nuoi cay mo lan

7/31/2019 nuoi cay mo lan

1/23

1

Bi 1 . K THUT C BN

V PHA CH MI TRNG NUI CY

1. K THUT C BN

1.1 K thut v trng

a. Dng c thu tinh

Bng 1.1: Thi gian kh trng dng c thu tinh bng nhit v nhit kh

trng

Nhit (oC) Thi gian kh trng

(pht)

160 45

170 18

180 7,5

190 1,5

b. Nt y

Hp kh trng cng vi cc dng c thu tinh 1210C, 1atm. Khng kh trng kh,

gy chy i vi nt bng v bin dng vi nt nha.

c. Mi trng

Bng 2: Thi gian kh trng dung dch v cc mi trng lng bng ni hp

(autoclave) 1210C, 1atm.

Th tch mi trng

(ml)

Thi gian hp kh trng

(pht)


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1.2 Kh trng m thc vt

Bng3:Ho cht kh trng, nng , thi gian x l v hiu qu thc nghim.

Tc nhn v trng Nng % Thi gian x l( pht) Hiu quCalci hypochlorit 9 10 5 30 Rt tt

Natri hypochlorit 2 5 30 Rt tt

Hydro peroxid 10 12 5 15 Tt

Nc Brom 1-2 2 10 Rt tt

HgCl2 0.1 1 2 10 Trung bnh

Cht khng sinh 4 50 mg/l 30 60 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).

1.3 Kh trng ni thao tc cy v dng c cy

-Sn v tng lau chi thng xuyn, trc khi a vo s dng, phng cy cnc x l hi formol. ng kn ca phng cy trong 24h, b formaldehyde i v kh hi

formaldehyde cn tha bng dung dch NH3 25% cng trong 24h.

-Kh trng trc t cy bng cch lau sch bng cn 700. Bt n UV kh trngphng cy 30pht trc khi s dng.

-Cc dng c mang vo phng cy u v trng trc: t o chong, m vi, khutrang ca ngi cy n dao, ko, kp (forceps), giy lc, bnh ng nc ct...

-Trn bn cy thng xuyn c mt n cn s dng trong khi cy v mt ccng cn 960 nhng cc dng c lm vic.


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-Trc khi cy, ngi cy cn ra tay bng x phng v lau k n khu tay bngcn 960.

-Cc dng c bng kim loi nh kp cy, dao m, que cy vng, kim mi nhn cth c kh trng bng cch t di ngn la n cn. Nhng dng c ny trc htphi c nhng vo cn tuyt i ri mi t. Nh ro i cc git cn tha ri mi

a vo ngn la n cn.

-Khi m np chai hoc np ng nghim mi trng nui cy, th dng ngn tay kt v ngn tay t cm ly np gn, v khng chm tay vo b mt bn trong ca np gn

cng nh khng th np gn xung bt c b mt no cho n khi gn n tr li chai mi

trng.

2. PHA CHMI TRNG:

2.1 Thnh phn mi trng dinh dng

Thnh phn mi trng

Nc ct ng, Acid amin, Vitamin (B1, B6, H, PP)

Cht hu c Auxin, Cytokinin,Gibberellin

Cc cht iu ha sinh trng thc vt a lng N P K Ca Mg S

Cht v c 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

Thnh phn mui khong c bn ca mi trng MS (Murashige v Skoog, 1962)

SKOOG I SKOOG II SKOOG III

NH4NO3 1650 mg/L FeSO4.7H2O 27,8 mg/L KI 0,83 mg/L

KNO3 1900 mg/L MnSO4.4H2O 22,3 mg/L Na2MoO4.2H2O 0,25 mg/L

KH2PO4 170 mg/L H3BO3 6,2 mg/L CuSO4.5H2O 0,025 mg/L

MgSO4.7H2O 370 mg/L ZnSO4.7H2O 8,6 mg/L CoCl2.6H2O 0,025 mg/L

CaCl2.2H2O 440 mg/L


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Na2EDTA 37,3 mg/L

Thnh phn vitamin ca Morel (MorelsVitamin)

