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Nguyen Bang Phi 02126078 BOVUd 20130705102132 3074 LTRU
Nguyen Bang Phi 02126078 BOVUd 20130705102132 3074 LTRU
B GIO DC V O TO
TRNG I HC NNG LM TP. H CH MINH
B MN CNG NGH SINH HC
***000***
Lun vn k s
Chuyn ngnh: Cng ngh sinh hc
Thnh ph H Ch Minh
-2006-
B GIO DC V O TO
TRNG I HC NNG LM TP. H CH MINH
B MN CNG NGH SINH HC
***000***
Lun vn k s
Chuyn ngnh: Cng ngh sinh hc
Thnh ph H Ch Minh
-2006-
Graduation thesis
Major: Biotechnology
Professor
Student
NGUYN BNG PHI
B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
B MN CNG NGH SINH HC
Thnh ph H Ch Minh
2006
B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
B MN CNG NGH SINH HC
Thnh ph H Ch Minh
2006
LI CM T
hon tt cun lun vn ny ti nhn c s hng dn, ging dy, gp
, gip ca cc thy c, bn b v gia nh. Ti xin c by t lng bit n su
sc n:
-Ba m dy cng nui dng, dy d con
-Cc Thy c B mn Cng Ngh Sinh Hc- trng i Hc Nng Lm TP.H
Ch Minh.
- TS.TRN TH DUNG, Trng B mn Cng Ngh Sinh Hc- Trng i
Hc Nng Lm TP.H Ch Minh tn tnh hng dn, gip ti trong sut qu
trnh thc hin ti.
- TS.NGUYN VN HNG, Trng B mn iu khin t ng khoa C kh
Trng i Hc Nng Lm TP.H Ch Minh
-KS.NG THANH HONG, Ging vin khoa C kh- Trng i Hc Nng
Lm TP.H Ch Minh
-Cc k s Trung Tm Cng Ngh Sinh Hc- Trng i Hc Nng Lm
TP.H Ch Minh.
-Tp th lp Cng Ngh Sinh Hc 28
Thng 08 nm 2006
Nguyn Bng Phi
TM TT
NGUYN BNG PHI, i hc Nng Lm Tp. H Ch Minh. Thng 08/2006.
MC LC
Trang
Tm tt lun vn ......................................................................................................... 7
Danh sch cc bng .................................................................................................... 11
Danh sch cc hnh ..................................................................................................... 12
Danh mc vit tt ........................................................................................................ 13
Phn 1 : Gii thiu
1.1. t vn ............................................................................................................ 14
1.2. Mc ch-yu cu ................................................................................................. 15
1.3. Gii hn ti ...................................................................................................... 15
Phn 2 : Tng quan ti liu
2.1. c im thc vt hc v sinh thi cy da ........................................................ 16
2.2.Phn loi .............................................................................................................. 17
2.3. Cc nhm da chnh v cc ging da ph bin Vit Nam ............................. 18
2.4. Tnh hnh sn xut v sn lng da ................................................................... 20
2.5. Gii thiu v nui cy m ................................................................................... 22
2.6. Cc yu t nh hng trong nui cy invitro ...................................................... 25
2.7. Tng quan v bioreactor ...................................................................................... 28
2.8. Gii thiu cc phng php nhn chi cy da .................................................. 34
2.9. Cc yu t nh hng trong qu trnh nui cy ngm chim
ky .................. 38
inh
2.10. nh hng h thng ngm chm nh k n cht lng cy trng ................. 40
2.11. nh hng ca h thng ngp chm n chi ph sn xut................................. 44
2.12. Cc nghin cu v ng dng bioreactor trn i tng cy da ...................... 44
Phn 3 : Vt liu v phng php nghin cu
3.1. Thi gian v a im .......................................................................................... 45
3.2 Ni dung nghin cu ............................................................................................ 45
3.3 Vt liu ................................................................................................................. 45
3.4. Phng php nghin cu ..................................................................................... 45
3.4.1Thit k h thng t ng cho bioreactor sc kh t to v ngm
chm nh k (TIB) ..................................................................................................... 45
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11
Trang
12
Trang
13
DANH MC CC CH VIT TT
BAP
: 6-benzylaminopurine
GA3
: Gibberellic acid
IAA
: Indole-3-acetic acid
IBA
: Indole-3-butyric acid
NAA
: -Naphthaleneacetic acid
TDZ
: Thidiazuron
MS
TIB
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Phn 1. M U
1.1 t vn
Nc ta thuc vng nhit i c kh hu nng m kh ph hp cho nhiu loi cy
trng khc nhau pht trin. Da l cy n qu nhit i, l mt trong ba loi cy n qu
hng u ca nc ta (chui-da-cam qut) dng n ti, c bit c th ch bin
xut khu. Chnh v vy, cy da c trng nhiu vng trong nc v vi iu kin
thch hp nhit v m cao th cy da c th sinh trng quanh nm. Do l loi
cy khng kn t, da c th trng vng g i, t dc (200 tr xung), t xu
ngho dinh dng. V vy chng ta c th ni cy da gip con ngi tn dng qu t
c thm sn phm v mang li hiu qu kinh t khi thu hoch. Da cn dng chit
xut enzyme bromelin dng trong cng nghip thuc da, vt liu lm film. Qu da
dng ch bin hp, lm ru, gim, nc p, nc c c, lm bt da dng cho
gii kht.. L da dng ly si (2-2.5% cellulose).
