LI CM N

Hon thnh lun n ny, trc ht chng ti xin by t lng bit n su sc

n GS. TS. Nguyn Hong Lc v PGS. TS. Cao ng Nguyn quan tm gip
v hng dn tn tnh.
Xin c by t lng bit n ti cc cn b, ging vin ca Phng th
nghim Cc hp cht th cp, Vin Ti nguyn-Mi trng v Cng ngh sinh hc,
i hc Hu; B mn Sinh l-Sinh ha, Khoa Sinh hc, Trng i hc Khoa hc,
i hc Hu gip chng ti trong sut thi gian thc hin ti.
Xin cm n Ban Gim c, Ban o to Sau i hc ca i hc Hu; Ban
Gim hiu, Phng Nghin cu Khoa hc v Hp tc Quc t, Phng o to Sau
i hc Trng i hc Khoa hc; Ban Ch nhim Khoa Sinh hc, Trng i hc
Khoa hc, i hc Hu; Ban Gim hiu, Khoa Sinh-Mi trng, Trng i hc
S phm - i hc Nng c nhiu gip qu bu, to mi iu kin tt nht
chng ti hon thnh lun n.
Xin cm n cc ng nghip, bn b nhit tnh ng vin, h tr chng ti
hon thnh lun n.
Cui cng, xin c by t lng bit n n nhng ngi thn trong gia nh
ng gp mt phn khng nh trong vic hon thnh lun n ny.
Hu, ngy 15 thng 02 nm 2014
Tc gi

V Chu Tun


LI CAM OAN


Ti xin cam oan y l cng trnh nghin cu ca ti. Cc s liu, kt qu
trnh by trong lun n l trung thc, khch quan, nghim tc v cha tng c ai
cng b trong bt k cng trnh no khc. Nu c g sai st, ti xin chu hon ton
trch nhim.
Tc gi

V Chu Tun



MC LC
LI CM N
LI CAM OAN
MC LC
BNG CH THCH CH VIT TT
DANH MC CC BNG
DANH MC CC HNH
M U....................................................................................................................1
1. TNH CP THIT CA TI ..............................................................1
2. MC TIU NGHIN CU ........................................................................3
3. NGHA KHOA HC V THC TIN.................................................3
4. NG GP MI CA LUN N............................................................4
Chng 1: TNG QUAN TI LIU......................................................................5
1.1. NUI CY T BO THC VT...................................................................5
1.1.1. S lc lch s nui cy t bo thc vt ...............................................5
1.1.2. Nui cy huyn ph t bo thc vt .....................................................6
1.1.2.1. Nui cy callus..................................................................................6
1.1.2.2. Nui cy huyn ph t bo................................................................7
1.1.2.3. Cc thng s nh gi kh nng sinh trng ca t bo...............10
1.1.2.4. Mt s yu t nh hng n qu trnh nui cy t bo ................12
1.1.2.5. Nui cy t bo thc vt qui m ln............................................16
1.2. S TCH LY CC HP CHT TH CP TRONG T BO THC
VT NUI CY IN VITRO...................................................................................19
1.2.1. Vai tr ca cc hp cht th cp thc vt .........................................19
1.2.2. Cc nhm hp cht th cp ch yu thc vt ................................19
1.2.2.1. Nhm terpene ..................................................................................20
1.2.2.2. Nhm phenol ...................................................................................20
1.2.2.3. Cc hp cht cha nitrogen............................................................20

1.2.3. Nhng nghin cu sn xut cc hp th cp t nui cy t bo thc
vt..............................................................................................................................21
1.2.3.1. Nhng nghin cu ngoi nc........................................................21
1.2.3.2. Nhng nghin cu trong nc ........................................................25
1.3. GII THIU V CY NGH EN..............................................................28
1.3.2. Thnh phn ha hc.............................................................................28
1.3.3. Cng dng .............................................................................................30
1.3.3.1. Cng dng c truyn.......................................................................30
1.3.3.2. Cc hot tnh sinh hc ....................................................................30
1.3.4. Tnh hnh nghin cu nui cy in vitro ca cy ngh en ................35
Chng 2: I TNG, NI DUNG V PHNG PHP NGHIN CU.37
2.1. I TNG NGHIN CU.........................................................................37
2.2. NI DUNG NGHIN CU............................................................................37
2.3. PHNG PHP NGHIN CU...................................................................38
2.3.1. Nui cy callus ......................................................................................39
2.3.2. Nui cy huyn ph t bo...................................................................39
2.3.2.1. Nui cy huyn ph t bo trong bnh tam gic.............................39
2.3.2.2. Nui cy huyn ph t bo trong h ln men..................................40
2.3.3. Xc nh kh nng sinh trng ca t bo ........................................40
2.3.4. nh lng tinh du .............................................................................41
2.3.5. nh lng curcumin...........................................................................41
2.3.6. nh lng polysaccharide ha tan trong nc ................................42
2.3.7. Xc nh sesquiterpene ........................................................................42
2.3.8. Xc nh hot tnh khng khun ca tinh du..................................43
2.3.9. X l thng k.......................................................................................43
Chng 3: KT QU NGHIN CU V THO LUN..................................44
3.1. NUI CY CALLUS NGH EN................................................................44
3.2. NUI CY HUYN PH T BO TRONG BNH TAM GIC..............47
3.2.1. nh hng ca c mu nui cy.........................................................47

3.2.2. nh hng ca tc lc ....................................................................49
3.2.3. nh hng ca cht HST .................................................................51
3.2.3.1. nh hng ca BA..........................................................................51
3.2.3.2. nh hng ca 2,4-D......................................................................52
3.2.3.3. nh hng ca 2,4-D v BA...........................................................52
3.2.4. nh hng ca ngun carbon.............................................................54
3.2.4.1. nh hng ca sucrose...................................................................54
3.2.4.2. nh hng ca glucose...................................................................56
3.3. NUI CY T BO HUYN PH TRONG H LN MEN.....................59
3.3.1. Kho st sinh trng ca t bo .........................................................59
3.3.2. nh hng ca iu kin nui cy......................................................61
3.3.2.1. C mu............................................................................................61
3.3.2.2. Tc khuy ...................................................................................62
3.3.2.3. nh hng ca tc sc kh .........................................................63
3.4. KHO ST S TCH LY MT S HP CHT C HOT TNH
SINH HC TRONG T BO NGH EN.........................................................65
3.4.1. Hm lng tinh du .............................................................................65
3.4.2. Hm lng polysaccharide ha tan trong nc tng s...................67
3.4.3. Hm lng curcumin...........................................................................68
3.4.4. Xc nh sesquiterpene ........................................................................73
3.5. HOT TNH KHNG KHUN CA TINH DU T BO NGH EN...77
KT LUN V NGH.....................................................................................80
NHNG CNG TRNH C CNG B LIN QUAN N LUN N
.................................................................................................................................. 82
TI LIU THAM KHO......................................................................................83
PH LC

BNG CH THCH CH VIT TT

BAP : 6-benzylaminopurine
BA : 6-benzyladenine
CIB : centrifugal impeller bioreator
cs : cng s
DMSO : dimethyl sulfoxide
C : i chng
HST : iu ha sinh trng
HPLC : high performance liquid chromatography
(sc k hiu nng cao p)
IAA : indoleacetic acid
IBA : indolebutyric acid
Kin : kinetin
L : lt
L-DOPA : L-3,4 -dihydrooxyphenylamine
LPS : lipopolysaccharide
MS : Murashige v Skoog (1962)
NAA : naphthaleneacetic acid
Nxb : nh xut bn
TNF- : tumor necrosis factor-alpha
2,4-D : 2,4-dichlorophenoxyacetic acid




DANH MC CC BNG

TT Tn bng Trang
1 Bng 3.1.Kh nng to callus t b l ca cy ngh en in vitro

44
2
Bng 3.2. nh hng ca cht HST ln sinh trng v pht
sinh hnh thi ca callus
46
3
Bng 3.3. nh hng ca c mu ln sinh trng ca t bo
nui cy huyn ph trong bnh tam gic
48
4
Bng 3.4. nh hng ca tc lc ln sinh trng ca t bo
nui cy huyn ph trong bnh tam gic
50
5
Bng 3.5. nh hng ca BA ln sinh trng ca t bo nui
cy huyn ph trong bnh tam gic
51
6
Bng 3.6. nh hng ca 2,4-D ln sinh trng ca t bo nui
cy huyn ph trong bnh tam gic
52
7
Bng 3.7. nh hng ca 2,4-D v BA ln sinh trng ca t
bo nui cy huyn ph trong bnh tam gic
53
8
Bng 3.8. nh hng ca sucrose ln sinh trng ca t bo
nui cy huyn ph trong bnh tam gic
54
9
Bng 3.9. nh hng ca glucose ln sinh trng ca t bo
nui cy huyn ph trong bnh tam gic
56
10
Bng 3.10. nh hng ca fructose ln sinh trng ca t bo
nui cy huyn ph trong bnh tam gic
57
11
Bng 3.11. nh hng ca c mu ln sinh trng ca t bo
nui cy huyn ph trong h ln men 10 L
62
12
Bng 3.12. nh hng ca tc khuy ln sinh trng ca t
bo nui cy huyn ph trong h ln men 10 L
63
13
Bng 3.13. nh hng ca tc sc kh ln sinh trng ca t
bo nui cy huyn ph trong h ln men 10 L
64

14
Bng 3.14. Hm lng tinh du ca t bo ngh en nui cy
trong h ln men 10 L
66
15
Bng 3.15. Hm lng polysaccharide ca t bo ngh en nui
cy trong h ln men 10 L
67
16
Bng 3.16. Hm lng curcumin ca t bo ngh en nui cy
trong h ln men 10 L
69
17
Bng 3.17. Chiu cao ph hp th (mAU) ca sesquiterpene
trong t bo nui cy h ln men 10 L v t bo c ngh t
nhin
74
18 Bng 3.18. Kh nng khng khun ca tinh du t bo ngh en 78














DANH MC CC HNH

TT Tn hnh Trang
1 Hnh 2.1. Cy ngh en nui cy in vitro 37
2 Hnh 2.2. S th nghim 38
3
Hnh 3.1. Callus hnh thnh t b l ca cy ngh en in vitro
(A) callus trng v xp, (B) callus trng v mng nc
45
4 Hnh 3.2. Callus c mu vng, rn, ri rc sau 14 ngy nui cy 47
5
Hnh 3.3. Dch huyn ph t bo ngh en sau 14 ngy nui cy
trong bnh tam gic trn mi trng c 3% sucrose
55
6
Hnh 3.4. Sinh trng ca t bo ngh en nui cy trong bnh
tam gic trn mi trng MS c 3% sucrose; 0,5 mg/L BA v
1,5 mg/L 2,4-D, lc 120 vng/pht
58
7
Hnh 3.5. Nui cy t bo ngh en trong bnh tam gic 250 ml
t trn my lc
59
8 Hnh 3.6. Nui cy t bo ngh en trong h ln men 10 L 60
9
Hnh 3.7. Sinh trng ca t bo ngh en trong h ln men
nui cy trn mi trng MS c 3% sucrose; 0,5 mg/L BA; 1,5
mg/L 2,4-D ; khuy 120 vng/pht; sc kh 2,0 L/pht, c mu
100 g
60
10
Hnh 3.8. Sinh khi ti (A) v sinh khi kh (B) ca t bo
ngh en sau 14 ngy nui cy trong h ln men 10 L
61
11
Hnh 3.9. Sinh trng ca t bo ngh en trong h ln men
nui cy trn mi trng MS c 3% sucrose; 0,5 mg/L BA; 1,5
mg/L 2,4-D ; khuy 150 vng/pht; sc kh 2,5 L/pht, c mu
200 g
65
12 Hnh 3.10. Ph HPLC ca curcumin chun (0,5 mg/ml) 71

13
Hnh 3.11. Ph HPLC curcumin ca c ngh en 01 nm tui
ngoi t nhin
72
14
Hnh 3.12. Ph HPLC curcumin ca t bo ngh en sau 2 n
18 ngy nui cy trong h ln men 10 lt
73
15
Hnh 3.13. Ph HPLC ca sesquiterpene. A: C ngh en t
nhin; B: t bo ngh en nui cy trong h ln men 10 L t 2
n 18 ngy
76
16 Hnh 3.14. Kh nng khng khun ca tinh du ngh en 77


1
MU

1. TNH CP THIT CA TI
Trong nhiu th k qua, loi ngi da ch yu vo thc vt nh l
ngun cung cp carbohydrate, protein v cht bo lm thc phm. Hn na, thc
vt cng l ngun cung cp phong ph cc hp cht t nhin dng lm dc
phm, ha cht nng nghip, hng liu, cht mu, thuc tr su sinh hc hoc
cc cht ph gia thc phm c gi tr [132]. Nhng sn phm ny c bit nh
l cc cht trao i th cp, c hnh thnh vi mt lng rt nh trong cy
(thng nh hn 1% khi lng kh) v chc nng trao i cht cha c bit
y . Chng c xem l sn phm ca cc phn ng ha hc ca thc vt vi
mi trng hoc l s bo v ha hc chng li vi sinh vt v ng vt [177].
Nhng nghin cu v cc hp cht th cp c ngun gc thc vt pht trin
t cui nhng nm 50 ca th k XX v n nay c khong hn 80.000 hp cht
th cp khc nhau thc vt uc cng b [19], [23].
Theo T chc Y t Th gii (WHO), c n 80% dn s th gii s dng
tho dc lm thuc cha bnh v chm sc sc khe. Vic khai thc ngun
dc liu t nhin t thc vt ang tr thnh mt vn quan trng mang tnh
ton cu v chng ngy cng c thng mi ha nhiu hn. Tuy nhin, vn
t ra hin nay l ni sng t nhin ca cc loi cy thuc ang b bin mt
nhanh chng do s bin i ca kh hu ton cu cng nh s khai thc ba bi
ca con ngi. Nh vy, sn xut cc hp cht th cp thc vt bng con ng
canh tc truyn thng v tng hp ha hc s c nhiu hn ch, kh c th p
ng nhu cu dc liu ngy cng tng trong tng lai [188]. iu ny buc
cc nh khoa hc cn phi tnh n cng ngh nui cy t bo thc vt nh mt
con ng tim nng cung cp nguyn liu cho ngnh cng nghip dc
phm [106].

2
Nui cy t bo thc vt c quan tm nghin cu t nhng nm
1950. Nhiu nghin cu cho thy, nui cy t bo thc vt l mt phng thc
c hiu qu trong sn xut cc hp cht c hot cht sinh hc hoc cc cht
chuyn ha ca chng [132]. u im ca nui cy t bo thc vt l c th
cung cp lin tc ngun nguyn liu di do tch chit quy m cng nghip
cc hot cht m khng ph thuc vo iu kin t nhin [106]; c th to ra cc
hp cht mi v ch ng nng cao kh nng sn xut chng bng cch thay i
cc iu kin nui cy [128]; cc hot cht thu c khng b nhim bn bi
thuc tr su, dit c, khng cn trng cng nh trnh c s khng ng nht
v ngun nguyn liu v nhng bin ng hm lng ca cc sn phm thc vt
ngoi t nhin. Bn cnh , nhiu nghin cu cng cho thy, hm lng cc
cht c hot tnh sinh hc tch ly trong t bo thc vt nui cy in vitro tng
ng hoc cao hn nhiu ln so vi c quan tch ly ca chng trong cy ngoi
t nhin [140], [167]. n nay, ngi ta thnh cng trong sn xut rt
nhiu loi hp cht c gi tr theo phng thc ny trn qui m ln
nh anthraquinone cy Rubia akane, vincristine cy da cn (Catharanthus
roseus), berberin cy Coscinium fenustratum, diosgenin cy Dioscorea
doryophora, taxol cc loi thuc chi Taxus, ginsenoside cc loi thuc chi
Panax [163].
Ngh en cn gi l nga trut thuc h Gng (Zingiberaceae) c ngun
gc t ng Bc n , c trng khp khu vc Nam , ng Nam ,
Trung Quc, i Loan, v Madagasca [9], [115]. C ca cy ngh en c cha
cc hot cht sinh hc ch yu l curcumin, terpenoid v tinh du. Ngoi ra n
cn c tinh bt, cht do v mt s cht c v ng nh tannin v flavonoid [82].
Cc nghin cu cho thy, curcumin c kh nng chng c s pht sinh khi u;
mt s dng ung th chut nh ung th rut kt, ung th d dy, ung th v v
ung th bung trng [55], [66], [152]; curcumin cng c tc dng chng ng
mu v h huyt p; curcuminoid v sesquiterpene l nhng cht c kh nng c

3
ch s hnh thnh TNF- ca i thc bo c hot ha, do c tc dng
chng vim nhim [152]; curcumin cn l mt cht chng oxy ha c kh nng
bo v t bo. Nhiu cng trnh nghin cu cng cho thy, tinh du ngh en c
tc dng khng khun v khng t bin rt cao [176]. Bn cnh ,
polysaccharide ca ngh en c ch hiu qu sinh trng ca cc bu tht
(sarcoma 180) c cy di da ca chut, ngn cn t bin nhim sc th, c
hot tnh kch thch i thc bo [75], [105], [169]. Trong t nhin, ngh en l
loi nhn ging bng thn r, phi mt mt thi gian di to c nn h s
nhn km; nng sut thu hoch thng thp, c bit l ph thuc rt ln vo
iu kin t ai, kh hu, ma v, chi ph nhn cng v vt t sn xut. Mt
khc, ngh en trong t nhin cn d mc cc bnh nh thi c v m l [29].
V vy, rt kh c ngun nguyn liu di do v n nh sn xut lng
ln cc hp cht c hot tnh sinh hc qu ca cy ngh en s dng trong bo
ch dc phm.
Xut pht t nhng c s trn, chng ti thc hin ti: Nghin cu
nui cy t bo cy ngh en (Curcuma zedoaria Roscoe) v kho st kh
nng tch ly mt s hp cht c hot tnh sinh hc ca chng
2. MC TIU NGHIN CU
Nghin cu thit lp cc iu kin v mi trng nui cy thch hp
sn xut nhanh sinh khi t bo, ng thi xc nh c kh nng tch ly v
hot tnh sinh hc mt s hp cht trong t bo ngh en nui cy huyn ph
trong h ln men 10 L.
3. NGHA KHOA HC V THC TIN
3.1. ngha khoa hc
Kt qu nghin cu ca lun n s cung cp cc dn liu khoa hc mi, c
tnh h thng v nui cy in vitro t bo cy ngh en v s tch ly mt s hp
cht c hot tnh sinh hc ca chng; ng thi l ngun ti liu tham kho hu

4
ch trong nghin cu, ging dy v nui cy t bo v sn xut cc hot cht sinh
hc c gi tr cao t nui cy t bo thc vt.
3.2. ngha thc tin
Kt qu ca lun n l c s khoa hc pht trin h thng nui cy
huyn ph t bo cy ngh en qui m ln nhm sn xut nhanh sinh khi,
cung cp ngun nguyn liu lin tc v n nh thu hi cc hp cht c gi tr
cao dng lm thuc, gp phn bo v v chm sc sc khe cng ng.
4. NG GP MI CA LUN N
- to ra dng t bo callus (rn v ri rc) t b l ca cy ngh en in
vitro thch hp nui cy huyn ph, ng thi xc nh mt cch c h thng
cc iu kin v mi trng nui cy thch hp cho s sinh trng nhanh v n
nh ca t bo ngh en nui cy trong bnh tam gic 250 ml v trong h ln
men 10 L.
- kho st s tch ly cc cht c hot tnh sinh hc nh: tinh du,
curcumin, sesquiterpene v polysaccharide trong t bo ngh en nui cy.
Nghin cu xc nh c hm lng v thi im tch ly cao nht ca cc
hp cht ny theo ng cong sinh trng v ng thi cho thy c s chuyn
ha sinh hc cc cht nh curcumin v sesquiterpene xy ra trong nui cy
huyn ph t bo cy ngh en.
- kho st c kh nng khng khun ca tinh du chit rt t t bo
cy ngh en nui cy in vitro v nhn thy, tinh du ca t bo c kh nng c
ch sinh trng mt s loi vi sinh vt gy bnh mc tng ng hoc cao
hn so vi tinh du chit rt t c ngh en 01 nm tui ngoi t nhin.

