Weed Biology and Management 2, 25–30 (2002)
RESEARCH PAPER
Natural hybridization in wild soybean (Glycine max
ssp. soja) by pollen flow from cultivated soybean
(Glycine max ssp. max) in a designed population
YUICHIRO NAKAYAMA* and HIROFUMI YAMAGUCHI
Conservation Ecology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai City, Japan
The frequency of hybridization through pollen flow from the cultivated soybean to the wild
soybean was evaluated for the purpose of assessment of the ecological risk of genetically
modified crops. The flowering habits of three soybean cultivars and one wild soybean
accession were monitored on an experimental farm. A cultivar and a wild accession, both of
which flowered at a similar period, were then planted alternately in 5 ¥ 12 arrays with 50 cm
spacing on the farm. The seedlings of progeny seeds gathered from individual plants of the
wild accession were used for an isozyme analysis to identify whether they were hybrid or
not. In 23 plants of the wild accession, four plants produced hybrids (the incidence of
hybridization = 17.4%). There was no directionality in hybridization. The hybridization rate
per maternal plant varied from 0 to 5.89% with a mean of 0.73% for all maternal plants.The
results indicate that natural hybrids are easily produced in a certain frequency by pollen flow
from the cultivated soybean to the wild soybean under their simultaneous flowering with
adequate pollinators.
Keywords: ecological risk, genetically modified crops, natural hybridization, pollen flow,
wild soybean.
INTRODUCTION
The diffusion of herbicide-resistant genes has become a
serious problem in the natural environment with the
development of herbicide-resistant crops (Raybould &
Gray 1993; Hancock et al. 1996; Morishima 2000). In
many genetically modified (GM) crops of which a gene
is inserted into the nucleus genome such as herbicideresistant soybean cultivars (Padgette et al. 1996), the GM
gene can spread to wild relatives through movement of
pollen grains (Raybould & Gray 1993; Hancock et al.
1996; Keeler et al. 1996). Pollen flow from the cultivated soybean [Glycine max (L.) Merr. ssp. max] to the
wild soybean [G. max (L.) Merr. ssp. soja (Sieb. et Zucc.)
Ohashi] is not studied or discussed in risk assessment as
*Correspondence to: Yuichiro Nakayama, Gakuen-cho 1-1, Sakai
City, Osaka Prefecture 599-8531, Japan
Email: [email protected]
Received 30 July 2001; accepted 22 November 2001
a natural hazard because there is no natural population
of the wild soybean near cultivated soybean fields in the
United States of America (Padgette et al. 1996). In contrast, the wild soybean is native to cool- and warmtemperate East Asia (Abe et al. 2000; Umemoto &
Yamaguchi 2000).
The cultivated soybean, a predominantly self-pollinating
species, usually shows an outcrossing rate of less than
1% (Garber & Odland 1926; Chiang & Kiang 1987);
however, the rate exceeds 2.5% in particular cultivars
under favorable conditions (Ahrent & Caviness 1994;
Cutler 1934). The wild soybean is also a self-fertile and
predominantly self-pollinating subspecies; however, it
shows a wide range in the outcrossing rate from 2.4 to
3.0% (Kiang et al. 1992), and in particular cases from 9
to 19% (Fujita et al. 1997).
The cultivated and wild soybeans have no crossing
barrier and produce highly fertile F1 hybrids by artificial crossing (Sekizuka & Yoshiyama 1960; Kwon et al.
1972; Oka 1983). There have been ‘circumstantial evi-
26
Y. Nakayama and H.Yamaguchi
dences’ of natural hybridization between the cultivated
and wild soybeans: the weedy or semiwild form, which
was referred to as Glycine gracilis Skvortz., is occasionally found in East Asia (Skvortzow 1927; Sekizuka &
Yoshiyama 1960; Kwon et al. 1972; Broich & Palmer
1980); introgression between the cultivated and wild
soybeans in Japan was reported (Abe et al. 1999); some
soybean cultivars have been bred by spontaneous
hybridization from the wild soybean to the cultivated
soybean in Japan and Russia (Mitsui 1967; Abe et al.