Pirydoxine(B6)

Biotin(H)

Meso-inositol

Nicotinic acid(P.P)

Thiamin HCl(B1)

PantotateCalci

1 mg/ L 0,01 mg/L 100 mg/L 1 mg/L 1 mg/L 1 mg/L

2.2 Cch pha cc dung dch m (stock)

*Stock a lng MS: SKOOG I (x10)

(Pha thnh 1Lvi nng s dng khi pha 1Lmi trng MS l 100ml/L)

Thnh phn Khi lng

NH4NO3 16500 mg

KNO3 19000 mg

KH2PO4 1700 mg

MgSO4.7H2O 3700 mg

CaCl2.2H2O 4400 mg

Cn v dng nc ct ho tan ln lt tng cht trong bcher cho

tan hon ton. Dng ng ong 1L iu chnh cho 1 lt.

* Stock st MS: SKOOG II (x100)

(Pha thnh 200ml vi nng s dng khi pha 1lt mi trng MS l 2ml/l)

Thnh phn Khi lngNa2EDTA 3730 mg

FeSO4.7H2O 2780 mg

Cn v ho tan tng cht bng 100mL nc ct trong mi bcher ring.


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t 2 bcher dung dch ln bp v gia nhit cho dung dch m ln va c hi nng

bc ln l c.

Khuy u v dung dch Na2EDTA vo ng ong 200 ml, ri cho t t dung dch

FeSO4 vo, va cho va khuy u. ngui ri cho vo bnh ti bo qun trong t lnh.

*Stock vi lng MS: SKOOG III (x100)

Trc tin cn chun b cc stock con:

Thnh phn Khi lng Th tch dd

KI 8300mg 100ml

Na2MoO4.2H2O 2500mg 100ml

CuSO4.5H2O 250mg 100ml

CoCl2.6H2O 250 mg 100ml

SKOOG III (x100): pha thnh 500ml vi nng s dng khi pha 1lt mi trng

MS l 5ml/l

Thnhphn Khi lng

MnSO4.4H2O 2230 mg

H3BO3 620 mg

ZnSO4.7H2O 860 mg

KI 83 mg/1 ml

Na2MoO4.2H2O 25 mg/1 ml

CuSO4.5H2O 2,5 mg/1 ml

CoCl2.6H 2,5 mg/1 ml

Cn 3 cht u tin v ho tan vi nc ct trong tng becher ring.Cho

tng dung dch theo th t vo ng ong 500ml.

Sau ht 1ml dung dch trong stock con ca 4 cht tip theo cho vo

ng ong, va cho va khuy u v thm nc ct cho 500ml.


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Thnh phn vitamin

Pha cc stock con

Dung dch Phng php

Pirydoxine (B6) (10mg/ml) Cn 1000mg B6 ha tan vi nc ct cho 100mlBiotin (H) (1mg/ml) Cn 100mg biotin ha tan vi nc ct cho 100 ml

Nicotinic Acid (P.P) (10mg/ml) Cn 1000 mg nicotinic acid ha tan vi nc ct cho

100ml

Thiamin-HCl (B1) (10mg/ml) Cn 1000 mg B1 ho tan vi nc ct cho 100 ml

Pantotate-Ca (10mg/ml) Cn 1000 mg Pantotate-Ca ha tan vi nc ct cho

100ml

Bng thnh phn vitamin Morel (x 100)

Thnh phn Khi lng th tch

Meso-inosito 100 g

Pirydoxine (B6) 10ml

Biotin (H) 1 ml

Nicotinic acid(P.P) 10ml

Thiamin HCl(B1) 10ml

Pantotate Calci 10 ml

Cn meso-inositol ri ha tan vi 100ml nc ct.

Cho th t tng cht vo ng ong c cha nc ct, va

cho va khuy uv thm nccho 200ml.

*Thnh phn cc cht iu ha sinh trng thc vt(tnh theo mg)

Dung dch cc cht Phng php thc hin

Dung dch BAP (1mg/ml) Cn 100 mg Benzylaminopurine (BAP) ho tan trong 5ml

NaOH 1N.


