Académique Documents
Professionnel Documents
Culture Documents
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BI 1:
M U
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1.CC THIT B C BN CA MT PHNG TH NGHIM NUI CY M V T
BO THC VT
a. Phng ra v ct nc
- My ct nc 1 ln
- My ct nc 2 ln
b. Phng hp sy
- Autoclave
- T sy 60 200oC
c. Phng chun b mi trng
- Cn phn tch (chnh xc n 0,0001 g)
- Cn k thut (chnh xc n 0,01 g)
- pH k
- My khuy t
- T lnh
- L vi sng (microwave)
d. Phng thao tc nui cy
- T cy v trng (laminar)
- Qut thng gi
- n t ngoi treo tng
e. Phng nui cy
- Cc gin k c gn n hunh quang
- My iu ha nhit
- My lc nm ngang
- T m
f. Phng th nghim:
(phng ny dng tin hnh cc phn tch sinh ha, phn t v di truyn)
- Knh hin vi 2 mt ( phng i 1000 ln)
- Knh lp 2 mt ( phng i 75 ln)
- Microtome
- My nh k thut s
- H thng n chiu
- Quang ph k
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Biotechnology
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Biotechnology
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b. Nt y
Thng dng nht l cc nt y lm bng bng gn 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
cc nhc im sau:
- Nu khi hp nt bng b t hoc dnh mi trng th v sau s rt d b nhim nm, nht
l vi nhng th nghim tin hnh trong mt thi gian di
- Thao tc lm nt bng chm, khng thun tin khi nui cy trn qui
m ln
- Ch dng c vi ln l phi b
Hin nay ngi ta s dng nhiu loi np y khc thay th nt bng. 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 v trng nhit 1210C m khng b bin dng. Mt s phng th nghim dng np
ng nghim bng
inox hoc cao su rt thun tin cho vic v trng kh hoc t. Cng c th s dng giy
nhm lm np y
c. Mi trng
Mi trng nui cy thng c hp kh trng trong ni hp (autoclave), kh trng bng
p sut hi nc bo ha. Thi gian hp t 15-30 pht p sut hi nc bo ha l 103,4
kPa (1atm) tng ng vi nhit 1210C. nhit 1210C, hu ht cc sinh vt c trong
mi trng u b tiu dit, k c dng bo t. Sau khi v trng cn phi lm kh np ng
nghim hoc nt bng trnh b nhim tr li.
Bng 2: Thi gian kh trng dung dch v cc mi trng lng bng
ni hp (autoclave) 121oC ti 103,4 kPa
Th tch mi trng (mL)
<50
15
75
20
250-500
25
1000
30
Vic hp kh trng bng ni hp th khng thch hp vi nhiu ho cht nhy cm vi nhit
nh: cc acid amin, cc vitamin, cc hormon tng trng, v cc cht khng sinh. Cc cht
nh vy thng phi c kh trng bng cch lc v trng. Mng lc c lm bng mng
polyethylen hoc sI cellulose. Cc l trn mng siu lc ny c thit k hiu qu cho vic
gi li cc vi sinh vt gy nhim.
d. Lc v trng
Phng php n gin nht l dng cc mng lc Millipore hoc dng cc phu lc thy
tinh xp s 5. Mt s loi mng lc v trng ca hng Millipore. y l loi mng lc c
kh trng bng chiu x v ch dng 1 ln
Phng php s dng mng lc Millipore: hng Millipore cung cp mng lc v gi
bng nha hu nhit. Di y m t mng lc loi Millipore Swinex c ng knh 25mm.
B lc gm c gi bng loi nha chu nhit gm np v , vng cao su v mng lc. t
mng lc (c kch thc l 0,25m) trn , t vng cao su ln v vn cht np vo . Gi
ton b b lc vo trong mt t giy nhm v kh trng trong autoclave 121 oC trong 15-20
Cng Ngh Sinh Hc Vit Nam
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Biotechnology
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pht. ng thi cng hp v trng mt bnh hu tinh hng dch lc. Dng ng tim ht
dch lc v bm qua b lc.
2.3.2 Kh trng m thc vt
M cy c th l hu ht cc b phn khc nhau ca thc vt nh ht ging, phi, non,
hoa, l, u r, thn ctu theo s tip xc vi mi trng bn ngoi, cc b phn ny cha
nhiu hay t vi khun v nm. ng la non khi cn trong b, m tht bn trong qu thng
t b nhim vi sinh vt; ngc li, l, thn c bit cc b phn nm su trong t nh r,
c c lng nm, khun tp rt cao. Hu nh khng th v trng m cy c nu nm,
khun nm su cc t bo bn trong m ch khng hn ch b mt. L khoai lang c th
v trng d dng trong ma kh nhng khng th lm c trong ma ma. Phng php v
trng m cy thng dng nht hin nay l dng cc cht ho hc c hot tnh dit nm khun.
