan. e. Coban (Co): Cobalt c mt trong khong mt na s lng mi trng nui cy m t bo thc vt . nng s dng l 1 m, i khi ngi ta c th s dng nng Cobalt cao gp 10 ln. Cobalt l thnh phn kim loi trong vitamin B12 c lin quan n s sinh tng hp acid nuclic nhng cha c bng chng no v tc ng ca n ln s tng trng v pht sinh hnh thi ca m trn mi trng nui cy.

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44 Mt trong nhng mc ch ca vic b sung cobalt vo mi trng nui cy c l l chng li s gy c ca cc chelat kim loi v c th ngn cn cc phn ng oxi ha gy ra bi ng v st. Coban ng vai tr quan trng trong qu trnh c nh nit r cy h u. Coban l thnh phn cn thit ca enzym cobalamin. Co (III) l thnh phn kim loi nh v gia 4 nguyn t nit trong cu trc porphyrin. Ba h thng enzym ca vi khun Rhizobium c bit ti c cha Co. Ngi ta thy rng c mi lin h gia nng Co vi s c nh nitrogen v s pht trin r c. Co c vai tr trong qu trnh tng hp methyonine vi khun, tng hp ribonucleotide v enzym methymalonyl-coenzyme A mutaza, mt enzym cn thit cho s tng hp leghemoglobin. Khng ai bit chc rng liu Co c gi vai tr g thc vt bc cao hay khng. Ch c mt enzym ph thuc cobalamin c bit ti l leucine-2,3-aminomutase khoai ty. i vi thc vt bc thp, Co l yu t cn thit v c mt trong mt s cu trc di t bo v thylakoid lc lp. f. Molybden (Mo): Thc vt hp thu Mo di dng MoO42- . Molydate thng c b sung vo mi trng nui cy vi nng n 1 m. Molybden ch yu tn ti dng MoO42-, mt dung dch ging nh nc. Trong mi trng axt yu, ion molybden tu thuc vo axt c th nhn 1 hoc 2 proton theo phng trnh sau: MoO42- ---> HMoO4- --->H2MoO4 Molybden c kh nng chuyn vn qua mch xylem v phloem nh ion MoO42-. Nitrogenase: Mt s enzym s dng Mo nh mt co-factor. Hai loi enzym c molybden c m t nhiu nht l nitrogenase v nitrate reductase. Nitrogenase lin quan n qu trnh c nh nit trong nt sn r cy h u nh vi khun Rhizobium: N2 + 8H+ + 8e- ---> 2NH3 + H2 Molybden lin quan trc tip n qu trnh kh N 2. Phn t nit c gn vi nguyn t molybden trong phc h nitrogenase. Mi phn t nit gn vi hai nguyn t molybden m chnh chng li l mt phn ca phn t protein st-molybden. Sau qu trnh hot ho phc h enzym nitrogenase s dng ATP, phc h st-molybden thay i cu trc ca n. Da trn s thay i hp l ny, qu trnh kh N2 xy ra. Nitrate reductase: Nitrate reductase kh nitrat thnh nitrit trong qu trnh ng ho nit t bo thc vt. Nitrate reductase cha 1 phn t heme-st v 2 nguyn t

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45 molybden. Enzym ny xc tc cho qu trnh kh nitrat thnh nitrit. S hot ng ca nitrat reductase b gim mnh khi thiu molybden nhng c th khi phc nhanh chng ngay khi thm molybden vo mi trng. g. Km (Zn): Thiu km th s sinh tng hp protein, acid nuclic v dip lc t s b gim i. Thc vt c t thn ngn, l nh, ng thi t bo trn cng km pht trin. Trong thc vt c mi quan h gn gi gia Zn v nng auxin ni sinh. Ngi ta cho rng , km l mt thnh phn trong enzim c lin quan n s tng hp tin cht ca IAA l trytophan. Nng ca km b sung vo trong mi trng nui cy thay i t 0,1-70 m, nh vy s d km trong mi trng nui cy t gy c cho m. Km c h r hp thu di dng Zn 2+. Trong mch xylem n c chuyn vn di dng ion Zn2+ hoc mui km ca mt axit hu c. Nguyn t ny l mt hp phn kim loi ca mt s enzym. N c th l mt cofactor cu trc cng nh cofactor iu ho ca phc h enzym. Cc enzym: Thc vt c mt s loi enzym cha km, bao gm c enzym dehydrogen ho ru trong vng m phn sinh ca cy. Trong phc h enzym SOD, Zn lin kt vi Cu thay cho nguyn t nit t histidine (xem thm v SOD trong phn ng). Enzym cacbon anhydraza c nh CO2, theo cn bng sau: CO2 + H2O <---> HCO3- + H+ Phn ng ny gip thc vt c th d tr CO 2 di dng HCO3- mt cch thun nghch. Sau khi chuyn thnh CO2, HCO3- c th s dng lm c cht cho enzym ribulose biphosphate carboxylase. Enzym ny c 6 tiu phn di n v, mi tiu phn c 1 nguyn t km gn vo v c th tm thy trong lc lp v t bo cht. Tng hp protein: Km rt quan trng trong qu trnh tng hp protein. Thiu km gy nh hng ln n qu trnh tng hp protein. S tp trung ribosom v s tch lu cc tin cht ca protein nh cc amino axt v cc amin, c th xy ra nh km. Zn cn cho hot ng ca RNA polymeraza. Di iu kin thng thng, RNA polymeraza cha 2 nguyn t Zn, to ra cu trc in hnh ca enzym. Hn na, c 1 mi quan h nghch tng ng gia nng Zn v hot ng ca RNAse . Nng Zn thp s lm tng hot ng ca RNAse. Tng hp IAA: Thiu km cng ph hng qu trnh tng hp indol acetic acid trong cy (Indol ---> Trytophan ---> Indol Acetic Acid)

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46 Km ng vai tr quan trng trong tng hp tryptophan, mt tin cht ca IAA. V d, thiu km trong ng c th thay th bng cch b sung tryptophan. h. Chlor (Cl): Ion Cl- cn thit cho s tng trng ca thc vt nhng n t c mt trong cc phn ng sinh hc v c vai tr vi mt lng rt nh. Thng thng trong mi trng nui cy m, nng Cl- cn thit l 3mM trung bnh l 6mM. Mt s loi thc vt nhy cm vi Cl-. Chlo c thc vt dao ng t 70 n 700 mM trong 1 kg trng lng kh (2000 n 20000 mg/kg trng lng kh). Chlo c hp thu di dng Cl - v rt c ng trong cy. Chc nng chnh ca ion ny l iu ho thm thu v b sung cc cht mang. Trong lc lp c cha hm lng chlo ln. Ngi ta cho rng chlo ng vai tr ht sc quan trng trong quang hp. Cc lc lp ca rau chn vt v c ci ng cha chlo nng xp x 100mM nhng trong l, ch c di 10mM , cho thy u th r rng ca chlo tp hp trong lc lp. Nng lc thm thu: Ion Cl- iu ho s ng, m kh khng. Tnh trng thiu chlo lm kh khng m mi, c th gy tnh trng mt nc nghim trng. Chlo rt quan trng trong iu ho nng lc thm thu ca cc khng bo v vi cc qu trnh lin quan n sc trng. S trao i nit: Chlo hot ho enzym tng hp asparagin, mt enzym quan trng trong qu trnh trao i nit. Enzym ny chuyn ho glutamin thnh asparagin v axit glutamic. Trong iu kin c Cl -, tc ca phn ng tng ln gp 7 ln. Bi th, chlo thc hin mt chc nng quan trng trong qu trnh trao i nit cc loi thc vt s dng asparagin nh cht mang. 2.6.1. 4.Cc vitamin Tt c cc t bo c nui cy u c kh nng tng hp tt c cc loi vitamin c bn nhng thng l vi s lng di mc yu cu. m c sc sinh trng tt phi b sung thm vo mi trng mt hay nhiu loi vitamin. Cc vitamin l rt cn thit cho cc phn ng sinh ho. Thng thng thc vt tng hp cc vitamin cn thit cho s tng trng v pht trin ca chng. Thc vt cn vitamin xc tc cc qu trnh bin dng khc nhau. Khi t bo v m dc nui cy in vtrro th mt vi vitamin tr thnh yu t gii hn s pht trin ca chng. Cc vitamin c s dng nhiu nht trong nui cy m l:

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47 thiamine (B1), acid nicotinic (PP), pyridoxine (B6) v myo-inositol. Thiamin l mt vitamin cn bn cn thit cho s tng trng ca tt c cc t bo. Thiamin thng c s dng vi nng bin thin t 0,1-10 mg/l. Acid nicotinic v pyridoxine thng c b sung vo mi trng nui cy nhng cng khng cn thit cho s tng trng ca t bo nhiu loi thc vt. Acid nicotinic thng c s dng vi nng 0,1-5 mg/l, pyridoxine c s dng vi nng 0,1-10 mg/l. Myo-inositol thng c pha chung vi dung dch m ca vitamin. Mc d y l mt carbohydrate ch khng phi l vitamin, n cng c chng minh kch thch cho s tng trng ca t bo a s loi thc vt. Ngi ta cho rng myo-inositol c phn tch ra thnh acid ascorbic v peptine v c ng ha thnh phosphoinositide v phosphatidylinositol c vai tr quan trng trong s phn chia t bo. Myo-inositol thng c s dng trong mi trng nui cy m v t bo thc vt nng 50-5000 mg/l. Cc vitamin khc nh biotin, acid folic, acid ascorbic, panthothenic acid, vitamin E (tocopherol), riboflavin v p-aminobenzoic acid cng c s dng trong mt s mi trng nui cy. Nhu cu vitamin trong mi trng nui cy ni chung khng quan trng v chng cng khng cn tr s tng trng ca t bo. Ni chung cc vitamin ny c thm vo mi trng ch khi nng thiamin thp hn nhu cu cn thit hoc cho huyn ph t bo c th tng trng khi mt t bo khi u thp. Cc vitamin sau y c s dng ph bin: inositol, thiamine HCl (B1), pyridoxine HCl (B6), nicotinic axt, trong vitamin B1 l khng th thiu v c s dng trong hu ht nhng mi trng nui cy m v t bo thc vt. Linsmaier v Skoog khng nh vitamin B1 l cn thit cho cho s sinh trng ca cy sau khi nghin cu k lng v s c mt ca n trong mi trng MS. Cc tc gi khc cng khng nh vai tr rt quan trng ca B1 trong nui cy m. Inositol thng c ni n nh l mt vitamin kch thch mt cch tch cc i vi s sinh trng v pht trin ca thc vt, mc d n khng phi l vitamin cn thit trong mi trng hp. Cc vitamin khc, c bit l nicotinic axit (vitamin B3), canxi pantothenate (vitamin B5) v biotin cng c s dng nng cao sc sinh trng ca m nui cy. nh hng ca cc vitamin ln s pht trin ca t bo nui cy in vitro cc loi khc nhau l khc nhau hoc thm tr cn c hi (gy c). a. Vitamin B1 (Thiamine.HCl, Aneurin) La mt cht b sung rt cn cho mi trng nui cy. Khi kh trung bng cach hp nhit cao vit B1 bi nhit phn thanh pyrimidin va thiazol la hai cu t cua vit B1, nhng

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48 t bao nui cy co kha nng tng hp li chung thanh phn t vit B1. Vi vy khng nht thit phai kh trung bng phng thc khac nh loc chng hn. b. Vitamin B2 (Riboflavin, Lactoflavin) Co th kh trung bng nhit, nhng lai d bi anh sang phn hu. i vi nui cy sang chi dung nng 0,01 ppm, nhng i vi nui cy trong ti co th tng ln 10-50 ppm. c. Vitamin B6 (Pyridoxine, Adermin) La tin cht cua pyridoxal-phosphate-cofactor cua cac nhom enzyme nh carboxylase va transaminase. Khi kh trng nhit cao phan ng xy ra nh sau: Pyridoxin + Phosphat Pyridoxalphosphate d. Myo-Inositol (Bios I) Co vai tro trong sinh tng hp thanh t bao, cu th la sinh tng hp acid polygalacturonic va pectine. Inosit la cht bn vng khi kh trung. Thng c s dung nng cao 100 ppm. Khi phn tich thanh phn cua nc da ngi ta thu c inosit trong mt phn oan trung tinh. e. Biotin (Bios II) Cn thit cho s phn bao cua mt s loai m. Chi s dung nng rt thp t 0,001-0,01 ppm. f. Acid pantothenic (Bios III, Vit B5) c s dung lam thanh phn cua coenzyme A . g. Nicotinic acid va pyridoxine thng c b sung vao mi trng nui cy nhng co th thay th bng cac vitamin khac cho s sinh trng cua t bao nhiu loai. Cac vitamin khac nh folic acid, ascorbic acid, vitamin E (tocophenol), va -aminobenzoic acid cung c s dung trong nui cy m va t bao, c bit khi t bao sinh trng mt qun th rt thp. Noi chung, cac vitamin nay c b sung trong khoang 0,1-10,0 ppm.