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Cho thm nc ct cho 100 ml.

Mi ml dung dch c cha 1mg BAP.

Dung dch IAA (1mg/ml) Cn 100mg Indolacetic acid (IAA) ha tan trong 5ml

NaOH 1N.Cho thm nc ct cho 100ml.

Mi ml dung dch c cha 1mg IAA.

Dung dch IBA (1mg/ml) Cn 100mg Indolebutyric acid (IBA) ha tan trong 5ml

NaOH 1N.

Cho thm nc ct cho 100ml.

Mi ml dung dch c cha 1mg IBA

Dung dch Kinetin (1mg/ml) Cn 100mg Kinetin (KIN) ha tan trong 5ml NaOH 1N.Cho thm nc ct cho 100ml.

Mi ml dung dch c cha 1mg KIN.

Dung dch NAA (1mg/ml) Cn 100mg Naphthaleneacetic acid (NAA) ha tan trong

5ml NaOH 1N.


Mi ml dung dch c cha 1mg NAA.

Dung dch 2,4-D (1mg/ml) Cn 100mg 2,4-D (2,4-dichlorophenoxyacetic acid) ha

tan trong 50ml ethanol 50%.


Mi ml dung dch c cha 1mg 2,4-D.

* Cc dung dch cht iu ha sinh trng thc vt(tnh bng mol/l)

BAP c trng lng phn t (MW) l 225.26

- Ho tan 225.26gr BAP trong 1l nc ct tc ta c 1l dung dch BAP1M hay

1mol/l

- Cn 225.26 mg BAP ho tan trong 1l nc ct tc ta c 1l dung dch BAP 1mM

hay 1mmol/l


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- Cn 225.26x10-3

mg BAP ho tan trong 1lt nc ct tc ta c 1l dung dch BAP

1M hay 1mol/l Dung dch BAP ( MW 225.26) (10mM)

Cn 225.26 mg BAP ho tan trong 5 ml NaOH 1N. Cho thm nc ct cho 100

ml, tc ta c 100 ml dung dch BAP 10mMV d: Cho 1ml dung dch BAP 10mM vo mi trng MS pha c 1lt mi

trng. Nh vy ta c 1lmi trng MS c cha 10M BAP. Mun pha 1lt mi trng

MS c cha 1M BAP, ta s dng 0.1mL dung dch BAP 10 mM.

Tng t nh vy pha cho cc loi kch thch t cn li, bit:

- IAA (MW 175.19)

- IBA (MW 203.24)

- Kinetin (MW 215.22)

- NAA (MW 186.21)

- 2,4-D (MW 221.04)

Thc hin pha cc mi trng nui cy sau:

-Mi trng nui cy phi cam chanh. -Mi trng nui cy nh sinh trng lanDendrobium.-Mi trng kho st s bit ha c quan t l Saintpaulia.-Mi trng tng sinh (m so).


9/23

9

Bi 2. NUI CY NH SINH TRNG LAN DENDROBIUM

1. MC TIU CA BI:

-Thc hin thao tc tch nh chi t chi bnDendrobium.-Theo di s thnh lp cm chi v nhn cm chi.- Tch chi con v nui cy cy con hon chnh

2. DNG C, HA CHT V MI TRNG NUI CY NH SINH

TRNG

2.1. Dng c

Bnh tam gic

Becher

ng nghim + mi trng

Kp inox

a petri v trng

Dao cy, n cn

Pipet thy tinh

Bnh nh mc, ng ong

Qu bp cao su

Giy cy v trng

2.2. Ha chtv mi trng

Khong a lng Knudson 100ml

Khong vi lng Heller 5mlSkoog II MS : 2ml

Vitamin Morel: 2ml

Glycerin :2ml

Inositol : 5ml


10/23

10

Vitamin B1: 5ml

ng: 30g

Nc da: 150ml

Khoai ty: 60gThan hot tnh: 0,25g

Agar: 6g

pH: 5,5

* Khong a lng ca KnudsonC (Morel,G.M.,1965)

