全 文 ：Received 7 Jul. 2003 Accepted 29 Oct. 2003
Supported by the National Special Program for Rasearch and Industrialization of Transgenic Plants in China (J99-B-001).
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A Novel System for Proliferation, Maintenance and Plantlet Germination
from Somatic Embryo of Soybean
WANG Ping, WANG Gang*, JI Jing, WU Ying
(Research Center for Plant Genetic Engineering, Changchun University of Agriculture and Animal Sciences,
Changchun 130062, China)
Abstract: Somatic embryogenesis was successfully induced with auxin in high concentration from
immature cotyledons of 18 genotypes of soybean (Glycine max (L.) Merr.) adaptable for planting in Northeast
China. The frequency of somatic embryogenesis varied from 0.29% to 77.62% among 18 genotypes of
soybean. The proliferative somatic embryos were obtained from 10 genotypes of soybean at rate of
5.2%-22.1%. For the first time, plantlets were obtained even after subcultured and maintained on solid
medium for over one year. This system provided a new and efficient receptor for genetic transformation of
soybean.
Key words: Glycine max ; somatic embryo; proliferation; maintainable culture; germinated plantlet
With genetic engineering developing, genetic transfor-
mation has been used for improving agricultural characters
of crops. Soybean is the important source of edible oil and
plant protein in China. However, research of genetic trans-
formation in soybean lagged when compared with other
crops since its tissue culture and plant regeneration are
extremely difficult. So far, it has been believed that the tis-
sue culture and genetic transformation of soybean are the
most difficult one among crops.
The path of tissue culture and plant regeneration in soy-
bean can be mainly divided into organogenesis and
embryogenesis. Most studies are focused on somatic em-
bryogenesis because chimaera is formed easily during ge-
netic transformation with the path of organogenesis in
soybean.
Christianson et al. (1983) firstly reported somatic em-
bryogenesis and regenerated plant from the immature em-
bryo axis of soybean with improved MS medium plus
2,4-D. Somatic embryogenesis and regenerated plant were
also obtained by Lazzeri et al. (1985) and Ranch et al. (1985)
with immature cotyledon and by Barwale et al. (1986) with
immature embryo. There have been some subsequent re-
ports on somatic embryogenesis and plant regeneration
corresponding to genotype, medium, hormone, concentra-
tion of sugar, organic component, length of cotyledon, pH,
light intensity, etc. However, these results are consistent
in different genotypes and experiment conditions. The fre-
quency of somatic embryogenesis was commonly low due
to genotype-dependent matter. Usually getting immature
cotyledon was limited by season. Also, somatic embryo
originating from immature cotyledon of soybean was not
maintained easily for a long period of time on solid medium.
Therefore, it is very important to establish the system of
somatic embryogenesis with high frequency and inducing
proliferative and to maintain somatic embryo on solid me-
dium for genetic transformation of soybean.
Finer and Nagasawa (1988) induced proliferative embry-
onic tissue that was maintained by routine subculture in
liquid medium from somatic embryo with genotype
“Fayette” of soybean. The embryos were transferred onto
solid medium and germinated to plantlets. However, such
an experiment was very difficult to be repeated and only
Bailey and Simmonds got proliferative embryonic cultures
respectively with very few genotypes by using this method.
Bailey et al. (1993) induced proliferative embryonic cul-
tures from eight genotypes. Simmonds and Donaldson
(2000) reported that 18 of 20 short-season soybean geno-
types screened for proliferative embryogenic capacity
formed secondary globular embryo at rates of 1%-70% of
cultured immature cotyledon. Five genotypes were cultured
for embryogenesis with liquid medium and were prolifer-
ated for at least six months. So far, there have been only
three reports about proliferative embryogenesis of soybean
in liquid. Maintainable embryonic cultures produced from
solid medium in soybean has not been reported.
The present study established a successful system of
rapid proliferation for somatic embryo on solid medium,
providing a competent receptor for genetic transformation
.Rapid Communication.