Chnh v kh nng ng dng kh ln ca cy da m cy da ngy cng chim
v th quan trng c th hin qua nm 1993 th kim ngch xut khu ch t 658 triu
la M v cho n nm 1998 th t 3450 triu la M. phc v nhu cu ngy
cng cao ca con ngi th vic tn dng li chi con ca cy da t cy m khng
mang li hiu qu kinh t. Do nhng cy ny thng cho qu nh dn trong cc ma v
sau. Vic cung ng ging da t cht lng v sch bnh l vn cp thit.
Nhn ging v tnh in vitro c th p ng c nhng yu cu v cht lng
ng u vi s lng ln. Tuy nhin vic nhn chi da bng phng php nui cy
m vi nhn ging tn nhiu chi ph v lao ng, phi cy tng chai mt trong iu kin
v trng nghim ngt, v phi cy chuyn sau khong 4-6 tun do cn kit mi trng
dinh dng. Vic sn xut ging quy m cng nghip kh c th thc hin mt cch
t ng ha c. T nhng thc trng , nm 1981 Takayama ln u tin ng dng
bioreactor nhn ging vi s lng ln trn i tng cy Begonia. Qua qu trnh
pht trin, nhiu kiu bioreactor khc nhau ra i nh : bioreactor sc kh, bioreactor
khuy, bioreactor ngm chm nh k....Trong , bioreactor ngm chm nh k trong
mi trng lng em li hiu qu cao hn phng php nhn ging thng thng trn
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mi trng thch v phng php nui cy trong dch lng c khuy v sc kh lin tc,
ng thi cy con khi ra vn cng tt hn so vi cy con nui mi trng thch.
Hin nay Vit Nam nhng nghin cu v bioreactor cn rt t, c bit l trn
i tng cy da. Do vy, ti : THIT K V NGHIN CU H THNG
BIOREACTOR CHO CY DA CAYENNE c tin hnh ti B Mn Cng
Ngh Sinh Hc i Hc Nng Lm Tp.HCM.
1.2 Mc ch - Yu cu
Mc ch
Xc nh c cu trc, cch vn hnh v m hnh ca h thng bioreactor
ngm chm nh k trong mi trng lng (temporary immersion bioreactor (TIB))
cho cy da.
So snh hiu qu nhn chi gia 2 kiu bioreactor : ngm chm nh k v sc
kh.
Xc nh thi gian ngm chm nh k trong TIB thch hp cho hiu qu
nhn chi cy da tt nht.
Yu cu
Nm c cu trc v cch vn hnh h thng bioreactor ngm chm nh k
trong mi trng lng(TIB)
nh gi hiu qu nhn chi ca h thng bioreactor ngm chm nh k(TIB)
1.3 Gii hn ti :
- Do gii hn v thi gian v iu kin trang thit b th nghim nn ti ch
tin hnh qu trnh nhn chi trong phng th nghim, cha thc hin th nghim ngoi
ng
- Do gii hn v kinh ph nn h thng iu khin cha kim sot cc yu t pH,
dinh dng, nh sng ... trong qu trnh nui cy mt cch t ng.
16
17
18
Lp: Liliopsida
B: Poales
H: Bromeliaceae
Ging: Ananas
Loi: A. comosus
2.3. Cc nhm da chnh v cc ging da ph bin Vit Nam
Cc nhm da chnh
Da gm khong 50 ging v 2000 loi phn b vng nhit i v cn nhit
i chu M . Cc ging da ang c trng trt hin nay c chia thnh 3 nhm:
nhm da Cayenne, nhm da Queen (cn gi l hong hu) v nhm Spanish (nhm
Ty Ban Nha).
