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
以叶盘为外植体的白桦的再生
石福臣　孔　瑾　吴双秀　聂明珠　祖元刚　
(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　从不同的激素组成(BA ,K T , 2 ,4 ～ D ,NAA ,GA3)、基本培养基(MS ,WP)、外植体放置的
方向性进行了实验 ,建立了以白桦叶盘为外植体的再生系统 。当叶盘向轴面朝下放置在培养基上
时 ,三周后 , 从叶盘边缘生出不定芽。不定芽的诱导率为 64%,平均每片叶盘可生出 6个不定芽。
叶盘再生系统的建立为白桦的遗传转化提供了前提。
关键词　白桦;再生;叶盘
REGENERATION OF BETULA PLATYPHYLLA BY LEAF EXPLANT
SHI Fu-chen　KONG Jin　WU Shuang -xiu　NIE Ming-zhu　ZU Yuan-gang
(Open Research Laboratory of Fo rest Plant Ecology Nor theast Forestry University , Harbin 150040)
Abstract　The regeneration system of birch Betula platyphyl la by leaf explant w as established.Dif-
ferent kinds of hormones(BA ,KT ,2 , 4-D ,NAA ,GA3), basal media(MS ,WP), explants orienta-
tion were experimented.The leaf explant w as placed on medium with the adaxial face downwards.
After three weeks , adventit ious buds sprouted from the edge of leaf explants.The percentage of the
adventi tious shoot induction is 64%.The number of adventitious shoots incuced per leaf is 6.The re-
generation system can be po tentially used for genetic t ransformation of w hite birch.
Key words　White Birch;Regeneration;Leaf explant
Int roduction
White birch is widely distributed in the northeast
of china.It g row s fast and is an important pioneer
tree species for forestation.Its wood is also mainly
used for const ruction and ornament.But its economic
use is limited by the poo r wood quality.
Genetic t ransformation is the most promising
tool to improve the individual trait without lost of any
desired t raits of the parent plant.Agrobacterium
tumefaciens-mediated t ransfo rmation is the most ac-
ceptable method.The establishment of a high f re-
quency regeneration sy stem is the foundat ion of t rans-
formation.It is still dif ficult to establish a regenera-
tion system appropriate for A.tumefaciens -medi-
ated t ransformation for many trees.Birch is one of
the earliest t ree species to be grown in vitro.Micro-
propagation of more than ten species and varieties of
birch has succeeded since 1979.But there are few re-
po rts on t ransgenic plants from birch because of the
difficulty in establishing a suitable regeneration sys-
tem for genetic t ransfo rmation.The establishment of
国家自然科学基金资助项目(39870634)
第一作者简介:石福臣(1966-),男 ,教授 ,从事分子生态学研究
收稿日期:2001-09-16
regeneration system of white bi rch provides the po-
tential of it s breeding for genetic transformat ion and
w ill make it possible to improve wood quali ty of w hite
birch by genetic t ransfo rmation.
1　Materials
1.1 　Explants preparation Leaves w ere t rimmed
from the subculture seedling that w ere regenerated
from buds , stem explants and hypocoty l explants.
1.2 　Leave from bud resources Seedlings ranging
from 14.5 to 25cm were t rimmed from trees with
good branching pattern in w inter and cultured in w a-
ter fo r burgeoning at 20℃±2℃。After 7 to 10 days ,
burgeoned buds w ere sterilized and cultured in initial
medium A(WPB5(WP[ 1] salt and B5[ 2] vitamin)sup-
plemented w ith 0.8mg/ l BA and 20g/ l sucrose).Af-
ter one month , sprouted buds w ere subcultured in
medium F(WPB5 supplemented w ith 0.5g/ l BA and
20g/l sucrose).Leaves can be trimmed from regen-
erated seedling af ter 10 weeks through 3 subcultures.
＊
1.3 　Leaves f rom hypocotyl explants Seeds were
sterilized and cultured in 1/2MS [ 3].Nine day s af ter
burgeoning , hypocotyl was cut into 8mm and cul-
tured in medium B(WP B5 supplemented w ith 1.0g/
l BA and 20g/ l sucrose).One month later , g reen cal-
lus formed and subcultured in medium B .Subcul-
tured callus w as t ransferred to medium F and differ-
enriated af ter one month.Leaves could be t rimmed
from the seedling that regenerated f rom hypocotyl ex-
plants.
