全 文 ：第 21 卷　第 4 期　　　　　　　　　　　　　植　　　物　　　研　　　究 2001 年　10 月
Vol.21　No.4　　　　　　　　　　　BULLETIN OF BOTANICAL RESEARCH Oct., 　2001
白桦下胚轴再生系统的建立
祖元刚　孔　瑾　吴双秀　聂明珠
(东北林业大学森林植物生态学开放研究实验室 ,哈尔滨 150040)
摘　要　本文对白桦下胚轴的再生系统作了研究。讨论了不同的基本培养基 、激素组成 、培养条
件及外植体的生理年龄在不同阶段产生的效应。通过诱导 、分化和生根阶段 ,得到了再生植株 ,提
出了最适的基本培养基 、激素组成 、培养条件及下胚轴外植体的生理年龄 ,为白桦的遗传转化奠定
了基础。
关键词　白桦;下胚轴;再生系统
THE ESTABLISHMENTOF HYPOCOTYL REGENETATION SYSTEM
OF BETULA PLATYPHYLLA
ZU Yuan-gang 　KONG Jin　WU Shuang-xiu　NIE Ming-zhu
(Open Research Laboratory of Fo rest Plant Ecology , Nor theast Forestry University , Harbin 150040)
Abstract　In this paper , a thorough study on hypocotyl regeneration system of Betula platyphy lla
was made.We discussed effects of dif ferent basal culture media , ho rmones , culture condi tions and
explants physiological ages on different culture stages.Through induction , differentiat ion and roo t-
ing stages , regenerat ive plants w ere got ten.Optimum basal culture media , hormone combinations ,
culture condi tions and hypocotyl explants physiological ages were put forw ard.These of fer a good
base fo r B .platyphyl la genetic transformation.
Key words　Betula platyphylla;Hypocotyl;Regeneration system
　　
1　Int roduction
Betula platyphy lla is a main t ree species of sec-
ondary forest in northeast of China.It covers a lot of
area and i t grows rapidly .But it has poor w ood quali-
ty and low use ef ficiency.As natural forest has been
lumbered g reat ly and consumption of wood increases ,
the contradiction betw een supplying enough wood fo r
indust ry and protecting natural forest is more acutely.
At the present , it is imperative to cultivate secondary
forest w ith good w ood quality .So improving B .
platyphyl la wood quali ty through genetic t ransfor-
mation has important economic value.
In some species , there are many reports about
gene transfer successfully using hypoco tyls as ex-
plants
[ 1] .The establishment of hypocotyls regenera-
tion sy stem has significance fo r B .platyphyl la ge-
net ic transformation using Agrobacterium tumefa-
ciens-mediated.
国家自然科学基金资助项目(39870634)
第一作者简介:祖元刚(1954-),男 ,教授 ,从事生态学研究
收稿日期:2001-09-15
2 M aterials and methods
2.1　Plant materials
The seeds of B .platyphy lla are f rom Xiaoxing
anling in China.Seeds w ere surface-sterilized by a
brief immersion in 75% ethanol for 30s , then dipped
into sterile w ater fo r 48 hours , then immersed into
5%NaClO for 12 ～ 17min (According to how long
seeds were saved), at last w ashed in sterile w ater fo r
three or four times.
2.2　Experiment methods
2.2.1　Acquirement of sterile plants
Sterile seeds w ere cultured in 1/2MS[ 2] medium.
Some are cultured in light w hile o thers are in dark.
2.2.2　Culture conditions
All explants are cultured in lighting incubator.
Light intensi ty is 1000 ～ 1500lux w ith 16h photoperi-
od.Culture temperature were kept at 22℃±1℃.
2.2.3　Selection of medium on induction of callus
2.2.3.1　Effect of dif ferent basal media and hor-
mones combination on induction of callus.
Ten day s old hypocotyls w ere cultured in I medi-
um (Hormone combination is listed in table 1).Basic
medium are IW and IM (IW contains WP[ 3] basal
salts and B5
[ 4]
vi tamins.IM contains MS basal salts
and B5 vi tamins).The concentration of sucrose is
20g/l.
　　 Table 1 Different hormone combination for callus-inducing
BA(mg/ l) KT(mg/ l)
0 0.2 0.5 0.8 1.0 1.0 1.5 2.0
NAA(mg/ l) 0 + + + + + + + +
0.2 + + + + + + +
0.5 + + + + +
2 , 4-D(mg/ l) 0.2 + + + + + +
0.5 + + + + +
1.0 + + + + +
2.0 + + + + + +
　　+　stand fo r hormone combination
2.2.3.2　Effect of sucrose content on induction of
callus
Twenty hypoco tyls were separately cultured in
IW medium with 1.0mg/ l BA and different concen-
tration sucrose of 20g/ l , 30g/ l , 40g/ l.
