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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)
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)
(pht)
<50
15
75
20
250-500
25
1000
30
Tc nhn v trng
Nng %
Hiu qu
Calci hypochlorit
9 10
5 30
Rt tt
Natri hypochlorit
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
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 cn
c 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 trng
phng cy 30 pht trc khi s dng.
- Cc dng c mang vo phng cy u v trng trc: t o chong, m vi, khu
trang 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 cc
ng cn 960 nhng cc dng c lm vic.
- Trc khi cy, ngi cy cn ra tay bng x phng v lau k n khu tay bng
cn 960.
- Cc dng c bng kim loi nh kp cy, dao m, que cy vng, kim mi nhn c
th c kh trng bng cch t di ngn la n cn. Nhng dng c ny trc ht
phi 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 k
t 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 CH MI TRNG:
2.1 Thnh phn mi trng dinh dng
Thnh phn mi trng
Nc ct
Cht hu c
Auxin, Cytokinin,Gibberellin
a lng N P K Ca Mg S
Cht v c
Vi l ng Fe Zn B Co N Mn Cu Al Mo 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
Na2EDTA
37,3 mg/L
Pirydoxine
Biotin
Meso-
Pantotate
(B6)
(H)
inositol
(P.P)
(B1)
Calci
1 mg/ L
0,01 mg/L
100 mg/L
1 mg/L
1 mg/L
1 mg/L
Thnh phn
Khi lng
NH4NO3
16500 mg
KNO3
19000 mg
KH2PO4
1700 mg
MgSO4.7H2O
3700 mg
CaCl2.2H2O
4400 mg
Thnh phn
Khi lng
Na2EDTA
3730 mg
FeSO4.7H2O
2780 mg
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
Thnh phn
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
Phng php
Pantotate-Ca (10mg/ml)
Thnh phn
Meso-inosito
100 g
Pirydoxine (B6)
10ml
Biotin (H)
1 ml
Nicotinic acid(P.P)
10ml
Thiamin HCl(B1)
10ml
Pantotate Calci
10 ml
3 CC BC TIN HNH:
+ Nguyn vt liu th nghim: Cc on chi con Dendrobium cao 10cm
- Ra sch chi Dendrobium 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
10
- 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 lan Dendrobium.
- 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.
11
2.2 Thit b
Autoclave
T sy
T cy (laminar)
T lnh
Cn k thut
12
My ct nc 2 ln
My khuy t
pH k
Bp t
2.3 Ho cht
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:
13
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.
14
- Agar
- Nc ct v trng
- Cn 960, 700
- Dung dch hypochloride calcium 10%
- X phng bt
Chun b 1lt 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
16
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.
17
3. CC BC THC HIN
18
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.
19
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
3. CC BC THC HIN:
- Cch chn v thu nhn mu: thu ly chi pht trin mnh. Nhng chi c th ly t
vn, 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 thnh cc 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 chi 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
21
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 cy
2. Cc cht kh trng
3. Ngun mu cy
4. Tp nhim vi sinh t bn trong mu cy
5. S nhim t ngi cy
6. T cy
7. Mi trng
8. Dng c
3. BO CO THC HNH
Quan st v ghi nhn tp nhim mu.
S lng cy con t yu cu khng nhim mi nhm.
22
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
23