全 文 ：Vol. 29 , No. 5
pp. 664～669 　Sept. , 2003
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 29 卷 第 5 期
2003 年 9 月 　664～669 页
Regeneration Study of Soybean Cultivars and Their Susceptibility to Agrobacterium
tumifaciens EHA 101
WANGLan1. 2 　T Clemente1 　WANGLian2Zheng2 　S S Sun3 　HUANG Qi2Man4
(1 Center for Biotechnology at University of Nebraska2Lincoln , Nebraska , USA , 6858820665 ; 　2 Crop Breeding and Cultivation Institute ; 　3 Chinese University of
Hong Kong ; 　4 Biotechnology Institute , Chinese Academy of Agricultural Sciences , Beijing ,100081 , China)
Abstract 　The soybean transformation procedure included the Agrobacterium2cotyledonary node system and the bar gene
as the selectable marker coupled with glufosinate as a selective agent . Cotyledonary nodes from 5 to 6 days germinated soy2
bean seeds were used as explants. The explants , were wound by slicing 5 to 6 times , inoculated with Agrobacterium tume2
f aciens EHA 101 with pPTN140 , then followed 3 days co2cultivation , washed the explants by wash medium which contain
antibiotics , put explants onto shoot initiation medium with 5 mgΠL glufosinate for selection. Regeneration rate of different
soybean cultivars was counted 2 weeks later , and their susceptibility to Agrobacterium tumefaciens was investigated 4 weeks
later by GUS assay. According to our experiments , Heinong 35 , Zhongzuo 975(Zhonghuang 13) , Hefeng 35 , Zhongzuo962
are better than Thorne for regeneration , William 82 , PI 361066 , Heinong 35 and Zhongzuo 975 were better than Thorne for
transformation.
Key words 　Soybean ; Regeneration ; Susceptibility to Agrobacterium
大豆品种的再生性能及对 EHA 101 农杆菌的敏感性
王　岚1 ,2 　T Clemente1 　王连铮2 　辛世文3 　黄其满4 Ξ
(1美国内布拉斯加林肯大学生物技术中心 ,6858820665 ; 　2中国农科院作物育种栽培研究所 ; 　3香港中文大学 ; 　4中国农科院生物技术研究
所 , 北京 100081)
摘 　要 　大豆转化可利用农杆菌和子叶节转化系统 , bar 基因作为选择标记 ,草丁膦作为选择试剂。用 526 d 发芽的种子
的子叶节作外植体 ,在子叶节处划 526 下 ,用含 pPTN 140 的农杆菌 EHA 101 感染后 ,共培养 3 d ,用含抗生素的洗液洗去
外植体上的农杆菌 ,将外植体放入 5 mgΠL 草丁膦的长芽培养基 ,两周后统计不同大豆品种的再生率 ,4 周后做 GUS 染色
对含 pPTN140 的农杆菌的敏感性进行统计。结果得知 :黑农 35、中作 975、合丰 35、Zhongzuo 962 是在再生方面比 Thorne 更
好的品种。William 82、黑农 35、中作 975、PI 361066 转化频率较高。
关键词 　大豆 ; 再生 ; 对农杆菌的敏感性
中图分类号 : S565 　文献标识码 : A
　　Soybean is a very important crop in China and in the
world. During the past 11 years the soybean production in2
creased 5817 %. Combination of traditional breeding method
with genetic engineering techniques for development of new
cultivars with herbicide resistance , insect resistance or good
seed quality is very important[124] . Agrobacterium2mediated
transformation of soybean using the cotyledonary node as the
explant for gene transfer was first achieved by Hinchee et al .
(1988) [2 ] . Zhang et al . described the soybean Agrobacteri2
um2mediated transformation system with bar gene as the se2
lectable marker coupled with glufosinate as a selective ag2
ent [3 ] . Zhou et al . introduced Bacillus thuringiensis cryIA
gene into soybean successfully with Agrobacterium2cotyledon2
ary node transformation system[4 ] . Genes were also trans2ΞFoundation item : This research was supported by Nobel Foundation in USA from 1999 —2000 and 948 project from MOA , PRC.
