全 文 ：第 24 卷　第 3 期　　　　　　　　　　　　　植　　　物　　　研　　　究 2004 年　7 月
Vol.24　No.3　　　　　　　　　　　　BULLETIN OF BOTANICAL RESEARCH July 　2004
This research has been supported by the grant of Project 973(G19990160)and part ly supported by g rant HKUST 6117/ 01M from the Re-
search Grant Council of Hong Kong.
First author:Wu Shuang-xiu(1972—), Female , Postdoctoral research associate , Research interests in plant biotechnology and molecular biology.
＊Corresponding author　Tel:86-451-82191517;Email:zygorl@public.hr.hl.cn
收稿日期:2003-12-01
一种快速有效的 741杨离体叶片再生芽方法
吴双秀1 , 2 　吴汪黔生2 　祖元刚1＊
(1.东北林业大学教育部森林植物生态学实验室 ,哈尔滨　150040)
(2.香港科技大学生物系 ,香港九龙清水湾)
摘　要　在对杂交品种 741杨(Populus alba (P .davidiana ×P .simonii)×P .tomentosa)进行
农杆菌介导法转基因的试验中发现了一种快速有效的叶片再生芽的方法 。首先叶片外植体在培
养基 I(MS 培养基添加 0.5 mg/ L BA 和 1.0 mg/L 2 ,4-D)上培养 2 ～ 3 d ,再转移到培养基 SH
(MS medium containing 2.0 mg/L of BA and 0.1 mg/L of NAA)上培养 10 d ,然后再转移到培养
基 II(MS medium with 0.5 mg/L of BA)上 ,培养大约 5 d之后 86.7%的叶片外植体产生的芽 ,每
片叶片外植体(1 cm×1 cm)可产生 40 ～ 50个芽 。但是 ,如果叶片外植体在培养基 I 上培养的时
间长于 5 d ,再依次转移到培养基 SH 和 II 上 ,则叶片会产生大量根 。
关键词　741杨;Populus alba (P .dav idiana ×P .simonii)×P.tomentosa;芽再生;再生
A new efficient method for rapid in-vitro shoot regeneration on leaf
explants of Poplar 741
WU Shuang-Xiu1 ,2　WU Madeline2　ZU Yuan-Gang1＊
(1.Key Labo rato ry of Forest Plant Ecolog y of the M inistry of Education , Nor theast Forestry University , Harbin　150040)
(2.Depar tment of Biology , The Hong Kong University of Science and Technology , Clear Water Bay , Kowloon ,
Hong Kong , SAR , People s Republic of China)
Abstract　In the experiments of Agrobacterium tumefaciens-mediated transformation of the hybrid
poplar 741 , Populus alba (P.dav idiana ×P.simonii)×P .tomentosa , we developed a rapid
and ef ficient method of shoot regeneration from the leaf explants in-vi tro on Murashige and Skoog
(MS)medium.The leaf explant segments w as f irst cultured on medium I(MS medium supplement-
ed w ith 0.5 mg/ L of BA and 1.0 mg/L of 2 ,4-D)for 2 ～ 3 day s and then transferred to medium SH
(MS medium containing 2.0mg/L of BA and 0.1 mg/L of NAA)to culture for 10 days.86.7% of
leaf segments produced shoots af ter the leaf segments w ere t ransferred f rom medium SH to medium II
(MS medium w ith 0.5 mg/ L of BA)for 1 w eek.The average shooting number w as 40 ～ 50 per
1 cm×1 cm of leaf segment.However , if the leaf explants incubated on medium I longer than 5
days , they produced more roots instead of shoots.
Key words　Poplar 741;Populus alba (P .davidiana ×P.simonii)×P .tomentosa;shooting;
regeneration
1　Introduction
As a model system fo r biotechnologies on w oody
species , poplar , the main deciduous tree species in
the forest ry , paper indust ry and ecological environ-
ment recovery , has widely been investigated in the
taxonomy , physiology , genomics , ecology , breed-
ing and propagat ion etc.A lo t of new hybrids of
poplar w ith precious characters have been created by
breeding techniques and asexual propagation tech-
niques in past a few decades.Such as poplar 741 is a
new hybrid aspen of Populus alba (P.dav idiana
×P .simonii) ×P.tomentosa , created in the
Hebei Forest ry College.I t has good qualities on high
g row th rate , fine wood tex ture , st raight stem and
w ide adaptation[ 1] .However , it show s poo r resis-
tance to w ater deficiency which limits i ts wide
spread and utilization
[ 2].With the development of the
molecular biological techniques and the deep under-
standing of poplar genome characters , the improve-
ment of the poplar qualities were at tempted by DNA
recombination techniques , such as Agrobacterium
tumefaciens-mediated transformation technique in
past 20 years.Poplar 741 has been transformed w ith
Bt gene to improve its resistance to insects
[ 3].Our re-
search on this species is to improve i ts tolerance to
w ater stress and cold stress by Agrobacterium
tumefaciens-mediated t ransfo rmation wi th CBFs (C
repeat/dehydration response elements Binding Fac-
tors)genes and desB(desaturase B)gene.The f irst
step to achieve this goal is to establish a high ef fi-
cient and stable in-vitro plant regeneration system of
poplar 741.Based on other previous w ork and the
results in our research [ 3], we found a new eff icient
method fo r rapid in-vitro shoo t regeneration from
the leaf explant of poplar 741.