2000). However, the frequency of the natural hybridization through pollen flow from the cultivated soybean to
the wild soybean has not been measured quantitatively.
An evaluation of a GM soybean cultivar, a glyphosatetolerant one, suggested no hybrid production in the
progeny of the wild soybean grown with the cultivar in
an isolated experimental field (Manabe et al. 1996);
however, the flowering time and pollinators of the soybeans were not mentioned in the report. For the risk
assessment of GM crops, it is important not to discuss
whether gene flow between GM crops and the wild
relatives will occur, but to clarify how frequently and
under what conditions the gene flow occurs.
This paper aims to estimate ‘directly’ the amount of
pollen-mediated gene flow from the cultivated soybean
to the wild soybean under the favorable conditions.
First, the overlap of the flowering time between the
cultivated and wild soybeans was examined because
the amount of pollen flow is affected by the overlap
of flowering between crops and their wild relatives
(Woodworth 1922; Lefol et al. 1996). Second, the frequency of the natural hybridization from the cultivated
soybean to the wild soybean was measured in an experimental plot using a cultivar and a wild accession of
soybeans, both of which flower at a similar period.
The possibility of natural hybridization from cultivated
fields to natural populations of soybeans was then
discussed.
MATERIALS AND METHODS
Flowering time
On an experimental farm in the Osaka Prefecture
University, we monitored the flowering habits of three
soybean cultivars, ‘Okuhara-wase’, ‘Tsurunoko-daizu’
and ‘Tambaguro’, which are local in Kyoto City, Japan,
and of one wild soybean accession, Gls/93-J-01, from
Kyoto Prefecture, Japan. Seeds of the cultivars were
sown on 9 May 1998 (‘Okuhara-wase’) and on 5 and
18 June 1998 (‘Tsurunoko-daizu’ and ‘Tambaguro’)
because precocious soybean cultivars are usually sown
in early May and late cultivars are usually sown in a
period from early to late June in the central or northern part of the Kinki district in Japan. Seeds of the wild
accession, of which the coats were abraded with nail
clippers, were sown successively on 1 May, 29 May and
19 June 1998 because the wild soybean emerges from
late April to late June in natural populations in the
central or northern part of the Kinki district (Y.
Nakayama & H. Yamaguchi, unpublished data, 1999).
Seedlings were grown in a greenhouse. Twenty days
after sowing, five seedlings per cultivar and per the wild
accession at each sowing date were individually transplanted into 18 cm unglazed pots with sandy loam, and
plants were grown under outdoor conditions on the
experimental farm. The flowering date of each plant
was recorded.
Natural hybridization by pollen flow from
cultivated to wild soybeans
To evaluate the frequency of the natural hybridization
in the wild soybean by pollen flow from the cultivated
soybean, ‘Tambaguro’, as paternal plants and Gls/93-J01 accession as maternal plants were used. The seeds
of the cultivar were sown on 18 June 1998, and the
abraded seeds of the wild accession were sown on 29
May 1998. Germinated seeds were individually placed
in 18 cm unglazed pots with sandy loam 5 days after
sowing, and plants were grown on the experimental
farm. A set of stays with rings for twining of vines was
put on each pot of the wild accession. On 18 July 1998,
30 plants of the ‘Tambaguro’ and 30 plants of the
Gls/93-J-01 accession were arranged alternately in 5 ¥
12 grids with 50 cm spacing on the experimental farm
(see Fig. 3).There was no natural population of the wild
soybean around the farm. In early November, mature
seeds were gathered from individual plants of the wild
accession and prepared for progeny tests to detect
hybrids.
In order to determine the genotypes of the parental
plants and to choose a suitable genetic marker, mature
leaf samples from parental plants of the ‘Tambaguro’
and the Gls/93-J-01 accession were used for an
isozyme analysis in early June 1998. Leaves of the
progeny seedlings obtained from the maternal Gls/93J-01 plants were analyzed for isozyme features to identify whether they were hybrid. An enzyme extraction
and gel staining procedure was adapted from Griffin
and Palmer (1987). Polyacrylamide-gel preparation and
electrophoresis were performed following the protocol of Shields et al. (1983). Genetic interpretation of
Natural hybridization in wild soybean
isozyme phenotypes (banding patterns) was performed
according to Candy and Beversdorf (1984), Griffin and
Palmer (1987) and Abe et al. (1992).