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49 2.6.1.5. Cc cht b sung vo mi trng cy m a. Amino acid v cc ngun cung cp nitrogen khc Mc d t bo c kh nng tng hp tt c cc amino acid cn thit nhng s b sung cc amino acid vo mi trng nui cy l kch thch s tng trng ca t bo. Vic s dng amino acid c bit quan trng trong mi trng nui cy t bo v nui cy t bo trn. Amino acid cung cp cho t bo thc vt ngun amino acid sn sang cho nhu cu ca t bo v ngun nitrogen ny c t bo hp thu nhanh hn nitrogen v c. Cc ngun nitrogen hu c thng s dng trong mi trng nui cy t bo thc vt l hn hp amino acid nh casein hydrolysate, L-glutamine, L-asparagine v adenine. Casein hydrolysate ni chung c s dng vi nng 0,05-0,1%. Khi amino acid c cung cp ring r th cn phi cn thn v n c th cn tr s tng trng ca t bo. Mt v d v amino acid trong mi trng nui cy lm tng s tng trng ca t bo l glysine 2 mg/l, glutamine n 8 mM, asparagine 100 mg/l, L-arginine v cysteine 10 mg/l v L-tyrosine 100 mg/l. Tyrosine cng c s dng kch thch s pht sinh hnh thi trong nui cy t bo nhng ch nn s dng trong mi trng c agar. Cung cp adenine sulfate vo mi trng nui cy c th kch s tng trng ca t bo nui cy v kch thch mnh s to chi. Cac m c nui cy vn co kha nng tng hp cac amino acid cn thit cho cac qua trinh trao i cht khac nhau. Mc du th, vic b sung cac amino acid vao mi trng vn cn thit kich thich sinh trng t bao trong nui cy protoplast va hinh thanh cac dong t bao. Khng ging nh cac N v c, cac amino acid c cac t bao thc vt hp thu nhanh hn. Casein hydrolysate (0,05-0,1%), L-glutamine (8 mmol/L), L-asparagine (100 mmol/L), L-glycine (2 mmol/L), L-arginine va L-cystein (10 mmol/L) la ngun N hu c thich hp c dung trong cac mi trng nui cy. Tyrosine (100 mmol/L) chi c dung khi c b sung agar vao mi trng. Cac amino acid c b sung ring le thng han ch s sinh trng cua t bao trong khi hn hp cua chung lai co hiu qua hn. B sung vao mi trng adenine sulphate co th kich thich sinh trng cua t bao hoc lam tng kha nng tao chi. b. Than hot tnh B sung than hot tnh vo trong mi trng nui cy s c li ch v c tc dng kh c. Khi b sung than hot tnh vo mi trng nui cy th s kch thch s tng trng v bit ha phong lan, hnh, c rt, c chua, cy trng xun nhng li c tc dng cn i vi thuc l, u nnh, tr mi. Than hot tnh ni chung nh hng trn 3 mt: ht cc hp cht cn, ht cc cht iu ha sinh trng hoc lm n mi trng.

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50 Ngi ta cho rng tc dng cn s tng trng ca m cy khi c s hin din ca than hot tnh trong mi trng l do n ht cht iu ha sinh trng c trong mi trng. NAA, kinetine, IAA, BAP, 2iP lin kt vi than hot tnh. Kh nng kch thch s tng trng ca than hot tnh l do n kt hp vi cc hp cht phenol c tit ra trong thi gian nui cy. Than hot tnh thng c b sung vo mi trng vi nng 0,5-3% (w/v). B sung AC vao mi trng nui cy a kich thich sinh trng va phn hoa cac loai hoa lan, ca rt, dy thng xun va ca chua. Ngc lai, no gy c ch thuc la, u tng va cac loai thuc chi Camellia. Noi chung AC c ra acid va trung hoa trc khi b sung no nng 0,5-3% vao mi trng nui cy. AC cung giup lam giam c t bng cach ao thai cac hp cht c (v d: phenol) c to ra trong qua trinh nui cy va cho phep t bao sinh trng ma khng bi tr ngai gi. c. Nc da Cng b u tin v s dng nc da trong nui cy m thuc v Van Overbeek v cng s (Van Overbeek cs, 1941,1942). Sau , tc dng tch cc ca nc da trong mi trng nui cy m, t bo thc vt c nhiu tc gi ghi nhn. Nc da c xc nh l rt giu cc hp cht hu c, cht khong v cht kch thch sinh trng (George, 1993; George, 1996). Nc da c s dng kch thch phn ha v nhn nhanh chi nhiu loi cy. Nc da thng c ly t qu ca cc ging v cy chn lc s dng ti hoc sau bo qun. Nc da c mt s cng ty ho cht bn di dng ng chai sau ch bin v bo qun. Thng thng nc da c x l loi tr cc protein, sau c lc qua mng lc kh trng trc khi bo qun lnh. Tn d protein trong nc da khng gy nh hng n sinh trng ca m hoc t bo nui cy, nhng c th dn ti kt ta dung dch khi bo qun lnh. Cht cn c th c lc b hoc lng di y bnh ri gn b phn cn. Nc da thng c s dng nng t 5 n 20 % (v/v). d. Bt chui Bt chui kh hoc bt nghin t qu chui xanh c s dng trong nui cy m mt s cy trng nh phong lan. Hm lng s dng vo khong 40g bt kh/l. trnh ng cc, cn b sung bt vo dung dch mt cch t t ri khuy u. Nc chit t khong 100 g tht qu chui xanh cng c s dng b sung vo mi trng.

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51 e. Mt s hn hp dinh dng hu c phc tp khc Nc ct c chua, dch chit khoai ty nghin, dch chit mch nha, dch chit nm men (yeast extract), casein thu phn (casein hydrolysate) cng c s dng lm tng s pht trin ca m so hay c quan nui cy. f. Dich chit nm men (yeast extract-YE) Vi dich nm men, White (1934) ln u tin nui thanh cng r ca chua trong ng nghim keo dai v thi han. Thanh phn hoa hoc cua dich nm men it c chu y phn tich. Chu yu cha: ng, nucleic acid, amino acid, vitamin, auxin, mui khoang. Tac dung cua YE vi r rt tt nhng vi callus th khng ro rang. g. Dich thuy phn casein (casein hydrolysate-CH) c s dung rng rai trong ky thut vi sinh vt, nui cy m va t bao thc vt chu yu c s dung lam ngun b sung amino acid. h. Hn hp amino acid nhn tao Da vo nhng kt qua phn tich cac hn hp cht t nhin ni trn nhiu tac gia a ra nhng cng thc pha ch hn hp amino acid nhn tao b sung vao mi trng dinh dng. Kt qua s dung cac hn hp nay con rt khac nhau, co th do yu cu amino acid cua tng loai t bao la khng ging nhau. Trong mi trng long nui callus lua va mi trng tai sinh cy lua t callus thi proline la mt thanh phn quan trong. i. Agar i vi nui cy tnh, nu s dng mi trng lng, m c th b chm v s cht v thiu xy. trnh tnh trng ny, mi trng nui cy c lm c li bng agar; mt loi tinh bt c ch t rong bin v m c cy trn b mt ca mi trng. Agar thng c s dng nng 0,6 n 1%. 2.6.1.6. Cc cht iu ho sinh trng Bn cnh cc cht cung cp dinh dng cho m nui cy, vic b sung mt hoc nhiu cht iu ha sinh trng nh auxin, cytokinin v giberellin l rt cn thit kch thch s sinh trng, pht trin v phn ho c quan, cung cp sc sng tt cho m v cc t chc. Tuy vy, yu cu i vi nhng cht ny thay i tu theo loi thc vt, loi m, hm lng cht iu ha sinh trng ni sinh ca chng. Cc cht iu ho sinh trng thc vt c chia thnh cc nhm chnh sau y:

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52 a. Nhm cc auxin Mi trng nui cy c b sung cac auxin khac nhau nh: 1H- indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 1H-indole-3-butyric acid (IBA), 2,4dichlorophenoxyacetic acid (2,4-D) va naphthoxyacetic acid (NOA). IAA la auxin t nhin co trong m thc vt; con lai NAA, IBA, 2,4-D va NOA la cac auxin nhn tao, thng thi cac auxin nhn tao co hoat tinh manh hn vi do c im phn t cua chung nn cac enzyme oxy hoa auxin (auxin-oxydase) khng co tac dung. Nhng auxin co hiu lc ring bit trong nui cy t bao thc vt la 4-chlorophenoxyacetic acid (4-CPA) hoc p-chlorophenoxyacetic acid (PCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-amino-3,5,6-trichloropicolinic acid (picloram), va 3,6-dichloro-2methoxybenzoic acid (dicamba). c im chung cua cac auxin la tinh cht phn chia t bao. Cac hormone thuc nhom nay co cac hoat tinh nh: tng trng chiu dai thn, long (giong), tinh hng (sang, t), tinh u th ngon, tao r, va phn hoa mach dn. Noi chung, cac auxin c hoa tan hoc trong ethanol hoc trong NaOH loang. Mc du, nhng chi tit mc phn t v hoat ng cua auxin vn cha c bit nhiu, nhng mt s nghin cu v di truyn va phn t a cung cp nhng kt qua mi thu vi. Trong nhng nm qua, mt s gen m ha cho cc protein lin kt auxin (auxin binding proteins) a c tao dong (cloning) va khao sat cac c im cua chung, va thng tin mi v gen cam ng auxin (auxin-induced gene) cung a thu c. Auxin co tac dung hoat hoa cac ion H+ trc tip hoc gian tip (thng qua s anh hng ln cac enzyme) lam tng tinh an hi cua thanh t bao, tng tinh gian n cua t bao trong phan ng vi ap sut trng. Auxin cung co anh hng mc biu hin gen va kich thich qua trinh tao r. Cc auxin c th l auxin t nhin hoc tng hp, thng c dng trong nui cy m v t bo kch thch s phn bo v sinh trng ca m so, c bit l 2,4-D, to phi v tnh, to r, Auxin l nhm cht iu ha sinh trng thc vt c s dng thng xuyn trong nui cy m t bo thc vt. Auxin kt hp cht ch vi cc thnh phn khc ca mi trng dinh dng kch thch s tng trng ca m so, huyn ph t bo v iu ha s pht sinh hnh thi, c bit l khi n c phi hp s dng vi cc cytokinin. S p dng loi v nng auxin trong mi trng nui cy ph thuc vo: - Kiu tng trng hoc pht trin cn nghin cu - Hm lng auxin ni sinh ca mu cy - Kh nng tng hp auxin t nhin ca mu cy - S tc ng qua li gia auxin ngoi sinh v auxin ni sinh