Hiu lc dit nm khun ca cc cht ny ph thuc vo thi gian x l, nng v kh nng
xm nhp ca chng vo cc k ngch li lm trn b mt m cy, kh n ng y ht cc bt
kh bm trn b mt m cy. tng tnh linh ng v kh nng xm nhp ca cht dit
khun, thng thng ngi ta x l m cy trong vng 30s trong ru ethylic 70% sau mi
x l dung dch dit khun. ng thi ngi ta thm cc cht gim sc cng b mt nh
Tween 80, Fotoflo, Teepol vo dung dch dit nm khun. c khi nim v nng v thi
gian s dng cc cht dit nm khun x l m cy, xin dn ti liu nghin cu ca Street
(1974) bng sau:
Tc nhn v trng
Nng %
Calci hypochlorit
Natri hypochlorit
Hydro peroxid
Nc Brom
HgCl2
Cht khng sinh
9 10
2
10 12
1-2
0.1 1
4 50 mg/l
Thi gian x l
( pht)
5 30
5 30
5 15
2 10
2 10
30 60
Hiu qu
Rt tt
Rt tt
Tt
Rt tt
Trung bnh
Kh tt
Trong thi gian x l, m cy phi ngp hon ton trong dung dch dit khun. i vi cc
b phn c nhiu bi ct, trc khi x l nn ra cn thn bng nc x phng bt v ra sch
li bng nc my. Khi x l xong, m cy c ra nhiu ln bng nc ct v trng (ti
thiu l 3 ln). Nhng phn trn m cy b tc nhn v trng lm cho trng ra cn phi c
ct b trc khi t m cy ln mi trng. trnh nh hng ca tc nhn v trng ln m
cy, nn ch li mt lp bc ngoi khi ngm m vo dung dch dit khun. Lp ngoi
cng ny s c lt b i trc khi t m cy ln mi trng. V trng m cy l mt thao
tc kh, t khi thnh cng ngay t ln u tin. Tuy vy, nu kin tr tm c nng v thi
gian v trng thch hp th sau vi ln th chc ch n s t kt qu. C th dng khng sinh
kim sot hoc loi b s nhim nm trn m cy. Hu ht cc khng sinh nhy cm vi nhit
do khng th hp v trng. Chng ho tan c trong nc hoc dung mi thch hp khc,
lc v trng v thm vo mi trng v trng khi mi trng ny c lm ngui cn 4550oC. Khng phi tt c cc khng sinh u thch hp cho s dng trong nui cy m thc vt.
Cc khng sinh nh Streptomycin, Kanamycin v Neomycin thng c cho m thc vt v
khng hot ng tt trong mt phm vi pH nht nh. Tetracylin cng l c t thc vt, c
khuynh hng c ch s tng trng ca m thc vt sau khi b x l trong mt thi giandi.
Chloramphenicol c ph hot ng rng nhng c cho thc vt (v ngi) nng thp.
Cng Ngh Sinh Hc Vit Nam
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Kh n ng hot ng
c ch s sinh tng hp protein bng cch
tc ng ln cc ribosome 30S hoc 50S
Gy tr ngi cho qu trnh sao chp DNA
bng cch c ch enzym DNA gyrase
c ch s tng hp vch t bo vi khun
Tetracyclin
Trimehtoprim v sulphonamide
Chloramphenicol
Macrolide v lincosamide
- Erythromycin
-Lincomycin
Glycopeptide
- Vancomycin
Polymixin
- Polymixin B
- PolymicinE
Rifampicin
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Biotechnology
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Biotechnology
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BI 2:
K THUT PHA CH MI TRNG
^!^
1.VN LA CHN MI TRNG
Khi khi s nui cy m v t bo i vi mt s i tng nht nh, vn t ra l chn
mi trng no v trn c s no phi hp t l cc cht dinh dng. Cch thng lm l qua
cc ti liu xut bn, cc cng trnh nghin cu v i tng hoc cng h tng ng
xem cc tc gi s dng mi trng loi no. Bc u c th gi nguyn mi trng ca tc
gi hoc trn c s m ci tin cho ph hp qua cc th nghim th m d.
