全 文 ：广 西 植 物 Guihaia 27(4)：622— 626 2007年 7月
Establishment of genetic transform ation system
of Populus euphratica and optimization
of antibiotic
LI W ei，CHEN Xiao—Yang，DING Xia，LI Hui
(KeyLaboratory ofGenetics and Breeding in Forest Trees and OrnamentalPlants，
MinistryofEducation，BeijingForestry University，Beijing 100083，China)
Abstract：Efects of hormones including NAA，IAA，6一BA on leaf differentiation of Populus euphratica and antibiot—
ies including Kanamycin(Kan)，G418，Carbenicillin(Cab)，Cefotaxime(Cef)on the growth and differentiation or
roofing of various explants of P．euphratica were studied．Genetic transformation system mediated by Agrobacterium
and optimum antibiotic concentrations for modified organism selection of P．euphratica were founded．Results shown
that：the optimal culture medium for leaf differentiation of P．euphratica was MS+BA 0．5 mg／L+N从 0．1～O．2
mg／L+sugar 25 g／L+agar5 g／L；at co-culture stage which leaves were used as explants，the optimum concentrations
of Kan，G418，Cab and Cef were 10 mg／L，7．5 mg／L，200~600 mg／L，200~400 mg／L respectively；at the stage of
subculture or rooting of resistance buds．the optimum concentrations were 15～2O mg／L，1O～15 mg／L，2oo～800
mg／L，200~600 mg／L respectively．
Key words：Populus euphratica；genetic transformation system；antibiotic concentration；optimization
CLC number：Q943．1 Document code：A Article ID：1000-3142(2007)04—0622-05
Populus euphratica，having extremely strong
stress resistance，is the only natural tall and big arbor
species distributed in the front aridity and desert north—
west area of China(Wang et a1．，1995；Wei，1990)．
For physiological reasons，P．euphratica has the char—
acteristic of few seeds，sexual reproduction handicap
and rooting difficulty by cutting。which restrict its pop—
ularization and application seriously。Genetic engineer—
ing has the advantages of specific purpose，shorting
breeding time and breaking
different species so that it
hybridization limitation of
has became an important
mean for genetic improving of P．euphratica． During
genetic transformation，screening genes were linked of—
ten for the selection of genetic modified organism．Npt
Ⅱgene which coding neomycin phosphoric acid shift
enzyme is fl screening gene used most extensively in
plant genetic engineering at present．It can phosphoric
the amino glucosidal type antibiotic such as Kan(ka—
namycin)，G418，Neo(neomycin)，etc．，thus enable ge—
netic transformed cells has the ability to resist above
antibiotics(Wang et a1．，1998)．But researches indiea-
ted that antibiotics ha s also some inhibitions to plant
tissues growth and differentiation(Guan et a1．，1994；
Wang et a1．，1996)．So the establishment of genetic
transformation system and the optimization of antibiot-
ic concentrations are the necessary precondition before
genetic transformation of P．euphratica．This paper fo—
cused on and studied the efects of 6一BA，IAA and NAA
on lear and calus differentiation of P．euphratica and
Kan，G4l8，Cab and Cef on the growt h and differentia—
tion or rooting ability of various explants of P．euphrati—
c口．Results of this research selected the optimal culture
mediulTt for leaf differentiation，screened and optim zed
the kinds and concentration of antibiotics，which estab-
lished the foundation for the genetic transformation of
P．euphratica with Agrobacterium tumefaciens．
Received date：2006-01—-09 necepted date：2006··10-27
Foundation：Supported by the National Natural Science Foundation of China(30271097)
First Author：LI Wei(1975一)，Male．Born in Dingzhou City，Hebei Province，Doctor，Lecturer．Major in Forestry Genetic Engineering．
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4期 李伟等：胡杨遗传转化体系的建立及抗生素浓度的优化 623
1 Materials and Methods
1．1 M aterials
Test—tube plantlets of P．euphratica for test were
from the key laboratory of genetics and breeding in
forest trees and ornament plants(Beijing Forestry U—
niversity)，Ministry of Education．Leaf and various ex—
plants for test were selected from test-tube plantlets
cultured after 20~ 30 d(Zhang et a1．，2001；Gu et a1．，
1999；Ding et a1．，2003)． The A．tumefaciens of
LBA4404 saved in our lab．Kan and G418 made in Ja—
pan．Other antibiotics brought from Dingguo corp．
1．2 Culture media
Aseptic seedlings come from adventitious buds of
P．euphratica cultured in vessels．The leaves were se—