Biography : WANGLan (1963 - ) , female , research assistant , Master of Sciences at University of Nebraska2Lincoln.
Received : 2002211208 , Accepted : 2003201231.

ferred to soybean protoplast by some researchers[526] . Soybean
shoot organogenesis could occur from tissues such as cotyle2
donary nodes[729] and primary leaves of seedlings[10] while so2
matic embriogenesis occurred from immature embryos and co2
tyledons[11212] of developing seeds.
Agrobacterium2mediated transformation was the best
method available for DNA transfer to tissue explants. In order
to produce soybean tumors with significant size , soybean cult2
ivars and Agrobacterium strains had been screened to find the
optimal compatible response. Successful transformation of in
vitro soybeans leaves , cotyledons , and protoplasts[13 ,14] were
demonstrated.
Agrobacterium was used as the biological vector to intro2
duce a portion of its DNA into the plant genome , resulting in
production of transformed plants. Cotyledonary nodes were
wounded and inoculated with Agrobacterium . The wounded
plant tissues gave off specific phenolic compounds which in2
duce Agrobacterium to express a set of vir genes. vir genes is
responsible for the excision and transfer of the T strand from
the bacterium into the recipient plant cell .
bar gene encodes for phosphinothricin acetyltransferase
(PAT) which detoxifies glufosinate. Glufosinate is the active
ingredient in the herbicide Liberty. Kanamycin was used as
selective agent in previous researches. In this paper glufosi2
nate was used as selective agent .
However , there is still some degree of cultivar specificity
of transformation efficiency ( Hinchee et al . , 1988) [2 ] . And
to study this cultivar specificity is the main task of this re2
search.
1 　Material and Methods
1. 1 　Material and Seed Sterilization
　　3 to 4 varieties were evaluated each experiment , Thorne
were used as control . To total 15 cultivars such as Zhongzuo
975 , Zhongzuo 962 , Zhongzuo 966 , Zhongzuo 965 , Zhong2
zuo M17 , Zhonghuang 4 , Heinong 35 , Heinong 37 , Hefeng
35 , NE3297 , William 82 , U9622208 , PI 361066 , A3237
and Thorne were screened for good regeneration and suscepti2
bility to EHA 101 with pPTN140. The seeds were surface
sterilized by two days exposure to chlorine gas by mixing 100
mL of a 5. 2 % sodium hypochlorite ( Chlorox bleach) with
313 mL of 12 molΠL HCl . The procedure should be conducted
within a fume hood. The seeds are ready for the germination
step [15225] .
1. 2 　Soybean germination
Sterilized seeds were germinated in 100 mm ×20 mm
Petri dishes on B5 medium supplemented with 2 % sucrose ,
pH 5. 8. The plates stacked 5 high and placed in plastic
bags , 5 small holes were made by scissors. Seed were for five
days in a growth room at 24 ℃, 18Π6 light regime , or for 6
days (at 5th day put the seeds to 4 ℃refrigerator) .
1. 3 　Plant transformation vectors
EHA101 containing binary vector pPTN140 were used ,
and the resistances to 25 mgΠL chloramphenicol [ EHA 101
chromosomal drug marker ] , 50 mgΠL kanamycin [ Ti plasmid
pEHA 101 drug marker ] , 100mgΠL spectinomycin [ binary
vector drug marker ] and 100 mgΠL streptomycin [ binary vec2
tor drug marker ] were included in the Agrobacterium and
vector[3 ] .
Fig. 1 Binary vector utilized in the experiments
Abbreviations : RB2right border , LB2left border , T35s and pAg7 are
polyadenylation signals , Pnos2nopaline synthase
promoter , aadA2bacterial drug resistance marker
for spectinomycin and streptomycin.