The frequency of hybridization from the cultivar to the
wild accession was evaluated by incidence and rate of
hybridization. Incidence was defined as the fraction of
plants of the wild accession having at least one hybrid
progeny. The rate was defined as the fraction of hybrid
progeny among the total progeny sampled from each
plant (Klinger et al. 1991).
RESULTS AND DISCUSSION
27
bore cleistogamous flowers from late September to early
October instead of chasmogamous flowers.
The flowering period of the ‘Tambaguro’ and Gls/93-J01 accession overlapped for a few weeks; however, the
‘Okuhara-wase’, ‘Tsurunoko-daizu’ and the wild accession showed no or only a few days of overlap in the
flowering time (Fig. 1). Therefore, the ‘Tambaguro’ and
Gls/93-J-01 accession were assumed to be suitable for
evaluating the frequency of the natural hybridization in
the wild soybean by pollen flow from the cultivated
soybean.
Frequency of natural hybridization in wild
soybean by pollen flow from cultivated soybean
Flowering period of the cultivated and
wild soybeans
The ‘Okuhara-wase’ plants sown on 9 May simultaneously started flowering on 6 June and continued
flowering until 19 June (Fig. 1). The ‘Tsurunoko-daizu’
plants sown on 5 June simultaneously started flowering
on 7 July, while plants sown on 18 June simultaneously
started flowering on 18 July. The ‘Tsurunoko-daizu’
plants sown on 5 and 18 June continued flowering for
approximately 3 weeks. The ‘Tambaguro’ plants sown
on 5 June started flowering from 29 July to 15 August,
while plants sown on 18 June started flowering from 3
August to 15 August.The ‘Tambaguro’ plants sown on 5
and 18 June continued flowering for approximately 4
weeks. The Gls/93-J-01 accession started flowering at
different times depending on the sowing date (Fig. 1).
The wild accession plants sown on 1 May started flowering from 31 July to 15 August, plants sown on 29
May simultaneously started on 15 August, and plants
sown on 19 June simultaneously started on 24 August.
They continued flowering until late September, and
The ‘Tambaguro’ plants started flowering from 4 August
to 18 August, and continued flowering in the middle
of September in the experimental plot. In contrast, all
plants of the Gls/93-J-01 accession started flowering on
15 August, and they gradually bore chasmogamous
flowers until late September. The flowering period
between the cultivar and the wild accession overlapped
for approximately 30 days.
All 30 of the ‘Tambaguro’ plants bore flowers with
purple petals; however, few of them fruited because of
infestation with stinkbugs and accidental drought. All
30 plants of the Gls/93-J-01 accession also bore flowers
with purple petals. Mature seeds were secured from 23
of the 30 plants. No seeds were obtained from seven
plants because of accidental events.
All parental plants of the cultivar and the wild accession
were homozygotes for a slow migration allele (b-allele)
and a fast migration allele (a-allele) at the endopeptidase
(Enp) locus, respectively, although the other loci had
cv. Okuhara-wase
Fig. 1. Flowering of three cultivars and a wild accession of
soybeans grown on the experimental farm of Osaka Prefecture
University, Sakai City, Japan. ()
Sowing; () the first flowering;
(......) vegetative growth; (—) flowering period; ( ) chasmogamous
flower; ( ) cleistogamous flower.
Five plants were grown at each
sowing date.
cv. Tsurunoko-daizu
cv. Tambaguro
Wild accession
(Gls/93-J-01)
April
May
June
Month
July
August
September
28
Y. Nakayama and H.Yamaguchi
–
➝
Fig. 2. Isozyme phenotypes and genotypes for the endopeptidase (Enp) locus of the cultivated soybean, the wild soybean
and hybrid progeny. (a) Cultivated soybean ‘Tambaguro’ (paternal plant, bb genotype), (b) wild Gls/93-J-01 accession
(maternal plant, aa genotype), (c) progeny of the wild accession, ( ) hybrid progeny, ab genotype.
the same alleles between the cultivar and the wild
accession. As monogenic and codominant inheritance
was known in the alleles for the Enp locus (Griffin &
Palmer 1987), the hybrid between the cultivar and the
wild accession was identified as a heterozygote for aballeles at the locus (Fig. 2).