NH4NO3: 500mg/l

NH4(SO4)2: 500mg/l

Ca(NO3)2: 241,3mg/l

KCl : 250 mg/l

KH2PO4: 250 mg/l

MgSO4: 122,15mg/l

* Khong vi lng ca Heller(1953)

AlCl3.6H2O : 0,054 mg/l

CuSO4.5H2O : 0,03 mg/l

H3BO3: 6,2 mg/l

KI : 0,01 mg/l

MnSO4.H2O : 0,08 mg/l

NiCl2.6H2O : 0,03 mg/l

ZnSO4.7H2O :1,0 mg/l

3 CC BC TIN HNH:

+ Nguyn vt liu th nghim: Cc on chi conDendrobium cao 10cm

- Ra sch chiDendrobium di vi nc chy. Dng dao m lt ht cc l non bao

xung quanh chi cho n khi l ra cc chi bn v chi ngn


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- Ngm chi trong nc x phng long v dng gn lau nh ln thn chi. Ra cho

sch x phng di vi nc chy

- Lc chi trong cn 700 trong 1 pht. Sau x l vi dung dch javel thng phm

theo t l 1:5 trong vng 25-30 pht- Trong t cy, dng kp v trng cho chi vo cc bcher c cha nc ct v trng.

Lc ra 3-5 ln cho ht mi javel.

- t cc chi ln giy v trng. Dng dao ct ring tng chi bn v chi ngn ra khi

chi m ban u. Nh ct b ht cc m cht b cht kh trng lm trng phn gc chi.

Cy tng chi vo ng nghim c cha mi trng chun b.

- Nui trong phng sng

- Ghi r tn nhm, ngy cy, ging cy vo tt c cc ng nghim

4. BO CO THC HNH

-Thc hin thao tc tch v nui cy v trng nh chi lanDendrobium.-Thu c cm chi v cy con v trng trong ng nghim.-Quan st v ghi nhn thi gian tng trng ca cy con hon chnh.


12/23

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Bi 3. NUI CY PHI CAM CHANH

1. MC TIU CA BIGip sinh vin lm quen vi thao tc v trng mu cy v k thut cy v trng i

vi mu ht.Thc hin pha mi trng gieo ht

Thc hin cc thao tc v trng trong nui cy v k thut gieo ht

2. DNG C, THIT B V HA CHT

2.1 Dng c:

Bnh tam gic

Becher

ng nghim + mi trng

Kp inox

a petri v trng

Dao cy, n cn

Pipet thy tinh

Bnh nh mc, ng ong

Qu bp cao su

Giy cy v trng

2.2 Thit b

Autoclave

T sy

T cy (laminar)

T lnh

Cn k thut


13/23

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My ct nc 2 ln

My khuy t

pH k

Bp t

2.3 Ho cht

Dung dch Skoog I, II v III ca mi trng MS

Stock vitamin Morel

Cn 90%, 70%

Stock glycine

ng saccharose

X phng bt

Dung dch hypochloride calcium 10%

Nc ct v trng

Agar

3. CC BC TIN HNH

3.1 Chun b mi trng(thnh phn cho 1l mi trng)

- Skoog I (MS):100ml

- Skoog II (MS): 2ml

- Skoog III (MS): 5ml

- Vitamin Morel: 2ml

- Glycine: 2ml

- Sucrose: 30g

- pH :5,8

- Agar:7g

3. 2 Nguyn liu thc vt:


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- Cc ht cam chanh cn nguyn vn, khng b h hi

3.3 Cc bc thc hin

- Cho ht vo bcher c cha nc x phng long, ra ht cho sch cht nht bm

xung quanh ht- Ra ht cho sch x phng bng nc ct. Ngm ht 2pht trong cn 70%

- Ra ht sch etanol bng nc ct. Sau ngm ht 10pht trong dung dch

hypochloride calcium 10%

- Trong t cy v trng, ra ht cho sch cht kh trng bng cch lc ht trong nc

ct v trng (3 ln)

- Tin hnh tch o ht bng kp v dao m v trng. Cho cc ht va tch b v o

vo a petri v trng c cha giy thm v trng v lm m giy thm bng mt t nc ctv trng.

