nguyen_bang_phi_02126078_bovud_20130705102132_3074_ltru
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B GIO DC V O TO TRNG I HC NNG LM TP. H CH MINH
B MN CNG NGH SINH HC ***000***
NGUYN BNG PHI
THIT K VA NGHIN CU H THNG BIOREACTOR
CHO CY DA CAYENNE
Lun vn k s
Chuyn ngnh: Cng ngh sinh hc
Thnh ph H Ch Minh
-2006-
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B GIO DC V O TO TRNG I HC NNG LM TP. H CH MINH
B MN CNG NGH SINH HC ***000***
THIT K VA NGHIN CU H THNG BIOREACTOR
CHO CY DA CAYENNE
Lun vn k s
Chuyn ngnh: Cng ngh sinh hc
Gio vin hng dn Sinh vin thc hin TRN TH DUNG NGUYN BNG PHI NGUYN VN HUNG
Thnh ph H Ch Minh
-2006-
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MINISTRY OF EDUCATION AND TRAINING
NONG LAM UNIVERSITY, HCMC
DEPARTMENT OF BIOTECHNOLOGY
************
DESIGN AND RESEARCH BIOREACTOR SYSTEM FOR
CAYENNE PINEAPPLE
Graduation thesis
Major: Biotechnology
Professor Student
TRAN THI DUNG NGUYN BNG PHI
NGUYN VN HUNG
Ho Chi Minh City
-2006-
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B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
B MN CNG NGH SINH HC
KHA LUN TT NGHIP
THIT K V NGHIN CU H THNG BIOREACTOR
CHO CY DA CAYENNE
NGNH: CNG NGH SINH HC
NIN KHA: 2002 2006
SINH VIN THC HIN: NGUYN BNG PHI
Thnh ph H Ch Minh 2006
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B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
B MN CNG NGH SINH HC
THIT K V NGHIN CU H THNG BIOREACTOR
CHO CY DA CAYENNE
GVHD: TS. TRN TH DUNG SVTH: NGUYN BNG PHI
TS. NGUYN VN HNG MSSV: 02126078
Thnh ph H Ch Minh 2006
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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
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TM TT
NGUYN BNG PHI, i hc Nng Lm Tp. H Ch Minh. Thng 08/2006.
THIT K V NGHIN CU H THNG BIOREACTOR CHO CY DA
CAYENNE
Hi ng hng dn:
TS. Trn Th Dung
TS.Nguyn Vn Hng
ti c thc hin ti B mn Cng ngh sinh hc-Trng i hc Nng
Lm Tp. HCM trn i tng cy da Cayenne in vitro. Tin hnh nhn chi da
Cayenne bng bioreactor. Mu cy l chi da 3 thng tui ca phng Nui cy m
thc vt, B mn Cng Ngh Sinh Hc- i Hc Nng Lm tp.HCM. Chi da nui
cy 6 tun bng bioreactor. Qu trnh thc hin bao gm 2 giai on :
+Giai on 1: Thit k h thng bioreactor sc kh t to v bioreactor ngm
chm nh k t to.
+Giai on 2: Nghin cu h thng bioreactor trn qua 2 th nghim :
Th nghim 1: Nghin cu nh hng ca yu t th tch dinh dng trong h
thng bioreactor sc kh i vi h s nhn chi
Th nghim 2: Thay i yu t thi gian i vi h thng bioreactor ngm
chm nh k(Temporary immersion bioreactor)
Nhng kt qu thu c :
+Bioreactor sau khi thit k hot ng tt.
+Hnh thc nui cy cy da bng bioreactor sc kh l khng ph hp cho s
pht trin ca cy da.
+Th tch mi trng 1,5L cho kt qu kh quan hn so vi th tch 1L khi nui
cy bng bioreactor sc kh lin tc.
+Bioreactor sc kh lin tc gip cho cy da pht trin tt hn so vi
bioreactor sc kh gin on.
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+Bioreactor ngm chm nh k l loi bioreactor ph hp cho s pht trin ca
cy da.
+Bioreactor ngm chm nh k vi thi gian ngm chm nh k 10/2h cho hiu qu nhn chi da tt nht.
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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 inh ky .................. 38
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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3.4.2 Tin hnh th nghim ......................................................................................... 48
3.5. X l s liu ........................................................................................................ 50
Phn 4 : Kt qu v tho lun
4.1 Thit k h thng t ng cho bioreactor sc kh t to v ngm chm nh k
(TIB) ........................................................................................................................... 51
4.1.1Thit k h thng t ng bioreactor sc kh t to ........................................... 51
4.1.2 Thit k h thng bioreactor ngm chm nh k t ng ................................ 51
4.2 Kt qu th nghim ............................................................................................... 53
4.2.1 Th nghim 1 Kho st hiu qu nhn chi da bng
bioreactor sc kh lin tc. .......................................................................................... 53
4.2.2 Th nghim 2 : nh hng ca thi gian ngm chm nh k n
kh nng nhn chi da bngTIB ............................................................................... 55
Phn 5 : Kt lun v ngh
5.1. Kt lun................................................................................................................ 60
5.2. ngh ............................................................................................................... 60
Ti liu tham kho ...................................................................................................... 61
Phn ph lc ............................................................................................................... 63
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DANH SCH CC BNG
Bng Trang
Bng 3.1 Cc nghim thc ca th nghim 1a ........................................................... 45 Bng 3.2 Cc nghim thc ca th nghim 1b ........................................................... 45
Bng 3.3 Cc nghim thc ca th nghim 2 ............................................................. 45
Bng 4.1- Cc ch tiu theo di ca th nghim 1a .................................................... 49
Bng 4.2- nh hng ca yu t th tch n h s nhn chi ca cy da Cayenne
trong bnh bioreactor sc kh lin tc ........................................................................ 49
Bng 4.3- Cc ch tiu theo di ca th nghim 1b .................................................... 51
Bng4.4- nh hng ca yu t th tch n h s nhn chi ca cy da Cayenne
trong bnh bioreactor sc kh gin on .................................................................... 51
Bng 4.5- Cc ch tiu theo di ca th nghim 2 ...................................................... 53
Bng 4.6- nh hng ca yu t thi gian ngm chm n h s nhn chi ca
cy da Cayenne trong bnh bioreactor TIB .............................................................. 54
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DANH SCH CC HNH
Hnh Trang
Hnh 2.1- Cu trc bioreactor ............................................................................ 26
Hnh 3.1- S lp rp h thng bioreactor sc kh hnh cu kiu Hn Quc .... 42
Hnh 3.2-S lp rp h thng bioreactor ngm chm nh k.......................... 43
Hnh 4.1-Bioreactor sc kh .................................................................................. 47
Hnh 4.2- S mch in thit k cho TIB t ng ........................................... 47
Hnh 4.3-Hp iu khin t ng TIB t ch ....................................................... 48
Hnh 4.4-Chi da sau 4 tun nui cy trong bnh bioreactor sc kh lin tc
v sc kh gin on .............................................................................................. 50
Hnh 4.5- Chi da sau 4 tun nui cy trong bnh TIB 10/2h ........................... 53
Hnh 4.6-Biu h s nhn chi ......................................................................... 55
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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 : Mi trng Murashige v Skoog, 1962
TIB : Temporary immersion bioreactor
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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.
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Phn 2. TNG QUAN TI LIU
2.1 c im thc vt hc v sinh thi cy da
c im thc vt hc
Da l cy thn tho lu nm, thuc lp n t dip. Sau khi thu hoch qu cc
mm nch thn tip tc pht trin v hnh thnh mt cy mi ging nh cy trc;
qu th hai thng b hn qu trc. Cy da trng thnh cao n 1 m v rng 0,5 m
trong khi cy da Smooth Cayenne trng thnh cao 1,5 m v c ng knh t 1,3
1,5 m. y l ging da c trng nhiu nht trn th gii.
Hoa
Hoa gm c 3 l i, 3 cnh hoa, 6 nh c xp thnh 2 vng trn, 1 nh ci c 3
tm b v bu h. Cnh hoa mu xanh, ta, gc c mu trng nht v trn mt cnh
hoa c nhng vy. Trng hoa dng ng di hi loe pha u, gia li ln 3 nm
nhy tm m ca vi nhy. Hoa t bt th (self-sterile) v pht trin qu khng ht;
th phn nh gi khng xy ra v sinh sn hu tnh him thy trong t nhin. Nhn
ging v tnh l hnh thc sinh sn tiu biu s dng chi bao gm chi nh, chi bn
v chi r.
Qu
Qu da thuc loi qu t do 100 200 qu nh hp li. Cc ging khc nhau th
hnh dng qu v mt qu cng khc nhau. B phn n c ca da l do trc ca
chm hoa v l bc pht trin nn. Sau khi hoa tn th qu bt u pht trin.
