Table of Contents
Ozminkowski, R.H. Jr. and P.S. Jourdan. 1993. Comparison of somatic and
sexual interspecific hybridization for the development of new Brassica
vegetable crops. p. 565-569. In: J. Janick and J.E. Simon (eds.), New crops.
Wiley, New York.
Comparison of Somatic and Sexual Interspecific Hybridization for the
Development of New Brassica Vegetable Crops
Richard H. Ozminkowski, Jr. and Pablo S. Jourdan
- Plant Material
- Production of Somatic Hybrids (R0) and Their F2 (= R1) Populations
- Production of Sexual Hybrids
- RESULTS AND DISCUSSION
- Somatic Hybrids
- Somatic F2 (= R1) Generation
- Sexual Hybrids
- Table 1
- Fig. 1
- Fig. 2
- Fig. 3
- Fig. 4
- Fig. 5
The success of an allopolyploid, whether natural (Brassica napus L.,
Nicotiana tabacum L., Triticum aestivum L.) or synthetic
(triticale, hakuran) is based in part on the variability within the original
parent individuals (Dewey 1980). The genus Brassica, is a classic
example of allopolyploid speciation (Fig. 1); it is extremely diverse in
morphology encompassing many economically important crops. The natural
allotetraploid B. napus has been produced from its diploid progenitor
species (B. oleracea L. and B. rapa L.) by sexual interspecific
hybridization for the development of the leafy vegetable crop 'Hakuran' (Nishi
1980), and by somatic hybridization (Jourdan et al. 1989) in attempts to
transfer various nuclear and cytoplasmic traits between the species. This
makes B. napus a model system for a comparison of sexual and somatic
hybridization systems for the development of new crops.
Diploid cells used in somatic hybridization are not subject to the allelic
segregation found in gamete formation nor the unilateral inheritance of
cytoplasm as generally seen in sexual hybridization; therefore, somatic hybrids
should retain any heterozygosity of the parents and allow for rearrangement of
cytoplasm. The use of highly heterozygous parents in somatic hybridizations
could incorporate high degrees of variation in the offspring (= F2) of a single
somatic hybrid not possible with sexual hybridization. Relatively homozygous
lines have generally served as parents to resynthesized B. napus, thus
impeding the exploitation of the high morphological variability within the
parental species. Such variation is essential for new crop development and
therefore requires that hybrids be produced from several different parent
combinations in order to provide sufficient variation for cultivar development
of a new crop.
The current study was designed to maximize the heterozygosity in a population
of resynthesized allotetraploid B. napus, so as to maintain high
variability in subsequent generations for the development and breeding of new
crops. The primary objectives are to provide novel and supplemental variation
for breeding vegetable-type B. napus, and to determine which method of
resynthesis, sexual or somatic, would be more effective for doing so.
Intraspecific F1 hybrids between cultigens within the diploid progenitor
species (B. oleracea and B. rapa) were produced by bud
pollination in the greenhouse (Fig. 2). Each vegetable parent [cauliflower
(B. oleracea ssp. botrytis), turnip (B. rapa ssp.
rapifera), and chinese cabbage (B. rapa ssp. pekinensis)]
was selected for its distinctive morphological character whereas purple
ornamental kale (B. oleracea ssp. acephala) was selected for its
excellent regeneration from protoplasts. All cultigens had yellow flowers
except cauliflower's which were white.
Production of the somatic hybrids followed standard protocols (Jourdan et al.
1989). Leaf tissue served as the protoplast source for somatic hybridization
involving individual B. rapa and B. oleracea species-parent
plants (Fig. 2). The latter's protoplasts were treated with iodoacetate prior
to fusion by the polyethylene glycol method.
Somatic F2 seed from bud self-pollinations were started in the greenhouse from
three Type 1 (see Results) somatic hybrids. Seeds of the diploid parental
lines 7212 (B. oleracea) and 7442 (B. rapa) were also started.
Five-week-old seedlings were transplanted at ca. 0.6 m intervals in rows 1.6 m
apart on raised beds. Plants were evaluated for various morphological
characters on a 1 to 5 rating scale [plant habit, branching, leaf shape,
undulation, vesture, leaf crinkle (internal bubbling of the leaf), leaf color,
and rib color]. Leaf shape was classified as described by Gomez-Campo (1980)
ranging from lyrate to heavily lobed (divided). Approximately, 350 F2 plants
from each of two somatic hybrids were used for frequency analysis.
Reciprocal interspecific bud pollinations were made between the same
species-parent individuals used in the fusion experiment (Fig. 2) during the
months of November and December, under natural lighting in the greenhouse.
