Nghiên Cứu Nhân Nhanh Loài Lan Kim Tuyến (Anoectochilus Roxburghii (Wall.) Lindl.) Của Việt Nam Bằng Phương Pháp Nuôi Cấy Mô Tế Bào

B GIO DC VO O TO
B NNG NGHIP V PTNT
VIN KHOA HC NNG NGHIP VIT NAM
-----------------------
------------------------
NGUYN TH LI
NGHIN CU NHN NHANH LOI LAN KIM

TUYN (ANOECTOCHILUS ROXBURGHII (WALL.)
LINDL.) CA VIT NAM BNG PHNG PHP
NUI CY M T BO
LUN VN THC S NNG NGHIP
H NI 2012
B GIO DC VO O TO B NNG NGHIP V PTNT

VIN KHOA HC NNG NGHIP VIT NAM
-----------------------
------------------------
NGUYN TH LI
NGHIN CU NHN NHANH LOI LAN KIM

TUYN (ANOECTOCHILUS ROXBURGHII (WALL.)
LINDL.) CA VIT NAM BNG PHNG PHP
NUI CY M T BO
Chuyn ngnh: Khoa hc cy trng
M s: 60620110
LUN VN THC S NNG NGHIP

Ngi hng dn khoa hc:
TS. Phm Hng Sn
H NI 2012
LI CAM OAN
Ti xin cam oan y l cng trnh nghin cu ca ring ti. Cc s

liu, kt qu nu trong lun vn l trung thc v cha tng c cng b
trong bt k cng trnh no khc, cc thng tin trch dn trong lun vn ny
u c ch r ngun gc.
H Ni, ngy
thng
nm 2012
Tc gi lun vn
Nguyn Th Li
Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip
LI CM N
c c kt qu ny, ti xin trn trng by t lng bit n su sc
nht n TS. Phm Hng Sn ngi tn tnh du dt, hng dn v to
mi iu kin gip ti hon thnh cng trnh nghin cu ny.
Ti xin chn thnh cm n cc thy c gio Ban o to sau i hc,
Vin Khoa hc Nng nghip Vit Nam to iu kin gip ti hon
thnh lun vn.
Ti xin chn thnh cm n s gip ca gia nh v cc bn b ng
nghip Trung tm Sinh hc Thc nghim, Vin ng dng Cng ngh cho
ti ng lc v to iu kin trong qu trnh hc tp v nghin cu.
H Ni, ngy
thng
nm 2012
Tc gi
Nguyn Th Li
ii
MC LC
M U .................................................................................................... 1
1. Tnh cp thit ca ti .......................................................................... 1
2. Mc ch v yu cu ca ti ............................................................... 3
3. ngha khoa hc v thc tin ca ti ................................................. 4
4. i tng v phm vi nghin cu ca ti ........................................... 5
CHNG 1 - TNG QUAN TI LIU V C S KHOA HC CA
TI....................................................................................... 6
1.1. C s khoa hc ca phng php nui cy m t bo thc vt............. 6
1.1.1. Tnh ton nng ca t bo thc vt................................................. 6
1.1.2. S phn ho v phn phn ho t bo ............................................ 6
1.1.3. Cc nhn t nh hng ti qu trnh nhn ging bng nui cy m
t bo....................................................................................................... 8
1.1.3.1. Mi trng nui cy ................................................................ 8
1.1.3.2. Cc cht iu ho sinh trng................................................ 11
1.1.3.3. Mi trng vt l................................................................... 13
1.1.3.4. Vt liu nui cy.................................................................... 14
1.1.3.5. iu kin v trng ................................................................. 14
1.1.4. Cc giai on chnh trong qu trnh nhn ging nui cy in vitro. 15
1.2. Mt vi nt v Lan kim tuyn............................................................. 17
1.2.1. Phn loi ...................................................................................... 17
1.2.2. c im hnh thi....................................................................... 17
1.2.3. Sinh hc v sinh thi .................................................................... 18
1.2.4. Phn b........................................................................................ 19
1.2.5. Gi tr dc liu ca A. roxburghii (Wall.) Lindl. ........................ 19
1.2.5.1. Hot tnh sinh hc ca A. roxburghii (Wall.) Lindl. ............... 19
1.2.5.2. Tc dng v cng dng ca A. roxburghii (Wall.) Lindl. ....... 22
1.2.6. Thc trng khai thc v pht trin cy Lan A. roxburghii (Wall.)
Lindl.).................................................................................................... 23
iii
1.3. Nui trng Lan kim tuyn .................................................................. 23

1.3.1. nh sng...................................................................................... 23
1.3.2. Nhit ....................................................................................... 24
1.3.3. m .......................................................................................... 24
1.3.4. Ngun nc ti.......................................................................... 24
1.3.5. Phn bn ...................................................................................... 24
1.4. Mt s nghin cu v nhn ging v bo tn cy Lan kim tuyn ....... 24
1.4.1. Nghin cu nhn ging v bo tn ngun gen cy Lan kim tuyn
trn th gii ............................................................................................ 25
Vit Nam ............................................................................................... 29
CHNG 2 - VT LIU, NI DUNG V PHNG PHP NGHIN
CU ............................................................................... 31
2.1. Vt liu nghin cu ............................................................................ 31
2.1.1. Ging nghin cu......................................................................... 31
2.1.2. Ho cht v thit b nui cy m .................................................. 31
2.1.3. Cc gi th s dng trong nghin cu .......................................... 32
2.1.4. Cc loi phn bn s dng ........................................................... 33
2.2. Ni dung nghin cu .......................................................................... 34
2.3. Phng php nghin cu.................................................................... 34
2.3.1. Phng php ly mu, kh trng v nui cy to vt liu khi u
Lan A. roxburghii (Wall.) Lindl. ............................................................... 34
2.3.2. Phng php nghin cu nhn nhanh chi cy Lan A. roxburghii
(Wall.) Lindl.......................................................................................... 36
2.3.3. Phng php nghin cu to cy Lan A. roxburghii (Wall.) Lindl.
hon chnh ............................................................................................. 39
2.3.4. Phng php a cy Lan A. roxburghii (Wall.) Lindl. ra vn
m ....................................................................................................... 40
2.4. Cc ch tiu theo di........................................................................... 42
iv
2.5. Phng php x l s liu.................................................................. 43

CHNG 3 - KT QU NGHIN CU V THO LUN................... 44
3.1. Kt qu nghin cu kh trng to vt liu khi u cho nui cy in
vitro loi Lan A. roxburghii (Wall.) Lindl. ................................................ 44
3.1.1. Kt qu nghin cu nh hng ca cht kh trng ti t l sng
ca mu cy trn mi trng MS........................................................... 44
3.1.2. nh hng ca mi trng khong n kh nng to chi t mu
cy ban u............................................................................................ 46
3.1.3. nh hng ca cht iu tit sinh trng BA n kh nng to chi
t mu cy ban u................................................................................ 49
3.2. Giai on nhn nhanh chi................................................................. 51
3.2.1. nh hng ca Kinetin (Kin) n kh nng nhn nhanh chi Lan
A. roxburghii (Wall.) Lindl.................................................................... 51
3.2.2. Nghin cu nh hng ca Thidiazuron (TDZ) n qu trnh nhn
nhanh chi Lan A. roxburghii (Wall.) Lindl........................................... 54
3.2.3. Nghin cu nh hng ca t hp (TDZ + IBA) n kh nng nhn
nhanh chi Lan A. roxburghii (Wall.) Lindl........................................... 56
3.2.4. Nghin cu nh hng ca t hp (BA + NAA) n kh nng
nhn nhanh chi Lan A. roxburghii (Wall.) Lindl. ................................. 59
3.2.5. Nghin cu nh hng ca dch nghin khoai ty n kh nng
nhn nhanh chi Lan A. roxburghii (Wall.) Lindl. ................................. 61
3.3. Giai on to cy hon chnh in vitro ................................................. 65
3.3.1. nh hng ca than hot tnh ...................................................... 65
3.3.2. nh hng ca NAA ti giai on to cy hon chnh .............. 67
3.4. Giai on a cy in vitro ra vn m ............................................. 70
3.4.1. nh hng ca gi th trng khc nhau n s sinh trng pht
trin ca cy Lan A. roxburghii (Wall.) Lindl. ....................................... 71
3.4.2. nh hng ca mt s loi phn bn khc nhau n s sinh trng
pht trin cy Lan A. roxburghii (Wall.) Lindl....................................... 73
KT LUN V NGH ......................................................................... 80

1. Kt lun ................................................................................................ 80
2. ngh ................................................................................................. 81
TI LIU THAM KHO ........................................................................... 82
Ting vit.................................................................................................. 82
Ting Anh................................................................................................. 82
PH LC ................................................................................................... 87
vi
DANH MC CC K HIU, CC CH VIT TT

2,4-D:
2,4-dichlorophenoxyacetic acid
BA:
Benzyladenine acid
CITES:
Cng c v bun bn quc t nhng loi ng, thc vt hoang

d nguy cp (Convention on International Trade in Endangered
Species of Wild Fauna and Flora)
CT:
Cng thc
CTTN:
Cng thc th nghim
CV% :
H s bin ng (Correlation of Variants)
/C:
i chng
IAA:
Indol-3-acetic acid
IBA:
Indolbutyride acid
NAA :
- Naphthalene acetic acid
Kin:
Kinetin (6-furfuryl-aminopurine)
LSD:
So snh theo gi tr khc bit c ngha nh nht mc 0,05

(Least Significant Difference)
MS:
Murashige and Skoog
RE:
Robert Ernst
TB:
Trung bnh
TDZ:
Thidiazuron (N-phenyl-N,-1,2,3-thiadiazol-5-ylurea)
VW:
Vacin and Went
WHO:
T chc y t th gii (World Health Organization)
WWF:
Qu Quc t Bo v Thin nhin (World Wide Fund For

Nature)
vii
DANH MC CC BNG
Bng 2.1. Nghin cu nh hng ca cht kh trng H2O2 v NaOCl......... 35
Bng 2.2. nh hng ca mi trng khong khc nhau n kh nng to
chi t mu cy ban u ............................................................. 35
Bng 3.1. nh hng ca cht kh trng H2O2 ln mu cy Lan A. roxburghii
(Wall.) Lindl............................................................................... 44
Bng 3.2. nh hng ca cht kh trng NaOCl ln mu cy Lan A.
roxburghii (Wall.) Lindl. ............................................................ 46
Bng 3.3. nh hng ca mi trng khong khc nhau n kh nng to
chi t mu cy ban u ............................................................. 47
Bng 3.4. nh hng ca cht iu tit sinh trng BA n kh nng to
chi t mu cy ban u ............................................................. 50
Bng 3.5. nh hng ca Kin n kh nng nhn nhanh chi Lan A.
Bng 3.6. nh hng ca TDZ n kh nng nhn nhanh chi ca Lan A.
Bng 3.7. Kt qu nh hng ca t hp (TDZ + IBA) n kh nng nhn
nhanh chi Lan A. roxburghii (Wall.) Lindl.............................. 57
Bng 3.8. nh hng ca t hp (BA + NAA) n kh nng nhn nhanh
chi Lan A. roxburghii (Wall.) Lindl. ....................................... 60
Bng 3.9. Nghin cu nh hng ca dch nghin khoai ty n kh nng
nhn nhanh chi Lan A. roxburghii (Wall.) Lindl. .................... 62
Bng 3.10. nh hng ca than hot tnh ti giai on to cy hon chnh . 66
Bng 3.11. nh hng ca NAA ti giai on to cy hon chnh ............ 68
Bng 3.12. nh hng ca gi th trng khc nhau n s sinh trng pht
trin ca cy Lan A. roxburghii (Wall.) Lindl. (sau 12 tun)..... 72
Bng 3.13. nh hng ca mt s loi phn bn khc nhau n s sinh
trng pht trin ca cy Lan A. roxburghii (Wall.) Lindl. (sau 12
tun) ........................................................................................... 74
viii
DANH MC CC HNH
Hnh 1.1. Cy Lan kim tuyn (A. roxburghii (Wall.) Lindl.) ....................... 18
Hnh 2.1. Cy Lan kim tuyn (A. roxburghii (Wall.) Lindl.) c a vo
nui cy m ................................................................................ 31
Hnh 2.2. Ru Sphagnum moss.................................................................... 32
Hnh 2.3. Dn t qu ................................................................................... 32
Hnh 2.4. HVP 1601 WP N: P: K = 30: 10: 10 ............................................ 33
Hnh 2.5. Growmore (M) N: P: K = 30: 10: 10 .......................................... 33
Hnh 2.6. B1 Thi Lan ................................................................................. 33
Hnh 3.1. nh hng ca mi trng khong khc nhau n kh nng to
chi t mu cy ban u ............................................................ 48
Hnh 3.2. Giai on nhn nhanh chi Lan A. roxburghii (Wall.) Lindl. ....... 64
Hnh 3.3. To cy hon chnh in vitro.......................................................... 70
Hnh 3.4. nh hng ca gi th trng khc nhau n t l sng ca cy Lan
A. roxburghii (Wall.) Lindl. (sau 12 tun)................................... 72
Hnh 3.5. nh hng ca mt s loi phn bn n s r mi xut hin ca
cy Lan A. roxburghii (Wall.) Lindl. (sau 12 tun) ..................... 75
Hnh 3.6. Cy Lan A. roxburghii (Wall.) Lindl. nui cy m sinh trng pht
trin vn m. ....................................................................... 76
ix
M U
1. Tnh cp thit ca ti
Theo t chc Y t th gii, hin nay khong 80% dn s ton cu s
dng cc loi tho dc truyn thng bo v sc khe. Phong tro dng
cy thuc phng v cha bnh trn th gii t ra mt vn cn quan
tm l 2/3 trong s 50.000 loi cy thuc c s dng l khai thc t cy
hoang di m khng h c trng b sung. Theo mt nghin cu ca nh
thc vt hc ngi Anh, Alan Hamilton, thnh vin ca qu Quc t Bo v
Thin nhin (WWF) c ti 4.000 10.000 loi cy c lm thuc c nguy c
b tuyt chng. Nguyn nhn khng phi hon ton do s pht trin ca y hc
c truyn m theo tc gi l do th trng dc tho Chu u v Bc M
tng 10% mi nm trong vng 10 nm nay. Trn quy m ton cu, doanh thu
mua bn cy thuc hng nm c tnh ln ti 16 t Euro. Trung Quc, mi
nm c khong 700.000 tn dc liu c a vo sn xut 6.266 mt hng,
mang li doanh thu khong 17,57 t USD; vic bun bn dc liu cng l
mt ngun thu ln ca n , mt hng ny em v cho quc gia ny trn 60
t rupi, cung cp 12% nhu cu th gii.
Theo Pawar V. M. v Puri S. N (2005) th trng tho dc tip tc
tng trng vi tc 15% /nm. Theo c tnh ca WHO th trng ton
cu v cc sn phm tho dc s t 5 nghn t USD. Trong Trung Quc
t 5 t USD t tho dc mi nm.
Vin Thc vt hc Trung Quc cho rng, cng vi Trung Quc, Lo,
Vit Nam l mt trong nhng nc c ti nguyn cy thuc phong ph nht.
Tuy nhin, ngun cy thuc ca Vit Nam ang cn kit v hot ng khai
thc ba bi v s yu km trong cng tc bo tn, ngay c cc khu bo tn
thin nhin v rng quc gia. C mt iu ng lu l vic khai thc ti

nguyn rng trong c cy thuc l sinh k ch yu ca cc dn tc t ngi
trong vng c cc thng nhn khai thc. Tuy vy, vic tn ph ti nguyn
rng cng ch mang li cho h mt mc sng rt thp. Trong khi , cc d
n trng rng ch yu mi ch quan tm n cy ly g, cy nguyn liu v
cy n qu m cha ch n cy dc liu, loi cy c gi tr hng ha rt
cao. Chng hn, s tin thu c t 0,2 ha sa nhn cng tng ng vi thu
nhp t 1,5 ha dong ring. V vy trong iu kin kinh t nh hin nay, vic
m rng khu vc trng cy thuc khng ch nhm pht trin ngun dc liu,
m cn l bin php hu hiu xa i gim ngho ti a phng.
Orchidaceae l mt trong nhng h thc vt rt phong ph v chng
loi. y khng nhng l mt h ni ting v c yu thch bi c hoa rt
p, nhiu mu sc v hnh dng, m c nhiu loi lan thuc h thc vt ny
cn l nhng v thuc cha bnh rt cng hiu. Vit Nam c chng loi Lan
rt phong ph gm tng s 125 chi v 902 loi lan, trong c nhiu loi Lan
rt qu, c nc ngoi bit n v t mua vi s lng ln nhng thc t
ch yu mi ch khai thc trong t nhin nn khng p ng c nhu cu
ca khch hng, ng thi li vi phm cc iu khon ca Hip c CITES l
khai thc ngy cng cn kit cc loi Lan, ng thi l cy dc liu ngun
gen qu him ca Vit Nam. V d nh: Thanh m (Coelogyne cristata
Lindl.), Thanh lan (Cymbidium cyperifolium Wall. ex Lindl.) (mt trong
nhng loi lan p nht th gii), Mc lan (Cymbidium ensifolium (L.) Sw.).
Trong c nhng loi Lan c s dng lm thuc cha bnh: Lan kim
tuyn (A. roxburghii (Wall.) Lindl.), Lan kim tuyn (Ludisia discolor (KerGawl.) A. Rich.), A. lylei Rolfe ex Downie Seidenf, A. lylei Rolfe ex Downie
Seidenf, A. formossanus, A. calcareu....c bit l loi Lan kim tuyn (A.
roxburghii (Wall.) Lindl.) hin s lng cn rt t.
Trn thc t cho thy Lan kim tuyn (A. roxburghii (Wall.) Lindl.) l
mt cy thuc quan trng thuc h Lan (Orchidaceae). N khng ch l mt
loi hoa c b l rt p m n cn l mt loi dc liu qu him ca Vit
Nam v cc nc Chu khc. N thng c gi vi ci tn l Lan kim
tuyn. A. roxburghii (Wall.) Lindl. phn b cc nc chu : Trung Quc,
Nht Bn, Sri Lanka, n , Nepal v Vit Nam... Vit Nam, A.
roxburghii (Wall.) Lindl. phn b ti Hu, Tam o, Lo Cai, Qung Tr,
Kon Tum, Gia Lai. Chng mc ri rc trong rng ni vi, ni m, dc theo
khe sui, cao 300 - 1800 m.
A. roxburghii (Wall.) Lindl. c coi l mt loi thn dc v c tc
dng cha bnh a dng nh: iu tr tiu ng, ung th, tr em suy dinh
dng, bnh gan, bnh tim mch, vim thn, cao huyt p v tr rn c cn
...(Xiao Xue Wang v cng s, 2011).
Hin nay, loi A. roxburghii (Wall.) Lindl. nc ta ang ng trc
nguy c b tuyt chng do nn khai thc ba bi v bun bn tri php ra
nc ngoi. Do vic nghin cu nhn nhanh loi Lan kim tuyn (A.
roxburghii (Wall.) Lindl.) ca Vit Nam l ht sc cn thit. V l , chng
ti tin hnh ti: Nghin cu nhn nhanh loi Lan kim tuyn
(Anoectochilus roxburghii (Wall.) Lindl.) ca Vit Nam bng phng php
nui cy m t bo nhm gp phn bo tn v pht trin loi Lan dc liu
qu him ca Vit Nam.
2. Mc ch v yu cu ca ti
Mc ch ca ti
Xy dng c quy trnh nhn nhanh loi Lan kim tuyn (A. roxburghii
(Wall.) Lindl.) bng phng php nui cy m t bo, nhm gp phn bo tn
v pht trin loi Lan dc liu qu him ca Vit Nam.
Yu cu ca ti
ti tp trung gii quyt mt s vn sau:
- Nghin cu to ngun vt liu khi u cho nui cy m t bo, tm ra
phng php kh trng ti u.
- Nghin cu cng on nhn nhanh chi.
- Nghin cu s sinh trng pht trin ca chi v to cy hon chnh.
- Nghin cu k thut a cy ra ngoi vn m, bao gm nghin cu v
cc gi th v mt s loi phn bn thch hp.
3. ngha khoa hc v thc tin ca ti
ngha khoa hc
- Bc u xy dng c quy trnh nhn ging bng phng php nui cy
m cy Lan kim tuyn.
- Gp phn trng trt v pht trin cy dc liu qu him m trc ch
khai thc trong t nhin, trn c s bo tn ngun gen qu him ang c
nguy c tuyt dit.
- Cung cp c s d liu cho vic xy dng quy trnh nhn ging in vitro cc
loi cy dc liu khc Vit Nam.
ngha thc tin ca ti
- A. roxburghii (Wall.) Lindl. l ngun gen qu him ca Vit Nam ang
ng trc nguy c tuyt chng, nhn ging Lan kim tuyn l nhm bo tn
a vo trng trt v pht trin ngun gen qu him ny.
- To ra c ngun cy ging vi s lng ln v sch bnh, gi c ph
hp, p ng nhu cu ca th trng, to ngun nguyn liu qu cho ngnh
Dc.
- Gp phn gip ng bo dn tc vng ni cao tng thu nhp, gp phn
xa i gim ngho v ng thi l bin php tt bo v rng.
4. i tng v phm vi nghin cu ca ti