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53 - c tnh ca auxin: Auxin c vai tr kch thch s tng trng v ko di t bo. Auxin c kh nng khi u s phn chia t bo.. c im chung ca cc auxin l tnh cht phn chia t bo. Cc hormone thuc nhm ny c cc hot tnh nh: tng trng chiu di thn, long (ging), tnh hng (sng, t), tnh u th ngn, to r v phn ha mch dn. Ni chung cc auxin c ha tan hoc trong ethanol hoc trong NaOH long (Razdan 1994) Cc auxin lin quan ti di ca thn, t, chi chnh, r i vi nui cy m, auxin c s dng cho vic phn chia t bo v phn ha r. Nhng auxin dng rng ri trong nui cy m l IBA (3-indolebutiric axid), IAA (3-indole acetic axid), NAA (Napthaleneaxetic axid), 2,4-D (2,4-D-Dichlorophenoxyaxetic axid) v 2,4,5-T (Trichlorophenoxyacetic axid). Trong s cc auxin, IBA v NAA ch yu s dng cho mi trng ra r v phi hp vi cytokinin s dng cho mi trng ra chi. 2,4-D v 2,4,5-T rt c hiu qu i vi mi trng to v pht trin callus. Auxin thng ha tan trong etanol hoc NaOH pha long. Vai tr ca cc cht thuc nhm auxin c khi qut di y: 1- Kch thch phn chia v ko di t bo 2- Chi nh cung cp auxin gy ra c ch sinh trng ca chi bn. u th chi nh lm c ch sinh trng ca chi nch. Nu ngt b chi nh s dn n s pht chi nch. Nu thay th vai tr ca chi nh ( b ngt b) bng mt lp cht keo c cha IAA th chi nch vn b c ch sinh trng. C ch c ch ca chi nh lin quan n mt cht iu ho sinh trng khc l ethylene. Auxin (IAA) kch thch chi bn sn sinh ra ethylen lm c ch sinh trng ca chi nh. 3- IAA ng vai tr kch thch s phn ho ca cc m dn (xylem and phloem). 4- Auxin kch thch s mc r cnh gim v kch thch s pht sinh chi ph trong nui cy m. 1- Auxin c cc nh hng khc nhau i vi s rng l, qu, s u qu, s pht trin v chn ca qu, s ra hoa trong mi quan h vi iu kin mi trng. 2- To v nhn nhanh m so (callus) 3- Kch thch to chi bt nh ( nng thp) 4- To phi soma (2,4-D) b. Nhm cc cytokinin Cac cytokinin la dn xut cua adenine, y la nhng hormone lin quan chu yu n s phn chia t bao, s thay i u th ngon va phn hoa chi trong nui cy m. Cac cytokinin c s dung thng xuyn nht la 6-benzylaminopurine (BAP) hoc 6Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

54 benzyladenin (BA), 6---dimethyl-aminopurine (2-iP), N-(2-furfurylamino)-1-H-purine-6amine (kinetin), va 6-(4-hydroxy-3-methyl-trans-2-butanylamino)purine (zeatin). Zeatin va 2-iP la cac cytokinin t nhin, con BA va kinetin la cac cytokinin nhn tao. Noi chung, chung c hoa tan trong NaOH hoc HCl loang. Mt s hp cht c phat hin trong thi gian gn y co hoat tinh ging cytokinin la N,N-diphenylurea (DPU), thidiaziron, N-2-chloro-4-puridyl-N-phenyl urea (CPPU) va mt s dn xut khac cua diphenyl urea. Hiu qua c bit cua cac hp cht gc urea ln s sinh trng cua m thc vt cn phai c nghin cu thm. Ty l auxin/cytokinin rt quan trong i vi s phat sinh hinh thai (morphogenesis) trong cac h thng nui cy. i vi s phat sinh phi (embryogenesis), tao callus va r cn co ty l auxin/cytokinin cao, trong khi trng hp ngc lai se dn n s sinh san chi va chi nach. Vn quan trong khng kem la nng cua hai nhom cht iu khin sinh trng nay. Chng han 2,4-D cung vi BA nng 5,0 ppm kich thich s tao thanh callus Agrostis nhng nu dung nng 0,1 ppm chung se kich thich tao chi mc du trong ca 2 trng hp ty l auxin/cytokinin la bng 1. C ch hoat ng cua cytokinin la cha c bit ro rang mc du co mt s kt qua v s co mt cua cac hp cht mang hoat tinh cytokinin trong RNA vn chuyn (transfer RNA). Cac cytokinin cung co hoat tinh tng hp RNA, tng hoat tinh enzyme va protein trong cac m nht inh. - Kinetin c phn lp t ch phm DNA cu hoc nucleic acid mi sau khi kh trng nhit cao hay un si. Trong c th sng khng co kinetin tn tai, san phm nay kich thich s phat sinh chi cua cy thuc la nui cy, nhng nu phi hp x ly cung auxin ty l nng thich hp thi se kich thich qua trinh phn chia t bao (do o co tn la kinetin) cac m khng phn hoa. Trong t nhin cung tn tai mt hormone phn bao khac, Letham la ngi u tin a phn lp, tinh ch va cho kt tinh thanh cng hormone phn bao t nhin o t ni nhu ang dang sa cua hat ng. Hp cht cytokinin t nhin o c goi la zeatin (zea: ng). - Tng t cac cytokinin khac, zeatin cung la mt dn xut cua adenin. Trong thc tin nui cy m ngi ta chi dung zeatin trong nhng trng hp c bit vi gia thanh rt t, thng thay th zeatin bng kinetin hoc mt san phm tng hp nhn tao khac, o la: - 6-Benzylaminopurine (BAP): Hoat lc cua BAP cao hn nhiu so vi kinetin va ban thn BAP bn vng hn zeatin di tac ng cua nhit cao. BAP co kha nng lam tng hinh thanh cac san phm th cp va tng kich thc cua t bao cac la mm, kich thich s nay mm cua hat va qua trinh trao i cht. Cytokinin lin quan ti s phn chia t bo, phn ha chi v.v Trong mi trng nui cy m, cytokinin cn cho s phn chia t bo v phn ha chi t m so hoc t cc c quan, gy to phi v tnh, tng cng pht sinh chi ph.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

55 Chc nng ch yu ca cc cytokinin c khi qut nh sau: 1- Kch thch phn chia t bo 2- To v nhn callus 3- Kch thch pht sinh chi trong nui cy m 4- Kch thch pht sinh chi nch v km hm nh hng u th ca chi nh 5- Lm tng din tch phin l do kch thch s ln ln ca t bo 6- C th lm tng s m ca kh khng mt s loi 7- To chi bt nh ( nng cao) 8- c ch s hnh thnh r 9- c ch s ko di chi 10- c ch qu trnh gi (ho vng v rng) l, kch thch to dip lc c. Gibberellin Gibberellin c phat hin vao nhng nm 1930. Lich s phat hin nhom hormone nay bt u t 1895 khi ngi Nht noi v bnh lua von. Nm 1926, xac inh c bnh o la do loai nm Gibberella fujikuroi gy ra. n nhng nm 30, mi phn lp va tinh ch c hoat cht, c goi la gibberellin. Mai sau chin tranh th gii th II nm 1950, ngi Anh va ngi My mi bit n cng trinh nay cua ngi Nht. Ti nay, ngi ta a phat hin c trn 60 loai thuc nhom gibberellic acid. Loai gibberellic acid thng dung nht trong nui cy m thc vt la GA3. Trong i sng thc vt gibberellin ong vai tro quan trong i vi nhiu qua trinh sinh ly nh: sinh ly ngu nghi cua hat va chi, sinh ly phat trin cua hoa, lam tng sinh trng chiu dai cua thc vt. Nhng trong nui cy m va t bao thc vt tac dung cua gibberellic acid cha tht ro rang. Nhiu tac gia co s dung va coi o la thanh phn khng th thiu cua mt loai mi trng chuyn dung nao o. Trong s hn 20 cht thuc nhm gibberellin, GA3 l cht c s dng nhiu hn c trong thc tin. GA3 kch thch ko di chi v ny mm ca phi v tnh. So vi auxin v cytokinin, gibberellin him khi c dng. GA3 c tnh ho tan trong nc. Gibberellin c cc chc nng c bn sau: 1- Cc m phn sinh tr, ang sinh trng, cc phi non, t bo u r, qu non, ht cha chn hoc ang ny mm u c cha nhiu gibberellic axit. 2- Kch thch ko di chi do tng cng phn bo v ko di t bo, v d ko di thn v ng la sau khi phun GA3, ko di t thn. Cc cy ln thng b thiu gibberellin. 3- Ph ng ht ging hoc c ging, v d ph ng khoai ty sau thu hoch.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

56 4- Kim sot s ra hoa ca cc cy 2 nm tui. Nm u thn mm nm in, sau ma ng mm hoa ko di t rt nhanh v phn ho hoa 5- c ch s hnh thnh r bt nh 6- Kch thch sinh tng hp ca -amylase ht cy ng cc ny mm, gip tiu ho cc cht d tr trong ni nh nui mm cy 7- Cc cht c ch tng hp kch thch qu trnh to c (thn c, thn hnh v c) 8- Kch thch s ny mm ca phn hoa v sinh trng ca ng phn 9- C th gy to qu khng ht hoc lm tng kch thc qu nho khng ht 10- C th lm chm s ho gi l v qu cy c mi e. Abscisic axt (ABA) ABA thuc nhom cac cht c ch sinh trng t nhin gy ra s ngu nghi cua chi, lam chm s nay mm cua hat va s ra hoa, ong khi khng. ABA con co tac dung tng cng kha nng chng chiu cua t bao thc vt i vi iu kin ngoai canh bt li, vi vy ABA c a vao mi trng nui cy va mang lai hiu qua nht inh Trong nui cy m v t bo, ABA c tc dng to phi v tnh, kch thch s chn ca phi, kch thch s pht sinh chi nhiu loi thc vt. Cc tc dng c bn ca ABA l: 1- Tham gia vo s rng l, hoa, qu hu ht cc cy trng v gy ra s nt qu 2- ABA thng c sn sinh khi c cc yu t c ch cy trng nh mt nc v nhit thp ng bng 3- Tham gia vo s ng ngh, ko di thi gian ng ngh v lm chm s ny mm ca ht 4- c ch s ko di thn v c s dng kim sot s ko di thn cnh 5- Gy ra s ng kh khng f. Ethylene 6 Cc chc nng c bn ca ethylene: 7- Gy gi ho l, kch thch s rng l v qu 1- Lm chn qu 1- Sinh tng hp ethylene c tng cng khi qu ang chn, cy ang b ng, lo ho, tn thng c gii v b nhim bnh 2- iu khin s chn ca mt s loi qu 3- Ethylene km hm s ra hoa ca a s cy. Tuy vy, s ra hoa ca xoi, da, mt s cy cnh li c kch thch bi ethylene. 4- Kch thch n hoa, kch thch s lo ho ca hoa v l