5
Chng 1
TNG QUAN TI LIU
1.1. NUI CY T BO THC VT
1.1.1. S lc lch s nui cy t bo thc vt
Nui cy m v t bo thc vt l nui cy v trng cc t bo, m, c
quan v cc b phn ca chng di cc iu kin v vt l v ha hc in vitro
[93]. Th nghim u tin v nui cy t bo bn ngoi mt c th thc vt hon
chnh c cng b vo nm 1902 bi Haberlandt - nh Sinh l thc vt ngi
c, ngi c bit n nh nh sng lp ra phng php nui cy t bo thc
vt. Trong bi vit ni ting vi tiu Nhng th nghim nui cy cc t bo
thc vt tch ri, ng m t nhng n lc trong thit lp c h thng nui
cy cc t bo tht l, biu b v lng ht. Mc d khng thnh cng trong nui
cy phn chia t bo, nhng ng d on s c kh nng t c s phn chia
t bo trong nui cy cc t bo ring r, iu ny nh hng mnh m n s
nghip ca nhiu nh nh khoa hc sau ny. Tip sau l nhng nghin cu
rng ri theo hng ci thin cc dung dch dinh dng v khm ph v cc cht
HST thc vt nhm kim tra li nhng d on ca Haberlandt [164]. Trong
nhng nm 1960, nhiu n lc hn na ci thin dung dch dinh dng
a n 2 cng b ng ch ca Skoog v cs, vi hai mi trng nui cy
c dng v m t l mi trng Murashige v Skoog [107] v mi trng
Linsmaier v Skoog [85].
Nui cy cc t bo n v cc khi nh t bo thnh cng u tin trong
nui cy t bo cy thuc l (Nicotiana tabacum) v cy Tagetes erecta trn my
lc. Nui cy t bo thc vt trn qui m ln u tin thnh cng t bo ca
cc cy bch qu, bt rui, c Lolium v hoa hng trong h ln men kiu ry
nc dng n gin vi dung tch 20 L [164].
Nhng th nghim u tin trong nui cy t bo n sn xut dc

6
phm c tin hnh trong nhng nm 1950 ti cng ty Charles Pfizer [164].
n nm 1987, c 30 h thng nghin cu nui cy t bo thc vt c kh
nng sn xut cc hp cht th cp cao hn trong cc thc vt tng ng [177].
n nay, mt th k sau nhng nghin cu ca Haberlandt, nhiu hp cht th
cp c sn xut thng mi bng con ng nui cy t bo thc vt nh
berberine, paclitaxel, ginseng saponin, polysaccharide [177].
1.1.2. Nui cy huyn ph t bo thc vt
Mi t bo thc vt l mt n v c lp, cha y thng tin di truyn
ca mt c th. Trong nhng iu kin nht nh, mi t bo c th hnh thnh
li c mt c th hon chnh. Hon ton c th tch ring v nui cy t bo
c lp nghin cu mi qu trnh sng xy ra trong [12].
1.1.2.1. Nui cy callus
Nui cy t bo thc vt c khi u bng vic hnh thnh cc t bo
khng phn ha, c gi l callus. Nui cy callus t c bng cch nui cy
cc mu m tch t thc vt trn mi trng dinh dng c bn c cht lm rn
l agar. Mi trng dinh dng c bn ny cha cc cht dinh dng khong a
lng, vi lng, ngun carbon v nhiu loi cht HST thc vt. nh gi chnh
xc cc nh hng ca thnh phn mi trng dinh dng hoc cht HST ln
kh nng sinh trng ca callus l yu cu quan trng xc nh mi trng ti
u cho nui cy. Cc thng s ph bin nht dng trong nh gi sinh trng
trong nui cy callus bao gm khi lng ti, khi lng kh v ch s sinh
trng [93].
Trong nui cy callus, cc t bo ca callus c th tri qua bin d dng
soma trong qu trnh cy chuyn. V vy, cc dng t bo n nh di truyn nn
c la chn trnh s sn xut tht thng cc cht trao i th cp trong
nui cy. Thng thng, sau mt s ln cy chuyn, callus c th c xem l
dng t bo ng nht khi cc thng s sinh trng ca dng t bo c lp li

7
trong qu trnh cy chuyn trn cng mt loi mi trng nui cy [35]. Bouque
v cs (1998) nghin cu nui cy 217 dng callus khc nhau t cc loi ca
chi Psoralea nhn thy, sau 16 ln cy chuyn (48 tun), c khong 90% s
dng callus sinh trng n nh [22]. Fett-Neto v cs (1994) nui cy t bo cy
Taxus cuspidate v thu c dng t bo n nh sinh trng sau 2 nm cy
chuyn [42].
1.1.2.2. Nui cy huyn ph t bo
Nui cy huyn ph t bo thng c khi u bng cch chuyn cc
khi callus vo nui cy trong mi trng lng c khuy bi my lc, quay
hoc mng lc xoay. M callus nui cy nn l loi m d v vn c th thit
lp c dch huyn ph t bo vi mc phn tn cao nht. Nui cy huyn
ph t bo trong mi trng lng cung cp mt h thng duy nht cho nhng
nghin cu chi tit v sinh trng v sn xut cc cht chuyn ha. So vi nui
cy callus, nui cy huyn ph sn xut ra lng ln sinh khi t bo m t
cc cht chuyn ha th cp c th d dng chit tch [35].
Nui cy dch huyn ph l s tin trin t thc vt n mu vt, n
callus v cui cng n dch huyn ph [7]. Trong qu trnh nui cy, cc t
bo s dn dn tch ra khi callus do nhng chuyn ng xoy ca mi
trng. Sau mt thi gian ngn nui cy, trong dch huyn ph l hn hp cc
t bo n, cc khi t bo vi kch thc khc nhau v cc t bo cht. Tuy
nhin, cng c nhng dch huyn ph tt, cha t l cao cc t bo n v t
l nh cc cm t bo. Kh nng tch ri ca cc t bo trong mi trng c
th ci thin bng cch thay i thnh phn mi trng nui cy [104]. Mc
d s kt khi ca t bo c th c loi b bi s thay i iu kin mi
trng nui cy, nhng thng trong cui pha lag ca qu trnh nui cy, cc
t bo tr nn kt dnh vi nhau nh l mt qui lut. thu c dch huyn
ph gm phn ln cc t bo n, ngi ta thng s dng cc enzyme ph
hy thnh t bo hoc dng cc sng (ry). Tuy nhin, cc dch huyn ph

8
ng nht c thit lp thng chng c xu hng quay tr li tnh trng
kt khi ban u (Fowler v cs 1982) [104].
Cho n nay, hu ht cc dch huyn ph t bo c nui cy c s
hin din ca c nhng t bo n v cc khi t bo. Nhn chung, mi trng
thch hp cho nui cy callus th cng thch hp cho nui cy huyn ph t bo.
Tuy nhin, trong mt s trng hp, nng ca cc auxin v cytokinin i hi
cao hn. Thng thng, ng hc sinh trng ca cc t bo nui cy huyn ph
l ng hm m; cc hp cht th cp ch yu to ra trong pha n nh, lin
quan vi trao i cht s cp v phn chia t bo [35].
Nui cy huyn ph t bo l tin hnh nui t bo trong mi trng lng
c dung tch nht nh thit lp h huyn ph t bo. Trong qu trnh nui
cy, bnh nui cy khng ch thng xuyn c s tng ln ca cc sn phm trao
i cht m cn lun lun din ra s trao i khng kh vi bn ngoi. Khi cc
cht dinh dng ca mi trng nui cy b tiu hao, cng vi s to thm mt
s sn phm trao i cht c hi, th s phn chia v sinh trng ca t bo s b
c ch. Khi , thng qua cy chuyn hoc thay i mi trng nui cy s kch
thch s sinh trng mnh m tr li ca huyn ph t bo [12]. Nhn chung, c
ba phng thc nui cy huyn ph t bo l nui cy m, nui cy m c b
sung cht dinh dng v nui cy lin tc.
- Nui cy m
Nui cy m l phng thc m trong sut thi gian nui cy khng thm
vo cht dinh dng cng nh khng loi b sn phm cui cng ca qu trnh
trao i cht. Do vy, cc iu kin mi trng lun thay i theo thi gian, mt
t bo tng ln cn nng c cht gim xung. Nui cy m c xem l
mt h thng ng, qun th t bo sinh trng v pht trin theo mt s pha
nht nh vi nhng iu kin c trng [7]. Mc d t bo thc vt v t bo vi
sinh vt c mt s im khc nhau nh kch thc ca t bo thc vt ln hn,
chu k sinh trng ca t bo thc vt di hn, s trao i cht chm hn

9
nhng nhn chung sinh trng ca t bo thc vt trong nui cy m cng tri
qua cc giai on nh t bo vi sinh vt, gm c bn pha. Pha lag (pha tim
pht): bt u t khi callus c a vo mi trng cho n khi c du hiu
phn chia t bo u tin; trong pha ny khng xy ra s tng v khi lng v
s lng t bo. Pha log (pha ly tha): pha ny, s phn chia v tng khi
lng t bo din ra vi tc ln nht (s lng t bo tng theo hm m); Pha
n nh: pha ny, kh nng phn bo gim mnh, s lng v khi lng t
bo n nh. Pha suy vong: s sinh trng ca t bo ra khi nh cao, gim
xung v dn n ngng sinh trng nu khng c cy chuyn [12].
- Nui cy m c b sung cht dinh dng
y l mt hnh thc khc ca phng thc nui cy m. Sau khi t bo
nui cy sinh trng cc i, cc cht dinh dng s dn cn kit, lc ny ngi
ta s cung cp thm cc cht dinh dng mi vo h ln men m khng loi b
dch nui c. Trong h ln men ny, c cc b phn iu khin hm lng cc
cht dinh dng c thm vo gip hn ch hay tng cng tc sinh trng
hoc s tch ly hp cht th cp. Tuy nhin, nh vy th tch h ln men s tng
ln, hn ch vic ny ngi ta thng s dng dung dch cht dinh dng
di dng m c. Phng thc ny vn c gi l nui cy m, v ton b
th tch ca h ln men cui cng c thu hi theo tng m [129].
- Nui cy lin tc
Nhng hn ch chnh ca nui cy m l tn thi gian nhiu cho qu
trnh kh trng, b sung vo v ly mi trng ra, lm sch h thng ln men
[48]. Nui cy lin tc l phng php kinh t v c th ko di thi gian nui
cy hay ko di pha log trong mt thi gian nht nh. Trong phng thc nui
cy ny, dng i vo (mi trng mi) bng vi dng i ra (gm mi trng, t
bo v cc cht chuyn ha) gi cho th tch bnh nui khng thay i, v
iu kin nui cy ca h thng lun n nh [138].

10
Nhn chung, cc phng thc nui cy c tnh truyn thng nh nui cy
m, nui cy m c b sung cht dinh dng v nui cy lin tc trong nui cy
vi sinh vt c th c dng trong nui cy t bo thc vt. V c bn, thit lp
mt phng thc nui cy t bo ph thuc bi (1) mi quan h gia sinh trng
v tng hp cc cht trao i th cp v (2) kh nng cc sn phm th cp tit
ra hoc khng tit ra mi trng [23].
1.1.2.3. Cc thng s nh gi kh nng sinh trng ca t bo
Trong nui cy huyn ph t bo thc vt, cn phi theo di cht ch s
sinh trng v sc sng ca t bo tng hiu sut nui cy t bo cng nh
ci thin iu kin nui cy [12]. Mt s ch tiu dng nh gi ng hc sinh
trng t bo thc vt trong nui cy, bao gm th tch lng, th tch ng gi,
khi lng ti v khi lng kh, mt , ch s sinh trng, thi gian nhn
i v mt s ch tiu khc ca t bo [135].
- Th tch lng v th tch ng gi ca t bo
Th tch lng ca t bo c xc nh bng cch cho cc huyn ph t
bo trm tch trong mt ci ng c chia vch v c biu th bng phn trm th
tch chung ca dch huyn ph, bao gm c sinh khi t bo. Th tch ng gi
ca t bo c xc nh bng cch tng t sau khi t bo c nn cht bi
quay ly tm. Hai thng s ny cho php nh gi nhanh sinh trng ca t bo
trong khi vn duy tr c iu kin v trng mu. Cc phng php ny thun
li gim st s sinh trng ca t bo sut mt chu k nui cy trong cc bnh
tam gic vi iu kin nui cy nh nhau, bi v dch huyn ph c th a tr
li cc iu kin nui cy trc . Tuy nhin, da vo th tch t bo c l
khng phi l cch chnh xc kim tra sinh trng v n ph thuc vo hnh
thi t bo [93].
- Khi lng ti v khi lng kh ca t bo
Khi lng ti v khi lng kh ca t bo cho php nh gi sinh

11
trng ca t bo chnh xc hn cc ch tiu v th tch t bo. Tuy nhin, yu
cu ca cc thao tc mu li tin hnh trong iu kin khng v trng. Vic xc
nh khi lng ti ca t bo t tn thi gian hn khi lng kh, nhng n
khng phn nh c s gia tng sinh khi thc ca t bo, c bit l cui giai
on nui cy, khi m hu ht s tng trng ca t bo trong nui cy l do s
hp thu nc [161].
- Mt t bo nui cy
thu c gi tr tin cy v s lng t bo trong nui cy huyn ph,
trc ht cc khi t bo phi c ph v bng cch dch huyn ph vi dung
dch chromium trioxide 8% hoc vi cc enzyme thy phn nh cellulase v
pectinase. Phng php s dng chromium trioxide th nhanh hn v t phc tp
hn dng cc enzyme thu phn, tuy nhin n gy tr ngi trong c tnh cc t
bo sng ca cng mt mu. Phng php dng enzyme duy tr c cc t bo
sng v nh gi v s lng cc t bo sng trong cng mt mu bi nhum
mu t bo c th thc hin c [122].
- Ch s sinh trng
Khi lng ti v kh ca t bo ch cho php nh gi sinh khi thun
ty ca m ti thi im ly mu, ch s sinh trng tng quan vi d liu sinh
khi ti thi im ly mu v nui cy ban u. N c tnh ton bng t l sinh
khi c tch ly vi sinh khi ban u nui cy [93].
- Thi gian t bo nhn i
Thi gian nhn i l thi gian lng sinh khi ca mt qun th t
bo t gp hai ln so vi ban u. Mt trong nhng im khc bit ln nht
gia sinh trng ca t bo thc vt v t bo vi sinh vt c lin quan ti tc
sinh trng ring ca chng. Mc d kiu sinh trng c th ging nhau, t bo
thc vt c thi gian nhn i hoc tc phn chia o theo ngy, trong khi
trong nhiu loi vi sinh vt th xc nh theo pht n gi. Thi gian nhn i

12
nhanh nht trong nui cy t bo thc vt c ghi nhn trong nui cy t bo
cy thuc l l 15 gi [24].
1.1.2.4. Mt s yu t nh hng n qu trnh nui cy t bo
- Mi trng nui cy
Thnh phn mi trng dinh dng nh hng ln n qu trnh sinh
trng v pht trin ca t bo. Trong giai on u ca qu trnh nui cy, cc
cht dinh dng gim nhanh, sn phm trao i cht mi bt u c tch t v
tng dn. Ba hp phn ca mi trng c nh hng ln n qu trnh nui cy t
bo thc vt l carbon, nitrogen v phospho. Tuy nhin, cng khng th coi nh
cc hp phn khc ca mi trng nui cy, c bit l cc nguyn t vi lng v
cc cht HST. Chng ng vai tr quan trng trong qu trnh trao i cht mc
d nng ca chng c trong mi trng mc rt thp [17].
+ Cc cht HST
Nhiu nghin cu cho thy, cc cht HST c nh hng ln n tc
sinh trng v kh nng sinh tng hp cc cht ca m thc vt trong nui cy
in vitro cng nh trong cy hon chnh. Zhao v cs (2001) nghin cu nh
hng ca 2,4-D v NAA phi hp vi BAP cc nng khc nhau ln sinh
trng v tch ly jaceosidin ca t bo cy hoa sen tuyt (Saussurea medusa).
Kt qu cho thy, cc mi trng c s phi hp gia BAP vi 2,4-D th khng
thch hp cho sinh trng ca t bo; khi nng 2,4-D gia tng ln, kh nng
sinh trng ca t bo gim i r rt. S dng cc mi trng b sung phi hp
gia BAP vi NAA, lng jaceosidin tch ly trong t bo tng t l thun vi
s gia tng nng ca NAA [189]. M ca cy c tri vng (Solanum
xanthocarpum) khi nui cy trn mi trng cha 2,4-D th c kh nng sn xut
c solasodine, nhng khi nui cy trn mi trng c cha IAA hoc IBA th
khng sn xut c cht ny [111]. Nm 1993, khi nghin cu sn xut
berberine t nui cy huyn ph t bo cy Thalictrum minus, Hara v cs nhn

13
thy, mi trng nui cy c b sung 2,4-D v NAA khng c tc dng kch
thch sn xut berberine, trong khi kh nng sn xut berberine ca t bo
tng khi s dng BAP [56].
+ Ngun carbon
M v t bo thc vt trong nui cy in vitro sng ch yu da theo
phng thc d dng. V vy, vic b sung vo mi trng nui cy ngun
carbon hu c l iu bt buc. Ngun carbon trong mi trng nui cy t bo
thc vt thng c cung cp di dng carbohydrate. Carbon va tham gia
tng hp cc thnh phn ca t bo va cung cp nng lng cho qu trnh sinh
trng v tn ti ca t bo. Ngoi ra, carbohydrate cng l ngun cung cp
carbon cn thit cho s hnh thnh cc sn phm trung gian thng qua trao i
cht [191]
Trong phn ln cc mi trng nui cy, ngun carbon c b sung ch
yu l ng sucrose v glucose vi nng 20 - 40 g/L. Gautheret (1959) cho
rng i vi phn ln cc m v t bo nui cy, ng sucrose v glucose l
ngun carbon tt nht, trong mt s trng hp khc, c th dng fructose,
galactose v maltose (Nguyn c Thnh, 2000) [14]. nh hng ca nng
sucrose trong mi trng nui cy c nghin cu nui cy t bo tam
tht (Panax notoginseng). Khi lng kh ca t bo tng t l thun vi s gia
tng nng sucrose t 20 n 40 g/L, nhng khi nng sucrose ln n 60
g/L th dng nh c ch s sinh trng ca t bo [140]. Theo Gunter v cs
(2003), nui cy t bo cy Silene vulgaris cc mi trng c nng ng
sucrose thp (di 30 g/L), kh nng sinh trng v tng hp polysaccharide ca
t bo khng cao. Khi tng nng sucrose ln dn 30 g/L kh nng tch ly
sinh khi t bo cng gia tng. Mi trng c cha 30 g/L sucrose hoc phi hp
gia ng sucrose (15 g/L) v glucose (15 g/L) l thch hp nht cho sinh
trng ca t bo [50].


14
- iu kin nui cy
+ C mu nui cy ban u
C mu a a vo nui cy ban u rt quan trng khi cy chuyn t bo
thc vt, v cng c th nh hng n s sn xut hp cht trao i th cp. Khi
lng mu ban u thp hn lng mu ti hn thp nht th t bo thng khng
sinh trng c [78]. Matsubara v cs (1989) trong nghin cu nui cy t bo
cy Coptis japonica nhn thy, khi lng mu a vo nui cy cao th sinh
khi t bo v berberine thu c cng cao. Lng mu a vo nui cy thch
hp nht cho sinh trng v tch ly berberine ca t bo l 8 g/L, t 55 g/L sinh
khi v 3,5 g/L berberine sau nui cy [97]. Lng mu nui cy nh hng ln
n sinh trng t bo c tm thy trong nui cy t bo cy Gymnema
sylvestre. Vi lng mu t bo a vo nui cy l 60 g/L th kh nng sinh
trng ca t bo l cao nht, nu lng mu a vo nui cy l 80 hoc 100 g/L
th sinh trng t bo b gim r rt [78]. Kt qu nghin cu nui cy mt
cao t bo ca cy ta t (Perilla frutescens ) sn xut anthocyanin cho thy,
lng mu a vo nui cy nh hng ng k ln ng hc ca qu trnh sinh
trng v tch ly anthocyanin ca t bo. Sinh trng v tch ly anthocyanin ca
t bo tt nht khi nui cy vi lng mu nui cy l 50 g/L, sinh khi t bo t
38,3 g/L sau 11 ngy nui cy, v anthocyanin t 5,8 g/L sau 10 ngy nui cy;
cao gp 3,3 ln (lng sinh khi) v 24 ln (lng anthocyanin) so vi nui cy
m lng mu ch s dng l 15 g/L [191].
+ Khuy trn
Khuy trn l iu cn thit phn b ng u t bo, m, cht dinh
dng trong pha lng [77]. Vic khuy trn thng c thc hin bng cch
sc kh hoc dng cnh khuy c hc hoc kt hp c 2 phng php trn.
Thy ng lc cho vic ha trn cn phi nh khng lm h hi m hay t
bo nhng phi kch thch chc nng ca t bo [116]. Hu ht cc cht
dinh dng u c kh nng ha tan cao trong nc, do trong thi gian ln

15
men nu ch phn b u mi trng th s khuy trn khng tht cn
thit. Tuy nhin, i vi oxy ha tan th phi c s khuy trn tt v kh nng
ha tan ca n trong mi trng ln men l rt km, trong khi yu cu oxy
cho s sinh trng ca cc vi sinh vt hiu kh (hoc t bo thc vt v ng
vt) li rt cao [7].
Nghin cu ca Wang v cs (2010) cho thy, nui cy t bo cy
Glycyrrhiza inflata trong h ln men, vi tc khuy t 150 vng/pht th
sinh khi t bo t cao nht, trong khi vi tc khuy 100 v 300
vng/pht th s tch ly sinh khi t bo km hn [171]. Nghin cu nh hng
ca tc khuy ln sinh trng ca t bo cy du c, Choi v cs (2008)
kho st cc tc khuy t 80 - 335 vng/pht. Kt qu cho thy, tc
khuy 120 v 225 vng/pht, sinh khi t bo tng 200%; tc khuy 335
vng/pht sinh khi t bo ch tng khong 7% so vi sinh khi t bo a vo
nui cy ban u [33].
+ Sc kh
Sc kh trong nui cy t bo thc vt nhm p ng ba chc nng chnh
l duy tr iu kin hiu kh, gim hp th cc sn phm bay hi v loi b
nhit sinh ra t qu trnh trao i cht. Tc tiu th oxy c trng ca t bo
thc vt ph thuc vo dng t bo, iu kin nui cy v giai on sinh trng,
nhng nhn chung l khong 6 x 10
4
g O
2
/g khi lng kh/pht [72].

