B GIO DC V O TO

B NNG NGHIP V PTNT

HC VIN NNG NGHIP VIT NAM

NGUYN TH THANH

NGHIN CU NHN GING V TO

C IN VITRO CY A HONG
LUN VN THC S
CHUYN NGNH
M NGNH

: CNG NGH SINH HC

: 60.42.02.01

Ngi hng dn 1

PGS.TS.NGUYN TH L ANH

Ngi hng dn 2

TS.NGUYN THANH HI

H NI - 2015

NI DUNG
PHN 1. M U

PHN 2: I TNG, NI DUNG

V PHNG PHP NGHIN CU

PHN 3: KT QU V THO LUN

PHN 4:

KT LUN V KIN NGH

PHN I: M U
1. t vn
Vit Nam nn y hc c truyn rt pht trin, nhiu cy dc liu qu
c s dng trong c cy a hong. Cy c ngun gc t Trung
Quc c khong cch y 2000 nm trc n nm 1958 nhp trng
nc ta, hin nay c pht trin trng nhiu ni t Bc n
Nam.R a hong thu hi v, ra nhanh, phn loi c to, nh ring,
em sy t t n khi c mm ri phi hay sy nhanh n kh. Loi c
to dng ngay (dng sng), tn thuc l sinh a. Dc liu sau khi c
ch bin c tn thuc l thc a.
Theo cc nghin cu gn y, a hong c rt nhiu tc dng tt c y
hc hin i quan tm. Nghin cu cng b, dch chit, c chit
t c a hong ti theo phng php hin i, dch chit ny c tc
dng cm mu, c ch t bo ung th, chng lot d dy, bo v gan,
tng cng min dch cho c th, iu chnh ni tit v chuyn ha.

2. Mc ch v yu cu

Mc ch
Xc nh c yu t k thut lm c s cho vic xy dng quy
trnh nhn nhanh v to c in vitro cy a hong phc v cho
nhu cu sn sut cy a hong nc ta
Yu cu
Xc nh c ch kh trng mu to ngun nguyn liu cho
nui cy
Xc nh c mi trng nui cy nhn nhanh in vitro
Xc nh c mi trng to r cho cc chi in vitro vn m
Xc nh mi trng to c in vitro
Xc nh c gi th v ch dinh dng cho cy in vitro vn
m
nh gi c sinh trng, pht trin v nng sut ca cy ging in
vitro trong iu kin t nhin

 ngha khoa hc, thc tin v tnh mi ca ti

ti nhn ging v to c ca cy a hong l ti u tin nc

ta.
Kt qu ca ti s cung cp thm ngun ti liu cho khoa hc v kh
nng s dng k thut mi trong nhn ging v tnh ca cy a hong
ni ring v cy dc liu ni chung.ng thi n l ti liu tham kho
v ng dng k thut nhn ging nhanh trong trng cy a hong cho
ngi dn trng cy tho dc v cc c s trng cy tho dc.
Kt qu nghin cu ca ti s gp phn tch cc vo hon thin quy
trnh nhn ging v to c ca cy a hong. Khi hon thin c quy
trnh nhn ging v to c cy a hong bng k thut in vitro s to
iu kin ch ng sn xut cy ging a hong vi cht lng tt v
gi thnh cnh pht huy ht tim nng nng sut, cht lng cy a
hong p ng ngun dc liu trong nc v xut khu.

2 I TNG, NI DUNG V PHNG PHP NGHIN CU

2.1 i tng nghin cu

Cy a hong (Rehmannia glutinosa (Gaertn) Libosch )
Ngun mu cy a hong ang sn xut huyn Vit Yn, tnh
Bc Giang
2.2 Vt liu nghin cu
Cc chi mm t c ca cy trng trong iu kin t nhin, cc chi
kch thc 0.8-2.5cm v c 1 n 3 l.
2.3 a im v thi gian nghin cu
a im nghin cu: Phng cng ngh sinh hc thc vt Vin Sinh hc Nng nghip tng 3 nh B Hc Vin Nng Nghip Vit
Nam. Thi gian nghin cu t 02/05/2014 n 20/05/2015

y.

