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GVHD: T Ngc Ly
1.1.1.1.
Trn th gii
Nm 1902: Haberlandt xut phng php nui cy m t bo n bi
tuy nhin cha mang li thnh cng m mn.
Nm 1934 m thc vt mi c cy bi A Carrel.
Nm 1939,1943,1945: Ln lt Nobeccourt, White, Gautheret cng b
thnh cng sm nht v phng php nui cy m trn mi trng thch.
Theo tin s White (1943) th lch s ca nui cy m t bo thc vt c
t lu v mang ngha quan trng trong lch s pht trin sinh hc, tim nng ca
n c ng dng trong vic ci tin cht lng v nhn nhanh ging cy trng.
Nm 1951, Skoog v Miller pht hin ra cc hp cht c th iu khin
s nhn chi.
Nm 1960: Tin s Morel l ngi bt u phng php nui cy nh sinh
trng. ng l ngi a ra k thut nui cy m a lan pht trin thnh qui
m cng nghip v nghin cu thnh cng cy nho sch bnh bng phng php
nui cy m(1945-1948).
Nm 1962, Murathige v Skoog ci tin mi trng nui cy nh du
mt bc tin trong k thut nui cy m. Mi trng ca h dng lm c s cho
vic nui cy nhiu loi cy v vn cn c s dng rng ri cho n nay.
Nm 1964, Ball l ngi u tin tm ra mm r t vic nui cy chi
ngn.
Nm 1981: Takayama v Misawa thnh cng trong t ng ho vi nhn
ging bng cc h thng bioreactor. n nay, ngnh nui cy m khng nh
vai tr quan trng ca mnh bng mt kho tng s cc cng trnh nghin cu
ca mnh v thc vt in vitro.
u th k 21, hng lot cc cng ty v vi nhn ging cy trng thng mi
vi qui m ln ln lt ra i trn khp th gii con s ln n hn 600 cng ty.
Nh vy, k thut nui cy m thc s m ra mt cuc cc mng trong nhn
ging thc vt.
1.1.12. Vit Nam
T nhng nm 1960 cng ngh nui cy m t bo thc vt c du
nhp vo nc ta bt u ti min Nam.
n u nhng nm 1970 th c ti min Bc.
Sau 1975, phng th nghim nui cy m v t bo u tin c xy dng
ti vin Sinh vt hc, vin Khoa hc Vit Nam do tin s L Th Mui ng u.
Hin nay, cc hng nghin cu ng dng nui cy m v t bo thc vt
c pht trin mnh: dung hp cy lai t bo cht v chuyn gen, nui cy bao
phn to dng thun, nui cy cc cy dc liu qu bo tn ngun gen v
to cc dng t bo c hm lng cc cht sinh hc.
Tuy nhin ch t cui nhng nm 1980 tr li y cng ngh m - t bo
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GVHD: T Ngc Ly
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GVHD: T Ngc Ly
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GVHD: T Ngc Ly
loi nm cng sinh trn nhiu ging lan khc nhau c bit:
Rhizoctonia repns c bit cho Cattleya, Laelie, Cypripedium
Rhizoctonia mucoroides cho Vanda v Phalaenopsis
Rhizoctonia lanuginosa cho Oncidium, Miltonia
Nm s cung cp ng nui cy, phn gii cc cht hu c kh hp thu.
B li cy s cung cp nc cho nm, ch , cc khong cht m n thu c t
sng.
Da trn nguyn tc ny, cc phng th nghim nui cy m to mi
trng gieo ht lan trn mi trng dinh dng nhn to c khong, ng v ht
lan ny mm vi t l rt cao. Hin nay, phng php nui cy m l phng
php duy nht c th nhn ging lan trn qui m cng nghip.
Tri lan khi hi phi gi v cha b nt c em kh trng. Mi
trng cy chuyn l mi trng gieo ht nhng b sung thm 10% nc da cho
protocorm pht trin nhanh.
Khi chi ln chuyn sang mi trng nh mi trng c bn, sau
khong 6 - 7 thng ty ging khi cy cao t 4-5 cm, ra r v c t 4 - 5 l l c th
mang ra ngoi trng.
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pT1 Electric-valve
GVHD: T Ngc Ly
() Pump
Filter
r
%
*
u
L J
=hf-<B
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GVHD: T Ngc Ly
Basket
U I
0u
solution
( 0
3
u.