1.4　Leaves f rom stem explants Stem explants f rom
seedlings regenerated f rom buds and hypocoty l ex-
plants were cultured in medium B.Green callus form
at thei r edge af ter one month and subcultured in
medium B.After 9 weeks through 3 subcultures , cal-
lus w as transferred to medium F fo r differentiation.
Leaves can be trimmed from the seedlings differenti-
ated f rom the callus form from stem explants
Young leaves(7mm ～ 10mm long )whose edge
were cut off were excised and cut transversely across
the midrib into three o r four sect ions.Each petri dish
was sealed w ith parafilm .30 day s later , adventitious
buds were induced and then transferred to medium G
(MS B5(MS salt and B5 vi tamin )supplemented with
0.1mg/ l GA3 , 0.4mg/ l BA and 30g/ l sucrose )fo r
sprouting .After 40 day s , the sprouted shoo ts longer
than 2.0cm were cultured in f resh medium for roo t-
ing .Cultures w ere maintained at 22℃±2℃.Under
cool w hi te f luo rescent tubes(60μEm -2s-1)w ith a
16h photoperiod.
2　Methods
2.1　Effect of hormones on adventitious bud in-
duction
All leaf explants w ere g row n on MS B5 basal
medium supplemented with 30g/ l sucrose , 6 ～ 6.5g/l
agar and different combination of cytokinin and auxin
(Table 1).20 leaf explants were cultured in each
combination.The number of leaf sections forming
shoots and the number of shoots per leaf w ere record-
ed.
　　Table 1 Percentage of adventitious shoot induced/ %
BA concentration in WPB5(mg/ l) BA concentration in MS B5(mg/ l)
0.5 1.0 1.5 0.5 1.0 1.5
NAA(mg/ l)
0 4 14 8 18 20 12
0.01 6 12 10 26 38 16
0.1 6 32 22 28 58 40
0.2 8 38 26 32 64 46
2.2　Effect of basal medium on adventit ious bud in-
duction
20 leaf explants were cultured in each basal
medium supplemented w ith 1.0mg/ l BA.0.2mg/l
NAA and 30g/ l sucrose.WP , WPB5 , MS , MSB5
were tested.
2.3　Effect of sucrose of adventitious bud induction
Each group of 20 leaf explants w as placed on the
612 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　21 卷
medium C(MS B5 +1.0mg/l BA+0.2mg/ l NAA)
supplemented w ith dif ferent concentration of sucrose
(20g/ l , 30 g/ l , 40 g/ l)(Figure 1).The ratio of
adventi tious buds induced w as recorded.
Figure 1　Effect of sucrose concentration
on adventitious shoot induction
2.4　Inf luence of the leaf explant o rientation on ad-
vent itious bud induction Half of the total 100 leaf ex-
plants were placed in medium C2(MSB5+1.0 mg/l
BA + 0.2 mg/ l NAA + 30g/ l sucrose)w ith the
adaxial surface upw ards , the other half w ith the
adaxial surface downwards.The ratio of adventitious
buds induced was recorded.
2.5　Effect of hormone on shoot elongation Shoots
induced from leaf explants were t ransferred to f resh
medium fo r elongation.Basal medium M SB5 supple-
mented wi th 30g/l sucrose and dif ferent combinations
of hormones were used(Table 2).20 buds w ere cul-
tured in each kind of medium.
2.6　Rooting f rom the shoo ts
Shoo ts longer than 2cm were t ransferred to
medium for rooting.Dif ferent basal media(MS , 1/2
M S )supplemented w ith different combinat ion of hor-
mones (0mg/l , 0.01mg/ l , 0.1mg/ l , 0.2 mg/l
NAA;0.02 mg/ l , 0.2 mg/l , 0.5 mg/l IBA )and
different concentration of sucrose (20 mg/ l , 30 mg/
l , 40 mg/ l)were used for roo ting.
3　Results and discussion
The adventitious shoo ts w ere induced three
w eeks after leaf explants w ere cultured.Three days
af ter t reatment , cuticle thickness occurred.Cuticle
thickness varied w ithin a leaf , regardless of t reat-
ment.A largest amount of cuticle development occurs
at the bases of multicellular trichomes and a smallest
amount on lamina aw ay from trichomes.The varia-
tion in cuticle thickness is common in many species.