- Gieo ht: gieo 4-5 ht vo cc bnh tam gic cha mi trng nui cy c hp

kh trng.

- y np bnh cy v ghi r h tn, ngy cy, s nhm, tn ging v mi trng.

4. BO CO THC HNH

- Tm tt phng php thc hin nui cy m ht cam chanh.- Thu c cc cy con v trng trong ng nghim

- Quan st v ghi nhn thi gian tng trng ca cy con hon chnh.


15/23

15

Bi 4. KHO ST S BIT HA C QUAN

T M L SAINTPAULIA (AFRICAN VIOLET)

1. MC TIU CA BI:

Chng minh nguyn tc vi nhn ging v kh nng ti to c quan hon chnh t mu

cy thc vt.

Theo di s bit ha phi sinh dng, chi, m so v r trn cc mu cy l

Saintpaulia.

2. DNG C, HA CHT V MI TRNG NUI CY.2.1 Dng c:

Bnh tam gic

Becher

ng nghim + mi trng

Kp inox

a petri v trng

Dao cy, n cn

Pipet thy tinh

Bnh nh mc, ng ong

Qu bp cao su

Giy cy v trng

2.2 Ho chtv mi trng- Dung dch Skoog I, II v III ca mi trng MS

- Stock vitamin Morel

- Stock glycine

- ng saccharose


16/23

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- Agar

-Nc ct v trng

- Cn 960, 700

- Dung dch hypochloride calcium 10%- X phng bt

Chun b1lt mi trng nui cy

-Cho 500ml nc ct vo bnh 2000ml-Ln lt cho nh sau:+ 10ml cc mui khong MS

+ 10ml dung dch m thiamine (40mg/l)

+ 30g ng+ 20ml dung dch m IAA (10mg/100ml)

+ 20ml dung dch m kinetin (10mg/100ml)

+ 10ml dung dch m NaH2PO4.H2O (17g/l)

+ 8ml dung dch m adenine sulfate (17g/l)

-Thm nc ct 1000ml. Chnh pH 5,7-Cho 8g agar.Nu tan-Chia 25ml vo mi ng nghim. y kn.-Hp kh trng 30 pht, 1210C, 1atm.- thch nghing.

2.3. Nguyn vt liu th nghim

L Saintpaulia

3. CC BC THC HIN:

Ct nhng l kho, non t cy m. ghi li tn ging mi cy.Ra mu l bng nc x phng m, ra sch. Cn thn v m l d b dp.

t vo cc 250ml, y li bng a petri

Kh trng bng thuc ty 19% trong 15 pht

Ra sch bng nc ct v trng 3 ln


17/23

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Ct mu l vi kch thc 1-2 cm2, mu cung 1cm.

t nhng mu ny ln mi trng

Nui cy trn k

Nui cy v kho st s thnh lp c quan trn cc mi trng c cc hormon khcnhau.

Nui cy chi c ti sinh t l.

To cm chi v nhn cm chi.

Tch chi con ra khi cm chi v to cy con hon chnh.

4. BO CO THC HNH

-Thc hin thao tc cy v trng m l Saintpaulia.-Quan st v ghi nhn kt qu v s ti to c quan v thi gian thnh lp.-Ghi nhn nh hng ca cc kt hp hormon thc vt ln s bit ha ca m l.


18/23

18

Bi 5: NHN GING, TO M SO TRNDENDROBIUM

1. MC TIU

Thc hin thao tc nhn ging in-vitro lan dendrobium bng m soKho st s thnh lp m so t cy non in-vitro

2. DNG C, HA CHT V MI TRNG NUI CY.

2.1 Dng c:

Bnh tam gic

Becher

ng nghim + mi trng

Kp inox

a petri v trng

Dao cy, n cn

Pipet thy tinh

Bnh nh mc, ng ong

Qu bp cao su

Giy cy v trng

2.2. Mi trng v ha cht nui cy

- Mi trng MS (20g/l ng) c b sung 0.1M 2,4-D v 1M 2,4-D.