Ht
Ht da nh, mu tm en, c v v ni nh rt cng, t l ny mm thp v bt
thng nu khng qua tin x l. Mi qu con ch c vi ht. Da thng khng ht
nu th phn t do. Ht da thng do th phn nhn to v c s dng trong
cc chng trnh lai to ging mi.
Thn
Thn cy da chia lm 2 phn: mt phn trn mt t v mt phn di mt t.
Phn trn thng b cc l che kn nn kh nhn thy. Khi cy pht trin n mc
nht nh, c th dng cc mm ng trn cc t nhn ging.
L
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L da mc trn thn cy theo hnh xon c. L thng dy, khng c cung,
hp ngang v di. B mt v lng l thng c mt lp phn trng hoc mt lp sp c
tc dng lm gim bc hi nc l. Cc ging da thng c gai nhn v cng
mp l, nhng cng c ging l khng gai nh Cayenne. Ty theo ging, mt cy da
trng thnh c khong 60 70 l.
R
R da gm r ci v r nhnh (mc ra t phi ht); r bt nh (mc ra t mm
r trn cc t ca cc loi chi da trc khi em trng). R da thuc loi n nng,
phn ln do nhn ging bng chi nn mc t thn ra, r nh v phn nhiu nhnh. B
r da thng tp trung tng t 10 26 cm v pht trin rng n 1 m.
Sinh thi cy da
Da l cy n qu nhit i thch nhit cao, nhit thch hp cho sinh
trng 28 320C, nhit gii hn 15 400C. Nhit c nh hng c bit quan
trng n qu trnh hnh thnh qu chn ca qu do l yu t u tin nh hng
n phm cht ca qu.
Yu t quan trng khng km l ch nc bao gm lng ma hng nm v
phn b ma. Da vo 2 ch tiu trn, cc vng sinh thi thch hp c thit lp m
bo vic trng da trn din rng t nng sut cao. Theo kinh nghim, lng ma
thch hp nht cho da l 1000 1.500 mm. Tuy nhin da vn pht trin tt nhng
vng c lng ma thp nhng thuc kh hu i dng, quanh nm m mt.
Cy da a nh sng tn x hn nh sng trc x. Lng chiu sng thch hp
lm tng nng sut v ci thin phm cht hng v qu. chiu sng cn nh hng
n mu sc qu.
Da c b r tp trung lp t mt do yu cu t phi ti xp, thong, c
kt cu ht, khng c nc ng vo ma ma. V pH, cc ging khc nhau c yu
cu khc nhau. pH 5,6 6,0 c th ln n 7,5 i vi ging Cayenne; nhm da
Queen c th sinh trng tt trn t phn pH 4,0 trong khi ging Spanish (cc
ging da ta) thch nghi vi pH t 4,5 5,0.
2.2.Phn loi
Da c tn khoa hc l Ananas comosus (L.) Merr thuc:
Phn lp: Magnoliophyta
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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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Nhc im: qu b, trng lng bnh qun ch t t 500 700 g. Dng qu hi
bu dc kh thao tc trong khi ch bin. Tht qu c nhiu khe h, khng cht nn kh
t tiu chun lm hp xut khu.
Nhm Spanish
L mm, mp l cong, hi ng v pha lng, hoa t c mu nht. Qu ngn,
kch thc to hn so vi nhm Queen nhng b hn so vi nhm Cayenne. Trng
lng qu trung bnh xp x 1kg. Khi chn v qu c mu nu , sm hn nhiu so
vi qu Cayenne v cng c dng hnh tr cn i. Tht qu mu vng trng khng
u, mt qu su, v hi chua. Chi ngn v c bit l chi cung nhiu, nh hng
n phm cht qu.
Nhn chung, cc ging da trong nhm Spanish tuy d trng, chu c bng
nhng phm cht km nn c trng ch yu qui m h gia nh, khng nn tp
trung thnh vng ln.
Ngoi ba nhm da trn, cn c nhm Abacaxi tch ra t nhm Spanish nhng
mc ph bin cn thp.
Cc ging da ph bin Vit Nam
Da hoa Ph Th
Cn c gi l Queen c in. N c nhng c tnh in hnh nht ca ging
Queen nh qu nh; mt nh, li; gai ra l nhiu v cngy l ging nhp ni
vo Vit Nam khong u th k XX, c trng ri rc cc tnh pha Bc v min
Trung.
u im ni bt ca da hoa Ph Th l tht vng gin, rt thm v hp dn nn
n c dng pha trn vo nc da p t cc ging khc hay cc loi qu khc
to ra mi thm c trng. Ging ny d trng, chu c t xu, t chua, d ra hoa
tri v.
Nhc im l qu nh, nng sut nhn chung thp, kh ch bin hp nn hiu
qu kinh t khng cao.
Da hoa Na Hoa (Hoa Bali)
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Ging da ny c c tnh ca nhm mt nh, li, khi chn v v tht qu u c
mu vng. So vi da hoa Ph Th, ging ny c l ngn v to, qu cng to hn. Bnh
qun trng lng t 0,9 1,2 kg/qu. Khi chn k, nc trong tht qu cng nhiu hn.
y l ging da kh ph bin cc vng trng tp trung vi u im d canh
tc, c th duy tr nng sut n v th 2, th 3 nu p dng k thut chm sc thch
hp; h s nhn ging tng i cao. Tuy nhin, do c mt su, qu hi bu dc nn
kh t t l ci cao khi ch bin hp, hiu qu kinh t thp.
Da Kin Giang v da Bn Lc (t a phng l khm)
Mt s tc gi lit k cc ging ny vo cng vi ging da Na Hoa. Trong iu
kin kh hu min Nam, cy sinh trng mnh, qu c kch thc ln hn so vi trng
min Bc, ng thi mt s c im cng khc i.
So vi da Bn Lc, da Kin Giang c dng hnh tr hn, mt qu to hn v
tht qu c nhiu nc hn. y l nhng ging trng kh ph bin vng ng bng
sng Cu Long.
Nhm da Cayenne
c im ca nhm da ny l l khng gai ngoi mt vi gai u mt l, l
dy, lng mng su, c nhiu phn mt di nht l pha gc.
Ging ny du nhp vo nc ta cui nhng nm ba mi, u nhng nm bn
mi mt s a phng min Bc ch yu trong nhng n in do ngi Php
qun l. Chn Mng (thuc Vnh Ph) l mt trong nhng ni tip nhn ging u tin,
v sau ngi ta quen gi l Cayenne Chn Mng.
Cc ging Cayenne c trng ph bin hin nay l ging Cayenne Thi Lan,
Cayenne Trung Quc v Cayenne Lm ng. Theo ti liu ca Vin cy n qu min
Nam , ging Cayenne Thi Lan v Trung Quc u cho tri to v pht trin tt; tuy
nhin l ging mi nhp ni nn cn c thi gian kt lun. Vi u im nng sut
cao, qu to v d thao tc trong ch bin lm hp, c cht lng cao c v ha sinh
ln t l ci nn ging Cayenne ang c ch ph bin ra din rng. Cc vng trng
da nguyn liu c hnh thnh nhm cung cp nguyn liu cho cc nh my ch
bin da.
2.4. Tnh hnh sn xut v sn lng da
Tnh hnh sn xut v sn lng da trn th gii
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Theo thng k nm 2001 sn lng da ca th gii t 13.739.000 tn, phn
b theo cc chu lc v khu vc nh sau: Chu Phi 2.229.000 tn, Bc M 1.512.000
tn, Nam M 2.556.000 tn, Chu 7.275.000 tn, Chu Phi 2.000 tn v Chu i
Dng 164.000 tn. Cc nc c sn lng da cao nh Thi Lan 2.300 tn, Philipin
1.572 tn, Brazil 1.442 tn, Trung Quc 1.284 tn, Nigeria 881 tn
Tnh hnh sn xut v sn lng da Vit Nam
Tnh hnh sn xut
Theo ti liu thng k , din tch trng da c nc nm 1995 l 25.734 ha n
nm 2000 ln n 36.541 ha. Tnh n nm 2000 din tch trng da ca min Bc l
9.675 ha chim 26,48% din tch trng da c nc, ch yu vng ng Bc v
ng bng sng Hng; trong khi din tch gieo trng ca min Nam l 26.866 ha
chim 73,52% din tch trng da c nc, ch yu tp trung vng ng bng sng
Cu Long.
