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Chapter2
Somatic En1bryogenesis From Poplar Leaf Tissue1
Young Goo Park and Sung Ho Son
Introduction
An efficient in vitro regeneration system with cell and
tissue cultures is a prerequisite for biotechnological applications to plant improvement programs. Although
whole plants have been regenerated by several techniques
from various species and explants, somatic embryogenesis is one of the most powerful morphogenetic schemes.
First reported using carrot (Steward et al. 1958}, somatic
embryogenesis was later defined by Haccius (1978) as a
nonsexual developmental process leading to the differentiation of zygotic embryo-like structures from somatic
cells. Two advantages of somatic embryogenesis are that:
1) genetically identical propagules are provided by avoiding genetic recombination that occurs during meiosis in
sexual reproduction; and 2) it provides a fast, reliable, reproducible method for mass production. In contrast to organogenesis, somatic embryogenesis exhibits a bipolar
structure with a closed vascular system and differentiated
shoot and root meristems.
Poplars, considered an economically important species
in many countries, are usually propagated by cuttings
and/ or root suckers. Thus, cloning techniques can propagate superior genotypes that show heterosis in the F1 progeny from artificial hybridization. For interspecific crosses,
some species from the Leuce (currently termed Populus)
section have superior traits such as a straight trunk and
rapid growth Giang et al. 1980). Nevertheless, most hybrids from this section have been difficult to propagate
asexually using standard techniques. Tissue culture systems, especially somatic embryogenesis are advantageous
1
Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S.
Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p.
10
for application to mass cloning. Techniques developed for
poplar may also be applied to related tree species such as
willow (Salix spp.).
Literature Review
Studies on poplar tissue culture began in the early 1930s
(Gautheret 1934). After 30 years, Mathes (1964}, Wmton
(1968}, and Wolter (1968) have established plant regeneration methods based on callus culture. In their systems,
shoots and roots were induced through organogenesis
from different callus sites at different developmental
stages. Since then, similar morphogenetic responses were
reported by other authors (Chen and Huang 1980; Sellmer
et al.1989; Whitehead and Giles 1977). Because of the potential applications associated with somatic embryogenesis, many studies have focused on crop, horticultural
species, and woody perennials (Gingas and Lineberger
1989; Tremblay 1989).
Pioneering studies on somatic embryogenesis in poplar were reported by Michler and Bauer (1987, 1991), Park
and Son (1988}, Cheema (1989). Somatic embryogenesis
at relatively high frequency was obtained directly and/
or indirectly by leaf-tissue and cell culture of hybrid aspen (Populus alba x P. grandidentata cl. 'NC5339'). With this
hybrid, short-term auxin treatment stimulated embryo
maturation and eventually allowed plants to harden under greenhouse conditions (Michler and Bauer 1991).
Cheema (1989) obtained callus and cell suspension cultures using semi-organized leaf culture from mature Himalayan poplars (P. ciliata). Similar to most other species,
somatic embryogenesis was induced in P. ciliata by exposure to a high level of 2,4-dichlorophenoxyacetic acid (2,40), followed by reduced auxin concentration. Callus
retained its embryonic potential over a year; however, embryogenic suspension cultures lost this ability after 6 subcultures. In another study, organogenesis and somatic
embryogenesis occurred simultaneously when punctured
leaves of hybrid poplar (Populus nigra x P. maximowicziz)
were cultured (Park and Son 1988).
Somatic Embryogenesis From Poplar Leaf Tissue
With Populus, embryogenic capacity has been associated with leaf-tissue explants. For most other species including woody plants, immature or mature zygotic
embryos are commonly used as explants to initiate somatic embryogenesis (Tremblay 1989). For many woody
plant species, immature embryos are available only once
a year for a very short time. Using leaf tissue, especially
that grown in vitro, for somatic embryogenesis provides
Populus species with a substantial advantage.
Poplar Somatic Embryogenesis
In poplar, the developmental process, morphology, and
physiology of somatic embryogenesis is similar to zygotic
embryogenesis. Because reports on somatic embryogenesis of poplar are limited, summarizing a general method
is difficult. However, based on our research and other reports, some critical steps for somatic embryogenesis in
Populus spp. are described. The steps in somatic embryogenesis from leaf explants of poplar are: 1) explant preparation and adjustment to culture conditions; 2) induction
of direct and/ or indirect embryogenesis; and 3) maturation and germination.
Explant Preparation and Culture Conditions
As donor plants, greenhouse- or field-grown plants can
provide explants to initiate in vitro plantlets or callus. Typically, stem-node sections with or without buds are disinfested
using 0.5 to 1 percent of sodium hypochlorite for 5 to 10
min. Murashigeand Skoog (1962) (MS)culture medium with
a low concentration of cytokinin (6-benzyladenine; BA),
alone and in combination with high levels of auxin (2,4-D),
is used to induce in vitro bud break and callus induction,
respectively. To multiply the shoots for source material, apices are excised from shoots before subculturing on proliferation medium containing 0.88 J.lM BA. A 16-h photoperiod
provided by cool-white, fluorescent light is used for shoot
elongation and direct embryogenesis. Complete darkness is
used to establish and maintain callus and embryogenic cell
suspension cultures. Suspension cultures are routinely agitated at 100 to 120 rpm using a gyratory shaker, and subcultured at 2-week intervals.