Trong hng trm mi trng do rt nhiu tc gi ngh cho nhiu loi cy khc nhau, nhiu
mc ch nui cy khc nhau. V c bn c th chia ra lm 3 loi:
- Mi trng ngho cht dinh dng: in hnh l mi trung White,Knop v Knudson C
- Mi trng c hm lng cht dinh dng trung bnh: in hnh lmi trng B5 ca
Gamborg
- Mi trng giu dinh dng: in hnh l mi trng MS (Murashige-Skoog)
V vy khi bt u nghin cu nui cy m mt s i tng mi, cha c ti liu trc th
nn thm d so snh 3 loi mi trng trn xem i tng nghin cu ph hp vi loi mi
trng no nht. Sau cn th tm t l NO3/ NH4+ thch hp. Cc tc gi phng Ty lm vic
vi cy trng cn thng khng a NH4+ vo mi trng. Nhng i vi nhng cy dinh dng
NH4+ mnh nh cy la, vic thm vo mi trng nui cy mt t l NH 4+ thch hp chc chn s
c li. Vic s dng mang tnh kinh nghim ch ngha i vi mt s mi trng cn tr kh
nhiu s tin b ca cng tc mt s phng th nghim nui cy m thc vt. Thuc l v cart
l 2 loi cy kinh in ca
nui cy m thc vt. Mi trng nui cy 2 loi cy ny c xy dng kh hon chnh.
Tuy vy, khng th dng nguyn cc mi trng nghin cu cc cy ho tho hoc cc cy
h u m khng c s ci tin, sa i. iu ny gii thch s tin b chm chp ca nui cy
m mt s cy ho tho so vi cy 2 l mm.
Hin nay, mi trng MS c coi nh l mi trng thch hp vi nhiu loi cy do giu v
cn bng v mt dinh dng. V vy, nhng ngi tp s lm nui cy m thng bt u vi
mi trng ny trc khi tm c mi trng ca ring mnh.
2.CHUN B MI TRNG
thun tin cho vic pha cc mi trng nui cy ngi ta khng cn ho cht mi ln pha
m thng chun b trc di dng cc dung dch m c (cn gi l cc stock), sau ch cn
pha long khi s dng. Cc stock ny thng c bo qun di ngy trong t lnh thng hoc
t lnh su.
2.1 Thnh phn mi trng dinh dng
Nc ct
Cht hu c - ng- Acid amin- Vitamin (B1, B6, H, PP)- Cc cht iu ha sinh trng
thc vt (Auxin, Cytokinin,Gibberellin)
Cht v c
Cng Ngh Sinh Hc Vit Nam
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a lng N P K Ca Mg S
Vi l ng Fe Zn B Co N Mn Cu Al Mo I
Cc hp cht khng bit r thnh phn
Nc da, nc khoai ty, nc chui, casein,hydrolysalt, trypton, pepton
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MorelsVitamin
Pirydox
ine (B6)
1 mg/L
Biotin
Meso(H)
inositol
0,01
100
mg/L
mg/L
2.2 Cch pha cc dung dch m (stock)
Nicotin
ic acid
(P.P)
1 mg/L
Thiami
n HCl
(B1)
1 mg/L
Pantota
te Calci
1 mg/L
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Biotechnology
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KI
83 mg/1 mL
H3BO3
620 mg
Na2MoO4.2H2O
25 mg/1 mL
ZnSO4.7H2O
860 mg
CuSO4.5H2O
2,5 mg/1 mL
CoCl2.6H2O
2,5 mg/1 mL
Cn 3 cht u tin v ho tan vi nc ct trong tng bcher ring.Cho tng dung dch theo
th t vo ng ong 500mL. Sau ht 1mL dungdch trong stock con ca 4 cht tip theo cho
vo ng ong, va cho va khuy u v thm nc ct cho 500mL
* Thnh phn vitamin
Pha cc stock con
Dung dch pirydoxine (B6) (10mg/mL)
Cn 1000mg B6 ha tan vi nc ct cho 100mL
Dung dch Biotin (H) (1mg/mL)
Cn 100mg biotin ha tan vi nc ct cho 100 mL
Dung dch Nicotinic Acid (P.P) (10mg/mL)
Cn 1000 mg nicotinic acid ha tan vi nc ct cho 100mL
Dung dch Thiamin-HCl (B1) (10mg/mL)
Cn 1000 mg B1 ho tan vi nc ct cho 100 mL
Dung dch Pantotheate-Ca (10mg/mL)
Cn 1000 mg Pantotheate-Ca ha tan vi nc ct cho 100mL
Vitamin Morel (x 100)
Meso-inosito l10 g/10g
Thiamin
Pirydoxine (B6)100g/10mL
Nicotinic acid(P.P)
HCl(B1)100mg/10mL
Biotin (H)1 mg/1 mL
100mg/10mL
Pantotate Calci100 mg/10
mL
Cng Ngh Sinh Hc Vit Nam