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

57 Bng 2.3. Cc cht thuc nhm Auxin Tn cc cht Phn t lng v tng ng Phn t S Dung lng M mi = 1mg /L 186.6 5.36 EtOH Chun b dung dch, bo qun v s dng Pha Bo Bo Kh Nng long qu qu trn s n n g dng kh lng (mg/ L) RT 2CA 0.180C 10.0 RT 280C 280C CA 0.016.0 0.016.0

pChlorophenoxyaceti c acid (4-CPA) 2,4Dichlorophenoxyacetic acid 2,4Dichlorophenoxy-

221

4.53

243

4.12

Nc

RT

CA

acetic acid Sodium salt Indole-3-acetic acid 175.2 Free acid (IAA) Indole-3-acetic acid Sodium salt Indole-3-acetic acid methyl ester Indole-3-acetyl-Laspartic acid Indole-3-butyric acid (IBA) 197.2 189.2 290.3 203.2

5.71

5.07 5.29 3.45 4.90

EtOH/ 1N NaOH Nc 0.5N NaOH EtOH/ 1N NaOH Nc

N c N c N c N c

-00C

-00C

CA/ F CA/ F F CA/ F

0.013.0 0.013.0 0.015.0 0.110.0

280C 280C -00C 280C 280C

-00C 280C -00C -00C

Indole-3-butyric acid 241.3 Potassium salt (KIBA) Alpha-Naphthalene- 186.2 acetic acid Free acid

4.14

00C 280C

CA/F 0.110.0 CA 0.110.0

5.37

1N NaOH

Nc RT

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

58 (NAA) Beta-Naphthoxy-

202.2

4.95

acetic acid Free acid (NOA) Phenylacetic acid 136.2 (PAA) Picloram 241.5 2,4,5Trichlorophenoxy 255.5

1N NaOH EtOH DMSO EtOH

Nc RT

280C 280C 280C 280C 00C

CA

0.110.0

7.34 4.14 3.91

RT RT RT

CA/F 0.150.0 CA 0.0110.0 CA 0.015.0 F 0.055.0

acetic acid (2,4,5-T) 2,3,5-Triiodobenzoic 499.8 acid Free acid (TIBA)

2.00

1N NaOH

Nc 00C

K hiu: CA = coautoclavable (Kh trng cng vi cc cht khc trong mi trng) F = filter sterlize: Kh trng bng lc qua phin lc CA/F: Kh trng cng vi cc thnh phn khc ca mi trng nhng c kh nng b mt hot tnh t nhiu, c th b p bng cch b sung thm hoc kh trng bng lc qua phin lc. Bng 2.4. Cc cht thuc nhm cytokinin Tn ha cht Phn t lng v tng ng Phn S M Dung t mi = ln 1mg/L g 135. 1 7.40 1.0 HCl Chun b dung dch, v s dng Pha Bo Bo lon qun qu g n lng N c RT 280 C 280 C 2bo qun Kh Nng trn s g dng (mg/ L) CA 50250 50250 0.1-

Adenine Free base

Adenine hemisulfate 184. Hemisulfate salt 2 6225.

5.43

Nc

RT

CA

4.44

1N

RT

CA/

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

59 Benzylaminopurine (BA) 6Benzylaminopurine Hydrochloride 6Benzylaminopurine (BA hoc BAP) N-Benzyl-9-(2tetrahydropyranyl)ad enine (BPA) N-(2-Chloro-4pyridyl)-N'phenylurea (4CPPU) 6-(gamma,gammaDimethylallylamino) purine (2iP) 6-(, Dimethylallylamino) purine (2iP) 1,3-Diphenylurea (DPU) Kinetin 3 261. 7 225. 3 309. 4 247. 7 3.82 NaOH Nc c RT 80 C 280 C 280 C 00 C 280 C 00 C 00 C 280 C 00 C 00 C 00 C 00 C 280 F CA/ F CA/ F CA/ F F 5.0 0.15.0 0.15.0 0.15.0 0.00 1-1.0

4.44

1N NaOH EtOH

N c

RT

3.23

-00C

4.04

DMSO

2-80C

203. 2 203. 2 212. 3 215. 2 215. 2 215. 2 251. 7 220. 2

4.92

1N NaOH 1N NaOH DMSO

N c

-00C

CA/ F CA/ F F

1.030.0 1.030.0 0.11.0 0.15.0 0.15.0 0.15.0 0.15.0 0.001 -0.05

4.92

Nc -00C

4.71

RT

4.65

1N NaOH 1N NaOH 1N NaOH Nc

Nc -00C

CA/ F CA/ F CA/ F CA/ F CA/ F

Kinetin

4.65

Nc -00C

Kinetin

4.65

Nc -00C

Kinetin Hydrochloride 1-Phenyl-3-(1,2,3thiadiazol-5-yl) urea

3.97

-00C

4.54

DMSO

RT

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

60 C 00 C 00 C 00 C 00 C

Trans-Zeatin base Zeatin

Free 219. 2 219. 2 255. 7 351. 4

4.56

1N NaOH 1N NaOH Nc

Nc -0 C

CA/ F CA/ F CA/ F F

0.015.0 0.015.0 0.015.0 0.015.0

4.56

Nc -00C

Trans-Zeatin Hydrochloride Trans-Zeatin riboside

3.91

-00C

2.85

1N NaOH

Nc -00C

K hiu: CA = coautoclavable (Kh trng cng vi cc cht khc trong mi trng) F = filter sterlize: Kh trng bng lc qua phin lc . CA/F : Kh trng cng vi cc thnh phn khc ca mi trng nhng c kh nng b mt hot tnh t nhiu, c th b p bng b sung thm hoc kh trng bng lc qua phin lc. Bng 2.5. Nhm cc cht iu ho sinh trng khc Tn ha cht Phn t lng v tng ng Ph S Dung n mi t M l = ng 1m g/ L 264. 3 256. 3 158. 1 221. 3.7 8 3.9 0 6.3 3 4.5 Chun b dung dch, bo qun v s dng Pha Bo Bo Kh Nng long qun qun trng s lng dng (mg/L)

(+-)-cis,trans-Abscisic acid (ABA) Ancymidol Chlorocholine chloride (CCC) 3,6-Dichloro-o-anisic

1N Nc NaOH DMS O Nc EtOH/

-00C 2-80C RT 2-80C

-00C

CA/ F -00C CA/ F 2-80C F 2-80C F

0.110.0 1.010.0 up to 500 0.01-

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

61 acid (Dicamba) Gibberellic acid (GA3) Gibberellic acid Potassium salt (KGA3) Gibberellin A4 Free acid (GA4) (+-)-Jasmonic acid Phloroglucinol 0 346. 4 384. 5 332. 4 210. 3 126. 1 169. 1 2 2.8 9 2.6 0 3.0 1 4.7 6 7.9 3 5.9 1 6.2 4 Nc EtOH Nc 10.0 0.015.0 0.015.0 0.015.0 0.01100.0 > 162

RT 2-80C

2-8 C CA/ F 0 -0 C CA/ F -00C -00C F F

EtOH EtOH Nc

-00C 2-80C RT RT

N(Phosphonomethyl)gl ycine (Glyphosate) Succinic acid 2,2- 160. dimethylhydrazide 2

1N Nc NaOH Nc

2-80C CA/ F 0 2-8 C F

2-80C

2-80C CA/ F

0.110.0

K hiu: CA = coautoclavable (Kh trng cng vi cc cht khc trong mi trng) F = filter sterlize: Kh trng bng lc qua phin lc . CA/F : Kh trng cng vi cc thnh phn khc ca mi trng nhng c kh nng b mt hot tnh t nhiu, c th b p bng b sung thm hoc kh trng bng lc qua phin lc. Bng 2.6. Gii thiu tm tt v mt s cht iu ho sinh trng chnh thc vt A 1 2 3 4 5 6 7 Nhm auxin Indole-3-acetic acid (IAA) Indole-3-butyric acid (IBA) 1-naphthaleneacetic acid (NAA) 2,4-dichlorophenoxyacetic acid (2.4D) (2,4,5-T) Picloram Dicamba Chc nng trong h thng nui cy m - Phn chia t bo - To v nhn callus - To r bt nh( nng cao) - To chi bt nh( nng thp) - To phi soma (2,4-D) - c ch chi nch

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

62 8 9 B 1 2 3 4 5 6 7 8 C 1 2 3 4 D 1 2 p-chlorophenoxyacetic acid (CPA) Phenylacetic acid (PAA) Nhm cytokinin Kinetin 6-Bezylaminopurine (BAP) Zeatin (Z) Zeatinriboside (ZR) Isopentenyladenosine (iPA) Isopentenyladenine (iP) Thidiazuron (TDZ) N-(2-chloro-4-pyridyl) -N-phenylurea (CPPU) Nhm gibberellin Gibberellic acid (GA3) Gibberllin 1 (GA1) Gibberellin 4 (GA4) Gibberellin 7 (GA7) Chc nng trong h thng nui cy m 1- Phn chia t bo 2- To v nhn callus 3- Kch thch bt chi nch. 4- To chi bt nh ( nng cao) 5- c ch s hnh thnh r 6- c ch s ko di chi. 7- c ch qu trnh gi (ho vng) l.

Chc nng trong h thng nui cy m - Ko di chi - Ph ng ht ging. - c ch s hnh thnh r bt nh. - Cc cht c ch tng hp kch thch qu trnh to c (thn c, thn hnh v c).

Nhm cc cht HST Chc nng trong h thng nui cy m khc Ethylene Abscisic acid - Gy gi ho l - Lm chn qu - S chn ca th phi - Kch thch s hnh thnh thn hnh v thn c - Thc y s pht trin ca tnh trng ng - Kch thch s t hnh thnh r - Kch thch s hnh thnh chi - y mnh s pht sinh th phi.