Tc
hp th oxy

phi ln cung cp y lng oxy cn thit cho nhu cu h
hp ca t bo v v th n h tr cho sinh trng t bo v sn xut cc hp
cht mong mun, tuy nhin khng nn qu cao bi v c vic cung cp d tha
hoc thiu oxy u c th c ch s sinh trng v trao i cht th cp ca t
bo. trnh nhng mt hn ch ca oxy, hm lng oxy ha tan trong mi
trng nui cy phi c gi trn mc hm lng oxy ha tan ti hn (thng
thng khong 15-20% bo ha khng kh) [72].


16
Lee v cs (2006) nghin cu kho st s nh hng ca mt s iu kin
nui cy n kh nng sinh trng ca t bo cy Gymnema sylvestre trong h
ln men 2 L. Kt qu cho thy, trong cc tc sc kh kho st t 0,1 n 0,4
L/L/pht th tc sc kh 0,1 L/L/pht l thch hp nht cho sinh trng ca t
bo [78]. Thng thng, tc dng kh thch hp cho nui cy huyn ph t
bo thc vt nm trong khong 0,1-0,5 L/L/pht [40]. Tuy nhin, vic tng dn
tc dng kh ph hp vi tnh trng sinh l t bo trong sut qu trnh nui
cy c th c li cho sinh trng ca t bo. Bng cch p dng kiu sc kh nh
vy (2 ngy u nui cy, sc kh tc dng 0,5 L/pht v sau l 1,0
L/pht) trong nui cy t bo cy hoa hng dng (Helianthus annuus) Haas
v cs (2008) nhn thy, sinh khi t bo tch ly cao v t n 15 g/L khi
lng kh [53].
nh hng ca nng oxy (20,8 - 50%) mi trng nui cy trong h
ln men ln kh nng sinh trng ca t bo Panax ginseng c nghin cu.
Nng oxy 40% th thch hp cho tch ly sinh khi v sn xut ginsenoside,
t 12,8 g/L sinh khi kh v 4,5 mg/g ginsenoside. Cc nng oxy: 20,8; 30
v 50% th khng thch hp cho s sinh trng cng nh tch ginsenoside ca t
bo nui cy [159]. Vronique v cs (1992) nghin cu nh hng ca nng
oxy ln sinh trng ca t bo cy c rt (Daucus carota) trong h ln men nhn
thy, nng oxy gim th tc sinh trng t bo cng gim theo. Kt qu
cng cho thy, phn ng ca cc loi t bo khc nhau i vi nng oxy cng
khc nhau. i vi nui cy to phi soma, nng oxy 75% c ch kh
nng sinh trng ca t bo [166].
1.1.2.5. Nui cy t bo thc vt qui m ln
Nhn chung, nui cy t bo thc vt ln qui m ln l thc hin qu trnh
sinh hc qui m phng th nghim lp li gn nh c th sn xut mt
lng ln hn cc sn phm mong mun. Trnh t ca mt qu trnh nng cp
in hnh bt u t nui cy t bo trong cc chai, l n bnh tam gic nui cy

17
lc c dung tch 1 L, sau n h ln men bng thu tinh t 1-10 L, v sau
nng cp n h ln men bng thp khng r t 30-150 L ri n 1000 L [181].
H ln men l mt h thng nui cy t ng m chc nng chnh ca n l ci
thin kh nng kim sot mi trng nui t c cc iu kin ti u cho
sinh trng ca t bo v/hoc hnh thnh sn phm. T bo thc vt c nui
cy trong h ln men u tin vo nhng nm 1960, s dng nhng thit b c
lm ph hp cho nui cy t bo thc vt t nhng thit b ln men thng mi
hoc khng thng mi dng trong nui cy t bo ng vt [123], [144].
Cc nghin cu trong h ln men l khu cui cng ca nui cy huyn
ph t bo thc vt thng mi ha cc cht trao i th cp. y l bc
quan trng v c nhiu vn c th pht sinh khi nng cp t nui cy lc trong
cc bnh tam gic n nui cy cc h ln men [181]. H ln men hot ng
nh mt nh my sinh hc sn xut cc sn phm c lng cao vi nhiu u
im nh sau: Cc sn phm c kim sot khng ph thuc vo ngun thc
vt sn c; Tng cc dung tch lm vic; Nui cy ng nht do c c ch khuy
trn bng c hc hoc sc kh; Kim sot tt hn mi trng nui cy v iu
kin vt l v vy c th d dng ti u ha cc thng s sinh trng nh: dinh
dng, pH, nhit trong sn xut cc cht chuyn ha; C th kim sot cc
sn phm cui cng di cc iu kin sinh trng; Ci thin kh nng hp th
cc cht dinh dng kch thch tc nhn nhanh v sn xut hm lng cao
hn cc hot cht sinh hc; Thu hi cc t bo n gin v nhanh hn; Thun li
trong cc kho nghim v sinh tng hp hoc/v chuyn ha sinh hc c lin
quan vi sn xut cc cht trao i bi cc enzyme sn c; D dng hn trong
phn tch cc hp cht mong mun bi tnh phc tp thp ca cc dch chit;
Kim sot tt hn trong nng cp sn xut trn qui m ln [44], [52].
Cc h ln men c dng ch yu cho nui cy trn qui m ln t bo
thc vt sn xut sinh khi t bo hoc cc cht chuyn ha [181]. Hiu sut
ca bt k h ln men no u ty thuc mt s kh nng sau: Duy tr nng

18
sinh khi phi cao; Duy tr iu kin v trng; Phn b ng u dinh dng v
cc nguyn liu sng trong ni ln men thng qua s khuy trn hiu qu; Tng
hoc gim nhit ty vo yu cu nui cy; To ra lc trt va phi. Mc
trt cao c th nguy hi cho t bo nui cy, nhng lc trt thp cng c th
khng mong mun bi v hnh thnh bt hoc bm dnh ca cc khi t bo ln
cnh khuy v thnh ca ni ln men [86].
C nhiu kiu ca h ln men c dng trong nui cy t bo thc
vt. Cc h ln men c tnh truyn thng nh cc h ln men khuy trn bng
cnh khuy, airlift reactors v si bt kh dng ct s dng trong nui cy in
vitro t bo thc vt c phin bn t cc h ln men dng trong cng ngh
sinh hc vi sinh vt [48]. Nhng n lc thit k h ln men dnh ring cho nui
cy t bo thc vt c thc hin vo cui nhng nm 1970. Vi s nhn thc
su v tnh mn cm ca cc t bo thc vt trong nui cy in vitro vi lc trt,
v vy trong hn mt thp nin, ch c dng h ln men airlift c xem l thch
hp cho nui cy t bo thc vt [165]. Tuy nhin, c nhiu nghin cu tin
hnh nui cy t bo thc vt trong h ln men khuy trn bng cnh khuy.
Ritterhaus v cs (1990) s dng cc h ln men khuy trn bng cnh khuy
vi dung tch 75, 750, 7.500, 15.000 v 75.000 L nui cy t bo cy
Echinacea purpurea, Rauwolfia serpentina v mt s thc vt khc trn qui m
ln [133]. Wang v Zhong (1996) pht trin mt loi h ln men mi dng
nui cy in vitro thc vt mn cm vi lc trt l h ln men khuy trn
bng cnh khuy ly tm (CIB) [172]. Zhang v Zhong (2002) cng dng h
ln men CIB (3 L) nui cy huyn ph t bo cy tam tht, vi tc khuy
145 vng/pht, sinh khi t bo tch ly t 22 g/L [186]. Hn na, cc tc gi
ny cng nui cy thnh cng t bo cy P. notoginseng khi nng cp dung
tch h ln men ny ln n 30 L, s tch lu sinh khi t trn 25 g/L, sn lng
saponin v polysaccharide cng cao hn (1,7 so vi 1,5 g/L v 2,9 so vi 2,7
g/L). T cc nghin cu ny, Zhang v Zhong (2002) cho rng, h ln men CIB

19
c tim nng ln trong sn xut sinh khi t bo chit tch cc cht mong
mun trn qui m ln [186]. Nm 2007, Prakash v Srivastava cng nui cy
thnh cng t bo cy Azadirachta indica sn xut sinh khi v azadirachtin
trong h ln men CIB v nhn thy, h men ln men ny to ra lc trt thp v
kh nng trao i oxy cng c ci thin [125].
1.2. S TCH LY CC HP CHT TH CP TRONG T BO THC
VT NUI CY IN VITRO
1.2.1. Vai tr ca cc hp cht th cp thc vt
thc vt, c nhiu loi hp cht hu c hoc cc cht chuyn ha c to
ra trong qu trnh trao i cht. Nhng cht chuyn ha ny c xp thnh nhm
cc cht trao i s cp v th cp. Cc hp cht th cp tm thy ch trong mt s
loi thc vt hoc trong nhm loi c lin quan nhau, trong khi cc hp cht s
cp c tm thy trong hu ht cc loi [153]. Trong vi thp k qua, nhng bng
chng t thc nghim v thc t cho thy, cc hp cht th cp thc vt c cc
chc nng c bn sau: Bo v c th chng li cc loi ng vt n c; Khng nm,
vi khun, virus; Chng li s canh tranh v nh sng, nc v cht dinh dng; Thu
ht cc loi ng vt trong qu trnh th phn v pht tn ht; To ra cc tn hiu
trong giao tip gia thc vt vi cc loi vi sinh vt cng sinh; Chng li tia t
ngoi v cc tc nhn vt l bt li khc [177]. Arnason v cs (1995) cng b
thc vt sn xut hn 80.000 hp cht khc nhau thng qua con ng trao i th
cp. Cc sn phm th cp c d tr ch yu trong cc cu trc c bit hoc
cc c quan d tr nh r, cc t bo d tr, khng bo, h thng mng[19]. Cc
hp cht ha hc ny c dng trong dc phm, ha cht nng nghip, thuc
nhum, gia v, cht to mi, thuc tr su; chng ng gp nhiu t la trong
sn xut cng nghip [47].
1.2.2. Cc nhm hp cht th cp ch yu thc vt
Cc hp cht th cp ca thc vt c th phn thnh ba nhm chnh l
terpene, phenol v cc hp cht cha nitrogen.

20
1.2.2.1. Nhm terpene
Terpene, terpenoid hoc isoprenoid l cc dimer, trimer hoc polymer ca
cc n v isoprene. Cc n v isoprene thng lin kt hnh thnh nn cc hp
cht mch thng hoc mch vng ph bin vi 10 carbon (monoterpenoid), 15
carbon (sesquiterpenoid), 20 carbon (diterpenoid) hoc 30 carbon (triterpenoid) v
rt him dng terpenoid c tm thy vi 25 carbon [153]. Cc monoterpene c
tm thy rng ri trong tinh du thc vt, v d nh menthol, borneol, linalool
Chng c s dng nh l cc cht khng cn trng v cc cht c hot tnh dc
l nh gim au v khng vim. Sesquiterpene l thnh phn ca tinh du nhiu
loi thc vt, nhng hp cht ny c hot tnh nh khng khun, khng nm, dit
tr giun sn, chng st rt[51]. Diterpennoid c tm thy trong nhiu loi thc
vt v ng vt. Mt s cht ca chng c ng dng trong iu tr bnh, chng
hn nh taxol v cc dn xut ca chng c dng lm thuc iu tr ung th,
forskolin chng tng huyt p. Triterpene l hp cht terpene cha 30 carbon,
chng cng c s dng nhiu trong dc phm [51].
1.2.2.2. Nhm phenol
Tt c cc hp cht nhm phenol c mt vng thm gn vi cc nhm chc
nh hydroxyl, methoxy v cc cu trc vng khng thm khc. Chng c cu trc
t n gin vi mt vng thm cho n cc polymer phc tp nh tannin, lignin.
Cc hp cht phenol c tng hp bng con ng shikimate hoc con ng
acetate. Chng c hot tnh dc l ph rng nh khng vim, gim au, khng
khi u, khng virus HIV, khng vi sinh vt, chng oxy ha, chng lot, bo v
gan [51], [177].
1.2.2.3. Cc hp cht cha nitrogen
Phn ln cc hp cht th cp ca thc vt u c cha nitrogen trong
cu trc ca chng. Cc hp cht ny c bit n nh l cc hp cht
chng li cc ng vt n c nh alkaloid v cyanogenic glycoside. Chng

21
c quan tm nhiu bi tnh c i vi con ngi v cc thnh phn dc
cht ca chng [153].
Cc alkaloid c dng tinh th, c hot tnh sinh l trn tt c cc ng vt
v c s dng trong cng nghip dc. Alkaloid thng c vi liu lng
ln nhng vi liu lng nh, chng c s dng lm thuc cha bnh [104].
Cc cyanogenic glycoside l v kh bo v ca thc vt. Chng khng c cho
chnh bn thn thc vt m sn sng hnh thnh cc phn t bay hi c tnh c
khi thc vt b e do. Mt s cht trong chng c s dng lm thuc nhum,
cht mi thc phm v dc phm [79].
1.2.3. Nhng nghin cu sn xut cc hp th cp t nui cy t bo
thc vt
1.2.3.1. Nhng nghin cu ngoi nc
Nghin cu v kh nng sinh tng hp ca cc t bo nui cy c
tin hnh bi cc nh khoa hc thc vt v vi sinh vt nhiu quc gia. Hu ht
cc ng dng nui cy t bo thc vt trong cng ngh sinh hc u nhm vo
mc ch sn xut cc hp cht th cp v gn y l sn xut cc cht iu tr
ung th quan trng nh taxol, vinblastine v vincristine [113]. Nhng thnh tu
t c trong lnh vc nui cy t bo thc vt sn xut cc hp cht dng
cha bnh to ra kh nng c th sn xut trn qui m ln cc cht
alkaloid, terpenoid, steroid, saponin, phenol, flavonoid v cc amino acid [167].
Taxol l mt trong nhng tc nhn khng ung th trin vng nht c bit
n bi dng hot ng c nht ca n trn h thng vi t bo hnh ng [113].
Hin nay, sn xut taxol nh nui cy t bo ca cc loi Taxus tr thnh mt
trong nhng ng dng rng ri trong nui cy t bo thc vt v ang lm ch cc
gi tr thng mi to ln ca taxol, mt cht ch c dng vt trong cy thy tng
v tng hp ha hc rt t tin [65]. Christen v cs (1989) cng b u tin v
sn xut taxol (paclitaxel) bi nui cy t bo Taxus [34]. Fett-Neto v cs (1995)

22
nghin cu nh hng ca cc cht dinh dng v cc yu t khc ln sn xut
trong nui cy t bo, kt qu sn xut paclitaxel t 0,02% khi lng kh [41].
Kim v cs (1995) cng to ra paclitaxel trong nui cy t bo cy Taxus
brevifolia sau 10 ngy trn mi trng ti u cha 6% fructose [74]. Bn cnh s
tch ly paclitaxel, mt s toxoid cng c tm thy c trong t bo v mi
trng nui cy t bo Taxus [71]. Parc v cs (2002) sn xut toxoid bng nui
cy callus ca cc cy Taxus c chn lc [121]. tng sn xut toxoid, b
sung cc amino acids vo mi trng nui cy cng c kho st, kt qu cho
thy, phenylalanine tng sn xut taxol ti a trong nui cy t bo cy T.
cuspidata [92].
T xa, r ca cy nhn sm c s dng rng ri nh mt v thuc b,
mt dc phm qu gi [148]. Ginseng c bit n nh l nhn t k diu
tng cng sc kho v ko di tui th. Thnh phn hot cht chnh c trong nhn
sm c xc nh l ginsenoside, thuc nhm saponin triterpenoid [167]. Furuya
(1988) nghin cu nh hng ca thnh phn mi trng v HST tng
lng ginsenoside [45]. Nui cy t bo cy nhn sm trn quy m ln c
Yasuda v cs (1972) cng b [180]. Sau , quy trnh nui cy quy m cng
nghip c cng ty Nitto Denko (Ibaraki, Osaka, Nht Bn) thc hin trong h
ln men c cnh khuy vi dung tch 2000 v 20.000 L, nng sut ginsenoside t
c l 500-700 mg/L/ngy [60]. Quy trnh ny c xem nh l mt bc ngoc
quan trng trong thng mi ha nui cy m v t bo quy m ln. Wang v
Zhong (2002) nhn thy b sung methyljasmonate hoc dihydro-methyl jasmonate
vo mi trng nui cy tng kh nng hiu sut ginsenoside ca t bo cy
nhn sm [173]. Nui cy r c gy nhim Agrobacterium rhizogenes c
tin hnh v hiu sut ginsenoside t c cao hn 3 ln so vi nui cy loi
callus to t r bnh thng [60]. Cc kt qu nghin cu ny cho thy, nui cy t
bo cy nhn sm vn l lnh vc thu ht nhm thng mi ha trn th gii v l
tim nng to ln cho cng nghip ha sinh [138].

23
L-DOPA l mt tin cht ca cc alkaloid, betalain v melanine; c
tch chit t cc cy Vinca faba, Mucuna, Baptisia v Lupinus [148]. N cn l
mt tin cht ca catecholamine ng vt v c s dng nh l mt loi
thuc tim nng cho iu tr bnh Parkinson [62]. L-DOPA c dng rng ri
trong iu tr bnh nn c nhu cu ln v gi thnh cao, chnh v th nui cy t
bo c xem nh l phng thc thay th sn xut L-DOPA nhiu hn [26].
Teramoto v Komamine (1988) to callus ca cy Mucuna hassjoo, M.
pruriense, M. deeringia v ti u ha iu kin nui cy. Kt qu nghin cu
cho thy, nng cao nht ca L-DOPA thu c khi nui cy t bo ca cy
M. hassjoo trn mi trng MS b sung 0,025 mg/L 2,4-D v 10 mg/L kinetin
[156]. Hm lng L-DOPA trong t bo nui cy t khong 80 mmol/g khi
lng ti cng c Vanisree v cs (2004) cng b [162].
Berberine l mt isoquinoline alkaloid c trong r ca cy Coptis japonica
v trong v ca cy Phellondendron amurense. Bng cch chn dng c nng
sut cao, nhm nghin cu ca Mitsui v cs to ra berberine trn quy m ln
vi nng sut 1,4 g/L sau hn 2 tun nui cy [167]. Vi vic s dng cht kch
khng polysaccharide ca nm men, Sarin (2005) thnh cng trong vic sn
xut vi mc tng i cao ca berberine trong nui cy t bo cy T. rugosum
[138]. nh hng ca permidine ln sn xut berberine trong nui cy t bo cy
T. minus cng c cng b bi Hara v cs (1991) [57].
Diosgenin l tin cht cho tng hp ha hc ca thuc steroidal v cc k
quan trng trong cng nghip dc phm [39]. Tal v cs (1983) nghin cu
sn xut diosgenin bng nui cy t bo cy D. deltoidea. H nhn thy rng
hm lng carbon v nitrogen nh hng ln n s tch ly diosgenin trong
mt dng t bo nui cy [154]. Ishida (1988) nhn thy sn xut diosgenin c
kch thch khi tin hnh nui cy c nh t bo cy D. deltoidea, vi sn lng
tng 25%. Kaul v cs (1969) nghin cu nh hng ca mt s yu t ln sn
xut diosgenin trong nui cy callus v huyn ph t bo cy D. deltoidea [70]

24
Campothecin l mt alkaloid khng ung th tim nng c trong cy
Camptotheca acuminate (Padmanabha, 2006). Sakato v Misawa (1974)
nghin cu to callus ca cy C. acuminata trn mi trng MS b sung 0,2
mg/L 2,4-D v l mg/L kinetin v nui cy lng vi s c mt ca gibberellin v
L-tryptophan; hm lng camptothecin thu c khong 0,0025% khi lng
kh [136]. Nghin cu ca Thengane v cs (2003) cho thy, khi nui cy t bo
cy C. acuminata trn mi trng MS c b sung 4 mg/L NAA th s tch ly
camptothecin t c l 0,998 mg/L [160].
Vincristine v vinblastine l cc indole alkaloid nh trng ngng tr
thnh thuc c gi tr trong ha tr liu ung th bi hot tnh khng ung th tim
nng ca chng chng li nhiu khi u rn v bch cu. Nhng hp cht ny
chit tch thng mi t s lng ln ca cy da cn. Do hm lng vincristine
v vinblastine cha trong cy thp (0,0005%), v vy nui cy t bo c
nghin cu nh mt s thay th cho sn xut mt lng ln cc alkaloid ny
[113]. Vinblastine hp thnh t catharanthine v vindoline. V vindoline c
nhiu catharanthin trong cy hon chnh nn n t t tin hn [20]. Nghin cu
nh hng ca nhiu hp cht nh vanadyl sulphate, abscisic acid v sodium
chloride ln sn xut catharanthin cng c cng b bi Smith v cs (1987)
[146]. nh hng ca nhiu yu t nh stress, b sung cht iu ha sinh
trng, cc cht kch khng v cc tin cht tng hp ln sn xut indole
alkaloid cng c Zhao v cs (2001a; 2001b) cng b [189], [190].
Cc cht tanshinone thuc nhm quinoid diterpenoid c cho l nhng
cht c hot tnh ca cy Salvia miltiorrhiza, chng c dng lm dc liu
truyn thng ca Trung Quc [174]. Nakanishi v cs (1983) thit lp c
mt dng t bo c cha mt lng ln cryptotanshinone t cy S. miltiorrhiza
[110]. Shimomura v cs (1991) nghin cu nui cy r bt nh cy S.
miltiorrhiza v kho nghim cc iu kin nui cy cho hiu sut tanshinone cao
[141]. Sn xut diterpenoid r t ca cy S. miltiorrhiza cng c Hu v
Alfermann (1993) nghin cu [61].