Cc tnh c din tch gieo trng da kh ln l Kin Giang (9.200 ha), Tin
Giang (7.803 ha), Bc Liu (3.625ha), Cn Th (1.338 ha), Long An (661 ha), Qung
Nam (2.320 ha), Bnh nh (500 ha), Thanh Ha (2.900 ha), Ninh Bnh (1.572 ha),
Ngh An (600 ha), Bc Giang (657 ha)
Bn cnh , cc tnh min ng Nam B c din tch da tng ng k trong
khong thi gian t nm 1998 n nm 2000: Tp. HCM tng t 76 ha ln n 150 ha,
ng Nai tng t 61 ha ln 123 ha, Bnh Thun tng t 12 ha ln 40 ha. Hin nay cy
da Cayenne c cc nh nng hc nh gi cao do ph hp vi t ai th nhng
ca cc tnh min Trung, Ty Nguyn v cc vng t nhim phn khc. Nhiu d n
m rng vng trng da Cayenne c thc hin khu vc ng Nam B nh
Chng trnh pht trin cy da Cayenne Tp. HCM thi k 2003 2005 , khu vc
Ty Nguyn (Gia Lai, aklak) . Mt s tnh min Trung nh Bnh nh, Ph Yn
cng ngh Tp. HCM cung cp ging da Cayenne nhn rng .
Sn lng da
Theo ti liu thng k sn lng da ca c nc nm 2000 l 291.428 tn phn
b trn cc khu vc trng da c nc nh sau: min Bc t 57.246 tn, khu vc Bc
trung b 25.218 tn, min Nam 234.164 tn trong cc tnh thuc khu vc Duyn
Hi Nam Trung B t 20.832 tn, Ty Nguyn 2.357 tn, khu vc ng Nam B
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22
1.417 tn v khu vc c sn lng da ln nht trong nc ng Bng Sng Cu
Long 209.558 tn. Cc tnh c sn lng da cao l Kin Giang (89.094 tn), Tin
Giang (79.880 tn), Bc Liu (24.860 tn), Ninh Bnh (20.315 tn), Qung Nam
(15.724 tn), Thanh Ha (11.107 tn), H Ty (1.725 tn), Bc Giang (1.495 tn), Ph
Th (1.159 tn)
2.5. Gii thiu v nui cy m
Nui cy m t bo thc vt
Nui cy m t bo thc vt hay cn gi l nui cy in vitro l cng c cn thit
trong nhiu lnh vc nghin cu c bn v ng dng ca ngnh cng ngh sinh hc.
Nh p dng k thut nui cy m, con ngi thc y thc vt sinh sn nhanh hn
gp nhiu ln so vi t nhin. Do to ra hng lot c th mi gi nguyn tnh trng
di truyn ca c th m, lm rt ngn thi gian a ging mi vo sn xut. Hn na
da vo k thut nui cy m c th duy tr v bo qun nhiu ging cy trng qu
him phc trng ging cy trng.
Phng php nui cy m t bo thc vt bt u t mt mnh nh thc vt v
trng c t trong mi trng dinh dng thch hp. Chi mi hay m so m mu
cy ny sinh ra bng s tng sinh c phn chia v cy chuyn nhn ging.
Lch s pht trin
Nm 1838, hai nh sinh vt hc c l Schleiden v Schwann xng hc
thuyt t bo v nu r: Mi sinh vt phc tp u gm nhiu sinh vt nh, cc t bo
hp thnh, cc t bo phn chia mang thng tin di truyn cha trong t bo u tin,
l trng sau khi th tinh v l nhng n v c lp t c th xy dng li ton
b c th.
Nm 1902, Haberlandt xng phng php nui cy m t bo thc vt
chng minh tnh ton nng ca t bo, ngha l mi t bo u mang y thng tin
di truyn ca c th. ng tin hnh trn cy h ha tha (cy mt l mm) mt lai cy
kh thc hin v ng b tht bi.
Nm 1922, Kote (hc tr Haberlandt) v Robbins (nh khoa hc ngi M)
lp li th nghim ca Haberlandt v nui cy c nh sinh trng tch ra t u r
ca mt loi cy thuc h ha tho to ra h r nh v c c r ph. Tuy nhin s sinh
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23
trng nh vy ch tn ti trong mt thi gian sau chm li v ngng hn mc d
tc gi chuyn sang mi trng mi.
Nm 1934, White nui cy thnh cng u r cy c chua (Lycopersicum
esculentum).
Nm 1937, Gautheret v Nobecout to ra v duy tr c s sinh trng m
so cy c rt trong mt thi gian di trong mi trng thch cng.
Nm 1941, Overbeck chng minh c vai tr ca cht kch thch sinh trng
trong nui cy phi h c. Trong thi gian ny cht hch thch sinh trng nhn to
thuc nhm auxin c nghin cu v tng hp ha hc thnh cng. V nm 1948
Steward xc nh c tc dng ca nc da trong nui cy m so cy c rt
Nm 1955, ngi ta tm ra tc dng kch thch phn bo ca kinetin.Sau cc
cht cytokinine khc nh BAP, 2 IP, Zeatin cng c pht hin.
Nm 1957, SKoog v Miller cng b kt qu nghin cu v ty l gia
kinetin/auxin i vi s hnh thnh cc c quan t m so trn cy thuc l.
T nm 1954, n nm 1959 k thut tch v nui cy t bo n c pht
trin, cc tc gi gieo t bo n v nui cy to c cy hon chnh.
Nm 1966, Guha v Mahheswari nui cy thnh cng t bo n bi t nui cy
ti phn cy c c dc.
Nm1967, Bougin v Nistsh to thnh cng cy n bi t ti phn cy thuc l.
ng dng
Nm 1986, mt s lng ln cy trng sn xut bng phng php nui cy m
c tiu th tn th trng thng mi vi hng chc triu dollar. K thut ny
th hin mt s u im c ng dng:
- Nhn ging v tnh vi tc nhanh.
- To cy sch bnh v khng bnh.
- Cm ng v tuyn la dng t bin.
- Sn xut cy n bi qua nui cy ti phn.
- Lai xa.
- Lai t bo soma v to dng protoplast.
- Gy bin tnh thc vt qua hp th DNA v ngoi lai.
- C nh nitrogen.
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24
- Ci thin hiu qu ca quang tng hp.
- Bo qun ngun gen qu.
Cc phng php nui cy in vitro
Nui cy nh sinh trng
Mu cy bao gm: nh sinh dng, chi nh, chi bn, c kch thc khong
0.58 - 1 cm. y l phng php d dng nht, mu sau khi v trng v c nui cy
trong mi trng thch hp cho loi cy th sau mt thi gian nui cy to thnh
mt hay nhiu chi. Sau , nui cy trong mi trng c b sung cht kch thch sinh
trng th s to thnh nhiu chi, r, to thnh cy hon chnh.
Nui cy m so
Mu cy l nhng t bo nh sinh trng hay nhu m c tch ra trong mi
trng giu auxin th m so c hnh thnh. M so l nhng t bo v t chc c
mu trng. Khi m so ny c kh nng ti sinh thnh cy hon chnh trong mi
trng khng c cht kch thch sinh trng to m so. Nui cy m so c thc
hin i vi nhng cy khng c kh nng nui cy t nh sinh trng. cy ti sinh t
m so c nhiu chi hn so vi cy ti sinh t nh sinh trng, tuy nhin mc
bin d t bo soma ca phng php ny rt cao.
Phng php nui cy t bo n
Nhng khi so c nui cy trong mi trng lng, t trong my lc th khi
so di tc dng c hc v ha hc s tch ra nhiu t bo n l gi l t bo n.
Nhng t bo n ny nui cy trong mi trng c bit th s tng sinh khi. Sau
mt thi gian nui cy trong mi trng lng t bo n tch ra v t tri trong mi
trng thch th s pht sinh thnh nhng t bo m so. Nhng t bo m so ny
c nui cy trong mi trng cytokinin/auxin thch hp th s ti sinh thnh cy
hon chnh. Trong chn ging cy trng ngi ta da vo phng php ny to ra
ging mi bng cch t bin t bo n bng ha cht hay phng x.
Nui cy protoplast- chuyn gen
Protoplast (t bo trn ), thc cht l t bo n c tch v cellulose, c sc
sng v duy tr chc nng sn c. Protoplast c th ti sinh trc tip t thn, l, r bng
c hc, hoc t nhng t bo n sn c. Trong mi trng thch hp cc protoplast c
kh nng ti sinh mng t bo, tip tc phn chia v ti sinh thnh cy hon chnh.
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25
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
Auxin l mt nhm cc cht c tng hp ch yu u thn, u r, c vn
chuyn n cc b phn khc nhau ca c th kch thch s tng trng ca t bo.