- Cn 960 v 700

-Nc ct v trng

- Dung dch hypochloride calcium 10%

- X phng bt

2.3. Nguyn liu thc vt

Cy con lan dendrobium in- vitro

3. CC BC THC HIN


19/23

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Cn thn gp cy con in-vitro ra khi bnh nui cy. Trnh kp qu mnh lm dp mu

cy.

- Dng dao cy ct on r, thn v l chuyn qua mt da cy khc x l.- L: 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.

- Thn: 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 giannui cy.

- Ghi r s nhm, tn mu cy, tn mi trng v ngy cy.

- t nui trong ti

Yu cu: Thao tc x l mu cy tt, lt ct dt khot cng mng cng tt; trnh dp

mu

4. BO CO THC HNH

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.


20/23

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Bi 6: NUI CY M HOA HNG

1. MC TIU CA BI:

Theo di s tng trng v nhn ging hoa hng bng cch tng sinh chi.

Thc hin nhn ging in-vitro cy thn g

2. DNG C, HA CHT V MI TRNG NUI CY.

2.1 Dng c:

Bnh tam gic

Becher

ng nghim + mi trng

Kp inox

a petri v trng

Dao cy, n cn

Pipet thy tinh

Bnh nh mc, ng ong

Qu bp cao su

Giy cy v trng

2.2. Ha cht v mi trng

- Mi trng MS b sung 30g/l ng, 100mg/l myo-inositol, vitamin MS, 8g/l agar,

v 1M BA- Cn 960 v 700

-Nc ct v trng

- Dung dch chlorine 10%

- X phng bt


21/23

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- Than hot tnh

- Tween 20

3. CC BC THC HIN:

-Cch chn v thu nhn mu: thu ly chi pht trin mnh. Nhng chi c th ly tvn, vn m hoc cy trong chu .

-Loi b l-Kh trng mu: kh trng 10-15 pht trong chlorine 10% (v/v) vi vi git tween 20

(2 git/ 100ml dung dch). Ra sch vi nc ct v trng 3 ln.

-Ct thn thnhcc on cha 1 chi ngn hay 1-3 t thn.-Cy mu vo mi trng- vi ngy (16-24 gi chiu sng)-Khi chi mc ln t cc mm cao khong 1cm, tch ly chng v cy ln mi trng

mi. Loi b phn ngn ca cc ch i s lm tng s to nhnh. Theo di s thay i v cht

lng chi (tnh n nh) khi s ln cy chuyn tng ln. (C th kho st thm s nh hng

ca nng BA ln s ti sinh chi: 0, 1, 4 v 10 M)

-Nhng chi cao khong 2cm c em ra ngoi mi trng trng.4. BO CO THC HNH

- Thao tc thc hnh th nghim

- Quan st v ghi nhn kt qu


22/23

22

BI 7: KHO ST TP NHIM

1. MC TIU

Ghi nhn cc kt qu th nghim. Xc nh nguyn nhn gy nhim mu . Cc nguyn nhn c

th gy nhim:

1. Mu cy2. Cc cht kh trng3. Ngun mu cy4. Tp nhim vi sinh t bn trong mu cy5. S nhim t ngi cy6. T cy7. Mi trng8. Dng c

2. PHNG PHP XL V HN CH TP NHIM

nh gi nh hng ca s tp nhim ln mc thnh cng ca cng ngh nui cy

m t bo.

Xem xt li thao tc thc hin k thut nui cy m.

3. BO CO THC HNH

Quan st v ghi nhn tp nhim mu.

S lng cy con t yu cu khng nhim mi nhm.


23/23

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BI 8: VIT BO CO TNG HP

1. MC TIU

nh gi tng hp cc kt qu thc hnh

2. YU CU

- 5 bi th nghim-Quan st v ghi nhn y cc kt qu

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