Nhm Cayenne
L di, khng c gai hoc c mt t u chp l, dy, lng mng l su, c th
di hn 100 cm, hoa c mu xanh nht, hi , qu c dng hnh tr, mt rt nng, qu
nng bnh qun 1,5 2,0 kg rt ph hp cho vic ch bin lm hp. Khi cha chn
qu mu xanh en, sau chuyn dn v n lc chn qu c mu hi pha da ng.
Cy yu, trung bnh ch cho 1 2 chi mt gc trong mt nm. Trong iu kin
chm sc km c th khng c chi cung.
Qu da Cayenne cha nhiu nc v v mng nn rt d thi khi vn chuyn.
V th vic chn vng, a im trng v qui hoch ng rung phi quan tm n c
im ny.
Nhm Queen
L hp, cng, c nhiu gai mp. Mt trong ca l c 3 ng vn trng hnh
rng ca chy song song theo chiu di, hoa c mu xanh hng. Qu c nhiu mt,
mt nh v li, cng do tng i d vn chuyn. Tht qu vng, t nc v c v
thm hp dn.
u im ca nhm da ny l khng kn t, c th trng trn cc loi t ngho
dinh dng, cy c h s nhn ging cao, trung bnh 4 6 chi/gc, c th chu c
bng rm. Tht qu dn, c mu sc v hng v ph hp n ti.
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Trong chn ging cy trng ngi ta s dng phng php ny ci tin ging cy
trng bng cch cho dung hp protoplast 2 protoplast cng loi hoc khc loi.
Protoplast c kh nng hp thu t bo ngoi lai ci thin c tnh ca mt s
loi cy trng m khng thng qua cc phng php chuyn gen khc.
Nui cy t bo n bi
Ht phn ca cy trng c nui cy trong mi trng thch hp to thnh m
so, nhng m so ny c kh nng ti sinh thnh cy hon chnh c b nhim sc th
n gi l cy n bi. Trong nui cy m thc vt ngi ta s dng nhng m so ny
x l colchicin to thnh cy a bi.
2.6. Cc yu t nh hng trong nui cy in vitro
Cht iu ho sinh trng
Cht sinh trng thc vt hay cn gi l cht iu ha sinh trng thc
vt l cc hp cht hu c (bao gm cc sn phm thin nhin ca thc vt v cc hp
cht tng hp nhn to) c tc dng iu tit cc qu trnh sinh trng v pht trin,
lm bin i mt qu trnh sinh l thc vt no , nhng nng rt thp. Chng
khng phi l cc cht dinh dng hay cc sinh t dng trong thc vt.
Mt s cht iu ho sinh trng thng dng:
Auxin
26
27
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Theo s phn tch thnh phn dinh dng ca T chc Y t Th gii, th trong
CW c cha protein, cacbohydrat, canxi, st v mt s vitamin nh thiamin,
riboflavin, niacin, acid ascorbic v ng.
nh hng ca than hot tnh
Phi c nhng thc nghim xc nh nng than hot tnh thch hp cho qu
trnh nui cy. Than hot tnh thm vo mi trng kch thch s pht trin phi ca
cy bp, jujube v u . Than hot tnh thng c b sung vo mi trng hp
th cc cht c ( dng phenol) do cy tit ra.
nh hng ca pH v Agar
pH ca mi trng nui cy thng khong 6, thp hn 4,5 hoc cao hn 7
u c ch s pht trin ca m (Nguyn Vn Uyn, 1993 v Bi B Bng, 1995).
nh hng ca cc iu kin vt l
nh sng cn thit cho s pht sinh hnh thi ca m cy. Trong to chi ban u
v nhn chi tip theo, cng nh sng ch cn trong khong 1.000 lux. Nhng trong
giai on to r, cy cn chiu sng cng cao t 3.000-10.000 lux kch thch
cy chuyn t giai on d dng sang t dng c kh nng quang hp. Di cng
nh sng cao, cy ln v c mu xanh hi gim nhng c t l sng st cao khi chuyn
sang mi trng t. Cha c nhiu nghin cu v ch sng trong mi trng cy
m, nhng thi gian chiu sng 16 gi/ngy ca bng n non hunh quang l thch
hp cho s pht trin m cy ca nhiu loi. Cy phi thng khng s dng nh sng
n. Theo Scozzoli v Pasini, 1992; Pinto v csv, 1994, cy phi o nn trong ti 14
ngy. Tng t, cy b nn 21 ngy trong ti nhit 210C (Lano v csv, 1995).
Ngoi ra, m cy pht trin tt th mi trng nui cy phi thng thong v
c nhit thch hp, nhit trong phng nui cy thng c gi 25-28oC
(Nguyn Vn Uyn, 1993).