Cc phn ng auxin v s tng trng c lin quan vi v s qu trnh sinh l v
trao i cht khc v mi quan h nhn qu gia auxin, ARN v chuyn ha protein
khng phi hon ton r rng. Phn ng ch yu v nhanh chng nht i vi vic x
l auxin l lm tng ko di ca t bo, iu ny xy ra ch mt vi pht sau khi x
l. Mt c trng quan trng l vch t bo, l mt v tr quan trng chu s tc ng
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26
ca cc cht iu ha sinh trng thc vt (Torry v csv, 1981). Auxin lm gim pH
do kch thch s bi xut proton H+, pH hot ha cc enzym tc ng ni lng vch t
bo v enzym tng hp vch t bo, nh khi ng qu trnh gin n t bo (Roger
Prat, 1993).
Auxin hot ha s sinh tng hp cc hp cht cao phn t (protein, xenluloza,
pectin, ) v ngn cn s phn gii chng (Grodzinxki, 1981).
Cytokinin
y l cht hot ha s phn chia t bo (Mitsuhashi v csv, 1969; Mai Trn
Ngc Ting, 1989), ng thi lm tng qu trnh chuyn ha acid nucleic v protein
(V Vn V v csv, 1993). Cytokinin c s dng kh nhiu trong k thut nui cy
m, nhng cht thng c dng l Kinetin v BA (Benzyl Adenin).
Cytokinin ph v trng thi ng ca ht, kch thch ht ny mm, lm tng s n
hoa. Cytokinin gy nn s hnh thnh chi mm trong nhiu m bao gm m so sinh
trng (cals) trong m nui cy, hay vic to thnh cc m bu cc cy g lu nm
(Nester v csv, 1985; Taiz L. v csv, 1991).
Cytokinin kch thch s tng hp mi enzym Rubisco (Ribulozo-1,5-biphotphat
cacboxylaza/oxygenaza) hoc mc sao chp (lm tng hot tnh ARN-polymeraza)
hoc mc dch m (thnh lp cc polyriboxom) (Parthier, 1985). nh hng ca
Cytokinin thy r khi phi hp s dng vi auxin (Meredith v csv, 1970). Skoog v csv
(1948) ghi nhn lng benzyl amino purine cao c tc dng kch thch s to chi, ng
thi c ch s phn ha to r.
Gibbrelline
Gibbrelline ni bt rt lu trc khi c nhn dng. Cht Gibbrelline u
tin c nhn dng l acid gibbrellique hoc l GA3, k n l GA4 + GA7 v GA7.
Tt c cc Gibbrelline th hin mt nhn ging nhau; chng c s khc nhau bi cht
lng v v tr ca cc cht gn trn nhn.
Tnh cht sinh l ca Gibbrelline
-Hot ng ko di cc t ca cy, hot ng ny cng c th p dng ln cc
cung hoa v iu ny cho php c mt s chn tt hoc nhng pht hoa pht trin
hn.
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27
Trong nui cy in vitro, Gibbrelline c tc ng i vi nhiu nh sinh trng,
nu thiu Gibbrelline nh sinh trng th hin mt dng hnh cu, to nn cc mt
cy.
-Hot ng phc tp trong s ra hoa.
-Tc ng ln s u tri ca cc tri khng ht.
-Tc ng gy thc gic cc chi, mm ng trn cc ht ging lm thun li cho
s ny mm.
-Trong lnh vc sinh to c quan thc vt, Gibbrelline cho thy cc hot ng
i khng: chng dng nh i ngc vi hin tng phn ha t bo. Trong nui
cy in vitro Gibbrelline khng c s dng vo mc ch ny, nhng chng c
cng dng vi cc m c t chc (nh sinh trng, chi ngn, chi thn).
nh hng ca ngun carbon
Trong mi trng nui cy, cc m khng c kh nng t dng do khng quang
hp y trong iu kin thiu s trao i kh vi bn ngoi, do vy cn cung cp
ng gip m, t bo thc vt tng hp cc cht hu c, gip t bo phn chia,
tng sinh khi. Cc loi ng thng c s dng l sucrose, d-glucose, d-fructose
(Doods v Roberts, 1987). Sucrose l ngun carbon c s rng rng ri nht cho cc
loi cy, nng sucrose thay i t 2%-12% hoc cao hn ty thuc vo ging v
tui phi cy.
nh hng ca nc da
Nc da (CW-coconut water) thm vo mi trng vi lng thch hp s kch
thch s pht trin ca chi bn cng nh s hnh thnh cy con (Urata v Iwanaga,
1965; Scully, 1966; Tanaka v Sakanishi, 1978).
T vic s dng CW, nhiu m thc vt c nghin tch dch chit v b sung
vo mi trng nui cy c tc dng kch thch s pht trin phi nh ni nh bp, ch
l, chui, mm u, mm la m, nc chit c chua nhng thng thng cc dch
chit ch c tc dng trn cc loi cy trng khng cng ngun gc (Trn Vn Minh,
2002).
Theo V Vn V v csv (1993), trong CW kh giu cc hp cht nit dng kh
nh cc acid amin, ngoi ra trong CW cn cha cc hormon sinh trng nh
cytokinine.
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28
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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29
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)
1. Ca np nguyn liu
2. ng dn kh vo
3. H thng sc kh
4. ng dn n c
5. ng thot n c
6. Motor
7. ai b o v
8. Thanh truyn n g
9. Cnh qut
10. B phn cm bin
11. Vch ngn
12. Lp gi nhit
13. Ca rt nguyn liu
Hnh 2.1- Cu trc bioreactor
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30
Thun li ca nui cy bng bioreactor
Theo Takayama v Akita (1994) th mt s thun li chnh ca bioreactor trong
vi nhn ging thc vt l:
- S tip xc tt hn gia sinh khi thc vt vi mi trng.
- Khng c s hn ch v trao i kh.
- C th iu khin sinh khi thc vt ty theo th tch mi trng.
- Tit kim c thi gian v nhn cng trong vic nui cy chuyn.
- D dng cho nhn ging s lng ln to nhiu sinh khi.
- D dng iu khin c thnh phn mi trng v iu kin nui cy.
- Tc sinh trng v pht trin c tng hn nu c tng cng
khng kh.
- Mu cy c tip xc y hn vi mi trng dinh dng nn lm
cho tc sinh trng v pht trin c tng nhanh.
- Nh vic lin tc di chuyn trong mi trng nui cy nn t xy ra hin
tng u th ngn, s ng ca chi bin mt v kt qu l to c nhiu chi
hn.
Kh khn ca nui cy bng bioreactor
Mc d phng php nui cy lng lc v bioreactor t ra vt tri hn so vi
cc phng php nui cy trn mi trng bn rn nhng bn cnh n vn cn c
nhng hn ch nht nh. Chng hn nh (Ziv, 2000):
- Tu vo tng i tng m thit k mt kiu bioreactor thch hp, kh
p dng ng lot cho nhiu ging khc nhau.
- Thng gp hin tng bt thng v pht sinh hnh thi nh: hin
tng thu tinh th (vitrification), hin tng bt thng ca phi, hin tng
stress t bo. Nhng hin tng trn lm gim hiu sut nhn ging v sn xut
sn phm trao i cht th cp.
Mt vn ln na thng gp trong nui cy mi trng lng l vic nhim
vi sinh vt. Nm, vi khun, nm mc v cn trng l nhng ngun gy nhim nghim
trng. Chng l nguyn nhn ch yu gy mt ngun mu thc vt trong cc phng th
nghim thng mi. V l mi trng lng nn s ly nhim vi sinh vt s xy ra rt
nhanh v gy hu qu nghim trng hn so vi cc loi mi trng khc (rn, bn
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31
rn). S ly nhim c th xut pht t cc giai on thao tc chun b v iu khin
thit b. Trong mt s phng th nghim hn ch nguy c b nhim th ngi ta
thng to mt khng gian v trng trong phng cy bng dng khng kh to p lc
dng (Ziv, 2000).
Phn loi bioreactor
Bioreactor khuy thong kh (Aeration-agitation bioreactor)
Kiu bioreactor ny thng c cp di dng nhng bnh phn ng khuy
trn (stirred tank bioreactors STRs) c cnh khuy chng hn nh: tua-bin, chn vt,
mi cho hay ruy-bng xon c. Mc d STRs c ngha trong nui cy to huyn
ph nhng n cng gy ra nhng lc khuy mnh. Tuy nhin, gn y STRs c
ci tin nhm khc phc cc bt li trn. Kiu cnh khuy ruy-bng xon c c xem
l c hiu qu i vi nui cy huyn ph t bo thc vt c mt cao v nui cy
to phi soma (Archambault et al., 1994). Ni chung th bioreactor dng ny thng
c s dng trong nui cy to phi. Nhng hin nay vn cn qu t cc nghin cu
tin hnh so snh hiu qu gia cc kiu bioreactor khc nhau.