Funnel
Filter
y Silicone tube
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GVHD: T Ngc Ly
cy trn thch thng thng trong bnh tam gic 500 ml, loi bnh ny thng
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Lan H ip
Lan H ip l mt trong nhng ging lan rt c yu thch khng ch
v mu sc, kiu dng m cn mang mt nt p rt sang trng v trang nh.
Chnh v vy, n nhanh chng tr thnh sn phm trng trt mang li hiu
qu kinh t khng ch Vit Nam m cn nhiu nc trn th gii nh i
Loan, Nht Bn, H Lan, M...
H ip l mt loi hoa rt kh nhn ging, thng cho h s nhn thp
trong iu kin vn m. c c s lng ln cy ging ng thi p ng
nhu cu th trng th cn phi kt hp cng ngh nui cy invitro v k thut
lai to cho t l sng st cao, hn na cng cho thm s a dng v hnh dng
v mu sc.
1.2.1. Tn khoa hc v phn loi
Lan H ip l ging lan c tn gi Phalaenopsis Blullle, 1825 , thuc h
ph Vandoideae Tng Vandeae. Lan H ip c tn khoa hc l Phalaenopsis
amabilis l loi lan c hoa ln, p, bn.
Ging Phalaenopsis gm 21 loi lan pht sinh, a nng c bn o M
Lai, Indnxia, Philipin, cc tnh pha ng n v Chu c.
_____________________________________________________________________
1.2.2. Mt vi c im ca Lan H ip
Lan H ip gm cc loi cO hoa ln p, cung ngan gom li thnh nhng
chm lng lo : n hay phn nhnh. L di v cnh hoa phng, tri rng, thng th
l i ging v gn bng cnh hoa. Mi lin tc, c gc trc ko di, v mang mt
im nh nh gc, phin bn tri rng hay hng ln mt t, phin gia tri ra
nguyn vn hay c 2 phin di, hp v c da, nhng b phn ph c dng thay i :
tr bn nguyt dy bn trn, thng hay hi cong.
Lan H ip c mu sc phong ph, khng thua km bt c ging lan no
khc t trng, hng, , vng, tm n cc loi h ip c sc nm ngang hoc thng
ng, hoc c m to hay nh v.v...
Nm
1996, anh Bch u t 2.000 cy lan ging. V u, mi tun ct hai ln, thu
li khong 5 triu ng/ thng. n nay, vi trn 1ha lan ct cnh c trn
50.000 cy, li nhun anh thu v mi nm gn t ng.
Ti vn lan ca anh Thanh, mi cy lan c gi trung bnh t 100.000 n
200.0 ng, c cy thuc loi hng c gi ln ti 4 - 5 triu ng.
T khi trng ti lc thu l khong 40 ngy; dng ko sc ct cnh, ng
gi, giao cho cc ch u mi. Gi bn t 4.000 - 14.000 /cnh; cy ging
25.0
- 45.000 /cy (tch t cy m). Qua vic trng lan - ct cnh, mi nm
gia nh ch Gm thu nhp trn di 500 triu ng, t vic bn hoa v lan
ging.
1.3.
Cc m hnh vn m Lan H ip
1.3.1.
Nh knh
Nh knh l mt khu vc sn xut bc cao ca vn m vi kt cu sau:
+ Khung gin lm bng st chng gi
+ Lp cc khung bng nhng tm knh trong sut hoc nha
polyethylene + C 1-2 ca ra vo v c ca s bng kch thc ca s ca
nh bp.
+ Nha bc nh knh l polyethylene t 3-5 lp, c ph li mu en
hoc xanh en.
Nh li
Trc y nh li c xy dng kh n gin vi nhng khung bng g
chng, bao quanh li thp v trn ph li nha nhng hin nay nhng ngi nng
dn s dng ct b tng, x st v u t mt cch chc chn. Chiu cao ca nh
li ti thiu l 2m. Li lp mi c 2 x 2 mm, li treo xung quanh c 1 x 1
mm. Nh li c th lp t cc h thng phun ti, lm mt nhng cn th cng
hn so vi nh knh.