- Cy Lan A. roxburghii (Wall.) Lindl. thu thp t Sa Pa c nui trng
trong nh li v a vo nui cy m ti phng nui cy m t bo thc vt
ca Trung tm Sinh hc Thc nghim - Vin ng dng Cng ngh - B Khoa
hc v Cng ngh. Thi gian tin hnh th nghim t thng 5/2011 n thng
10/2012
- Quy trnh nhn ging in vitro cy Lan kim tuyn n giai on a cy ra
vn m.
CHNG 1 - TNG QUAN TI LIU V C S KHOA HC

CA TI
1.1. C s khoa hc ca phng php nui cy m t bo thc vt
1.1.1. Tnh ton nng ca t bo thc vt
Nm 1902, nh sinh l thc vt hc ngi c Haberlandt, tin
hnh nui cy cc t bo thc vt chng minh t bo l ton nng.
Haberlandt cho rng mi t bo ca bt k sinh vt no cng u c kh nng
tim tng pht trin thnh mt c th hon chnh. ng nhn thy rng, mi
t bo ca c th a bo u pht sinh t hp bo thng qua qu trnh phn
bo nguyn nhim. iu c ngha l mi t bo ca mt sinh vt s cha
ton b thng tin di truyn cn thit ca mt c th hon chnh. Khi gp iu
kin thun li nht nh, nhng t bo c th s pht trin thnh mt c th
hon chnh.
Nm 1953, Miller v Skoog thnh cng khi thc nghim ti sinh cy
con t t bo l, chng minh c tnh ton nng ca t bo. Thnh cng trn
to ra cng ngh mi: cng ngh sinh hc ng dng trong nhn ging v
tnh, to ging cy trng v dng chng chu.
Tnh ton nng ca t bo m Haberlandt nu ra chnh l c s l lun
ca phng php nui cy m t bo thc vt. Cho n nay, con ngi
hon ton chng minh c kh nng ti sinh mt c th thc vt hon chnh
t mt t bo ring r (Nguyn Quang Thch, 2003).
1.1.2. S phn ho v phn phn ho t bo
Qu trnh pht sinh hnh thi trong nui cy m l kt qu phn ho v
phn phn ho t bo. C th thc vt trng thnh l mt chnh th thng
nht bao gm nhiu c quan chc nng khc nhau, trong c nhiu loi t
bo khc nhau, thc hin chc nng c th khc nhau. Cc m c c cu

trc chuyn mn ho nht nh nh vo s phn ho.
Phn ho t bo l s chuyn ho cc t bo phi sinh thnh cc t bo
ca m chuyn ho, m nhn cc chc nng khc nhau trong c th. Qu
trnh phn ho c th biu din nh sau:
T bo phi sinh
T bo gin
T bo phn ho chc nng
Khi t bo phn ho thnh m chc nng, chng khng hon ton

mt kh nng phn chia ca mnh. Trong trng hp cn thit, iu kin
thch hp, chng li c th tr v dng ging nh t bo phi sinh v tip tc
thc hin qu trnh phn ho, qu trnh ny gi l s phn phn ho ca t
bo.
Phn ha
t
TboT
bo phi sinh
T bo dn
T bo chuyn ha
Phn phn ha
V bn cht th s phn ho v phn phn ho l mt qu trnh hot ho
phn ho gen. Ti mt thi im no trong qu trnh pht trin c th, c
mt s gen c hot ho cho ra tnh trng mi, mt s gen khc li b c
ch hot ng. Qu trnh ny xy ra theo mt chng trnh c m ha
trong cu trc ca phn t DNA ca mi t bo. Khi t bo nm trong c th
thc vt, chng b c ch bi cc t bo xung quanh. Khi tch t bo ring r,
gp iu kin bt li th cc gen c hot ho, qu trnh phn chia s c
xy ra theo mt chng trnh nh sn trong DNA ca t bo (V Vn V,
1994).
iu khin s pht sinh hnh thi ca m nui cy, ngi ta thng

b sung vo mi trng nui cy hai nhm cht iu tit sinh trng thc vt
l Auxin v Cytokinin. T l hm lng hai nhm cht ny trong mi trng
khc nhau s to ra s pht sinh hnh thi khc nhau theo quy lut. Khi trong
mi trng nui cy c t l nng Auxin (IAA, IBA, NAA, 2,4D)/Cytokinin (BAP, Kinetin, Zeatin, TDZ) thp th s pht sinh hnh thi ca
m nui cy theo hng to chi, ngc li nu t l cao th m nui cy s
theo hng to r cn t l cn i s pht sinh theo hng to m so
(callus).
1.1.3. Cc nhn t nh hng ti qu trnh nhn ging bng nui cy m
t bo
Nhn ging bng nui cy m t bo gm nhiu giai on k tip nhau,
qu trnh ny c th chia thnh cc nhn t sau:
1.1.3.1. Mi trng nui cy
Trong nui cy in vitro, mi trng nui cy v iu kin bn ngoi
c xem l vn quyt nh s thnh bi ca qu trnh nui cy. Mi
trng nui cy c xem l phn m cung cp cc cht dinh dng cn
thit cho s tng trng v phn ho m trong sut qu trnh nui cy in vitro.
Cho n nay, c nhiu mi trng dinh dng c tm ra (MS, WPM,
VW, RE, N6, B5, LS) tu thuc vo i tng v mc ch nui cy.
Mi trng nui cy bao gm thnh phn sau:
+ Ngun cc bon: Trong nui cy m, cc t bo cha c kh nng quang
hp tng hp nn cht hu c do vy ngi ta phi a vo mi trng
mt lng hp cht cc bon nht nh cung cp nng lng cho t bo v
m. Ngun cc bon y l cc loi ng khong 20-30 mg/L c tc dng
gip m t bo thc vt tng hp cc hp cht hu c, gip t bo tng sinh
khi, ngoi ra n ng vai tr l cht thm thu chnh ca mi trng. Ngi

ta thng s dng 2 loi ng l saccarose v glucose (Trn Vn Minh,
1994).
+ Ngun Nit: T l ngun nit tu thuc vo loi cy v trng thi pht trin
m. Thng thng, ngun nit c a vo mi trng hai dng l NH4
-
v NO3 . Trong , vic hp th NO3 ca cc t bo thc vt t ra c hiu qu

hn so vi NH4 + .
-
+ Nhng i khi NO3 gy ra hin tng kim ha mi trng v vy gii

php s dng phi hp c 2 ngun nit vi t l hp l c s dng rng ri
nht.
+ Cc nguyn t a lng: L nhng nguyn t khong nh: N, P, K, S, Mg,
Ca cn thit v thay i tu i tng nui cy. Nhn chung, cc nguyn t
ny c s dng nng trn 30 ppm.
C nhiu mi trng vi thnh phn, t l cc cht khc nhau c th
la chn s dng. Ni chung, mi trng giu nit v kali thch hp cho vic
hnh thnh chi, cn mi trng giu kali s thc y qu trnh trao i cht
mnh hn.
+ Nhm nguyn t vi lng: Fe, Cu, Bo, Zn, Mn, Co, I l cc nguyn t rt
quan trng do chng ng vai tr quan trng trong cc hot ng ca enzym.
Chng c dng nng thp hn nhiu so vi cc nguyn t a lng
m bo sinh trng v pht trin bnh thng ca cy (Nguyn Vn Uyn,
1993).
+ Cc vitamin: Mc d cy nui cy m c th t tng hp c vitamin,
nhng khng cho nhu cu (Czocnowki, 1952). Do , cy sinh trng
ti u mt s vitamin nhm B c b sung vo mi trng vi lng nht
nh tu theo tng h m v giai on nui cy. Cc vitamin B1 (Thiamin) v
B6 (Pyridocin) l nhng vitamin c bn nht thng dng trong mi trng

nui cy vi nng thp khong 0,1-1mg/L (Trn Vn Minh, 1994).
+ Dung dch hu c: C thnh phn khng xc nh nh nc da, dch chit
nm men, c rt, chui, khoai ty... c b sung vo mi trng c tc dng
kch thch sinh trng m so v cc c quan.
* Nc da: Thng cha cc axt amin, axt hu c, ng, ARN, ADN.
c bit trong nc da c cha nhng hp cht quan trng cho nui cy in
vitro l: myo - inositol, cc hp cht c hot tnh auxin, cc glucosit ca
cytokinin. Do vy, khi b sung vo mi trng nui cy nc da kch thch
s phn chia, sinh trng v pht sinh hnh thi ca t bo, m nui cy.
Nc da thng c s dng rt hiu qu trong vic thay th cc hp cht
iu tit sinh trng tng hp nhn to khi nui cy hoa lan (Mamoru
Kusomoto, 1980).
Nc da c s dng nh l mt cht truyn thng trong nui cy
m nhn ging cy trng, trong nc da c nhiu cc thnh phn c o
ca cc loi ng, vitamin, khong cht, axt amin v cht iu tit sinh
trng. Cc loi ng c trong nc da nh: saccaroza, glucoza, fructoza,
mannitol, sorbitol, myo-inositol, scyllo-inositol; cc vitamin nh: C, B1,
B2, B3, B5, B6, folate, folic axt, biotin, niacin; khong cht nh: Ca, Fe,
Mg, P, K, Na, Zn, Cu, Mn, Se, Cl, S, Al...; Nhm auxin c trong nc da
ch yu l indole-3-acetic acid 40 chim 150,6 nM; nhm cytokinin c: N6isopentenyladenine chim 0,26 nM, dihydrozeatin chim 0,14 nM, transzeatin chim 0,09 nM, kinetin chim 0,31 nM...(Jean W.H. Yong v cng s,
2009).
* Dch nghin ca khoai ty: C cha cacbonhydrat di dng saccaroza,
glucose v fructose, amino axt (21 loi bao gm c lysine l mt axt amin
thng khng c trong protein thc vt), cc mui khong (K, Fe, Mg) v
10
c bit l cc vitamin (C, B1, B6). Dch nghin khoai ty thng c b

sung vo mi trng vi nhn ging hoa lan v c bit c hiu qu vi mt s
loi nh: Phalaenopsis, Doritaenopsis (Thorpe v cng s, 2008).
+ Than hot tnh: B sung than hot tnh vo trong mi trng nui cy s c
li ch v c tc dng kh c. Khi b sung than hot tnh vo mi trng
nui cy s kch thch s tng trng. Than hot tnh ni chung nh hng
trn 3 mt: ht cc hp cht cn, ht cc cht iu ha sinh trng v cc cht
lm en mi trng.
Ngi ta cho rng tc dng ngn cn s tng trng ca m cy khi c
s hin din ca than hot tnh trong mi trng l do n ht cht iu ha
sinh trng c trong mi trng. NAA, kinetin, IAA, BAP, 2iP lin kt vi
than hot tnh. Kh nng kch thch s tng trng ca than hot tnh l do n
kt hp vi cc hp cht phenol c tit ra trong thi gian nui cy. Than
hot tnh thng c b sung vo mi trng vi nng 0,5-3%, cn c
ngha kch thch sinh trng v phn ha cc loi hoa Lan.
Than hot tnh cng gip lm gim c t bng cch o thi cc hp
cht c (v d: phenol) c to ra trong qu trnh nui cy v cho php t
bo sinh trng m khng b tr ngi g.
+ Cht lm ng cng mi trng: Agar (thch) l mt loi Polysacharid ca
to c kh nng ngm nc kh cao 6-12g/L. thong kh ca mi trng
thch c nh hng r rt n sinh trng m nui cy. Nng thch dao
ng trong khong 6-10g/L tu thuc mc tiu nui cy.
1.1.3.2. Cc cht iu ho sinh trng
Cc Phytohormon l nhng cht c tc dng iu ho sinh trng v
pht trin ca thc vt. Chng ng vai tr quan trng trong qu trnh sinh
trng v pht trin ca thc vt nh: phn chia, bit ho t bo ngoi ra
cn c nh hng n qu trnh lo ho m v nhiu qu trnh khc. Cc
11
phytohormon c th chia thnh 5 nhm: Auxin, Cytokinin, Giberillin,

Ethylen, Abscisic axt. Chng l yu t quan trng nht trong mi trng
quyt nh n s thnh cng ca kt qu nui cy.
+ Auxin: Nhm ny gm c cc cht chnh l: IBA (3-Indol butyric acid),
IAA (Indol acetic acid), NAA (Nathyl acetic acid), trong nui cy m thc
vt Auxin thng c s dng kch thch s phn chia t bo, bit ho r,
hnh thnh m so, km hm s pht trin chi v to ra cc r ph (Nguyn
Vn Uyn, 1993).
+ Cytokinin: c b sung vo mi trng ch yu kch thch s phn chia
t bo v quyt nh s phn ho chi bt nh t m so v c quan. Cc hp
cht thng s dng l: Kinetin (6-Furfuryl aminopurine C10H 9NO5), BAP
(6-Benzyl amino purine), Zip (Izopentenyl adenin), Zeatin Trong cc cht
ny th Kinetin v BAP c s dng ph bin nht v chng c hot tnh cao
v gi thnh r. nng thp (10-7- 10-6M) chng c tc dng kch thch s
phn bo, nng 10-6 - 10-5M chng kch thch s phn ho chi. Trong
nui cy m kch thch s nhn nhanh ngi ta thng x dng Cytokinin
vi nng 10-6- 10-4 M (L Vn Chi, 1992).
+ Gibberellin: Nhm ny c khong 20 loi hormone khc nhau nhng quan
trng nht l GA3 (Gibberellin acid 3). GA3 c tc dng kch thch ny mm
ca cc loi ht khc nhau, ko di cc lng t thn cnh. Bn cnh GA3
cn c tc dng ph ng ca cc phi, c ch to r ph cng nh to chi
ph (Street, 1974). Ngoi ra, c cn c tc dng nh hng n s ra hoa ca
mt s thc vt v c tc dng rt ngn thi gian sinh trng sinh dng ca
cy.
+ Axt Abscisic (ABA): L cht c ch sinh trng t nhin nhng vn c
dng trong nui cy t bo in vitro. ABA c nh hng m tnh n m nui
12
cy, khi ABA tng tc vi BAP cho h s nhn chi cao hn khi dng BAP
ring r (L Vn Chi, 1992).
+ Ethylen: C biu hin tc ng hai chiu, n km hm s hnh thnh chi
giai on sm nhng li kch thch s pht trin chi giai on mun. Trong
mt s trng hp, Ethylen c tc dng kch thch hnh thnh r nhng mt s
trng hp n li km hm qu trnh ny (Nguyn Vn Uyn, 1993).
Ngoi ra, cn phi ch ti pH ca mi trng. pH thng c
s dng trong nui cy m t bo thc vt ni chung t 5,6 - 6.
1.1.3.3. Mi trng vt l
Trong nui cy m t bo cc yu t ca mi trng vt l c quan
tm l nh sng, m, nhit .
+ nh sng: y l yu t cn thit cho s pht trin v pht sinh hnh thi
ca cc m nui cy. nh sng c nh hng ti mu cy thng qua thi gian
chiu sng, cng nh sng v cht lng nh sng. Thi gian chiu sng
c vai tr quan trng trong qu trnh pht trin ca m nui cy. Vi a s cc
loi cy, thi gian chiu sng thch hp l 8-12h/ngy. Cng nh sng
nh hng n qu trnh pht sinh hnh thi m nui cy.
Cng nh sng cao kch thch sinh trng ca m so trong khi
cng thp gy nn s to chi. Nhn chung, cng nh sng thch hp
cho m nui cy l t 1000 - 7000 lux.
Bn cnh thi gian chiu sng, cng nh sng th cht lng nh
sng cng nh hng kh r ti s pht sinh hnh thi ca m nui cy. nh
sng lm tng chiu cao ca thn chi hn so vi nh sng trng, cn nh
sng xanh th c ch s vn cao ca chi nhng li nh hng tt ti s sinh
trng ca m so. Chnh v vy m trong phng th nghim thng s dng
nh sng ca n hunh quang vi cng 2000 - 3000 lux.
13
+ Nhit : L nhn t c nh hng r rt n s phn chia t bo v cc

qu trnh trao i cht ca m nui cy, ng thi n c nh hng ti s
hot ng ca auxin, do lm nh hng n kh nng ra r ca cy m.
Theo kt qu nghin cu ca Vonanorld (1982) th nu nhit ngy/m l
200C/150C hoc 200C/180C t l ra r t c khong 33%, thm ch cn
thp hn. nhit trung bnh th hot ng trao i cht tt hn. Cn
nhit cao li to nhiu t bo khng c t chc. Trong nui cy m, nhit
thng c duy tr n nh, ban ngy t 25 - 300C v ban m t 17 200C.
+ m: Trong cc bnh nui cy th m tng i lun bng 100%
m bo s pht sinh pht trin bnh thng ca cy nui cy m.
1.1.3.4. Vt liu nui cy
Vic la chn vt liu nui cy quyt nh n s thnh bi ca qu
trnh nhn ging in vitro. V nguyn tc th mi t bo ca cc m chuyn
ho u c tnh ton nng, ngha l u c th nui cy thnh cng. Thc t
cho thy cc loi t bo v cc loi m khc nhau c mc nui cy thnh
cng khc nhau. Mt nguyn tc c bn trong nui cy m t bo l cc t
bo lm vt liu nui cy cng non th kh nng nui cy thnh cng cng
cao. Nh vy, t bo v m phi non l trin vng nht, ri n cc t bo ca
nh sinh trng nh: m phn sinh nh ngn, u r, l non, tng tng
sau l cc t bo sinh dc nh non bo v t bo ht phn giai on non
(Nguyn c Thnh, 2000); (Nguyn Quang Thch, 1995).
1.1.3.5. iu kin v trng
y l iu kin c bn u tin quyt nh s thnh bi ca qu trnh
nui cy in vitro. Nu iu kin ny khng c m bo th mu nui cy
hoc mi trng s b nhim, m nui cy s b cht, cc th nghim giai
14
on sau s b ngng li. Do , trong ton b qu trnh nui cy in vitro cn

m bo iu kin v trng tuyt i. Mun m bo iu kin v trng cn
c phng php kh trng mu thch hp, phng tin kh trng hin i,
bung v bn nui cy v trng.
Chn ng phng php kh trng s cho t l sng cao, mi trng
dinh dng thch hp s t tc sinh trng nhanh.
1.1.4. Cc giai on chnh trong qu trnh nhn ging nui cy in vitro
Theo Georger (1993) qu trnh nhn ging v tnh in vitro bao gm cc
bc sau:
Bc 0: Chn lc v chun b cy m
Trc khi tin hnh nhn ging in vitro cn chn lc cn thn cc cy
m (cy cho ngun mu nui cy). Cc cy ny cn phi sch bnh, c bit
l bnh virus v giai on sinh trng mnh. Vic trng cc cy m trong
iu kin mi trng thch hp vi ch chm sc v phng tr su bnh
hiu qu trc khi ly mu cy s lm gim t l mu nhim, tng kh nng
sng v sinh trng ca mu cy in vitro.
Bc 1: Nui cy khi ng
L giai on kh trng a mu vo nui cy in vitro. Giai on ny
cn m bo cc yu cu: T l nhim thp, t l sng cao, m tn ti v sinh
trng tt. Khi ly mu cn chn ng loi m, ng giai on pht trin ca
cy. Quan trng nht l nh chi ngn, nh chi nch v sau l nh chi
hoa v cui cng l on thn, mnh l...
Bc 2: Nhn nhanh
L giai on kch thch m nui cy pht sinh hnh thi v tng nhanh
s lng thng qua cc con ng: hot ha chi nch, to chi bt nh v
to phi v tnh. Vn l phi xc nh c mi trng v iu kin ngoi
cnh thch hp c hiu qu cao nht. Theo nguyn tc chung mi trng c
15
nhiu cytokinin s kch thch to chi. Ch nui cy thng l 25 - 270C v

