Principles of Plant Genetics and Breeding

TISSUE CULTURE AND THE BREEDING OF CLONALLY PROPAGATED PLANTS 193
Somaclonal variation
A variety of mechanisms have been implicated in this
phenomenon. Chromosomal changes, both polyploidy
and aneuploidy, have been observed in potato, wheat,
and ryegrass. Some research suggests mitotic crossovers
to be involved whereas cytoplasmic factors (mitochondrial
genes) have been implicated by others. Further,
point mutation, transposable elements, DNA methylation,
and gene amplification are other postulated mechanisms
for this phenomenon.
As a breeding tool, breeders may deliberately plan and
seek these variants by observing certain factors in tissue
culture. Certain genotypes are more susceptible to
genetic changes in tissue culture, polyploid genotypes
generally being more so than diploid. Also, holding the
callus in an undifferentiated state for prolonged periods
of time enhances the chances of somaclonal variation
occurring. Not unexpectedly, the tissue culture environment
(medium components) can induce heritable
changes in the callus. The inclusion of auxin 2,4-D
enhances the chances of somaclonal variation.
Somaclonal variation from calli with disease resistance
(e.g., Helminthosporium sacchari in sugarcane
and Fusarium in Apium graveolens) has been found.
Somaclones with resistance to various abiotic stresses
have also been reported.
Directed selection
Rather than allowing the variation to arise spontaneously,
sometimes plant breeders apply selection pressure
during the in vitro cultural process to influence the
variability that might arise.
Selection for disease resistance
Various toxin metabolites have been included in tissue
culture for use as the basis for selection, assuming that
such metabolites have a role in pathogenesis. The culture
filtrates from various fungi (Fusarium, Helminthosporium
maydis) have been used to exert selection pressure for
cells that are resistant to the pathogen. The main constraint
to the use of directed selection in plant breeding
for disease resistance is the inability of the in vitro system
to be used to select for hypersensitivity, a major strategy
in disease resistance.
Selection for herbicide tolerance
Mutants with about 10–100 times the level of resistance
to herbicides (e.g., imidazilinone in sugar beet) have
been successfully isolated, characterized, and incorporated
into commercial cultivar development. Many of the
recorded successes with in vitro selection have been with
herbicide tolerance.
Selection for tolerance to abiotic stresses
Selection for tolerance to salinity, metals (Zn, Al) and
temperature (cold tolerance) has been attempted with
varying degrees of success.
Single-cell selection system
Some researchers use single-cell tissue culture systems
(suspension culture, protoplast culture) for in vitro
selection. The advantages of this approach include a
lack of chimerism, higher chances of isolation of true
mutants, and an ability to more effectively apply microbial
procedures to the large number of individual cells
than can be screened in a small space. Selection for
biotic stress resistance, herbicide tolerance (the author
did this for chlorsulfuron), and aluminum tolerance, are
among successful applications of direct selection using a
single-cell selection system.
Germplasm preservation
Germplasm preservation in tissue culture was discussed
in Chapter 6. This method of germplasm storage is often
used for vegetatively propagated species.
Breeding vegetatively (clonally)
propagated species
Because seed is produced via the process of meiosis,
cultivars propagated by seed always have some heterozygosity,
the degree of it varying with the method of
breeding. Asexual or clonal propagation entails the
use of parts of the plant other than the seed as the
propagule. Clones have identical genotypes because
they reproduce via mitosis not meiosis.
Clones, inbred lines, and pure lines
As previously discussed, plants may be naturally sexually
or asexually propagated. Further, sexually propagated
species may be self-fertilized or cross-fertilized. These
natural modes of reproduction have implications in the
genetic structure and constitution of plants and breeding
implications. Plant breeders are able to manipulate