Thirteen to 18 days after pollination, ovules were aseptically removed from the
silique and placed in the liquid medium (MSQ) of Quazi (1988) with the omission
of tri-potassium phosphate. Cultures were incubated in dim light (60 µmol
m-2s-1) on a gyratory shaker (33 rpm). Two to three
weeks later, torpedo stage embryos were transferred to solid B5G1 medium [B5
media with 0.1 mg/liter gibberellic acid (GA3), 0.8% washed agar] and grown at
4°C with 8/16 h light/dark periods for 10 days. Once developed, shoots were
proliferated on a modified solid Murashige and Skoog (MS) medium (B5 vitamins)
with 0.2 mg/liter 6-benzylamino purine (BA) and then maintained on hormone-free
MS medium until placement into soil. Sexual hybrids were identified by
intermediate morphology and glucose phosphate isomerase (GPI) and
phosphoglucomutase (PGM) isoenzyme banding patterns using cellulose acetate
electrophoresis (Hebert and Beaton 1989).
Two types of somatic hybrids were produced (Fig. 2) and have been characterized
previously (Ozminkowski and Jourdan 1991). Type 1 hybrids were uniform,
vigorous, near-rosette in habit, with divided pubescent leaves, and white,
fertile flowers. Flow cytometry analysis of nuclear DNA content indicated that
these plants had a genome size similar to natural B. napus. Type 2
hybrids were variable in appearance, rugose, less vigorous, with distinct
internodes, and near lyrate leaf shape; mature plants were nearly glabrous.
These plants had low fertility and were chimeric (white and yellow) for flower
color; flow cytometry data suggested that these plants had higher ploidy levels
than natural B. napus making them less useful in a breeding program. No
somatic hybrids were found to contain only the B. oleracea RFLP markers
for either mitochondria or chloroplasts while many contained only the B.
rapa markers. Several hybrids were found to contain the chloroplast and/or
mitochondria RFLPs of both parents, suggesting either mixed or recombinant
The somatic hybrids did not appear to be of any economic value per se.
However, every somatic hybrid contained an allele at each locus from four
diverse cultivated forms of Brassica allowing extensive segregation in
the F2 generation; the production of F2 seed of the Type 1 hybrids was not a
Field-grown F2 plants of the Type 1 hybrids were uniformly vigorous. Distinct
morphologies became apparent about 4 weeks after planting. The morphological
variability seen in the F2 populations was sufficiently large to permit further
intrapopulation crossing and selection for forms appealing to the consumer
(e.g. glabrous, heading, good color). Several morphologies were found in the
somatic F2 population which show potential for new plant development (Fig. 3).
Variation in leaf morphology is illustrated in Fig. 4.
When individual plant characters were evaluated, most of the plants were
intermediate between the two species-parents though the frequencies were skewed
in favor of B. rapa; this may be a contribution of the predominantly
B. rapa cytoplasm or of more dominant B. rapa nuclear alleles.
However, somatic hybrids containing only B. oleracea cytoplasm were not
obtained for comparison. Most F2 plants exhibited less extreme pubescence than
the B. rapa parent, although several were seen with higher levels.
Several glabrous plants were found, a character more favorable for marketing.
Many plants displayed leaf undulation and crinkle more extreme than either
species-parent. Plants with rib color that was much darker purple than either
parent line were also found in this population. Leaf color was the only
evaluated character to favor the blue-green B. oleracea parent. Only
three F2 plants flowered within five months after planting, thus preventing the
analysis of fertility and segregation of flower color (all three had white
Twenty-seven embryos produced sexual hybrid plants (Table 1). Most (19 of 27)
hybrid embryos produced greater than 20 shoots allowing clones of 11 to be
placed in the field. Each sexual hybrid was unique in morphology (Fig. 2 and
5), as would be expected since allelic segregation occurred during gamete
formation. Hybrids were very vigorous both in the greenhouse and field. All
sexual hybrids were pubescent. Only four hybrids, all field-grown, have
flowered five months after transplanting and are segregating for flower color
though all flowers were small and sterile. Analysis of organelle-specific
RFLPs suggested only maternal inheritance of cytoplasm.
DNA quantification through flow cytometry indicate these plants are
allodiploids and therefore must have their genomes doubled to restore
fertility. The result should be fully homozygous, inbred lines containing only
one allele from each locus of each species. Thus, only those types produced in
the hybrid generation are available for continued development without the
production of additional interspecific hybrids. Few if any sexual hybrids
displayed an economically favorable morphology per se; no glabrous or heading
type plants were produced. This may be due to the limited number of sexual
hybrids produced, an inherent difficulty to sexual hybridization between these
The parents used in the synthesis of a novel or natural allopolyploid crop
should contain maximum levels of variation to provide a germplasm resource
sufficient for the development of new forms with a minimal number of
resynthesis experiments. Using Brassica, we have compared two methods
of producing allopolyploid species, sexual and somatic interspecific
hybridization, for the development of new vegetables. Many independent sexual
hybrids were necessary to represent only a fraction of the variability observed
in the F2 population of a single somatic hybrid produced from the same
heterozygous parents. Since interspecific hybrids are often difficult to
produce, maximal variability in fewer individual hybrids can be a tremendous
savings in resources. Somatic hybridization can result in highly heterozygous
hybrids and, in addition, offers the opportunity for novel organelle
rearrangements which can lead to unique cytoplasmic male sterility systems
(Jourdan et al. 1989); sexual hybridization generally produces hybrids
containing maternal cytoplasm. Organelle analysis of the somatic and sexual
hybrids produced in this study did support this hypothesis.