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 154-158
WANG Ping et al.: A Novel System for Proliferation, Maintenance and Plantlet Germination from Somatic Embryo of Soybean 155
of soybean.
1 Materials and Methods
Seeds of soybean (Glycine max (L.) Merr.) were sown
in Changchun during 2000-2002. About 20 d after flowering,
immature pods were removed and surface-sterilized for 30 s
in 70% ethanol，followed by 10-15 min in 0.1% HgCl2
and two to three times rinses with sterile water. Cotyledon
explants were excised by removing the embryonic axis and
pushing the cotyledons 3-5 mm in length out of the seed
coat. Cotyledon halves were placed on MSD inducing me-
dium which consisted of MS plus 10-40 mg/L 2,4-D at 25
℃ under dark conditions in order to induce somatic
embryogenesis. The somatic embryos were subcultured and
maintained in MSD medium in weak light, and developed
and matured in MSC medium (MS plus 0.5%-1.0% activity
charcoal and 5%-10% sucrose). The germinated somatic
embryo consequently developed to plantlets in MS medium.
The number of callus formation and somatic embryo-
genesis from immature cotyledon of different soybean geno-
types were recorded after cultured for four and six weeks
respectively. Frequency of callus formation (number of
cotyledon with callus/number of cultured cotyledon×
100%) and frequency of somatic embryogenesis (number
of cotyledon with somatic embryogenesis/number of cul-
tured cotyledon×100%) were calculated. The number of
proliferative somatic embryo were evaluated and inducing
frequency of proliferative somatic embryo (number of pro-
liferative somatic embryo / number of cotyledon with
somatic embryogenesis) were calculated after cultured for
eight to ten weeks. Frequency of germination (number of
germinated cotyledon embryo/number of total cotyledon
embryo) was scored when somatic embryo developed into
plantlet.
2 Results and Analysis
The callus was first observed from immature cotyledon
4-10 d after initiative cultures depending on different geno-
types of soybean. The somatic embryogenesis was formed
after 15-20 d of culture (Fig.1). The frequency of somatic
embryogenesis for different genotypes was similar among
different years. The result of experiment in 2001 is shown in
Table 1.
Nineteen genotypes formed callus and frequency of
callus formation varied from 1.82% to 100% depending on
different genotypes of soybean. In total 19 genotypes, 18
genotypes were responsible for embryogenesis with a range
of 0.29%-77.62%, but Dongnong 40567 did not form so-
matic embryos. Ten of 19 genotypes of soybean could form
proliferative somatic embryos (Fig.2) and frequency of pro-
liferative somatic embryos was from 5.2% to 22.1%.
Meanwhile, we found that the response of soybean geno-
types to initiation of somatic embryogenesis and to the
formation of proliferative somatic embryos were different.
Some of soybean genotypes were easier to inducing so-
matic embryogenesis and others could form proliferative
somatic embryos although they formed somatic embryo-
genesis with lower frequency. The frequency of callus
Table 1 Initiation of somatic embryogenesis and germination in soybean

Number Genotypes
Number of Frequency of Frequency of somatic Frequency of prolifer- Frequency of
immature callus formation embryogenesis ative somatic embryo germination
cotyledon (%± SE) (%± SE) (%± SE) (%± SE)
1 Heinong 35 98 85.71± 7.32 25.51± 7.12 10.2± 2.91 3.5± 1.21
2 Heinong 40 21 100.00 57.14± 14.25 15.0± 4.21 16.7± 4.53
3 Hefeng 25 162 83.95± 1.65 41.36± 8.78 18.4± 5.15 46.5± 8.65
4 Hefeng 39 247 96.76± 0.77 55.87± 13.54 15.6± 3.73 14.0± 3.14
5 Dongnong 40 233 99.57± 0.4 40.34± 7.22 16.5± 10.03 11.0± 4.38
6 Dongnong L13 391 100.00 42.71± 10.73 22.1± 11.90 22.0± 5.63
7 Dongnong 163 345 38.26± 11.76 0.29± 0.28 0 -