Four maternal plants of the wild accession segregated
hybrid offspring. The four maternal plants appeared
to be randomly located in the experimental plot, indicating no directionality in hybridization (Fig. 3). The
incidence of hybridization from the cultivar to the
wild accession (17.4%) was in the range from 3 to 33%,
which was found in predominantly self-pollinating
and wind-pollinated sorghum cultivar and wild
sorghum (Arriola & Ellstrand 1996).This incidence was
lower than the incidences found in mixed mating and
insect-pollinated tobacco cultivars (ranged from 48 to
96%; Paul et al. 1995), and those found in outcrossing
and insect-pollinated radish cultivar and weedy radish
(reached at 100%; Klinger et al. 1992). This indicates
that a reasonable outcrossing syndrome was found in
predominantly self-pollinating and insect-pollinated
soybean cultivar and wild soybean.
Five hybrids were detected from 686 progeny, and
the mean rate of hybridization was 0.73%, which was
similar to the outcrossing rates of less than 1% obtained
for cultivated soybeans in the studies by Chiang and
Kiang (1987), and Garber and Odland (1926).The variance of the outcrossing rates is important information
on the risk evaluation of gene flow from GM crops
to the wild relatives as well as the mean rate. The
hybridization rate per maternal plant was variable
(Fig. 3), 2.33% (one hybrid of 43 progeny), 4.76% (two
of 42), 5.26% (one of 19) and 5.89% (one of 17). The
rates per maternal plant indicate that higher outcrossing
Fig. 3. Arrangement of cultivated ( ) and wild () soybeans in the experimental plot, and the frequency of
hybrids in the progeny of wild soybeans. Numerals: No. of
hybrids/no. of progeny analyzed.
that surpasses the prediction from the self-pollinating
nature of soybeans would occur depending on ecological conditions.
The cultivated and wild soybeans are entomophilous
(Erickson 1984; Fujita et al. 1997). In the experimental
plot, the leaf-cutter bee (Megachile turugensis Cockerell)
frequently visited the wild soybean, cut the leaves and
carried the pieces into her underground nest beside the
plot in late June. From middle to late July, a certain kind
of halictid bee (Halictus sp.) visited the ‘Tsurunokodaizu’ flowers of those plants which were placed
approximately 10 m apart from the experimental plot.
In the ‘Tambaguro’ and Gls/93-J-01 flowers, only a
certain kind of thrip (a species of Thripidae) was frequently observed during their flowering season. In contrast, there are more pollinators and visitors to the wild
soybean flowers in natural sites of rural environments
(Fujita et al. 1997; Y. Nakayama & H. Yamaguchi unpublished data, 1998). Although there was no natural
Natural hybridization in wild soybean
population of wild soybean around the farm, and the
fauna of insects visiting the cultivated and wild soybeans seemed to be poor, natural hybridization from
the cultivated soybean to the wild soybean occurred.
Pollen-feeding thrips, although they are pests to crops,
also cause outcrossing in predominantly self-pollinating
species (Miyazaki & Kudo 1988; Namai 1989). They
have been used for mediating pollen in a breeding
program through spontaneous hybridization from the
wild soybean to the cultivated soybean in Russia (Abe
et al. 2000). Halictid bees are considered to be pollinators of soybeans as well as honeybees and bumblebees
(Namai 1989). In addition, leaf-cutter bees are effective
pollinators of leguminous crops such as alfalfa and
clover (Namai 1989; St. Amand et al. 2000). The
thrips, the halictid bee or the leaf-cutter bee, thus
might mediate pollen during the hybridization from
‘Tambaguro’ to Gls/93-J-01 accession.