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Biotechnology
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BI 3:
GIEO HT IN-VITRO
^!^
I.CHN M CY V X L M CY
1.1 Chn la m cy
Khng c nhng hng dn c th trong vic chn m cy. V nguyn tc, tr nhng m cy
ha g, cc m khc trong c th thc vt u c th dng lm m cy. Tuy vy c th nhn
xt chung l cc m ang pht trin, tht qu non, l non, cung hoa, hoa, m phn sinh khi
t vo mi trng c cha mt lng cht sinh trng thch hp u c kh nng phn chia v
phn ha. bt u nghin cu nhn ging v tnh mt cy nht nh, trc tin ngi ta ch
n cc chi nch v m phn sinh ngn. Cn bit rng tuy mang mt lng thng tin di truyn
nh nhau, cc m khc nhau trn cng mt cy c th sinh trng v pht trin vi kh n ng ti
sinh chi, r hay cy hon chnh rt khc nhau. V vy, khi khi s chn ging, nhn ging mt
cy c th bngphng php nui cy mv t bo thc vt, trc ht cn th nghim tm hiu
phn ng ca cc b phn khc nhau ca cy trong nui cy cc nng cht sinh trng khc
nhau. Sau khi cy, m cy cn c t trong iu kin nhit v nh sng n nh. Tu vo
cc mc ch nghin cu m c cc ch chiu sng khc nhau, chng hn qu trnh to m so
c th cn bng ti hay nh sng, nhng qu trnh ti sinh v nhn ging v tnh nht thit phi
c nh sng. Nhit phng nui nn gi n nh 25+2oC bng my iu ha nhit . Cng
chiu sng khong t 2000 3000 lux.
1.2 X l v trng mu cy
Mu a vo nui cy invitro c nhiu ngun gc khc nhau. C th l nhng mu cy v
trng nh cy con invitro cho ny mm trong iu kin v trng; cng c th l cc mu cy
cha v trng, ly trc tip t bn ngoi t nhin nh chi non, l, thn, c, r Thun li nht
l s dng cc mu cy v trng bi l cc phng php v trng mu cy trc tip thng t
nhiu gy hi cho mu cy do cht kh trng gy ra.
C nhiu phng thc v trng mu cy:
a. V trng ht:
- Ra ht bng x phng, lc u 2-3 pht. Vi cc loi ht nh, thng
ng ht trong ti vi mng.
- Ra sch x phng bng nc my di vi nc chy mnh
- V trng s b bng cn 70%, lc u 1-2 pht
- Ra sch cn bng nc v trng
- Cho ht vo dung dch kh trng NaOCl 1-15%. Thm vi git bm
dnh Tween20. Kh trng trong 15-20 pht tu mu.
- Ra sch cht kh trng nhiu ln bng nc v trng (khong 5 ln)
cho n khi ht mi javel.
- Ht v trng c th nui cy trn mi trng to mu v trng.
b. Cc phng thc khc v trng ht
- Kh trng ln th nht vi NaOCl 5,25%
- Kh trng ln th hai vi HgCl2 0,1 1%, thm vi git HCl 0,5% trong 10 pht.
- Ra mu vi H2O2 6% v trng trc khi ra li bng nc v trng
Cng Ngh Sinh Hc Vit Nam
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- T lnh
- Cn k thut
- My ct nc 2 ln
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- My khuy t
- pH k
Biotechnology
2.2.3 Ho cht
- Dung dch Skoog I, II
v III ca mi trng MS
- Stock vitamin Morel
- Stock glycin
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- ng saccharose
- Agar
- Nc ct v trng
- Cn 90%, 70%
- Dung dch
hypochloride calcium 10%
- X phng bt
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BI 4:
NUI CY NH SINH TRNG
^!^
1.GII THIU
Trong nui cy invitro, mt phng thc n gin v thng hay c s dng ti sinh chi
invitro l nui cy nh sinh trng. Hiu mt cch ng ngha th nui cy nh sinh trng l s
dng phn m phn sinh ngn vi 3-4 tin pht khi l, tc l cc nh sinh trng c kch thc
t 0,1 0,15mm tnh t chp sinh trng. K thut ny kh phc tp, phi thc hin di knh lp
v kh nng sng st ca mu cy c kch thc nh nh th thng khng cao, do ch c
tin hnh khi cn nui cy vi mc ch to cc cy con invitro sch virus.