25
Podophyllotoxin l nguyn liu khi u cho vic iu ch cc dn xut
bn tng hp nh toposide, teniposide v c dng rng ri trong iu tr
khng khi u [31]. Cc cy Podophyllum peltatum v Podophyllum hexandrum
sinh trng rt chm, vic thu hi podophyllotoxin t t nhin cng gp kh
khn. Do , nhng hn ch trong vic cung cp podophyllotoxin t t nhin cn
phi c khc phc [117]. Nui cy t bo cy P. peltatum sn xut
podophyllotoxin c nghin cu u tin bi Kadkade (1982) [69]. tng sn
lng ca podophyllotxin, Woerdenberg v cs (1990) s dng phc cht
conifery1 alcohol v b-cyclodextrin nui cy huyn ph t bo cy P.
hexandrum [178].
1.2.3.2. Nhng nghin cu trong nc
nc ta, cng ngh nui cy t bo thc vt pht trin vo nhng nm
1970 v n nay t c mt s thnh cng. Mt trong nhng kt qu nui
cy t bo thnh cng nht l nui cy t bo cy sm Ngc Linh (Panax
vietnamensis). Sm Ngc Linh c tc dng phng chng ung th, bo v t bo
gan, kch thch h min dch, chng stress v trm cm, chng oxy ha, lo ha.
Hin nay, loi cy ny b khai thc cn kit v c nguy c tuyt chng [157].
Thanh v cs (2007) nghin cu nh hng ca thnh phn mi trng ln sn
xut sinh khi v ginsenoside trong nui cy huyn ph t bo cy sm Ngc
Linh. Kt qu cho thy, mi trng MS v MS thch hp cho c sn xut sinh
khi cng nh ginsenoside, sinh khi v ginsenoside t ln lt l 9,8 g/L v
6,81 mg/g khi lng kh. Nng sucrose tng t 20-50 g/L tng sn xut
sinh khi, tuy nhin khi sucrose tng n 70 g/L th c ch c sinh trng v tch
ly ginsenoside ca t bo. Nng nitrogen thch hp cho c sinh trng v
tch ly ginsenoside l 30 mM [157]. Nghin cu ca Thanh v cs (2008) cng
cho thy, NH
4
NO
3
nng 0,5 g/L cho sinh khi ln nht v hm lng
ginsenoside t cao nht l 6,5 mg/g khi lng kh. Nng 1,0 g/L ca KNO
3

thch hp cho sinh trng t bo nhng ginsenoside thu c ln nht (6,1 mg/g

26
khi lng kh) li nng 2,0 g/L KNO
3
. Nghin cu cn cho thy, sinh
trng cng nh hm lng ginsenoside ca t bo tng ng k khi b sung
CaCl
2
vo mi trng nui cy [158].
Nghin cu sn xut taxol t cc h thng t bo in vitro cng v ang
c quan tm bi cc nh khoa hc trong nc. L Th Thu Tin v cs ( 2006)
nghin cu nui cy t bo cy thng (Taxus wallichiana). Callus hnh
thnh v sinh trng tt trn mi trng B
5
c b sung auxin v cytokinin. Mi
trng c 4 mg/L 2,4-D v 1 mg/L kinetin thch hp cho s hnh thnh v sinh
trng callus t thn non. Callus t l non sinh trng tt trn mi trng c 3
mg/L NAA v 0,25 mg/L kinetin. Nui cy t bo c thit lp trn mi trng
lng c cng thnh phn vi mi trng nui cy callus; phn tch cho thy, c
s hin din ca taxol trong callus v huyn ph t bo, v khng tm thy taxol
trong mi trng nui cy [16]. nh hng ca cc ngun carbon ln sinh
trng ca t bo cy thng c Nhut v cs (2006) nghin cu. Kt qu
cho thy, mi trng nui cy c b sung 30g/L glucose v 30 g/L fructose l
thch hp nht cho sinh trng ca t bo [112]. Dng Tn Nht v cs (2007)
cng nghin cu thit lp iu kin nui cy t bo cy thng v ti sinh
callus t t bo dch huyn ph. Cc t bo v callus thu nhn c c th s
dng nhn, nh lng v tch chit taxoid [11].
C gai leo (Solanum hainanense) l mt cy thuc qu, r ca chng
thng c dng lm thuc cha cc bnh phong thp, au nhc xng,
ho[4]. R cy c gai leo l ni tch ly ch yu glycoalkaloid, tuy nhin chng
c hm lng thp v ph thuc nhiu vo ngun nguyn liu t nhin [15]. L
Th H Thanh v cs (2009) nghin cu sn xut glycoalkaloid t t bo cy c
gai leo. Kt qu cho thy, mi trng MS rn c 30 g/L sucrose, b sung thm
0,1 mg/L BAP v 1,0 mg/L 2,4-D thch hp nht to callus. Huyn ph t bo
c gai leo sinh trng tt nht trn mi trng MS c 40 g/L sucrose; 1,0 mg/L
BAP v 1,0 mg/L 2,4-D, vi tc lc 150 vng/pht. Hm lng glycoalkaloid

27
ton phn tch ly cao nht trong t bo sau 4 tun nui cy t 48,41 mg/g khi
lng kh, cao gp 5,9 ln hm lng glycoalkaloid trong r cy c gai leo t
nhin 1 nm tui [13]. Loc v cs (2010) cng kho st s tch ly
glycoalkaloid trong nui cy callus ca cy c gai leo [88].
Cy rau m (Centella asiatica) l loi dc tho c cha asiaticoside v
madecassoside. Asiaticoside c hot tnh chng oxy ha, cha bng, bo v t
bo thn kinh v sinh tng hp collagen [9]. Nguyn Hong Lc v cs (2008)
nghin cu to callus v kho st kh nng tch ly asiaticoside trong callus cy
rau m. Kt qu cho thy, mi trng MS c b sung 1,0 mg/L NAA v 2,0
mg/L BAP thch hp nht cm ng to callus; mi trng c 1,5 mg/L NAA
v 0,5 mg/L kinetin kch thch callus sinh trng mnh. Hm lng asiaticoside
tch ly cao nht trong callus l 3,12 mg/g tng ng vi 0,31% khi lng
kh [8]. Thit lp nui cy huyn ph t bo cy rau m v kho st s tch ly
asiaticoside trong t bo nui cy cng c nghin cu Loc v An (2010)
nghin cu [87].
Cy da cn l mt trong nhng loi cy dc liu cha nhiu alkaloid,
din hnh l cc cht cha ung th rt tt nh vincristine v vinblastine. Bi Vn
L v Nguyn Ngc Hng (2006) nghin cu sinh trng v tch ly alkaloid
ton phn ca t bo cy da cn. Kt qu cho thy, trn mi trng b sung 1,0
mg/L NAA v 0,5 mg/L Kin, sinh khi t bo v alkaloid ton phn t cao nht;
nng sucrose 60 g/L ti u cho s tch ly alkaloid ton phn. Khi kt hp
nng cht HST ti u. Cc tc gi thu c lng vincristin cao trong t
bo khi nui cy trn mi trng c 1 mg/L NAA; 0,5 mg/L Kin v 60 g/L
sucrose, trong kh l ca cy da cn t nhin khng tm thy loi alkaloid
ny [5]. Cy inh lng (Polyscias fruticosa) l mt loi thc vt cha nhiu
saponin. Phm Th T Lin v V Th Bch Mai (2007) to callus t cc mu
cy con trn mi trng MS b sung 2 mg/L 2,4-D. Callus to thnh sau 14 tun
c chuyn sang nui cy trn mi trng lng b sung 1,0 mg/L 2,4-D v
20% nc da thu sinh khi v chit rt saponin [6].

28
Nh vy, nghin cu nui cy t bo thc vt sn xut cc hp cht th
cp nc ta cng c c mt s kt qu nht nh. Tuy nhin, nhng nghin
cu ni trn vn cn quy m phng th nghim. Pht trin cc k thut nui
cy trong cc h ln men quy m cng nghip sn xut cc hp cht c hot
tnh sinh hc vn cn l mt con ng y tim nng cha c khai ph ht
ca nn cng ngh nui cy t bo ca Vit Nam.
1.3. GII THIU V CY NGH EN
1.3.2. Thnh phn ha hc
Ngh en l loi cy tho dc khng c, cha nhiu cc hp cht ha
hc c gi tr dc liu cao. Nhiu nghin cu cng b v thnh phn ha hc
ca cy ngh en. T nm 1928, Rao v cs kho st s b thnh phn ha hc
tinh du t thn r ca ngh en v tm thy cc hp cht nh -pinen, camphen,
cineol, camphor v borneol bn cnh cc sesquiterpene, tuy nhin khng phn lp
v xc nh c mt sesquiterpene no [131]. Shiobara v cs (1985) tch chit
t c ngh en c 3 loi sesquiterpenoid l curcumeone, curcumanolide-A,
curcumanolide-B [143]. Xingyi (1999) nghin cu cho thy, tinh du ngh en
c cha 37 thnh phn khc nhau, trong ch yu l curzerenone, curcumenol,
b-elemene, isocurcumenol [179]. Singh v cs (2002) kho st thnh phn tinh
du ca mt s loi ngh ca n cho thy, n c cha cc thnh phn chnh
nh: 1,8 cineol, cymene, -phellandrene (14,9%) [145]. Mau v cs (2003) xc
nh c 36 hp cht t ngh en gm 17 terpenes, 13 alcohol, v 6 ketones
[100]. Garg v cs (2005) nghin cu v thnh phn ha hc ca tinh du t l
cy ngh en n . Kt qu cho thy, tinh du c cha 23 hp cht khc nhau
[46]. Makabe v cs (2006) xc nh c hn 10 loi sesquiterpene t c ngh
en [94]. Champakaew v cs (2007) phn tch tinh du bay hi ca c ngh en
nhn thy, thnh phn chnh ca tinh du bao gm -tumerone, 1,8-cineole v 7-
zingiberene [28].

29
Bn cnh cc nghin cu v thnh phn ha hc ca tinh du ngh en,
cc nghin cu v thnh phn hp cht mu vng c trong ngh en cng
c tin hnh. Syu v cs (1998) nghin cu nhn thy dch chit ethanol ca
c ngh en c cha curcumin, demethoxycurcumin v bisdemethoxycurcumin
[152]. Paramapojn v Gritsanapan (2007) nghin cu v thnh phn
curcuminoid trong c ngh en cc vng sinh thi khc nhau ca Thi Lan.
Kt qu cho thy, hm lng curcumin, demethoxycurcumin v
bisdemethoxycurcumin ln lut t 1,46% n 5,73% w/w (trung bnh 2,73%
w/w); t 3,15% n 10,98% w/w (trung bnh 7,37%) v t 0,49% n 2,99%
w/w (trung bnh 1,40% w/w) [119].
nc ta, kho st thnh phn ha hc ca ngh en cng ang rt
c quan tm. Tuy nhin, cc nghin cu ch yu tp trung vo nh gi
thnh phn ha hc ca tinh du ngh en trng cc vng khc nhau. Phan
Minh Giang v cs (1997) cng b thnh phn chnh trong tinh du thn r
ngh en Sc Sn (H Ni) l zurumbon (chim 79,08%) [2]. L Qu Bo
v cs (2004) cng b thnh phn tinh du trong thn r ngh en
Lng (Ngh An) v Hng Sn (H Tnh) [1]. Trn Th Vit Hoa v cs
(2007) nghin cu thnh phn ha hc ca tinh du c ngh en trng
Lt c trch ly theo phng php gia nhit thng thng v phng
php gia nhit bng l vi sng. Kt qu nghin cu cng ch ra s khc
bit ln v thnh phn sesquiterpene ca ngh en Vit Nam v cc nc
khc [3].
Tm li, ngh en l cy tho dc c cha cc nhm cht nh tinh du
bao gm cc cht thuc sesquiterpene v monosesquiterpene; curcuminoid bao
gm: curcumin, demethoxycurcumin v bisdemethoxycurcumin. Ngoi ra, ngh
en cn cha cc cht nh tinh bt, cht do v mt s cht c v ng khc nh
tannin v flavonoiod [82].

30
1.3.3. Cng dng
Ngoi ngh vng, loi cy c nghin cu lin tc v c xem l loi
tho dc tim nng c nhiu hot cht sinh hc, ngh en cng ang c quan
tm ngy cng nhiu nh cc hot cht mi c xc nh t n [32].
1.3.3.1. Cng dng c truyn
Cy ngh en c trng rng ri dng lm rau hoc gia v cc quc
gia ng, Nam v ng Nam bao gm Trung Quc, Vit Nam, n , Nht
Bn v Thi Lan [137], [58]. T lu, ngh en c con ngi dng trong bi
thuc ng y c truyn cha bnh. Ngh en c trong Dc in XIII ca
ngi Nht Bn v s dng trong thuc c truyn ca ngi Trung Quc. Ngh
en tng c k trong cc n thuc dng cha bnh d dy, iu kinh,
iu tr hi chng Oketsu gy ra do tt ngn mch mu v ci thin kinh
nguyt trong nhiu dng thuc pha ch khc nhau [98], [99], [182]. T lu
Thi Lan, ngh en c dng lm du cn au d dy, chng tiu chy,
chng nn ma v st. N cng c dng ngoi da nh cht lm se cc vt
thng [137]. Ngi n dng c ngh en tr chng vim da, bong
gn, ung nht v vt thng. C ngh en giu tinh bt, c dng nh l ngun
thay th cho tinh bt ca cy hong tinh, la mch v c ch dng lm thc
phm cho tr s sinh, ngi ang dng bnh. Bt mu gi l Abir lm t
c ngh en kh x l vi nc sc cy t mc c dng trong nghi l tn gio
Hindu. N cng c dng sn xut ri, nhiu loi nc hoa, m phm v
cc loi hng liu khc [36]. Ngoi ra, c ngh en cn c dng cha
giun sn tr em; bt ngh dng chng d ng; l ngh c tc dng cha
bnh ph [108], phong hi [68].
1.3.3.2. Cc hot tnh sinh hc
- Hot tnh gim au
Shin v cs (1994) kho st hot tnh dc l ca 2 sesquiterpene:

31
cuzerenone (I) v curcumenol (II) t c ngh en. Kt qu cho thy, c hai cht
ny u th hin hot tnh gim au tng i. Nghin cu khc cng cho thy,
hp cht curcumenol c tc dng gim au cao gp my ln so vi cc loi thuc
gim au thng thng. Dch chit dichloromethane t c ngh en thu c
trong ma thu v ma ng u c tc dng c ch s co tht bng [142].
- Hot tnh khng ung th
Nghin cu ca Hong v cs (2002) cho thy, dch chit ngh en c hot
tnh chng ung th v khng vim [58]. Dch chit bng nc ca c ngh c
hot tnh chng li di cn phi ca cc t bo khi u c tnh B16 [139].
Priosoeryanto v cs (2001) nghin cu kh nng c ch sinh trng cc dng t
bo ung th ca dch chit ethanol v chloroform c ngh en nhn thy, dch
chit chloroform c ch sinh trng t bo myeloma v t bo carcinoma;
dch chit ethanol c ch sinh trng t bo myeloma v t bo carcinoma [126].
Syu v cs (1998) nghin cu nhn thy cc curcuminoid c hot tnh chng cc
t bo ung th bung trng OVCAR-3 ngi [152]. Jang v cs (1997); Hanif
v cs (1997) cng nhn thy, cc curcuminoid phn lp t ngh en c kh
nng c ch sinh trng khi u v gy c cho cc dng t bo ung th rut kt
v ung th biu m gan ngi.
Moon v cs (1985) nghin cu hot tnh khng khi u ca polysaccharide
ngh en. Kt qu cho thy, polysaccharide c ch dng t bo ung th sarcoma
180 vi t l l 61,1% [105]. Theo Kim v Kim (2000), polysaccharide ngh en
lm gim kch thc khi u trn chut c cy t bo sacrom 180 vi t l 52%
v ngn chn t bin nhim sc th [75]. Wang v cs (2004) th hot tnh kch
thch min dch, chng oxy ha v ung th trn chut ca polysaccharide ngh en.
Kt qu nghin cu cho thy, polysaccharide c hot tnh khng ung th, oxy ha
v tng cng min dch [170].
Lee v cs (2002) nghin cu nh hng ca mt s sesquitertene phn lp
t cc cy h Gng ln hot tnh ca cc enzyme: cyclooxygenase (COX-2) v

32
nitric oxide synthase (iNOS) trong nui cy i thc bo chut c kch hot
bi lipopolysaccharide. Cc sesquiterpenoid ca ngh en c kh nng c ch
mnh hot tnh cc enzyme: cyclooxygenase v nitric oxide synthase. Cc tc gi
ny cng cho rng, cc sesquiterpene phn lp t ngh en c th pht trin
thnh cc cht c ch COX-2 v iNOS, sn xut cc tc nhn phng nga ung
th v khng vim [80]. Carvalho v cs (2010) nghin cu nhn thy c s tng
ng k lng t bo hng cu v bch cu tng s; gim s t bo mng bng
v kch thc khi u trn chut c tim dch chit ca ngh en so vi i
chng [27].
- Hot tnh bo v gan
Matsuda v cs (1998) nhn thy dch chit acetone-nc ca c ngh
en c hot tnh bo v gan. Cc sesquiterpene v curcumin bo v chng li s
hnh thnh D-galactosamine/lipopolysaccharide, gim tn thng gan chut
[99]. Kt qu nghin cu ca Kim v cs (2005) cho thy, ngh en th s dng
nh thuc tim nng cho iu tr chng x gan mn tnh [73].
- Hot tnh khng lot
Ngh en c s dng nh l phng thuc ch yu trong vic iu tr
cc ch lot trong h tiu ha. Tc ng ca bt r ngh en ln dch d dy, pH
d dy, acid t do v acid tng s trong h thng tiu ha ca chut c
nghin cu. Kt qu nghin cu ca
Raghuveer v cs (2003) cho thy, dch chit ngh en c kh nng chng
li tnh trng tit nhiu acid v vim lot trong d dy [127]. Watanabe v cs
(1986) nghin cu hot tnh khng lot ca 8 dch chit t ngh en trn
chut gy vim lot d dy cp tnh. Cc hp cht furanogermenone v (4S, 5S)-
(+) germencrone 4,5-epoxide ca tinh du ngh c hot tnh c ch s hnh
thnh vt lot trn chut thc nghim [175].
- Hot tnh khng vim
Jang v cs (2001) nhn thy 3 hp cht 7-bis (4-hydroxyphenyl)-1,4,6-

33
heptatrien-3-one, procurcumenol v epiprocurcumenol t dch chit c ngh en
c hot tnh khng vim bi kh nng c ch s gii phng yu t TNF- i
thc bo chut [63]. Makabe v cs (2006) nghin cu kh nng khng vim ca
cc sesquiterpene phn lp t dch chit ethanol ca c ngh en cho thy, hai
hp cht curzenone v dehydrocurdione c tc dng c ch s hnh thnh cht
12-O-tetradecanoylphorbol-13-acetate (cht to thnh khi chut b vim tai) vi
hiu qu c ch tng ng l 75% v 53% [94]. Yoshioka v cs (1998) dng
cht dehydrocurdione, mt sesquiterpene chit tch t cy ngh en kho st
hot tnh khng vim in vivo v in vitro chut. Kt qu cho thy,
dehydrocurdione, thnh phn chnh ca ngh en c tim nng khng vim i
cng vi tc dng chng oxy ha [182].
- Hot tnh chng oxy ha
Mau v cs (2003) nghin cu hot tnh chng oxy ha ca tinh du ngh
en. nng 20 mg/ml, tinh du ngh en c hot tnh tt trong vic qut gc
t do 1,1-diphenyl-2-picrylhydrazyl [100]. Cc hp cht curcuminoid phn lp
t ngh en c cng b c hot tnh chng oxy ha v khng vim tng
ng vi cc cht ny tch t cy ngh vng [96]. Kt qu nghin cu ca
Paramapojn v cs (2009) chng minh rng, dch chit ethanol c ngh en c
hot tnh qut gc t do. Kh nng qut gc t do ca cc curcuminoid ngh en
cao nht l curcumin ri n demethoxycurcumin v thp nht l
bisdemethoxycurcumin [120].
nc ta, Trn Th Vit Hoa v cs (2007) cng kho st hot tnh
chng oxy ha v nhn thy tinh du ngh en trng Lt nng 20
mg/ml c kh nng chng oxy ha tng i cao t 74,8-77,8%. Cao ether du
ha ca c ngh en c kh nng chng oxy ha cao nht t 61,4-84,5%, vi nng
t 5-20 mg/ml [3].
- Hot tnh khng khun v khng nm
Hot tnh khng cc loi vi khun v nm gy bnh ngi v thc vt

34
ca ngh en c nhiu nghin cu cp. Wilson (2005) kho st
hot tnh khng khun, khng nm ca dch chit t c ngh en trn 6 loi vi
khun v 2 loi nm. Kt qu cho thy, cc loi dch chit ca c ngh en
u c kh nng c ch sinh trng ca cc loi vi khun v nm kim nh
(tr Staphylococcus aureus) [176]. Nghin cu ca Ficker v cs (2003) v kh
nng khng nm gy bnh ngi ca cc loi dch chit t 11 loi cy h
Gng cho thy, dch chit t c ngh en c kh nng chng li cc loi nm
gy bnh ngi, gm nhng loi nm chng chu vi cc cht dit nm ph
bin nh amphotericin B v ketoconazole [43]. Joshi v cs (1989) nghin cu
nhn thy, tinh du ca ngh en cha methyl-p-methoxy-cinnamate l thnh
phn khng cc nm gy bnh ngi [67]. Philip v cs (2009) nghin cu
kh nng khng khun 32 loi dch chit t 8 loi dc liu trong cc bi
thuc c truyn Malaysia trn cc loi vi khun nhn thy dch chit ca c
ngh en c ch sinh trng ca cc loi vi khun Pseudomonas aeruginosa,
S. aureus, Bacillus. subtilis v khng c ch sinh trng ca vi khun E. coli
[124]. Dch chit ngh en Thi Lan c hot tnh khng 3 chng vi khun E.
coli, 2 chng vi khun Salmonella typhimurium, B. cereus, Listeria
monocytogenes, S. aureus, 1 chng vi khun B. subtilis, Vibrio
parahaemolyticus, Yersinia enterocolitica, v 2 chng vi khun Pseudomonas
sp. [155]. Hiu qu khng khun ca dch chit ngh en tng ng vi cc
sn phm khng khun thng mi v s phi hp dch chit ngh en vi cc
sn phm khng khun ci thin hiu qu ca cc sn phm ny [25]. Singh
v cs (2002) nh gi hot tnh khng khun ca tinh du bay hi ca c cy
ngh en nhn thy, tinh du c ch hon ton kh nng sinh trng ca cc
si nm Colletotrichum falcatum [145]. Srvidya v cs (2009) nghin cu hot
tnh khng khun ca dch chit c ngh en. Kt qu cho thy, dch chit
hydro ethanolic ca ngh en c kh nng khng B. cereus v c ch tng
i vi K. pneumonia v C. albicans [149].