3 a b c 4

Nhm Polyamine Putrescin Spermidine Sspermine Jasmonic acid (Ja)

- Kch thch s hnh thnh thn c v thn hnh Methyl jasmonate (MeJa) - y nhanh s hnh thnh nh sinh trng.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

63 Bng 2.7. Hng dn cc phng php pha ho cht, kh trng v bo qun cc cht dng trong nui cy m v t bo thc vt Tn ho cht AUXIN 2,4-D IAA IBA NAA NAA Solution CYTOKININ Adenine BA BA Commercial Grade Ba solution 1,3-Diphenylurea 2Ip 2iP Solution 2iP Riboside Kinetin Kinetin thng mi Dung dch kinetin Kinetin Riboside Zeatin Riboside (trans) Cc cht HST khc () ABA GA3 Phloroglucinol Succinic Acid 2,2-Dimethydrazide Dung mi ho tan Cch kh trng Nhit qun Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Phin lc Hp kh trng Hp kh trng Hp kh trng Phin lc Phin lc Phin lc Hp kh trng Hp kh trng Hp kh trng bo

50% EtOH 1N NaOH 1N NaOH 1N NaOH

Nhit phng -00C 0-50C Nhit phng 0-50C Nhit phng Nhit phng Nhit phng 0-50C Nhit phng -00C -00c -00c -00c -00c -00c -00c -00c -00c Nhit phng Nhit phng 0-50C

H2O 1N NaOH 1N NaOH DMSO 1N NaOH -

1N NaOH 50%EtOH EtOH/H2O H2O

Khng sinh (ANTIBIOTIC) Amphotericin B DMF/H2O Phin lc 0-50C Tn ho cht Dung mi ho tan Cch kh trng Nhit bo qun Carbenicinllin H2O Phin lc 0-50C
ThS. Vu Ngc Dung

Cng ngh nui cy m t bo thc vt

64 Cefotaxime Chloramphenicol Gentamicin sulfate Kanamycin monosulfate Nystatin Rifampicin Vancomycin. HCL Cc ho cht khc Colchicine Folic Acid Riboflavin Mannitol Sorbitol L-Tryptophan MES Ca-Pantothenate Pepton l-Ascorbic Acid Biotin Citric Acid EDTA (Na2) EDTA (FeNA2) Ferrric Sulfate Ferric Tartrate Ferrous Sulfate Manganese Potasssium Phosphate H2O EtOH/H2O H2O H2O Phin lc Phin lc Phin l Phin lc 0-50C Nhit phng 0-50C Nhit phng -00C -00C 0-50C Nhit phng 0-50C Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng 0-50C Nhit phng Nhit phng -00C Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng

Insoluble Phin lc EtOH/H2O/ DMSO Phin lc H2O Phin lc H2O 1N NaOH H2O H2O H2O 1N NaOH H2O H2O H2O H2O H2O H2O H2O/ Nhit H2O/ Nhit H2O/ Nhit H2O/ Nhit H2O/ Nhit H2O/ Nhit H2O/ Nhit Phin lc Hp kh trng Phin lc Hp kh trng Hp kh trng Phin lc Hp kh trng Phin lc Hp kh trng Phin lc Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng Hp kh trng

Bng 2.8. Phng php tnh tng ng gia nng phn triu (ppm= part per million= 1 mg/l) v nng phn t gam ca mt s cht Nng ppm = mg/l 2,4-D 1 2 5. 371 10. NAA 4. 524 9. 048 IAA 5. 708 11. BA 4. 439 8. 879 kinetin 4. 439 9. 293 GA3 2. 887 5. 774
ThS. Vu Ngc Dung

Nng phn t gam (x 10-6M)

Cng ngh nui cy m t bo thc vt

65 741 16. 112 21. 483 26. 853 32. 223 37. 594 42. 965 48. 339 417 17. 125 22. 834 28. 542 34. 250 39. 959 45. 667 51. 376 175.18

3 4 5 6 7 8 9

13. 572 18. 096 22. 620 27. 144 31. 668 36. 193 40. 717 211.04

13. 318 17. 757 22. 197 26. 636 31. 075 35. 515 39. 954 225.26

13. 940 18. 586 23. 231 27. 880 32. 526 37. 173 41 820

8. 661 11. 548 14. 435 17. 323 20. 210 23. 097 . 25. 984 346.37

Phn t lng (mol Wt) 186.20

215.21

V d, cch tra bng nng ca 5 ppm (5 mg/l) ca IAA s tng ng vi nng phn t gam trn bng l 28.542 x 10-6M: Nng IAA: 5 ppm = 28,542 x 10-6M Nng BA: 8 ppm = 35,515 x 10-6M Nng NAA: 10 ppm = 10 x 5,371 x 10-6M = 53,71 M Chuyn i n v o: 10-6 M = 10-3mM = 1M 53 Bng 2.9. Cch tra cu t nng phn t gam tnh sang nng phn triu (ppm) hoc (1 mg/l) ca mt s cht (ppm = 1 mg/l) Nng phn t gam (10-6M) Nng ppm = mg/l NAA 1 2 3 4 0.186 2 0.372 4 0.558 6 0.744 2,4-D 0.221 0 0.442 1 0.663 1 0.884 IAA 0.175 2 0.350 4 0.525 5 0.700 BA 0.225 3 0.450 5 1.675 8 0.901 Kinetin GA3 0.2152 0.4304 0.6456 0.8608 0.346 4 0.692 7 1.039 1 1.385

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

66 8 0.931 0 1.117 2 1.303 4 1.489 6 1.675 8 186.2 0 2 1.205 2 1,326 2 1.547 3 1.768 3 1.989 4 211.0 4 7 0.875 9 1.051 1 1.226 3 1.401 4 1.576 6 175.1 8 0 1.126 3 1.351 6 1.576 8 1.802 1 1.027 3 225.2 6 5 1.731 9 2.078 2 2.424 6 2.771 0 3.117 3 346.3 7

5 6 7 8 9 Mol Wt

1.0761 1.2913 1.5065 1.7217 19369 215.21

V d: Nng kinetin 3 x 10-6 M = 0.6456 ppm Nng IAA 9 x 10-6M = 1.5766 ppm Cch chuyn i n v o khi nng phn t gam l mt s l: V d: Nng BA 4.5 x 10-6M = 0.9010 ppm + 1.1263 - 0.9010 ppm = 0.9010 ppm + 01127 ppm = 1.0137 ppm =ppm 2.6.1.7. Cc cht khng sinh Cht khng sinh l cht c sn xut bi cc loi vi sinh vt khc nhau, hoc l cc cht khng sinh tng hp. Chng c kh nng ngn chn s sinh trng ca cc vi sinh vt khc v cui cng lm tiu hu chng. Cac khang sinh (antibiotics) B sung cac khang sinh vao mi trng nui cy noi chung thng c tranh do s hin din cua chung trong mi trng lam chm sinh trng cua m va t bao. Tuy nhin, mt s t bao thc vt bi nhim mt cach h thng cac vi sinh vt. ngn chn sinh trng cua bon vi sinh vt nay, iu khng th thay th la lam giau mi trng bng khang sinh. Streptomycin hoc kanamycin nng thp kim soat hiu qua hin tng nhim co tinh h thng va mi trng c b sung cac khang sinh nay khng gy bt li cho s sinh trng cua t bao nui cy. Vai tr ca cc loi thuc khng sinh trong nui cy m v t bo thc vt:

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

67 1 2 3 - Ngn chn s ly nhim ca cc vi khun vo mi trng nui cy t bo v m thc vt - Ngn chn nm mc v nm men ly nhim vo m, t bo nui cy - Loi tr cc chng vi khun Agrobacterium dng trong chuyn gen ra khi mi trng v m nui cy (sau nui cy, hn hp Agrobacterium vi t bo thc vt chuyn gen hon thnh). - S dng khng sinh trong mi trng chn lc chn cc t bo hoc m c chuyn gen (mang gen ch th khng khng sinh). Cc t bo khng c chuyn gen s b cht trong mi trng c khng sinh nng thch hp. 2.6.1.8. Cc cht kh trng

S pht trin ca nm v vi khun rt bt li cho qu trnh nui cy. trnh iu ny mu cy phi c kh trng b mt bng cht kh trng trc khi cy. Nhng dung dch dng kh trng vt liu c k bng 15 vi nng v thi gian thch hp v tiu dit s nhim nm khun ng thi khng lm cht m nui cy. Quy trnh kh trng b mt 11. Ra mu bng cht ty nh trc khi thao tc vi dung dch kh trng. 22. Ra mu di vi nc chy t 10 - 30 pht. 33. Nhng ngp mu vo dung dch kh trng trong iu kin v trng. y np l ri lc nh trong thi gian kh trng. 44. Cht dung dch kh trng i ri ra vi ln bng nc ct v trng. Quy trnh kh trng c th c tng cng thm bng cch: 11. Trng vt liu trong dung dch cn 70% trc khi kh trng bng mt loi dung dch kh trng khc. Phng php hai bc (hai ngun) ny t ra c hiu qu vi mt s loi. 22. S dng thm dung dch xc tc nh Tween 20 hay 80 vo dung dch kh trng lm gim trng thi cng b mt v lm b mt mu c kh nng tip xc tt hn vi cht kh trng. 33. Kh trng trong iu kin chn khng c tc dng i vi vic kh bt kh v tng thm hiu qu cho qu trnh kh trng. Quy trnh kh trng thng c p dng: 11. Ra mu di vi nc chy khong 30 pht. 22. Nhng ngp mu trong cn 70%. 33. Ngm mu trong dung dch CLOROX 10% cng vi 2 - 3 git Tween 20 trong 15 pht.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

68 44. Ngm mu trong dung dch CLOROX 5% cng vi 2 - 3 git Tween 20 trong 10 pht. 55. Ra li mu 3 ln vi nc ct v trng. Bng 2.10. Nhng dung dch kh trng ph bin dng cho nui cy m t bo thc vt Cht kh trng Calcium hypocholorite Sodium hypocholorite Hydrogen peroxide Bromine water Ethyl alcohol Silver nitrate Murcuric choloride Benzalkonium choloride Antibiotics Nng (%) Thi gian (pht) 9 - 10 0.5 - 5 3 - 12 1-2 70 - 95 1 0.1 - 1.0 0.01 - 0.1 4 - 50 mg/l 5 - 30 5 - 30 5 - 15 2 - 10 0.1 - 5.0 5 - 30 2 - 10 5 - 20 30 - 60 kh trng

2.6.2. pH mi trng T bao va m thc vt oi hoi pH ti u cho sinh trng va phat trin trong nui cy. Trong khi chun bi mi trng, pH co th c iu chinh n gia tri cn thit cua thi nghim. pH anh hng n s di chuyn cua cac ion va i vi hu ht cac mi trng nui cy pH 5,0-6,0 trc khi kh trung c xem la ti u. pH cao hn se lam cho mi trng rt rn trong khi pH thp lai giam kha nng ng c cua agar. Hu ht cac mi trng nui cy ngheo m, vi th chung lam dao ng gia tri pH, s giao ng nay co th gy bt li cho thi nghim nui cy dai ngay va s sinh trng cua cac t bao n hoc cac qun th t bao mt thp. pH cua mi trng dinh dng anh hng trc tip ti qua trinh thu nhn cac cht dinh dng t mi trng vao t bao. Vi vy, i vi tng mi trng nht inh va tng trng hp cu th cua cac loai cy phai chinh pH cua mi trng v mc n inh ban u. Nui cy callus cua nhiu loai cy, pH ban u thng la 5,5-6,0 sau 4 tun nui cy pH at c gia tri t 6,0-6,5. c bit khi s dung cac loai phu gia co tinh kim hoc tinh acid cao nh amino acid, vitamin thi nht inh phai phai dung NaOH hoc HCl loang chinh pH mi trng v t 5,5-6,5.