2 I TNG, NI DUNG V PHNG PHP NGHIN

CU

2.4 Ni dung nghin cu

2.4.1 Xc nh ch kh trng ca mu cy
Th nghim 1: Nghin cu nh hng ca HgCl2 0, 1% kt hp
Sodium dichloroisocyanuric axit (NaDCC 25%) n hiu qu kh
trng
2.4.2 Xc nh mi trng nhn nhanh in vitro
Th nghim 2: Nghin cu nh hng ca BA n nhn nhanh
chi in vitro
Th nghim 3: Nghin cu nh hng BA+ -NAA n nhn
nhanh chi in vitro
Th nghim 4: Nghin cu nh hng ca nc da n kh
nng nhn nhanh chi
Th nghim 5: nh hng ca pepton n kh nng nhanh nhanh
chi

2 I TNG, NI DUNG V PHNG PHP NGHIN CU

2.4.3. Nghin cu chng thy tinh ha mu cy

Th nghim 6: Nghin cu nh hng ca cht khng Ethylene
( Thiosunfat Bac ) n s thy tinh ha ca cy.
2.4.4. To cy in vitro hon chnh
Th nghim 7: Nghin cu nh hng ca -NAA n kh nng ra r
ca cy a hong in vitro
Th nghim 8: Nghin cu nh hng ca IBA n kh nng ra r ca
cy a hong in vitro

2 I TNG, NI DUNG V PHNG PHP NGHIN CU

2.4.5 Nghin cu mi trng to c in vitro

Th nghim 9: Nghin cu nh hng ca -NAA n kh
nng to c cy a hong in vitro.
Th nghim 10: Nghin cu nh hng ca hm lng ng
n kh nng to c in vitro ca cy a hong
Th nghim 11: Nghin cu nh hng ca ng v NAA
n kh nng to c in vitro ca cy a hong
2.4.6. Nghin cu xc nh gi th v phn bn l thch hp
cho cy a hong in vitro ti vn m
Th nghim 12: Nghin cu nh hng ca gi th n kh
nng sng v sinh trng ca cy a hong in vitro
Th nghim 13: Nghin cu nh hng ca dinh dng qua l
n sinh trng ca cy in vitro ca vn m

Cc th nghim trong phng c b

tr theo kiu hon ton ngu nhin
CRD,

3 KT QU V THO LUN
3.1.nh hng ca HgCl2 0,1% kt hp Sodium dichloroisocyanuric axit
(NaDCC 0.5%) n hiu qu kh trng.
Bng 3.1. : nh hng ca HgCl2 0,1% kt hp Sodiumdichloroisocyanuric axit
(NaDCC 0.5%) n hiu qu kh trng.

Cng

Thi gian

T l

T l mu

thc

(pht)

mu

sch(%)

T l mu sch

HgCl2

NaDC

nhim

T l mu

T l mu

0,1%

C 0.5%

(%)

sng(%)

cht (%)

CT1

70,2

29,8

80,5

19,5

CT2

10

34,3

65,7

63,2

36,8

CT3

15

21,7

78,3

31,6

68,4

3.2. Nhn nhanh in vitro

3.2.1.nh hng ca BA n nhn nhanh chi
Bng
Bng3.2.
3.2.nh
nhhng
hngca
caBA
BAn
nkh
khnng
nngto
tochi
chiininvitro
vitro(sau
(sau44tun)
tun)
Cng
thc

BA
(mg/l)

T l
Chiu cao S l trung
S chi ti
mu to sinh(chi/mu)
TB
bnh(l/mu
chi(%)
chi(cm)
)

CT1
(C)

10

1,0

3,21

7,43

++

CT2

0,5

100

3,85

3,35

8,32

+ ++

CT3

1,5

100

3,50

4,16

5,85

+ ++

CT4

100

6,87

5,65

7,97

+ ++

CT5

100

2,42

3,47

4,17

+ ++

Ghi ch: (+ +) Chi pht bnh thng

(+ + +) Chi xanh, mp, pht trin cn i, l to xanh m

Cht
lng
chi

Kt lun: mi trng MS b sung 1mg/l BA tt nht cho

kh nng to chi t mu cy trong th nghim ny

CT1:C MS

CT3:MS + 1mg BA

3.2.2. nh hng ca t hp BA v NAA

n kh nng nhn nhanh chi
Bng 3.3. nh hng ca t hp BA v NAA n
kh nng nhn nhanh chi(sau 4 tun nui cy)
Cng thc