The availability of efficient techniques for protoplast regeneration and fusion
in an increasing number of crops will allow plant breeders to choose between
sexual and somatic hybridization for the production of interspecific hybrids
based upon the individual breeding program objectives. We have shown in this
study that, when a primary objective involves the production of new
allopolyploid crops or to substantially increase the variability within
existing allopolyploid species, somatic hybridization is the preferred method
because high variability can be obtained among progeny from a single fertile
somatic hybrid. The use of parent plants heterozygous for quantitative trait
loci (QTL) involving yield or stress resistance permits combination of several
alleles at each QTL in every somatic hybrid which would allow high genetic gain
from subsequent selection. Conversely, if the objective is to introgress a
simply-inherited trait into an existing crop, sexual hybridization may be
- Dewey, D.R. 1980. Some applications and misapplications of induced polyploidy
to plant breeding, p. 445-470. In: W.H. Lewis (ed.). Polyploidy: biological
relevance. vol 13. Basic life sciences. Plenum Press, NY.
- Gomez-Campo, C. 1980. Morphology and morpho-taxonomy of the tribe
Brassiceae, p. 3-31. In: S. Tsunoda, K. Hinata, and C. Gomez-Campo
(eds.). Brassica crops and wild allies: biology and breeding. Japan
Scientific Societies Press, Tokyo.
- Hebert, P.D.N. and M.J. Beaton. 1989. Methodologies for allozyme analysis
using cellulose acetate electrophoresis: a practical handbook. Helena
Laboratories Inc. Beaumont, TX.
- Jourdan, P.S., E.D. Earle, and M.A. Mutschler. 1989. Synthesis of male
sterile, triazine-resistant Brassica napus by somatic hybridization
between cytoplasmic male sterile B. oleracea and atrazine-resistant
B. campestris. Theor. Appl. Genet. 78:445-455.
- Nishi, S. 1980. Differentiation of Brassica crops in Asia and the breeding of
'hakuran', a newly synthesized leafy vegetable, p. 133-150. In: S. Tsunoda, K.
Hinata, and C. Gomez-Campo (eds.). Brassica crops and wild allies:
biology and breeding. Japan Scientific Societies Press, Tokyo.
- Ozminkowski, R.H., Jr. and P.J. Jourdan. 1991. Characterization of
Brassica napus resynthesized by interspecific somatic hybridization from
highly heterozygous parents. HortScience 26:740 (Abstr. 425).
- Quazi, M.H. 1988. Interspecific hybrids between Brassica napus L. and
B. oleracea L. developed by embryo culture. Theor. Appl. Genet.
- U, N. 1935. Genomic analysis in Brassica with special reference to the
experimental formation of B. napus and peculiar mode of fertilization.
Japan. J. Bot. 7:309-452.
Table 1. Sexual F1 hybrid plants produced by hybridizations between
B. oleracea and B. rapa via ovule culture.
|Cross ||No. of pollinations made ||No. of siliques cultured ||No. of ovules cultured ||No. of embryos developed ||No. of hybrids produced ||No. of maternal escapes|
|B. rapa x B. oleracea ||603 ||176 ||~600 ||17 ||8 ||4|
|B. oleracea x B. rapa ||188 ||12 ||114 ||40 ||19 ||3|
Fig. 1. Nuclear genomic relationships among selected Brassica species (adapted from U 1935). Species at intersections of the triangle are the diploid progenitor species of the respective allotetraploid species upon each side. Lower case letters represent the species nuclear genome composition.
Fig. 2. Pedigree of (c) somatic and (d) sexual interspecific hybrids
between (a) B. oleracea and (b) B. rapa.
Fig. 3. Four somatic F2 individuals exhibiting promising morphologies
for development of new vegetables.
Fig. 4. Leaf variability of two parent lines a) 7212 and b) 7442 and
the F2 population (all others) of 2 somatic hybrids between these lines (stake
= 30 cm).
Fig. 5. Leaf variability among sexual hybrids between heterozygous
B. oleracea a) and B. rapa lines. b) Type 1 somatic hybrid
Last update September 17, 1997