2.2.3.3　Effect of phy siological ages of hypocoty ls
on induction of callus.Each f if ty hypoco tyls of differ-
ent ages(3 days old , 6 day s old , 9 day s old ,12 days
old , 15 day s old , 20 days old)were cultured in IW
with 1.0mg/ l BA and 20g/ l sucrose .
2.2.4 Subculture of callus
The callus w ere exercised w ith 0.5cm2 and
maintained in C medium (Table 2).Twenty explants
were cultured in each medium.After subcultured two
times , results were statisticed.
　　Table 2 Different hormone content for subculture of callus
Medium code
BA(mg/ l)
0.5 1.0 1.5 2.0
C C1 C2 C3 C4
　　Basic medium is IW w ith 20g/ l sucrose.
2.2.5　Differentiation of callus
The callus were cultured in F medium(Table 3).
Twenty explants w ere cultured in each medium.Af-
ter forty days , results w ere statisticed.
616 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　21 卷
　　Table 3 Different hormone combination for differentiation of cultures
Medium code
BA(mg/ l)
0.2 0.5 0.8 1.0
0.2 0.5 0.8 1.0
F F1 F2 F3 F4
　　Basic medium is IW contained with 20g/ l sucrose.
2.2.6　Culture of regenerative plants
Regenerative plants w ere cul tured in Z medium
(Table 4).Twenty explants w ere cultured in each
medium.After fo rty days , results were statisticed.
　　Table 4 Different hormone combination for culture of regenerative plants
Medium code
BA(mg/ l)
0.2 0.5 0.8 1.0
Z Z1 Z2 Z3 Z4
　　Basic medium is IW w ith 20g/ l sucrose .
2.2.7 Rooting of regenerative plants
Regeneration plants w ere cultured in medium fo r
rooting (Table 5)
　　Table 5 Different medium for rooting
Sucrose content in solid medium(g/ l) Sucrose content in liquid medium(g/ l)
20 30 40 20 30 40
NAA(mg/ l) 0+ + + + + +
0.01 + + + + + +
0.1 + + + + + +
0.2 + + + + + +
IBA(mg/ l) 0.2 + + + + + +
0.5 + + + + + +
　　+stand for hormone combination
3　Results and discussions
3.1　Selection of medium for induction of callus
3.1.1 　The optimum ho rmone combination for in-
duction of callus
The experiment results show ed that BA is the
key ho rmone for inducing callus.Only BA can induce
green callus and NAA has no stimulation for inducing
callus.When BA content is 0.2 ～ 1.5 mg/ l , they all
can induce g reen callus.When BA is 1.0mg/ l , in-
duction efficiency is the highest.While other hor-
mone combinations induce the callus which are diffi-
cult for dif ferentiation.
　　Table 6 Effect of different hormone combination on callus-inducing ratio/%
BA(mg/ l)
0 0.2 0.5 0.8 1.0
NAA(mg/ l) 0 0 30 58 86 92
0.2 0 20 52 80
0.5 0 32
3.1.2　The optimum basal medium for callus induc-
tion
M edium WP is the more useful medium for stem
apex s elongation of woody plant [ 5].Our experiment
results also proved that Cultures in IW can form callus
earlier than IM for three or five days.Furthermore
callus in IW proliferates more rapidly than those in
IM.After thirty day s culture , average diameter of
6174 期　　　　　　　　　　　　　　祖元刚等:白桦下胚轴再生系统的建立
callus in IW is 1cm around.But in IM , it is only
about 0.5cm.
3.1.3　The optimum content of sucrose
Callus-inducing efficiency is similar in different
content of sucrose.So 20g/ l sucrose is optimum con-
tent.
3.1.4　Optimum medium for callus-inducing
According to above experiment results , IW+1.
0mg/l BA+20g/ l sucrose+6.0g/ l agar(pH5.8)is
the best medium for callus-inducing.
3.2 　Effect of physiological ages of hypoco tyls on
callus-inducing
Physiological age has important ef fect on callus-
inducing.The mo re younger explants are , the more
easilier explants dedifferentiated.But hopycotyl is
very gentle , they can not survive if suf fered ex ternal
harm .So selection of physiological age of hypocoty l is
very important for callus-inducing.Experiments re-
sults show that induction ration of 9 day s old
hypoco tyl reaches highest , and i t decreases f rom
when age of hypocoty l age is 15 day s old.According
to principle of selection younger in tissue culture[ 6] , 9
days old hypocotyls w ere used to induce callus.
3.3　Optimum callus subculture medium
Callus w ere t rimmed in 0.5cm2 and continuly
subcul tured in medium C for two times.The results
show that w ith subculture times increasing , hormone
will be accumulated in callus.Higher ho rmone con-
tent w ill lead to loss of callus s differentiation abili-
ty[ 7] .And regenerative plants is easy to vit rify..So
i t is very important to select hormone content for sub-
cultures.When BA content is low er , callus is in the
stage of differentiation.When BA content is higher ,
callus become brow n and thei r differentiation ability
decrease and regenerative plants g row w eakly.Medi-
um C2 can make callus proliferate rapidly and have
higher dif ferentiation efficiency.So C2 is the opti-
mum subculture medium.