BA

NAA

T l mu

H s nhn

Chiu

S l TB

Cht

(mg/l)

(mg/l)

to

(chi/mu)

cao chi

(l/mu)

lng

chi(%)

CT1(C)

TB (cm)

chi

10

0,5

5,76

7,5

++

0,1

100

4,78

2,57

5,27

+ ++

0,3

100

6,53

3,38

6,9

+ ++

0,5

90

3,07

1,94

5,73

+++

0
CT2
CT3
CT4

Ghi ch: (+ +) Chi pht bnh thng

(+ + +) Chi xanh, mp, pht trin cn i, l to xanh m

CT3

CT2

CT1:C

Bng 3.4: nh hng ca nc da n kh nng nhn nhanh chi

Cng thc

CT1(C)

BA
(mg/l)

CT2
CT3
CT4
CV(%)
LSD0.05

Nc
da
(mg/l)

T l
mu to
chi (%)

H s nhn
(chi/mu)

Chiu
cao
chi
TB
(cm)

S l TB
(l/mu)

7,43c

1,95b

10,77a

50

8,00b

1,96b

11,17a

100

8,23b

1,97a
b

11,43a

150

8,77a

2,02a

11,57a

2,31

1,4

4,4

0,35

0,05

0,93

Cht
ln
g
chi

Bng 3.5: nh hng ca pepton

n kh nng nhanh nhanh chi

Cng thc

H s nhn
(chi/mu)

Chiu cao
chi TB (cm)

S l
TB(l/mu)

7.43b

1,95b

10,77b

0,5

6,9c

1,91b

10,77b

1,0

6,9c

1,94b

10,77b

1,5

8,43a

1,84c

11,4ab

2,0

8,77a

2,02a

11,57a

CV(%)

3,1

1,26

3,8

LSD0.05

0,44

0,04

0.76

CT1(
C)

BA
(mg/l)

CT2
CT3
CT4
CT5

Pepton
g/l)

T l mu
to chi (%)

Cht
lng
chi

3.3.3.Th nghim chng thy tinh ha

Bng 4.6: nh hng ca manitol n s thy tinh ha
ca cy
Cng thc

CT1(C)

BA

Manito

(mg/l)

(g/l)

T l mu
to
chi(%)

H s nhn
(chi/mu)

Chiu cao
chi TB
(cm)

T l

S l

cy b

TB(l/mu)

thy
tinh

CT2

CT3

10

CT4

15

KHNG CO TRONG WORD SAO LI C TRONG

POWERPOIN

Cht
lng
chi

3.3.3.Th nghim chng thy tinh ha

Bng 3.6:nh hng ca cht khng Ethylene ( Thiosunfat Bac )

T l

T
Cng thc

BA

Thiosunfat Bac

(mg/l)

(M/l)

H s

mu

nhn

to

(chi/mu)

chi(%)

Chiu

cy

cao

S`l TB

khng

chi TB

(l/mu)

b thy

(cm)

tinh(%
)

CT1(C)

85,5

2,73a

3,30bc

7,50a

4,5

CT2

10

100

2,23b

3,50b

6,70b

22,4

20

93,2

2,80a

3,93a

6,67b

31,2

30

100

2,20b

3,13bc

7,03ab

26,3

CV(%)

4,78

4,93

4,34

LSD0.05

0,22

0,32

0,57

CT3
CT4

Bng 3.7:nh hng ca n tm n s thy tinh ha.

Cng thc

BA

T l mu

H s nhn

Chiu

S l

T l cy b

Cht

(mg/l)

to chi

(chi/mu)

cao chi

TB(l/mu)

Thy tinh

lng

(%)
CT1:n

TB (cm)

chi

thng
CT2: n
tm

KHNG CO TRONG WORD SAO LI C TRONG

POWERPOIN

3.4 .To cy in vitro hon chnh

3.4.1. nh hng ca NAA (Axit -naphtylaxetic) n kh nng
ra r cu cy a hong
NAA (Axit -naphtylaxetic) l cht iu ha sinh trng thuc nhm
auxin. Hp cht ny c tc dng nhiu mt ln qu trnh sinh trng
ca t bo, thc y s phn bo v phn ha t chc c bit l
dng kch thch hnh thnh r nhnh v cc r l, dng tng
nhanh tc ra r. NAA c s dng nng t 0,1mg/l 2,0mg/l v c hiu qu sinh l nng thp.
Trong th nghim ny, chng ti b sung NAA vo mi trng nui
cy vi nng ln lt l 0,5 mg/l, v 1,0 mg/l. Kt qu sau 4 tun
nui cy c th hin bng 5.0