Possibility of natural hybridization from
cultivated fields to natural populations
of soybean
An evaluation report of a glyphosate-tolerant GM
soybean cultivar asserted the environmental safety of the
cultivar on the grounds that no hybrid was obtained
from the progeny of the wild soybean grown with the
cultivar in an isolated experimental field under uncertain
flowering behavior and pollination ecology of the soybeans (Manabe et al. 1996). Our results, however, indicate
that natural hybrids are easily produced in a certain frequency by pollen flow from the cultivated soybean to the
wild soybean under sympatric occurrence of the cultivated and wild soybeans, their simultaneous flowering,
and under the presence of adequate pollinators.
There are many natural populations of the wild soybean
around large-scale cultivated soybean fields in Japan,
with the exception of Hokkaido. In general, the size of
the area planted with crops, or the pollen ‘load’ affects
the dynamics of pollen flow into natural populations,
and the outcrossing frequency for large-scale cultivated
fields was much greater than that of small-scale experimental plots (St. Amand et al. 2000). The wild
soybean usually flowers later than the cultivated
soybean; however, their flowering times fluctuate with
their genotypes (Sekizuka & Yoshiyama 1960; Fukui &
Kaizuma 1971) and with the timing of seedling emergence (Fig. 1). The flowering period of the cultivated
and wild soybeans may overlap especially in the southwestern part of Japan where late-flowering soybean cultivars are usually planted (Saito 1981).This tendency has
been clearly observed in the examination of the specimens of cultivated and wild soybeans deposited in the
29
herbarium of Kyoto University to estimate the flowering time of cultivated and wild soybeans in Japan (Y.
Nakayama & H. Yamaguchi, unpublished data, 1999).
Many insect species such as pollinators usually visit
natural populations and cultivated fields of soybeans in
many countries (Beard & Knowles 1971; Erickson
1984; Fujita et al. 1997). The outcrossing rate of both
the cultivated and wild soybeans can be increased under
the condition where pollinators are frequently visiting
flowers (Beard & Knowles 1971; Fujita et al. 1997).
Under these particular environments, natural hybridization through pollen flow from cultivated fields to
natural populations of soybeans may occur more
frequently.
Although it is unknown how to establish and regenerate the hybrids between the cultivated and wild soybeans under natural habitats, artificial hybrid derivatives
with various life history traits can regenerate their
offspring under seminatural sites (Oka 1983). When
herbicide-resistant soybean cultivars are released and
cultivated in East Asia, including Japan, the gene flow
from the cultivars to the wild soybean may cause ecological risks, as was pointed out in rice (Langevin et al.
1990; Suh et al. 1997; Akimoto et al. 1999; Morishima
2000): introgression will occur from cultivated to wild
soybeans, and consequently a few alleles on particular
loci of the wild soybean will be gradually replaced by
cultivar alleles (genetic erosion of wild populations); in
some cases a genotype similar to weedy soybean will
evolve as was suggested by Oka (1983) (evolution of
weedy type). Thus, much more information regarding
the ecological and genetic relationship between the
cultivated and wild soybeans, as well as other crop-weed
and crop-wild complexes, is needed in order to make
monitoring-systems for diffusion of the transgene.
ACKNOWLEDGMENTS
We wish to thank Dr M. Kato of Kyoto University and
Dr K. Gokon of Tohoku Gakuin University for their
advice about pollination ecology. We would also like to
thank Dr J. Abe of Hokkaido University for his invaluable information on the cultivated and wild soybeans.
This study was supported by Grants-in-Aid for scientific research from the Ministry of Education, Culture,
Sports and Science of Japan (No. 5876), and from
the Japan Society of the Promotion of Science (No.
12780427).
REFERENCES
Abe J., Hasegawa A., Fukushi H., Mikami T., Ohara M. and Shimamoto Y.
1999. Introgression between wild and cultivated soybeans of Japan
30
Y. Nakayama and H.Yamaguchi
revealed by RFLP analysis for chloroplast DNAs. Econ. Bot. 53,
285–291.
Abe J., Kanazawa A. and Shimamoto Y. 2000.Wild soybean in Far-East
Russia: Distribution, ecology and genetic structure of populations.
In: Genetic Diversity and in situ Conservation of Wild Soybeans: Report
of Grants-in-Aid for Scientific Research from the J.S.P.S., No. 0904135
(ed. by Abe J.). Hokkaido University, Sapporo, 3–24.