Hin nay c cc kiu nh li ph bin trong sn xut nh:
GVHD: T Ngc Ly
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GVHD: T Ngc Ly
MS2
MS3
MS4
MS5
KNO3
KH2PO4
NH4NO3
MgS04.7H20
CaCl2.2H2O
H3BO3
MnSO4.4H2O
CoCl2.6H2O
CuSO4.5H2O
ZnSO4.4H2O
Na2MoO4.2H2O
KI
FeSO4.7H2O
Na2-EDTA
myo-Inositol
Thiamine. HCl
Pyridoxine .HCl
Nicotinic acid
Glycine
1900
170
1650
370
440
6,2
22,3
0,025
0,025
8,6
0,25
0,83
27,8
37,3
100
0,1
0,5
0,5
2
trng
20 ml
10 ml
10 ml
10 ml
10 ml
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GVHD: T Ngc Ly
Nc da 15%
NAA 1 mg/l
BA 10 mg/l
D
D
Adenine 10 mg/l
Thm 20 g saccharose vao va khuy tan.
Ly 8 g agar cho vao 1 cc thuy tinh khc , b sung 300 ml nc ct va un
si cho n khi agar tan ht.
dung dich MS cc th nht vao cc th hai ng agar ri thm nc
ct, nh mc n 1l ri khuy u
mi trng a chun bi vao cac bnh nui cy, th tch 40 ml/l bnh. Cn
chia xong mi trng tht nhanh trc khi dung dich ngui xung di 50 oC.
Dng nt bng khng thm nc y kn lai ri em hp kh trung nhit
121oC, ap sut 1 atm/15 - 30 phut.
ngui va rao nc trn b mt mi trng thi mi dung nui cy
(thng thng mi trng sau khi nu 2 ngay la cO th cy c).
Sau 10 tun nui cy, cc protocorm c hnh thnh ch yu t cc mnh
l phn gc v t cc mnh l phn nh.
Sau mt thi gian, cc chi xut hin xung quanh mp l ( ni c vt thng
) tip tc pht trin trong khi phn m l ban u b hoi i. Phin l ban u
c s dng nh ngun dinh dng khi u cho protocorm v cho chi sau ny
nhng h thng mch ca chi c hnh thnh th hon ton c lp vi h thng
mch ca m m.
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GVHD: T Ngc Ly
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GVHD: T Ngc Ly
Hm lng trong 1 lt mi
Ca(NOs)2.4H2O
trng
1
g
KH2PO4
FeSO4.7H2O
MgSO4.7H2O
(NH4)2SO4
ng saccharose
Agar
Nc ct va
0,25 g
0,025 g
0,25 g
0,50 g
20 g
10 g
1 lt
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GVHD: T Ngc Ly
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GVHD: T Ngc Ly
Cy ly hoa:
c = 300.000 cy/1 nm
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Nui cy m
GVHD: T Ngc Ly
hp ra
T l hao ht
Ra hoa
Chuyn cy t Chuyn cy t
nh knh
nh knh
sang chu
10 %
3%
13 %
5%
315.789,474 (cy)
100 - 5
S cy trng thnh ly hoa cn: 315.790 (cy)
S cy t nh knh chuyn sang chu T l hao ht
khi chuyn sang chu l 3 %.
315.790
c2 =
--------------------- x 100 =
325.556,701 (cy)
100 - 3
S cy t nh knh chuyn sang chu: 325.557 (cy)
S cy t hp nui cy m chuyn sang nh knh
T l hao ht khi chuyn t hp ra ngoi l 10 %.
S cy chuyn sang vn m:
325.557
c3 =
--------------------- x 100 =
361.729,066 (cy)
100 - 10
S cy chuyn sang vn m: 361.730 (cy)
S cy cn nui cy m
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GVHD: T Ngc Ly
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GVHD: T Ngc Ly
555.556
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GVHD: T Ngc Ly
b
2
100 - 13
S cy con 3 thng tui cn nui cy m trong 1 nm: 638.571 (cy)
S cy con 3 thng tui cn nui cy m trong 1 qu:
b3 = 638.571 : 4 = 159.642,75 (cy)
o Vy s cy 3 thng tui cn nui cy trong 1 qu: 159.643 (cy)
S chi ng cn nui cy ban u
Trong mt cm chi c th phn lm 6 phn, mt phn ti sinh cm chi v
5 phn nhn tip.
5 phn ny tip tc tin hnh cy chuyn nhn nhanh s lng, tin hnh
nhn tip 5 ln na. M c 1 phn li nhn ra lm 5 phn. Vy sau 5 ln c
s protocom nh l: 5 x 55 = 15.625 (protocom nh).