2.7.Tng quan v bioreactor
H thng bioreactor c thit k v mc ch ci thin v nng cao hiu qu
cng nh nng sut ca qu trnh vi nhn ging. M hnh bioreactor c thit k theo
dng fermentor trong nui cy vi sinh vt, ty vo i tng cy m c nhiu kiu
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bioreactor khc nhau nhm mc ch nhn s lng ln t bo, m hay c quan trong
mi trng lng c h thng lm thong kh.
T bo thc vt khc vi t bo nm men. Nu c bo qun trong iu kin
ging nhau th t bo thc vt khng sinh trng n c m cng khng xy ra s
sinh trng ng thi ging nh nm men. Tuy nhin bng k thut nui cy m th
vic nhn sinh khi, nghin cu dinh dng v sinh ha trong mi trng lng th t
ra hiu qu(Gamborg, 1966; Gamborg, et al, 1968; Gamborg and Shyluk, 1970).
Vic s dng bioreactor cho nhn ging thc vt c thc hin u tin vo
nm 1981 do Takayama thc hin trn i tng cy Begonia (Takayama v
Miasawa,1981). H thng ny cn c ng dng c hiu qu trn nhiu loi thc vt
khc(Takayama,1991). S dng mt bioreactor c nh ( t 4-10l) trong khong 1-2
thng c th to ra n 4000-20.000 cy con. iu ny cho thy mt trin vng ln
trong vic p dng bioreactor thng mi ng thi m ra mt hng mi trong sn
xut cc sn phm c hot tnh sinh hc c ngun gc thc vt(Takayama,1991). Mc
ch ca nui cy lng lc v bioreactor l tng nhanh s lng ln cc chi ng nht
v ng thi gip gim chi ph trong nhn ging thc vt(Nhut et al,2004)
6. Motor
7. ai b
1. Ca np nguyn liu
8. Thanh truyn
3. H thng sc kh
4. ng dn n
5. ng thot n
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nhm t bo ang tng trng ph hp cho s pht trin to phi bt c lc no. Giai
on th hai ca bioreactor l to phi theo chu k. S pht trin ca phi c th c
iu khin thng qua ngun dinh dng thit yu hoc cc thnh phn kh. Vic to ra
cc cy con thng qua con ng to phi soma c nhn thy l thch hp nht i
vi qu trnh nhn sinh khi bng cch s dng bioreactor mng lc xoay (Honda et
al., 2001).
Bioreactor c khuy v khng khuy bng nn kh (Pneumatically
agitated and non-agitated bioreactor)
y l kiu bioreactor n gin, c thit k vi mt b phn si bt
kh pha di y bnh, n c nhim v l khuy trn mi trng v cung cp
oxygen (i vi bioreactor sc kh n gin, bioreactor to bt dng hnh ct). Trong
mt s trng hp, bnh nui cy c th gn thm cc ng thng (i vi bioreactor
air-lift).
Bioreactor air-lift
Kiu bioreactor ny cng tng t vi bioreactor c khuy trn bng dng
xoy-STRs (nhng y th khng c cnh khuy). Bioreactor air-lift khc phc c
hai nhc im ca bioreactor khuy bng cnh khuy l: t tn nng lng cho
vic khuy trn mi trng v t gy ra lc x rch cc t bo, nh nhng dng kh
nh di chuyn nh nhng t pha di ln. S ha trn dng kh vo trong pha lng tr
nn c hiu qu hn do c s lu tr cc bong bng kh trong mi trng. c th
to c cc bt kh nh mn th dng kh phi c thi qua mt mng lc vi nhng
l c kch thc rt nh 0,01 0,1 mm. Thng s dng mng ceramic. Chnh nh s
nh mn ca cc bong bng kh lm cho cc t bo gim ng k s c st nn t b
tn thng, nht l i vi nhng t bo c nhy cm cao (Paek v Debasis, 2003)
Bioreactor sc kh dng n gin v bioreactor si bt kh dng hnh ct
(Simple aeration bioreactor and bubble column bioreactor)
Cng ging nh bioreactor air-lift, bioreactor si bt hnh ct cng to ra t s c
st. im khc bit ch yu gia bioreactor air-lift v bioreactor si bt hnh ct l h
thng tun hon v ch thy ng lc hc. Loi bioreactor ny thch hp cho nui
cy nhiu loi cy khc nhau thng qua qu trnh nui cy chi, thn c, r c
(Takayama, 1991). Hn na, vic chia bioreactor si bt hnh ct thnh nhiu phn v
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trnh dng ln v h xung. C mt h thng van 3 cng dng so-le c dng trong
trng hp iu khin ng m ny. H thng bioreactor ny c bo co l nui
cy thnh cng trn mt s cy nh: cy l (Damiano et al., 2000), cy da (Esscalo et
al., 1999) v cy coffea arabica (Etienne et al., 1999).