Cng ngh nui cy m t bo thc vt

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69 Nhng thi nghim nui cy t bao n hay t bao trn thi vic chinh pH la bt buc. pH mi trng thng c iu chnh t 5,8 - 6,0 trc khi kh trng. Nhn chung nu PH cao hn 6 s lm mi trng b cng v nu thp hn 5 th agar kh ng. 2.6.3. Cac tac nhn lam rn mi trng Cac tac nhn lam rn hoc tao gel c s dung ph bin chun bi cac mi trng nui cy m dang rn (solid) hoc dang st (semi-solid). Trong nui cy dich long m hoc t bao bi ngp trong mi trng va cht do thiu oxy. Cac gel tao mt gia cho m sinh trng trong iu kin tinh (static conditions). Agar la mt loai polysaccharide thu c t mt s loai tao (nganh tao oRhodophyta), chung co u im hn cac tac nhn tao gel khac. Trc tin, gel cua agar khng phan ng vi cac thanh phn cua mi trng. Th hai, chung khng bi thuy phn bi cac enzyme thc vt va duy tri s n inh tt ca cac nhit nui cy c tin hanh. Binh thng, t 0,5-1% agar c dung trong mi trng tao gel rn chc pH c trng cho mi trng nui cy m v t bao thc vt. Trong nhng nghin cu v dinh dng, vic s dung agar c tranh bi vi agar thng phm khng sach do co cha mt s ion Ca, Mg, K, Na va mt s nguyn t khac dang vt. Tuy nhin, cac cht bn noi trn cung co th c loai bo bng cach ra agar vi nc ct hai ln it nht la 24 gi, trang trong cn va lam kh 60oC trong 24 gi. Noi chung, 80oC agar ngm nc chuyn sang trang thai sol va 40oC tr v trang thai gel. Kha nng ngm nc cua agar cao t 6-12 g/L nc. Gelatin nng cao (10%) cung co hiu qua tao gel nhng bi han ch s dung bi vi no nong chay nhit thp (25 oC). Cac hp cht khac a c th nghim thanh cng bao gm methacel, alginate, phytagel va gel-rite. Cng ty FMC Corp. gn y a phat trin mt loai agarose c tinh sach cao goi la Sea Plaque( k), loai nay co th c dung phuc hi cac protoplast n (single protoplast) trong nui cy. Cellophane uc l (perforated cellophane), cu giy loc (filter paper bridge), bc giy loc (filter paper wick), bot polyurethane (polyurethane foam) va xp polyester (polyester fleece) la cac phng thc thay i gia th c dung trong mi trng nui cy m hoc t bao. iu thun li khi lam vic vi cac hp cht tao gel nhn tao la chung tao ra cac gel sach cac nng tng i thp (1,25-2,5 g/L) va no co th giup phat hin s nhim bn c phat trin trong sut thi gian nui cy. Cac mu vt sinh trng tt hn trn agar hoc cac tac nhn tao gia th khac phu thuc vao loai m va tng loai khac nhau.
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70

2.7. Mt s loai mi trng c ban

Thanh phn c ban cua mt s loai mi trng nui cy m va t bao thc vt c trinh bay trong bang 2.1. Mi trng Murashige-Skoog (MS) la mt trong nhng loai mi trng c s dung rng rai nht trong nui cy m va t bao thc vt. Mi trng MS thich hp cho ca thc vt hai la mm va mt la mm. Ti nay, co rt nhiu cng thc cai tin mi trng MS trn c s cng thc gc do Murashige va Skoog cng b nm 1962. Mi trng B5 c thit k u tin cho nui cy callus hoc nui cy dich huyn phu t bao, sau o c cai tin va tr thanh mi trng thich hp cho nui cy protoplast. Mi trng nay cung c s dung tai sinh cy t protoplast. Mi trng Chu (N6) la loai mi trng rt hiu qua trong nui cy bao phn cua lua, c phat trin c bit cho nui cy bao phn cac loai hoa thao, mc du trong cac thi nghim nui cy bao phn mi trng c phat minh bi Nitsch (1969) vn c dung ph bin hn. Mi trng Nitsch ngay cang thich hp va ph bin trong nui cy cy u tng, co ba la o (red clover) va cac loai legume khac. Thanh phn dinh dng cua mi trng nay a giup tng sinh trng cua t bao trong qua trinh phat sinh phi va nui cy protoplast. Thnh phn ho hc ca mi trng ng vai tr quyt nh i vi thnh cng ca nui cy t bo v m thc vt. Mi loi cy, thm ch mi kiu gen, cc kiu nui cy khc nhau (nui cy m so, huyn ph t bo, t bo trn, bao phn, ht phn) c nhng i hi khc nhau v thnh phn mi trng. Khi bt u nui cy m mt loi mi hoc mt ging mi, cn phi la chn cho i tng nghin cu mt loi mi trng c bn ph hp. Cho n nay, cc nh khoa hc to ra mt s lng rt ln cc mi trng thch hp vi tng i tng v mc tiu nghin cu. Lch s ra i ca cc mi trng nui cy m v t bo thc vt c lit k bng 2, trong c mt s mi trng c bn c s dng rt ph bin (bng 3), c bit l cc mi trng nh MS, LS, B5 v WP. Thnh phn ho hc ca cc mi trng ny c th hin cc bng 4, 5, 6 v 7. Cc cht iu ho sinh trng ng vai tr quyt nh i vi pht sinh hnh thi, phn chia v phn ho t bo, hnh thnh m v c quan nh pht chi v to r. i khi thay i nng cc cht khong ca mi trng cng ng vai tr quan trng trong vic iu khin cc giai on pht trin khc nhau ca qu trnh nui cy. Bng 2.11. Mt s mi trng c bn thng c s dng trong nui cy m v t bo thc vt Thnh phn Muras White(1 Gamborg( hige & 963 1968) Skoog Nits Hell Sche ch er nk (195 (195 & Nits ch & Kohlen Kno bach & p Schmidt (186

Cng ngh nui cy m t bo thc vt

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71 (1962) 1) 3) Hild eb (197 2) 400 200 2,50 0 Nits (1975) ch (196 7) 125 125 500 185 166 950 5)

(NH4)2 SO4 MgSO4 7H2O KC1 CaC12 2H2O NaNO3 KNO3 Ca(NO 3)2 4H2O NH4N O3

370

720 200 65 80 300

134 500 150 3,000 -

250 1,50 0 25 2,00 0 -

250 750 75 600 -

250 250 1,00 0 250 -

Na2SO4 440 1,900 -

1,650

16.5 7 3

150 27.8 37.3 10 (1 H2O) 2

250 3 0.5

125 _ 0.1 1

300 15 20 10 0.1

125 27.8 5 37.2 5 25 10

720 68 27.85 37.25 25 10

NaH2P O4 H2O NH4H2 PO4 KH2PO

4

170 27.8 1 22.3 8.6

FeSO4 7H2) Na2ED TA MnSO4 4H2O ZnSO4

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72 7H2O CuSO4 5H2O H2SO4 Fe2(SO 4)3 NiC12 6H2O CoC12 6H2O A1C13 FeC13 6H2O FeC6O5 H7 5H2O K1 H3BO3 Na2M0 O4 2H2O 0.025 0.025 2.5 0.025 0.025 0.02 5 0.5 10 0.03 0.03 0.03 1 0.2 0.1 0.02 5 0.02 5 0.025 -

0.83 6.2 0.25

0.75 1.5 -

0.75 3 0.25

0.5 0.5 0.25

0.01 1 -

1.0 5 0.1

10 0.25

10 0.25

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73 Bng 2.12. Mi trng MS (Murashige & Skoog, 1962) Murashige T. and Skoog F., 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum. 15, 473-497. Mui khong a lng Potasium nitrate (KNO3) Amonium nitrate (NH4NO3) Calcium chloride (CaCl2.H2O) Nng (mg/l) 1,900 1,650 440 Nng Vi lng (mg/l) Manganese sulfate (MnSO4. 4H2O) 22.3 Boric Acid (H3BO3) Colbalt chloride (CoCl2.6H2O) Cupric Sulfate (CuSO4.5H2O) Ferrous sulfate (FeSO4.7H2O) 0.83 0.25 8.6 37.2 100 mg/l 0.5 mg/l 1-30 mg/l 2.0 g/l 20 g/l Nicotinic aAcid Thiamine HCl Kinetin Edamine Agar 0.5 mg/l 0.1 mg/l 0.04-10 1.0 g/l 10 g/l 6.2 0.025 0.025 27.8

Magnesium sulfate (MgSO4.7H2O) 370 Potassium phosphate (KH2PO4) Potassium Iodine (KI) Sodium molybdate (Na2MoO4.2H2O) Zinc Sulfate (ZnSO4.7H2O) Na2EDTA.2H2O Cc cht hu c Myo-inositol Pyridoxine HCl IAA Glycine (recrytallized) Sucrose 170

Bng 2.13 . Mi trng LS (Linsmaier & Skoog, 1965) Linsmaier, E.M. and Skoog, F., 1965. Organic growth factor requirements of tobacco tissue cultures. Physiologia Plantarum. 18,100-127.

Mui khong a lng Amonium nitrate (NH4NO3)

Cng ngh nui cy m t bo thc vt

Nng (mg/l) 1,650

Nng Vi lng Boric acid (H3BO3) (mg/l) 6.2

ThS. Vu Ngc Dung

74 Calcium chloride (CaCl2.H2O) Magnesium (MgSO4.7H2O) 440 Colbalt (CoCl2.6H2O) chloride 0.025 0.025 27.8 22.3

sulfate 370 1,900 170

Cupric sulfate (CuSO4.5H2O) Ferrous sulfate (FeSO4.7H2O) Manganese (MnSO4.4H2O) 0.83 0.25 8.6 37.3 sulfate

Potassium nitrate (KNO3) Potassium phospate (KH2PO4) Potassiom iodine (KI)

Sodium molybdate (Na2MoO4.2H2O) Zinc sulfate (ZnSO4.7H2O) Na2EDTA Cc cht hu c myo-Inositol IAA Sucrose Aminobenzoic acid Gibberellic acid Adenine sulfate Guanylic acid L-Glutamine Bng 2.14. Mi trng B5 (Gamborg cs., 1976) 100 mg/l 1-30 mg/l 30g/l 0.1 mg/l 1.0 mg/l 40 mg/l 200 mg/l

Thiamine.HCl Kinetin Agar Tyrosine Casein hydrolysate Cytidylic acid L-Asparagine 500 mg/l

0.4 mg/l 0.001-10 mg/l 10g/l 100 mg/l 1-3 g/l 200 mg/l 500 mg/l

Cc cht b sung (thay i tu theo mc ch v i tng nghin cu)

Gamborg, O.L., Murashige, T., Thorpe, T.A., and Vasil, I.K., 1976. Plant tissue culture media in vVitro, 12, 473-478. Mui khong a lng Amonium sulfate ((NH4)2SO4) Calcium chloride (CaCl2.H2O) Nng mg/l 134 150 Vi lng Boric acid (H3BO3) Colbalt chloride (CoCl2.6H2O) Cupric Sulfate (CuSO4.5H2O) Ferrous sulfate (FeSO4.7H2O)
ThS. Vu Ngc Dung

Nng mg/l 3.0 0.025 0.025 27.8

Magnesium sulfate (MgSO4.7H2O) 246 Potassium nitrate (KNO3)