16 gi chiu sng/ngy, cng nh sng 2000 - 4000 lux.
Bc 3: To cy in vitro hon chnh
to r cho chi, ngi ta chuyn chi t mi trng nhn nhanh
sang mi trng to r. Mi trng to r thng c b sung mt lng
nh auxin. Mt s chi c th pht sinh r ngay sau khi chuyn t mi trng
nhn nhanh giu cytokinin sang mi trng khng cha cht iu tit sinh
trng. Theo Murashige, giai on ny l giai on u tin cho s to r ca
chi sau y c th chuyn chng sang mi trng trng trt t nhin. To
r cho chi l mt phn rt quan trng ca bt k quy trnh nhn in vitro no.
i khi mt s chi cn c ko di trc khi to r. gim chi ph nhn
ging, nhiu phng th nghim chuyn chi khng r t mi trng in vitro v
to r t bn ngoi phng nui cy.
Bc 4: Thch ng cy in vitro ngoi iu kin t nhin
a cy t ng nghim ra vn m vi t l sng cao, cy sinh
trng tt cn m bo mt s yu cu:
+ Cy trong ng nghim t c nhng tiu chun hnh thi nht nh (s
l, s r, chiu cao cy).
+ C gi th tip nhn cy in vitro thch hp: Gi th sch, ti xp, thot
nc.
+ Phi ch ng iu chnh c m , s chiu sng ca vn vm cng
nh ch dinh dng ph hp.
Trong nhng nm gn y, nhn ging in vitro c p dng nhiu
trong nhn ging cy trng. Nhng c im ni bt ca nhn ging in vitro
l cho h s nhn ln (thng t c cc loi cy khc nhau trong phm
vi t 36 1012/ nm) trong mt thi gian ngn, d dng sn xut cy sch bnh,
khe mnh; Phng php nhn ging v tnh bng nui cy in vitro nhanh v
16
hiu qu, c bit c p dng cho cc loi hoa, cy thuc, cy c sn qu

him c gi tr kinh t cao.
1.2. Mt vi nt v Lan kim tuyn
Chi Anoectochilus thuc h lan c hn 40 loi c mt hu khp cc
nc nhit i tri di t dy Himalaya ca n n vng ng Nam v
ko di ti Hawai. Trong chi Anoectochilus c mt s loi c s dng
lm thuc Trung Quc nh: A. formosanus Hayata, A. koshunensis Hayata,
v A. roxburghii (Wall.) Lindl. Trong cc loi ny th hai loi A. formosanus
Hayata v A. koshunensis Hayata ch phn b i Loan Trung Quc v
Okinawa Nht Bn. A. roxburghii phn b min nam Trung Quc, Nht
Bn, Srilanka, n , Nepal v Vit Nam (Zhong HJ v cng s, 2000).
1.2.1. Phn loi
Lan kim tuyn c tn khoa hc l: Anoectochilus roxburghii (Wall.)
Lindl. (1832)
Synonym: Chrysobaphus roxburghii Wall. (1826); Anoectochilus
setaceus Blume (1825)
Tn khc: Kim tuyn t; Gii thy t; Gii thy roxburgh; Sa hng
Chi: Kim tuyn (Anoectochilus)
H: H phong lan (Orchidaceae)
1.2.2. c im hnh thi
Cy thn tho, mc t, c thn r mc di; thn trn t mng nc
v c nhiu lng mm, mang 2 - 4 l mc xe st t. L hnh trng, gn trn
gc, chp hi nhn v c mi ngn, c 3 - 4 x 2 - 3cm, c mu khc nhau
vi mng gn thng nht hn (mu lc sm vi mng gn mu lc nht hay
mu nu - vi mng gn mu vng - lc hay hng); cung l di 2 3cm. Cm hoa di 10 - 15cm, mang 4 - 10 hoa mc tha. L bc hnh trng,
17
chp thp nhn, di 8 - 10mm, mu hng. Hoa thng mu trng, di 2,5 3cm; cc mnh bao hoa di khong 6mm.
Hnh 1.1. Cy Lan kim tuyn (A. roxburghii (Wall.) Lindl.)

1.2.3. Sinh hc v sinh thi
Ma hoa n vo thng 10 - 12. Ma qu chn t thng 12 n thng 3
nm sau. Ti sinh bng chi t thn r v ht, sinh trng rt chm. Mc di
tn rng nguyn sinh, hu ht l rng nguyn thy, thng xanh nhit i,
rng cy l rng trn sn ni granit, rilit, phin st, cao 500 1600m ri rc thnh tng nhm vi ba cy trn t m, rt giu mn.
18
1.2.4. Phn b
Trong nc: Lo Cai (Sa Pa: Phng Xi png, Vn Bn: Lim Ph), H
Tnh (Hng Sn: Ro n), Qung Tr, Kom Tum (k Glie: ni Ngc Linh,
Sa Thy: ni Ch Mom Ray), k Lk (Knng Bng: ni Ch Yang Sinh),
Lm ng (Lc Dng: ni B p).
Th gii: n , NePan, Butan, Trung Quc, Mianma, Thi Lan, lo,
Campuchia, Malaixia, Innxia.
1.2.5. Gi tr dc liu ca A. roxburghii (Wall.) Lindl.
1.2.5.1. Hot tnh sinh hc ca A. roxburghii (Wall.) Lindl.
Trn th gii vic xc nh cc cht c hot tnh sinh hc bng cc
phng php khc nhau Lan kim tuyn c rt nhiu nh khoa hc s
dng.
He CN v cng s (2005) s dng phng php sc k phn lp
c 5 hp cht l: p-Hydroxybenzaldehyde (I), ferulic acid (II), quercetin
(III), daucosterol (IV), cirsilineol (V).
Trong Lan kim tuyn cn cha cc hp cht chng oxy ha l dn xut
ca
phenol
nh:
8hydroxybenzylquercetin,
isorhamnetin7OD
glucopyranoside, isorhamnetin3ODglucopyranoside, kaempferol3

ODglucopyranoside, kaempferol7Dglucopyranoside, 5hydroxy
3',
4',
7trimethoxyflavonol3Drutinoside,
isorhamnetin3D
rutinoside v quercetin7OD[6''O(transferuloyl)]glucopyranoside
cc hot cht ny c kh nng t tm ra gc t do DPPH.
Theo nghin cu ca Chun-Nian He v cng s (2006), trong A.
roxburghii (Wall.) Lindl. c cha quercetin-7-O--D-[6''-O-(trans-feruloyl)]glucopyranoside (compound 1), 8-C-p-Hydroxybenzylquercetin (compound
19
2), isorhamnetin-7-O--Dglucopyranoside (compound 3), isorhamnetin-3-O-D-glucopyranoside (compound 4), kaempferol-3-O--Dglucopyranoside

(compound 5), kaempferol-7-O--D-glucopyranoside (compound 6), 5hydroxy-3',4',7- trimethoxyflavonol-3-O--D-rutinoside (compound 7), and
isorhamnetin-3-O--D-rutinoside (compound 8).
Liying Huang v cng s (2007) dng cc phng php sc k
HPLC/APCI/MS nhn dng v xc nh c 3 hp cht l ergosterol,
stigmasterol v -sitosterol vi nng o c ln lt l 0,5 150g/ml
vi h s tng quan r = 0,9999 ergosterol, 5 400g/ml vi h s r = 0,9999
stigmasterol v 10 - 2000g/ml vi h s r = 0,9998 -sitosterol.
Liying Huang v cng s (2007) cng dng phng php sc k LCAPCI-IT-MS phn tch v phn lp c hai hp cht l Oleanolic acid
(OA) v axit ursolic (UA) rt quan trng c trong A. roxburghii(Wall.) Lindl
v c s dng nh mt loi thuc truyn thng ca Trung Quc vi
nng tng ng ln lt l 0,5 80 (r = 0.9992) v 0,5 50g/ml (r =
0,9989).
Mei-Hua Han v cng s (2008) tch chit c 2 ester mi l
sorghumol 3-O-Z-p-coumarate v sorghumol 3-O-E-p-coumarate, v mt
alkaloid mi l Anoectochine t ton b cy A. roxburghii cng vi
triterpenoid, sorghumol.
Theo Cai Jin yan v cng s (2008) s dng phng php sc k
xc nh cc hp cht c trong tinh du v petrol ether (PE) chit xut t cy
Lan kim tuyn (A. roxburghii) v xc nh c tng s 72 hp cht trong
c 69 hp cht c xc nh t PE.
Li Chun-yan v cng s (2009) dng phng php sc k lng (HPLC)
v phng php Soxhlet xc nh hai axit ferulic v axit cinnamic c trong
20
cy Lan kim tuyn vi kt qu tng ng ln lt l 4,0 110,0g/ml vi h

s tng quan r = 0,9985 v 2,5 50g/ml vi h s tng quan r = 0,9995,
bng phng php Soxhlet xc nh c 0,189mg/g axit ferulic, 0,091mg/g
axit cinnamic.
Theo Hu Guo-hai v cng s (2010), dng phng php phn tch
quang k ngn la (FAAS) nh gi v xc nh hm lng ca cc
nguyn t vi lng (Zn, Mn, Cu, Fe, Ni, Co, Cr, Cd, Pb) c trong Lan kim
tuyn nui trng v Lan kim tuyn trong t nhin. Kt qu cho thy xc
nh c s c mt ca cc nguyn t vi lng l 99,8% vi gi tr RSD =
0,66%. Cc nguyn t Fe, Mn, Zn, Co v Cu trong Lan kim tuyn nui trng
thp hn so vi cc nguyn t ny Lan kim tuyn trong t nhin. Tuy nhin
nguyn t Cr trong Lan kim tuyn nui trng li cao hn so vi Lan kim
tuyn trong t nhin.
Zhong Tian-hua v cng s (2011) xc nh cc axit amin t do c
trong Lan kim tuyn bng phng php sc k anion xc nh c 17 axit
amin vi nng t 0,23 3,37 pmol c ng tuyn tnh vi cao im 2
3 v gi tr RSDs ca 5 ng c gi tr t 0,96 4,2%.
Liying Huang v cng s (2011) nghin cu s dng HSCCC (highspeed counter-current chromatography) tch chit v tinh sch c
ergosterol v stigmasterol l hai hp cht sterol ch yu ca A. roxburghii
(Wall) Lindl. vi nhiu hot tnh sinh hc quan trng. Nhm nghin cu
tch chit c 36,5mg ergosterol v 43,6mg stigmasterol t 100g of A.
roxburghii vi tinh khit 92,0 95,5%.
Xiao Xue Wang v cng s (2011) bng phng php cng hng t
v phng php sc k lng cng u d khi ph (NRM/LRC-MS PDS)
xc nh c 13 hp cht cng cu trc phn t ca cc hp cht l: p
21
hydroxybenzaldehyde (1), trans4hydroxycinnamic acid (2), trans3

hydroxycinnamic acid (3), trans2 hydroxycinnamic acid (4), Isorhamnetin
3O-[6 rhamnosyl(16)] glucopyranoside (5), 5hydroxyferulic acid (6),
3, 5dihydroxy3, 4, 7 trimethoflavone (7), 5, 6, 3, 4 - tetrahydroxy7,
5-trimethoflavonol3Oglucoside
(8),
5,
4dihydroxy6,
7,
trimethoflavone (9), Quercetin (10), Kaempferol (11), Isorhamnetin (12),

Quercetin 3, 4dimethyl ether (13), trong cc hp cht 3, 4, 6, 8, 11 l cc
hp cht c phn lp ln u tin.
1.2.5.2. Tc dng v cng dng ca A. roxburghii (Wall.) Lindl.
A. roxburghii (Wall.) Lindl. vi s phong ph ca cc hot cht trong
cy c coi l mt loi thn dc v c tc dng cha bnh a dng nh:
iu tr tiu ng, ung th, tr em suy dinh dng, bnh gan, bnh tim mch,
vim thn, cao huyt p v tr rn c cn ...(Xiao Xue Wang v cng s,
2011).
Theo tc gi Cai Wen-yan (2008) nh gi hot ng khng khun
v nng c ch ti thiu ca nc chit ton cy Lan kim tuyn kt qu
cho thy nc chit xut ca ton cy c kh nng c ch s tng trng ca
4 loi vi khun v 2 loi nm th nghim, tng t tc dng c ch trn vi
khun mnh hn trn nm vi nng khng cao hn 12,5mg/ml i vi vi
khun v 25mg/ml i vi nm.
Shi-Chao Cui v cng s (2012) nghin cu tc dng h ng
huyt v chng oxy ha bng cht alloxan c chit xut t A. roxburghii
th nghim trn nhng con chut mc bnh tiu ng. Vi liu t 2g/kg
trong 14 ngy lm gim ng k lng ng trong mu, gim
22
cholesterol. Ngoi ra lm tng cc hot ng ca enzym cht chng oxy ha

v mc vitamin E trong gan v thn ca chut b tiu ng.
Yonghui Zhang v cng s (2007) th nghim trn chut, vi vic
dng hot cht kinsenoside c trong Lan A. roxburghii liu lng 15mg/1kg
trng lng c th, iu khin c hot ng ca cc enzym chng oxy ha,
-
loi b cc gc t do v gim hm lng NO , lm tng cc t bo nguyn

vn trong tuyn ni tit sn sinh insulin chut c iu tr so vi chut
khng c iu tr bng hot cht kisennoside.
1.2.6. Thc trng khai thc v pht trin cy Lan A. roxburghii (Wall.)
Lindl.)
Vit Nam Lan kim tuyn c phn b rng nhng vi s lng c th
khng nhiu, ti sinh chm i hi iu kin sng ngt ngho ngy cng
him. Do b thu hi nhiu bn lm thuc t rt lu, nn loi Lan kim tuyn
ang b e da nghim trng, rt c th s b tuyt chng ngoi t nhin nu
khng c bin php bo tn hu hiu. Hin nay, Lan kim tuyn c xp
trong nhm IA ca Ngh nh 32/2006/N-CP, nghim cm khai thc v mc
ch thng mi v nhm thc vt ang nguy cp EN A1a,c,d (Sch Vit
Nam, 2007).
1.3. Nui trng Lan kim tuyn
Lan kim tuyn l cy sinh trng rt chm, tuy nhin trong iu kin
thun li chng vn pht trin tt. Nhng iu kin l: nhit , m v
che bng thch hp. Ngoi ra Lan kim tuyn pht trin trn cc gi th nh l:
ru, dn, t qu, x da, than, gch vn, v cy...
1.3.1. nh sng
Lan kim tuyn pht trin nhng ni c nh sng yu. nh sng thch
hp cho Lan kim tuyn 30 40% so vi nh sng t nhin.
23
1.3.2. Nhit
Nhit thch hp cng ty thuc vo tng loi, ty thuc vo a
im pht sinh ca chng. Ni chung, nhit l tng cho Lan kim tuyn
13 - 16C vo ban m, 24 - 30C vo ban ngy. Cy s chu c nhit
100C v ln ti 380C trong mt thi gian ngn, nhit cao hn cn to s
thng gi tt trong vn.
1.3.3. m
m khng kh trong vn, m ca gi th nui cy u phi thch
hp, cy mi sinh trng pht trin tt. Lan kim tuyn yu cu m cao.
m khng kh thch hp cho Lan kim tuyn 70 80%. Nu m thp cy s
sinh trng km, m qu cao cy d b nhim bnh.
1.3.4. Ngun nc ti
Dng bt c ngun nc ti no cho Lan kim tuyn cng c, min
l nc sch, khng mn, khng vi, khng c clo v c pH 5,5 7. i
vi nhng cy lan con trng trong chu yu cu ti phun sng nh. Lan
kim tuyn cn m cao, nhng cng cn s thng thong to ra khng gian
thch hp lm cho cy sinh trng pht trin.
1.3.5. Phn bn
Lan kim tuyn khng yu cu lng phn bn nhiu. Vo ma xun v
ma thu bn phn 10 15 ngy/ln. Vo ma ng v ma ma ch bn phn
30 ngy/ln. Bn vi lng phn bn c t l N:P:K (15-15-15) vi tha c
ph/4,5 lt nc. Phn bn c s dng bng cch phun phn ha tan trong
nc, phun bng bnh c tia mn, phun u ton cy l v r. Ti cy sng
sm v chiu mun.
1.4. Mt s nghin cu v nhn ging v bo tn cy Lan kim tuyn
24