2.8. Gii thiu cc phng php nhn chi cy da
H thng nui cy in vitro bn rn
L h thng nui cy in vitro s dng gi th cha cht dinh dng gi cho
cy ng vng ng thi cung cp cht dinh dng cho cy.
Khi nim v gi th
Gi th l mt loi hay mt hn hp vt liu c s dng lm vt nng , l
ch
cho r bm vo trong sut qu trnh sinh trng v pht trin. Vt liu lm gi
th
phi chc chn v n nh trong cc iu kin khc nhau, chu c hp v trng
m khng b bin tnh, tr vi cc cht trong mi trng nui cy. Ngoi ra, vt
lm gi th phi sch v khng c cha cc cht l do thnh phn ca mi trng
dinh dng l xc nh v cn kim sot c trong tng trng hp nui cy. Cht
to ng thng dng nht l agar, nhng theo thi gian, nhiu loi gi th khc c
pht trin vi nhng tnh nng u vit hn nh rockwool, oasis, v.v.
Agar
Agar l mt polysaccharide chit xut t rong bin. Agar c s dng nh mt
tc nhn to ng trong hu ht cc mi trng nui cy m in vitro. Agar l loi gi
th s dng ph bin nht trong cc phng th nghim.
Alginate
Gi th ny c c tnh tng t nh agar nhng c nng ion calcium cao
hn.
Phytagel
c tng hp t glucuronic acid, glucose v rhamnose. Mi trng to ra t
loi gel ny rt sch, bn cao, trong sut nn d pht hin ra hin tng nhim vi
sinh vt.
Agargel
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dung tch nh (140 ml). Monette (1983) ch ra rng khi nui cy chi cy nho trong
nhng bnh cha ln s thu c chi c kch thc di hn. Chi cy nho tng trng
rt nhanh trong mi trng lng s dng h thng bioreactor nghing lc (trong nhng
hp vung Mason 910 ml, ming rng) so vi trong bnh tam gic dung tch 125 ml do
mu khng b dn qu nhiu mt ch, d dng thu mu sau nui cy v ngn chn
c s thiu ht cht dinh dng.
Vic s dng nhng vt cha ln nui cy cho php s dng nhiu mi
trng, iu ny c nh hng tch cc n s hnh thnh v pht trin ca thc vt
nui cy bng bioreactor.
S thong kh t nhin v thong kh cng bc
Th nghim vi nhn ging cy chui trong mi trng lng chng minh rng
s thiu ht oxygen khi nui cy lng l nguyn nhn chnh dn n s hnh thnh
nhng cy con c kch thc nh. Cung cp oxygen bng phng php sc kh c th
kch thch cy tng trng nhng phng php ngp chm tm thi t ra c hiu qu
nht trong trng hp ny.H thng ngp chm tm thi c thay mi trng mi bng
p lc kh s to ra iu kin thong kh cng bc v lm mi bu kh quyn trong
bnh cy sau mi chu k chm ngp. Theo Teisson v Alvard (1995), khng kh c
thay mi trong qu trnh cht lng di chuyn v do mt lng kh mi c cp vo t
my bm kh. Trong iu kin cp kh bt buc, hm lng kh v m tng i
sinh ra trong bnh cy c th c nh hng tch cc n vic nui cy. m tng
i sinh ra do nh hng ca trao i kh c tc dng kch thch s thot hi nc
thc vt, gip cy in vitro c kh nng thch nghi tt hn khi chuyn ra mi trng ex
vitro
2.10. nh hng h thng ngm chm nh k n cht lng cy trng
c im hnh thi ca thc vt sn xut t cc bioreactor ngp chm tm
thi :
L ca nhng chi pht sinh trong h thng ngp chm tm thi c kch thc
nh hn l t chi nui cy trong mi trng lng. Nhng cm chi pht trin t cc
chi bn trong bioreactor thng c hnh cu v chi c xu hng pht trin ta trn
quanh tm. Do , mt s chi con c kch thc khng ln v cn c ko di trong
cng h thng trc khi cho ra r ngoi ng nghim. Ngc li, chi cy cow tree c
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v cng s (1998) chng minh c rng s dng h thng ngp chm tm thi
gip gim c 46% chi ph so vi quy trnh nui cy chun trn mi trng thch.