Cng ngh nui cy m t bo thc vt

2,528

75 Manganese sulfate, monohydrate (MnSO4.H2O) Potassiom iIodine (KI) Sodium molybdate (Na2MoO4.2H2O) Sodium phosphate (NaH2PO4.H2O) Zinc sulfate (ZnSO4.7H2O) Na2EDTA.2H2O Cc cht hu c myo-Inositol PyridoxineHCl 2,4-D Sucrose Bng 2. 15. Mi trng WPM - Woody Plant Medium (Lloyd and McCown, 1980) Lloyd, G. B. and McCown, B. H., 1980. Commercial Feasible Micropropagation of Mountain Laurel, Kalmia latifolia, by use of shoot-tip culture. Proceeding of Int. Plant. Propagators Society, 30, 421-427. Mui khong a lng Amonium nitrate (NH4NO3) Calcium nitrate (Ca(NO3)2.4H2O) Calcium chloride (CaCl2. 2H2O) Nng mg/l 400 556 96 Nng Vi lng Boric Acid (H3BO3) Zinc sulfate (ZnSO4.7H2O) Na2EDTA.2H2O Cupric Sulfate (CuSO4.5H2O) Ferrous sulfate (FeSO4.7H2O) mg/l 6.2 8.6 37.2 0.25 27.8 100 mg/l 1.0 mg/l 0.1-1.0 mg/l Nicotinic Acid ThiamineHCl Kinetin 20 g/l 1.0 mg/l 10 mg/l 0.1 mg/l 10 0.75 0.25 150 2.0 37.2

Magnesium sulfate (MgSO4.7H2O) 370 Potassium sulfate (K2SO4) Potassium phospate (KH2PO4) 990 170

Manganese sulfate (MnSO4.4H2O) 22.3 0.25

Sodium molybdate (Na2MoO4.2H2O) Cc cht hu c Myo-inositol

Cng ngh nui cy m t bo thc vt

100 mg/l

Nicotinic acid

0.5 mg/l
ThS. Vu Ngc Dung

76 PyridoxineHCl Glycine Sucrose 0.5 mg/l 2.0 mg/l Thiamine HCl Agar 20 g/l 1.0 mg/l 6.0 g/l

2.8. Mt s thut ng c ban trong nui cy m va t bao thc vt

- Nui cy m (tissue culture) l thut ng dng ch qu trnh nui cy v trng in vitro cc b phn tch ri khc nhau ca thc vt. K thut nui cy m dng cho c hai mc ch nhn ging v ci thin di truyn (v d: ging cy trng), sn xut sinh khi cc sn phm ha sinh, bnh hc thc vt, duy tr v bo qun cc ngun gen qu Cc hot ng ny c bao hm trong thut ng cng ngh sinh hc (biotechnology). - Thut ng nhn ging in vitro (in vitro propagation) hay cn gi l vi nhn ging (micropropagation) c s dng c bit cho vic ng dng cc k thut nui cy m nhn ging thc vt, bt u bng nhiu b phn khc nhau ca thc vt c kch thc nh, sinh trng iu kin v trng trong cc ng nghim hoc trong cc loi bnh nui cy khc. Trong thc t, cc nh vi nhn ging (micropropagators) dng thut ng nhn ging in vitro v nui cy m thay i cho nhau ch mi phng thc nhn ging thc vt trong iu kin v trng. Thut ng ng ngha (synonymous) l nui cy in vitro (in vitro culture). Nhn ging in vitro v nui cy m bt u bng cc mnh ct nh ca thc vt, sch vi sinh vt, v c nui cy v trng. Thut ng u tin dng trong qu trnh nhn ging l explant (mu vt) tng ng vi cc phng thc nhn ging khc l cutting (cnh gim), layer (cnh chit), scion (cnh ghp) hoc seed (ht). Nm thut ng khc c dng ch cc loi ti sinh sinh dng (vegetative or somatic regeneration) c bn trong nhn ging in vitro v nui cy m: - Nui cy nh phn sinh (meristem-tip culture) Phng thc nhn ging bng cch dng cc b phn rt nh ca nh chi (shoot-tip) bao gm m phn sinh nh ring r (single apical meristem) v mm l non (young leaf primordia) ko di chi (shoot elongation) ngay sau . Kiu nui cy ny c dng ln u tin lm sch virus (virus-free) thc vt. Nu dng nh phn sinh khng th sng st v to r mt cch c lp, th c th thay th bng phng thc vi ghp (micrografting). -Sinh sn chi nch (axillary shoot proliferation) Kiu nui cy ny s dng chi ca cc im sinh trng bn v ngn ni m s ko di ca chi ngn (elongation of terminal shoot) b km hm v s sinh sn chi nch c y mnh. S iu khin ny cho php nhn nhanh c cc chi in vitro (microshoots), l cc chi c th tch ra v to r in vitro hnh thnh cy trong ng nghim (microplants), hoc n c th c ct ra ring bit to thnh cc cnh gim in vitro (microcuttings) to r bn ngoi in vitro. - To chi bt nh (adventitious shoot induction)

Cng ngh nui cy m t bo thc vt

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77 Loi nui cy ny cho php hnh thnh cc chi bt nh hoc trc tip trn mu vt hoc gin tip t m callus, m m callus ny hnh thnh trn b mt vt ct ca mu vt. -Pht sinh c quan (organogenesis) Thut ng ny dng m t qu trnh pht trin cc chi, r bt nh t cc khi t bo callus. Qu trnh ny xy ra sau thi im m mu vt c t vo mi trng nui cy v s bt u cm ng to callus. - Pht sinh phi v tnh (somatic embryogenesis) Thut ng ny dng cho s pht trin ca cc phi hon chnh t cc t bo sinh dng c sn xut t cc ngun mu vt khc nhau sinh trng trong nui cy in vitro. Thut ng tng ng i vi s pht trin phi thc vt sinh trng trong iu kin t nhin l pht sinh phi hu tnh (zygotic embryogenesis) v pht sinh phi v tnh (apomitic embryogenesis). - Bao quan lanh su (cryopreservation): Bao quan t bao, m, phi, hat nhit siu lanh, thng la -100oC. - Bin nap bng in (electroporation): Ky thut dung dong in tao nhng l thung trn mang t bao chuyn nap nhng vt la, c bit la DNA t ngoai vao trong t bao. - Bin i dong giao t (gametoclonal variation) : Bin i kiu hinh khng do kiu nhn hoc ngoai bin ma do ngun gc giao t gy nn. - Bin nap (transformation): Qua trinh a vao va dung nap mt cach chc chn DNA la vao trong t bao thc vt bt lun bng cach gi co c mt bin i di truyn thi u c coi la bin nap thanh cng. - Bin nap in vitro (in vitro transformation): Bin i di truyn t bao nui cy trong iu kin in vitro di tac ng cua cac tac nhn khac nhau nh cht gy ung th, chiu xa, virus, vi khun gy bin i di truyn. - Bi th ung (euploid): Trang thai bi th ma t bao co s lng nhim sc th ung bng bi s cua s nhim sc th n bi. - Cam ng (induction): Hormone gy tao mt loai cu truc, b phn hay mt qua trinh nao o trong iu kin in vitro. - Cy chuyn (passage hoc subculture) : Chuyn t bao, m hay mu vt nui cy sang binh nui co cha mi trng mi pha kt hp vi tach nho hoc lam loang mt nhn s lng. - Chi hoc r bt inh (adventitious roots or shoots) : La nhng chi hoc r phat sinh t nhng vung khac thng, khng phai la hp t. - Chung phu (substrain): c tach va nhn t mt nhom t bao cua mt chung vi nhng tinh trang ma chung b me o khng co. - Chung t bao (cell strain): Chung t bao gm nhng t bao co c im ring bit c chon t nui cy khi sinh hay dong t bao co trc. Thng phai nu ro ngun gc cua chung t bao trong cac cng b khoa hoc nht la khi cac chung o co ngun gc t cac phong thi nghim khac.
Cng ngh nui cy m t bo thc vt ThS. Vu Ngc Dung

78 - Con lai t bao soma (somatic cell hybrid): T bao hoc cy hoan chinh tao c do lai t bao trn (protoplast) vi c tinh di truyn khac nhau. - Di bi (heteroploid): Tinh trang cac t bao trong mt thi nghim nui cy co b nhim sc th nhiu mc bi th khac nhau. Khai nim nay dung cho mt c th a bao hay nui cy gm nhiu t bao. - Di nhn (heterokaryon): Mt t bao co hai hay nhiu nhn khac nhau trong mt t bao cht chung, thng thng la do dung hp t bao tao thanh. - Di truyn t bao cht (cytoplasmic inheritance) : Hin tng di truyn do cac gen ngoai nhn quyt inh, vi du: gen cua luc lap, ty th hay plasmid. - Di truyn hoc t bao soma (somatic cell genetics) : Nganh khoa hoc chuyn nghin cu v di truyn cua t bao soma, phn ln la t bao trong nui cy in vitro. - inh sinh trng chi ngon (shoot apical meristem): M cha phn hoa hinh chop nm trong cac mm la chi ngon, khi tach khng ln qua 0,1 mm. - xp long (friability): Tinh trang khng lin kt cua cac t bao thc vt trong khi m seo. M seo xp long rt kho tai sinh cy hoan chinh. - Dong (clone): Tp hp cac ca th nhn c bng phng thc nhn ging v tinh t mt ca th duy nht. - Dong giao t (gametoclone): Nhng thc vt c tao ra t giao t, bao t giam nhim hoc th giao t. - Dong t bao (cell line): Khai nim chi s nui cy cua nhng t bao co ngun gc chung t ln cy chuyn u tin. - Dung hp t bao trn (protoplast fusion): Ky thut lam cho hai hay nhiu protoplast dung hp vi nhau thanh mt t bao. - t bin (mutation): Bin i kiu hinh do thay i gen hay do gen mi. - ng nhn (homokaryon): T bao co cha hai hay nhiu nhn ng nht v mt di truyn trong mt t bao cht chung. Thng la san phm hinh thanh sau dung hp t bao. - Gia hoa (senesence): Biu hin mt kha nng phn chia cua t bao va m trong nui cy. - Giai oan I (stage I): Trong nhn ging in vitro giai oan I la thi ky khi u tao nguyn liu v trung cho nui cy. - Giai oan II (stage II): Trong nhn ging in vitro giai oan II la thi ky nhn nhanh v s lng m, phi, r trong binh nui. - Giai oan III (stage III): Trong nhn ging in vitro giai oan III la thi ky chun bi cho cy ging u iu kin chuyn ra ngoai t. Thi ky nay bao gm vic tao r cho cy, thich nghi iu kin ngoai canh va bt u chuyn t trang thai di dng sang trang thai t dng. - Giai oan IV (stage IV): Trong nhn ging in vitro y la giai oan chuyn cy t iu kin binh nui sang iu kin t nhin trng trn t. Co nhiu loai cy co th chuyn trc tip t giai oan II sang giai oan IV. - Hin tng b sung di truyn (complementation) : Hin tng hai khuyt tt di truyn co kha nng h tr ln nhau lam cho tinh trang khim khuyt o mt i.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