2　Materials and Methods
2.1　Plant materials
The in-vitro plant lets culture of poplar 741 w as
kindly provided by the Research Institute of
Forest ry , the Chinese Academic of Forest ry.The
routine subculture w as carried out every month on
M urashige and Skoog (MS)medium supplemented
wi th 30% glucose , 0.7% agar and 0.1 mg/L of
benzylaminopurine(BA)and 0.1 mg/L of indole-3-
butyric acid(IBA)[ 4].The cultured w as kept at 24℃
with the light :dark cycle of 16 :8 hr in an envi-
ronment chamber (Conviron CMP3244)with the
light intensi ty of 150 μE·m-1·s-1provided by cool
w hite f luo rescent lamps during the light period.
2.2　Tissue cultures of the leaf explants
The sterile leaf were excised and cut into
1 cm ×1 cmsegments and cultured on MS medium
supplemented w ith 30% glucose , 0.7% agar and
various concentrations and combination of BA , IBA ,
NAA and 2 , 4-D respectively as bellow .Medium I ,
for callus induction , is MS medium supplemented
wi th 0.5 mg/L of BA and 1.0 mg/L of 2 , 4-D.
Medium SH , for shoot regeneration , is MS medium
containing 2.0 mg/L of BA and 0.1 mg/L of
NAA.Medium II , for shoot grow ing up , is MS
medium w ith 0.5 mg/ L of BA.All the culture
t reatments w ere carried out under the same condi-
tion as the subculture.The formations of shoots ,
callus and roots w ere examined and recorded every
day .Each treatment included 30 ～ 40 pieces of ex-
plants.All the t reatments were triplicates.
3　Results and Discussion
3.1　Effects of the explant phenotypes
During the experiments of callus induction and
shoot induction f rom the leaf , petiole and stem ex-
plants of poplar 741 on M S medium containing vari-
ous exogenous BA , IBA , NAA and 2 ,4-D combina-
tions , we found that the leaf explants more easily
gave shoo t regeneration along the midveins at the
cutting sites than the pet iole explants and stem ex-
plants (Table 1).Furthermore , the leaf explants
wi th different grow th appearance show ed the differ-
ent shooting abili ty (Table 1).Thicker and dark
green color leaf segments produced more shoo ts than
the thinner and yellow green color ones.The leaf
explants , pet iole explants and stem explants had the
same potential on callus fo rmation w hen cultured on
the medium I (Table 1).
3.2 　Effects of incubation time on medium I on
shoot regeneration
Zheng et al .reported that the shoo ting on the
348 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　24 卷
Table 1　The effects of explant phenotypes on the shooting induction of poplar 741 on MS medium supplemented
with BA and IBA.Callus induction was carried on the medium I
Explant phenotypes
S hoot ing f requency
(%)
Shoot numbers per leaf explant
segment of 1cm×1cm
Callus format ion f requency
(%)
Leaf
Thicker and dark green color 51.7～ 86.7 40～ 50 100
Thinner and yellow greencolor 0 0 <14.5
Petiole 43.3 6～ 12 100
S tem 33.3 6～ 15 100
Table 2　The effects of incubation time on medium I on the callus formation and shoot regeneration
from the leaf explants of poplar 741
Incubat ion time on
medium I(day)
Callus formation frequency
on medium I(%)
Shoot ing f requency
on medium II (%)
Shooting number per leaf
segment on medium II
Rooting f requency on
medium II (%)
0 0 4.5 1～ 3 5.0
1 0 33.3 8～ 10 12.8
2 0 75.0 14～ 24 66.7
3 0 , leaf swell 86.7 40～ 50 76.7
4 0 , leaf sw ell 27.7 12～ 15 91.3
6 95 0 — 100
9 100 0 — 100
Fig.1　The pho to pictures of the shoot regeneration(A)and root formation
(B)from the leaf explant o f poplar 741 on the medium I I.
leaf explants of poplar 741 was succeeded on M S
medium supplemented w ith 3.0 mg/L of BA and 0.
3 mg/L of NAA[ 3] .In our experiments , we found
that the exogenous 2 , 4-D not only significant ly in-
duced the callus formation but also stimulated the
shoot regeneration on the leaf explants of poplar 741
(Table 2).