Trn thc t ngi ta thng nui c nh chi non vi kch thc khong vi mm. c th
l nh chi ngn hoc nh chi nch. Mi nh sinh trng nui cy iu kin thch hp s
to ra mt hay
nhiu chi v mi chi s pht trin thnh cy hon chnh. Xt v ngun gc ca cc cy ,
c th c ba kh nng:
- Cy pht trin t chi ngn
- Cy pht trin t chi nch ph ng
- Cy pht trin t chi mi pht sinh, v d: nui cy on tr h dip ca cy mng cu
(Annona squamosa) s cho rt nhiu mm (buds) trn m cy v mt s mm sau s pht trin
thnh chi v sau s thnh cy invitro hon chnh. Tuy nhin thng thng rt kh phn bit
chi ph ng v chi mi pht sinh. Cc phng thc pht trin cy hon chnh t nh sinh
trng nui cy nh sau:
* Pht trin cy trc tip:
Ch yu cc i tng hai l mm (dicotyledon) nh khoai ty, thuc l, cam chanh, hoa
cc V d: Khoai ty (Solanum tuberosum):
Mm (nh sinh trng) Chi nch Cy
* Pht trin cy thng qua giai on protocorm
Ch yu gp cc di tng mt l mm (monocotyledon) nh phong lan, da, hu Cng
mt lc nh sinh trng to hng lot protocorm (proembryo) v cc protocorm ny c th tip
tc phn chia thnh cc protocorm mi hoc pht trin thnh cy hon chnh. Bng phng thc
ny trong mt thi gian ngn ngi ta c th thu c hng triu c th
V d:Hoa lan (Orchidaceae):
nh sinh trng Protocorm Cy
Theo Champagnat (1977) v Fast (1980), cc chi non ang tng trng di 10-15cm, ca mi
nh l thng c dng lm vt liu cho vic nui cy nh sinh trng. t bn c di sch
i vi nc chy v l c lt sch cho n khi thy r cc chi bn. Chi non lc ny c
nhng vo cn 70% v c kh trng trong dung dch kh trng 10%(v/v). Cc chi bn c
ly ra v ra trong nc ct v trng. Sau , chng c kh li thm 10 pht na trong dung
dch kh trng 3% c cha Tween80 v c ra li trong nc ct v trng. Trong t cy v
trng, phn gc ca nhng
chi nh nht c ct b v vic cy c tin hnh. Trn cc chi ln hn, trc ht tch b
cc l v phn gc b cht do tc ng ca cht kh trng, sau cy vo mi trng. Phi mt
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2.THC HNH
2.1 Mc ch
- Kh trng mu nh chi
- kho st s ti sinh cy trc tip t nui cy nh chi
- Kho st s thnh lp protocorm t nui cy nh chi
2.2 Nui cy pht trin thnh cy trc tip
2.2.1 Nguyn vt liu
on thn non cy cam hoc chanh
2.2.2 Mi trng nui cy
Mi trng MS b sung BA 2ppm v NAA 0,2ppm
2.2.3 Tin hnh:
- Cc cnh mu non ly t vn m v c ct b ht l, ct thnh on 2-3cm, cho vo
bcher
- Ra sch cnh mu bng nc x phng long, sau ra sch x phng bng nc my
nhiu ln di vi nc chy mnh.
- a cnh mu vo t cy v trng, ngm trong cn 70% trong 2-3 pht
- Ra sch cn bng nc ct v trng 1 ln
- X l mu bng Ca(OCl)2 6% trong 10 pht sau thay bng dung dch Ca(OCl)2 5% trong 5
pht
- Ra sch mu bng nc ct v trng 6-7 ln cho ht mi javel
- Cnh mu c ct b cung l v 2 u ca phn thn b cht kh trng ty trng. Chia
cnh mu thnh cc t 1cm
- Cm cc t vo mi trng nui cy chun b, cho phn cung l hng ln trn, chi
ng phi nm trn mt thong ca mi trng.
- Nui mu trong iu kin sng 2000lux/16h/ngy 25oC.