Bng 3.7: nh hng ca NAA n kh nng

to r cho chi a hong (sau 4 tun)

Cng thc

MS0+30g/l
saccaroza
MS0+30g/l
saccaroza
MS0+30g/l
saccaroza
MS0+30g/l
saccaroza
CV(%)
LSD0.05

-NAA
(mg/l)

T l ra r
(%)

Chiu
cao TB
(cm)

S l TB
(l/cy)

S`r
TB(r/cy)

Chiu
di`r TB
(cm)

94,5

3,57c

6,40ab

5,00c

2,23d

0,1

100

3,77c

6,37ab

5,33c

2,57c

0,3

100

5,47a

6,57a

6,30a

3,57a

0,5

100

4,27b

6,13b

5,77b

3,13b

4,74
0,38

2,60
0,31

3,30
0,35

5,59
0,30

CT1

CT3

3.4.2. nh hng ca IBA n kh nng ra r

cu cy a hong
Bng 3.8: nh hng ca IBA n kh nng to
r cho chi a hong (sau 4 tun)

Cng thc

IBA (mg/l)

CT1 (C)
CT2
CT3
CT4
CV(%)
LSD0.05

0
0,1
0,3
0,5

Chiu
T l ra
cao TB
r (%)
(cm)

90,20
95,74
100
100

3,53b
3,80b
4,77a
3,70b
4,13
0,31

S l
trung bnh
(l/cy)

S r TB
(r/cy)

Chiu di
r TB (cm)

6,50b
6,47b
7,27a
5,87c
2,76
0,34

2,30c
3,67b
4,53a
4,77a
4,72
0,34

3,47d
4,63c
5,90a
5,43b
3,25
0,30

3.4.3. nh hng ca -NAA n kh nng to c in vitro

cy a hong

Bng 3.9: nh hng ca -NAA n kh nng to c in vitro cy a hong (Sau 4 tun nu

Cng thc

MS0 + 30g /l
saccaroza + 0mg/l
-NAA
MS0 + 30g /l
saccaroza +0,5
mg/l -NAA
MS0 + 30g /l
saccaroza 1mg/l NAA
MS0 + 30g /l
saccaroza 1,5
mg/l -NAA
MS0 + 30g /l
saccaroza 2 mg/l
-NAA
CV(%)

T l ra r Chiu cao
(%)
TB (cm)

S l
trung
bnh
(l/cy)

S c TB
(c/cy)

Chiu
di c
TB (cm)

Khi lng
TB c(g)
Khi lng
ti

100

3,10c

5,33b

3,20d

2,13d

0,87c

100

3,40b

5,57b

3,70c

2,60c

1,23b

100

4,53a

5,60b

4,27b

3,63a

1,33b

100

4,43a

5,93a

4,60a

3,20b

1,54a

100

3,53b

5,47b

3,53c

2,80c

1,22b

3,11

2,81

3,60

4,02

4,66

3.4.4. nh hng ca hm lng ng n kh nng

to c in vitro ca cy a hong
Bng 3.10: nh hng ca hm lng ng n kh nng to c
in vitro ca cy a hong (sau 6 tun theo di)

Cng thc

Chiu
cao TB
(cm)

S l
trung
bnh
(l/cy)

S c TB
(c/cy)

Chiu di
c TB
(cm)

Khi lng TB c
(g)
Khi
lng
ti

Khi
lng
kh

MS0 + 30g /l saccaroza 2,87b 7,13b

5,50b

2,50b

1,12c

0,19b

MS0 + 60g /l saccaroza 3,27a 7,67a

5,90a

2,77a

1,54a

0,26a

MS0 + 90g /l saccaroza 2,93b 7,20b

5,63ab

2,70ab

1,34b

0,20b

CV(%)
LSD0.05

2,75
0,31

3,97
0,21

1,66
0,04

5,37
0,02

3,82
0,23

1,87
0,27

3.4.5. nh hng ca t hp ng v -NAA n kh nng

to c in vitro cy a hong
Bng 3.11: nh hng ca t hp ng v -NAA n kh nng to c in vitro
cy a hong. (Sau 6 tun theo di).