Bioreactor hnh trng quay (Rotating drum bioreactor)
Kiu bioreactor ny gm c mt bnh cha c dng hnh trng c gn
trn mt trc quay, trc ny c nhim v nng v quay bnh cha. Bnh c quay
vi tc khong 2 6 vng/pht nhm hn ch ti a lc x lm tn thng t bo.
Mt bnh phn ng khng c tm ngn cch c th c s dng trong nui cy to
cy con thng qua qu trnh to chi v to phi (Takayama v Akita, 1994).
Bioreactor mng lc xoay (Spin filter bioreactor)
Kiu bioreactor ny c mt mng lc xoay c nhim v ha trn huyn ph nui
cy v ng thi ly i mi trng s dng v b sung mi trng mi vo. Cc
mng lc quay khuy trn mi trng khng lm x rch mng t bo chnh l nh
vo vic to ra c nhng ng khuy mng. c th duy tr c lu s tng
trng lin tc ca m so v s pht trin ca phi t c s nhn nhanh, ngi ta
thit k mt bioreactor c hai mng lc cng quay. Mt chin lc nhm nui cy
hiu qu hn l trong giai on tng sinh t bo, ngi ta tin hnh nui cy lin
tc. Nhm to s ng nht, n nh iu kin nui cy cho cc t bo v duy tr c
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32
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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ci t nhiu b phn si bt kh s y nhanh tc tng sinh khi (Buitelaar et al.,
1991).
Nhng hn ch chung ca c air-lift bioreactor v bioreactor si bt hnh ct l:
a) c bt ni ln do tng cng mt lng kh ln, b) cc t bo c khuynh hng b
tng ra khi dung dch bi bt kh, c) t bo ln ln trn thnh ca bnh nui cy
(trong bt). S lng t bo bn trong bnh biu th tng sinh khi ca t bo. Hin
tng ni bt v tng trng trn thnh bnh l do ng knh ca bnh v np bnh c
cng kch thc. Vn ny c gii quyt bng cch ci tin bnh nui cy nh
ming bnh c ng knh ln hn, hay bnh c kiu dng cu (Paek et al., 2001).
Bioreactor si bt dng cu (Ballon type bubble bioreactor BTBB)
Bioreactor dng ny c hnh cu v c np trn nh. gn y ca bnh c
mt kha hnh ch Y hoc ch T c nhim v chm thm mi trng v l cng thu
sn phm. Bng vic s dng mt thit b si bt vi cc l ng tm t y bnh,
iu ny lm cho lng bt c gim. Ngoi ra, trn np bnh cn c gn thm cc
thit b o pH, oxy ha tan. Nhng bioreactor dng ny c s dng trong vi nhn
ging to phi soma (Son et al., 1999; Kim, 1999; Lian, 2001; Paek et al., 2001).
Bioreactor thi kh trn b mt (Overlay aeration bioreactor)
Dng bioreactor ny thi kh t trn b mt xung dung dch lng v i khi kt
hp vi sc kh nh nhng h s oxy ha tan kh thp (nh hn 1). Kiu bioreactor
ny c bo co l cha thnh cng lm trong nui cy m (Ishibashi et al., 1987).
Bioreactor ngp chm gin on t ng (Automated temporary
immmersion bioreactor)
H thng ngp chm t ng ny c thng mi di tn gi l RITA do
Teisson v Alvard thit k (1995). H thng bioreactor ny gm hai bnh cha, mt
bnh dng cho s tng trng ca thc vt, mt bnh dng cha mi trng lng.
Hai bnh ny c ni vi nhau bng ng silicon v thy tinh. Khng kh nn t mt
thit b bm kh s y mi trng lng t bnh cha th nht sang bnh cha th hai,
lm ngp chm hon ton mu thc vt. Sau th kh s rt khi bnh cha mi
trng, lm cho mi trng bnh nui cy h xung. Trong mi trng hp nh vy
th khng kh c thi qua mt mng lc v trng vi kch thc l 0,2 mm. Mt
thit b iu khin c th hn gi c dng n nh khong thi gian cho mt chu
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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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L hn hp ca agar v phytagel. u im ca gi th ny l c tc dng gim
c hin tng thy tinh th (mng nc) trong nui cy in vitro. Hn na, loi gel
ny cng c trong cao nn d dng pht hin vi sinh vt nhim trong mi trng.
Transfergel
c s dng lm ng mi trng nui cy chi mm, chi nh, phi soma.
Cc cht to gel khc nh bacto agar, gellam gum, v.v, c tc dng tng t nh agar
nhng gi thnh cao hn do c tinh khit cao hn v c nhiu cng dng h tr
khc cho nui cy m. Tuy vy, nui cy m trn mi trng bn rn vn bc l nhiu
khim khuyt:
- Cht to gel bin mi trng dinh dng thnh mt h kn, hn ch kh nng di
chuyn ca cc thnh phn dinh dng, do mu cy ch hp th c mt phn
dinh dng v tr gn n nht. Hn na, nhng cht c do chnh thc vt tit ra b
tch ly xung quanh mu cy v c ch tr li s sinh trng v pht trin ca thc vt
(thng gp nht l hin tng tch t phenol gy cht mu).
- Cht dinh dng c b sung cng vi agar trc khi hp, kt qu l cc cu
t dinh dng phn b u khp cc l, trong khi m cy ch s dng c mt lng
nh cht dinh dng phm vi hp quanh mu nn h s hp thu cht dinh dng ca
mu cy trong mi trng thch thp. Khi cy s dng ht cht dinh dng xung
quanh, cy kh c th hp thu phn dinh dng phn b nhng v tr xa mu, do vy
kh nng sinh trng v pht trin ca mu b gim st.
- Mi trng bn rn ch s dng c mt ln, khng cho php thay i thnh
phn mi trng cho nhng giai on pht trin khc nhau ca thc vt vi nhn ging,
i hi mu cy phi c cy chuyn lin tc.
- ha tan agar, ta cn s dng mt nhit lng nng nhit ln 60oC, kt
hp vi gi thnh agar hin nay tng i cao lm gim hiu qu kinh t ca vic
s dng agar vo nhn ging in vitro.
- Khi t mu vo mi trng thch, mu s pht trin khng ng u do c
nhng v tr trn mu khng c tip xc vi mi trng. V d nh khi nui cy m
so trn mi trng thch, s tng trng ca m so s khng u do s trao i kh,
gradient ca cc hp cht gy c, cht c ch m thc vt tit ra mi trng khng
ging nhau nhng v tr tip xc khc nhau gia khi m so v mi trng Nhng
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nhc im trn a n yu cu v mt h thng nui cy mi ph hp hn, p
ng nhng i hi ngy cng cao ca cng tc nhn ging in vitro. Trc tnh hnh ,
h thng nui cy s dng mi trng lng t ra c trin vng hn c.
H thng nui cy in vitro s dng mi trng lng tnh
Nui cy lng l mt cch thc l tng cho mc tiu gim chi ph v t ng
ha vi nhn ging thc vt. Nui cy lng cung cp iu kin nui cy ng nht hn,
mi trng c th thay mi m khng cn phi chuyn mu sang bnh cha khc, c
th thc hin kh trng bng vi lc v vic c ra chai l sau khi nui cy cng n
gin hn. So vi vic nui cy trn thch, nui cy lng cho php s dng nhng loi
bnh nui cy dung tch ln t gim c s ln cy chuyn. M ca mt s loi
thc vt biu hin tt hn trn mi trng lng so vi trn mi trng c, chng hn
nh chi cy o (Prunus persica), phi soma la m (Triticum aestivum), v phi
soma cy bng (Gossypium hirsutum). Nui cy lng thc ra c t rt lu i.
Trong thi k u, Haberlandt tin hnh nui cy m l ca cc cy n t dip
bng mi trng lng. Vic nghin cu nng khong v cc cht iu ha sinh
trng thc vt cng c thc hin vi mi trng lng. Tuy nhin, mi trng lng
c s dng trong thi k ny n gin ch l mi trng lng tnh. H thng nui
cy lng c nhiu li im khng th ph nhn:
- Mi trng nui cy lng c ng nht cao do cc cht tinh dng khuch
tn u trong dung mi lng, to iu kin mu cy s dng trit cht dinh
dng sn c. Hn na, ton b b mt mu c tip xc trc tip vi mi trng
nn vic hp thu cht dinh dng hiu qu hn, cy sinh trng tt hn, thi gian nui
cy c th c rt ngn.
- Nui cy lng cho php kim sot cc thng s ha l mt cch ch ng, y
l mt iu kin cn c thc hin t ng ha. Do vy, nui cy lng rt ph hp
khi trin khai ra quy m cng nghip.