It was also observed that cuticle thickness w as g reater
on the abaxial surface of the leaf than the adaxial sur-
face in all cases.It is common in other species such as
Europe white birch
[ 4] .It is related exactly to the
amount of multicellar trichomes.Desiccation of leaf
explant w hich g reatly causes reduction of cuticle de-
velopment can be avoided by carefully strong leaf se-
lection.
　　Table 2 Different combinations
of hormones on shoot elongation
BA Concentration in MSB5 medium(mg/ l)
0.2 0.4 0.8 1.0
NAAmg/ l 0 + + + +
0.01 + +
0.1 + +
GA3mg/ l 0.1 + + + +
0.2 + +
0.5 + +
　　+　stand fo r hormone combination
　　It was show ed that maximum shoots induced
were observed at medium C2 (Table 3).The per-
centage of the adventit ious shoots induction can be as
high as 64%.The average number of the shoo ts per
leaf is 6.There w as a tendency for white birch on
media supplemented wi th BA and NAA to have
greater amounts of shoo t inducing .But the deg ree of
vi trif ication appeared to increase as the concentration
of BA was increased.Compared wi th other basal me-
dia , higher induction percentage w as got at MSB5 and
WPB5.MSB5 was the optimum basal medium among
the media used (Table 3).The concentration of su-
crose is an important factor that influences the adven-
titious shoots induction.It w as show ed that maxi-
mum shoots w ere induced when the concentration of
sucrose is 40g/l , fo r the economic aim ,we use 30g/
l(Figure 1).
Explant o rientation effects have been observed in
shoot - producing cultures of several woody
species
[ 5] .It has been reported that the number of
6134 期　　　　　　　　　　　　　　石福臣等:以叶盘为外植体的白桦的再生
　　Table 3　Effect of different hormones and different basal medium on adventitious shoot induction/%
BA Concentration in WPB5(mg/ l) BA Concentration in MS B5(mg/ l)
0.5 1.0 1.5 0.5 1.0 1.5
NAA(mg/ l)
0 4 14 8 18 20 12
0.01 6 12 10 26 38 16
0.1 6 32 22 28 58 40
0.2 8 38 26 32 64 46
shoot induced can be as tw ice as much wi th the adaxi-
al face downw ards than wi th the adaxial face up-
wards.It w as also observed that the percentage of
shoot induction w ith adaxial face contacted with
medium is much higher than that w ith abaxial face
contacted w ith medium in our experiment(Figure 2).
Figure 2　Effect of leaf explants o rientation
on adventitious shoot induction
Adventitious shoo ts were t ransferred to f resh
medium for elongation 30 day s af ter the shoo ts were
induced.The use of GA3 and cy tokinin is not com-
mon for inducing elongation of fo rest t ree species
[ 6] .
It is thought that GA3 stimulates elongation by in-
hibiting the action of auxins in meristematic regions.
Our experiment proves that enhanced shoot elonga-
tion occurs when GA3 is combined w ith BA (Table
4).It showed that maximum percentage of elonga-
tion was obtained when shoots w ere cultured at medi-
um G.
　　Table 4　Percentage of the adventitious
shoot elongation＊
BA concentration in MS B5 medium(mg/ l)
0.2 0.4 0.8 1.0
NAA(mg/ l) 0 14 20 10 0
0.01 14 26 10 0
0.1 16 32 14 0
GA3(mg/ l) 0.1 28 50 28 0
0.2 20 42 22 0
0.5 10 26 18 0
　　＊ the result unit is percentage of shoot elonga tion %
　　Table 5　Effect of hormones on rooting
Percentage of
rooting/ %
Num.of
roots
Quality of
roots
IBA(mg/ l)
0 94 1 ～ 4 +
0.02 95 1 ～ 5 +
0.2 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.
Shoo ts were t ransferred to f resh medium fo r
rooting w hen they w ere higher than 2.5cm.Roots
forms one w eek later.It w as proved by the experi-
ment that R1 (1/2M S supplemented with 0.1mg/l
NAA and 40g/ l sucrose )and R2 (1/2MS supple-
mented w ith 0.2 mg/ l NAA and 40g/ l sucrose)were
the optimum media for roo ting in the media used
(Table 5).
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