2.3 Nui cy pht trin cy thng qua giai on protocorm
2.3.1 Nguyn vt liu
Cc on chi con Dendrobium cao 10cm
2.3.2 Mi trng nui cy nh sinh trng (cho 1L mi trng)
- Khong a lng Knudson
- Vitamin Morel: 2mL
- Nc da: 150mL
100mL
- Glycin :2mL
- Khoai ty: 60g
- Khong vi lng Heller
- Inositol : 5mL
- Than hot tnh: 0,25g
5mL
- Vitamin B1: 5mL
- pH: 5,5
- Skoog II MS : 2mL
- ng: 30g
- Agar: 6g
* Khong a lng ca KnudsonC (Morel,G.M.,1965)
- NH4NO3: 500mg/L
- KCl : 250 mg/L
- NH4(SO4)2: 500mg/L
- KH2PO4: 250 mg/L
- Ca(NO3)2: 241,3mg/L
- MgSO4: 122,15mg/L
* Khong vi lng ca Heller (1953)
- AlCl3.6H2O : 0,054 mg/L
- KI : 0,01 mg/L
- CuSO4.5H2O : 0,03 mg/L
- MnSO4.H2O : 0,08 mg/L
- H3BO3: 6,2 mg/L
- NiCl2.6H2O : 0,03 mg/L
Cng Ngh Sinh Hc Vit Nam
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BI 5:
NUI CY M SO
^!^
1. GII THIU
Nui cy m so l khu rt quan trng trong nui cy m t bo. M so l nguyn liu khi
u cho cc nghin cu quan trng khc nh: phn ha m v t bo, chn dng t bo, nui cy
t bo trn, nui cy t bo n, nui cy phi soma, sn xut cc cht th cp c hot tnh sinh
hcM so l mt khi t bo khng c t chc, hnh thnh t cc m v cc c quan phn ha
di cc iu kin c bit (c vt thng, x l cc cht iu ho sinh trng thc vt). Cc
t bo thuc cc m hoc c quan ny phi chu mt s phn phn ha trc ln phn chia u
tin. Nhn chung s to m so invitro (nh auxin tc ng) do 3 qu trnh:
- S phn phn ha t bo nhu m (t nhiu su bn trong c quan) bao gm cc t bo nhu
m mc v libe, nhu m v hay li.
- S phn chia ca cc tng tng: cc t bo tng tng ca phn ln STD d dng phn chia
di tc ng ca auxin thm ch khng cn auxin ngoi sinh nh cc loi cy c hay dy leo.
- S xo trn ca cc m phn sinh s khi (chi hay r) qu trnh ny c u tin p dng
TD, v cc cy ny tng tng thiu v nhu m kh phn phn ho so vi STD Mu sc ca m
so khng ging nhau trn cc mi trng nui cy khc nhau hay trn cc b phn khc nhau v
chng thng c mu vng, trng, nu hay trng xanh
Nng v loi kch thch t s dng trong mi trng nui cy l nhng yu t c nh hng
n s hnh thnh v pht trin m so. Thng m so c hnh thnh trn mi trng giu
auxin; c th dng auxin ring r hay kt hp vi nhau hoc c th kt hp vi cytokinin tu tng
loi cy.
Hm lng hormon ni sinh v chiu di chuyn ca cc hormon ny trong mu cy c nh
hng n s pht sinh m so. V vy ngun mu cy, vic ly mu cy, cch t mu cy trn
mi trng nui cy s nh hng n s pht sinh m so dn n nhng phn ng khc nhau
ca mu cy.
Vi mt s cy th vn ny khng quan trng nhng cng c mt s cy chu nh hng rt
ln.
2. THC HNH
2.1. Mc ch:
Kho st s pht sinh m so t cc b phn khc nhau cy thuc l
2. 2 Vt liu
2.2.1 Mi trng nui cy
MS (20g/l ng) c b sung 0.1M 2,4-D v 1M 2,4-D
2.2.2 Nguyn liu thc vt
Cy con thuc l in- vitro
2.2.3 Ho cht v dng c
- Nuc ct v trng
- Dao, kp, a cy, giy cy
2. 3. Cc bc thc hin
Cn thn gp cy con in-vitro ra khi bnh nui cy. Trnh kp qu mnh lm
Cng Ngh Sinh Hc Vit Nam
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Biotechnology
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dp mu cy (hnh A)
- Dng dao cy ct on r, lng thn v l chuyn qua mt da cy khc
x l mu (hnh B)
- L: ct b gn l v ra l. Phn l cn li c ct thnh nhiu mnh
nh vi kch thc 0,8 -1mm x 8 10mm. t cc mnh l ny nui trn cc a petri cha mi
trng MS + 1M 2,4-D BC
- Lng thn: chn cc on lng thn c ng knh 2-2,5mm c ct lt mng 0,05 0,1mm
bng li dao tht sc. Cc lt ct c t nm trn cc a petri cha mi trng MS + 1M
2,4-D
R: Ra sch agar bng nc ct v trng, ct nh thnh tng on 1-1,5mm t ln cc a
petri c cha mi trng MS + 0.1M 2,4-D
- Dng nha nylon cun quanh mp a petri m bo s v trng
trong thi gian nui cy.
- Ghi r s nhm, tn mu cy, tn mi trng v ngy cy.
- t nui trong ti
4. Yu cu:
- Thao tc x l mu cy tt, lt ct dt khot cng mng cng tt; trnh
dp mu
- Ghi nhn v so snh thi gian pht sinh so, hnh dng v mu sc khi
m so, v tr pht sinh so t cc mu cy khc nhau.