trn th gii
Nhng nm gn y, thuc ng dc tr thnh mt ti quan trng
mang tnh ton cu. Mc d cc nc pht trin ngi ta thng s dng
tn dc trong iu tr nhng cc loi thuc c ngun gc tho mc vn
c dng ph bin do yu t lch s v vn ha. Theo cc nh gi v mt
khoa hc, nhiu loi tho mc c th ng dng trong y hc. Vn t ra l
vng sinh trng ca cy thuc ang bin mt nhanh chng do s khng n
nh ca iu kin mi trng v cc yu t khc. Nh vy, tht kh c mt
ngun nguyn liu ln tch chit cc hp cht th cp dng trong
dc phm. iu ny cnh bo cho ngnh cng nghip cng nh cc nh
khoa hc cn tnh n tim nng ca k thut nui cy t bo thc vt nh
mt s thay th khc cung cp nguyn liu cho ngun dc phm ny.
Trong nhng nm gn y, trn th gii c nhiu c s nghin cu
tm cc bin php nhn ging loi Lan kim tuyn qu him c nguy c b
tuyt chng.
Gangaprasad A v cng s (2000) nghin cu vi nhn ging hai loi Lan
A. sikkimensis v A. regalis. Hai loi Lan kim tuyn ny c nui cy trn
mi trng WPM sau 12 tun t l callus c to ra 4,8 - 5,6 v s chi
tng ng 95 98%. Khi mu c cy chuyn sang mi trng tng t
trn th s chi bn tng 21,4 v chi nh l 8,2 i vi loi A. regalis, sai
khc c ngha v mt thng k. Cn i vi loi A. sikkimensis s bt chi
chm hn, s chi bn t 12,3 v chi nh l 4,3. Cc chi di (4 - 6cm)
c cy sang mi trng c cha (2,70 M) NAA v 0,2% than hot tnh.
Cc cy c kh nng sng 95 - 98% trong cc chu. Sau 6 thng trng trong
nh knh, Lan A. regalis c a ra mi trng t nhin vi t l sng l
95% v sau 12 thng t l sng cn 70%. Cc cy trng trong chu hay
25
ngoi mi trng t nhin u khng b thay i v hnh thi v kh nng

sinh trng.
Yih-Juh Shiau v cng s (2001) bo tn A. formosanus Hayata
bng phng php th phn nhn to v nui cy ht in vitro. Cc tc gi
ti u ha phng php tng sinh khi cc ht ging A. formosanus qua th
phn cho v ny mm ht ging khng cng sinh vi nm. T l thnh cng
nh th phn bng tay t 86,7%, cc ht ging sau khi th phn 7 tun th
c ny mm trn mi trng MS v MS c b sung 0,2% than hot tnh
v 8% dch chui sau 4 thng nui cy. Cc ht ging ny mm c nui cy
trong mi trng MS lng c cha 2,0mg/L BA trong 2 thng nui cy.
Cy ging trc khi a ra ngoi vn m, c nui trng trn mi trng
1/2MS c b sung 0,2% than hot tnh, 8% dch chui, 2,0mg/L BA v 0,5
mg/L NAA cho thn v r pht trin tt hn. C khong 90% cy nui cy t
ht 2 thng tui c a ra bu vi hn hp ru + than bn.
Vi cng trnh nghin cu gieo ht in vitro cy A. roxburghii tc gi
Huang H v cng s (2002) tm ra c mi trng Knudson C c b sung
0,5mg/L BA + 20% dch khoai ty l thch hp nht cho s pht trin
protocorm t ht ca A. roxburghii.
Vi nhn ging cy A. formossanus c tc gi Ket N.V v cng s
(2004) nghin cu nhm mc tiu bo tn ngun gen qu c nguy c b tuyt
dit. tm ra c mi trng nhn nhanh cm chi thch hp nht l mi
trng Hyponex c b sung 1g dm3Benzyl adenin hoc 12mg dm3
Thidiazuron (TDZ). B sung 1g dm3 than hot tnh vo mi trng c cha
TDZ thc pht trin nhiu chi (11,1 chi/m cy). Tuy nhin, tc chi ti
sinh chm v khng ko di thn. Trn mi trng H3 b sung 2% ng v
0,5g dm3 than hot tnh thch hp cho s pht trin ca cm chi v t l cy
ra r l 100%.
26
Y.J. Shiau v cng s (2005) nghin cu nhn ging loi Lan kim
tuyn (Haemaria discolor (Ker) Lindl. var.) bng phng php gieo ht in
vitro. Cc ht c cy trn mi trng MS c b sung 3% sucrose and
0,85% thch. Cc ht sau khi ny mm cy chuyn trn mi trng MS b
sung 0,2% hot tnh, 8% dch chui, 0,1mg dm3 (TDZ) v 1,0mg dm3
(NAA) cho cy sinh trng v pht trin tt. C ti 96% cy sng sau khi
chuyn ra nh knh.
X F Xu (2008) tin hnh nghin cu nhn nhanh v nui trng cy A.
formosanus. Cc kt qu cho thy, mi trng B5 + 5mg/L BA + 0,3mg/L
NAA l thch hp nht cho nhn chi. Mi trng MS + 5mg/L IBA l thch
hp nht cho s ra r. Cc cy ra ngoi vn m, phun 0,2% carbamide,
0,2% KH2PO4 4 ln v na thng/ln. Sau 4-5 thng cy pht trin n chiu
di 10cm, 1-2g trng lng ti c th thu hoch.
Zhang F v cng s (2009) ng dng phn mm SPSS trong nui
cy m cy A. roxburghii, cc cy c thu hoch sau khi nui cy 60 ngy.
Cc th nghim ca ng nhm nghin cu nh hng ca NAA v BA n s
l, chi nh, chi bn v chiu cao ca chi. Tt c s liu c s l bng
phn mm SPSS. Kt qu cc nng NAA khc nhau t <1mg/L hoc
>1mg/L u c nh hng n s sinh trng v pht trin ca A. roxburghii.
Trn mi trng MS c b sung 0,5mg/L NAA v 1mg/L BA thch hp nht
cho pht trin cc chi nh, vi nng 1mg/L NAA v 2mg/L BA thch
hp nht cho s pht trin ca chi bn v h s nhn chi. Cc nng khc
nhau ca NAA v BA u c nh hng khc nhau n s tng trng gia
chi nh v chi bn ca A. roxburghii.
Zhou YuMei v cng s (2009) nghin cu xy dng quy trnh nhn
nhanh ging cy A. formosanus. Kt qu cho thy: Mi trng ti u cho
nhn nhanh chi l mi trng MS b sung 0,1mg/L TDZ, 5% nc da v
27
3% ng. Mi trng thch hp cho r pht trin l mi trng MS b sung

0,1mg/L BA + 0,5mg/L IBA v 2g/L than hot tnh. Gi th ra cy trong vn
m tt nht l ru v s dng dinh dng Hyponex 2 (20: 20: 20) bn tt
nht cho cy A. formosanus trong vn m.
Qiu Yue v cng s (2010) tin hnh nghin cu ny mm ca ht
ging v nhn nhanh protocorm loi A. roxburghii. Th nghim nghin cu
v ny mm khng cng sinh vi nm ca ht ging A. roxburghii v nh
hng ca 4 yu t (mi trng nui cy, cht BA, ZT v NAA) n vic
nhn nhanh protocorm ca loi A. roxburghii. Kt qu cho thy rng: kiu
th phn l mt nhn t quan trng i vi s ny mm ca ht ging khng
cng sinh vi nm A. roxburghii, t l ny mm ca ht ging giao phn
t 78,53% v ht ging th phn khc hoa cng gc t 69,62% cn ht
ging t th th t l ny mm ch t 39,87%. Qu nang pht trin 150 ngy
l thi gian thnh thc nht, t l ny mm ca ht ging c th t 78,59%.
Sc sng ca ht ging b nh hng bi thi gian bo qun lnh v khi tng
thi gian bo qun trong kho lnh ln th t l ny mm ca ht ging gim.
X l ht ging bng NaOCl 10% th t l ny mm ca ht ging khng
thay i so vi vic khng x l. NAA nh hng rt tt n s tng nhanh
protocorm, mi trng nui cy thch hp nht cho nhn nhanh protocorm l
MS + 0,5mg/L ZT + 1,0mg/L NAA.
Nghin cu k thut nui cy nh sinh trng nhm mc tiu bo tn
v pht trin cy A. elatus Lindley, tc gi N. Ahamed Sherif v cng s
(2012) s dng chi nh v chi bn lm vt liu nui cy. Trn mi
trng MS c b sung 3,0mg/L TDZ v 3,5mg/L Kin thch hp nht cho nhn
nhanh cm chi. T l ra r t cao nht khi b sung vo mi trng 0,3g/L
than hot tnh.
28

Vit Nam
Vit Nam c xp th 16 trong s cc nc c a dang sinh hc cao
nht th gii. Nhng s a dng sinh hc ca nc ta ang b suy thoi mc
bo ng. Din tch rng b gim rt nhanh ko theo nguy c tuyt chng ca
nhiu loi ng thc vt qu him trong c cc loi Lan ni chung v Lan
kim tuyn ni ring. Con ngi ang khai thc t nhin mt cch qu mc,
thm ch hu dit ngun ti nguyn thin nhin phc v nhu cu trc mt
m qun mt nhng him ha ko theo sau trong tng lai. Vic duy tr a
dng sinh hc c vai tr rt quan trng trong i sng vt cht v tinh thn
ca con ngi, cng nh trong vic duy tr cc chu trnh t nhin v s cn
bng sinh thi.
Cc kt qu nghin cu nhn ging Lan kim tuyn Vit Nam cn rt
hn ch. Ch trong mt s nm gn y mi c mt vi tc gi cng b cc kt
qu nghin cu v nhn ging loi Lan ny. R rt nht l tc gi Phng Vn
Ph v cng s (2010), theo ng mi trng ph hp nht nhn nhanh chi
Lan kim tuyn (A. roxburghii (Wall.) Lindl.) in vitro l Knud. Th chi 8 tun
tui t phi ht chn v chi t th chi cao t 2 - 3cm l ph hp nht
nhn nhanh trong mi trng thch hp Knud b sung 0,5mg/L BAP +
0,3mg/L Kinetin + 0,3mg/L NAA + 100ml/L nc da + 100g/L dch chit
khoai ty + 20g/L ng + 7g/L thch + 0,5g/L than hot tnh.
Gn y nht theo Nguyn Quang Thch v cng s (2012), cho bit c
quan vo mu ph hp nht l th chi v mt t ngang thn i vi loi Lan
kim tuyn Anoectochilus setaceus c kh trng v a vo cc mi trng
nn khc nhau (MS, Knud, Knudson). Cc chi v mt t c chuyn sang
mi trng nn thch hp c b sung BA, Kinetin, NAA trong 4 tun. Mi
trng thch hp nht nhn nhanh th chi v mt t ngang thn l Knud
29
+ 0,5mg/L BAP + 0,3mg/L Kinetin + 0,3mg/L NAA + 20g/L sucrose +

0,5g/L than hot tnh + 7g agar/L cho h s nhn chi l 6,55 chi/mu. Cc
chi c chiu cao t 3 - 4cm c s dng ra r in vitro. T l ra r l
100% v s r/chi (4,21 r/chi) t cao nht trn mi trng c b sung
1mg/L NAA.
R rng rng, cc kt qu nghin cu trn cho thy cng tc nhn
ging v loi Lan A. roxburghii (Wall.) Lindl. cn rt hn ch Vit Nam.
Do vic nghin cu nhn nhanh loi Lan (A. roxburghii (Wall.) Lindl.) tr
nn cn thit hn bao gi ht. Nhim v ny song song gii quyt hai vn
trn i tng cy Lan (A. roxburghii (Wall.) Lindl.) qu him ca Vit
Nam l: Va c ngha bo tn li va ng thi nhn nhanh to ra mt khi
lng cy con ging vi cht lng cao trong mt thi gian ngn. Trn c s
cy Lan (A. roxburghii (Wall.) Lindl.) c em vo trng trt vi din
tch ngy cng m rng, gp phn cung cp ngun dc liu qu him cho
ngnh dc Vit Nam.
30
CHNG 2 - VT LIU, NI DUNG V PHNG PHP

NGHIN CU
2.1. Vt liu nghin cu
2.1.1. Ging nghin cu
Nguyn liu c dng trong th nghim l cc cy Lan kim tuyn (A.
roxburghii (Wall.) Lindl.) c chiu cao khong 20 - 30cm, thu thp t Sa Pa
v c nui trng trong nh li Trung tm Sinh hc Thc nghim - Vin
ng dng Cng ngh - B khoa hc v Cng ngh a vo nui cy m.
Hnh 2.1. Cy Lan kim tuyn (A. roxburghii (Wall.) Lindl.) c a vo

nui cy m
2.1.2. Ho cht v thit b nui cy m
Cc dng c v ho cht cn thit thc hin qu trnh nui cy do
phng Nui cy m t bo, Trung tm Sinh hc Thc nghim - Vin ng
dng Cng ngh - B Khoa hc v Cng ngh cung cp.
* Dng c
- Bung cy v trng (Bioiogical safety Cabinets)
- Ni hp kh trng (Auto Clave) ca hng Tomy Nht Bn
31
- Cn in t ca c
- My o pH
- T sy
- Bnh tam gic c kch thc t 250ml - 500ml, pipet loi 1ml, dng c nui
cy...
* Ho cht
Thnh phn ho cht ca mi trng Murashige & Skoog (MS) c
trnh by ph lc bng 1 (trang 84); mi trng Vacin Went (VW) c
trnh by ph lc bng 2 (trang 85); mi trng Robert Ernst (RE) c
trnh by ph lc bng 3 (trang 85).
* Cc cht iu tit sinh trng
1.BA: (Benzyladenine acid )
2.Kin: Kinetin (6-furfuryl-aminopurine)
3.TDZ: Thidiazuron (N-phenyl-N,-1,2,3-thiadiazol-5-ylurea)
4. NAA: ( -naphthalene acetic acid)
5. IBA ( Indolbutyride acid)
* Dch nghin hp cht hu c t nhin : Khoai ty, chui,...
* Cc yu t nh hng n nhn nhanh in vitro: ng, than hot tnh,
nc da...
2.1.3. Cc gi th s dng trong nghin cu
Hnh 2.2. Ru Sphagnum moss
Hnh 2.3. Dn t qu
32
2.1.4. Cc loi phn bn s dng
Hnh 2.4. HVP 1601 WP Hnh 2.5. Growmore

N: P: K = 30: 10: 10
(M) N: P: K = 30: 10: 10
Hnh 2.6. B1 Thi

Lan
a) Phn bn HVP 1601 WP (30:10:10) phn bn qua l v r, gip cy

lan sinh trng v pht trin tt, c xut x t Vit Nam.
Thnh Phn:
+ N tng s: 30%; P2O5: 10%; K2O: 10%; Ca: 0,05%; B: 0,02%; Cu: 0,05%
Fe: 0,15%; Mg: 0,015%; Mg: 0,015%; Mo: 0,005%; S: 0,2%; Zn: 0,15%
+ Alkylphenoxypolyethoxyenthanol: 0,05%
b) Phn bn Growmore c t l N: P: K = 30: 10: 10 l loi phn bn
qua l v r, gip cy lan sinh trng v pht trin tt, c xut x t M.
Thnh phn:
+ N tng s: 30% (NH4+: 2,0%; NO3: 3,2% ); P2O5: 10%; K2O: 10%
33
+ Cu : 0,05%; Cu EDTA: 0,05%; Fe: 0,10% ; Fe EDTA: 0,10%; Mn:

0,05%; Mn EDTA: 0,05%; Mo: 0,0005%; Zn: 0,05%; Zn EDTA : 0,05%
c) B1 Thi Lan l loi phn bn nc phun qua l v r, gip cy lan
sinh trng v pht trin tt, c xut x t Thi Lan.
Thnh phn:
P2O5: 2,0%; Fe: 0,10%; Fe EDTA: 0,10%; B1: 0,10 %; NAA: 0,04%.
2.2. Ni dung nghin cu
t c mc ch nghin cu, ti thc hin cc ni dung chnh
sau y:
2.2.1 Nghin cu to vt liu khi u cho nui cy in vitro
2.2.2. Nghin cu nhn nhanh chi cy Lan A. roxburghii (Wall.) Lindl.
2.2.3. Nghin cu to cy Lan A. roxburghii (Wall.) Lindl. hon chnh
2.2.4. Nghin cu a cy Lan A. roxburghii (Wall.) Lindl. ra vn m
2.3. Phng php nghin cu
2.3.1. Phng php ly mu, kh trng v nui cy to vt liu khi u
Lan A. roxburghii (Wall.) Lindl.
- Cc chi v thn c ly t cy m khe mnh, sch bnh c ra
sch, ct thnh tng on di 2 3 cm. Cc mu c lau bng cn 70o, sau
kh trng bng oxy gi (15% H2O2), hypochlorite-Na (3% NaOCl) v ra
li nhiu ln bng nc ct v trng ri cy trn mi trng Murashige &
Skoog (MS)
- Cc cng thc c b tr theo kiu ngu nhin hon ton vi 3 ln
nhc li, mi ln nhc li 30 mu/CT
Cc th nghim tin hnh:
34
Th nghim 1: Nghin cu nh hng ca cht kh trng H2O2 v NaOCl ti

t l sng v trng ca mu cy
Bng 2.1. Nghin cu nh hng ca cht kh trng H2O2 v NaOCl
Cng
thc
Thi gian v nng

x l nc oxy gi
(H2O2 )
Cng
Thi gian v nng x
thc
l (NaOCl)
CT1
15% H2O2 + 5 pht
CT1
3% NaOCl + 5 pht
CT2
15% H2O2 + 10 pht
CT2
3 % NaOCl + 10 pht
CT3
15% H2O2 + 15 pht
CT3
3 % NaOCl + 15 pht
CT4
15% H2O2 + 20 pht
CT4
3 % NaOCl + 20 pht
CT5
15% H2O2 + 25 pht
CT5
3 % NaOCl + 25 pht
CT6
3 % NaOCl + 30 pht
Mi trng MS + 20g/L ng + 100ml/L nc da + 6,5 g/L thch

Th nghim 2: nh hng ca mi trng khong khc nhau n kh nng
to chi t mu cy ban u
Bng 2.2. nh hng ca mi trng khong khc nhau n kh nng
Cng thc
Mi trng khong c
bn
Cht b sung
Murashige & Skoog (MS)
20g ng + 100ml nc da
Vacin Went (VW)
+ 6,5g thch trong 1lt mi
Robert Ernst (RE)
trng, pH 5,5
35
Tt c cc cng thc u b sung (20g/L ng + 100ml/L nc da +

6,5 g/L thch)
Th nghim 3: nh hng ca cht iu tit sinh trng BA n kh nng to
chi t mu cy ban u
CT1(/C): Nn (MS + 20g/L ng + 100ml/L nc da + 6,5 g/L thch)
CT2: Nn + 0,5mg/L BA
2.3.2. Phng php nghin cu nhn nhanh chi cy Lan A. roxburghii
(Wall.) Lindl.
Cc chi in vitro thu c trong nui cy ban u chuyn sang mi
trng nhn nhanh chi MS + 20g/L ng + 100ml/L nc da + 6,5g/L
thch pH 5,5 v b sung cc cht kch thch sinh trng Kinetin (Kin),
Thidiazuron (TDZ), Benzyladenine acid (BA), Naphtyl axetic axit (NAA)
nng dao ng t 0,1 3,0mg/L ty theo mc ch th nghim. Ngoi ra
trong mi trng nui cy cn b sung hp cht hu c t nhin nh dch
nghin khoai ty...
Cc cng thc c b tr theo kiu ngu nhin hon ton vi 3 ln
nhc li, mi ln nhc li 30 mu/CT.
Th nghim 4: Nghin cu nh hng ca Kin n kh nng nhn nhanh chi
CT1(/C): Nn (MS + 20g/L ng + 100ml/L nc da + 6,5 g/L
thch)
36
CT2: Nn + 0,5mg/L Kin