Abe J., Ohara M. and Shimamoto Y. 1992. New electrophoretic mobility
variants observed in wild soybeans (Glycine soja) distributed in Japan
and Korea. Soybean Genet. Newsl. 19, 63–72.
Ahrent D.K. and Caviness C.E. 1994. Natural cross-pollinaion of twelve
soybean cultivars in Arkansas. Crop Sci. 34, 376–378.
Akimoto M., Shimamoto Y. and Morishima H. 1999.The extinction of
genetic resources of Asian wild rice, Oryza rufipogon Griff.A case study
in Thailand. Genet. Res. Crop Evol. 46, 419–425.
Arriola P.E. and Ellstrand N.C. 1996. Crop-to-weed gene flow in the
genus Sorghum (Poaceae): spontaneous interspecific hybridization
between johnsongrass, Sorghum halepense, and crop sorghum, S. bicolor.
Am. J. Bot. 83, 1153–1160.
Beard B.H. and Knowles P.F. 1971. Frequency of cross-pollination of
soybeans after seed irradiation. Crop Sci. 11, 489–492.
Broich S.L. and Palmer R. 1980.A cluster analysis of wild and
domesticated soybean phenotypes. Euphytica 29, 23–32.
Candy B.J. and Beversdorf W.D. 1984. Identification of soybean cultivars
using isoenzyme electrophoresis. Seed Sci.Technol. 12, 943–954.
Chiang Y.C. and Kiang Y.T. 1987. Geometric position of genotypes,
honeybee foraging patterns and outcrossing in soybean. Bot. Bull.
Acad. Sinica 28, 1–11.
Cutler G.H. 1934.A simple method for making soybean hybrids. J.Am.
Soc.Agron. 26, 252–254.
Erickson E.H. 1984. Soybean pollination and honey production – a
research progress report. Am. Bee J. 124, 775–779.
Fujita R., Ohara M., Okazaki O. and Shimamoto Y. 1997.The extent of
natural cross-pollination in wild soybean (Glycine soja). J. Hered. 88,
124–128.
Fukui J. and Kaizuma N. 1971. [Morphological and ecological
differentiation of various characters of the Japanese wild soybean,
Glycine soja.] J. Fac.Agr. Iwate Univ. 10, 195–208 (in Japanese with
English abstract).
Garber R.J. and Odland T.E. 1926. Natural crossing in soybeans. J.Am.
Soc.Agron. 18, 967–970.
Griffin J.D. and Palmer R.G. 1987. Inheritance and linkage studies with
five isozyme loci in soybean. Crop. Sci. 27, 885–892.
Hancock J.F., Grumet R. and Hokanson S.C. 1996.The opportunity for
escape of engineered genes from transgenic crops. Hortscience 31,
1080–1085.
Keeler K.H.,Turner C.E. and Bolick M.R. 1996. Movement of crop
transgenes into wild plants. In: Herbicide-Resistant Crops (ed. by Duke
S.O.). CRC Press, Boca Raton, 303–330.
Kiang Y.T., Chiang Y.C. and Kaizuma N. 1992. Genetic diversity in
natural populations of wild soybean in Iwate Prefecture, Japan. J. Hered.
83, 325–329.
Klinger T.,Arriola P.E. and Ellstrand N.C. 1992. Crop-weed
hybridization in radish (Raphanus sativus): effect of distance and
population size. Am. J. Bot. 79, 1431–1435.
Klinger T., Elam D.R. and Ellstrand N.C. 1991. Radish as a model system
for the study of engineered gene escape rates via crop-weed mating.
Conservation Biol. 5, 531–535.
Kwon S.H., Im K.H. and Kim J.R. 1972. [Studies on diversity of seed
weight in the Korean soybean.]. Korean J. Breed 4, 70–74 (in Korean
with English abstract).
Langevin S.A., Clay K. and Grace J.B. 1990.The incidence and effects of
hybridization between cultivated rice and its related weed red rice.
Evolution 44, 1000–1008.
Lefol E., Danielou V. and Darmency H. 1996. Predicting hybridization
between transgenic oilseed rape and wild mustard. Field Crop Res. 45,
153–161.