35
1.3.4. Tnh hnh nghin cu nui cy in vitro ca cy ngh en
Mello v cs (2001b) thnh cng trong ti sinh chi t callus ngh
en trn mi trng lng c b sung 13,4 mM NAA v 2,2 mM BAP sau 70
ngy nui cy [102]. Miachir v cs (2004) nghin cu nhn ging in vitro
cy ngh en t chi ca thn c [103]. Bharalee v cs (2005) cng
nghin cu nhn ging bng chi c ca cy ngh en, cc cy to r in vitro
c th a ra trng ngoi t [21]. Loc v cs (2005) thnh cng trong nhn
ging in vitro cy ngh en v c hn 95% cy in vitro hnh thnh r sinh
trng tt trong chu [90]. Anisuzzaman v cs (2008) nghin cu to c in
vitro cy ngh en Bangladesh. Chi in vitro khong 10-12 tun tui c
nui cy trn mi trng MS vi nng khc nhau ca BA, NAA v cc
ngun carbon khc nhau. C hnh thnh tt nht trn mi trng c 4,0 mg/L
BA v 6% sucrose sau 7-9 tun nui cy [18]. Stanly v cs (2010) nghin cu
nhn ging in vitro cy ngh en Malaysia thng qua nhn nhanh chi in
vitro trn mi trng rn; nui cy lc trong bnh tam gic v nui cy ngp
chm tm thi [150].
Ngoi cc nghin cu nhn ging in vitro, nghin cu nui cy callus
v huyn ph t bo cy ngh en cng c cng b. Mello v cs (2001a,
b) to callus t on r ca cy ngh en in vitro trn mi trng MS c
3% sucrose, 13,4 M NAA v 2,2 M BAP trong iu kin ti [101], [102].
Theo Miachir v cs (2004), callus khng hnh thnh t m l v r ca cy
ngh en in vitro trn mi trng c 2,4-D. Callus hnh thnh tt nht t on
r trn mi trng MS b sung 1 mg/L NAA v nui cy trong ti [103 ].
Mello v cs (2001a) nghin cu nui cy huyn ph t bo cy ngh en.
Kt qu cho thy, vi 0,5 g t bo trong bnh tam gic 125 ml cha 10 ml
mi trng MS c 3% sucrose, 13,4 M NAA v 2,2 M BAP, tc lc 60
vng/pht thu c khong 6 g sinh khi ti ca t bo sau 35 ngy
nui cy. Kt qu cng cho thy, sucrose l ngun carbon thch hp cho sinh

36
trng ca t bo ngh en, cc ngun carbon khc nh galactose glycerol v
sorbitol khng c vai tr ng k trong kch thch sinh trng ca t bo
[101]. Miachir v cs (2004) cng thit lp nui cy huyn ph t bo cy
ngh trong bnh tam gic 250 ml trn my lc. Nui cy 1 g t bo trong 75
ml mi trng MS c b sung 1 mg/L NAA thu c sinh khi t bo cao
nht l 8 g sau hn 20 ngy nui cy tc lc 100 vng/pht [103].


37
Chng 2
I TNG, NI DUNG
V PHNG PHP NGHIN CU

2.1. I TNG NGHIN CU
i tng nghin cu l cy ngh en (Curcuma zedoaria Roscoe) thuc
chi ngh (Curcuma), h Gng (Zingiberaceae), b Gng (Zingiberales).
Nguyn liu nghin cu l cc t bo callus c to ra t b l ca cy
ngh en in vitro (Hnh 2.1) [90].

Hnh 2.1. Cy ngh en nui cy in vitro
2.2. NI DUNG NGHIN CU
- Nui cy to callus t nguyn liu b l ca cy ngh en in vitro.
- Nui cy huyn ph t bo ngh en trong bnh tam gic 250 ml v
trong h ln men 10 L.
- Kho st s tch ly cc hp cht t nhin nh tinh du, curcumin,
sesquiterpene v polysaccharide trong t bo ngh en nui cy trong h ln
men 10 L.
- Th nghim nh gi hot tnh khng khun ca tinh du t bo ngh
en nui cy trong h ln men 10 L.

38
2.3. PHNG PHP NGHIN CU





Hnh 2.2. S th nghim
Cy ngh en t nhin
Cy ngh en in vitro
Callus c ngun gc t b l
Chn loi callus thch hp cho nui
cy huyn ph
Nui cy huyn ph t bo trong bnh tam gic cc iu
kin v mi trng nui cy khc nhau
Mi trng v iu kin nui cy thch hp
C mu
C mu
iu kin nui cy thch hp
Kho st s tch ly ca tinh du, curcumin,
polysaccharide, v sesquiterpene
Phn tch hot tnh khng khun ca tinh du
Tc lc Ngun carbon Cht HST
Nui cy TB trong h ln men cc iu kin nui
cy khc nhau
Tc khuy Tc sc kh

39
Cc th nghim ca lun n c tin hnh ti Phng th nghim Hp cht
th cp, Vin Ti nguyn, Mi trng v Cng ngh sinh hc, i hc Hu.
2.3.1. Nui cy callus
B l (0,21,0 cm) ca cy ngh en in vitro c nui trn mi trng
MS [107] c 2% sucrose v 0,8% agar, b sung 0,5-4,0 mg/L 2,4-D v 0,5-4,0
mg/L BA to callus. Cc callus s cp hnh thnh t b l (c mu trng, xp)
c ct thnh cc khi nh (ng knh 2-3 mm) cy chuyn ln mi trng MS
c b sung 0,25-4,0 mg/L 2,4-D v 0,25-4,0 mg/L BA pht trin thnh callus
th cp. Cc callus th cp c mu vng, rn v ri rc c tch thnh nhng
khi nh (ng knh 1-2 mm) nui cy trn trn cng mi trng nhn sinh
khi. Callus c cy chuyn 2 tun/ln to ngun nguyn liu nui cy huyn
ph t bo.
2.3.2. Nui cy huyn ph t bo
2.3.2.1. Nui cy huyn ph t bo trong bnh tam gic
Thit lp nui cy huyn ph t bo bng cch cy chuyn 2 g callus (14
ngy tui) vo bnh tam gic 250 ml, cha 50 ml mi trng MS c 2% sucrose, b
sung 2,4-D 0,5 mg/L v BA 0,5 mg/L, nui trn my lc vi tc lc 100
vng/pht. Sau 10 ngy nui cy, 2 g t bo c chuyn sang mi trng mi c
thnh phn tng t v nui trong cng mt iu kin cho n khi thu c dch t
bo huyn ph ng nht.
Sau 3 ln cy chuyn, dch huyn ph t bo ng nht, sinh khi ca t
bo 10 ngy tui c s dng nui cy trong bnh tam gic 250 ml, cha 50 ml
mi trng MS c 2% sucrose, b sung 0,5 mg/L 2,4-D v 0,5 mg/L BA vi cc
iu kin khc nhau nh: c mu 2-5 g, tc lc t 80-180 vng/pht nh
gi nh hng ca iu kin nui cy n kh nng sinh trng ca t bo.
Sau khi kho st mt s iu kin nui cy thch hp cho sinh trng ca t
bo trong bnh tam gic, trn my lc (c mu, tc lc), s dng 3 g sinh khi

40
t bo (10 ngy tui) t th nghim thm d iu kin nui cy thch hp tip tc
nui cy trong bnh tam gic 250 ml, cha 50 ml mi trng MS b sung cc
ngun carbon khc nhau: sucrose, fructose v glucose (nng : 20-70 g/L), v
cc cht HST khc nhau: BA (0,25-2,5 mg/L) v 2,4-D (0,25-2,5 mg/L) dng
ring r hoc phi hp. Nui tc lc 120 vng/pht nh gi nh hng
ca yu t mi trng nui cy n kh nng sinh trng ca t bo.
2.3.2.2. Nui cy huyn ph t bo trong h ln men
100 g sinh khi t bo (10 ngy tui) nui cy trn bnh tam gic 250
ml trong mi trng c bn MS c 3% sucrose, b sung thm BA 0,5 mg/L v
2,4-D 1,5 mg/L c a vo h ln men c tng th tch 14 L, th tch lm
vic 10 L (BioFlo 110, New Brunswick Scientific, USA) cha mi trng MS
lng c 3% sucrose, b sung 2,4-D 1,5 mg/L v BA 0,5 mg/L, c mu 100-
250 g, tc khuy 100-200 vng/pht, tc sc kh 1,5-3,5 l/pht. Sinh
khi t bo c thu 2 ngy mt ln trong sut 18 ngy kho st kh nng
sinh trng ca chng.
Tt c mi trng nui cy c iu chnh pH 5,8 trc khi kh
trng 121
o
C trong 15 pht. Cc th nghim nui cy in vitro c duy tr
nhit 25 2
o
C, iu kin chiu sng khong 2000 lux (cho cc th nghim
nui cy callus) v khong 500 lux (cho cc th nghim nui cy huyn ph
t bo trong bnh tam gic 250 ml v h ln men 10 L), thi gian chiu sng
8-10 gi/ngy.
2.3.3. Xc nh kh nng sinh trng ca t bo
Kh nng sinh trng ca t bo nui cy huyn ph c nh gi thng
qua khi lng ti, khi lng kh, ch s sinh trng ca t bo.
Sinh khi ti ca t bo thu c bng cch lc chn khng dch
huyn ph t bo, sau ra bng nc ct loi b mi trng, cn xc
nh khi lng ti.

41
Sinh khi ti ca t bo c sy kh trong t sy 50
0
C cho n khi
khi lng khng i, cn xc nh khi lng kh ca t bo.
Ch s sinh trng (G
I
) c tnh theo cng thc:
I
F
I
W
W
G
Trong , W
F
: khi lng ti t bo sau mt thi gian nui cy, W
I
: khi
lng ti t bo lc a vo nui cy.
2.3.4. nh lng tinh du
Xc nh tinh du c thc hin theo phng php ca (Manzan v cs
2003) [95]. 4 g kh ca t bo ngh en c nghin mn v cho vo bnh nt
mi cha 50 ml dung mi petrol ether, lc 180 vng/pht 40C trong 6 gi.
Qu trnh chit lp li 3 ln. Sau khi chit, dung dch c c trn my c quay
chn khng (Heidolph, c) loi b dung mi v thu tinh du.
2.3.5. nh lng curcumin
Tch chit curcumin c tin hnh theo phng php ca Paramapojn v
Gritsanapan (2009) [120]. Ha 2 g bt mn ca t bo ngh en trong 20 ml
ethanol 70%. Hn hp c ph bng sng siu m trn my T 700/H (Elma,
c) trong 30 pht 30
o
C. Sau , th ni c lc bng giy Whatman (No.
1), b c chit li vi cng dung mi cho ti khi ht hon ton (dch chit cui
cng l khng mu). Dch chit nhiu ln c trn li v lm giu bng my c
quay chn khng (Heidolph, c) .
Mu sau khi c c ha tan vi ethanol 70% n 10 ml, lc bng mng
Millipore 0,25 m (Sartorius, c) v pha long dch chit 5 ln. 20 l ca dch
chit c a vo my sc k lng hiu nng cao (HPLC) bng Hamilton
syringe. iu kin chy HPLC nhit phng: ct Vertisep GES C18 (5 m;
4,6150 mm), tc chy 1,2 ml/min, thi gian chy 10 pht, detector c
bc sng 420 nm, pha tnh l silica gel (pha ngc) v pha ng l
acetonitrile:acetic acid 2% (50:50, v/v).

42
Phn tch HPLC c thc hin trn my Spectra System (Thermo
Electron, M) bng chng trnh ChromQuest (ver. 4.2.34). Tt c dung mi t
tiu chun phn tch c mua t cc hng Sigma (M) v Merck (c). Ha
tan curcumin chun ( 94%) bng ethanol 70% cc nng t 0,1-1 mg/ml
dng ng chun. Hm lng curcumin (% khi lng kh) ca t bo c
tnh theo phng trnh ng chun y = 10805,46 x (r
2
= 0,966).
2.3.6. nh lng polysaccharide ha tan trong nc
Tch chit polysaccharide c thc hin theo phng php ca Sun v
cs (2005) [151]. 1 g bt mn ca t bo ngh en c chit Soxhlet (Selecta,
Ty Ban Nha) trong 3 gi vi ethanol 80% loi b cht mu, lm bt hot cc
enzyme, v bin tnh cc protein t do. Phn cn c cho vo bnh tam gic 150
ml, thm vo 30 ml nc ct, sau chit trong thit b lm sch bng sng siu
m T 700/H (Elma, c) 60
0
C trong 30 pht. Dch chit c thm vo 40 ml
ethanol 95%, qua m 4
0
C kt ta polysaccharide. Ly tm thu kt ta v
ra sch bng hn hp diethyl ether v acetone (1:1 v/v) thu polysaccharide.
Ha tan kt ta polysaccharide trong 3 ml nc ct, sau thm vo 1 ml
phenol v 7 ml H
2
SO
4
, trong b iu nhit 40
0
C trong 30 pht. Lm lnh n
nhit phng trong 20 pht. So mu trn my quang ph BioMate 3 UV-Vis
(Thermo Spectronic, M) bc sng 485 nm. Mu trng c thnh phn ging
nh mu phn tch nhng khng c polysaccharide. Hm lng polysaccharide
c xc nh da vo ng chun D-glucose (4-20 mg/ml) (Chaplin v cs
1994) [30] v tnh ton theo h s chuyn i sang polysaccharide l 3,168 (Li
v cs 2007) [83]. Phng trnh ng chun D-glucose nh sau: y = 0,0668x vi
R
2
= 0,996.
2.3.7. Xc nh sesquiterpene
Xc nh sesquiterpene theo phng php ca Jang v cs (2004) [64]. Ly
1 g mu bt mn ca t bo ngh en chit trong 3 gi vi dung mi methanol.

43
Qu trnh chit lp li 3 ln. Dch chit trn li, lc v c bng h thng c quay
chn khng (Heidolph, c) cho ti kh. Mu sau c ha tan trong hn
hp ethyl acetate v nc (1:1 v/v). Phn ha tan trong ethyl acetate c c cho
ti kh, sau ha tan trong methanol, lc bng mng Millipore 0,25 m
(Sartorius, c), ly 50 l chy HPLC pha ngc. iu kin chy HPLC
nhit phng nh sau: ct Vertisep GES C18 (5 m, 4,6 150 mm), pha ng
methanol 60%, tc dng 1 ml/min, hp thu quang o bc sng 254 nm.
Phn tch HPLC c thc hin trn my Spectra System (Thermo
Electron, M) bng chng trnh ChromQuest (ver. 4.2.34). Tt c dung mi t
tiu chun phn tch c mua t hng Merck (c).
2.3.8. Xc nh hot tnh khng khun ca tinh du
Hot tnh khng khun ca tinh du t bo ngh en c xc nh theo
phng php khuch tn a thch ca Lehrer v cs (1991) [81]. S dng dch
huyn ph (10
6
- 10
8
CFU/ml) ca cc chng vi khun Bacillus cereus ATCC
11778, Staphylococcus aureus ATCC 6538, v Escherichia coli ATCC 25922
(Bansal, 2003). 100 l dch huyn ph ca mi chng c b sung ln a Petri
cha 20 ml mi trng dinh dng dng rn, dng que cy dn u t bo vi
khun trn mt thch. Khoan 3 l ng knh 6 mm trn a mi trng, sau
cho vo mi l 70 l tinh du (50 mg/ml), dung dch DMSO c dng lm i
chng. t a mi trng trong t lnh khong 8 gi, sau nui trong t m
37
o
C, sau 24 gi xc nh ng knh v khun D - d (D: ng knh vng v
khun; d: ng knh l khoan).
2.3.9. X l thng k
Mi th nghim c b tr lp li 3 ln. S liu thc nghim c tnh
trung bnh mu sai s chun v phn tch Ducans test (p<0,05) bng chng
trnh SAS.


44
Chng 3
KT QU NGHIN CU V THO LUN

3.1. NUI CY CALLUS NGH EN
Callus thng c dng lm ngun nguyn liu khi u thit lp
nui cy huyn ph t bo ca thc vt. V vy, tm ra mi trng dinh dng
thch hp cho nui cy callus l rt cn thit. Bng 3.1 trnh by kt qu to
callus t b l ca cy ngh en in vitro trn cc mi trng khc nhau sau 90
ngy nui.
Bng 3.1. Kh nng to callus t b l ca cy ngh en in vitro
2,4-D
(mg/L)
BA
(mg/L)
% mu vt
to callus
Sinh trng
ca callus
Hnh thi callus
- - - - -
0,5 0,5 15,74
c
++ Trng, mng nc
1,0 1,0 46,01
a
++++ Trng, xp
2,0 2,0 40,10
b
++++ Trng, xp
3,0 3,0 10,20
d
+++ Trng, mng nc
4,0 4,0 8,20
cd
+ Trng, mng nc

Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k p<0,05
(Duncans test).
++++: sinh trng mnh; +++: sinh trng kh; ++: sinh trng trung bnh; +: sinh trng
km; - : khng xut hin callus.

Cc mi trng c b sung BA v 2,4-D u kch thch to callus, trong
khi mi trng khng b sung cht HST li khng to callus. Mi trng c
b sung 1,0 mg/L 2,4-D v 1,0 mg/L BA hoc 2,0 mg/L 2,4-D v 2,0 mg/L BA

45
l thch hp nht (t l to callus t 40,1-46,01%), callus c mu trng, xp v
c kh nng sinh trng tt (Hnh 3.1A). cc mi trng cn li t l to
callus thp (8,2-15,74%), callus c mu trng v mng nc (Hnh 3.1B).







Hnh 3.1. Callus hnh thnh t b l ca cy ngh en in vitro. (A) callus trng v
xp, (B) callus trng v mng nc.