`

Th nghim 5: Nghin cu nh hng ca TDZ n kh nng nhn nhanh

chi Lan A. roxburghii (Wall.) Lindl.
CT1(/C): Nn (MS + 20g/L ng + 100ml/L nc da + 6,5g/L
thch)
CT2: Nn + 0,2mg/L TDZ
Th nghim 6: Nghin cu nh hng ca t hp (TDZ + IBA) n kh nng
nhn nhanh chi Lan A. roxburghii (Wall.) Lindl.
ti tm hiu nh hng ca Auxin n qu trnh pht sinh chi c s
tng tc vi TDZ. ti tin hnh b sung IBA vi cc nng khc nhau
vo mi trng l MS + 20g/L ng + 100ml/L nc da + 6,5 g/L thch +
0,6 mg/L TDZ, pH 5,5 (mi trng ny xc nh l mi trng c h s nhn
chi cao nht th nghim trn), nng IBA thay i l:
CT1(/C ): Nn (MS + 20g/L ng + 100ml/L nc da + 6,5g/L
thch + 0,6mg/L TDZ )
CT2: Nn + 0,5mg/L IBA
37
Th nghim 7: Nghin cu nh hng ca t hp (BA + NAA) n kh

nng nhn nhanh chi Lan A. roxburghii (Wall.) Lindl.
T mi trng tt nht th nghim nghin cu nh hng ca BA n
kh nng to chi t mu cy ban u, ti tin hnh b sung NAA cc
nng thay i nh sau:
thch + 2,0 mg/L BA)
CT2: Nn + 0,5mg/L NAA
Th nghim 8: Nghin cu nh hng ca dch nghin khoai ty n kh nng
T cc th nghim trn, chng ti thu c mi trng MS + 20g/L
ng + 100ml/L nc da + 6,5g/L thch + 2,0mg/L BA + 1,0mg/L NAA,
pH 5,5 l tt nht cho kh nng nhn nhanh chi. Tuy nhin, tm ra mi
trng thch hp hn cho nhn nhanh chi. Chng ti tin hnh nghin cu
nh hng phi hp ca dch nghin khoai ty vi t hp BA v NAA. Vi
dch nghin khoai ty c b sung vo mi trng vi hm lng thay i
l:
thch + 2,0mg/L BA + 1,0mg/L NAA)
CT2: Nn + 20g/L dch nghin khoai ty
38
2.3.3. Phng php nghin cu to cy Lan A. roxburghii (Wall.) Lindl.

hon chnh
Khi cc chi t chiu cao khong 3 - 5cm vi khong 3 - 4 l th
chuyn sang giai on to cy hon chnh. Cc th nghim c t trn mi
trng nn (MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch, pH 5,5 ).
Chng ti tin hnh cc th nghim xc nh nh hng ca than hot tnh c
hm lng (0,5 2,0g/L), cht iu tit sinh trng NAA (0,3 - 1,5mg/L) ti
giai on to cy hon chnh.
Cc cng thc c b tr theo kiu ngu nhin hon ton vi 3 ln
nhc li, mi ln nhc li 20 mu/CT.
Th nghim 9: nh hng ca than hot tnh
Than hot tnh c b sung vo mi trng MS + 20g/L ng +
100ml/L nc da + 6,5g/L thch, pH 5,5, hm lng than hot tnh thay i
nh sau:
thch)
CT2: Nn + 0,5g/L than hot tnh
Th nghim 10: nh hng ca NAA
T th nghim 9 chng ti tm ra hm lng than hot tnh 1,0g/L l
tt nht cho kh nng to cy hon chnh. tm ra mi trng thch hp hn
cho kh nng to cy hon chnh, chng ti tip tc tin hnh nghin cu nh
hng NAA b sung vo mi trng MS + 20g/L ng + 100ml/L nc
39
da + 6,5g/L thch + 1,0g/L than hot tnh, pH 5,5 vi nng NAA thay
i trong mi cng thc th nghim l:
thch + 1,0g/L than hot tnh)
iu kin nui cy: Mi trng c v trng 1210C v 1at trong 20 pht.
Cng nh sng: 2400 - 3000 lux, nhit phng nui: 25
20C, s gi
chiu sng: 16 gi/ngy.

Cc th nghim trn c thc hin phng nui cy m t bo thc
vt ca Trung tm Sinh hc Thc nghim - Vin ng dng Cng ngh - B
Khoa hc v Cng ngh.
2.3.4. Phng php a cy Lan A. roxburghii (Wall.) Lindl. ra vn
m
Cc cy sau khi nui cy trong phng th nghim t tiu chun chiu
cao khong 5 - 7cm, c khong 3 - 4 l, 3 - 5 r tin hnh ra cy. Trc khi
a cy con ra trng ngoi t nhin, tin hnh hun luyn cy quen dn vi
iu kin mi trng bn ngoi. Thi gian ny ko di 7 ngy v tng dn
cng vo nhng ngy cui tng nhanh kh nng thch nghi ca cy.
Cc cy con trong ng nghim ly ra c ra ht thch, trnh dp nt. Sau
nhng vo dung dch thuc nm ri trng vo cc gi th khc nhau ty
vo mc ch th nghim. Chng ti ra cy vo ma thu v tin hnh cc th
40
nghim nghin cu nh hng ca gi th, mt s loi phn bn khc nhau

c t l cy sng cao v cy sinh trng, pht trin tt.
Cc th nghim trn c nui trng trong iu kin nh li vi cc iu
kin:
- m gi th: 70 80%
- m khng kh: 90 95%
- Nhit nui trng: 20-25 0C
- nh sng: che sng > 70%
- Cc cy con khi trng c ti nc 1-2 ln/ngy ty theo iu kin thi
tit.
- Cc cng thc c b tr theo kiu ngu nhin hon ton vi 3 ln nhc
li, mi ln nhc li 5 cy/chu.
Th nghim 11: nh hng ca gi th
CT1: Ru Sphagnum moss
CT2: Dn t qu
CT3: Ru Sphagnum moss + Dn t qu (1:1)
Phun phn bn Growmore (30:10:10), phun 1tun/ln, liu lng
0,5g/L.
Th nghim 12: nh hng ca mt s loi phn bn khc nhau
CT1: HVP (30:10:10)
CT2: Growmore (30: 10:10)
CT3: B1 Thi Lan
Cc phn bn c phun 1tun/ln, liu lng 0,5g/L.
Cc th nghim ngoi vn m: c t trong nh li ca Trung tm
Sinh hc Thc nghim - Vin ng dng Cng ngh - B Khoa hc v Cng
ngh.
41
2.4. Cc ch tiu theo di

* Cc ch tiu theo di trong nui cy in vitro
Tng s mu nhim
- T l mu nhim (%) =
x 100
Tng s mu a vo
Tng s mu sng
- T l mu sng (%) =
x 100
Tng s mu a vo
Tng s mu cht
- T l mu cht (%) =
x 100
Tng s mu a vo
Tng s chi sau 8 tun nui cy
- H s nhn chi (ln ) =

Tng s chi ban u
Tng chiu cao chi
- Chiu cao trung bnh ca chi (cm) =
Tng s cy th nghim
- S l trung bnh/chi (l) = Tng s l/Tng s chi th nghim
- Cht lng chi bt:
Chi tt: chi mp, l xanh thm
Chi kh: chi bnh thng, l xanh
Chi trung bnh: Chi hi gy, l xanh.
42
Chi km: Chi gy, l xanh nht, hoc chi b d dng

Tng chiu cao cy
- Chiu cao trung bnh ca cy (cm) =
Tng s cy th nghim
- S l trung bnh/cy (l) = Tng s l/Tng s cy th nghim
- S r trung bnh (r) = Tng s r/Tng s cy th nghim
- S nhnh trung bnh (nhnh) = Tng s nhnh/Tng s cy th nghim
* Cc ch tiu theo di th nghim giai on a cy ra vn m (theo
di sau 12 tun):
Tng cy sng (cy)
- T l cy sng (%) =
x 100
Tng cy ban u (cy)
Tng chiu cao cy
- Chiu cao trung bnh ca cy (cm) =

Tng s cy th nghim
- S l trung bnh (l) = Tng s l/Tng s cy th nghim
- S r trung bnh mi xut hin (r) = Tng s r mi ra/Tng s cy th
nghim
2.5. Phng php x l s liu
S dng phng php thng k sinh hc phn tch cc s liu th
nghim trn chng trnh IRRISTAT for Windows (5.0).
43
CHNG 3 - KT QU NGHIN CU V THO LUN

3.1. Kt qu nghin cu kh trng to vt liu khi u cho nui cy in
vitro loi Lan A. roxburghii (Wall.) Lindl.
y l giai on a i tng nui cy t ngoi vo iu kin nui
cy v trng in vitro. V vy, i vi tt c cc loi cy trng khc nhau, vic
la chn loi ha cht, nng v thi gian kh trng thch hp c ngha
i vi thnh cng ca qu trnh nui cy in vitro. Giai on ny cn t c
cc yu cu sau: T l nhim thp, t l sng cao, m tn ti, phn ha v
sinh trng tt.
3.1.1. Kt qu nghin cu nh hng ca cht kh trng ti t l sng
ca mu cy trn mi trng MS
Bng 3.1. nh hng ca cht kh trng H2O2 ln mu cy Lan
A. roxburghii (Wall.) Lindl.
Thi gian v nng
CTTN
x l nc oxy gi
(H2O2 )
T l
T l mu
T l mu
cht (%)
nhim (%)
mu sng
(%)
CT1
15% H2O2 + 5 pht
3,6 e
80,8 a
15,6 e
CT2
15% H2O2 + 10 pht
5,4 d
70,6 b
24,5 c
CT3
15% H2O2 + 15 pht
20,9 c
35,9 c
43,2 a
CT4
15% H2O2 + 20 pht
40,5 b
24,0 d
35,7b
CT5
15% H2O2 + 25 pht
57,2 a
22,1 e
20,7 d
LSD 0,05
1,06
1,67
0,34
CV (%)
2,3
2,0
2,1
44
Ghi ch: Nn mi trng MS + 20 g/L ng + 100ml/L nc da + 6,5g/L thch, pH 5,5

a, b, c, d, e: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%
Cc mu nui cy l chi nh, chi bn c chiu di 20 - 30cm ca loi

Lan A. roxburghii (Wall.) Lindl. c a vo kh trng bng H2O2, NaOCl
l nhm cht t gy c hi cho c th con ngi. Sau 4 tun nui cy, kt qu
c trnh by bng 3.1.
Qua bng 3.1 cho thy vic to vt liu khi u kh trng t chi bn
v chi nh ca cy Lan A. roxburghii (Wall.) Lindl. l tng i kh, mu
c t l nhim cao.
Kh trng bng H2O2 vi nng nh nhau (15%) song thi gian kh
trng tng t 5 pht n 25 pht t l mu nhim gim, nhng s mu cht
tng do thi gian kh trng lu. T l mu sng v trng cao nht l sau 15
pht (43,20%), t l mu nhim cao nht l sau 5 pht (80,80%). Khi tng
thi gian kh trng ln 25 pht th t l mu cht tng ln 57,20%, bn cnh
khi tng thi gian kh trng cho thy nh hng yu n t l mu sng.
T kt qu c nhn xt H2O2 l cht kh trng b mt yu i vi mu cy
Lan A. roxburghii (Wall.) Lindl. a vo nui cy in vitro, chnh v vy chng
ti tip tc th nghim vi loi cht kh trng khc NaOCl. Kt qu c
trnh by bng 3.2
T bng 3.2 cho thy: Khi s dng cht kh trng NaOCl vi nng nh
nhau 3%, thi gian x l tng t 5 pht n 30 pht cho thy t l mu nhim
gim t 80,70% xung 5,60%; s mu cht tng t 2,1% n 76,0%. T l ti
sinh ca mu v mu sng tng khi thi gian kh trng tng ln 15 pht v
t t l mu sng cao nht l 65,5% v t l mu sng gim xung 18,40%
thi gian kh trng l 30 pht.
45
Bng 3.2. nh hng ca cht kh trng NaOCl ln mu cy Lan

Cng thc
th nghim
Thi gian v nng

x
l (NaOCl)
T l mu
T l mu
T l mu
cht (%)
nhim (%)
sng (%)
CT1
3% NaOCl + 5 pht
2,1 f
80,7 a
17,2 e
CT2
3% NaOCl+10 pht
13,4e
51,0 b
35,6 c
CT3
3%NaOCl+15 pht
18,8 d
15,7 c
65,5 a
CT4
3% NaOCl+20 pht
31,4 c
13,2 d
55,4 b
CT5
3% NaOCl+25 pht
61,2b
10,3 e
28,5 d
CT6
3% NaOCl+30 pht
76,0 a
5,6 f
18,4 e
LSD 0,05
1,0
1,8
1,2
CV (%)
1,7
2,3
1,8
Ghi ch: Nn mi trng MS + 20 g/L ng + 100ml/L nc da + 6,5g/L thch, pH 5,5

a, b, c, d, e, f: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%.
T kt qu trn, chng ti la chn NaOCl nng 3% vi thi gian 15

pht cho kt qu tt nht khi a mu A. roxburghii (Wall.) Lindl. vo nui
cy in vitro.
3.1.2. nh hng ca mi trng khong n kh nng to chi t mu
cy ban u
m bo thnh cng i vi nhn ging bng phng php nui cy
m, nhn thy giai on quan trng l xc nh mi trng nui cy thch
hp cho i tng cn nhn ging. Cc cng b v mi trng nhn ging
loi cy Lan A. roxburghii (Wall.) Lindl. cn rt hn ch. Do vy, ti th
nghim mt s mi trng khong khc nhau: MS, VW, RE nhn ging.
T chn ra mi trng thch hp nht nui cy loi Lan A. roxburghii
46
(Wall.) Lindl. Trong iu kin nui cy m t bo, h s nhn v cht lng

chi ph thuc vo thnh phn mi trng, nng cht dinh dng c trong
mi trng nui cy. Cc nghin cu giai on ny nhm tm ra c mi
trng thch hp cho giai on to chi t mu cy ban u, giai on ny c
vai tr quyt nh n hiu qu ca qu trnh nhn ging. Kt qu c trnh
by bng 3.3 v hnh 3.1.
Qua bng 3.3 cho thy: Cc mi trng khong khc nhau, kh nng
nhn nhanh chi v chiu cao trung bnh ca chi l khc nhau.
Trn mi trng RE cho h s nhn chi thp nht, h s nhn chi ch
t 1,06 ln xp mc c v chiu cao cy t 0,21cm c xp mc b
theo so snh LSD. Qua quan st hnh thi, chi pht trin chm hn, chi c
mu xanh nht v nh.
Trn mi trng VW cho h s nhn chi l 1,30 ln v chiu cao chi
t 0,25cm c xp mc b theo so snh LSD. Qua quan st hnh thi
chi c mu xanh nhng chi yu.
Bng 3.3. nh hng ca mi trng khong khc nhau n kh nng
Mi
H s nhn
Chiu cao TB
Cht lng
trng
chi (ln)
ca chi (cm)
chi
CT1
RE
1,06 c
0,21b
CT2
MS
1,49 a
0,42 a
+++
CT3
VW
1,30 b
0,25 b
++
LSD 0,05
0,17
0,05
CV (%)
4,6
8,3
CTTN
Ghi ch: Cc cng thc u c b sung (20g/L ng+100ml/L nc da+6,5g/L

thch, pH 5,5)
47
a, b, c: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%

+: Chi sinh trng km
++ : Chi sinh trng mc trung bnh
+++: Chi sinh trng mc kh
++++: Chi sinh trng tt, mp
Trn mi trng MS c h s nhn chi t cao nht l 1,49 ln v

chiu cao trung bnh ca chi t 0,42cm, c xp mc a trong so snh
LSD. Qua quan st hnh thi, nhn thy cc chi pht trin u v sinh trng
mnh hn. Cc kt qu ca chng ti ph hp vi nhiu tc gi khc khi
nui cy in vitro cho nhiu loi Lan khc nhau. Tc gi N. Ahamed Sherif v
cng s (2012) s dng chi nh v chi bn ca cy Lan A. elatus
Lindley lm vt liu nui cy trn mi trng MS. Zhou YuMei v cng s
(2009) nghin cu xy dng quy trnh nhn nhanh ging cy Lan A.
formosanus trn mi trng MS.
T kt qu nghin cu trn, chng ti chn mi trng MS lm mi
trng c bn cho cc qu trnh nghin cu sau ny ca cy A. roxburghii
(Wall.) Lindl.
RE
MS
VW
Hnh 3.1. nh hng ca mi trng khong khc nhau n kh nng

