Plant Cell Rep (2005) 24: 507–512
DOI 10.1007/s00299-005-0971-7
CELL BIOLOGY AND MORPHOGENESIS
Zhihui Shan · Krit Raemakers ·
Emmanouil N. Tzitzikas · Zhengqiang Ma ·
Richard G. F. Visser
Development of a highly efficient, repetitive system
of organogenesis in soybean (
Glycine max
(L.) Merr).
Received: 20 October 2004 / Revised: 16 March 2005 / Accepted: 17 March 2005 / Published online: 17 June 2005
C
Springer-Verlag 2005
Abstract A highly efficient, repetitive system of organo-
genesis was developed in soybean. Seeds of soybean cv.
‘White hilum’ pretreated with TDZ formed multiple bud
tissue(s) (MBT) at the cotyledonary nodes. MBT initiation
occurred only if the axillary buds were not removed
from the cotyledonary node. The best MBT formation
was achieved by pretreating the seeds for 1 week on
medium supplemented with 0.1 mg/l TDZ, followed by
culture of the cotyledonary node on medium supplemented
with 0.5 mg/l BA for 4 weeks. Culture of the MBT on
medium supplemented with 0.1 mg/l TDZ resulted in
the proliferation of MBT. MBT was maintained in this
way for 12 months. Three hundred thirty six shoots were
obtained when 1 g of MBT was subcultured on medium
supplemented with 0.5 mg/l BA. Plants were rooted on
medium without growth regulators. The regenerated plants
grew normally in the greenhouse. Unfortunately, they did
not set seeds because of the long-day conditions during
growth. This system was successfully applied in three
other genotypes.
Keywords Soybean (Glycine max (L.) Merr)
.
Cotyledonary node
.
Regeneration
.
Thidiazuron
.
TDZ
Introduction
Soybean (Glycine max (L.) Merr.) is grown on more
areas worldwide than any other dicotyledonous crop.
Z. Shan · K. Raemakers · E. N. Tzitzikas · R. G. F. Visser
Laboratory of Plant Breeding, Wageningen University,
386, 6709 AJ Wageningen, The Netherlands
e-mail: krit.raemakers@WUR.NL
Z. Shan · Z. Ma (
)
National Key laboratory of Crops Genetics & Germplasm
Enhancement, Nanjing Agriculture University,
Nanjing Jiangsu 210095, Peoples’ Republic of China
e-mail: zqm2@njau.edu.cn
Tel.: +86-25-84396029
Fax: +86-25-84396707
Commercial breeding is still very important for the genetic
improvement of the crop. However, breeding is difficult
due to the fact that soybean is a self-pollinating crop
and the genetic variation among soybean varieties is
narrow. Genetic modification has been used for producing
varieties with traits that are not available in the soybean
gene pool. The regeneration system used to produce
genetically modified plants was somatic embryogenesis
from immature seeds (Christou et al. 1989) or organo-
genesis from cotyledonary nodes (Finer and McMullen
1991).
Somatic embryogenesis is an efficient system to produce
genetic modified plants, however, it is genotype specific
and accompanied with a high level of somaclonal variation
in the regenerated plants (Finer and Nagasawa 1988; Parrot
et al. 1989; Finer and McMullen 1991). Organogenesis is
less genotype dependent and has become routine in several
laboratories (Wright et al. 1986, 1987a, 1987b; Barwale
et al. 1986; Dan and Reighceri 1998). The regeneration is
based on proliferation of meristems in the cotyledonary
node. However, recovery of transgenic plants capable of
transmitting the target genes to R1 is very low (Christou
et al. 1990). Olhoft et al. (2001) stated that the efficiency
of soybean transformation has to be improved 5–10 times
before one person can produce 300 transgenic lines per
year. The efficiency of the organogenic system for genetic
modification could be improved if the number of shoots
per explant is increased or if the number of meristematic
cells in explants is increased.
In pea, where genetic modification is also based on
organogenesis from cotyledonary nodes, a highly effi-
cient regeneration system was obtained by subculturing
nodes on thidiazuron (TDZ) supplemented medium. As
a result tissues were formed covered with very small
buds. These bud containing tissues were maintained by
repetitive subculture (Tzitzikas et al. 2004) and were
successfully used in genetic modification. This report
describes the establishment of a highly efficient, repetitive
organogenic system in soybean based on the use of
TDZ.