lected from top to bottom of No．1 to 3 and the obverse
side of leaves laid upwards to the following culture
media of number l to 8．Every group contained 6 ex—
plants and repeated 4 times．
The antibiotics were added to the sterilized culture
medium when the temperature of medium reduces to
under 50 degree and mixed well before solidify．
1．3 Effects of antibiotics on the growth。differentiation
and rooting of explants
The explants of leaf．stem and virus-free seedlings
were planted in culture medium containing different
kinds and concentrations of antibiotics and then their
growth situations were observed．Investigation and sta—
tistics of index carried on 30 days later．Concentration of
Kan and G418 were divided into 0，2．5，5，7
． 5，10，15
mg／L group and Cab and Cef were divided into 0、200、
400、600、800 mg／L group also．Every concentration
group included 5 culture vessels which planted 5 pieces
(clones)under 28℃ and repeated 3 times．40／tmol·m
· of illumination intensity．12～14 h／d for illumina—
tion．
2 Results and Analysis
2．1 Effects of different hormone concentration group on
leaf regeneration of P．euphratica
Co nclusions from table 1 showed that：on the cul—
ture media with NAA．at the concentration range of BA
from 0．5 to 1．0 mg／L．the leaf regeneration efficiency
of al the groups reached up to 100 ．Whereas on the
culture media with IAA，at the concentration range of
BA from 0．1 to l_5 mg／L．the highest leaf regenera—
tion efficiency reached up to 83．3 9／6 and each adventi—
tious bud of leaf only 5．7 at most on average．Within
the specific limi ts，the quantity of the adventitious bud
of leaf relates to BA and NAA (IAA)ratio．When the
ratio was 5，regeneration frequency and quantity of ad—
ventitious bud of leaf on average reached the maximum
leve1．Among them，the culture media containing anti—
biotic concentration of BA 0．5 mg／L and NAA 0．1 to
0．2 mg／L were relatively favorable to the leaf regener—
ation．Variance analysis and multiple comparing to the
quantity of adventitious bud indicated that the culture
medium numbers of 2 and 4 were the best．Therefore。
the best culture medium prescription for leaf regenera—
tion of P．euphratica was：MS+BA 0．5 mg／L+NAA
0．1～0．2 mg／L+sugar 25 g／L+agar 5 g／L
Table 1 Effects of different BA，NAA and IAA level
on adventitious bud regeneration
Note：The small letter means significant difference at 0
． 05 leve1．The
same below．
2．2 Effects ofKan onleaf regeneration-stem differentia—
tion and rooting of P．euphratica
Results from the Fig．1 showed that all the culture
media containing Kan had inhibition to the regeneration
and differentiation or rooting of P．euphratica ex—
plants．With the rising of Kan concentration，the re—
generation and differentiation frequency of leaf and
stem were reduced gradually．Kan had more influences
to the regeneration and differentiation frequency of ex—
plants．When the concentration of Kan was at 2． 5 rag／
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624 广 西 植 物 27卷
120
100
80
60
40
20
0
0 2．5 5
Kan concentrat i on(mg·L一 )
Fig．1 Effects of kanamycin on morphogenesis
of Populus euphratica in tissue culture
L，the regeneration and differentiation frequency of leaf
and stem reached to more than 90 ；when the concen—
tration of Kan equaled to 5 mg／L，it reduced to 38％
from 100 of contrast，stem differentiation reduced to
56 rapidly tOO；as the concentration of Kan was more
than 7．5 mg／L，the leaf regeneration for adventitious
bud were totaly suppressed． The inhibition of Kan
still behaved as organization albino and differentiation
delaying．Higher Kan concentrations accompanied with
higher rate of albino seedlings．W hen Kan concentra—
tion equaled to 2 mg／L，only a smal amount of albino
seedlings were produced；when Kan concentrations
were more than 5．0 mg／L，the color of adventitious
bud or axilary bud appeared yellowish-white，and only
appeared leaf extend without growth point．W hen cul—
ture continued，buds stop growing and become albino
gradually．In the culture of adding Kan，the acquisition
time of the bud was postponed 5 days than contrast．It
was obvious that Kan had strong inhibition on the re—
generation and differentiation of leaf and stem．
The rooting ability of P．euphratica was very sensi—
tiveness to Kan too．It was found that explants which