Direct Embryogenesis
Isolated leaves (< 2 em in diameter) from in vitro cultured plantlets are cultured on MS medium supplemented
with BA and 2,4-D. Leaves are further prepared by puncturing with a pin or slicing into leaf disks with a cork borer
(1 em in diameter). Amendments such as coconut milk,
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
malt extract, and glutamine may be used to stimulate somatic embryogenesis. In this method, direct and indirect
embryogenesis and organogenesis can occur simultaneously. To maintain somatic embryos or embryogenic
callus for further development, embryoids and I or embryogenic callus must be selected at an early stage.
Indirect Embryogenesis
Isolated embryogenic callus or embryogenic callus-derived cell suspension cultures can be used for indirect somatic embryogenesis. A simple, reliable method for indirect
somatic embryogenesis is obtained by altering the 2,4-D
level. A semi-solid agar medium can support further development of embryos harvested at an early developmental stage. Overall, the culture media for maintenance of
embryogenic cell lines are similar to induction media except that plant growth regulator levels are lower.
Maturation and Germination
Harvested embryos require several washes in MS liquid medium without plant growth regulators. Somatic
embryos originating directly or indirectly from poplar leaf
cultures typically do not exhibit dormancy. Maturation of
somatic embryos in poplar can be triggered by the changing plant growth regulators. In 2 cases, the maturation
medium included BA, alone or combined with anaphthaleneacetic acid. Increased levels of osmoticum
may effectively stimulate maturation.
Limitations
Although somatic embryogenesis was successfully
demonstrated in Populus species, the induced somatic
embryo is strikingly plastic in its development. Many of
the induced embryos exhibited abnormal growth characteristics including: 1) bearing multiple secondary embryoids among the embryonic axes; 2) rapid growth of
nonembryonic callus during embryogenesis; 3) abnormal
cotyledons growth; and 4) growth failure of either cotyledon or root radical. This plasticity may result from the
altered gene expression during development. Another
problem associated with poplar embryogenesis is its low
success rate and poor reproducibility in comparison with
carrot. Because all stages from induction to maturation
are very labor intensive, large-scale propagation through
somatic embryogenesis is impractical without improving
overall efficiency. A critical limitation is lack of knowledge on the somatic embryogenic processes of poplar.
Continued intensive studies are needed.
11
Section I In Vitro Culture
Conclusion and Prospects
Somatic embryogenesis can be induced from leaf explants of poplar. Many factors such as basal media composition, undefined substances, amino acids, plant growth
regulator dosage, and treatment methods influence somatic embryogenesis. Among these factors, 2,4-0 plays a
paramount role as an auxin source. However, a phytohormone imbalance usually causes the formation of abnormal or incomplete structures. Because the role of auxins
in embryoid formation is complex, purification and identifi~ation of various auxin receptors and understanding
the1r molecular action will contribute to our knowledge
of cellular differentiation mechanisms related to somatic
embryogenesis (LoSchiavo 1995). Using leaf explants of
poplar, the somatic embryogenic response was examined
using inverted stereo microscopy (Michler and Bauer 1991;
Park and Son 1988). When embryos were produced from
cultured leaves, they appeared to arise directly or via
callogenesis from wounded portions or from sliced leaf
ends.
In recent years, embryogenesis through cell suspension
culture has provided opportunities for gene transformation by microparticle bombardment of intact cells or direct gene transfer using protoplasts. Liquid plating of
protoplast allows stringent selection of transformed cells.
Cell suspension culture-based somatic embryogenesis is
also applicable to germplasm preservation and mutant selection. If successfully developed, these technologies can
be employed in bioreactor systems for mass production.
Atte~pts at. large-scale embryogenesis are being applied
to vanous btoreactor systems such as air-lift, impeller, and
hybrid types.
Induction of somatic embryogenesis and organogenesis
have usually occurred simultaneously, but embryoid formation efficiency was relatively low. Abnormalities in
m?rphology and growth were evident. It is our hope that
th1s s~mmary of somatic embryogenesis methods for poplar wtll be useful to future studies. Continued studies are
needed to improve culture systems and for further understanding of somatic embryogenesis for application to poplar and other woody plants.
Acknowledgments
Part of this manuscript was reported in: Bajaj, Y.P.S., 1995.
ed ..Biotechnology in agriculture and forestry 30. Berlin:
Spnnger-Verlag Publication: 446-454.
12
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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.