79 - Hiu sut bam (attachment efficiency) : Phn trm s t bao bam c ln b mt mi trng nui cy trong mt thi gian nht inh. - Hiu sut nui trai (plating efficiency): Khai nim nay ng nghia vi hiu sut tao dong, noi ln phn trm s t bao phat trin thanh dong khi nui trai trn b mt mi trng. - Hiu sut tao dong (cloning efficiency) : Phn trm s t bao a tao c dong khi nui trai trn b mt mi trng. - Hp bao sinh cht (cybrid): T bao sng tao c khi dung hp th sinh cht (khng co nhn) vi mt t bao. o la th lai t bao cht. - Hp bao recon (recon, reconstituted cell hay reconstructed cell): Hp bao sng binh thng c tao khi dung hp th nhn cua t bao nay vi th nguyn sinh cht cua t bao khac. - Ky thut v trung (aseptic technique) : Qui trinh ngn nga s nhim nm, vi khun, siu vi khun hoc cac loai vi sinh vt khac i vi nui cy m va t bao. - Lai t bao (cell hybridization): S dung hp hai hay nhiu t bao khng ging nhau tao mt th t bao hn hp (synkaryon). - Lai t bao soma (somatic cell hybridization): Qua trinh dung hp protoplast cua t bao soma ng vt hay thc vt co c tinh di truyn khac nhau. - Ln cy chuyn (passage number): S ln t bao, m hay mu vt nui cy c cy chuyn, qua o co th tinh tui va h s ng trng cua chung. - Lch bi (aneuploidy): Tinh trang b nhim sc th cua t bao mang s lng nhim sc th khac vi bi s cua b nhim sc th n bi. Co th la d hoc thiu mt hay nhiu nhim sc th. - Lng bi gia (pseudodiploid): Lng bi hay nhi bi gia la th nhi bi, nhng do sp xp mi nhim sc th mt kha nng tao i trong khi phn bao. - Mt qun th (population density): S lng t bao trn n vi din tich nui cy hay trn n vi th tich nui cy. - Mu vt (explant): M c tach t nguyn liu ban u dung duy tri hoc nui cy. - Mi trng nhn tao (chemically difined medium): Dung dich dinh dng dung nui cy chi cha nhng thanh phn ma cu truc hoa hoc a c bit. - M seo (callus): Khi m thc vt gm nhng t bao khng phn hoa, co kha nng phn chia, c phat sinh t cac t bao a phn hoa it nhiu. Khi thc vt bi thng tn thng tao loai m nay trn vt seo, vi th co tn goi la m seo. - Ngoai bin (epigenetic variation): Hin tng bin i kiu hinh, nhng khng bin i kiu gen. Ngoai bin co th do s thay i cua qua trinh methyl hoa DNA trong qua trinh phin ma va dich ma hay s bin i sau dich ma. - Nhn dong (clonal propagation): Nhn ging v tinh nhng dong thc vt co ngun gc t mt ca th hay mt manh ct duy nht, am bao hoan toan ng nht v di truyn. - Nhn ging in vitro (in vitro propagation) : Nhn ging mt loai thc vt trong ng nghim (binh thuy tinh, binh plastic, hp plastic,...) trn mi trng nhn tao va trong iu kin v trung. ng nghia vi khai nim micropropagation.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

80 - Nhn ging v tinh (vegetative propagation): Nhn ging khng thng qua qua trinh sinh san hu tinh, bao gm cac ky thut nh: nhn ging in vitro, gim cac b phn canh, manh la, oan r, chit, ghep, tach gc,... - Nhi bi (diploid): Tinh trang bi th ma t bao co tng i tt ca cac nhim sc th, tr nhim sc th gii tinh va hoan toan ging b nhim sc th cua loai o ngoai t nhin. - Nhim bin (transfection): Khai nim nay trc y c dung trong vi sinh vt hoc chi qua trinh bin nap gen trc tip bng DNA cua virus vao t bao. Ti nay khai nim nay c m rng chi qua trinh bin nap gen bng DNA tinh khit khng phn bit ngun gc. - Nui cy inh ngon (shoot tip (apex) culture) : S dung inh sinh trng chi ngon cung vi mt hai mm la vi tng kich thc t 0,1 n 1,0 mm. - Nui cy c quan (organ culture) : Duy tri va phat trin toan b hay mt phn c quan ng, thc vt trong iu kin in vitro. - Nui cy inh sinh trng (meristem culture) : Nui cy mu m hinh chop khng ln hn 0,1 mm. Thng c tach t r ngon di kinh hin vi. - Nui cy huyn phu (suspension culture) : Phng thc nui t bao n hay cum nhiu t bao (cell aggregate) trang thai l lng trong mi trng long. - Nui cy m tissue culture): Duy tri va sinh trng cac loai m trong iu kin in vitro nhm iu khin phn hoa v hinh thai va chc nng cua chung. - Nui cy m thc vt (plant tissue culture): Duy tri va nui dng t bao, m, c quan, hay cy hoan chinh cua thc vt trong iu kin in vitro. - Nui cy khi u, nui cy s cp (primary culture) : Nui cy u tin khi tach t bao, m hoc mu vt t c th ban u tinh n khi cy chuyn hu hiu ln u, t o se thu c dong t bao. - Nui cy phi (embryo culture): Duy tri va phat trin phi non hoc a trng thanh c phn lp t hat. - Nui cy t bao (cell culture): Khai nim chi nhng nui cy trong ng nghim ( in vitro) cua nhng t bao k ca t bao n khng phn hoa thanh m. - Pht sinh phi v tnh (somatic embryogenesis) : Thut ng ny dng cho s pht trin ca cc phi hon chnh t cc t bo sinh dng c sn xut t cc ngun mu vt khc nhau sinh trng trong nui cy in vitro. Thut ng tng ng i vi s pht trin phi thc vt sinh trng trong iu kin t nhin l pht sinh phi hu tnh (zygotic embryogenesis) v pht sinh phi v tnh (apomitic embryogenesis).

- Phn hoa (differentiation): Qua trinh chuyn mn hoa cac t bao v chc nng va hinh thai tao ra cac loai m, c quan va c th hoan chinh. - Phn hoa hinh thai (morphogenetic differentiation) : Phn hoa ring v mt hinh thai, chu yu noi n s hinh thanh chi va r t m seo. ng nghia vi phat sinh hinh thai. - Phn hoa phi (embryogenesis): Qua trinh hinh thanh phi (phi hoa) va phat trin phi.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

81 - Phat sinh c quan (organogenesis): Hin tng cac c quan ring bit nh chi, la, r hinh thanh trong nui cy t bao, m seo hoc m khac. Hin tng nay cung xay ra trong nui cy t bao ng vt. y la kt qua cua qua trinh phn hoa hinh thai hay phn hoa chc nng hay ca hai. - Phat sinh hinh thai (morphogenesis): Hin tng phat sinh va phat trin cac cu truc ging hay khng ging cac c quan nh chi, r, la t t bao, m seo hay mu vt nui cy. - Sach bnh (pathogen free): c kim inh la khng mang mm bnh. - Sach virus (virus free): c kim tra bng phep th c hiu chng to khng mang loai virus c trng cn phat hin. - Sinh sn chi nch (axillary shoot proliferation) :Kiu nui cy ny s dng chi ca cc im sinh trng bn v ngn ni m s ko di ca chi ngn (elongation of terminal shoot) b km hm v s sinh sn chi nch c y mnh. S iu khin ny cho php nhn nhanh c cc chi in vitro (microshoots), l cc chi c th tch ra v to r in vitro hnh thnh cy trong ng nghim (microplants), hoc n c th c ct ra ring bit to thnh cc cnh gim in vitro (microcuttings) to r bn ngoi in vitro. - Tai sinh (regeneration): Hin tng t bao hoc m nui cy chiu tac ng kich thich phn hoa thanh m, c quan hoc cy hoan chinh. - Tng nui dng (feeder layer) hoc t bao nui dng (nurse cells) : Lp t bao co th a bi chiu xa lam mt kha nng phn bao c trai bn di cung cp mt s cht cn thit cho lp t bao khac nui bn trn. - Tao phi soma (somatic embyogenesis): Qua trinh hinh thanh phi t t bao khng phai la t bao sinh san hay giao t th trong nui cy in vitro. - To chi bt nh (adventitious shoot induction) : Loi nui cy ny cho php hnh thnh cc chi bt nh hoc trc tip trn mu vt hoc gin tip t m callus, m m callus ny hnh thnh trn b mt vt ct ca mu vt.

- T bao lai (hybrid cell): La t bao co mt nhn c hinh thanh sau dung hp hai t bao dn n s hinh thanh mt nhn hn hp (synkaryon). - T bao tiu phn (microcell): Mt phn nho t bao cha vai ba nhim sc th, xut hin trong khi tin hanh ky thut pha bo nhn t bao. - T bao trn (protoplast): T bao bi lam mt toan b thanh t bao. Khai nim nay dung cho ca thc vt, vi khun va nm, ng nhin hai trng hp cui khi thanh t bao cha bi loai hoan toan ngi ta dung khai nim "t bao trui" (spheroplast). - Th bao cht (cytoplast): T bao nguyn ven sau khi lam mt nhn. - Th lai t bao cht (cytoplasmic hybrid): ng nghia vi hp bao sinh cht (cybrid). - Th nhn (karyoplast): Nhn t bao thu c khi phn lp, c boc bi mt lp nguyn sinh cht rt mong va mang nguyn sinh.

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung

82 - Thi gian tng i qun th (population doubling time) : Thi gian ma s lng t bao cua dong hay chung nui cy tng n gp i k t khi bt u nui. S ln gp i qun th trong mt t nui cy c tinh bng cng thc sau: N 333 n = log , No Trong o, N: s t bao trong binh nui cy khi kt thuc t nui, No: s t bao trong binh khi bt u nui. Lu y nn ly s lng t bao sng hay bam c tinh. Thi gian tng i qun th la thi gian cn thit s lng t bao cua qun th o tng gp i, vi du: t 1.106 thanh ra 2.106. - Tinh toan nng (totipotency): Mt c tinh cua t bao la co kha nng phat trin thanh moi kiu t bao co trong c th trng thanh ma t o no c tach ra, tc la co kha nng tai sinh thanh mt c th hoan chinh. - Trang thai non tre (juvenile): Mt giai oan phat trin trong chu trinh sinh san hu tinh cua thc vt, phn bit vi giai oan trng thanh la khng phan ng vi cac tac nhn kich thich ra hoa. - Trang thai t sinh (habituation): Trang thai phat trin khng cn b sung cht iu khin sinh trng ngoai sinh cua mt qun th t bao. Phn bit vi t dng. - T dng (autotrophy): Kha nng phat trin nh quang hp khng cn cung cp mt ngun dng cht cha carbon hu c. Ngc vi di dng la bt buc phai b sung ngun dng cht hu c vao mi trng sng. - Tui th h t bao (cell generation time) : Thi gian gia hai ln phn chia cua t bao. Khai nim nay khng ng nghia vi thi gian gp i qun th. - c ch sinh trng phu thuc mt (density-dependent inhibition of growth): hin tng c ch sinh trng bi mt t bao tng ln. - V trung (asepsis): Khng bi tp nhim cac loai vi khun khac. - Vi nhn ging (micropropagation) hay nhn ging (in vitro propagation) c s dng c bit cho vic ng dng cc k thut nui cy m nhn ging thc vt, bt u bng nhiu b phn khc nhau ca thc vt c kch thc nh, sinh trng iu kin v trng trong cc ng nghim hoc trong cc loi bnh nui cy khc. Trong thc t, cc nh vi nhn ging (micropropagators) dng thut ng nhn ging in vitro v nui cy m thay i cho nhau ch mi phng thc nhn ging thc vt trong iu kin v trng. Thut ng ng ngha (synonymous) l nui cy in vitro (in vitro culture). Nhn ging in vitro v nui cy m bt u bng cc mnh ct nh ca thc vt, sch vi sinh vt, v c nui cy v trng. Thut ng u tin dng trong qu trnh nhn ging l explant (mu vt) tng ng vi cc phng thc nhn ging khc l cutting (cnh gim), layer (cnh chit), scion (cnh ghp) hoc seed (ht).

Cng ngh nui cy m t bo thc vt

ThS. Vu Ngc Dung