- Chi ph sn xut v gi thnh sn phm thp hn so vi nui cy bn rn, tit
kim c thi gian v lao ng. Bn cnh nhng mt mnh, vic nui cy lng cng
vp phi nhiu kh khn, nht l kh nng thong kh km, hin tng thy tinh th v
i hi nhiu thit b phc tp. hn ch nhng nhc im ny, mt s phng
php c xut nh b sung thm cc gi mu nui cy trong mi trng
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lng tnh, b sung mi trng lng vo mi trng thch s dng trc , v s
dng cc bioreactor sc kh. Mt vn ln thng gp trong nui cy mi trng
lng l s nhim vi sinh vt. Nm mc, vi khun v cn trng l nhng ngun gy
nhim nghim trng. Chng l nguyn nhn gy mt ngun mu thc vt ch yu
trong cc phng th nghim thng mi. V l mi trng lng nn s nhim vi sinh
vt s l rt nhanh v nghim trng hn c so vi cc loi mi trng khc. S gy
nhim c th xut pht t cc giai on thao tc chun b v iu khin thit b. Trong
mt s phng th nghim, hn ch nguy c b nhim, ngi ta thng to mt
khng gian v trng bng dng khng kh c lc qua cc lp mng lc. Trc hai
mt c v cha c ca h thng ny, cc nh khoa hc tch cc nghin cu pht
trin nhng h thng mi va tn dng c cc u im ca nui cy lng, va
khc phc nhng yu im c hu ca n.
H thng nui cy lng s dng gi th
Gi th c s dng vi mc ch nng mt phn hoc ton b mu cy ln
khi mi trng lng m vn m bo dinh dng cho cy nh s thm thu ca cc
cu t dinh dng qua gi th n mu. Cc loi vt liu lm gi th rt a dng, t
nhng vt liu d kim nh bng gn, giy lc n nhng vt liu cao cp nh
rockwool, oasis, v.v. in hnh cho gi th cao cp l rockwool. Bn cht ca
rockwool l th si (wool) c ngun gc t (rock). , ch yu l basalt, c
un chy nhit 1600oC, c vo cc ng v quay vi tc cao, bin dch
lng thnh cc si. Chiu di v ng knh si c kim sot thng qua tc quay,
nhit nung v mt s cc yu t khc. Khi nghin cu trn i tng cy chui
Nam nh, Nhut (2002) chng minh rng cy nui cy trn rockwool v oasis c
chiu cao thn v s lng l nhiu hn hn cc h thng s dng agar truyn thng.
H thng nui cy lng lc
Mu cy c t trong mi trng lng v c lc trn my lc trn vi tc
thch hp. Chuyn ng lc to iu kin cho khng kh t do khuch tn vo mi
trng tt hn, tng lng oxygen ha tan trong mi trng, tha mn nhu cu h hp
ca cy. Tewary P.K. v Oka S. (1999) s dng h thng lng lc nhn ging
chi bn cy du tm vi tc lc 120 vng/pht. Kt qu thu c sau 6 tun rt tt:
90% mu cm ng to chi mi vi chiu di mi chi khong 3,3cm.
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H thng nui cy lng trong bioreactor
Khi nui mu trong dung dch lng kt hp vi tng cng thong kh, mu
cy vn h hp tt d b ngp trong dung dch. C th m rng quy m ca h thng
ny bng cch s dng cc bnh cha dung tch ln (h thng bioreactor cp kh cng
bc) nhn sinh khi hay thu hp cht th cp t thc vt. Cc phng php to iu
kin thong kh khi nui cy lng trong cc bioreactor gm c: sc kh cng bc,
ngp chm tm thi, v.v.
Hin nay c rt nhiu h thng bioreactor sc kh c thit k nhm nhn ging
cy hoa lily vi s lng ln. Trong cc h thng ny, mc d vy c lily chm xung
y bnh nhng s tng trng ca chng vn rt tt.
E. Jimnez v cng s (1999) chng minh rng h thng ngp chm tm thi
cho hiu qu to c khoai ty bi tt hn so vi mi trng bn rn truyn thng: c bi
c hnh thnh trn tt c cc t thn sau 9 tun nui cy, trung bnh khong 3,1
c/t. Trng lng ti v kch thc c cng ln hn c hnh thnh trn mi trng
thch. Tm li, cc h thng ci tin u c nhng tin b v hu ht u tp trung gii
quyt vn thong kh khi nui cy trong mi trng lng. Ngoi ra, mt yu t
khng th thiu cn cn nhc khi thit k cc h thng mi l hiu qu kinh t ca h
thng. C nh vy, h thng mi c nhanh chng thng mi ha v c kh nng
cnh tranh vi nhng h thng nhn ging truyn thng khc. Cc h thng bioreactor
nui cy in vitro c tm hiu trong ti ny cng hng n cc yu t vi mc
ch chn la c h thng nui cy ph hp cho mt s i tng thc vt c th.
2.9. Cc yu t nh hng trong qu trnh nui cy ngm chm nh k
Thi gian ngp chm
Thi gian mu ngp trong mi trng dinh dng c ngha rt quan trng
do n nh hng n s hp thu cht dinh dng ca m thc vt cng nh tham gia
iu khin hin tng thy tinh th. Yu t ny thay i theo i tng nui cy, mc
ch nui cy v loi h thng ngp chm c s dng. Thi gian ngp chm di (1
gi ngp trong chu k 6 gi) t ra hiu qu cho s hnh thnh c khoai ty bi, trong khi
thi gian ngp chm rt ngn (1 pht ngp trong chu k 12 gi) li tt cho s hnh
thnh phi soma cy c ph (C. arabica) v cao su. Ngoi ta, tn s ngp chm cao
(30 giy ngp, 30 giy rt) cho hiu qu nhn chi cao khi nui cy chi nho trong h
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thng bioreactor nghing lc. 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 5 pht/60 pht. Tuy nhin, ch
x l trc (5 pht/30 pht) li tt cho s nhn v s lng chi. T kt qu ny, tc
gi ngh s dng chu k 5 pht/30 pht giai on u nui cy nhn s lng
v p dng chu k sau 5pht/60 pht 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. Khi chuyn t tn s cao sang tn s thp, mt s chi b stress v b
cht, nhng phc hi tr li sau .
Th tch mi trng dinh dng
Th tch mi trng cn c ti u ha khi nui cy bng bioreactor ngp chm
m khng thay mi trng mi (bioreactor nghing lc, bnh i hoc RITA).
Lorenzo v cng s (1998) xc nh th tch mi trng 50 ml cho mt mu cy l ti
u khi nhn chi Saccharum spp. trong h thng bioreactor bnh i BIT. 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. Tuy nhin, th tch mi trng s dng khng
nh hng ln chiu di chi. Th tch nhiu hn t ra khng hiu qu cho nhn chi.
Gii thch hin tng ny, tc gi cho rng trong qu trnh nui cy, thc vt tit ra
ngoi t bo nhng hp cht c tc dng kch thch to chi, v vi th tch nui cy
ln, lng cht ny c th b pha long i. Cng s dng h thng nui cy tng t,
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 trong trng hp
ny.
Th tch bnh nui cy
Tt c cc h thng bioreactor ngp chm u c th tch v khng gian thong
kh bn trn ln hn cc bnh nui cy truyn thng. Hn na, c th p dng cc loi
bnh cha khc nhau vi th tch t 1 n 20 l thit lp h thng. Krueger v cng
s (1991) chng minh rng th tch bnh cha ln (7 l) c nh hng tch cc ln hiu
qu vi nhn ging cy serviceberry, c bit l h thng gip trnh c hin tng
mu cy tp trung qu nhiu, ng thi kch thch ko di chi so vi cc bnh cy
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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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thn di hn v ra nhiu l hn khi nui cy trong h thng ngp chm tm thi so vi
nui cy trn thch. Chi cy serviceberry nui cy trong h thng ny cng di v
nng hn chi nui cy trn mi trng bn rn. Chi cy nho v Amelanchier
alnifolia khi nui cy trong h thng bioreactor nghing-lc c kch thc ln v ra r
nhanh hn so vi khi nui cy trn thch. Nhng nh hng c li t h thng ngp
chm tm thi ln s pht trin chi c th l kt qu ca vic s dng nhng bnh
cha dung tch ln. H thng ngp chm tm thi c nh hng rt tt n s pht
trin phi soma ca cc cy Citrus, cho php hnh thnh l mm v v phn sinh ngn
(protoderm) ngay trong h thng. iu ny khng xy ra c trong nui cy huyn
ph t bo. Hnh thi ca cc phi ny rt ging vi phi nhn. Mt khc, cc cy c
ph, cao su, chui, phi soma to ra trong h thng RITA c cht lng tt hn phi
hnh thnh trn mi trng thch hay lng trong cc bnh erlen. cao su, t l phi
soma bt thng gim i phn na khi nui cy bng h thng ngp chm tm thi so
vi nui cy trn thch. H thng ny cng c p dng thnh cng ln u tin
khi cho phi soma C. arabica ny mm trong mi trng lng. Mt 1600 phi/1
nh hng tt ln hnh thi ny mm ca phi do kch thch s ko di nh phi (4-5
mm), tng trng lng ti (100%) v gim din tch l mm. Ba yu t ny s c lin
quan n t l pht trin thnh cy con cng nh h s tng trng ca cy con sau
ny.