grew in culture media including no kan，shoot and rot
wound can produce phenomenon of expanding after 10
d。and had production of adventitious rot quickly which
can reach 3 cnl finally，and there was more figure．When
Kan concentration was at 5．0 mg／L，it can suppress vi—
ms-free seedlings of P．euphratica to take ro t obvious—
ly．Though some shots can produce adventitious rot
to ，the rot was relatively short，shorter than 1 ClTI，rot
quantity less，and plant high grow suppressed~when the
concentration of Kan equaling to 7．5 mg／L，the rot
produced was shorter，the suppressed degree of high
growth of the plant increases；when Kan concentration
was more than 10 mg／L，no taking rot phenomenon ap—
peared。the plant was short and small and the top leaf
yellowy seriously．So，stem section of P．euphratica can
not take ro t in screening culture which Kan concentra—
tion is greater than 10 mg／L．Only the adventitious bud
transformed through genetics en~neering ma y take rot,
that was the measure to select the transgenic adventi—
tious bud or not．
2．3 Effec~of G418 on leaf regeneration，stem diferentia-
tion and aseptie se~ irCs rooting of P．euphrafica
Rg．2 showed tha t the inhibition of G418 on regen—
eration and differentiation or rooting of P．euphratica
explants were more obvious than Kar1．With the rising
of concentration，the regeneration and differentiation fre—
quency of leaf and stem，the taking rot rate of shot
were reduced rapidly．In the treatment of adding G418
with 5 mg／L，the blade loses green by a large scale，only
split up a sma l amount of adventitious bud in the petiole
and blade notch place．The regeneration frequency of the
blade was dropped to 18 and the induce frequency ro —
ting and division of stem to reduce by a large margin，
lower to 15 and 25 separately；as G418 concentra—
tion equaled to 7．5 mg／L，adventitious bud regeneration，
stem differentiation and ro t inducing inhibited totally
and no blade and petiole growing phenomenon appeared．
The whole blade loses green and not differentiation of
the adventitious bud． There was no adventitious bud to
split up even if lengthen culture time，and then the blade
withered． The apical domina nce and induced roting of
virus-free seedlings were suppressed seriously． The
plants appeared short，sma l，yelow top blade and out of
shape to twist．So 7．5 mg／L can be as the critical con—
centration of G418 that screening the transgenic virus-
free seedlings of P．euphratica．
2．4 Effects of Cef on leaf regeneration。stem differentia-
tion and aseptic seedlings rooting
The experimental results indicated：Cef had little
effects on root inducing of aseptic seedlings of P．eu—
phratica；the root inducing rate of al groups were a—
bove 80 at different concentration level；when the
一 一 oc∞丁叮∞ co— 町一 c∞ ∞ 一凸
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4期 李伟等：胡杨遗传转化体系的建立及抗生素浓度的优化 625
0 2．5 5 7．5
G41 8 concent r at i on(rng．L )
Fig．2 Effects of G41 8 on morphogenesis
of Populus euphratica in tissue culture
concentrations were lower than 400 mg／L，the differ—
entiation of the blade and stem section was influenced
little also；but with the rising of the concentration，the
division of the blade and stem section were suppressed，
more inhibition to the section of the stem；when Cef
concentration equals to 800 mg／L，the rate of blade and
stem regeneration were dropped to under 80 9／5 and
6O respectively；when Cef concentration was more
than 800 mg／L，adventitious bud that foriD wound
blade was no obvious extending and growi ng with cul—
ture time extension，which prove the function of Cef on
adventitious bud mainly to suppress the extending and
growing．When Cef concentration exceeds 800 mg／L，
the production time of the blade adventitious bud post—
pones 2 to 3 days more than contrast and the root and
division time of stem postpone 3 to 5 days．
1 20
1 00
80
60
40
20
0
0 2 0 0 4 00
C ef cO nc e nt r at
60 0 800
o n(mg．L-1)
Fig．3 Effects of Cef on morphogenesis of
Populus euphratica in tissue culture
2．5 Effec~ of Cab on leaf regeneration，stem differentia-
tion and aseptic seedlings rooting
Compared with Cef，carboxyl benzyl penicillin had
less influence on leaf regeneration，stem differentiation
and aseptic seedlings rooting．Acting as the concentra—
tion of benzyl penicillin of carboxyl from 0 to l 000