Qu trnh tin hnh tip tc l c 1 protocom nh s to nn 5 cy con. Vy
s cy nhn ra c l: 15625 x 5 = 78.125 (cy con)
Nh phn tnh ton trn th s cy cn nui cy trong mt nm p ng
nhu cu cy hon chnh l 638.569,604 cy th s chi ng cn cy lc ban u l:
B = 638.571: 78.125 = 8,17 (chi)
Vy s chi ban u l: 9 chi ng t cc pht hoa
3.3.3. S cy con trong ng nghim
S cy con trong ng nghim: a = 800.000 cy/1 nm.
S cy cn nui cy m
T l nhim khi nui cy l 13 %
S
100 -
13
o S
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GVHD: T Ngc Ly
52 tun
7
S ngy lm vic trong 1 nm l:
n = 365 - (52 X 2 + 8) = 253 ngy S cy con trong ng nghim cn
nui cy hng ngy:
919.541: 253 = 3.634,549 (cy)
<=> Vy s cy trong ng nghim cn nui cy hng ngy: 3.635 (cy)
S chi ng cn nui cy ban u
Trong mt cm chi c th phn lm 6 phn, mt phn ti sinh cm chi v
5 phn nhn tip.
5 phn ny tip tc tin hnh cy chuyn nhn nhanh s lng, tin
hnh nhn tip 5 ln na. M c 1 phn li nhn ra lm 5 phn. Vy sau 5 ln
c s protocom nh l: 5 x 55 = 15.625 (protocom nh).
Qu trnh tin hnh tip tc l c 1 protocom nh s to nn 5 cy con. Vy
s cy nhn ra c l: 15625 x 5 = 78.125 (cy con)
Nh phn tnh ton trn th s cy cn nui cy trong mt nm p ng
nhu cu cy hon chnh l 919.540,23 cy th s chi ng cn cy lc ban u l: A
= 919.541 : 78.125 = 11,77 (chi)
Vy s chi ban u l: 12 chi ng t cc pht hoa
3.3.4. Lng ha cht trong mi trng MS c b sung cc cht iu ha sinh
trng
1 hp nui cy cha khong 40 ml mi trng, 12 cm chi.
S cy ly hoa cn nui cy m trong 1 nm: 415.782 cy
o 34.649 cy/thng.
1 cm chi to 5 cy con nn s cm chi:
34.649 : 5 = 6.930 (cm chi)
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GVHD: T Ngc Ly
= 2.741,58 (hp)
12
Vy s hp mi trng to cm chi s dng trong 1 thng: 2.742 (hp) Lng
mi trng MS c b sung cc cht iu ha sinh trng:
V = 2.742 X 40 = 109.680 ml = 109,68 lt
MS1:
KNOs
KH2PO4
1.900
170
g
208.392
18.645,6
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GVHD: T Ngc Ly
1.650
370
440
6,2
22,3
0,025
0,025
8,6
0,25
0,83
27,8
37,3
100
0,1
0,5
0,5
2
108.972
40.581,6
48.259,2
680,016
2.445,864
2,742
2,742
943,248
27,42
91,0344
3.049,104
4.091,064
10.968
10,968
54,84
54,84
219,36
NAA 1 mg/L:
m= 1 X 109,68 = 109,68 mg
BA 10 mg/l:
m = 10 X 109,68 = 1096,8 mg
Adenine 10 mg/l:
m = 10 X 109,68 = 1096,8 mg
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GVHD: T Ngc Ly
Knudson C
------------------
= 13.707,75 (hp)
12
Vy s hp mi trng ti sinh cy s dng trong 1 thng: 13.708
(hp) Lng mi trng Knudson C:
V = 13.708 X 40 = 548.320 ml = 548,32 lt
Bng 3.3. Lng ha cht mi trng Knudson C cn s dng trong 1 thng
Thnh phn
Ca(NOs)2.4H2O
Hm lng trong 1 lt
mi 1trng
g
trng
548,32 g
0,25 g
0,025 g
0,25 g
0,50 g
20 g
10 g
1 lt
137,08 g
13,708 g
137,08g
274,16 g
10.966,4g
5.483,2 g
548,32 lt
KH2PO4
FeSO4.7H2O
MgSO4.7H2O
(NH4)2SO4
ng saccharose
Agar
Nc ct va
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GVHD: T Ngc Ly
Hnh
5.7.