Mello v cs (2001b) to c callus ngh en t on r trn mi
trng MS c 3% sucrose, b sung 13,4 M NAA v 2,2 M BA, nui trong
iu kin ti. Nghin cu ca Miachir v cs (2004) cho thy, callus ngh en
hnh thnh t on r sinh trng trn mi trng MS c b sung 1,0 mg/L
NAA, nhng nu nui cy trn mi trng MS c b sung 2,4 D trong iu kin
ti th callus s khng xut hin [102].
Trong nghin cu ca chng ti, cc HST nh NAA v KIN cng
c thm d khi nui cy b l, tuy nhin chng cho kt qu rt km, callus
sinh trng yu trn mi trng c NAA v ha nu trn mi trng c KIN.
Chng ti cng kho st kh nng hnh thnh callus t on r trn cc mi
trng c b sung 2,4-D, BA, KIN, v NAA. Trn hu ht cc mi trng th
nghim, m r to callus rt km hoc callus c mu trng, mng nc v hi
nhy, sinh trng rt chm.
Trong vic chn dng t bo callus thit lp nui cy huyn ph, cc
dng callus rn v d v vn (friable) thng c la chn lm nguyn liu
B A

46
nui cy huyn ph t bo do chng c kh nng sinh trng nhanh v n nh
trong mi trng lng [165]. Trong nghin cu ca chng ti, callus iu c kh
nng sinh trng mnh trn mi trng MS b sung 1,0 mg/L 2,4-D v 1,0 mg/L
BA hoc 2,0 mg/L 2,4-D v 2,0 mg/L BA. Tuy nhin, cc callus ny c dng
xp v kh v vn nn khng thch hp lm nguyn liu nui cy huyn ph
t bo. V th, cn phi chn cc dng t bo callus thch hp hn thit k
nui cy huyn ph.
Callus s cp c mu trng, xp v sinh trng mnh c cy chuyn ln
mi trng MS c b sung 2,4-D v BA t 0,25-4 mg/L. Kt qu cho thy, cc
callus sinh trng trn mi trng c 2 mg/L 2,4-D v 2 mg/L BA dn dn ha
nu v cht sau khi cy chuyn. Cc callus trn mi trng c 1 mg/L 2,4-D v 1
mg/L BA pht trin thnh dng callus th cp c kh nng sinh trng tt nht
trn mi trng c cha 0,5 mg/L 2,4-D v 0,5 mg/L BA (Bng 3.2). Cc mi
trng cn li khng c tc dng kch thch sinh trng callus (callus sinh
trng km hoc ha nu v cht) sau 4 tun cy chuyn.
Bng 3.2. nh hng ca cht HST ln sinh trng v pht sinh hnh thi ca
callus
2,4-D
(mg/L)
BA
(mg/L)
Sinh trng
ca callus
Hnh thi callus
0,25 0,25 + Trng v mng nc
0,50 0,50 ++++ Vng sng, rn v ri rc
1,00 1,00 ++ Trng v xp
2,00 2,00 ++ Trng v mng nc
4,00 4,00 - Ha nu v cht

Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
++++: sinh trng mnh; +++: sinh trng kh; ++: sinh trng trung bnh; +: sinh
trng km; - : khng sinh trng.

47







Hnh 3.2. Callus c mu vng, rn v ri rc sau 14 ngy nui cy
Cc calus th cp c mu vng sng, rn v d v vn (Hnh 3.2) thu c
trn mi trng MS c b sung 0,5 mg/L 2,4-D v 0,5 mg/L BA sau 4 tun c
cy chuyn 2 tun mt ln ln mi trng c thnh phn tng t nhn sinh
khi callus, to nguyn liu cho nui cy huyn ph t bo trong bnh tam gic.
3.2. NUI CY HUYN PH T BO TRONG BNH TAM GIC
3.2.1. nh hng ca c mu nui cy
Sinh khi t bo (10 ngy tui) nui cy trn mi trng MS c 2%
sucrose, b sung 2,4-D 0,5 mg/L v BA 0,5 mg/L c dng lm nguyn liu
kho st. T bo vi cc c mu ban u khc nhau (2, 3, 4, 5 g t bo) c nui
cy trn mi trng lng c thnh phn ging nh mi trng sinh trng tt
nht ca callus, tc lc 100 vng/pht. Kt qu nh hng ca c mu nui
cy ln sinh trng ca t bo trong khong thi gian t 2 n 18 ngy c trnh
by bng 3.3.
Kt qu cho thy, vi c mu 2 g, sinh khi t cao nht sau 16 ngy l
4,12 g ti (0,37 g kh) v ch s sinh trng l 2,06. Vi 3 g t bo nui cy th
sinh trng ca t bo t cc i ch sau 14 ngy l 5,61 g ti (0,41 g kh) v
ch s sinh trng l 1,87. Khi tng c mu ln 4-5 g, sinh khi t cc i sau
12 ngy, tng ng l 6,12 g ti (0,46 g kh) v 7,22 g ti (0,51 g kh) nhng
ch s sinh trng thp ch t 1,54 v 1,44.

48
Bng 3.3. nh hng ca c mu ln sinh trng ca t bo nui cy huyn ph
trong bnh tam gic
2 g t bo 3 g t bo 4 g t bo 5 g t bo
TN
F
W
D
W
G
I
F
W
D
W
G
I
F
W
D
W
G
I
F
W
D
W
G
I

2 2,22
c
0,18
d
1,11 3,14
d
0,28
c
1,05 4,07
c
0,32
b
1,02 5,20
d
0,39
c
1,04
4 2,52
c
0,24
c
1,26 3,52
c
0,32
c
1,17 4,34
c
0,35
b
1,09 5,32
d
0,42
c
1,06
6 3,00
b
0,24
c
1,50 4,00
c
0,35
b
1,33 4,94
c
0,37
b
1,24 5,61
c
0,44
c
1,12
8 3,30
b
0,30
b
1,65 4,53
b
0,38
b
1,51 5,65
b
0,40
b
1,41 6,09
c
0,47
b
1,22
10 3,50
b
0,33
b
1,75 4,92
b
0,40
a
1,64 6,04
a
0,42
b
1,51 7,02
b
0,48
b
1,40
12 3,70
a
0,35
b
1,85 5,15
b
0,40
a
1,72 6,15
a
0,44
a
1,54 7,22
a
0,51
a
1,44
14 4,00
a
0,35
b
2,00 5,61
a
0,41
a
1,87 6,12
a
0,46
a
1,53 5,68
c
0,41
c
1,14
16 4,12
a
0,37
a
2,06 5,21
b
0,37
b
1,74 5,20
b
0,35
b
1,30 5,00
d
0,32
cd
1,00
18 4,01
a
0.23
c
2,01 4,67
b
0,29
c
1,56 4,56
c
0,27
c
1,14 4,12
e
0,22
d
0,82
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05
TN: thi gian nui cy (ngy); F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
:
ch s sinh trng
Nghin cu cng cho thy, mc d vi c mu nui cy l 2 g, sau 14 ngy
ch s sinh trng ca t bo c cao (2,0), nhng cht lng dch huyn ph t bo
km hn (dch t bo c mu vng nht, t ng nht) so vi khi s dng 3 g t
bo nui cy (dch huyn ph t bo c mu vng m, ti v ng nht). V th,
theo nhn nh ca chng ti, s dng 3 g t bo callus nui cy trong bnh tam
gic c th tch 250 ml cha 50 ml mi trng l thch hp hn c.
Ni chung, xc nh c mu l rt cn thit m bo cho qu trnh sinh
trng din ra hiu qu. Khi mt t bo trong bnh nui cy qu cao hoc qu
thp u lm cho t bo sinh trng km [111]. Mt ban u thp c th dn
n ko di pha sinh trng hm m; trong khi , nu mt t bo ban u
cao hn c th nhanh chng dn n pha t vong (Ling v cs 2008) [84]. nh

49
hng ca c mu nui cy ban u ln sinh trng ca t bo nui cy huyn
ph c kho st trn nhiu loi thc vt khc nhau. Gou v Zhang (2005),
khi nghin cu nui cy huyn ph t bo cy gng nhn thy, nu lng mu
ban u thp hn 0,5% (w/v) th t bo s sinh trng chm, trong khi vi lng
mu cao hn 2,0% (w/v) th t bo s sinh trng nhanh [49]. Ling v cs (2008)
nui cy t bo cy Ficus deltoidea vi c mu l 2,5-10 ml dch t bo huyn
ph, kt qu t bo sinh trung tt nht 10 ml v sinh khi t cc i sau 12
ngy. Nu lng mu thp hn 2,5 ml th pha thch nghi (pha lag) ca t bo s
ko di n ht ngy th 9 [84]. Nagella v Murthy (2010) cng kho st
nh hng ca c mu ban u t 2,5- 20 g/L ln tch ly sinh khi ca t bo
cy Withania somnifera nhn thy, nui cy vi lng mu khong 10 g/L l
thch hp nht cho sinh trng ca t bo [109].
T kt qu kho st nh hng ca c mu ln sinh trng ca t bo
ngh en cho thy, t bo ngh en sinh trng tt vi c mu nui cy ban u
l 3 g trong bnh tam gic th tch 250 ml, cha 50 ml mi trng v sau 14 ngy
sinh khi s t cc i. Trong cc nghin cu tip theo chng ti s dng c
mu nui cy l 3 g v thu hoch sinh khi sau 14 ngy nui cy kho st nh
hng cc iu kin nui cy khc ln sinh trng ca t bo ngh en.
3.2.2. nh hng ca tc lc
T bo c nui cy trn mi trng MS c BA 0,5 mg/L v 2,4-D
0,5 mg/L, sucrose 20 g/L, c mu ban u l 3 g vi tc lc thay i t 80
n 180 vng/pht. Kt qu nghin cu nh hng ca tc lc ln sinh
trng ca t bo sau 14 ngy nui cy c trnh by bng 3.4.
Nhn chung, tc lc nh hng ln sinh trng ca t bo ngh
en. Vi tc lc tng t 80 n 120 vng/pht, sinh khi t bo tng dn
ln v t cc i l 5,91 g ti (0,44 g kh), ch s sinh trng l 1,97 v
dch huyn ph t bo c mu vng sng, ng nht. Khi tng tc lc ln
cao hn (150-180 vng/pht), t bo sinh trng chm li, c bit b c ch

50
mnh tc lc 180 vng/pht, sinh khi cao nht ch l 4,75 g ti (0,35 g
kh), dch huyn ph t bo chuyn sang mu nu sau 14 ngy nui cy.
Bng 3.4. nh hng ca tc lc ln sinh trng ca t bo nui cy huyn
ph trong bnh tam gic
Tc lc (vng/pht) F
W
D
W
G
I

80 5,00
ab
0,34
c
1,67
100 5,61
b
0,41
b
1,87
120 5,91
a
0,44
a
1,97
150 5,60
b
0,42
b
1,87
180 4,75
ab
0,35
c
1,58

Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng
Tc lc nh hng nhiu n sinh trng t bo do lin quan n vic
cung cp oxy v cht dinh dng. Lng oxygen trong mi trng nui cy
ng vai tr quan trng qu trnh trao i cht ca t bo thc vt. Lng oxy
ny ch yu ph thuc vo lp khng kh trn b mt mi trng. Mi trng
lng c trn u s tng tc ha tan ca oxy, ng thi to iu kin d
dng cho t bo hp th cht dinh dng. V vy, tc sinh trng ca t bo
s tng khi tng tc lc n mt gii hn thch hp, tuy nhin khi tc lc
qu cao s c ch sinh trng do t bo b bin dng gy nn hin tng t phn
lm cht t bo (Narayanaswany 1994) [111], (Ziv 2000) [193]. Theo nghin cu
ca Choi v cs (2008), tc lc thch hp cho nui cy huyn ph t bo cy c
du nm trong khong t 120-300 vng/pht. Nu tc lc thp hoc cao hn
(80 v 335 vng/pht) u bt li cho sinh trng ca t bo [33]. Nghin cu
ca chng ti nhn thy, sinh trng ca t bo ngh nui cy trong bnh tam
gic 250 ml cng chu nh hng ng k khi thay i tc lc, t bo sinh
trng tt nht khi nui cy trn my lc tc 120 vng/pht.

51
3.2.3. nh hng ca cht HST
Kho st nh hng ca cht HST ln sinh trng ca t bo cy
ngh en bng cch nui 3 g t bo trn mi trng MS c 20 g/L sucrose, b
sung cc cht 2,4-D v BA cc nng khc nhau, tc lc 120
vng/pht.
3.2.3.1. nh hng ca BA
Kh nng sinh trng ca t bo ngh en trong mi trng c b sung
BA t 0,25-2,5 mg/L sau 14 ngy nui cy c trnh by bng 3.5. Kt qu
nghin cu cho thy, mi trng c b sung BA 0,25 mg/L v 0,5 mg/L c hiu
qu tng ng (p<0,05) trong kch thch sinh trng ca t bo, khi lng
ti ca t bo t t 2,06-2,08 g (0,45-0,46 g kh) v ch s sinh trng xp x
2,1 l cao nht. Trn cc mi trng c nng BA cao hn (1,0 -2,5 mg/L),
sinh trng ca t bo gim nhanh v b c ch mnh nng BA 2,5 mg/L,
sinh khi t bo ch t 4,55 g ti (0,32 g kh), ch s sinh trng l 1,52 sau
14 ngy nui cy.
Bng 3.5. nh hng ca BA ln sinh trng ca t bo nui cy huyn ph
trong bnh tam gic
BA (mg/L) F
W
D
W
G
I

0,25 6,17
a
0,45
a
2,06
0,5 6,25
a
0,46
a
2,08
1,0 5,76
b
0,42
ab
1,92
1,5 5,92
b
0,37
b
1,97
2,0 5,41
bc
0,35
b
1,80
2,5
4,55
bc
0,32
b
1,52
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng

52
3.2.3.2. nh hng ca 2,4-D
Kh nng sinh trng ca t bo ngh en trong mi trng c b sung
2,4-D t 0,25-2,5 mg/L sau 14 ngy nui cy c trnh by bng 3.6. Kt qu
nghin cu cho thy, nng 2,4-D 1,5 mg/L c nh hng tt nht ln sinh
trng ca t bo, khi lng ti ca t bo t 6,75 g (0,52 g kh) v ch s
sinh trng l 2,25, cao hn cc mi trng cn li.
Bng 3.6. nh hng ca 2,4-D ln sinh trng ca t bo nui cy huyn ph
trong bnh tam gic
2,4 D (mg/L) F
W
D
W
G
I

0,25 5,10
c
0,42
b
1,70
0,5 5,77
b
0,43
b
1,92
1,0 6,02
b
0,45
b
2,01
1,5 6,75
a
0,52
a
2,25
2,0 6,04
b
0,46
b
2,01
2,5 5,35
c
0,36
c
1,78

Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng

3.2.3.3. nh hng ca 2,4-D v BA
T bo ngh en c nui cy trn cc mi trng c b sung 2,4-D
0,5 mg/L v BA 0,25-2,5 mg/L; BA 0,5 mg/L v 2,4-D 0,25-2,5 mg/L. Kt
qu sinh trng ca t bo sau 14 ngy nui cy c trnh by bng 3.7.

53
Bng 3.7. nh hng ca 2,4-D v BA ln sinh trng ca t bo nui cy huyn
ph trong bnh tam gic
2,4 D (mg/L) BA (mg/L) F
W
D
W
G
I

0,5 0,25 5,82
b
0,43
b
1,94
0,5 0,5 5,91
b
0,44
b
1,97
0,5 1,0 5,32
c
0,40
c
1,77
0,5 1,5 5,00
c
0,39
c
1,67
0,5 2,0 4,45
cd
0,38
c
1,48
0,5 2,5 4,00
d
0,35
c
1,33
0,25 0,5 5,12
c
0,39
c
1,71
0,5 0,5 5,91
b
0,44
b
1,97
1,0 0,5 6,20
b
0,46
b
2,07
1,5 0,5 7,22
a
0,55
a
2,41
2,0 0,5 6,03
b
0,43
b
2,01
2,5 0,5 6,01
b
0,42
b
2,00
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng
Nhn chung, sinh trng ca t bo trn cc mi trng c b sung ng thi
2,4-D v BA cho kt qu tt hn cc mi trng ch b sung ring r hai cht ny.
Khi lng t bo v ch s sinh trng t cao nht trong mi trng c 2,4-D 1,5
mg/L v BA 0,5 mg/L, tng ng l 7,22 g ti (0,55 g kh) v 2,41. mi trng
ny dch t bo huyn ph c mu vng sng v ng nht. Nhng mi trng cn
li khng thch hp lm cho sinh trng ca t bo, hu ht dch t bo huyn ph
ng sang mu nu v mt s t bo b cht sau 14 ngy nui cy.
Tm li, sau khi kho st trn nhiu mi trng c b sung cht HST
cc nng khc nhau, chng ti nhn thy mi trng MS c b sung 2,4-
D 1,5 mg/L v BA 0,5 mg/L l mi trng nui cy huyn ph t bo ngh
en thch hp nht, sinh khi t bo cao nht l 7,22 g ti (0,55 g kh), gp khong
2,5 ln so vi sinh khi t bo a vo ban u sau 14 ngy nui cy.

54
Kittipongpatana v cs (1998) khi nghin cu nui cy t bo huyn ph cy
Solanum aviculare nhn thy rng t bo sinh trng tt nht trn mi trng
lng MS b sung 4,52 M 2,4-D v 4,44 M BAP, ch s sinh trng ti v
kh cao nht tng ng l 10,38 v 4,43.
3.2.4. nh hng ca ngun carbon
kho st nh hng ca ngun carbon ln sinh trng ca t bo ngh
en, chng ti nui cy 3 g t bo (10 ngy tui) trong mi trng MS c b
sung 2,4-D 1,5 mg/L, BA 0,5 mg/L vi cc nng khc nhau ca sucrose,
glucose v fructose t 20 n 70 g/L , tc lc 120 vng/pht.
3.2.4.1. nh hng ca sucrose
nh hng ca sucrose ln sinh trng ca t bo ngh en sau 14 ngy
nui cy c trnh by bng 3.8.
Bng 3.8. nh hng ca sucrose ln sinh trng ca t bo nui cy huyn ph
trong bnh tam gic
Sucrose (g/L) F
W
D
W
G
I
20 7,22
c
0,55
b
2,41
30 10,44
a
0,66
a
3,48
40 8,85
b
0,64
a
2,95
50 8,80
b
0,65
a
2,93
60 8,75
b
0,70
a
2,92
70 6,75
c
0,60
b
2,25
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng
Kt qu nghin cu cho thy, nng sucrose thp (20 g/L) t bo sinh
trng chm, ch t 7,22 g khi lng ti (0,55 g kh). Sinh trng ca t bo
tng mnh t 10,44 g khi lng ti (0,66 g kh) vi ch s sinh trng l
3,48 trong mi trng c b sung sucrose 30 g/L, dch huyn ph c mu vng

55
ti v kh ng nht (Hnh 3.3). nng sucrose 40-70 g/L, sinh trng ca
t bo gim, c bit l 70 g/L (ch t 6,75 g ti/0,60 g kh).
Sucrose c xem l ngun carbon thch hp nht cho sinh trng ca t
bo thc vt, nng thng dng khong t 20 n 30 g/L (Suzuki v cs
1984). Sucrose va l ngun cung cp nng lng va l mt thnh phn nguyn
liu trong sinh tng hp cc cht th cp. Tc tng trng sinh khi ca t
bo lun lun lin quan trc tip vi s tiu th sucrose (Rao v Ravishankar
2002) [132]. Theo Omar v cs (2004) trn mi trng m tt c cc ngun dinh
dng mc d tha, s gia tng nng sucrose s dn n tng sinh khi kh
[114]. Mt s nghin cu khc nh nui cy t bo cy Solanum eleagnifolium
(Nigra v cs 1990), t bo cy Solanum chrysotrichum (Villarreal v cs 1997)
[168], t bo cy Psoralea corylifolia (Shinde v cs 2009) cng nhn thy t bo
tng sinh khi kh cng vi vic tng nng sucrose. Tuy nhin, khi nng
sucrose qu cao s dn n p sut thm thu vt gii hn cho php ca t bo,
v th nh hng xu ln sinh trng ca chng (Bi Vn L v cs 2006) [5].