48
3.1.3. nh hng ca cht iu tit sinh trng BA n kh nng to

chi t mu cy ban u
BA l mt cht iu tit sinh trng nhn to thuc nhm Cytokinin, l
nhm c s dng rng ri nht trong k thut nui cy m hin nay. Tc
dng ch yu ca n l kch thch s phn chia t bo, thc y s hot ng
ca chi bn. Nng s dng khc nhau ty i tng thc vt nui cy,
cc kt qu nghin cu cho thy: nng 22M thch hp vi s pht trin
chi Cymbidium aloifolium, nhng i vi Dendrobium aphyllum v
Dendrobium moschatum nng thch hp nht cho s pht trin ca chi li
l 44M (N.R.Nayak v cng s, 1997). tm nng ph hp ca BA ln
s to chi ban u cho mu cy cy Lan A. roxburghii (Wall.) Lindl., trong
th nghim ny chng ti b tr 7 nng khc nhau: 0; 0,5; 1,0; 1,5; 2,0; 2,5
v 3,0mg/L. Sau 8 tun nui cy kt qu c trnh by bng 3.4.
Qua kt qu thu c bng 3.4 cho thy: h s nhn chi dao ng t
1,49 n 4,44 ln. H s nhn chi c xp theo th t t cao xung thp
trong so snh (LSD) l a, b, c, d, e cho thy. nng 2,0mg/L c h s
nhn chi t cao nht l 4,44 ln mc a. Trong khi cng thc khng
b sung (/C) c h s nhn chi t thp nht l 1,49 ln xp mc e;
nng 2,5mg/L c h s nhn chi l 3,6 ln mc b; nng 3,0mg/L
v 1,5mg/L c h s nhn chi tng ng l 2,61 v 2,65 ln mc c;
nng 0,5mg/L v 1,0mg/L c h s nhn chi tng ng l 2,15 v 2,36
ln mc d.
i vi ch tiu chiu cao trung bnh ca chi cho thy: Tt c cc cng
thc c b sung BA u cho kt qu cao hn cng thc khng b sung BA
vo mi trng nui cy. Chiu cao trung bnh ca chi dao ng t 0,38cm
n 2,53cm v c xp vo cc nhm phn mc a, ab, c, trong so snh
(LSD). Trong : nng 1,0 2,5mg/L c chiu cao trung bnh ca chi
49
t cao nht l 2,21 2,53cm nhm a; cng thc (/C) khng b sung
BA c chiu cao trung bnh ca chi thp nht l 0,38cm mc c; nng
0,5mg/L c chiu cao trung bnh ca chi l 2,05cm mc bc. Nng
3,0mg/L c chiu cao trung bnh ca chi l 1,75cm mc b. nng
BA t 1,0 n 2,5mg/L chi mp, l xanh thm.
Bng 3.4. nh hng ca cht iu tit sinh trng BA n kh nng to
chi t mu cy ban u
Chiu cao
TB ca
Cht
lng
chi (cm)
chi
1,49 e
0,38 c
0,5
2,15 d
2,05 ab
++
CT3
1,0
2,36 d
2,21 a
+++
CT4
1,5
2,65 c
2,32 a
+++
CT5
2,0
4,44 a
2,53 a
++++
CT6
2,5
3,60 b
2,43 a
+++
CT7
3,0
2,61 c
1,75 b
LSD 0,05
0,21
0,33
CV (%)
4,3
4,7
Nng
BA (mg/L)
H s nhn
chi (ln)
CT1(/C)
0,0
CT2
CTTN
Ghi ch: /C: Nn mi trng MS + 20 g/L ng + 100ml/L nc da + 6,5g/L

thch, pH 5,5.
a, b, c, d: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%.
+: Chi sinh trng km
50
Tuy nhin khi tng nng BA vt qu 2,5mg/L cht lng chi c

xu hng gim, chi gy, l xanh nht v b d dng (loi km).
T phn tch trn cho thy: nng BA thch hp nht b sung vo
mi trng MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch, pH 5,5
vi mc ch to chi t mu cy ban u l 2,0mg/L. Trong mi trng ny
c h s nhn chi t 4,44 ln, chiu cao trung bnh ca chi t 2,53cm chi
mp, xanh thm.
3.2. Giai on nhn nhanh chi
Giai on nhn nhanh chi l giai on quan trng to ra mt khi
lng ln cy con in vitro t mu cy ban u trong mt thi gian ngn.
giai on ny s sinh trng v pht trin ca m nui cy ph thuc cht ch
vo iu kin nui cy (nhit , nh sng), mi trng nui cy (thnh phn
dinh dng, cht iu tit sinh trng v t l gia chng). Ty theo tng loi
cy, c th b sung vo mi trng nui cy cc cht iu tit sinh trng vi
nng v t l thch hp c c h s nhn chi v cht lng chi cao
nht.
Vi mc ch nhn nhanh chi A. roxburghii (Wall.) Lindl., chng ti
tin hnh nghin cu nh hng mt s cht iu tit sinh trng v hp cht
t nhin n kh nng nhn nhanh chi ca Lan A. roxburghii (Wall.) Lindl.
3.2.1. nh hng ca Kinetin (Kin) n kh nng nhn nhanh chi Lan
Kinetin thuc nhm Cytokinin chng c b sung vo mi trng ch
yu kch thch s phn chia t bo v phn ha chi bt nh, tng cng
kh nng tng hp cht dip lc ca cy. nh gi kh nng nh hng
ca Kin ln qu trnh tng h s nhn chi v chiu cao trung bnh ca chi.
51
Chng ti tin hnh th nghim trn mi trng (MS + 20g/L ng +

100ml/L nc da + 6,5g/L thch, pH 5,5) vi cc nng Kin thay i l
/C(0); 0,5; 1,0; 1,5; 2,0; 2,5mg/L. Kt qu c trnh by bng 3.5.
Bng 3.5. nh hng ca Kin n kh nng nhn nhanh chi
CTTN
Nng
Kin (mg/L)
H s
Chiu cao
Cht
nhn chi
TB ca chi
lng
(ln)
(cm)
chi
CT1(/C)
0,0
1,50 f
0,39 cd
CT2
0,5
2,88 a
0,82 a
+++
CT3
1,0
2,42 b
0,60 b
++
CT4
1,5
2,16 c
0,49 c
++
CT5
2,0
1,89 d
0,40 cd
CT6
2,5
1,69 e
0,34 d
LSD 0,05
0,12
0,1
CV (%)
3,2
4,5

thch, pH 5,5.
a, b, c, d, e, f: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%.
+ : Chi sinh trng km
52
Sau 8 tun nui cy kt qu thu c bng 3.5 cho thy. Nhn chung,
khi b sung Kin vo mi trng nui cy cc cng thc trong th nghim
u c h s nhn v chiu cao trung bnh ca chi tng khng ng k. Song
cng th hin s sai khc gia cc cng thc so snh theo gi tr khc bit c
ngha nh nht (LSD). C th: cng thc i chng khng b sung Kin cho
t l h s nhn chi v chiu cao trung bnh ca chi thp nht l 1,50 ln v
0,39cm. Khi b sung 0,5mg/L vo mi trng h s nhn chi v chiu cao
trung bnh ca chi t cao nht tng ng l 2,88 ln v 0,82 cm. Tip tc
tng nng Kin t 1,0mg/L n 2,5mg/L, h s nhn chi v chiu cao
trung bnh chi c xu th gim dn. nng 2,5mg/L h s nhn chi gim
xung thp ch l 1,69 ln, trong khi chiu cao trung bnh ca chi li gim
thp hn so vi i chng 0,34cm.
Kt qu nghin cu ca chng ti cho thy: h s nhn chi ca loi
Lan A. roxburghii (Wall.) Lindl. khng tng theo t l thun vi mc tng ca
nng Kin t 0,5 2,5mg/L. Vi cy A. roxburghii (Wall.) Lindl., ngng
nng Kin thch hp cho h s nhn chi v chiu cao trung bnh ca
chi l 0,5mg/L. Vt qu ngng ny s lm c ch s nhn chi. la
chn ng nng Kin thch hp cho nhn nhanh chi ngoi ch tiu h s
nhn chi, chiu cao trung bnh ca chi cn phi ch n cht lng chi.
Ti cng thc th nghim c nng Kin 0,5mg/L chi pht trin bnh thng
sau 8 tun nui cy c nh gi l mc kh (chi khe, l xanh). Tuy
nhin nng Kin tng ln cao qu 1,0 - 2,5mg/L th s lng v cht
lng chi c xu th gim chi gy v l xanh nht.
Vy vi tin cy 95% cho chng ti nhn xt l: Nng Kinetin
thch hp nht cho h s nhn chi v chiu cao trung bnh ca chi l
0,5mg/L.
53
3.2.2. Nghin cu nh hng ca Thidiazuron (TDZ) n qu trnh nhn

nhanh chi Lan A. roxburghii (Wall.) Lindl.
Mc d b sung Kinetin trong th nghim trn c kh nng nhn nhanh
chi v chiu cao chi nhng h s nhn cn thp, cht lng chi cha cao.
Nn chng ti tip tc tin hnh nghin cu b sung TDZ vo mi trng
nui cy.
TDZ c xem l mt Cytokinin c tc ng rt hiu qu n vic
nhn chi ca nhiu loi cy. Mt nghin cu ch ra rng, nng t 1,0
2,0mg/L TDZ l thch hp cho s nhn chi cy Lan A. formosanus (N.V.
Ket v cng s, 2004). i vi cy Lan A. roxburghii (Wall.) Lindl., nh
hng ca TDZ n qu trnh nhn chi nh th no, kt qu nghin cu ca
chng ti c th hin qua bng 3.6.
TDZ c tc ng r rt n qu trnh nhn chi v chiu cao chi. H
s nhn chi dao ng t 1,50 ln n 3,97 ln. Theo so snh (LSD): nhn
thy cng thc 4 c nng 0,6mg/L, t h s nhn chi cao nht l 3,97 ln
mc a so vi cng thc i chng khng b sung TDZ c h s nhn chi
thp nht l 1,50 ln mc e; cng thc 3 c h s nhn chi l 3,58 ln
mc b; cng thc 5 c h s nhn chi l 3,24 ln mc c; cng thc 2
v cng thc 6 c h s nhn chi tng ng l 2,86 ln v 2,79 ln mc
d.
thc c b sung TDZ u cho kt qu cao hn cng thc i chng khng b
sung TDZ vo mi trng nui cy. Chiu cao trung bnh ca chi dao ng
t 0,40cm n 2,51cm v c xp vo cc nhm phn mc a, ab, b, c trong
so snh (LSD). Trong : cng thc 4 v cng thc 5 tng ng l 0,6 v 0,8
mg/L TDZ c chiu cao trung bnh ca chi t cao nht l 2,51cm v 2,26cm
nhm a v ab; cng thc i chng khng b sung TDZ c chiu cao
54
trung bnh ca chi thp nht l 0,40cm mc c; cng thc 2, cng thc 3
v cng thc 6 c chiu cao trung bnh ca chi t tng ng l 1,95cm;
2,20cm v 2,01cm mc b trong so snh (LSD).
Bng 3.6. nh hng ca TDZ n kh nng nhn nhanh chi ca Lan
Nng
CTTN
TDZ
H s nhn
(mg/L)
chi (ln)
Chiu cao
TB ca chi
(cm)
Cht
lng chi
CT1(/C)
0,0
1,50 e
0,40 c
CT2
0,2
2,86 d
1,95 b
CT3
0,4
3,58 b
2,20 b
+++
CT4
0,6
3,97 a
2,51 a
++++
CT5
0,8
3,24 c
2,26 ab
++ ++
CT6
1,0
2,79 d
2,01 b
++
LSD 0,05
0,24
0,3
CV (%)
4,5
0,9

thch, pH 5,5.
Bn cnh ch tiu v h s nhn chi, chiu cao trung bnh ca chi, ch

tiu cm quan v cht lng cho thy: Khi b sung TDZ vo mi trng nui
55
cy nng t 0,6 0,8mg/L th hin cht lng chi rt tt (chi xanh v

mp) hn so vi i chng khng b sung TDZ.
T phn tch trn cho thy: Nng TDZ thch hp nht b sung
vo mi trng MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch, pH
5,5 vi mc ch nhn nhanh chi l 0,6mg/L. Trong mi trng ny c h s
nhn chi t 3,97 ln v chiu cao trung bnh ca chi t 2,51cm, chi mp,
xanh thm.
Theo Zhou YuMei v cng s (2009) cho rng b sung nng
0,1mg/L TDZ vo mi trng nui cy l ti u cho nhn nhanh chi loi Lan
A. formosanus. Cng b ca nhm tc gi cho thy nng TDZ vi cng
mt loi cc ch tiu khc nhau l khng ging nhau. Tuy nhin cc kt qu
ca chng ti ch ra c s sai khc ca cc nng TDZ khi b sung
vo mi trng nui cy trn loi Lan A. roxburghii (Wall.) Lindl. nghin cu
n h s nhn, chiu cao trung bnh ca chi v cht lng chi.
3.2.3. Nghin cu nh hng ca t hp (TDZ + IBA) n kh nng nhn
Trong nui cy m t bo thc vt, t l Auxin/Cytokinin c vai tr
quan trng trong s bin ho c quan v quyt nh s sinh trng v pht
trin ca cy. Nhiu nghin cu cho thy t hp gia Auxin v Cytokinin hu
ht u c hiu qu tt hn so vi vic s dng ring l Cytokinin trong mi
trng nui cy. tm hiu nh hng ca Auxin n qu trnh nhn nhanh
chi c s tng tc vi TDZ. ti tin hnh b sung IBA vi cc nng
khc nhau vo mi trng: MS + 20g/L ng + 100ml/L nc da + 6,5g/L
thch + 0,6mg/L TDZ, pH 5,5 (mi trng ny xc nh l mi trng c h
s nhn chi v chiu cao trung bnh ca chi t cao nht th nghim
56
nghin cu nh hng ca TDZ n kh nng nhn nhanh chi), kt qu

c trnh by bng 3.7.
Bng 3.7. Kt qu nh hng ca t hp (TDZ + IBA) n kh nng
Cht iu tit sinh
H s
Chiu cao
Cht
trng (mg/L)
nhn chi
TB ca
lng
CTTN
TDZ
IBA
(ln)
chi (cm)
chi
CT1(/C)
0,6
0,0
3,96 d
2,51 b
CT2
0,6
0,5
9,16 a
2,85 a
++++
CT3
0,6
1,0
7,08 b
2,56 b
+++
CT4
0,6
1,5
6,36 c
2,28 c
++
CT5
0,6
2,0
6,18 c
2,15 c
LSD 0,05
0,42
0,19
CV (%)
3,5
4,0
Ghi ch: /C: Nn mi trng MS + 20 g/L ng + 100ml/L nc da + 6,5g/L thch+

0,6mg/L TDZ, pH 5,5
a, b, c, d: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%
Chng ti nhn thy nh hng ca t hp gia hai loi cht kch thch
sinh trng ny rt tt i vi vic nhn Lan A. roxburghii (Wall.) Lindl..
tt c cc cng thc th nghim, h s nhn chi cng nh s sinh trng ca
57
chi u c s thay i r rt. Cc cng thc c h s nhn chi dao ng t

3,96 n 9,16 ln. Kt qu th nghim c xp theo 4 mc phn nhm trong
so snh LSD theo th t t cao xung thp l a, b, c, d, cho thy l: H s
nhn chi cng thc 2 t cao nht l 9,16 ln mc a, trong khi cng
thc i chng h s nhn chi ch t 3,96 ln c xp mc d.
Khi tng nng IBA t 1,0 n 2,0mg/L kt hp vi 0,6mg/L TDZ
nhn thy h s nhn chi, chiu cao trung bnh ca chi gim xung theo
chiu tng ca IBA. iu ny c th do nng IBA cao qu gy c ch
n kh nng nhn nhanh chi Lan A. roxburghii (Wall.) Lindl. C th: H s
nhn chi cng thc 3 l 7,08 ln mc b; h s nhn chi cng thc 4
v cng thc 5 t tng ng l 6,36 ln v 6,18 ln mc c.
i vi ch tiu chiu cao trung bnh ca chi, so snh theo LSD cho
thy: Cng thc 2 (0,6mg/L TDZ + 0,5mg/L IBA) c chiu cao trung bnh
ca chi t cao nht l 2,85 cm mc a, trong cng thc i chng ch
t l 2,51 cm mc b. Khi tng IBA t 1,0 n 2,0mg/L kt hp vi
0,6mg/L TDZ nhn thy chiu cao trung bnh ca chi c xu hng gim
dn. cng thc 3 chiu cao trung bnh ca chi t 2,56cm. Trong khi
cc cng thc 4 v 5 c chiu cao trung bnh ca chi ch t 2,28cm v
2,15cm v mc c.
i vi ch tiu cht lng chi: cng thc c b sung t hp
0,6mg/L TDZ + 0,5mg/L IBA cho cht lng rt tt, chi xanh v mp.
Nh vy: T hp nng TDZ + IBA thch hp nht b sung vo
mi trng MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch, pH 5,5
nhn nhanh chi c h s nhn nhanh v chiu cao chi tt l 0,6mg/L
TDZ + 0,5mg/L IBA.
58
3.2.4. Nghin cu nh hng ca t hp (BA + NAA) n kh nng

S pht sinh pht trin ca chi chu tc ng cn bng ca cc cht
iu tit sinh trng Auxin v Cytokinin. ci thin h s nhn chi v cht
lng ti sinh chi, ti tin hnh b sung NAA vi cc nng khc
nhau vo mi trng: MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch
+ 2,0mg/L BA, pH 5,5 (mi trng ny xc nh l mi trng c h s nhn
chi v chiu cao trung bnh ca chi t cao nht th nghim nghin cu
nh hng ca BA n kh nng to chi t mu cy ban u), kt qu c
trnh by bng 3.8.
Qua bng 3.8 cho thy: Tt c cc cng thc c b sung t hp NAA
+ BA u cho kt qu cao hn cng thc khng b sung t hp BA + NAA
vo mi trng nui cy. H s nhn chi dao ng t 4,44 n 11,56 ln
chng t s phi hp gia BA + NAA c tc dng tch cc n nhn nhanh
chi A. roxburghii (Wall.) Lindl.
H s nhn chi cng thc 3 c nng (2,0mg/L BA + 1,0mg/L
NAA) t cao nht l 11,56 ln mc a. Trong khi cng thc i
chng (khng b sung NAA) h s nhn chi ch t 4,44 ln mc d.
Tuy nhin tng nng NAA vt qu 1,0mg/L th h s nhn chi, chiu
cao chi v cht lng chi c xu hng gim.
C th: H s nhn chi cng thc 4 (1,5mg/L NAA) ch t l 8,05
ln mc b v cng thc 5 (2,0mg/L NAA ) 6,75 ln l mc c.
i vi ch tiu chiu cao trung bnh ca chi cho thy: Khi b sung t
hp BA + NAA vo mi trng nui cy. Chiu cao trung bnh ca chi dao
ng t 2,22cm n 3,05cm, trong : cng thc 3 (2,0mg/L BA + 1,0mg/L
NAA) c chiu cao trung bnh ca chi t cao nht l 3,05cm c xp
mc a, trong khi cng thc i chng (2,0mg/L BA + 0,0mg/L NAA)
59
ch t 2,53cm xp mc c. Tip tc tng nng ca t hp, chiu cao

trung bnh ca chi c xu hng gim mnh ti mc thp hn so vi i
chng; cng thc 5 (2,0mg/L BA + 2,0mg/L NAA) c chiu cao trung bnh
ca chi t thp nht l 22,2 cm xp mc d. Cc cng thc 2 c chiu
cao trung bnh ca chi l 2,76cm xp mc b v cng thc 4 c chiu cao
trung bnh ca chi l 2,41cm xp mc c.
Bng 3.8. nh hng ca t hp (BA + NAA) n kh nng nhn
Cht kch thch sinh
H s
Chiu cao
Cht
trng (mg/L)
nhn chi
TB ca
lng
CTTN
BA
NAA
(ln)
chi (cm)
chi
CT1 (/C)
2,0
0,0
4,44 d
2,53 c
CT2
2,0
0,5
9,12 b
2,76 b
+++
CT3
2,0
1,0
11,56 a
3,05 a
++++
CT4
2,0
1,5
8,05 b
2,41 c
+++
CT5
2,0
2,0
6,75 c
2,22 d
LSD 0,05
1,14
0,13
CV (%)
7,8
2,8
Ghi ch: /C: Nn mi trng MS + 20 g/L ng + 100ml/L nc da + 6,5g/L thch+