Chi ph tit kim c ch yu nh vo vic gim c nhn cng v khng gian s
dng cho nui cy m. cy da, chi c nhn trong h thng bioreactor ngp
chm tm thi c t l nhn gp 100 ln so vi nui cy trn thch trong khong thi
gian 4 thng. Quy trnh ny gip gim 20% chi ph sn xut tnh cho 1 cy so vi
phng php nui cy lng truyn thng. Theo tc gi, yu t dn n thnh cng l
vic gim c s lng bnh cha, dng c thao tc; gim thiu cc cng on cy,
trng; loi b cng on ra r in vitro v hn ch c mc nhim vi sinh vt. Kh
nng duy tr mu chi Pinus radiata trong thi gian di (18 thng) m khng cn cy
chuyn ca h thng bioreactor ngp chm tm thi c s thay mi mi trng gip
gim chi ph lao ng v m ra kh nng t ng ha sn xut, kt qu cui cng l
gim c gi thnh cho cy vi nhn ging . T h thng ny (s dng bnh cha dung
tch 600 ml), chi c thu hoch vi sn lng 672 chi/gi so vi khong 1100 chi
thu hoch t 1 m2 din tch mi trng thch trong 1 thng.
Theo t l ny, h thng ch tiu tn 1/7 chi ph so vi quy trnh sn xut Pinus
radiata theo phng php cy chuyn trn thch thng thng. Trong sn xut phi
soma, cng on cho phi ny mm l tn km nht do mt nhiu lao ng trong vic
cy chuyn. H thng nui cy ngp chm c th c s dng va nhn phi, va
cho phi ny mm. Sn xut phi soma ny mm trong h thng kt hp vi gieo mm
trc tip ra t gip gim 13% cho ph thao tc v 6,3% din tch so vi chi ph khi s
dng k thut a cy nui cy t mi trng thch ra vn m. Ngoi ra, h thng
ny gip rt ngn giai on cho phi trng thnh v ny mm ba thng.
H thng ngp chm tm thi hin nay ang tng bc c thng mi ha.
CIRAD s dng h thng RITA sn xut mt lng ln cy ging lai C.abrabica
F1 Trung M v Tanzania. Mt nhm cc nh nghin cu thuc Trung tm Sinh hc
Thc vt Centro de Bioplantas Ciego de Avila, Cuba ang pht trin mt quy trnh
nhn ging thng mi trn hai i tng thc vt l cy ma v cy da s dng
phng php nhn m phn sinh trong h thng bnh i BIT.
Tm li, h thng ngp chm tm thi c nhng tc ng tch cc ln tt c cc
giai on nui cy chi v phi soma ca thc vt. H s nhn v s tng trng
43
thc vt nui cy trong h thng ny thng cao hn nui cy trn thch hoc trong
cc h thng bioreactor khc. Cy con ti sinh v phi soma to thnh c cht lng
tt hn, kh nng thch ng vi mi trng t nhin v t l sng st ngoi vn m
cng cao hn. H thng ny kt hp c hai u im ca nui cy lng v nui cy
rn. Trong mi trng lng, m thc vt hp thu cht dinh dng d dng hn, trong
khi kh nng trao i kh ca thc vt tt hn trn mi trng rn.
Khi s dng h thng nui cy m thc vt, thi gian v tn s ngp chm l
cc thng s quan trng nht cn c xc nh. Vic ti u ha cc thng s ny trn
tng i tng thc vt v tng i tng nui cy s cho hiu sut nui cy cao
hn, cho php iu khin tt hn s pht sinh hnh thi ca thc vt cng nh khng
ch c hin tng mng nc thng xy ra khi nui cy lng. Ngoi ra, thnh
phn mi trng dinh dng s dng cng nh mt mu cy cng l nhng yu t
quan trng, tuy nhin nhng yu t ny vn cha c hiu cn k v do cn c
nhiu nghin cu, tm hiu su hn na.
Khng nhng ng dng tt trong nui cy m, h thng ngp chm tm thi cn
c th tr thnh cng c c lc cho vic nghin cu c ch ca cc qu trnh trao i
cht. Chng hn nh nhng nghin cu trn cy c ph (Coffea spp.) cho thy rng
thi gian ngp chm c nh hng n hin tng mng nc v trng thi nc ca
m so v phi soma. Chnh xc hn, cc thng s v nc (hm lng, th nc) c
lin quan mt thit n hin tng mng nc v chu nh hng mnh m ca tn s
v thi gian x l ngp. cy cao su, h thng cho php nghin cu nhng cm ng
ca hin tng stress oxygen ha trong giai on ngp khi nui cy phi. Cc dng h
thng ngp chm khc nhau c th c s dng thch hp trong vic nghin cu nh
hng ca thnh phn khng kh trong kh quyn n s sinh trng ca thc vt.