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Biotechnology
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BI 6:
KHO ST S PHT SINH CHI T CC B PHN
KHC NHAU CA CY THUC L
^!^
1.GII THIU
Nhn ging cy trng bng phng php nui cy m t bo invitro c gi l vi nhn ging.
C nhiu phng php vi nhn ging khc nhau to chi t to cy con invitro hon chnh.
Tu thuc vo tng i tng khc nhau m s dng phng php ph hp v tt nhin mi
phng php s c nhng u v nhc im ring
* Vi nhn ging bng phng php ct t gim cnh
- H s nhn thp
- Thng ng dng cc i tng kh to cm chi (v d nh lan
s)
* Vi nhn ging bng cch tch chi t cm chi
- H s nhn cao
- Phc to hn trong vic kch thch chi pht trin cao thnh cy con
hon chnh
Cm chi c th c hnh thnh t nhiu con ng khc nhau:
- Ti sinh t m so (kh nng t bin cao hn nn thng c s dng trong nui cy chn
lc ging mi)
- Pht sinh chi bt nh t cc c quan khng sinh sn ca cy nh lng thn, l, cung l, r,
trc pht hoa, l i, cnh hoa (mu cy trI qua giai on phn phn ha to cc t bo sinh
m; tip n l giai on to c quan vi giai on trung gian to m so m ta c th quan st
thy hoc khng ri t mi pht sinh chi bt nh)
- Ph trng thi tim sinh ca cc chi ng mu cy l 1 t chc nh t thn, nh sinh
trng. Cn kim sot hormon tng trng chn ng s pht trin ca chi to nhiu chi
(cm chi) V nguyn tc c th kch thch s thnh lp chi bt nh t tt c cc c quan khng
sinh sn ca thc vt. Tuy nhin trn thc t thng ch thnh cng trn i tng l l v cung
l ngoi tr mt s i tng kinh in, d lm nh thuc l, c chua Hm lng v loi kch
thch t b sung vo mi trng c nh hng quyt nh n s thnh lp chi (thng s dng
kch thch t nhm cytokinin vi nng cao kt hp vi nhm auxin c nng thp). Kh
nng ti to chi cn ph thuc vo kch thc ca mu cy. Mu cy qu nh c th khng p
ng c vi cc iu kin nui cy v s ho nu.
2. THC HNH
2.1. Mc ch
Chng minh nguyn tc vi nhn ging v kh n ng ti to chi bt nh
t cc b phn khc nhau ca thc vt
2.2. Vt liu
2.2.1 Mi trng
MS (20g/l ng) c b sung 10M BA
Cng Ngh Sinh Hc Vit Nam
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Biotechnology
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Biotechnology
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BI 7:
KHO ST S TI SINH CHI T M SO
^!^
1.GII THIU
Qu trnh hnh thnh c quan trong m xy ra qua 2 giai on:
- Giai on th nht l ti phn ha. Trong giai on ny xy ra qu trnh chuyn cc t bo
bit ha thnh m so
- Giai on th hai l hnh thnh cc mm mng c quan. Bng phng php phng x t bo
thy rng nhng t bo ca cc mm mng nhu m m y c hnh thnh mm mng c
quan, tng hp DNA v protein xy ra rt mnh, hm lng ng cng tng. Trong qu trnh
phn ha, cc m so khng c t chc c hnh thnh cc cu trc hnh thi dn n vic to
chi, r, cnh, hoa v cy hon chnh. Qu trnh phn ha ny c th thc hin bng cch thay i
mt s cht v cc cht iu ha sinh trng trong mi trng nui cy i vi m so, xu th
to c quan gim dn khi m cy chuyn nhiu ln v khi cy chuyn nhiu ln nh th thng
hnh thnh cc t bo a bi
v lch bi, ngoi ra c th mt cc yu t di truyn.
Theo V Vn V (1999) m so khi hnh thnh thng c 2 loi:
Loi xp: cha nhiu t bo xp vi nhn nh, t bo cht lng v khng bo to.
Loi cng: Cc t bo cng, chc thnh khi, nhn to, t bo cht m c v khng bo nh.
Dng m so cng c nh hng n kh nng ti sinh c quan ca khi m. Kh nng ti sinh
chi sm mt i m so xp nhng vn duy tr m so cng. Nguyn nhn c th do cc t
bo m so s mt i kh nng tng hp mt s cht thit yu cho s ti sinh ca n khi s ln
cy chuyn tng ln ( Gautht, 1962). V vy khi nui cy m so nhm mc ch ti sinh chi,
nht thit phi c gng tm iu kin mi trng thch hp cho s hnh thnh cc khi m so
cng, chc; cc m so xp cn c loi b trong cc ln cy chuyn v i khi dng m so ny
pht trin rt nhanh v ln t c cc m so cng c kh n ng ti sinh phi.