2,0 mg/L BA, pH 5,5.
a, b, c, d: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%.
60
la chn ng nng t hp BA + NAA thch hp cho nhn

nhanh chi A. roxburghii (Wall.) Lindl., th ngoi ch tiu h s nhn chi,
chiu cao trung bnh ca chi cn phi ch n cht lng chi. Trong cc
cng thc th nghim c nng NAA tng t 0 n 1,0mg/L + 2,0mg/L
BA chi u pht trin rt tt, chi mp v l xanh thm. Tuy nhin nng
NAA cao qu 1,0mg/L kt hp vi 2,0mg/L th cht lng chi c xu th
gim, chi c hin tng vng nht v gy.
Nh vy: Nng phi hp gia 2,0mg/L BA + 1,0mg/L NAA vo
mi trng nn (MS b sung 20g/L ng + 100ml/L nc da + 6,5g/L
thch, pH 5,5) l thch hp nht cho nhn nhanh chi A. roxburghii (Wall.)
Lindl.(chi xanh, khe v mp). Kt qu ca chng ti cng ph hp vi
nghin cu ca Zhang F v cng s (2009) cho rng b sung vo mi trng
2mg/L BA v 1mg/L NAA thch hp nht cho s pht trin ca chi bn v
h s nhn chi i vi cy A. roxburghii.
3.2.5. Nghin cu nh hng ca dch nghin khoai ty n kh nng
Trong qu trnh nui cy in vitro vic b sung cc hp cht hu c t
nhin vo mi trng s c hiu qu trong qu trnh sinh trng, pht trin
ca mu cy. Ty vo cc loi cy trng khc nhau m b sung cc hp cht
hu c t nhin nh: Khoai ty, chui, to, c rt... l khc nhau.
Theo Thorpe v cng s (2008) trong dch nghin khoai ty c cha
cacbonhydrat di dng saccaroza, glucose v fructose, amino axit (21 loi
bao gm c lysine l mt axt amin thng khng c trong protein thc vt),
cc mui khong (K, Fe, Mg) v c bit l cc vitamin (C, B1, B6). Cng
theo tc gi th dch nghin khoai ty thng c b sung vo mi trng vi
61
nhn ging hoa lan v c bit c hiu qu vi mt s loi nh: Phalaenopsis,

Doritaenopsis.
xc nh hm lng dch nghin khoai ty ph hp ln qu trnh
nhn nhanh chi cy Lan A. roxburghii (Wall.) Lindl., t cc kt qu nghin
cu trn, chng ti la chn c mi trng (MS + 20g/L ng +
100ml/L nc da + 6,5g/L thch + 2,0mg/L BA+ 1,0mg/L NAA, pH 5,5)
c h s nhn v chiu cao trung bnh ca chi t cao nht. Trn c s
ti tip tc nghin cu b sung dch nghin khoai ty vo mi trng nui cy
vi hm lng khc nhau tip tc nhn nhanh cc cm chi. Kt qu c
trnh by qua bng 3.9 v hnh 3.2.
Bng 3.9. Nghin cu nh hng ca dch nghin khoai ty n kh
nng nhn nhanh chi Lan A. roxburghii (Wall.) Lindl.
CTTN
Cht iu tit
sinh trng
Hm
lng dch
(mg/L)
H s
nhn
Chiu
cao TB
chi
(ln)
ca chi
(cm)
Cht
lng
BA
NAA
nghin
khoai ty
(g/L)
CT1 (/C)
2,0
1,0
0,0
11,53 e
3,05 e
CT2
2,0
1,0
20
13,36 d
3,58 d
++
CT3
2,0
1,0
40
16,83 c
3,94 c
+++
CT4
2,0
1,0
60
18,08 b
4,09 b
++++
CT5
2,0
1,0
80
21,83 a
4,31 a
++++
CT6
2,0
1,0
100
17,58 bc
4,08 b
+++
LSD 0,05
1,33
0,12
CV (%)
4,5
1,8
chi
62
Ghi ch: /C: Nn mi trng MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch+

2,0 mg/L BA + 1,0 mg/L NAA, pH 5,5
+++ +: Chi sinh trng tt, mp
Kt qu nghin cu cho thy khi b sung dch nghin khoai ty

vo mi trng mi cy c tc dng tch cc n h s nhn nhanh chi,
chiu cao trung bnh ca chi, so vi mi trng khng b sung dch nghin
khoai ty.
S liu qua bng 3.9 cho thy: cng thc 5 b sung 80g/L dch nghin
khoai ty c h s nhn chi t cao nht l 21,83 ln mc a so vi cng
thc i chng khng b sung dch nghin khoai ty ch t 11,53 ln mc
e. Khi tip tc tng hm lng dch nghin khoai ty ln 100g/L th h s
nhn chi gim dn. cng thc 4 (60g/L dch nghin khoai ty) c h s
nhn chi l 18,08 ln mc b; cng thc 6 (100g/L dch nghin khoai ty)
c h s nhn chi l 17,58 ln mc bc; cng thc 3 v cng thc 2 tng
ng 40g/L, 20g/L dch nghin khoai ty c h s nhn chi l 16,83 ln mc
c v 13,36 ln mc d.
thc c b sung dch nghin khoai ty u c kt qu cao hn cng thc
khng b sung dch nghin khoai ty vo mi trng nui cy. Chiu cao
trung bnh ca chi dao ng t 3,05cm n 4,31cm v c xp vo cc
nhm phn mc a, b, c, d, e trong so snh LSD. Trong : cng thc 5 c
chiu cao trung bnh ca chi t cao nht l 4,31cm c xp mc a.
Trong khi cng thc i chng c chiu cao trung bnh ca chi t thp
nht l 3,05cm xp mc e. cng thc 4 v cng thc 6 c chiu cao
chi l 4,09cm v 4,08 xp mc b. Cng thc 3 c chiu cao trung bnh
63
ca chi l 3,94cm xp mc c; cng thc 2 c chiu cao chi t 3,58cm

xp mc d.
V ch tiu cht lng cho thy: Tt c cc cng thc c b sung dch
nghin khoai ty u cho cht lng chi cao hn so vi i chng. Cng
thc 5 cht lng chi t im cao nht, chi mp, khe v l xanh bng.
Nh vy: B sung dch nghin khoai ty vo mi trng nhn nhanh
c tc dng rt tt ti h s nhn chi, chiu cao trung bnh ca chi cng nh
cht lng ca chi. Hm lng dch nghin khoai ty b sung vo mi
trng MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch + 2,0mg/L
BA + 1,0mg/L NAA, pH 5,5 thch hp nht trong nhn nhanh l 80g/L.
Kt lun chung cho giai on nhn nhanh chi: Trong giai on nhn
nhanh cc Cytokinin kt hp vi Auxin nh: TDZ + IBA, BA + NAA u
c kh nng nhn nhanh chi v h s nhn cng nh cht lng chi kh
cao. Tuy nhin, s dng 2,0mg/L BA + 1,0mg/L NAA+ 80g/L dch nghin
khoai ty b sung vo mi trng MS + 20g/L ng + 100ml/L nc da +
6,5g/L thch, pH 5,5 cho h s nhn chi cao nht, cht lng chi tt nht,
ko di tui th ca chi trong nhn nhanh.
Hnh 3.2. Giai on nhn nhanh chi Lan A. roxburghii (Wall.) Lindl.
64
3.3. Giai on to cy hon chnh in vitro

y l giai on cui cng ca qu trnh nhn ging in vitro to cy
hon chnh (c , thn, l, r), cy khe mnh, c th sng, sinh trng, pht
trin tt khi ra vn m v vn sn xut.
Vi mc ch tm ra mi trng ti u, thch hp cho cy Lan A.
roxburghii (Wall.) Lindl. sinh trng v pht trin thun li, sm t tiu
chun cy a ra vn m v vn sn xut, chng ti tin hnh th nghim
b sung hm lng than hot tnh, NAA i vi cy Lan A. roxburghii
(Wall.) Lindl. trong giai on to cy hon chnh.
3.3.1. nh hng ca than hot tnh
Theo Misson v cng s (1983), Kelin v Bopp (1971), b sung than
hot tnh vo mi trng nui cy c tc dng rt tt vi mt s loi lan v
than hot tnh c kh nng hp thu cht thi c mu nu en to iu kin
cho qu trnh phn ha v sinh trng ca r cy in vitro. tng cng s
sinh trng v pht trin ca cy, chng ti b sung hm lng than hot tnh
khc nhau vo mi trng nui cy. Kt qu c trnh by bng 3.10 cho
thy:
Vic b sung than hot tnh vo mi trng nui cy c tc dng tch
cc n s sinh trng ca chi th hin chiu cao, s l, s r, hnh thi r,
so vi mi trng khng b sung than hot tnh.
i vi chiu cao trung bnh ca cy: Chiu cao trung bnh ca cy dao
ng t 4,90cm n 5,70cm. Theo so snh LSD: cng thc 3 hm lng
1,0g/L than hot tnh c chiu cao trung bnh ca cy t cao nht l 5,70cm
mc a. Trong khi cng thc i chng ch t 4,90cm mc c; cng thc
2 v cng thc 4 tng ng 0,5g/L; 1,5g/L c chiu cao trung bnh ca cy l
65
5,25cm v 5,34cm mc b; cng thc 5 (2,0g/L than hot tnh) c chiu

cao trung bnh ca cy 5,03cm c xp mc bc.
Bng 3.10. nh hng ca than hot tnh ti giai on to cy hon
chnh
CTTN
Hm lng
than
hot tnh
(g/L)
Chiu
cao TB
ca cy
(cm)
S l
TB(l)
S r
TB (r)
CT1(/C)
0,0
4,90 c
3,20 cd
1,87 d
CT2
0,5
5,25 b
3,51 b
2,25 c
CT3
1,0
5,70 a
3,90 a
3,62 a
CT4
1,5
5,34 b
3,25 c
3,42 ab
CT5
2,0
5,03 bc
3,04 d
3,23 b
LSD 0,05
0,25
0,19
0,23
CV (%)
2,7
3,1
4,5
Hnh thi r
u r nu
u r nu,
ngn
u r trng,
mp v u
u r trng,
di
u r trng,
mnh
Ghi ch: /C: Nn mi trng MS + 20 g/L ng + 100ml/L nc da + 6,5g/L thch,

pH 5,5.
a, b, c, d: so snh theo gi tr khc bit c ngha nh nht nht mc xc sut 95%.
i vi ch tiu s l: cng thc 3 c hm lng 1,0g/L than hot

tnh c s l trung bnh t cao nht l 3,90 l mc a so vi cng thc i
chng khng b sung than hot tnh 3,20 l xp mc cd. Khi tng hm
lng ln 2,0mg/L th s l c hin tng gim hn so vi i chng v s l
66
trung bnh gim xung 3,04 l xp mc d. Cc cng thc 2 (0,5g/L than

hot tnh) v cng thc 4 (1,5g/L than hot tnh) c s l ch t 3,51 l xp
mc b v 3,25 l xp mc c.
S r trung bnh cng thc 3 c hm lng 1,0g/L than hot tnh cng
t cao nht l 3,62 r c xp mc a; trong khi i chng (khng b
sung than hot tnh) ch t 1,87 r xp mc d. Cn cc cng thc 4, cng
thc 5 v cng thc 2 tng ng 1,5; 2,0; 0,5g/L than hot tnh s r trung
bnh t tng ng l: 3,42 r mc ab, 3,23 r mc b v 2,25 r
mc c.
V cht lng r cho thy: Cng thc c b sung than hot tnh hm
lng 1,0 g/L 2,0mg/L cho cht lng r rt tt. V tt nht hm lng
1,0 g/L than hot tnh, ti hm lng ny cy sinh trng pht trin tt, u r
trng, mp v u.
Nh vy: cng thc 3 c b sung 1,0g/L than hot tnh vo mi
trng (MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch, pH 5,5) l
thch hp cho s sinh trng pht trin ca cy.
Theo Zhou YuMei v cng s (2009) nghin cu trn loi cy A.
formosanus v vic b sung 2g/L than hot vo mi trng nui cy l thch
hp nht cho r pht trin.
Kt qu ca chng ti ch ra c sai khc ca cc hm lng than
hot tnh khi b sung vo mi trng nui cy trn loi Lan A. roxburghii
(Wall.) Lindl. n kh nng to cy hon chnh.
3.3.2. nh hng ca NAA ti giai on to cy hon chnh
NAA l cht iu ho sinh trng thuc nhm Auxin, thng c s
dng hu ht trong nui cy m t bo thc vt, c tc dng to r cho chi.
67
N c s dng khc nhau ph thuc vo loi cy trng c th v thng

c s dng dao ng t 0,5 - 5mg/L.
T kt qu nghin cu bng 3.10 cho thy ti 1,0g/L than hot tnh c
tc dng rt tch cc ti vic to cy hon chnh ca cy lan, nhng s r ca
cy vn cha c khe v nhiu. V vy, chng ti chn than hot tnh c
hm lng 1,0g/L kt hp vi cc nng NAA khc nhau: 0; 0,3; 0,6; 0,9;
1,2; 1,5mg/L vo mi trng MS + 20g/L ng + 100ml/L nc da +
6,5g/L thch + 1,0g/L than hot tnh, pH 5,5 tip tc cc th nghim nghin
cu to cy hon chnh. Sau 8 tun theo di c kt qu c trnh by
bng 3.11.
Bng 3.11. nh hng ca NAA ti giai on to cy hon chnh
CT1(/C)
0,0
Chiu
cao TB
ca cy
(cm)
5,68 d
CT2
0,3
5,90 cd
3,96 b
3,89 b
u r nu, ngn
CT3
0,6
6,59 a
4,38 a
4,11 a
u r trng, nhiu
CTTN
Nng
NAA
(mg/L)
S l
TB
(l)
S r
TB (r)
3,80 b
3,54 c
u r nu
Hnh thi r
lng ht nh, mp
v di u
CT4
0,9
6,21 b
4,17 ab
3,85 b
u r trng, di
CT5
1,2
5,92 c
4,01 b
3,60 c
u r trng, mnh
CT6
1,5
5,30 e
3,82 b
3,57 c
u r nu, yu
LSD 0,05
0,22
0,24
0,15
CV (%)
2,1
3,3
2,2
Ghi ch: /C: Nn mi trng MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch +

1,0g/L than hot tnh, pH 5,5.
a, b, c, e: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%
68
Kt qu nghin cu cho thy NAA c nh hng r rt n s sinh

trng v pht trin ca cy Lan A. roxburghii (Wall.) Lindl. Chiu cao cy,
s l, s r tng ln rt nhiu khi b sung vi nng 0,6mg/L NAA c th:
chiu cao trung bnh ca cy t 6,59cm, s l trung bnh t 4,38 l, s r
trung bnh t 4,11 r c xp mc a. Trong khi cng thc i chng
(khng b sung NAA) cc ch tiu theo di ch t l: chiu cao trung bnh
ca cy 5,68cm xp mc d, s l trung bnh t 3,80 l xp mc b, s
r trung bnh t 3,54 r xp mc c. Tuy nhin khi tng nng NAA
ln qu 0,9mg/L cc ch tiu trn li c xu hng gim dn. cng thc 4
(0,9mg/L NAA) chiu cao trung bnh ca cy t 6,21cm xp mc b, s
l trung bnh t 4,17 l xp mc ab v s r trung bnh t 3,85 xp
mc b. cng thc 5 (1,2mg/L NAA) chiu cao trung bnh ca cy t
5,92cm, s l trung bnh 4,01 l xp mc b v s r trung bnh t 3,60 r
xp mc c. cng 6 c nng NAA cao nht 1,5mg/L th cc ch tiu
theo di iu gim v cn thp hn so vi i chng c th: Chiu cao trung
bnh ca cy ch t 5,30cm xp mc e, s l trung bnh t 3,82 l xp
mc b, s r trung bnh t 3,57 r c xp mc c, trong so snh LSD.
V cht lng r cho thy: B sung 0,6mg/L NAA cht lng r rt
tt, r ra nhanh hn, u r trng, mp v di u hn cc cng thc th
nghim khc. Khi tng nng NAA ln cao 1,5mg/L s c ch s sinh
trng ca r th hin l s r v chiu di r gim, r c hin tng nu ha
v yu.
Nh vy: cng thc 3 c b sung 0,6mg/L NAA vo mi trng
(MS + 20g/L ng + 100ml/L nc da + 6,5g/L thch + 1,0g/L than hot
tnh, pH 5,5) l thch hp nht cho s sinh trng pht trin ca cy trc khi
ra ngoi vn m.
69
Hnh 3.3. To cy hon chnh in vitro

3.4. Giai on a cy in vitro ra vn m
y l giai on c ngha v cng quan trng, quyt nh kh nng
ng dng ca ton b qu trnh nui cy in vitro vo thc tin sn xut. y
cng l giai on gp phi rt nhiu kh khn cho vic chuyn cy t ng
nghim ra vn m ko theo s thay i t ngt cc iu kin sng t mi
trng nhn to sang iu kin t nhin c nhiu yu t t nhin kh iu
khin c nh: nh sng, nhit , m, nm, bnh...
c t l sng cao, cy sinh trng tt khi a ra ngoi vn m,
chng ti tin hnh nghin cu nh hng ca mt s nn gi th v mt s
loi phn bn khc nhau i vi cy Lan con nui cy in vitro.
70
3.4.1. nh hng ca gi th trng khc nhau n s sinh trng pht

trin ca cy Lan A. roxburghii (Wall.) Lindl.
Cy Lan con ra r tt trong iu kin in vitro trn mi trng MS b
sung 0,6 mg/L NAA v 1,0 g/L than hot tnh, c dng lm th nghim
tip theo.
Mi mt gi th c c tnh khc nhau, s ph hp vi mi loi cy
trng cng nh trong giai on vn m chng c yu cu khc nhau i
vi iu kin ngoi cnh. Nhn chung, gi th tt l gi th c kh nng gi
m v thot nc tt, c kh nng cung cp dinh dng cho cy con giai
on u d dng tip cn vi mi trng sng t nhin. Cy Lan A.
roxburghii (Wall.) Lindl. sau nui cy m c yu cu rt cht ch i vi iu
kin mi trng. C th: yu cu m cao nhng khng b ng, nhit
mi trng khng qu cao, gi th sch v c kh nng cung cp dinh dng
cho giai on cy con trong vn m. xc nh gi th ph hp, chng
ti tin hnh th nghim ra cy vo v thu v trng trn 3 loi gi th khc
nhau, kt qu c trnh by qua bng sau 3.12 v hnh 3.4.
Qua bng cho thy: Cc gi th khc nhau cho kh nng sng v
sinh trng ca cy con in vitro l khc nhau. Trn gi th dn t qu c t l
sng ca lan con t thp nht 60,87%; chiu cao trung bnh ca cy t
5,93cm, s r mi trung bnh xut hin 1,58 r c xp mc c. Cn s l
trung bnh v s nhnh trung bnh l 3,47 l v 1,09 nhnh xp mc c
trong so snh LSD. iu ny c th gii thch l do loi gi th ny khng c
cht dinh dng cung cp cho cy trng ban u, kt hp vi thng thong
ca gi th thp, kh nng gi nc qu cao, gy ra hin tng thi r. Qua
quan st, cho thy hnh thi cy v mu sc ca l cy yu v nh.
71
Bng 3.12. nh hng ca gi th trng khc nhau n s sinh trng

pht trin ca cy Lan A. roxburghii (Wall.) Lindl. (sau 12 tun)
Ch tiu theo di
Cng thc
th nghim
Chiu cao
TB ca cy
S l TB
S nhnh TB
(l)
(nhnh)
(cm)
S r mi
TB xut hin
(r)
CT1
6,14 b
4,22 a
1,21 b
1,75 b
CT2
5,93 c
3,47 b
1,09 b
1,58c
CT3
6,38 a
4,36 a
1,46 a
1,99 a
LSD 0,05
0,11
0,14
0,19
0,13
CV (%)
0,9
1,7
7,4
3,6
Ghi ch: a, b, c: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%
Cng thc 1: Ru Sphagnum moss
Cng thc 2: Dn t qu
Cng thc 3: Ru Sphagnum moss : Dn t qu
88,17
T l sng (%)
82,07
90
80
70
60
50
40
30
20
10
0
60,87
CT1
CT2
CT3
Cng thc th nghim
Hnh 3.4. nh hng ca gi th trng khc nhau n t l sng ca cy