3.4　Callus differentiation
Callus w as cultured in F differentiation medium.
Lower cytokinin is helpful for regenerative plants e-
longation.After 25 days , statistical result showed
that F2 is the optimum medium for callus dif ferentia-
tion(Table 7).
　　Table 7 Effect of different hormone on callus differentiation
Medium code Differention ratio/ % Differention time/day s
Average regenerant
plants number (the
number of per cm2
callus)
Regenerant plants
grow th state
F1 70 About 25 4 Stronger
F2 80 About 15 10 Very strong
F3 35 About 15 11 Stronger
F4 25 About 15 10
Escessively g row , w hich
need to become strong
3.5　Regeneration plants subculture
The regeneration plants from callus through sev-
eral subcultures are of ten weak w ith small leaf and
thin stem , so it needs to become sturdy.Regenerative
plants(1.0cm high)were cultured in Z medium , the
statistical results af ter twenty days were show ed in
table 9.We found that the reason caused regenerative
plants g row weakly is that their higher endogenous
hormone.The experiment results show that Z2 is the
best subculture medium for regenerative plant(Table
8).Regenerative plants in medium Z2 grow rapidly
and st rongly.After 20 ～ 30 days , thei r height can
reach 2.5cm .They can be directly transferred into
rooting medium.
　　Table 8 Effect of different hormone content of regenerative plants subculture
Medium code Z1 Z2 Z3 Z4
Regenerate g row th state ++ +++ ++ +
Regenerate g rowth speed + ++ ++ +++
　　+ means good , ++means better , +++means best.
3.6　Culture of regenerative plants rooting
3.6.1　Effect of different ho rmone on roo ting
100%percent roo t percent was got when IBA0.
5mg/l and NAA0.01 ～ 0.2mg/ l independently.
618 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　21 卷
From table 9 , we can see that root ratio and average
root number increased w ith the content of this two
hormones increasing , and roo t quality is more bet ter
w ith hormones content increasing.This showed that
this tw o ho rmones all favor to root.From the consid-
erat ion of low ing experiment expense , NAA is the
best hormone for roo t and its optimum content is 0.
01mg/ l.
　　Table 9 Effect of different hormone on rooting
Rooting ratio/ % Root number Roo t quality
IBA mg/ l 0 94 1 ～ 4 +
0.02 95 1 ～ 5 +
0.02 97 2 ～ 5 ++
0.5 100 2 ～ 7 +++
NAA mg/ l 0.001 97 2 ～ 6 +
0.01 100 2 ～ 8 ++
0.1 100 2 ～ 9 +++
0.2 100 2 ～ 9 +++
　　+ means good , ++means better , +++means best.
3.6.2　Effect of sucrose content on rooting
3.6.2.1　Effect of sucrose content on rooting in sol-
id medium
Table 10 show that w hen sucrose content is 30g/
l , roots number reaches the maximum efficiency , and
their quality is the best.So the optimum content of
sucrose for roo ting is 30mg/ l.
3.6.2.2　Effect of sucrose content on roo ting in liq-
uid medium.
From table 10 , we can see that w hen the content
of sucrose is 40g/ l , roots number is max imum and
their number is f rom 3 to 9.Roots become st ronger
w ith the content of sucrose increasing.In liquid
medium , because of sucrose play a big role in adjust-
ing penetrability , so it s content variation bring great
effect on rooting speed.When sucrose content is 40g/
l , root ing speed is the fastest.After seven day s cul-
turing , root began to form.When the content of su-
crose is 20g/ l and 30 g/ l , root ing time w ill delay five
days and three day s respectively.
3.6.2.3　The optimum medium for rooting
Considering all factors , especially the cost of re-
generation plants , R1(1/2MS liquid medium , 30g/l
sucrose , pH 5.8)and R2(1/2MS liquid medium , 0.
01mg/l NAA , 30g/l sucrose , pH 5.8)is the best
medium for rooting of regenerative plants.
　　Table 10 Effect of sucrose content on rooting
Content of sucrose(g/ L) Rooting ratio/ % Rooting number Roo t quality Rooting time/days
Solid medium 10 100 1 ～ 5 + About 17
20 100 2 ～ 6 ++ About 15
30 100 2 ～ 8 +++ About 12
Liquid medium 20 100 2 ～ 7 ++ About 12
30 100 2 ～ 8 +++ About 10
40 100 3 ～ 9 ++++ About 7
　　+means normal , ++ means good , +++means better , ++++means best.
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6194 期　　　　　　　　　　　　　　祖元刚等:白桦下胚轴再生系统的建立