8 Dongnong 434 317 69.72± 11.70 3.15± 2.35 0 -
9 Dongnong 1168 277 99.28± 0.69 77.62± 7.66 18.3± 3.84 18.2± 3.27
10 Dongnong 32310 388 95.10± 1.88 32.47± 7.24 6.5± 1.85 5.5± 2.73
11 Dongnong 40567 662 52.11± 11.49 0 0 -
12 Jilin 26 160 100.00 2.50± 2.40 0
13 Jilin 35 53 92.45± 11.1 9.43± 8.40 0 -
14 Jilin 36 264 97.64± 0.78 16.54± 2.01 16.8± 7.46 19.1± 4.39
15 Jilin 38 138 100.00 58.70± 20.90 0 -
16 Jinong 9 159 99.37± 0.64 23.27± 10.95 0 -
17 Kaiyu 10 265 89.81± 1.39 35.47± 13.18 0 -
18 Kaiyu 11 127 51.18± 16.39 2.36± 1.39 5.2± 1.71 4.2± 2.06
19 Tiefeng 29 100 1.82± 1.77 18.00± 11.4 0 -
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004156
formation, somatic embryogenesis and proliferative somatic
embryos depended on genotypes of soybean to some extent.
The proliferative somatic embryo of globular stage de-
veloped into heart stage (Fig.3), torpedo stage (Fig.4), coty-
ledon stage (Fig.5) and matured embryos (Fig.6) in MSC
medium after six to eight weeks. Frequency of germination
varied from 3.5% to 46.5% depending on different geno-
types of soybean. The plantlets normally developed and
fertilized (Fig.7). Matured plants with the seeds were ob-
tained (Fig.8).
Figs.1-8. 1. The somatic embryogenesis in soybean. 2. Proliferation of somatic embryos at globular stage. 3. Somatic embryo at heart
stage. 4. Somatic embryo at torpedo stage. 5. Somatic embryo at cotyledon stage. 6. Matured somatic embryos. 7. Germinated plantlet.
8. Matured plant from proliferative somatic embryos.
WANG Ping et al.: A Novel System for Proliferation, Maintenance and Plantlet Germination from Somatic Embryo of Soybean 157
3 Discussion
For the first time proliferative somatic embryo was main-
tained on solid medium more than one year and germinated
plantlets were also obtained in our laboratory. Such a diffi-
cult problem for tissue culture and genetic transformation
of soybean was improved. Simmonds and Donaldson (2000)
obtained five genotypes that were eligible for proliferative
embryogenic formation from 20 genotypes with a liquid
medium by using the method of Finer and Nagasawa (1988).
In our study, proliferative somatic embryos were induced
from 10 of 19 genotypes and the frequency of initiation of
somatic embryos reached 77.62%. The system possesses
simplicity, high frequency and good repetition. The results
were consistent over three growth seasons (2000-2002).
We obtained and maintained proliferative somatic embryos
of soybean from 40 genotypes.
Long term tissue culture system has been available for
many crops in which mature or immature embryos were
used as explants and MS or N6 (plus auxin and cytokinin)
was applied. The embryonic callus could be subcultured,
proliferated and maintained for a long time, which was
proved to be a convenient receptor for genetic
transformation. However, such a kind of embryonic callus
could not be induced in soybean as effective as other crops.
Also, the somatic embryo will be licked up by callus around
if somatic embryo was transferred to developmental and
mature MSC germination medium in time. The system could
solve this difficulty and might be used as a better receptor
for genetic transformation of soybean.
Inducing and maintenance of proliferative somatic em-
bryo in soybean was mainly determined by medium
(including growth regulator of different categories and
concentrations), light conditions, types of somatic embryo
and genotypes of soybean. However, the functions of these
factors on regulating somatic embryogenesis and its main-
tenance were unclear so far.
Acknowledgements: The authors thank Dr. LI Wen-Bin
who works in Northeast Agricultural University as a pro-
fessor for his careful reading and correcting of the
manuscript.
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(Managing editor: WANG Wei)