Principles of Plant Genetics and Breeding

186 CHAPTER 11
Products of somatic hybridization may be true hybrids
or parasexual hybrids with the complete genomes of two
parents, partial genomes of the parents (called asymmetric
hybrids), or cybrids (combination of the nuclear
genome of one parent and the cytoplasm of another).
Protoplasm has been isolated from numerous species
(e.g., barley, carrot, cassava, cotton, pea, soybean). One
of the earliest successes was the pomato (potato–
tomato fusion product). Intergeneric protoplast fusion
has been accomplished in carrot × petunia, maize ×
sorghum, and soybean × barley. Interspecific hybrids
include Dacus carrota × D. capillifolius and Nicotiana
tabacum × N. sylvestris. When closely related species are
used, polyploidy somatic hybrids are formed. For example,
a somatic hybrid of potato was produced that combined
the genomes of Solanum tuberosum (2n = 48) and
S. brevidens Phil. (a wild non-tuberous variety; 2n = 24)
to produce a somatic hybrid with 72 chromosomes.
This hybrid has resistance to potato leafroll virus.
Protoplast fusion has also been used for a one-step cross
(rather than via a backcross) to transfer cytoplasmic male
sterility (CMS) to different fertile varieties.
Production of haploids
Haploids contain half the chromosome number of
somatic cells. Anthers contain immature microspores
or pollen grains with the haploid (n) chromosome
number. If successfully cultured (anther culture), the
plantlets resulting will have a haploid genotype. Haploid
plantlets may arise directly from embryos or indirectly
via calli, as previously discussed. To have maximum
genetic variability in the plantlets, breeders usually use
anthers from F 1 or F 2 plants. Usually, the haploid plant
is not the goal of anther culture. Rather, the plantlets
are diplodized (to produce diploid plants) by using
colchicine for chromosome doubling. This strategy
yields a highly inbred line that is homozygous at all loci,
after just one generation.
Methods used for breeding self-pollinated species
generally aim to maintain their characteristic narrow
genetic base through repeated selfing over several generations
for homozygosity. The idea of using haploids to
produce instant homozygotes by artificial doubling has
received attention. Haploids may be produced by one of
several methods:
1 Anther culture to induce androgenesis.
2 Ovary culture to induce gynogenesis.
3 Embryo rescue from wide crosses.
Anther culture
Flower buds are picked from healthy plants. After
surface sterilization, the anthers are excised from the
buds and cultured onto an appropriate tissue culture
medium. The pollen grains at this stage are in the uninucleate
microspore stage. In rice the late uninucleate
stage is preferred. Callus formation starts within 2–6
weeks, depending on the species, genotype, and physiological
state of the parent source. A high nitrogen
content of the donor plant and exposure to a low
temperature at meiosis reduces albinos and enhances the
chance of green plant regeneration. Pretreatment (e.g.,
storing buds at 4–10°C for 2–10 days) is needed in
some species. This and other shock treatments promote
embryogenic development. The culture medium is
sometimes supplemented with plant extracts (e.g.,
coconut water, potato extract). To be useful for plant
breeding, the haploid pollen plants are diplodized (by
artificial doubling with 0.2% colchicine, or through
somatic callus culture).
Applications
1 Development of new cultivars. Through diplodization,
haploids are used to generate instant homozygous
true-breeding lines. It takes only two seasons to
obtain doubled haploid plants, versus about seven
crop seasons using conventional procedures to attain
near homozygous lines. The genetic effect of doubling
is that doubled haploid lines exhibit variation
due primarily to additive gene effects and additive ×
additive epistasis, enabling fixation to occur in only
one cycle of selection. Heritability is high because
dominance is eliminated. Consequently, only a small
number of doubled haploid plants in the F1 are
needed, versus several thousands of F2 plants for
selecting desirable genotypes.
2 Selection of mutants. Androgenic haploids have
been used for selecting especially recessive mutants.
In species such as tobacco, mutants have been
selected that are resistant to the methionine analogue
(methionine sulfoxide) of the toxin produced by
Pseudomonas tabaci.
3 Development of supermales in asparagus. Haploids
of Asparagus officinalis may be diplodized to produce
homozygous males or females.
Disadvantages
1 The full range of genetic segregation of interest to
the plant breeder is observed because only a small