Cng thc

MS0 + 60g /l saccaroza +

0mg/l -NAA
MS0 + 60g /l saccaroza
+0,1 mg/l -NAA
MS0 + 60g /l saccaroza
+0,3 mg/l -NAA
MS0 + 60g /l saccaroza
+0,5 mg/l -NAA
CV(%)
LSD0.05

S c
Chiu
TB(c/cy di c
)
TB (cm)

Chiu cao
TB (cm)

S l trung
bnh(l/cy)

3,33d

5,37b

3,10d

3,60c

5,70a

4,83a

Khi lng TB
c(g)
Khi
lng
ti

Khi
lng
kh

2,30d

0,93d

0,10d

3,57c

2,70c

1,33c

0,17c

5,67a

4,17b

3,77a

1,60a

0,24a

4,53b

5,87a

4,70a

3,37b

1,47b

0,22b

2,45

1,98

2,78

3,56

5,30

5,10

1,19

0,21

0,20

0,20

0.13

0,18

3.5. Xc nh gi th v dinh dng thch hp cho cy in vitro ti vn m

3.5.1. nh hng ca gi th n kh nng sng v sinh trng ca cy in vitro
Bng 3.12 nh hng ca gi th n kh nng sng v sinh trng ca cy in vitro
(sau 4 tun theo di)
Thi gian hi
xanh sau trng

T l sng

Chiu cao TB

S l trung

(ngy)

(%)

(cm)

bnh(l/cy)

t ph sa

86,67

2,75b

7,37b

t ph sa + tru hun (1:1)

93,33

2,98a

8,13a

t ph sa + mn da ( 1:1)

92,00

2,94a

8,20a

Mn da + tru hun (1:1)

96,67

3,01a

8,37a

CV(%)

2,82

4,37

LSD0.05

0,16

0,66

Cng thc

3.5.2. nh hng ca dinh dng qua l n sinh trng ca cy in v

ngoi vn m
Bng 3.13: nh hng ca dinh dng qua l n sinh trng ca cy in vitro
ngoi vn m (sau 4 tun theo di)
Thi gian hi
xanh sau

T l sng

Chiu cao

S l trung

trng (ngy)

(%)

TB (cm)

bnh(l/cy)

C( phun nc l)

93,33

13,77c

6,03d

Yogen 30-10-10

96,67

13,50b

7,17b

Atonik

96,67

17,47a

7,73a

u tru 502

93,33

14,40c

6,73c

CV(%)

2,35

2,77

LSD0.05

0,72

0,38

Cng thc

4. KT LUN V NGH
4.1. Kt lun
i vi mu chi a hong, cht kh trng thch hp l HgCl 2 0,1% thi gian kh trng l 5
pht kt hp NaDDC 10 pht vi t l mu sch sng l 63,2 %.
- Mi trng nui cy khi ng thch hp to chi t mu cy ban u mi trng MS
b sung 1mg/l BA tt nht cho kh nng nhn nhanh chi invitro cy a hong.
- Vi mi trng MS b sung 1mg/l BA kt hp vi 15% nc da cho h s nhn chi
cao nht t 5,43 chi/mu.
- Cht khng Ethylene ( Thiosunfat Bc ) vi hm lng 20 M/l khi cho MS + 1BA c h
s nhn chi cao nht l 93,2%, chi khng b thy tinh l 31,5%
- Gi th gim cy thch hp nht cho cy a hong invitro l gi th tru hun + mn da
1:1. Trn gi th ny t l sng t 96,67%, chiu cao cy 3,01 cm, s l 8,37 l/cy.
- S dng phn bn l atonik thch hp cho s sinh trng cy a hong invitro ngoi
vn m. Kt qu so vi i chng th khi phun phun bn l Atonik chiu cao t
17,47cm.

4.2. ngh:
Tip tc nghin cu hon thin quy trnh to c invitro cy a hong, nghin cu cc bin
php chng hin tng thy tinh ha cy a hong invitro.