1. 4 　Preparation of Agrobacterium
Agrobacterium was streaked from frozen glycerol stocks
onto solid YEP medium with appropriate antibiotics , and grew
for 3 days. One day before co2cultivation 2 mL Agrobacterium
were cultured in 10 —15 mL tube for about 10 to 12h , then
subcultured 015 mL Agrobacterium to 150 mL to 250 mL with
appropriate antibiotics for about 12 to 16 h until the OD650 =
1. 1 to 1. 2 at 27 ℃. The bacterial cultures were centrifuged
then at 3 ,500 rΠmin for 10 min , and the pellet was resus2
pended to a final OD650 = 016 - 018 in 1Π10 Gamborg’s B5
medium amended with 1. 67 mgΠL BAP , 0. 25 mgΠL GA3 ,
566　5 期 　WANG Lan et al : Regeneration Study of Soybean Cultivars and Their Susceptibility to Agrobacterium tumifaciens EHA 101 　　　

200μmolΠL Acetosyringone (AS) and 3 % sucrose. The me2
dium was buffered with 20 mmolΠL MES , pH 5. 4. All growth
regulators , vitamin components and AS were filter sterilized
by using syringe after autoclaving.
1. 5 　Plant transformation
1. 5. 1 　Explant slicing
Green seeds were only chosen from the 52day or 62day
old soybean seedlings and cotyledonary explants were pre2
pared for making a horizontal slice on the hypocotyl region.
The embryonic axis was removed , about 5 —6 vertical slice
was made on the adaxial surface of the explant at the cotyle2
don and hypocotyl junction by blade. The slicing was 0. 5 mm
deep and 3 —4 mm long.
1. 5. 2 　Inoculation and Co2cultivation
Co2cultivation medium contained 1Π10 the salts and vita2
mins of Gamborg’s medium amended with 1. 76 mgΠL BAP ,
0. 25 mgΠL GA3 , 200 μmolΠL AS , 3 % sucrose and 20
mmolΠL MES buffer , pH 5. 4(Note : filter sterilize all growth
regulators and AS) , and it should be prepared fresh the day
before the inoculation of soybean explants.
Explants were put in Agrobacterium solution for 30 min
to 1 h , and then on 100 mm×15 mm Petri plates containing
the co2cultivation medium solidified with 0. 5 % purified
agar. The co2cultivation plates were overlaid with a piece of
Whatman # 1 filter paper. The explants ( 5 per plate) were
placed adaxial side down on the co2cultivation plates for 3
days at 24 ℃, under an 18Π6 h light regume.
1. 5. 3 　Wash and shoot initiation
After the co2cultivation period the explants were briefly
washed with B5 medium containing 1. 67 mgΠL BAP , 3 % su2
crose , 50 mgΠL ticarcillin 50 mgΠL cefotaxime and 50 mgΠL
vancomycin. The medium was buffered with 3 mmolΠL MES ,
pH 516. Growth regulator , vitamins and antibiotics were filter
sterilized by using syringe after autoclaving. After the washing
step , explants were cultured in 100 mm ×20 mm Petri
plates , adaxial side up with the hypocotyl imbedded in the
medium , containing the washing medium solidified with 0.
8 % purified agar amended with 4 or 5 mgΠL glufosinate. This
medium was referred as shoot initiation medium(SI) . The ex2
plants were cultured under the same conditions as the seed
germination.
1. 5. 4 　Regeneration rate
After 2 weeks of culture , regeneration rate was counted
by the ratio of regular shoot and total explants set up . Regular
shoots referred to a lot of shoots regenerated from cotyledonary
node. If on the middle of cotyledonary node a big shoot was
growing , this kind of shoots referred to axillary shoot . Axil2
lary shoot occurrence was because the explant didn’t wound
enough. No regeneration referred to nothing growing on the
cotyledonary node , this was because the explant wounded too
much.
The bottom of the hypocotyl region was excised from
each of the explant . The regular explants , cotyledon with dif2
ferentiation nodes were subsequently subcultured on fresh SI
medium.