2.THC HNH
2.1. Mc ch:
Chng minh kh nng ti sinh chi t m so
2.2. Vt liu:
2.2.1 Mi trng
MS (30g ng) + BA 1,5M + NAA 0,5 M
2.2.2 Nguyn liu thc vt
M so t lng thn, l v r ca bi 3 sau 4 tun nui cy
2.2.3 Ho cht v dng c
- Nc ct v trng
- Dao cy, kp, a cy v giy cy v trng
2. 3. Cc bc thc hin
- Dng kp gp m so t bnh mu cho vo a petri
- Dng dao cy ly sch phn agar cn bm vo mu
Cng Ngh Sinh Hc Vit Nam
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Biotechnology
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Phinl
Chi ti
sinh
- 25 -
M so
Biotechnology
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BI 8:
A CY RA VN M
^!^
1.GII THIU
Cc k thut chit cnh, gim cnh v ghp l cc phng php nhn ging v tnh thc vt
hay cn gi l nhn ging in vivo. Ngoi ra cn c k thut nhn ging in vitro hay nui cy m
l mt phng php nhn ging v tnh thc vt cho h s nhn cao hn cc k thut trn Cy
con in vitro khi pht trin hon chnh c y thn, l v r s c chuyn ra vn m.
y l giai on kh khn nht trong k thut nhn ging v tnh bng nui cy m. Cy in vitro
c nui cy trong iu kin n nh v dinh dng, nh sng, nhit , m nn khi
chuyn ra t vi cc iu kin t nhin hon ton khc hn nh dinh dng thp, nh sng c
cng mnh, nhit cao, m thp cy con d b stress, d mt nc v mau b ho. Mt
khc trong mi trng t nhin c rt nhiu vi khun v nm gy bnh lm thi v cht cy.
khc phc tnh trng ny, ngi ta c cc bin php nh sau:
- Vn m cy cy m phi mt, cng chiu sng thp, nhit khng kh thp, m
thp
- Cy con c trng trn lung m cy c c cht d thot nc, ti xp, gi c m.
C cht v cy con cn c x l vi thuc chng nm hoc thuc tm phng nga nm bnh
cho cy
- Trc khi a cy ra vn m nn cc bnh mu ngoi vn m trong 1-2 tun cho cy
thch nghi dn vi iu kin t nhin - Trong nhng ngy u a cy ra vn m, lung m
cy cn c ph nylon gim qu trnh thot hi nc l ( thng 7-10 ngy sau khi ra cy).
Mi ngy nn ti nc 2 ln km theo phun sng thng xuyn khi tri nng nhm duy tr
m.
-Cy con sau khi trng 7-10 ngy cn phi bn phn cy pht trin ng thi phun thuc
nga nm bnh.
2. THC HNH
2.1. Mc ch
Lm quen cc k thut a cy ra vn m
2.2. Vt liu
2.2.1.i tng cy ging:
- Cy con Lan Hong Tho ( Dendrobium sp.) in-vitro
- Cy con Thuc l in-vitro
2.2.1 Nguyn liu v dng c
- X da
- Dy thun
- Thau nha
- Tro tru
- Ti m 12x17cm
- R nha
- Ct xy dng
- Kp
- Bnh phun
2. 3. Cc bc thc hin
-Cho mt t nc vo cc bnh mi trng c cy ging, lc mnh mi trng v ra.
-Dng kp gp nh nhng cy ging ra khi bnh trnh lm t,dp thn, l hoc r cy
-Ra sch agar bm r cy bng 1 tm nhn; cn thao tc nh nhng khng lm t v dp
r ; nu khng khi em trng vm m cy s d b nhim nm bnh v cht.
Cng Ngh Sinh Hc Vit Nam
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- Ngm cy trong dung dch thuc tm 10/00 trong 1 pht sau ra li bng nc sch ro.
- i vi Thuc l (gi th trng l hn hp x da, tro tru v ct xy dng): Dng 1 cy a
xm cc l m trn ti m vI su khong 5cm. t gn r cy con vo l m v dng tay
m cht t li gi cy ng vng. m 5 cy cho 1 ti m
- i vi Lan hong tho (gi th trng l x da): Gi gn phn r trong ming x da, dng
thun buc nh nh hnh cy. Xp vo r nha, ti phun sng nh trc khi a ra vn
m.
4. Yu cu:
- Thc hnh thao tc a cy con ra vn m, trnh lm dp v
t r qu nhiu
- Quan st t l cy con sng st sau khi ra vn m.
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Biotechnology