T khi Harris v Mason (1983) cng b h thng ngp chm u tin, c mt
s h thng bn t ng hot ng theo nguyn l tng t c ch to, v vn
ny ngy cng thu ht nhiu s quan tm. H thng c n gin ha rt nhiu so
vi nhng h thng bioreactor u tin v c th tr thnh mt phng php nui cy
thc vt in vitro ph bin. Cc h thng hin nay gip gim c chi ph sn xut
v hon ton thch hp a vo sn xut ln. Nhng nghin cu trn h thng ny
44
45
da
3.3. Vt liu
Trang thit b
T cy, ni hp,my o pH, cn in t, n hunh quang, timer Omron, , my
bm kh (Resun, TQ),....
Dng c
Bnh erlen 2l, kp, ko, dy silicon, film lc v trng Satorius(0,2 m), adapter
220-110V, pipet 5ml, co L....
Mu cy
Mu cy l cy da c nui cy trong phng tng trng ca b mn CNSH
trong thi gian 6 thng. Mu da invitro c nui cy trong mi trng MS+30g/l
ng sucrose + 2mg/l BA
3.4 Phng php nghin cu
3.4.1Thit k h thng t ng cho bioreactor sc kh t to v ngm chm
nh k (TIB)
Thit k h thng t ng bioreactor sc kh t to
H thng c thit k vi yu cu m bo v trng v t ng
- Dng nt cao su s 9 c 2 l c kch thc va vi pipet 5ml v u co
L( ng knh 1cm). Dng pipet 5ml v co L t v 2 l trn nt cao su.
- Dng dy silicone to thnh vng trn, bt kn mt u. Trn ng dy silicone
dng kim c c l vi kch thc <=2mm. Ni u cn li vi pipet 5ml
46
47
48
49
Th tch mi trng(lt)
1,5
Th tch mi trng(lt)
1,5
Nghim thc
10 pht/1gi
10 pht/2gi
50
51
Hnh 4.1-Bioreactor sc kh
4.1.2 Thit k h thng bioreactor ngm chm nh k t ng
Da trn nguyn l hot ng ca bioreactor ngm chm nh k(TIB) mch
in c thit k vi mc ch t ng ho qu trnh trn :
CH THCH
K
T1
T4A
T1A
T2B
T1B
T3A
Bo m
T2
T3
T2B
Van
T3A
T3B
T4
-T: timer
-1,2,3,4:s th t timer
-K hiu A :cng ra ca
timer
-K hiu B : cng ra ca
timer
-Bom : my bm sc kh Van : van solenoid 3 l
-K : cng tc ngun
-K hiu
:thng ng
-K hiu
:thng m
-K hiu
timer.
: cun dy ca
52
53
4.2 Kt qu th nghim.
4.2.1 Th nghim 1 : Kho st hiu qu nhn chi da bng bioreactor sc kh
lin tc.
Th nghim 1a:nh hng ca th tch mi trng n kh nng nhn chi
da bng bioreator sc kh lin tc
Bng 4.1- Cc ch tiu theo di ca th nghim 1a
Nghim thc
MT lng sc kh lin
MT lng sc kh lin
tc 1L
tc 1,5L
Trng lng ti
trung bnh(g)
3,12
3,75
0,3
0,35
12,5
13,5
2,15
2,32
tch
nhiu
nc, tch
nhiu
nc,
thy tinh th
H s nhn chi
SC LIN TC TH TCH 1L
10,97a
14,10b
54
Nhn xt :
-Kt qu nhn c cho thy vi cng mt mu cy da nh nhau khi x l vi
th tch khc nhau cho kt qu tng chi trung bnh khc nhau. Khi phn tch trn
thng k cho thy c s khc bit c ngha gia 2 phng php.
-Mu chi da trong bnh th tch 1,5 lt c s chi tng hn so vi th tch 1 lt.
Gii thch iu ny l do trong th tch 1,5 lt lng cht dinh dng cn thit cho vic
nhn chi nhiu hn so vi th tch 1 lt. Vi h thng ngm chm nh k, Escalona
v cng s (1999) chng minh rng th tch ti u cho nhn chi da l 200 ml/mu
cy. S dng th tch ln hn lm gim h s nhn chi. Lorenzo v cng s (1998)
khi nhn chi Saccharum spp. trong h thng bioreactor bnh i BIT cng chng
minh khi tng dn th tch mi trng t 5,0 n 50,0 ml/mu cy, h s nhn chi
tng t 8,3 chi ln n 23,9 chi sau 30 ngy nui cy.