1. 5. 5 　GUS assay
After 4 weeks of culture , histochemical GUS assay was
carried out to see how many GUS sectors and how many GUS
differentiating shoots were developed. Soybean tissues were
vertically sacrificed on differentiating region for 5 to 6 times
and incubated in the X2Gluc substrate plus GUS assay solu2
tion for 8 h at 37 ℃, then taking the solution away , storing
the tissues in 70 % ethanol prior to observation under micro2
scope.
1. 5. 6 　Shoot elongation
The cotyledonaries were cut away from the differentiating
shoots . The multiple shoots were subcultured on shoot elonga2
tion medium ( SE) composed of Murashige and Skoog(MS)
(1962) basal salts , B5 vitamins , 1 mgΠL ZR , 0. 5 mgΠL GA3
and 0. 1 mgΠL IAA , 3 % sucrose and 3 mmolΠL MES ,
pH 516. The SE medium was amended with 3 mgΠL glufosi2
nate.
1. 5. 7 　Rooting
When reached 3 cm , the shoots were cut at the bottom
and rooted on MS salts with B5 vitamins , 1 % sucrose , 0. 5
mg ΠL NAA without further selection in Sundae cups ( Indus2
trial Soap Company. St Louis MO) .
1. 5. 8 　Transfer to soil
When the roots reached 0. 5 - 1. 0 cm , the plantlet were
transferred onto soil after carefully washing with water.
2 　Results
After putting the explants into SI mediumfor 2 weeks re2
generation rate was checked. Experiment 1 showed that 62
explants of Zhongzuo 975 were set up , 51 explants with good
regeneration. Another 2 weeks later , the number of GUS sec2
tors , buds and shoots calculated. GUS sector referred to GUS
expression bigger than 1mm in size. Several GUS sectors
666 　　　作 　　物 　　学 　　报 29 卷 　

could be obtained from one explant since one sector might be
sacrificed several times. Buds or shoots were observed from
62 explants in Zhongzuo 975. GUS differentiation shoots
referred to GUS expression , either chimeric or clonal , within
differentiating structures with leaf development . Susceptibility
to Agrobacterium tumefaciens EHA 101 depended on GUS as2
say results. The more GUS differentiating shoots showed , the
better susceptibility to EHA 101 was. Good experiments
should show at least 4 % GUS + budΠshoots and 40 % GUS +
sector among differentiation and nondifferentiation tissues , re2
spectively. [16225]
So as a result of experiment 1 , Heinong 35 and Zhong2
zuo 975 gave better regeneration rate and better susceptibility
than Thorne. The regeneration rate of Heinong 35 exceeded
17. 29 % over Thorne.
In experiment 2 of Zhongzuo 975 , Zhongzuo 962 and
Thorne used , among 124 explants of Zhongzuo 975 , 109 ex2
plants produced regular shoots , 30 explants gave GUS sector ,
6 explants showed GUS differentiating shoots. Of 91 explants
from Thorne , 60 became regular shoot , 1 explant showed
GUS differentiation shoot . So as a result of experiment 1 and
2 , regeneration rate of Zhongzuo 975 exceeded Thorne by
14102 %. Regeneration rate of Zhongzuo 962 exceeded
Thorne by 8. 36 %. It was suggested Zhongzuo 975 be better
susceptibility than Thorne.
In experiment 3 , regeneration rate of Hefeng 35 exceed2
ed Thorne by 9. 67 % , Hefeng 35 was more susceptibility
than Thorne or Heinong 37.