Hnh 3.3. Dch huyn ph ca t bo ngh en sau 14 ngy nui cy trong bnh
tam gic trn mi trng c 3% sucrose
Trong nghin cu ca chng ti nng sucrose 30 g/L l thch hp nht
cho sinh trng ca t bo ngh en. Kt qu ny cng tng t vi kt qu nui
cy t bo cy sm Ngc Linh (Panax vietnamensis Ha Grushv.) (Thanh v cs
2007) [157], cy nhn sm (Panax ginseng) (Lian v cs 2002), cy Gynema

56
sylvestre (Lee v cs 2006) [78], v cy Withania somnifera (Nagella v Murthy
2010) [109].
3.2.4.2. nh hng ca glucose
nh hng ca glucose ln sinh trng ca t bo ngh en sau 14 ngy
nui cy c trnh by bng 3.9. Kt qu cho thy, t bo sinh trng tng
i thp khi c nui trong mi trng c glucose 20-30 g/L. nng
glucose t 40-60 g/L, sinh trng ca t bo tng ln v sinh khi t cc i l
7,32 g ti (0,59 g kh), ch s sinh trng l 2,44. Glucose nng 70 g/L
c ch sinh trng ca t bo.
Mt s kt qu nghin cu cho thy, bn cnh ng sucrose, glucose
cng l ngun carbon thch hp thng c s dng trong nui cy m v t
bo ca nhiu loi thc vt (Kato v cs 2007, Sturn v cs 1999, Koch 2004). Tuy
nhin, trong nghin cu ca chng ti, glucose khng phi l ngun carbon thch
hp cho sinh trng ca t bo cy ngh en, kt qu ny cng tng t nh
nui cy t bo cy Ficus deltoidea (Ling v cs 2008) [84] v cy Psoralea
corylifolia (Shinde v cs 2009).
Bng 3.9. nh hng ca glucose ln sinh trng ca t bo nui cy huyn ph
trong bnh tam gic
Glucose (g/L) F
W
D
W
G
I

20 3,15
cd
0,22
c
1,05

30 6,05
c
0,42
bc
2,02

40 7,12
a
0,53
b
2,37

50 7,20
a
0,55
b
2,40

60 7,32
a
0,59
a
2,44

70 6,65
b
0,53
b
2,22
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng

57
3.2.4.3. nh hng ca fructose
nh hng ca fructose ln sinh trng ca t bo ngh en c trnh by
bng 3.10. Kt qu nghin cu cho thy, fructose c nh hng ng k ln sinh
trng ca t bo hn glucose. Khi tng nng fructose trong mi trng t 20-
60 g/L, sinh khi t bo tng theo v t cc i l 8,40 g ti (0,62 g kh). Tuy
nhin, khi fructose b sung ln n 70 g/L th sinh trng ca t bo chm li v
ch cn 7,65 g ti (0,57 g kh).
Bng 3.10. nh hng ca fructose ln sinh trng ca t bo nui cy huyn
ph trong bnh tam gic
Fructose (g/L) F
W
D
W
G
I

20 6,72
b
0,35
c
2,24
30 7.34
b
0,55
b
2,45
40 7,70
ab
0,57
b
2,57
50 7,72
ab
0,59
b
2,57
60 8,40
a
0,62
a
2,80
70 7,65
b
0,57
b
2,55
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
F
W
: khi lng ti (g); D
W
: khi lng kh (g), G
I
: ch s sinh trng
Mc d sucrose v glucose c s dng ph bin trong nui cy t bo
thc vt. Tuy nhin, mi loi li thch hp vi mt ngun carbon khc nhau cn
cho qu trnh hnh thnh cc sn phm trao i cht ca chng [101]. Trong
nui cy t bo thc vt, nhiu loi ng n cng c s dng nh
glucose, fructose, sorbitol, galactose... Abdullah v cs (1998) khi nui cy t
bo cy Morinda elliptica cho thy, fructose 5% gip tng kh nng sinh
trng ca t bo. Felker v cs (1989) khi nui cy t bo cy ng nhn thy,
fructose c vn chuyn nhanh nht, tip n l glucose v sau cng l

58
sucrose. Mt s nghin cu khc cng thu c kt qu tng t, chng hn
t bo ca cc cy Solanum eleagnifolium (Nigra v cs 1990), Ficus deltoide
(Ling v cs 2008) v Tinospora cordifolia [130].
Mc d fructose c tc dng tt cho sinh trng t bo ca nhiu loi
thc vt, tuy nhin nu s dng nng cao n s gy ra c ch chng hn
nh trng hp nui cy t bo cy thuc l v cy Cinchona succirubrum
(Nigra v cs 1990). Kt qu nghin cu ca chng ti cng cho thy, fructose
l ngun carbon kh thch hp cho sinh trng ca t bo cy ngh en, h tr
sinh trng t bo mnh hn glucose nhng thp hn sucrose.
T cc kt qu nghin cu nh hng ca mt s iu kin (c mu, tc
lc) v mi trng (cht HST, ngun carbon) nui cy ln sinh trng ca t bo
cy ngh en, chng ti tin hnh nui cy v thit lp ng cong sinh trng
ca t bo trong iu kin thch hp trn mi trng MS c 30 g/L sucrose, 2,4-D
1,5 mg/L v BA 0,5 mg/L, tc lc 120 vng/pht, c mu 3 g trong 18 ngy
(Hnh 3.4). Sinh khi cc i ca t bo thu c ch sau 14 ngy nui cy l
10,44 g ti; cao gp hn 3 ln so vi lng sinh khi t bo ban u.
0
0.2
0.4
0.6
0.8
0
2
4
6
8
10
12
0 2 4 6 8 10 12 14 16 18
K
h

i

l

n
g

k
h

o

(
g
)
K
h

i

l

n
g

t

i

t

o

(
g
)
Thi gian nui cy (ngy)
Khi lng ti t bo
Khi lng kh t bo

Hnh 3.4. Sinh trng ca t bo ngh en nui cy trong bnh tam gic trn mi
trng MS c 3% sucrose; 0,5 mg/L BA v 1,5 mg/L 2,4-D, lc 120 vng/pht

59







Hnh 3.5. Nui cy t bo ngh en trong bnh tam gic 250 ml t trn my lc
Nghin cu ca Mello v cs (2001a) ngh en cho thy, khi b sung 0,5
g t bo trong 10 ml mi trng MS c 3% sucrose, 13,4 M NAA v 2,2 M
BA, tc lc 60 vng/pht s thu c 6 g sinh khi ti sau 35 ngy [101].
Trong khi Miachir v cs (2004) nui cy 1 g t bo ngh en trong 75 ml mi
trng MS c 1 mg/L NAA, tc lc 100 vng/pht, sau 20 ngy thu c 8 g
sinh khi ti [103]. Mt s nghin cu khc, thi gian t bo sinh trng
cc i di hn t bo ngh en, chng hn nh t bo cy Solanum
eleagnifolium l 26 ngy (Nigra v cs 1989), Solanum aviculare 28 ngy
(Kittipongpatana v cs 1998), Taxus willichiana 32 ngy [112], Solanum
chrysotrichum 22 ngy (Rodrguez-Monroy v cs 2004) [134], Psoralea
corylifolia 20 ngy (Shinde v cs 2009), Solanum hainanense 28 ngy (Loc v
cs 2009), Withania somniera 28 ngy (Nagella v Murthy 2010) [109].
3.3. NUI CY T BO HUYN PH TRONG H LN MEN
3.3.1. Kho st sinh trng ca t bo
Sinh khi t bo (10 ngy tui) nui cy lc trn bnh tam gic 250 ml
trong mi trng c bn MS c 3% sucrose, b sung thm BA 0,5 mg/L v 2,4-
D 1,5 mg/L c s dng lm nguyn liu kho st qu trnh sinh trng ca
t bo ngh en khi nui cy h ln men th tch 14 L, th tch lm vic 10 L
(Hnh 3.6).

60

Hnh 3.6. Nui cy t bo ngh en trong h ln men 10 L
Kt qu nghin cu trnh by hnh 3.7 cho thy, sinh khi t bo t
cc i ngy th 14 vi 241,67 g ti (22,28 g kh), sau bt u gim
dn. Sinh khi ti t bo ch cn li 132,67 g (10,08 g kh) sau 18 ngy nui
cy. Lc ny, quan st thy dch huyn ph t bo t mu vng chuyn sang
nu, nguyn nhn c th lin quan n s xut hin cc sn phm phenol
trong mi trng do qu trnh chuyn ha trao i cht ca t bo to ra.
0
5
10
15
20
25
0
50
100
150
200
250
300
0 2 4 6 8 10 12 14 16 18
K
h

i

l

n
g

k
h

o

(
g
)
K
h

i

l

n
g

t

i

t

o

(
g
)
Thi gian nui cy (ngy)
Khi lng ti t bo
Khi lng kh t bo

Hnh 3.7. Sinh trng ca t bo ngh en trong h ln men nui cy trn mi
trng MS c 3% sucrose; 0,5 mg/L BA; 1,5 mg/L 2,4-D ; khuy 120 vng/pht;
sc kh 2,0 L/pht, c mu 100 g

61
Nh vy, thi gian v ng thi sinh trng ca t bo cy ngh en
nui cy huyn ph trong h ln men 10 L tng t nui cy trong bnh tam
gic 250 ml (Hnh 3.4). Thi gian sinh trng cc i ca t bo ngh en
trong h ln men l 14 ngy, kt qu ny cng tng ng vi sinh trng
ca t bo cy Gymnema sylvestre (15 ngy) (Lee v cs 2006) [78] v
Solanum chrysotrichum (16 ngy) (Rodrguez-Monroy v cs 2004) [134] khi
nui cy trong h ln men.

Hnh 3.8. Sinh khi ti (A) v sinh khi kh (B) ca t bo ngh en sau 14 ngy
nui cy trong h ln men
Da trn kt qu kho st ng cong sinh trng ca t bo ngh en,
chng ti tin hnh kho st nh hng ca mt s iu kin nui cy trong h ln
men (c mu, tc khuy, tc sc kh) ln sinh trng ca t bo sau 14 ngy.
3.3.2. nh hng ca iu kin nui cy
3.3.2.1. C mu
Lng mu a vo nui cy trong h ln men l mt trong nhng yu
t quan trng c th nh hng n s tch ly sinh khi v cc sn phm
trao i cht. Kt qu nghin cu ca chng ti cho thy, c mu nui cy
ban u nh hng r rt n kh nng sinh trng ca t bo. Sau 14
ngy nui cy, kh nng tch ly sinh khi ca t bo tng ln cng vi s

62
tng lng mu nui cy t 100 n 200 g t bo v t cc i l 514,64 g
ti (47,97 g kh). Tuy nhin, khi tng lng mu ln 250 g th sinh trng
ca t bo b c ch do tng mt t bo, v th ch t 441 g ti (38,89 g
kh) (Bng 3.11).
Bng 3.11. nh hng ca c mu ln sinh trng ca t bo nui cy huyn ph trong
h ln men 10 L
C mu (g) Khi lng ti (g) Khi lng kh (g)
100 241,67
c
22,28
d

150 358,33
bc
32,68
c

200 514,67
a
47,97
a

250 441,00
b
38,89
b


Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k p<0,05
(Duncans test).
Lee v cs (2006) khi nui cy huyn ph t bo cy Gymnema sylvestre
cho thy, c mu ban u khong 60 g/L thch hp cho sinh trng t bo v tc
sinh trng s gim nu a vo nui cy 80 hoc 100 g/L [78]. Matsubara
v cs (1989) nghin cu cy Coptis japonica nhn thy lng t bo nui cy
ban u 8 g/L thch hp cho s tch ly sinh khi [97].
3.3.2.2. Tc khuy
Khuy trn c tc dng lm tng tc ha tan ca oxy v cc cht
dinh dng trong mi trng gip t bo sinh trng tt hn. Nghin cu ca
chng ti cho thy, khi tng tc khuy t 100 n 150 vng/pht th sinh
khi t bo cng tng theo v cc i t l 561 g ti (50,84 g kh). Tuy
nhin, khi khuy trn trn 150 vng/pht th sinh trng t bo bt u gim
v ch t c 437 g ti (37,42 g kh) 200 vng/pht (Bng 3.12).


63
Bng 3.12. nh hng ca tc khuy ln sinh trng ca t bo nui cy
trong h ln men 10 L
Tc khuy
(vng/pht)
Khi lng ti (g)
Khi lng kh (g)
100 435,67
c
38,19
c
120 514,67
bc
47,97
b
150 561,00
a
50,84
a
180 547,33
b
50,03
a
200 437,00
c
37,42
c
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
Paek v cs (2005) cho rng, trong qu trnh nui cy huyn ph, thy lc
sinh ra do khuy trn cn phi nh khng gy nguy hi cho m hay t bo
nhng phi ln kch thch cc chc nng ca chng [116]. Nguyn nhn
ca s c ch sinh trng t bo khi nui tc khuy cao c th l do cc t
bo b ph v c hc v gy ra hin tng t phn lm cht t bo (Ziv, 2000)
[193]. Wang v cs (2010) khi nui cy t bo cy Glycyrrhiza inflata trong h
ln men nhn thy sinh khi t bo t cao nht 150 vng/pht; tc
khuy 100 v 300 vng/pht, kh nng sinh trng ca t bo km hn [171].
Nghin cu ca Choi v cs (2008) cho thy, tc khuy 120 v 225 vng/pht
tng sinh khi t bo ca cy c du ln n 200%, nhng khi khuy vi tc
335 vng/pht th sinh khi t bo ch tng 7% [33].
3.3.2.3. nh hng ca tc sc kh
Kt qu ca chng ti cho thy, tc sc kh cng nh hng ln n sinh
trng t bo ngh en. tc sc kh 1,5-2,5 L/pht, sinh trng ca t bo tng
dn v t cc i vi 603 g ti (53,25 g kh). Nu tc sc kh tip tc tng ln
t 3-3,5 L/pht th sinh trng ca t bo s gim. tc sc kh 3,5 L/pht, khi
lng ti ca t bo ch t 441 g (33,17 g kh) (Hnh 3.9 v Bng 3.13).
Hm lng oxy ha tan tan trong mi trng nh hng ln n sinh
trng ca t bo nui cy, khi tc sc kh tng, tc ha tan ca oxy s

64
tng. Tuy nhin, sinh trng ca t bo khng phi lun lun t l thun vi tc
sc kh m n b gii hn trong mt mc nht nh. Vic sc kh v khuy
trn khng ch cung cp oxy cho t bo m cn ngn chn s ng cn sinh khi
t bo. Tuy nhin, nu tc sc kh v tc khuy qu cao s lm cho t bo
b bin dng v ph v thnh t bo. Nhng t bo khi v s gii phng
polysaccharide to thnh bt, kt dnh cc mnh v t bo li vi nhau v bm
ln thnh bnh nui hoc to thnh lp vng trn b mt mi trng. Lp ny
ngn cn s lu thng kh cn thit gy ra s thiu khng kh cho sinh trng t
bo (Ziv 2000) [193]. Ngoi ra, khi vng bt ni ln trn mi trng s cun
theo cc t bo lm cho cc t bo ny khng tip xc vi mi trng dinh
dng v cht do thiu ht dinh dng.
Bng 3.13. nh hng ca tc sc kh ln sinh trng ca t bo nui cy
trong h ln men 10 L
Tc sc kh (l/pht) Khi lng ti (g) Khi lng kh (g)
1,5 489,67
c
36,9
b
2,0 561,00
b
50,84
a
2,5 603,00
a
53,25
a
3,0 583,33
b
50,33
a
3,5 441,00
d
33,17
b
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
nh hng ca s sc kh trong h ln men ln tch ly sinh khi t bo
cng c nghin cu nhiu loi thc vt. Lee v cs (2006) khi nui cy t
bo cy Gymnema sylvestre trong h ln men 2 L cho thy, tc sc kh 0,1
L/L/pht l thch hp nht [78]. Thanh v cs (2005) kho st nh hng ca
nng oxy (20,8-50%) n sinh trng ca t bo cy nhn sm nui cy trong
h ln men 5 L. Kt qu cho thy, nng oxy 40% l thch hp nht cho sn
xut sinh khi v cht ginsenoside, trong khi nng oxy 20,8%, 30% v 50%
l khng thch hp cho sinh trng ca t bo [159]. Nh vy, nhu cu oxy ca

65
mi loi thc vt l khng ging nhau v n ph thuc vo nhiu yu t nh
lng mu nui cy; tc sinh trng; hot tnh sinh l ca t bo; iu kin
mi trng xung quanh nh nng ng, cc cht dinh dng, v s tch ly
cc sn phm c ca qu trnh trao i cht t bo.
0
10
20
30
40
50
60
0
100
200
300
400
500
600
700
0 2 4 6 8 10 12 14 16 18
K
h

i

l

n
g

k
h

o

(
g
)
K
h

i

l

n
g

t

i

t

o

(
g
)
Thi gian nui cy (ngy)
Khi lng ti t bo
Khi lng kh t bo

Hnh 3.9. Sinh trng ca t bo ngh en trong h ln men nui cy trn mi
trng MS c 3% sucrose; 0,5 mg/L BA; 1,5 mg/L 2,4-D ; khuy 150 vng/pht;
sc kh 2,5 L/pht, c mu 200 g
3.4. KHO ST S TCH LY MT S HP CHT C HOT TNH
SINH HC TRONG T BO NGH EN
3.4.1. Hm lng tinh du
Hm lng tinh du ca t bo ngh en nui trong h ln men 10 L c
trnh by bng 3.14. Nhn chung, hm lng tinh du (% khi lng kh) ca
t bo tng dn t 2-14 ngy nui cy v t gi tr cc i l 2,57% cao hn ca
c ngh en t nhin (1,61%) khong 1,6 ln (p<0,05). Hm lng tinh du tch
ly trong t bo gim nhanh sau 16 n 18 ngy; c bit sau 18 ngy nui cy,
hm lng tinh du ch cn 1,09% khi lng kh. iu ny c th gii thch l
do sinh trng ca t bo ngh en sau khi t cc i vo ngy th 14, t bo
c pha sinh trng n nh rt ngn, sau t bo bc nhanh vo pha suy vong

66
sau 16 n 18 ngy nui cy (Hnh 3.9); lc hin tng phn hy (t phn)
ca t bo din ra mnh m dn n kh nng tch ly tinh du trong t bo cng
gim theo rt nhanh.
Bng 3.14. Hm lng tinh du ca t bo ngh en nui cy trong h ln men 10 L
Thi gian nui cy (ngy) Hm lng tinh du (%)
2 1,41
c
4 1,49
c
6 1,57
bc
8 1,98
c
10 2,32
b
12 2,42
b
14 2,57
a
16 1,68
bc
18 1,09
d
MTN 1,61
c
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
MTN: c ca cy ngh en t nhin 01 nm tui
Nghin cu s tch ly tinh du trong t bo thc vt nui cy in vtro
cng c nhiu tc gi cng b. Chng hn, Figueiredo v cs (1995) nui
cy t bo cy Achillea millefolium L. ssp. mille thu c hm lng tinh du
0,002% cao gp 2 ln tinh du c trong cy t nhin, v thnh phn tinh du
cng nhiu hn. Nui cy callus v huyn ph t bo cy Origanum vulgare L.
cho thy, s tch ly tinh du t 0,095% (trong callus) v 0,06% (t bo nui
cy huyn ph) cao hn hm lng tinh du tch ly trong t bo ca cy t
nhin (ch t 0,042%) (Arafeh v cs 2006).
Ngh en l cy tho dc c cha tinh du bao gm cc cht thuc nhm
sesquiterpene v monosesquiterpene [82]. Hm lng tinh du trong c ca cy
ngh en t nhin l 0,22% khi lng ti [1] hoc xp x 1,5% khi lng kh

67
[10]. Mau v cs (2003) nhn thy, tinh du ngh en c hot tnh tt trong vic
qut gc t do 1,1-diphenyl-2-picrylhydrazyl [100]. Nghin cu ca Trn Th
Vit Hoa v cs (2007) nhn thy, tinh du ngh en trng Lt, Vit Nam c
kh nng chng oxy ha tng i cao [3]. Vi kt qu nghin cu ny, chng ti
nhn thy nui cy huyn ph t bo ngh en l mt phng thc trin vng
trong vic thu hi ngun tinh du c gi tr cao t loi tho dc ny.
3.4.2. Hm lng polysaccharide ha tan trong nc tng s
Bng 3.15 trnh by hm lng polysaccharide ha tan trong nc tng s
(% khi lng kh) trong t bo ngh en ti cc thi im nui cy khc nhau.
Nhn chung, hm lng polysaccharide tng t 2 - 10 ngy nui cy v t cc
i l 6,55% thp hn khong 1,4 ln so vi c ngh en (9,46%) (p<0,05). Hm
lng polysaccharide ca t bo gim sau 12 ngy nui cy v gim rt nhanh
ch t khong 1,6-2,01% trng lng kh sau 16 n 18 ngy.
Bng 3.15. Hm lng polysaccharide ha tan trong nc tng s ca t bo ngh
en nui cy trong h ln men 10 L
Thi gian nui cy (ngy) Hm lng polysaccharide (%)
2 1,73
e
4 2,23
d
6 3,53
cd
8 5,15
bc
10 6,55
a
12 5,76
b
14 4,53
c
16 2,01
d
18 1,60
e
MTN 9,46
g
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
MTN: c ca cy ngh en 01 nm tui

68
Trong nhng thp k gn y, cc polysaccharide c ngun t thc vt
c ch c bit trong lnh vc y sinh hc bi chng c nhiu hot tnh sinh
hc tt trong iu tr bnh v khng c tnh c (Schepetkin v cs 2006). Nui
cy t bo thc vt c xem l ngun b sung c hiu qu cc sn phm t
nhin t thc vt, bao gm polysaccharide. Cho n nay, c hn 130 loi thc
vt c s dng trong nui cy t bo sn xut cc polysaccharide c hot
tnh sinh hc (Mahmoudifar v cs 2000). Gunter v cs (2003) nghin cu nui
cy t bo cy Silene vulgaris nhm sn xut cc polysaccharide [50]; Zhang v
Zhong (2002) cng dng h ln men CIB (th tch 30 L) nui cy huyn ph
t bo cy tam tht thu cc cht hp cht nh saponin, polysaccharide vi
hm lng t c ln lt 1,7 v 2,9 g/L [186].
Nhiu nghin cu cho thy, polysaccharide ca cc loi thuc chi ngh
nh C. longa, C kwangsiensis, C. xanhthorrhiza c cc hot tnh qu: tng
cng kh nng min dch, c ch khi u, chng oxi ha(Zeng v cs 2002;
Yuea v cs 2010). cy ngh en, mt s nghin cu cho thy, cc
polysaccharide ca n c kh nng c ch sinh trng ca t bo khi u, ngn
cn t bin nhim sc th, kch thch chc nng ca i thc bo [75], [105],
[169]. Wang v cs (2004) tch chit ca c ngh en t nhin bng sc k ct
v xc nh hm lng polysaccharide tng s l 33,12% [169]. Kt qu nghin
cu ca chng ti th hm lng polysaccharide ha tan trong nc tng s ca t
bo ngh en nui cy in vitro t 6,55% sinh khi kh, sau 10 ngy nui cy l rt
c trin vng trong sn xut cc polysaccharide c hot tnh sinh hc ca ngh en
bng con ng nui cy t bo.
3.4.3. Hm lng curcumin
Da trn kt qu nghin cu sinh trng ca t bo ngh en trong h ln
men 10 L, chng ti kho st s tch ly curcumin ca chng trong qu trnh
nui cy. Kt qu phn tch HPLC c trnh by cc hnh 3.10-3.12 v bng
3.16. Ph HPLC ca curcumin chun c 1 nh (peak) duy nht thi gian lu l