mg／L，all the culture media can form adventitious bud，
auxiliary bud，root，and the division frequency of ad—
ventitious bud and root were comparatively steady．
The division frequency of adventitious bud，axilary bud
and the rooting frequency of aseptic seedlings were
higher than corresponding concentration of Cef gener—
aly．When the Cab concentration equaled to 600 mg／
L，leaf regeneration and stem differentiation rate were
9l 9／5，76 9／5 respectively，aseptic seedlings rooting rate
was 100 ． New blade grown fast than that of Cef
treated usually and all blades kept green；when the Ca b
concentration was at 600 to 800 mg／L，the blade be—
gins to taken off green gradualy；when the Ca b con—
centration was at l 000 mg／L，leaf regeneration and
stem root inducing frequency were all above 90 ，re—
generation bud displayed slight glass symptom．
3
1 20
1 00
80
60
40
20
0
0 2 00 4 00 600 800 1 000
C a b c O n c e nt r at i o n(mg·L。’)
Fig．4 Effects of Cab on morphogenesis of
Populus euphratica in tissue culture
Lx)nclus10ns and k)lSCUSSlOnS ，、 1 0 11卜、● 0
While the plant genetic transformation，nptI~USU—
aly regarded as the screening ma rk gene，the expres—
sion result of this gene give the resisting of transgenic
plant to kanamycin or G418．Understanding the exper—
imental result of this research wi ll help to adopt the
proper choice pressure in plant transgenic studies and
control the quantity of no-transforming body under the
minimum limit．Kan and G418 have strong inhibition
on the leaf regeneration，stem diferentiation and asep—
tic seedlings rooting of P．euphratica． The inhibition
of G418 was more remarkable． Culture medium con一
∞ ∞ ∞ ∞ 0
一 一 oc∞3 ∞L co一 一 c∞L∞ 一凸
一 一 uc∞3叮∞L c0一 一 c∞L∞ 一凸
一 一 uc∞3 ∞L c0一 一 c∞L∞ 一凸
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626 广 西 植 物 27卷
taimng Kan concentration of 10 mg／L or G418 concen—
tration of 7．5 mg／L，can suppress leaf differentiation of
P．euphratica totally and can t emerge adventitious
bud，axillary bud or adventitious root．So，Kan or G418
were relatively suitable for and regarded as the screen-
ing antibiotic． In the course of choosing，in order to
prevent more none transgenic buds inducing，its con—
centration can be raised properly to Kan 15~20 mg／L
or G418 l0w 15 mg／L．However，even use higher Kan
or G418 concentration，there are specific plants to as—
cape antibiotic selection and survive．So ，as the recep—
tor of gene transformation，there also exist the phe—
nomenon of not transforming adventitious bud after
screen of antibiotic pressure．
Cef and Cab were the bactericide which used ex—
tensively during plant genetic transformation mediated
by agricultural bacillus(Zhang eta1．，2000；Lin eta1．，
1995)．But both Cef and Cab have different impacts on
different plant tissues．In this experiment，Ca f and Cab
were relatively less inhibition on leaf regeneration，stem
differentiation and aseptic seedlings rooting of P．eu—
phratica．But it can suppress the blade regeneration
and stem section to split up under high concentration
and can suppress the excessive reproduction of the ag—
ricultural bacillus completely．Confirm tentatively from
above that the suitable concentration of antibiotic at
every stage during genetic transfolrmation of P．euph—
ratica were：during transgenic buds selection stage of
blade(or stem section)the suitable concentration of
Cef or Cab were the range of 200 to 400 mg／L~during
rooting selection stage of the transgenic aseptic seed—
lings the suitable concentration of Cef or Ca b were the
range of 200 to 600 mg／L．
References：
Ding X，Chen XY，Li Y，eta1．2003．
1eaves of Populus euphratica[J]．
28— 31
Bud regeneration system from
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(1)：29—33
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胡杨遗传转化体系的建立及抗生素浓度的优化
李 伟，陈晓阳，丁 霞，李 慧
(北京林业大学 林木、花卉遗传育种教育部重点实验室，北京 100083)
摘 要：探讨了不同激素浓度对胡杨叶片分化以及卡那霉素、G418、羧苄青霉素和头孢霉素4种抗生素对胡杨
不同培养阶段外植体生长、分化或生根的影响，确定了由农杆菌介导的胡杨遗传转化研究中抗生素种类和转化
体的筛选浓度，建立了适于胡杨叶片转化的遗传转化体系。结果表明：胡杨叶片再生的最佳培养基为 MS+BA
0．5 rag／L+NAA 0．1t0．2 mg／L+白砂糖25 L+琼脂5 g／L；在叶片转化筛选阶段，卡那霉素和G418的适宜
浓度分别为 10 mg／L和7．5 mg／L，羧苄青霉素和头孢霉素的适宜浓度为200~600 mg／L和200~400 mg／L；在
抗性芽生根培养时，卡那霉素和G418分别为15～2O mg／L和10~15 mg／L，羧苄青霉素和头孢霉素为200~800
rag／I 和 200~600 mg／L。
关键词：胡杨；遗传转化体系；抗生素浓度；优化
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