Table 1 Regeneration rate and GUS assays under 5 mgΠL glufosinate selection
Experiment Variety
Number of
explants set up
Regeneration rate
after putting explants
onto SI medium for 2 wk
GUS assays after putting explants
onto SI medium for 4 weeks
GUS sector GUS differentiating shoot
Exp 1 Zhongzuo 975 62 51Π62 (82. 26 %) 99 10 (16. 13 %)
Heinong 35 92 86Π92 (93. 48 %) 61 11 (11. 96 %)
Zhongzuo 962 147 116Π147 (78. 91 %) 2 0
Thorne (CK) 44 32Π44 (76. 19 %) 17 5 (11. 36 %)
Exp 2 Zhongzuo 975 124 109Π124 (87. 90 %) 30 6 (4. 84 %)
Zhongzuo 962 175 130Π175 (74. 29 %) 12 2 (1. 14 %)
Thorne (CK) 91 60Π91 (65. 93 %) 26 1 (1. 10 %)
Exp 3 Hefeng 35 68 58Π68 (85. 29 %) 30 5 (7. 35 %)
Heinong 37 74 40Π74 (54. 05 %) 17 1 (1. 35 %)
Thorne (CK) 18 13Π18 (72. 22 %) 0 1 (5. 56 %)
Exp 4 Hefeng 35 73 65Π73 (89. 04 %) 150 3 (4. 11 %)
Heinong 37 59 46Π59 (77. 97 %) 92 5 (5. 43 %)
Thorne 9 8Π9 (88. 89 %) 15 0
Exp 5 Heinong 37 73 55Π73 (75. 34 %) 139 9 (12. 33 %)
Hefeng 35 74 62Π74 (83. 78 %) 87 4 (5. 40 %)
Thorne 50 34Π50 (68. 00 %) 44 2 (4. 00 %)
Exp 6 William 82 30 18Π30 (60. 00 %) 7 6 (20. 00 %)
U9622208 183 136Π183 (74. 32 %) 56 8 (4. 37 %)
Thorne (CK) 87 65Π87 (74. 71 %) 14 3 (3. 45 %)
Exp 7 Zhonghuang 4 125 92Π125 (73. 60 %) 46 1(0. 80 %)
Zhongzuo 966 33 23Π33 (69. 70 %) 11 0
Thorne 80 59Π80 (73. 75 %) 14 3(3. 75 %)
Exp 8 Zhongzuo M17 18 14Π18 (77. 78 %) 5 2 (11. 11 %)
Zhongzuo M965 20 12Π20 (60. 00 %) 13 2 (10. 00 %)
Thorne 33 28Π33(84. 85 %) 21 0
Exp 9 NE 3297 61 49Π61 (80. 33 %) 16 1 (1. 64 %)
Thorne 70 64Π70 (91. 43 %) 9 0
Exp 10 PI 361066 129 119Π129 (92. 25 %) 138 16 (12. 40 %)
Thorne 104 92Π104 (88. 47 %) 29 4 (3. 85 %)
766　5 期 　WANG Lan et al : Regeneration Study of Soybean Cultivars and Their Susceptibility to Agrobacterium tumifaciens EHA 101 　　　

　　In experiment 4 , Hefeng 35 had higher regeneration rate
and more susceptibility than control .
In experiment 5 , Heinong 37 , Hefeng 35 had higher re2
generation rate and more susceptibility than Thorne.
In experiment 6 ,William 82 was more susceptibility than
U9622208 or Thorne.
In experiment 7 , Thorne was more susceptibility than
Zhonghuang 4 and Zhongzuo 966.
In experiment 8 , Zhongzuo M17 and Zhongzuo M965
was more susceptibility than Thorne.
In experiment 9 , NE 3297 was more susceptibility than
Thorne.
In experiment 10 , regeneration rate of PI 361066 ex2
ceeded Thorne by 3. 78 % , and PI 361066 was more suscepti2
bility than Thorne.
As the result of GUS differentiating shoot assay William
82 was the best cultivar for transformation , with 20 % of GUS
differentiating shoots , PI 361066 with 12. 40 % , Heinong 35
with 11. 96 % , Zhongzuo 975 with 10. 48 %. All of them
were better than control Thorne.
3 　Discussion
It is still a difficult event for most institutions in China to
transform soybean routinely using Agrobacterium mediated
transformation system.
3. 1 　Choices of Agrobacterium tumefaciens strains and
vectors , were proven to be critical . Agrobacterium tumefa2
ciens EHA101 was an ideal strain for soybean transformation.