Hin tng thu tinh th
Vi nhn ging trong mi trng lng gip cho s hp thu cht dinh dng thc
hin d dng hn v do kch thch cy tng trng, tuy nhin, hin tng mng
nc cng thng xy ra. Hin tng ny xut hin khi cho tip xc lin tc vi cht
lng. c trng ca thc vt b mng nc l m c nhng ri lon v hnh thi v
sinh l nh biu hin mng nc, c mu trong nh thy tinh, chi sinh trng bt
thng, c bit l nhng d thng l. C th gim bt hin tng mng nc bng
cch tng cng thong kh, cho m tip xc gin on vi mi trng lng. C hai
yu t ny u c tha mn vi h thng nui cy ngp chm tm thi
2.11. nh hng ca h thng ngp chm tm thi n chi ph sn xut
Nhng tnh ton ban u cho thy h thng c tc dng lm tng hiu qu ca
vic nhn ging trong mi trng lng. Khi nhn chi ma (Saccharum spp.), Lorenzo
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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
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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
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44
vn tip tc c thc hin gim chi ph ng thi ti u ha iu kin sn xut
nhng chng loi thc vt khc nhau
2.12. Cc nghin cu v ng dng bioreactor trn i tng cy da
Nghin cu v ng dng bioreactor trn i tng cy da trong nc
Cha thy c c quan no nghin cu v ng dng bioreactor trn i tng cy
da c cng b
Nghin cu v ng dng bioreactor trn i tng cy da ngoi nc :
Nm 1999, Esscalo v cng s nghin cu trn i tng cy da. Nhiu
nghin cu tip theo cho thy hiu qu ca vic s dng bioreactor trn i tng
cy da : s chi thu c trn 1 chi ban u l 30 (Plant Cell Rep,2003), gi thnh
h, chi ph nhn cng thp, cy trng khi a ra vn cng pht trin tt hn (Plant
Cell Rep, 2005),
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45
Phn3. VT LIU V PHNG PHP NGHIN CU
3.1 Thi gian v a im
Th nghim c tin hnh t thng 2 n thng 7 nm 2006 ti phng nui cy
m ca B Mn Cng Ngh Sinh Hc Trng i Hc Nng Lm Tp.HCM
3.2 Ni dung nghin cu
- Thit k h thng iu khin t ng cho bioreactor ngm chm nh k : Da
trn nguyn l hot ng ca bioreactor TIB (dng dung dch c chuyn qua li
gia bnh mi trng v bnh nui cy theo mt quy trnh).
- So snh hiu qu nhn chi da gia TIB v nui cy bng bioreactor sc kh
- Kho st thi gian ngm chm nh k thch hp cho s pht trin chi ca cy
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
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46
-Gn mt u dy silicone vo ng pipet 5ml, u dy silicone cn li c bt
kn nn kh i vo s thot qua theo ng cc l kim trn thn ng silicone.
- Kh c sc qua u lc 2 m , kh ra cng c a qua u lc 2 m to
thnh h thng kn v trng.
Hnh 3.1- S lp rp h thng bioreactor sc kh hnh cu kiu Hn Quc
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47
Thit k h thng bioreactor ngm chm nh k t ng
H thng c thit k gm 2 bnh thng nhau, mi trng dinh dng c di
chuyn qua li mt cch t ng. Ton b h thng phi m bo v trng.
Hnh 3.2-S lp rp h thng bioreactor ngm chm nh k
*Gii thch qu trnh vn hnh: bm a dng kh theo hng mi tn qua u
lc v trng to mt p sut trong bnh, p sut tc ng ln b mt cht lng v y
cht lng theo ng pipet qua bnh cha mu. Tt my bm kh ( hnh i), dung dch
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48
c trong bnh mu mt thi gian quy nh. Sau , bt my bm to dng kh
theo hnh mi tn(hnh h) to p sut ln b mt cht lng trong bnh mu, y dung
dch t bnh mu tr v bnh mi trng ban u, gi dung dch trong bnh mi trng
trong mt thi gian quy nh.
3.4.2 Tin hnh th nghim
B tr th nghim
Th nghim c b tr theo kiu hon ton ngu nhin (Completely Randomized
Design, CRD), mt yu t, 2 nghim thc, mi nghim thc 3 ln lp li. Mi nghim
thc gm 8 chi da hy nh sinh trng.
Cc bc tin hnh : Cc bc tin hnh sau u ging nhau cc th nghim
- Bc 1: Hp kh trng ring tng b phn ca bnh bioreactor
- Bc 2: Hy nh sinh trng ca 8 chi da v a chi da hy nh sinh
trng b vo bnh bioreactor.
- Bc 3 : Gn u lc vo 2 u dy silicone to thnh h thng kn v trng
- Bc 4 : Dng bng keo qun quanh nt cao su tht cht.
T bc 2 n bc 4 thc hin trong t cy v trng.
*Cc ch tiu theo di :
-Trng lng ti trung bnh(g) : Trng lng ti ca tt c cc mu sau khi
loi b ht nc bm quanh mu/8
-Trng lng kh trung bnh(g) : Trng lng ti tt c cc mu c sy
nhit 37oC trong 72 gi/tng s mu
-H s nhn chi: tng s chi to ra/8.
-Chiu cao chi(cm) : tnh t r cho n l cao nht ca chi da.
Th nghim 1:Kho st hiu qu nhn chi da bng bioreactor sc kh.
Th nghim 1a: nh hng ca th tch mi trng n kh nng nhn chi
da bng bioreator sc kh lin tc
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49
Bng 3.1 Cc nghim thc ca th nghim 1a
Nghim thc Th tch mi trng(lt)
1 1
2 1,5
Th nghim 1b: nh hng ca th tch mi trng n kh nng nhn chi
da bng bioreactor sc kh gin on (10pht/1chu k)
Bng 3.2 Cc nghim thc ca th nghim 1b
Nghim thc Th tch mi trng(lt)
1 1
2 1,5
* Ngoi cc bc tin hnh chung trn cn tin hnh gn thm timer vo h
thng, bm sc kh gin on 10 pht/1 chu k ( 10 pht sc kh, 10 pht ngh)
Th nghim 2: Kho st thi gian ngm chm nh k thch hp cho vic nhn
chi cy da.
Bng 3.3 Cc nghim thc ca th nghim 2
Nghim thc Thi gian ngm
chm(pht/gi)
1 10 pht/1gi
2 10 pht/2gi
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50
* Ngoi cc bc tin hnh chung trn cn tin hnh gn bnh TIB vo h
thng iu khin t ng c thit k.
3.5 X l s liu :
S liu c x l trn phn mm Statgraphic 7.0
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51
Phn 4. KT QU V THO LUN
4.1 Thit k h thng t ng cho bioreactor sc kh v ngm chm nh k
(TIB)
4.1.1Thit k h thng t ng bioreactor sc kh
H thng c thit k hot ng tt. Kh c sc qua cc l kim trn thnh
ng silicone to thnh cc bt kh, cung cp oxy cho mi trng.
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 :
T4A
T2B
T1
T1A T3A
T2B
T1BT2
T3
T3A
T3BT4
Bom
B
Van
V
K
CH THCH
-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 : cun dy ca
timer.
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52
Hnh 4.2- S mch in thit k cho TIB t ng
*Gii thch mch in : Khi ng ngun th T1 hot ng bm kh vo bnh 1
trong mt thi gian quy nh, sau T1 tt v T2 m lm cho my bm ngng hot
ng trong mt thi gian nh. Tip T2 ngh v T3 hot ng ng thi valve 3
chiu cng m theo lm dng kh i qua bnh 2(thay v qua bnh 1 nh lc trc) sau
trong mt thi gian nh. Tip , T3 ngh v bm ngng hot ng ng thi T4
hot ng lm cho my bm ngng hot ng trong mt thi gian nh . Cui cng
T4 ngh v T1 li hat ng, lp li chu k nh trn.
H thng t ng ny tit kim c 1 my bm thay v 2 my bm nh s dng
van solenoid 3 l, ng thi tit kim c sc lao ng trong qu trnh canh thi gian
chy my bm, bn cnh c th ch ng c thi gian cho mi trng qua li
gia 2 bnh mt cch hiu qu.