69
2,05 pht. Mt peak c thi gian lu tng ng cng c tm thy dch
chit ca c ngh en t nhin (2,08 pht) v t bo nui cy cc thi gian
khc nhau t 2-18 ngy (1,97-2,09 pht). Phn tch HPLC cng nhn thy, y l
peak c din tch ln nht trong ph HPLC ca t bo in vitro cng nh c ngh
t nhin. Da vo ph HPLC ca curcumin chun, c th xc nh rng, thnh
phn chnh curcuminoid trong dch chit ca t bo ngh den nui cy l
curcumin, ging trong c ngh en t nhin.
Ngoi ra, ph phn tch HPLC dch chit ca c ngh en t nhin cn c
thm 2 peak khc, mt peak c thi gian lu nh peak th hai t bo nui cy
(1,79 pht), peak cn li c thi gian lu l 1,21 pht. Dch chit t bo nui cy
c mt peak th ba rt nh vi thi gian lu khong 1,6 pht. S bin mt ca
peak th ba 1,21 pht ( dch chit c ngh en t nhin) trong t bo in vitro c
th l do s chuyn ha sinh hc ca curcuminoid xy ra trong nui cy t bo.
Bng 3.16. Hm lng curcumin ca t bo ngh en nui cy trong h ln men 10 L
Thi gian nui
cy (ngy)
Thi gian
lu (pht)
Din tch peak
(mAU)

Hm lng
curcumin (%)
2 2,09 1909 0,44
g
4 1,98 2226 0,52
e
6 2,05 2285 0,53
e
8 2,00 2442 0,56
e
10 1,97 4244 0,98
b
12 1,99 4448 1,03
b
14 2,07 5034 1,16
a
16 1,99 3631 0,84
c
18 2,07 2573 0,60
d
MTN 2,06 1868 0,43
g
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
MTN: c ca cy ngh en 1 nm tui

70
Kt qu nghin cu bng 3.16 cho thy, hm lng curcumin (theo %
khi lng kh) tch ly trong t bo tng dn t ngy th 2 n ngy th 14, t
cc i l 1,16%, sau gim t ngy 16-18 cng vi sinh trng ca t bo
(Hnh 3.9). Nhn chung, hm lng curcumin trong t bo cc thi gian nui
cy khc nhau u cao hn trong c ngh en t nhin (0,43%), lc cao nht gp
khong 2,7 ln.
Thng thng, cc hp cht th cp tch ly nhiu nht vo cui giai on
sinh trng cc i. c nhng bng chng r rng cho thy c mi quan h
ngc gia tc sinh trng v kh nng sn xut cc cht th cp. Khi tc
sinh trng cao, cc qu trnh s cp ca t bo l phn chia t bo v sn xut sinh
khi t bo din ra mnh m. Ngc li, trong pha tnh khi s sinh trng gim,
lc ny hot ng sn xut v tch ly cc cht th cp mi tng ln [7]. Nghin cu
qu trnh sinh trng ca t bo ngh en nui trong h ln men cho thy, pha sinh
trng n nh ca t bo rt ngn, sau khi t bo sinh trng cc i th sinh khi
gim ngay (t ngy th 14 n 16) (Hnh 3.9). V vy, hm lng curcumin c tng
hp cao nht vo cui giai on t bo sinh trng cc i (14 ngy) l hp l. Kt qu
ny ca chng ti cng tng t kt qu nghin cu kh nng sn xut jaceosidin ca
t bo S. medusa. Hm lng jaceosidin v sinh khi t bo u t cc i vo ngy
th 14 ca qu trnh nui cy [189].
Ngoi ra, qu trnh tch ly cc hp cht th cp khng ging nhau gia cc
loi, khng phi loi thc vt no cng tng tch ly cc hp cht th cp khi sinh
trng ca t bo i vo pha n nh. Khi nui cy t bo T. cordifolia, Rao v cs
(2008) nhn thy rng trong khi khi lng kh t bo t cc i (9,9 g/L) sau 36
ngy nui cy, th hm lng berberine trong t bo li thu c cao nht vo ngy
24 trong pha log ca t bo [130]. Mt khc, nhiu nghin cu cho thy, t bo
nui cy huyn ph c kh nng tch ly cc hp cht th cp cao hn so vi cy
t nhin, chng hn nh t bo in vitro ca cy Solanum chrysotrichum c hm
lng sprirostanol saponin khong 14 mg/g, cao gp 50 ln so vi hm lng

71
ca n trong l cy t nhin (Villarreal v cs 1997) [168], hoc t bo cy da
cn nui cy in vitro cng c lng vincristin cao hn l t nhin [5].
Nhiu nghin cu chng minh rng, curcumin c kh nng chng
c s pht sinh khi u; ung th rut kt, ung th d dy, ung th v v ung
th bung trng [55], [66], [152]; chng ng mu v h huyt p; chng
vim nhim, khng oxy ha v bo v t bo [152]. Syu v cs (1998) nghin
cu nhn thy, dch chit ethanol ca c ngh en c cha curcumin,
demethoxycurcumin v bisdemethoxycurcumin [152]. Paramapojn v
Gritsanapan (2007) phn tch HPLC thnh phn curcuminoid trong c ngh
en ca Thi Lan cho thy, hm lng curcumin, demethoxycurcumin v
bisdemethoxycurcumin ln lut t 1,46% n 5,73% w/w (trung bnh 2,73%
w/w); t 3,15% n 10,98% w/w (7,37%) v t 0,49% n 2,99% w/w (1,40%
w/w) [119]. Kt qu nghin cu ca chng ti cho thy, curcumin tch ly
trong t bo ngh en nui cy in vitro vi hm lng kh cao (1,16%), iu
ny c kh nng gi t bo ngh en in vitro c trin vng l ngun nguyn
liu thch hp sn xut curcumin, v ch cn trong thi gian ngn khong 2
tun l c th to c nguyn liu sn xut thay v trong t nhin trng
phi mt hng nm mi c.

Hnh 3.10. Ph HPLC ca curcumin chun (0,5 mg/ml)

72

Hnh 3.11. Ph HPLC curcumin ca c ngh en 01 nm tui ngoi t nhin



73


Hnh 3.12. Ph HPLC curcumin ca t
bo ngh en sau 2-18 ngy nui cy
trong h ln men 10 L

3.4.4. Xc nh sesquiterpene
Sesquiterpennoid l cc hp cht C
15
c to thnh bi lin kt ca 3 n
v isoprenoid. Chng c mt trong nhiu t chc sng bao gm thc vt bt cao.
Khong 140 hp cht sesquitertene khc nhau c tch chit t chi Curcuma v
c chia thnh 10 nhm cu trc khc nhau. Tuy nhin, hu ht cc hp cht
ny nm trong 3 nhm chnh l bisabolane, germacrane v guaiane.

74
S phn b ca sesquiterpene trong t bo ngh en c phn tch bng
HPLC. Kt qu cho thy, s hin din ca cc peak phn tch trong c ngh t
nhin nhiu hn trong t bo huyn ph (Hnh 3.13). Nhn chung, cc peak c
thi gian lu lin quan vi cc hp cht sesquiterpene (c nh s t 1-9)
trong c ngh v t bo huyn ph c chiu cao ph hp th (mAU) khc nhau,
c bit l peak s 1 v 4 (Bng 3.17). Chng hn, peak s 1 (thi gian lu 4,7
pht) c hp th l 6,05 mAU trong c ngh nhng ch t 2,55 mAU trong t
bo huyn ph, ngc li peak s 4 (thi gian lu 7,5 pht) t bo huyn ph
c hp th l 7,33 mAU cn c ngh l 2,23 mAU. Mt s peak hin din
trong c ngh (5-8) th khng c hoc ch c dng vt (< 1 mAU) trong t bo
huyn ph. Tuy nhin, t bo huyn ph li xut hin nhng peak mi (khng
nh s) vi cc thi gian lu xp x 6,8; 7,8; 8,2 v 9,2 pht tng ng vi
hp th 1,38-4,10; 5,62-7,54; 1,70-8,41 v 1,06-8,38 mAU. Kt qu ny cho
thy, trong qu trnh nui cy t bo xut hin s chuyn ha sinh hc ca cc
sesquiterpen, to ra nhng hp cht sesquiterpen khc so vi c ngh t nhin.
Bng 3.17. Chiu cao ph hp th (mAU) ca sesquiterpene trong t bo nui cy
h ln men 10 L v t bo c ngh t nhin
Hp cht sesquiterpene (s)/thi gian lu (pht)
Thi gian
nui cy
(ngy) 1/4,7 2/5,1 3/6,0 4/7,5 5/12,7 6/13,8 7/19,2 8/20,5 9/26,7
2 2,11 - 2,49 5,99 - vt vt vt -
4 1,93 - 2,23 5,76 - vt vt vt -
6 1,15 vt 2,81 3,17 - vt - - -
8 1,06 - 1,54 3,57 - - - - -
10 1,22 - 1,02 4,05 - vt vt - -
12 1,04 vt 2,14 3,29 - vt - vt -
14 2,55 1,59 - 7,33 - vt - vt -
16 1,58 - 3,82 4,04 - vt - - -
18 2,36 1,15 1,15 5,11 - vt - vt -
MTN

6,05 2,59 2,15 2,23 6,23 1,46 3,59 1,29 1,19
MTN: c ca cy ngh en 01 nm tui

75



76


Hnh 3.13. Ph HPLC ca sesquiterpene. A: C ngh en t nhin; B: t bo
ngh en nui cy trong h ln men 10 L t 2 n18 ngy
Sakui v cs (1992) nui cy t bo callus ngh en hn 2 nm kho
st s chuyn ha sinh hc ca sesquiterpene v chng minh c s chuyn ha
sesquiterpen mch vng thnh vin s 10 thuc nhm germacrone thnh
sesquiterpene thuc nhm guaiane. Chuyn ha sinh hc trong nui cy in vitro
t bo thc vt cng c thng bo nhiu loi chng hn, Sakamoto v cs
(1994) nui cy t bo ca cc cy Lonicera japonica, Bupleurum falcatum,
Polygonum tinctorium v Solidago altissima nhn thy c s chuyn ha
germecrone thnh guaiane, eudesmane v secoguaiane; Hashimoto v cs (1999)
nui cy t bo cy Heteroscyphus planus nhn thy c s chuyn ha cubenene
thnh 7-hydroxycalamene; Chuyn ha geranyl acetate v linalyl acetate thnh
geraniol, linalool, a-terpineol cng xut hin trong nui cy t bo cy
Peganum harmala [192]. Nh vy, nui cy t bo thc vt c xem l mt
phng thc hiu qu sn xut cc hp cht th cp cng nh cc cht chuyn ha
ca chng (Rao v Ravishankar, 2002). Kt qu nghin cu ca chng ti cho thy
c s tch ly mt s hp cht sesquiterpene trong t bo ngh en nui cy in vitro
ging nh trong c ngh t nhin, ng thi trong t bo cng xut hin mt s
sesquiterpene khc vi c t nhin (c th do chuyn ha sinh hc); kt qu ny c
th gi quan trng trong sn xut sesquiterpene cng nh cc hp cht th cp
mi c hot tnh sinh hc qu thng qua nui cy huyn ph t bo cy ngh en.

77
3.5. HOT TNH KHNG KHUN CA TINH DU T BO NGH EN
Kt qu trnh by bng 3.18 cho thy, tinh du tch chit t t bo v
c ngh en t nhin u c kh nng c ch sinh trng ca c 3 loi vi
khun: Escherichi coli, Staphylococcus aureus v Bacills cereus. Nhn chung,
tinh du thu c t t bo ngh en c kh nng khng khun cao nht th
hin qua ng knh vng c ch (Hnh 3.14). Tinh du t bo nui cy c
ch mnh nht i vi S. aureus (ng knh v khun 31 mm), sau n B.
cereus (22,33 mm) v km nht i vi E. coli (18,33 mm). Trong khi ,
kh nng c ch sinh trng ca tinh du c ngh en t nhin i vi B.
cereus v E. coli l tng ng nhau (16 mm v 17,67 mm), km nht l i
vi S. aureus ch t 14 mm.

Hnh 3.14. Kh nng khng khun ca tinh du ngh en. A: E. coli ATCC 25922.
B: B. cereus ATCC 11778. C: S. aureus ATCC 6538. C: i chng. TN: tinh du c
ngh en t nhin. TB: tinh du ca t bo ngh en


78
Bng 3.18. Kh nng khng khun ca tinh du t bo ngh en
ng knh vng v khun D-d (mm)
Vi khun
i chng Tinh du t bo
Tinh du c ngh
B. cereus ATCC 11778 0 22,33
b
16,00
a
E. coli ATCC 25922 0 18,33
b
17,67
a
S. aureus ATCC 6538 0 31,00
a
14,00
a

i chng: khng c tinh du ngh en
Theo nghin cu ca nhiu tc gi, vi khun Gram m

thng t nhy cm
vi hot tnh ca nhiu loi tinh du hn vi khun Gram dng

(Dorman 2000;
Ruberto v cs 2000; Senatore v cs 2000; Tsigarida v cs 2000). iu ny c th
do s khc nhau v cu trc thnh t bo gia vi khun Gram m v Gram
dng. Vi khun Gram m

c lp mng bao quanh thnh t bo, chnh lp ny
ngn cn s khuch tn ca cc hp cht k nc thng qua lp cht
lipopolysaccharide (LPS). Ngc li, cc vi khun Gram dng khng c lp
mng ny nn tinh du c th tip xc trc tip vi lp LSP thnh t bo vi
khun, do lm tng kh nng thm ca cc ion, s tht thot cc hp cht ni
bo hoc lm suy yu h thng enzyme ca vi khun (Cowan, 1999; Wendakoon
v Sakaguchi, 1995).
Mt s nghin cu ch ra rng, cc hp cht thu c t t bo thc vt
nui cy in vitro c kh nng khng cc loi vi sinh vt. Kt qu nghin cu ca
Par v cs (1991) cho thy, hp cht aurone tch chit t t bo cy
Cephalocereus senilis nui cy huyn ph c kh nng c ch sinh trng ca
cc vi khun Gram m. Robles-Zepeda v cs (2009) cng nhn thy, cc cht
khng vi sinh vt (phytoalexin) tch chit t t bo cy xng rng (Mamillaria
huitzilopochitli) nui cy huyn ph c kh nng c ch sinh trng ca 8 loi
nm gy bnh thc vt.
Trn i tng cy ngh en, c mt s cng trnh nghin cu kh
nng khng khun ca tinh du, tuy nhin vic kho st hot tnh khng khun

79
ch yu t tinh du chit rt c ngh en ngoi t nhin. Srvidya v cs (2009)
nghin cu cho thy, tinh du ca c ngh en n c tc dng c ch mt
s vi khun, trong tc dng c ch sinh trng mnh i vi vi khun B.
cereus nhng tng i vi E. coli v S. aureus [149]. Wilson v cs (2005)
nghin cu tnh khng khun ca tinh du ngh en t nhin Nam n cng
cho thy, n c ch sinh trng nhiu loi vi sinh vt nhng khng c ch S.
aureus v E. coli [176]. Kt qu nghin cu ca chng ti cho thy, tinh du thu
c t t bo ngh en nui cy in vitro c kh nng c ch sinh trng ca c
3 loi vi khun kim nh l S. aureus, B. cereus v E. coli.

80
KTLUNVNGH
KT LUN
Trn c s cc kt qu thu c, chng ti rt ra nhng kt lun sau y:
1. Mi trng c bn MS c 20 g/L sucrose, 0,8% agar, b sung thm BA
0,5 mg/L v 2,4-D 0,5 mg/L thch hp cho nui cy callus t b l cy ngh en
in vitro. Callus c to thnh c mu c mu vng, rn v ri rc c dng
lm nguyn liu thit lp nui cy huyn ph.t bo.
2. Mi trng c bn MS c 30 g/L sucrose, b sung thm BA 0,5 mg/L
v 2,4-D 1,5 mg/L, tc lc 120 vng/pht thch hp cho sinh trng ca t
bo ngh en trong bnh tam gic 250 ml. Sinh khi t cc i l 10,44 g ti
(0,66 g) sau 14 ngy khi nui cy vi c mu l 3 g.
3. Mi trng c bn MS c 30 g/L sucrose, b sung thm BA 0,5
mg/L v 2,4-D 1,5 mg/L, tc khuy 150 vng/pht, tc sc kh 2,5
L/pht thch hp cho sinh trng ca t bo ngh en trong h ln men 10 L.
Sinh khi t cc i l 603 g ti (53,25 g kh) sau 14 ngy nui cy vi
c mu l 200 g.
4. Hm lng tinh du trong t bo t cao nht l 2,57% khi lng kh
sau 14 ngy nui cy, gp khong 1,6 ln tinh du c cy ngh en t nhin 1
nm tui. Hm lng curcumin trong t bo t cao nht l 1,16% sau 14 ngy
nui cy, gp khong 2,7 ln curcumin c cy ngh en t nhin 1 nm tui.
c chuyn ha sinh hc curcuminoid trong nui cy t bo. Hm lng
polysaccharide trong t bo cao nht l 6,55% khi lng kh sau 10 ngy nui
cy, thp hn c ngh t nhin khong 1,4 ln. C s tch ly cc hp cht
sesquiterpene trong t bo nui cy. Thnh phn sesquiterpene hin din trong t
bo huyn ph t hn so vi c cy ngh 1 nm tui. Trong t bo c 3-4 peak
ca sesquiterpene ging nh t bo c ngh. c chuyn ha sinh hc
sesquiterpene trong nui cy t bo.

81
5. Tinh du t bo cy ngh en c kh nng c ch mnh sinh trng
ca 3 chng vi khun B. cereus ATCC 11778 (ng knh v khun 31 mm),
S. aureus ATCC 6538 (22,33 mm) v E. coli ATCC 25922 (18,33 mm). Nhn
chung, kh nng khng khun ca tinh du thu hi t t bo nui cy in vitro
kh cao.
NGH
1. Nghin cu ci thin kh nng tch ly cc hot cht sinh hc trong t
bo ngh en.
2. Nghin cu thnh phn v hot tnh ca curcuminoid, sesquiterpenes,
polysaccharides c sn xut t t bo ngh en.

82
NHNG CNG TRNH C CNG B
LIN QUAN N LUN N

1. Nguyen Hoang Loc, Vo Chau Tuan, Doan Huu Nhat Binh, Truong Thi
Bich Phuong, Tae-Geum Kim and Moon-Sik Yang (2009), Accumulation of
sesquiterpenes and polysaccharides in cells of zedoary (Curcuma zedoaria
Roscoe) cultured in a 10 L bioreactor, Biotechnology and Bioprocess
Engineering 14, pp. 619-624.
2. V Chu Tun, Nguyn Hong Lc (2010), Sn xut curcumin t t
bo ngh en (Curcuma zedoaria Roscoe) nui cy trong h ln men 10 L, Tp
ch Cng ngh Sinh hc, 8(3B), tr. 1459-1464.
3. Nguyn Th Phc Lc, V Chu Tun, Nguyn Hong Lc (2010),
Kho st hot tnh khng khun ca tinh du chit xut t t bo ngh en
(Curcuma zedoaria Roscoe) nui cy trong h ln men 10 L, Tp ch Cng
ngh Sinh hc 8(3B), pp. 1465-1471.
4. Vo Chau Tuan, Vu Duc Hoang, Nguyen Hoang Loc (2011), Cell
suspension culture of zedoary (Curcuma zedoaria Roscoe), VNU Journal of
Science, Natural Sciences and Technology 27, pp. 64-70.






83
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84
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85
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PH LC

Ph lc 1: Mi trng c bn MS (Murashige v Skoog, 1962)
Mui khong Nng (mg/l)
Potasium nitrate (KNO
3
) 1900
Amonium nitrate (NH
4
NO
3
) 1650
Calcium chloride (CaCl
2
.H
2
O) 440
Magnesium sulfate (MgSO
4
.7H
2
O) 370
Potassium phosphate (KH
2
PO
4
) 170
Na
2
EDTA.2H
2
O 37,2
Manganese sulfate (MnSO
4
.4H
2
O) 22,3
Ferrous sulfate (FeSO
4
.7H
2
O) 27,8
Zinc Sulfate (ZnSO
4
.7H
2
O) 8,6
Boric Acid (H
3
BO
3
) 6,2
Potassium Iodine (KI) 0.83
Sodium molybdate (Na
2
MoO
4
.2H
2
O) 0,25
Colbalt chloride (CoCl
2
.6H
2
O) 0,025
Cupric Sulfate (CuSO
4
.5H
2
O) 0,025
Vitamin Nng (mg/l)
Myo-inositol 100
Nicotinic acid 0,5
Pyridoxine HCl 0,5
Thiamine HCl 0,1
Glycine 2
Sucrose 30 g/l
Agar 8 g/l






Ph lc 2: Hnh n h th nghim


Hnh: Nui cy t bo ngh en trong bnh tam gic 250 ml, trn my lc

Hnh: T bo ngh en lng trong bnh tam gic 250 ml

Hnh: Sinh khi ti ca t bo ngh en thu c sau kh nui cy 14 ngy
trong h ln men 10 L