Sc kh lin tc
Sc kh gin on 10/1ln
55
Sc kh gin on 1L
Trng lng ti
Sc kh gin on 1,5L
0,71
0,96
0,078
0,11
H s nhn chi
11
7,25
1,3
1,63
c im hnh thi
H s nhn chi
SC KH GIN ON 1L
11,13a
SC KH GIN ON 1,5L
7,24b
Nhn xt :
-Kt qu trn cho thy phng php sc kh gin on vi 2 th tch khc nhau
cho kt qu thng k khc nhau.
56
57
nh k 10'/1h
nh k 10'/2h
2,07
4,73
0,15
0,287
14,83
21,25
2,27
2,76
c im hnh thi
58
Hnh 4.5- Chi da sau 4 tun nui cy trong bnh TIB 10/2h
59
Nghim thc
H s nhn chi
TIB 10/1H
14,78a
TIB 10/2H
21.32b
Nhn xt:
Hiu qu nhn chi 2 nghim thc l khc nhau. H s nhn chi trung bnh
ca nghim thc 10/2h(10 pht trong chu k 1h) c kt qu vt tri so vi nghim
thc 10/1h. Kt qu trn cng l mt iu hp l. V cy da vn l loi thc vt
thch hp vi iu kin kh ro thong kh. Do vy khi ngm chm vi tn s 10/1h
lm cho cy da pht trin khng tt, hiu qu nhn chi khng cao. Krueger v cng
s (1991) chng minh c tm quan trng ca tn s ngp chm ln hiu qu nhn
chi cy serviceberry. Cy b mng nc khi x l ch ngp chm 5 pht/30
pht (5 pht ngp trong chu k 30 pht), v khng b mng nc khi x l ngp chm
5pht/60pht. Tuy nhin, ch x l 5 pht/30 pht li tt cho s nhn v s lng
chi. T , tc gi ngh s dng chu k 5pht/30pht giai on u nui cy
nhn s lng v p dng chu k sau 5pht/60pht duy tr cht lng chi. Qua th
nghim ny, tc gi cng quan st c khong thi gian thch nghi ca thc vt khi
thi gian ngp chm b thay i.
-Th nghim cho thy cy da thch hp vi iu kin ngm chm 10pht/2gi
hn so vi 10pht/1h.
-Trong qu trnh tin hnh th nghim c mt s khc bit so vi mt s tc gi
khi thit lp h thng : s dng bng gn lt mt di y gip cho vic gi mi
trng dinh dng lu hn. Cn th nghim ca mt s tc gi : s dng mt ming
li lt pha di dn ti cht dinh dng c rt ht ton b.
60
H S NHN CHI
25
20
15
10
5
0
SKLT 1L SKG
1L
SKLT
1,5L
SKG
1,5L
TIB
10'/1h
TIB
10'/2h
61
62
63
64
PHN PH LC
Bng ANOVA phn tch h s nhn chi trong phng php sc kh lin tc
vi th tch khc nhau :
One-Way Analysis of Variance
---------------------------------------------------------------------Data: SUCLT.nhanchoi
Level codes: SUCLT.T
Labels:
Means plot: LSD
Confidence level: 95
Analysis of variance
---------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio
Sig. level
---------------------------------------------------------------------Between groups
14.726667
1
14.726667
10.322
.0325
Within groups
5.706667
4
1.426667
---------------------------------------------------------------------Total (corrected)
20.433333
5
0 missing value(s) have been excluded.
65
Bng ANOVA phn tch h s nhn chi trong phng php sc kh gin on
vi th tch khc nhau :
One-Way Analysis of Variance
---------------------------------------------------------------------Data: SUCGD.NHANCHOI (pp sc kh gin on)
Level codes: SUCGD.T
Labels:
Means plot: LSD
Confidence level: 95
Analysis of variance
---------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio
Sig. level
---------------------------------------------------------------------Between groups
22.698150
1
22.698150
86.805
.0007
Within groups
1.045933
4
.261483
---------------------------------------------------------------------Total (corrected)
23.744083
5
0 missing value(s) have been excluded.
66
Bng ANOVA phn tch h s nhn chi trong phng php ngm chm nh
k vi thi gian ngm chm khc nhau :
One-Way Analysis of Variance
---------------------------------------------------------------------Data: TIB.NHANCHOI (pp ngm chm nh k)
Level codes: TIB.T
Labels:
Means plot: LSD
Confidence level: 95
Analysis of variance
---------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio
Sig. level
------------------------------------------------------------------------------Between groups
64.137782
1
64.137782
31.206
.0050
Within groups
8.221259
4
2.055315
---------------------------------------------------------------------Total (corrected)
72.359041
5
0 missing value(s) have been excluded.