3. 2 　13 soybean cultivars had been tested , and regeneration
rates of 5 cultivars were better than control . Cultivars whose
regeneration rate exceeded control by 10 % were Heinong 35 ,
Zhongzuo 975 , Hefeng 35 , Zhongzuo 962 , PI 361066. The
regeneration rate of other 8 cultivars didn’t exceed control .
Compared with Thorne , Heinong 35 , Zhongzuo 975 ( Zhong2
huang 13) , Hefeng 35 , Zhongzuo 962 and PI 361066 were
best genotypes for regeneration. However , more soybean
cultivars need to be screened for higher transformation effi2
ciency. [22225]
3. 3 　Agrobacterium mediated soybean cotyledonary node
transformation can result directly shoot organogenesis , in
which bar gene can be used as selectable marker.
3. 4 　The best combination of co2cultivation was 24 ℃ tem2
perature ,1Π10 strength of B5 salt and addition of 200μmolΠL
acetosyringone.
3. 5 　Satisfactory wounding during explant preparation played
an essential role. Wounding influenced not only shoot differ2
entiation but also Agrobacterium infection. We hope to get
more regular shoots of regeneration instead of axillary shoots
or non2regenerated shoots. If the explant was not wound
enough , axillary shoots(one big shoot grows) would grow. If
the explant was wound too much , no shoot might grow , and
this should be regarded as non2regenerated shoot .
3. 6 　Herbicide selection schemes needed to be optimal .
Glufosinate can be used as the selective agent for bar gene.
Typical problems associated with low selection pressure were
the increased escapes , chimerism events. While the problems
from high selection pressure were subsequent failure of trans2
genic recovery. Under standard culture conditions 5 mgΠL
glufosinate during shoot initiation period and 2. 5 mgΠL during
shoot elongation were optimal .
3. 7 　Antibiotics such as ticarcillin , cefotaxime and vanco2
mycin were helpful for satisfactory results. If some of them
were not available , Carbenicillin 300 —500 mgΠL plus some
of above available antibiotics can be used as replacement .
3. 8 　Use of L2cysteine as a strong antioxidant might enhance
Agrobacterium infection on soybean substantially according to
some research. The recommended concentration was 400 mgΠ
L in co2cultivation medium.
Acknowledgements
This work was mostly conducted at the Plant Transforma2
tion Core Research Facility , Center for Biotechnology of the
University of Nebraska2Lincoln. I’d like to express sincere
thanks to my advisor Dr. T Clemente for his help and finan2
cial Support from Nobel Foundation. Some works conducted at
Soybean laboratory of Institute of Crop Breeding and Cultiva2
tion Institute , Chinese Academy of Agricultural Sciences.
Thanks to Prof . XIN Zhi2Yong and all the colleagues at the
soybean department . Thanks to Prof . Samuel S Sun from Chi2
nese University of Hong Kong , Dr Zhangyuan Zhang from Mi2
ssouri University and SHAO Qi2Quan from Institute of Genet2
ics , Chinese Academy of Sciences for their help and support .
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966　5 期 　WANG Lan et al : Regeneration Study of Soybean Cultivars and Their Susceptibility to Agrobacterium tumifaciens EHA 101 　　　

WANGLan et al . : Study on Regeneration Rate of Soybean Cultivars and Their Susceptibility
to Agrobacterium tumefeciens EHA 101 with pPTN140 Plate I
A : Sterilized germinated on B5 medium. B : Cotyledonary explants put in the Agroba. C : How to slice the explants. D : Embryonic axis was removed. E : Verti2
cal slice on cotyledonary node through the hypocotyl region about 5 - 7 times. F : Shoot initiation of Zhongzuo 975.

WANGLan et al . : Study on Regeneration Rate of Soybean Cultivars and Their Susceptibility
to Agrobacterium tumefeciens EHA 101 with pPTN140 Plate II
G: Shoot elongation of Heinong 35. H: Shoot reached 3 cm , the root reached 0. 5 - 1. 0 cm , the plantlet can be transfer to soil . I : Gus assay result of William
82. J : Gus assay of T1 (Left) and T0 (right) . K: Gus assay result of William 82. L : Gus assay result of Heinong 35.