Lan A. roxburghii (Wall.) Lindl. (sau 12 tun)
72
Vi gi th ru Sphagnum moss, c t l sng ca lan con t tng i

cao 82,07 %; chiu cao trung bnh ca cy t 6,14cm, s nhnh trung bnh l
1,21 nhnh, s r mi trung bnh t 1,75 r c xp mc b trong so
snh LSD. gi th ru Sphagnum moss tuy s l trung bnh t kh cao 4,22
l/ cy c xp mc a, nhng qua quan st chng ti thy gi th ny
cy pht trin mc kh, b r li khng bm chc vo gi th.
Trn loi gi th phi trn ru Sphagnum moss : dn t qu (1:1)
cho t l sng cao nht t 88,17%. Cc ch tiu sinh trng khc: chiu cao
trung bnh ca cy t 6,38cm; s l trung bnh t 4,36 l; s nhnh trung
bnh l 1,46 nhnh v s r mi trung bnh cng t cao nht 1,99 r c xp
mc a trong so snh LSD. Trn gi th ny do s phi hp ca hai gi th
ru Sphagnum moss + dn t qu b sung cho nhau to iu kin cung
cp y dinh dng cho cy con, c kh nng gi v thot nc tt. Qua
quan st chng ti thy cy cng cp, l xanh bng, r khe v bm chc vo
gi th.
Nh vy, gi th thch hp nht cho cy Lan A. roxburghii (Wall.)
Lindl. trong giai on vn m l gi th phi trn ru Sphagnum moss :
dn t qu (1:1) cho t l cy sng cao v gi thnh r hn ru ngoi.
3.4.2. nh hng ca mt s loi phn bn khc nhau n s sinh trng
pht trin cy Lan A. roxburghii (Wall.) Lindl.
Phn bn l mt trong nhng yu t quan trng quyt nh n qu
trnh sinh trng, pht trin ca cy trng. Cy Lan rt nhy cm vi yu t
dinh dng, nu dinh dng y khng nhng gip cy trng sinh trng,
pht trin tt m cn cho nng sut, cng nh cht lng cao.
Da vo kt qu nghin cu v loi gi th thch hp, ti chn
loi gi th ru Sphagnum moss : dn t qu t l (1:1) tip tc nghin cu
nh hng ca mt s loi phn bn khc nhau nhm tm ra loi phn bn
73
thch hp nht cho sinh trng ca cy lan con sau khi ra vn m. nh

hng ca phn bn ln sinh trng ca cy lan con c th hin ti bng
3.13 v hnh 3.5.
Sau 12 tun theo di v phun loi phn bn HVP c cc ch tiu u
thp hn so vi phn bn Growmore v B1- Thi Lan c th l: Chiu cao
trung bnh ca cy ch t 6,92cm, s l trung bnh t 4,53 l; s nhnh l
1,29 nhnh xp mc b, s r mi trung bnh xut hin 2,22 r xp mc
c. Cng nh hnh thi, mu sc ca l nh v xanh nht.
Bng 3.13. nh hng ca mt s loi phn bn khc nhau n s sinh
trng pht trin ca cy Lan A. roxburghii (Wall.) Lindl. (sau 12 tun)
Ch tiu theo di
Chiu cao
TB ca cy
(cm)
S l TB
(l)
S nhnh TB
(nhnh )
6,92 c
4,53 c
1,29 b
7,21 b
4,89 b
1,61 b
7,53 a
5,24 a
1,86 a
LSD 0,05
0,12
0,18
0,13
CV (%)
0,8
1,9
4,1
Loi phn
bn
HVP
(30:10:10)
Growmore
(30:10:10)
B1 -Thi
Lan
Ghi ch: a, b, c: so snh theo gi tr khc bit c ngha nh nht mc xc sut 95%).
Ti cc cng thc phun loi phn bn Growmore, nhn thy cy sinh

trng v pht trin kh tt, th hin cc ch tiu nh: chiu cao trung bnh
ca cy t 7,21cm; s l trung bnh t 4,89; s nhnh trung bnh l 1,61
nhnh; s r mi trung bnh t 2,36 r c xp mc b trong so snh
74
LSD. V cht lng cy qua quan st nhn thy cy tng i khe v l

xanh.
2,7
2,62
S r mi TB xut hin (r)
2,6
2,5
2,4
2,36
2,3
2,2
2,22
2,1
2
HVP (30:10:10)
Growmore (30:10:10)
B1 -Thi Lan
Loi phn bn
Hnh 3.5. nh hng ca mt s loi phn bn n s r mi xut hin

ca cy Lan A. roxburghii (Wall.) Lindl. (sau 12 tun)
Vi loi phn bn B1- Thi Lan cho cy sinh trng v pht trin tt
nht so vi hai loi phn bn trn. Chiu cao trung bnh ca cy t cao nht
7,53cm; s l trung bnh t 5,24 l; s nhnh trung bnh l 1,86 nhnh v s
r mi trung bnh cng t cao nht 2,62 r c xp mc a trong so snh
LSD. Khi quan st hnh thi cy, chng ti cng thy phun loi phn bn ny
cy mp, b l v r pht trin tt hn 2 loi cn li.
Nh vy, giai on vn m c th s dng phn bn l B1 Thi
Lan phun 1tun/ln vi liu lng 0,5g/L. Khi phun phn bn ny cho cy
sinh trng v pht trin tt nht, cy xanh khe v c b r mp v di.
75
Hnh 3.6. Cy Lan A. roxburghii (Wall.) Lindl. nui cy m sinh trng

pht trin vn m.
76
T cc kt qu nghin cu thu c, chng ti i n xut quy trnh

nhn ging in vitro vi loi Lan A. roxburghii (Wall.) Lindl. nh sau:
Vt liu nui cy (1)
Chi bn, chi nh
Kh trng mu (2)
NaOCl 3% + 15 pht
To vt liu khi u (3)

MS + 20g/L ng + 100ml/L nc da + 2,0mg/L BA
+ 6,5g/L thch, pH 5,5
(4 tun)
Nhn nhanh cm chi (4)

MS + 20g/L ng + 100ml/L nc da + 2,0mg/L BA
+ 1,0mg/L NAA + 80g/L dch nghin khoai ty + 6,5g/L
thch, pH 5,5
(8 tun)
To cy hon chnh in vitro (5)

MS + 20g/L ng + 100ml/L nc da + 1,0g/L than hot tnh
+ 0,6mg/L NAA + 6,5g/L thch, pH 5,5
(8 tun)
Chuyn cy con in vitro ra vn m (6)

Gi th: ru Sphagnum moss + dn t qu (1:1)
Bn phn B1 Thi Lan, phun 1tun/ln vi liu lng 0,5 g/L
(12 tun)
77
Bc 1: Chn nguyn liu khi u

Chi bn, chi nh ly t cy m trng thnh nhm hn ch ti a
mu nhim v s bin d soma v sau.
Bc 2: Kh trng mu
Cc mu cy c kh trng bng NaOCl 3% trong 15 pht c t l
sng cao nht (65,5 %).
Bc 3: To vt liu khi u (4 tun)
Trn mi trng MS + 20g/L ng + 100ml/L nc da + 2,0mg/L
BA + 6,5g/L thch, pH 5,5 cho h s nhn chi t 4,44 ln; chiu
cao trung bnh ca chi t 2,53cm
Bc 4: Nhn nhanh cm chi (8 tun)
Chi hnh thnh trong bc (3) tip tc cy chuyn sang mi trng
MS + 20g/L ng + 100ml/L nc da + 2,0mg/L BA + 1,0mg/L
NAA + 80g/L dch nghin khoai ty + 6,5g/L thch, pH 5,5 cho h
s nhn chi cao nht 21,83 chi; chiu cao trung bnh ca chi t
4,31cm; cht lng chi tt nht, ko di tui th ca chi trong
nhn nhanh.
Bc 5: To cy hon chnh in vitro (8 tun)
Tch cc chi t cm chi cy sang mi trng MS + 20g/L ng +
100ml/L nc da + 1,0g/L than hot tnh + 0,6mg/L NAA +
6,5g/L thch, pH 5,5 cho chiu cao trung bnh ca cy t cao nht
6,59cm; s l trung bnh 4,38 l; s r trung bnh t 4,11 r.
Bc 6: Chuyn cy con in vitro ra vn m (12 tun)
78
Cy con sau khi rt khi mi trng in vitro cn ra sch thch bm

vo r, sau trng vo khay hay chu t nung vi mt 55cm
- Gi th: ru Sphagnum moss + dn t qu (1:1)
- m gi th: 70 80%
- m khng kh: 90 95%
- Nhit nui trng: 20-250C
- nh sng: che sng > 70%
- Ti phun sng u 3 - 4 ln/ngy
- Bn phn B1 Thi Lan, phun 1tun/ln vi liu lng 0,5g/L
t t l cy sng cao nht 88,17% v sinh trng pht trin tt
vn m
79
KT LUN V NGH
1. Kt lun
Trong qu trnh nghin cu nhn nhanh loi Lan kim tuyn (A.
roxburghii (Wall.) Lindl.) qu him ca Vit Nam, t cc kt qu thu
c chng ti c nhn xt nh sau:
1.1. Kh trng mu lan nui cy thch hp nht l 3% NaOCl + 15 pht, t l
mu sng t ti 65,5%.
1.2. Mi trng khi ng to vt liu khi u thch hp nht trn mi
trng MS + 20g/L ng + 100ml/L nc da + 2,0mg/L BA + 6,5g/L
thch, pH 5,5 cho h s nhn chi t 4,44 ln; chiu cao trung bnh ca
chi t 2,53cm sau 4 tun nui cy cho chi mp, xanh thm.
1.3. Mi trng nhn nhanh cm chi thch hp trn mi trng MS + 20g/L
ng + 100 ml/L nc da + 2,0mg/L BA + 1,0mg/L NAA+ 80g/L
dch nghin khoai ty + 6,5g/L thch, pH 5,5 cho h s nhn chi cao
nht 21,83 chi; chiu cao trung bnh ca chi t 4,31cm sau 8 tun
nui cy cho cht lng chi tt nht, ko di tui th ca chi trong
nhn nhanh.
1.4. To cy hon chnh in vitro thch hp nht trn mi trng MS + 20g/L
ng + 100ml/L nc da + 1,0g/L than hot tnh + 0,6mg/L NAA +
6,5g/L thch, pH 5,5 cho chiu cao trung bnh ca cy t cao nht
6,59cm; s l trung bnh 4,38 l; s r trung bnh t 4,11 r.
1.5. Kh nng sng v sinh trng pht trin ca cy Lan khi a t ng
nghim nui cy m trong vn m ph thuc cht ch vo gi th
trng v loi phn bn.
+ Gi th ra cy thch hp nht l gi th ru Sphagnum moss : dn t
qu (1:1) cho t l cy sng v cc ch tiu sinh trng pht trin t cao
80
nht t 88,17%; chiu cao trung bnh ca cy t 6,38cm; s l trung

bnh t 4,36 l; s nhnh trung bnh l 1,46 nhnh v s r mi trung
bnh 1,99 r.
+ giai on vn m c th dng phn bn B1 Thi Lan phun
1tun/ln vi liu lng 0,5g/L cho cy sinh trng pht trin tt nht
cy xanh khe v c b r mp v di. Chiu cao trung bnh ca cy t
cao nht 7,53cm; s l trung bnh t 5,24 l, s nhnh trung bnh l 1,86
nhnh v s r mi trung bnh cng t cao nht 2,62 r.
2. ngh
2.1. Trn c s cc kt qu nghin cu thu c, chng ti bc u xut
ng dng cc kt qu thu c trong nhn ging in vitro cho loi Lan
Anoectochilus roxburghii (Wall.) Lindl., ng thi lm tin cho cc
nghin cu nhn ging trn cc loi Lan Anoectochilus dc liu khc.
2.2. Tip tc nghin cu tip v giai on cy sinh trng ngoi vn m v
giai on chuyn cy con in vitro tiu chun em trng ngoi vn
sn xut hon thin quy trnh.
2.3. Nghin cu s n nh di truyn v kh nng tng hp hot cht sinh hc
cy Lan kim tuyn (Anoectochilus roxburghii (Wall.) Lindl.) nhn
ging bng phng php nui cy m.
81
TI LIU THAM KHO

Ting vit
1. B Khoa hc v Cng ngh (2007), Sch Vit Nam (phn thc vt),
NXB Khoa hc t nhin & Cng ngh, H Ni, tr. 404 -405.
2. L Vn Chi (1992), Cch s dng cht iu ho sinh trng v vi lng
hiu qu cao, NXB Khoa hc - K thut, H Ni.
3. Trn Vn Minh (1994), Nui cy m t bo thc vt, Phn vin Cng ngh
sinh hc Thnh ph H Ch Minh, tr. 58-97.
4. Phng Vn Ph v cng s (2010), Nghin cu k thut nhn nhanh chi
In vitro loi Lan kim tuyn Anoectochilus roxburghii (Wall.) Lindl., Tp
ch Khoa hc HQGHN, Khoa hc T nhin v Cng ngh, 26, tr. 248253.
5. Nguyn Quang Thch (1995), Cng ngh sinh hc thc vt, NXB Nng
nghip, H Ni.
6. Nguyn Quang Thch v cng s (2003), Bi ging cng ngh sinh hc
thc vt, i hc NN I - H Ni.
7. Nguyn Quang Thch v cng s (2012), Nghin cu k thut nhn ging
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86
PH LC
I. Thnh phn c bn ca cc loi mi trng nui cy
Bng 1. Mi trng Murashige & Skoog (1962)
Thnh phn
a lng
Vi lng
CaCl2.2H2O
440
KH2PO4
170
KNO3
1900
MgSO4.7H2O
370
NH4NO3
1650
MnSO4.4H2O
22,3
ZnSO4.7H2O
8,6
H3BO3
6,2
KI
0,83
Na2MoO4.2H2O
0,25
FeSO4.7H2O
27,8
Na2EDTA
37,5
CuSO4.5H2O
0,025
CoCl2.6H2O
0,025
Glyxin
Vitamin v cc
thnh
phn hu c
Nng (mg/L)
Myo-inositol
100
Acid nicotinic (Niaxin)
0,5
Thiamin HCl (vitamin B1)
0,1
Pyridoxine HCl (vitamim B6)
0,5
87
Bng 2. Mi trng Vacin - Went (1949)

Thnh phn
a lng
Vi lng
Vitamin
Nng (mg/L)
KNO3
52500
(NH4)2SO4
50000
Ca3 (PO4)2 . 4H2O
10000
KH2PO4
12500
MnSO4 .4H2O
370
Na2EDTA
7450
FeSO4 .7H2O
5570
Glyxin
2000
Myo-inositol
100
Thiamin HCl (vitamin B1)
100
Pyridoxine HCl (vitamim B6)
500
Bng 3. Mi trng Robert Ernst (1979)

Thnh phn
a lng
Vi lng
Nng (mg/L)
KNO3
400
(NH4)2SO4
150
Ca (NO3)2 .4H2O
150
KH2PO4
300
NH4 NO3
400
Mg(NO3)2 .6H2O
100
Na2EDTA
37
FeSO4 .7H2O
25
88

Nghiên Cứu Nhân Nhanh Loài Lan Kim Tuyến (Anoectochilus Roxburghii (Wall.) Lindl.) Của Việt Nam Bằng Phương Pháp Nuôi Cấy Mô Tế Bào

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Nghiên Cứu Nhân Nhanh Loài Lan Kim Tuyến (Anoectochilus Roxburghii (Wall.) Lindl.) Của Việt Nam Bằng Phương Pháp Nuôi Cấy Mô Tế Bào

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B GIO DC VO O TO

B NNG NGHIP V PTNT

VIN KHOA HC NNG NGHIP VIT NAM

NGHIN CU NHN NHANH LOI LAN KIM

LUN VN THC S NNG NGHIP

B GIO DC VO O TO B NNG NGHIP V PTNT

NGHIN CU NHN NHANH LOI LAN KIM

LUN VN THC S NNG NGHIP

Ti xin cam oan y l cng trnh nghin cu ca ring ti. Cc s

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

1.3. Nui trng Lan kim tuyn .................................................................. 23

2.5. Phng php x l s liu.................................................................. 43

KT LUN V NGH ......................................................................... 80

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

DANH MC CC K HIU, CC CH VIT TT

Cng c v bun bn quc t nhng loi ng, thc vt hoang

Cng thc th nghim

H s bin ng (Correlation of Variants)

- Naphthalene acetic acid

So snh theo gi tr khc bit c ngha nh nht mc 0,05

Murashige and Skoog

Vacin and Went

T chc y t th gii (World Health Organization)

Qu Quc t Bo v Thin nhin (World Wide Fund For

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

thin nhin v rng quc gia. C mt iu ng lu l vic khai thc ti

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

4. i tng v phm vi nghin cu ca ti

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

CHNG 1 - TNG QUAN TI LIU V C S KHOA HC

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

bo khc nhau, thc hin chc nng c th khc nhau. Cc m c c cu

T bo phn ho chc nng

Khi t bo phn ho thnh m chc nng, chng khng hon ton

iu khin s pht sinh hnh thi ca m nui cy, ngi ta thng

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

khi, ngoi ra n ng vai tr l cht thm thu chnh ca mi trng. Ngi

v NO3 . Trong , vic hp th NO3 ca cc t bo thc vt t ra c hiu qu

+ Nhng i khi NO3 gy ra hin tng kim ha mi trng v vy gii

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

B6 (Pyridocin) l nhng vitamin c bn nht thng dng trong mi trng

c bit l cc vitamin (C, B1, B6). Dch nghin khoai ty thng c b

phytohormon c th chia thnh 5 nhm: Auxin, Cytokinin, Giberillin,

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

+ Nhit : L nhn t c nh hng r rt n s phn chia t bo v cc

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

on sau s b ngng li. Do , trong ton b qu trnh nui cy in vitro cn

nhiu cytokinin s kch thch to chi. Ch nui cy thng l 25 - 270C v

hiu qu, c bit c p dng cho cc loi hoa, cy thuc, cy c sn qu

Hnh 1.1. Cy Lan kim tuyn (A. roxburghii (Wall.) Lindl.)

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

glucopyranoside, isorhamnetin3ODglucopyranoside, kaempferol3

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

2), isorhamnetin-7-O--Dglucopyranoside (compound 3), isorhamnetin-3-O-D-glucopyranoside (compound 4), kaempferol-3-O--Dglucopyranoside

cy Lan kim tuyn vi kt qu tng ng ln lt l 4,0 110,0g/ml vi h

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

hydroxybenzaldehyde (1), trans4hydroxycinnamic acid (2), trans3

trimethoflavone (9), Quercetin (10), Kaempferol (11), Isorhamnetin (12),

Trng i hc Nng nghip H Ni Lun vn thc s khoa hc nng nghip

cholesterol. Ngoi ra lm tng cc hot ng ca enzym cht chng oxy ha

loi b cc gc t do v gim hm lng NO , lm tng cc t bo nguyn