Manabe T., Kashiwara Y.,Yamada S., Harada J. and Matsumura T. 1996.
[Environmental safety evaluation of transgenic soybean with
glyphosate-tolerance in the environmentally isolated field.] Breed. Sci.
46 (Suppl. 1), 261 (in Japanese).
Mitsui K., ed. 1967. [Handbook of Forage Crops and Grasslands.] Yokendo,
Tokyo (in Japanese).
Miyazaki M. and Kudo I. 1988. [Feeding habit of thrips. In: Pest Thrips in
Japan (ed. by Umeya K., Kudo I. and Miyazaki M.).] Zenkoku Noson
Kyoiku Kyokai Publishing,Tokyo, 62–76 (in Japanese).
Morishima H. 2000. [Harbicide-resistant crops: a review.] Chem.
Regulation Plants 35, 86–94 (in Japanese).
Namai H. 1989. [Pollinators and pollination, fertilization and
fructification. In: Collected Data of Plant Genetic Resources, Vol. 1
(ed. by Matsuo T.).] Kodansha Scientific,Tokyo, 297–302
(in Japanese).
Oka H.I. 1983. Genetic control of regenerating success in semi-natural
conditions observed among lines derived from a cultivated ¥ wild
soybean hybrid. J.Appl. Ecol. 20, 937–949.
Padgette S.R., Re D.B., Barry G.F., Eichholtz D.E., Delannay X., Fuchs
R.L. et al. 1996. New weed control opportunities: Development of
soybeans with a Roundup Ready Gene. In: Herbicide-Resistant Crops
(ed. by Duke S.O.). CRC Press, Boca Raton, 53–84.
Paul E.M., Capiau K., Jacobs M. and Dunwell J.M. 1995.A study of gene
dispersal via pollen in Nicotiana tabacum using introduced genetic
markers. J.Appl. Ecol. 32, 875–882.
Raybould A.F. and Gray A.J. 1993. Genetically modified crops and
hybridization with wild relatives: a UK perspective. J.Appl. Ecol. 30,
199–219.
Saito M. 1981. [Ecological Features of Soybean Cultivars. In: Compendium
of Crops: the Pulses.] Nosangyoson Bunkakyokai,Tokyo, 106–390 (in
Japanese).
Sekizuka S. and Yoshiyama T. 1960. [Studies on the native wild grasses for
fodder (IV). Crop-scientific studies on wild species of Glycine soja in
Japan.] J. Kanto-Tosan Agr. Exp. Stn. 15, 57–73 (in Japanese with English
abstract).
Shields C.R., Orton T.J. and Stuber C.W. 1983.An outline of resource
and procedures for the electrophoretic separation of active enzymes
from plant tissue. In: Isozymes in Plant Genetics and Breeding. Part A (ed.
by Tanksley S.D. and Orton T.J.). Elesvier Science Publishers B.V.,
Amsterdam, 443–481.
Skvortzow B.W. 1927.The soybean – wild and cultivated in Eastern Asia.
Proceedings of Manchurian Res. Soc. Publ. Ser.A. Nat. Hist. Sec. 22, 1–8.
St.Amand P.C., Skinner D.Z. and Peaden R.N. 2000. Risk of alfalfa
transgene dissemination and scale-dependent effects. Theor.Appl. Genet.
101, 107–114.
Suh H.S., Sato Y.I. and Morishima H. 1997. Genetic characterization of
weedy rice (Oryza sativa L.) based on morpho-physiology, isozymes
and RAPD markers. Theor.Appl. Genet. 94, 316–321.
Umemoto S. and Yamaguchi H. 2000. [Vegetations in natural habitats
of wild soybean in Southern China. In: Genetic Diversity and in situ
Conservation of Wild Soybeans: Report of Grants-in-Aid for Scientific
Research from the J.S.P.S., No. 0904135 (ed. by Abe J.).] Hokkaido
University, Sapporo, 25–44 (in Japanese).
Woodworth C.M. 1922.The extent of natural cross-pollination in
soybeans. J.Am. Soc.Argon. 14, 278–283.
TM