Mi
kh
trng
GVHD: T Ngc Ly
Cng sut: 10 kW
Ngun in: 220V-50 Hz
p sut : 0,235 Mpa
Nhit ln nht: 135 0C
Thi gian kh trng: 0-250 pht/ln.
Mi ngy ta cn hp 2 ln nn s ni cn hp tit trng l:
300 : (180 x 2) = 0,833 (ni hp)
Ta cng cn thm 1 ni kh trng hp cha mi trng sau khi cy nui cy
m c cy chuyn sang hp mi trng mi.
Vy ta cn 02 ni hp kh trng
4.1.3. T sy
S lng hp cn sy trong 1 ngy: 748 (hp)
Din tch 1 hp: 0,65 x 0,65 = 0,43 (dm2) = 0,0043 (m2)
Ta chn t sy nhn hiu: MOV-212 do hng SANYO sn xut Th tch:
157 lt
Kch thc (LXWXH): 730X645X870 mm
Trng lng: 63 kg
Cng sut: 1300 W
Ngun in: 220V- 50Hz
Nhit : 40-250 0C
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GVHD: T Ngc Ly
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GVHD: T Ngc Ly
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4.2.2. Cn k thut
c s dng cn cc loi ha cht pha mi tru
trnh nui cy vi lng cn nh nht l 0,01 g/1 ln cn.
Ta chn cn nhn hiu : 572-35 do hng KERN-c
Kh nng cn ti a : 1210 g
c : 0,01 g
lp li: 0,01 g
tuyn tnh : 0,03 g
Khi lng mu nh nht : 0,03 g
My ct nc
Nc ct c s dng pha ch mi trng nui cy, trng hp cho nui cy
(lt)
- Nc cung cp:
Lu lng: 2 lt/pht p lc nc:
3 n 100 psi
- Kch thc (W X D X H): 550 X 410 X 410 mm
- Ngun in s dng: 01 phase, 220V, 50/60 Hz
- Cng sut: 6 kW
S lt nc ct sn xut c trong 1 ngy: 4 x 8 = 32 (lt)
S lng my cn phc v trong nui cy: 180 : 32 = 5,625 (my)
Vy s lng my ct nc 1 ln cn dng l: 06 (my)
4.2.5.
My khuy t
c dng khuy khi pha ch mi trng nui cy.
Lng mi trng cn c pha ch trong 1 ngy:
(109,68 + 548,32 ) : 22 = 30 (lt)
Ta c th chn my khuy t gia nhit c th tch ln Mode CB302
Hillh
My khl|y t
GVHD: T Ngc Ly
Bp un
Hnh 4.10. My o pH
,1 ___________/V __________4*
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GVHD: T Ngc Ly
4.2.8. Pipette
c dng ht chnh xc cc dung dch
L-
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GVHD: T Ngc Ly
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Hnh 4.13. T cy
Mt ngi cy trung bnh 1 ngy c 600 mu, ta chn 2 ngi cng cy 1
t nn s t cy cn s dng: 9.216 : (600 x 2) = 7,68 (t)
Vy s lng t cy cn s dng l: 08 (t)
4.3.2. Cc dng c cy khc
Gm n cn, giy thm, ng ong cha cn, kp, dao m, ko, a
petri, a khuy bng thy tinh.
GVHD: T Ngc Ly
Hnh 4.19. Ko
Hnh 4.20. n cn
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GVHD: T Ngc Ly
IbDg gi
Qut thi
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Thng s k thut
in p: 220V
Cng sut: 48W
Lu lng: 18 m3/pht
Kch thc: 500 mm X 500 mm
ng knh cnh qut: 450 mm
Mi nh knh cn 2 ci, vy s lng qut cn c: 19 x 2
= 38
ci
4.5.3. Qut thi
Chn qut Model VF36CS2 ca hng J & D [27]. Thng s k
thut
- ng knh cnh qut: 915 mm
- Kch thc ngoi: A = B = 1016 mm
- Tc : 317 rpm
-
GVHD: T Ngc Ly
4.6.2. Gi ch u
Gi
hi
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GVHD: T Ngc Ly
315.790 :11.760=27 nh
o Chn 27 nh
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