We developed 3-step pro tocol fo r shoot regener-
ation f rom the leaf explant of poplar 741.First step ,
the leaf explants were cultured on the medium I for
callus ini tiation for 0 , 1 , 2 , 3 , 4 , 6 , 9 days respec-
tively.During the f irst 1 ～ 2 days on the medium I ,
there is no different appearance on the leaf explants
compared wi th those directly cultured on medium SH
(0 day).On the day 3 on medium I , the leaf ex-
plants begun to swell.From the day 6 on medium I ,
callus appeared on the leaf explants along the cut ting
sites.Second step , all above leaf segments were
transferred onto medium SH respectively to culture
for 10 day s for shoo ting induction.During this peri-
od , there w as no shoots appearing on the leaf seg-
ments.Third step , all these leaf explants were t rans-
ferred to the medium I I for g row ing.In about 5 days
on medium II , bo th shoot ing and root ing appeared
3493 期　　　　　　　　　　　　　吴双秀等:一种快速有效的 741杨离体叶片再生芽方法
on the leaf explants which w ere first incubated on
medium I fo r 1 ～ 4 days(Table 2 and Fig.1A).The
maximum shooting f requency was 86.7% achieved
on which incubation time on medium I w as 3 day s
and the co rresponding shoo ting number was 40 ～ 50
per leaf segment.The shoots were keeping on
sprouting w ithin a month on medium II.However ,
instead of shoo ting , a lo t of roots formed on the leaf
explants w hich w ere o riginally incubated on medium
I fo r 6 , 9 days(Table 2 and Fig.1B).
4　Disccusion
In this study , the shoo ting regeneration poten-
tial of the leaf explant of poplar 741 was demonst rat-
ed to be not only related to the pheno type of the leaf
explants but also related to the 2 , 4-D induction.
Dark green color and thicker leaf explants had higher
shooting ability (Table 1).This phenomenon was al-
so found in another hybrid aspen , P.langfangensis
3 in our w ork(in another paper).The mechanism of
this phenomenon was also studied in P .nigra , P.
del toids and P. tomentosa[ 5 ,6] . The cellular
polyamine contents in different development stage
and the activities of their main biosynthetic enzymes
might play important the role in the shoot regenera-
tion f rom the leaf segments[ 7 , 8] .The genes involved
in this process have been caused attention recently.
2 , 4-D was repo rted to be significant in callus in-
duction for a lot of plant species
[ 9] .Our study
demonstrated that it is necessary for the callus induc-
tion for poplar 741.The procedure of put ting the leaf
explants on the callus induction medium (medium I)
befo re shooting induction (on medium SH)was
demonstrated to be determinat ive for shoot o r root
formation f rom the leaf explant of poplar 741.The
mechanism of this phenomenon is under studies in
our laboratory.However , this pro tocol is efficient
for shoot regeneration f rom the leaf explant of polar
741 for the usage of Agrobacterium tumefaciens-me-
diated t ransformation.
Abbreviation:MS , Murashige and Skoog ;BA ,
6-benzylaminopurine; IAA , indole-3-acetic acid;
IBA , indole-3-butyric acid;NAA , 1-naphthale-
neacetic acid;2 , 4-D , (2 , 4-dichlorophenoxy)acetic
acid)
Acknowledgment　Sincerely thank for Professor Dr.Made-
line Wu s support on experiments and Professor Nie Shaoquan s
suggestion on taxonomy of poplar.
Reference
1.Jiang H M .The characteristics analysis of the new hybrid
poplar 741.Forestry Science , 1991 , 27(6):589 ～ 594
2.Fei B H , Chen S G , Guo J Q.Eco-physiological Studies of
741 poplar.Journal of Hebei Fo restry Colleg e , 1994 , 9:52～
62
3.Zheng J B , Zhang Y M , Yang W Y , et al.Plant regenera-
tion of excised leaf from 741 poplar and transfo rmation with
insect-resistant B t.tox in gene.Journal of Agricultural Uni-
versity of Hebei , 1995 , 18(3):20～ 25
4.Murashig e T , Skoog F.A revised medium fo r rapid grow th
and bioassays with tobacco tissue cultures.Physiol Plant ,
1962 , 15:473～ 497
5.Srivastava S , Glock H , Zhang Z.Short note:T issue culture
studies on Chinese poplar Populus Tomentosa.Silvae Genet-
ica, 1991 , 40(5 , 6):247～ 248
6.Mofidabadi A J K , Mazik-Tokei K , Gergacz E , et al.Callus
induction and haploid plant regenera tion from anther culture
of two poplar species.Silv ae Genetica , 1995 , 44(2 , 3):141
～ 145
7.Kim S H , Kim M W , Kang Y H , et al.Correlations between
poly amine and adventitious shoo t regenera tion from Popu-
lus leaf segments.Korean Journal of Bo tany , 1993 , 36(1):1
～ 8
8.Coleman G D , Ernst S G.Protein differences among popu-
lus deltoids internodal stem explants determined fo r shooting
regeneration or callus g rowth.Plant Science (Limerick),
1991 , 75(1):83～ 92
9.Bhojw ani S S , Razdan M K.Plant Tissue Culture:theory
and practice , a revised edition.ELSEVIER Amsterdam ,
1996.483 ～ 537
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