Hnh 4.3-Hp iu khin t ng TIB t ch
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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
tc 1L
MT lng sc kh lin
tc 1,5L
Trng lng ti
trung bnh(g) 3,12 3,75
Trng lng kh trung
bnh(g) 0,3 0,35
H s nhn chi 12,5 13,5
Chiu cao chi(cm)
2,15 2,32
c im hnh thi
L b en, cng, dn
tch nhiu nc,
khng c hin tng
thy tinh th
L b en, cng, dn
tch nhiu nc,
khng c hin tng
thy tinh th
Bng 4.2-nh hng ca yu t th tch n h s nhn chi ca cy da
Cayenne trong bnh bioreactor sc kh lin tc
Nghim thc H s nhn chi
SC LIN TC TH TCH 1L 10,97a
SC LIN TC TH TCH 1,5L 14,10b
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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
Hnh 4.4-Chi da sau 4 tun nui cy trong bnh bioreactor sc kh lin tc v
sc kh gin on.
Th nghim 1b: nh hng ca th tch mi trng n kh nng nhn chi
da bng bioreactor sc kh gin on (10pht/1chu k)
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55
Bng 4.3-Cc ch tiu theo di ca th nghim 1b
Bng4.4-nh hng ca yu t th tch n h s nhn chi ca cy da
Cayenne trong bnh bioreactor sc kh gin on
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.
Nghim thc Sc kh gin on 1L Sc kh gin on 1,5L
Trng lng ti
trung bnh (g)
0,71 0,96
Trng lng kh
trung bnh (g)
0,078 0,11
H s nhn chi 11 7,25
Chiu cao chi(cm) 1,3 1,63
c im hnh thi L b en, cng, dn tch
nhiu nc, khng c
hin tng thy tinh th
L b en, cng, dn tch
nhiu nc, khng c
hin tng thy tinh th
Nghim thc H s nhn chi
SC KH GIN ON 1L 11,13a
SC KH GIN ON 1,5L 7,24b
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56
-Trong phng php sc kh gin on lng oxy cung cp cho mu cy da
khng . Do vy, bnh 1,5L c th tch ln hn nn lng oxy trung bnh cung cp
cho mu da khng bng lng oxy trung bnh cung cp cho bnh 1L. Kt qu l mu
cy da khng th pht trin cng nh s dng cht dinh dng mt cch hiu qu.
Oxygen ch ha tan mt phn trong nc (0,25 mmol l-1 25oC, 1 atm, oxygen
chim 21% th tch khng kh) nn p ng nhu cu oxygen cho s pht trin ca
m thc vt, mt lng ln oxygen cn c khuch tn vo mi trng nui cy.
Nhng yu t lin quan n vn ha tan oxy gm c thong kh, tc khuy
o, hnh dng cnh khuy, ha trn khng kh v kt cu ca bioreactor. Ngoi ra,
mc o trn ca mu cy cng l mt yu t cn quan tm do oxygen ha tan
trong mi trng cn nhanh chng c m, t bo hp thu. Tm li, p ng tt
nht nhu cu oxygen cho s pht trin ca m v t bo thc vt, lng oxygen ha
tan trong dung dch nui cy lun phi ln hn ngng oxygen ha tan cc i m t
bo m hp thu (DO2 htc). Trong thc t, ngi ta cn c vo gi tr DO2htc thit
k bioreactor v gi tr ny cng l yu t c quan tm hng u khi tin hnh
phng i h thng ln quy m ln hn (Leather, 1995)
4.2.2 Th nghim 2 : nh hng ca thi gian ngm chm nh k n kh nng
nhn chi da bngTIB.
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57
Bng 4.5-Cc ch tiu theo di ca th nghim 2
Nghim thc
MT lng ngm chm
nh k 10'/1h
MT lng ngm chm
nh k 10'/2h
Trng lng ti trung
bnh(g) 2,07 4,73
Trng lng kh trung
bnh(g) 0,15 0,287
H s nhn chi 14,83 21,25
Chiu cao chi (cm)
2,27 2,76
c im hnh thi
Cy con pht trin tt, l
xanh ,mm,khng tch
nc, khng c hin
tng thy tinh th
Cy con pht trin tt, l
xanh ,mm,khng tch
nc, khng c hin
tng thy tinh th
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58
Chi da sau 4 ngy nui Chi da sau 4 tun nui
Hnh 4.5- Chi da sau 4 tun nui cy trong bnh TIB 10/2h
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59
Bng 4.6-nh hng ca yu t thi gian ngm chm n h s nhn chi ca
cy da Cayenne trong bnh bioreactor TIB
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.
Nghim thc H s nhn chi
TIB 10/1H 14,78a
TIB 10/2H 21.32b
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60
H S NHN CHI
0
5
10
15
20
25
SKLT 1L SKG
1L
SKLT
1,5L
SKG
1,5L
TIB
10'/1h
TIB
10'/2h
Hnh 4.6-Biu h s nhn chi
CH THCH : SKLT 1L : sc kh lin tc 1lt
SKG 1L : sc kh gin on 1 lt
SKLT 1,5L : sc kh lin tc 1,5 lt
SKG 1,5L : sc kh gin on 1,5 lt
TIB 10/1h : ngm chm nh k 10 pht/1 gi
TIB 10/2h : ngm chm nh k 10 pht/2 gi
*Nhn xt : Biu trn cho thy phng php nui cy ngm chm nh k
(TIB) cho hiu qu nhn chi cao hn hn so vi phng php nui cy sc kh. c
bit, tn s ngm chm nh k 10/2h cho hiu qu cao nht.
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61
Phn 5.KT LUN V NGH
5.1 Kt Lun :
H thng bioreactor chng t kh nng nhn sinh khi vi nhiu u im. Tuy
nhin, mi loi cy cn c mt kiu bioreactor thch hp.Trong cc kiu bioreactor ,
TIB cho thy nhng u im ph hp vi s pht trin ca cy da. T nhng kt
qu t c c th kt lun nh sau :
+Bioreactor sau khi thit k hot ng tt
+Hnh thc nui cy cy da bng bioreactor sc kh l khng ph hp cho s pht
trin ca cy da.
+Th tch mi trng 1,5L cho kt qu kh quan hn so vi th tch 1L khi nui cy
bng bioreactor sc kh lin tc.
+Bioreactor sc kh lin tc gip cho cy da pht trin tt hn so vi bioreactor sc
kh gin on.
+Bioreactor ngm chm nh k l loi bioreactor ph hp cho s pht trin ca cy
da.
+Thi gian ngm chm nh k 10 pht/2h l thch hp hn cho cy da.
+Cy con bioreactor ngm chm nh k c hnh thi p (l xanh non, khng b
en) hn so vi bioreactor sc kh.
5.2. ngh :
-Cn kho st thm thi gian ngm chm nh k thch hp nht cho kh nng ra chi
ca cy da.
-Nghin cu qu trnh thay i thnh phn mi trng trong qu trnh nui cho ph
hp vi s tng sinh chi ca cy da.
-Nghin cu ci tin thm h thng bioreactor ngm chm nh k, khc phc cc
nhc im ca h thng, kim sot v iu khin mt cch t ng cc yu t nh
hng ti s pht trin ca cy da(pH, khng kh, dinh dng...)
-Tip tc nghin cu qu trnh a cy da ra ngoi vn sau thi gian nui cy thch
hp.
-Nghin cu ng dng h thng bioreactor ngm chm nh k cho cc i tng khc
nh : chui, tiu...
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62
TI LIU THAM KHO
TI LIU TING VIT
1.Dng Cng Kin, 2002. Nui cy m thc vt. NXB i hc Quc gia Tp.HCM.
2.Dng Tn Nht v csv, 2004. Nui cy lng v nui cy thong kh trong vic gia
tng sinh khi v nng cao cht lng cy hoa lily (Lilium longiflorum). Tp Ch
Cng Ngh Sinh Hc, 2(4), 487 499
3.Nguyn Thnh Hi, 2005. Nghin cu ch to bioreactor m hnh phc v cng tc
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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 Range test: LSD
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.
Bng Multiple Range Analysis ca PP sc kh lin tc(h s nhn chi) Multiple range analysis for SUCLT.nhanchoi by SUCLT.T
----------------------------------------------------------------------
-
Method: 95 Percent LSD
Level Count Average Homogeneous Groups
----------------------------------------------------------------------
-
1L 3 10.966667 X
1,5L 3 14.100000 X
----------------------------------------------------------------------
-
contrast difference +/- limits
1 - 2 -3.13333 2.70869 *
----------------------------------------------------------------------
-
* denotes a statistically significant difference.
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65
Bng ANOV