Principles of Plant Genetics and Breeding

previously described. Sometimes, to increase the success
of viral elimination, researchers may include chemicals
(e.g., Ribavirin®, Virazole®) in the tissue culture
medium. The plants produced must be tested to confirm
virus-free status.
The virus-free plants are used to produce more
materials (by micropropagation) for planting a virusfree
crop. It should be pointed out that virus elimination
from plants does not make them virus resistant. The
producer should adopt appropriate measures to protect
the crop from infection.
Applications in wide crosses
TISSUE CULTURE AND THE BREEDING OF CLONALLY PROPAGATED PLANTS 185
Embryo rescue
Sometimes, the embryo formed after fertilization in
wide crosses fails to develop any further. The breeder
may intervene in the development process by dissecting
the flower to remove the immature embryo. The
embryo is then nurtured into a full plant by using the tissue
culture technology. This technique is called embryo
rescue. The fertilized ovary is excised within several
days of fertilization to avoid an abortion (due to, for
example, abnormal endosperm development). Normal
embryogenesis ends at seed maturation. The development
of the embryo goes through several stages with
certain distinct features. The globular stage is undifferentiated,
while the heart stage is differentiated and
capable of independent growth. The torpedo stage
and cotyledonary stage of embryo development follow
these early stages. Prior to differentiation, the developing
embryo is heterotrophic and dependent on the
endosperm for nutrients. Excising the embryo prematurely
gives it less of a chance of surviving the embryo
rescue process. Just like all tissue culture work, embryo
rescue is conducted aseptically and cultured on the
medium appropriate for the species.
Somatic hybridization
The first step in somatic hybridization is to isolate
intact protoplasts. Mesophyll protoplasts are preferred
to protoplasts from other sources. Young tissues from
healthy and well-watered and shaded plants are used.
The cell wall is removed enzymatically using commercial
enzyme preparations (e.g., pectinase, cellulose) to
digest it. The excised leaves are sterilized prior to subjection
to about a 16-hour digestion. Protoplasts may
also be obtained from suspension culture. Protoplasts
are uniformly negatively charged and hence repel each
other, a force that must be overcome for fusion to
occur.
A protoplast is all the cellular component of a cell
excluding the cell wall. Protoplasts from two different
plants can be fused to create a hybrid. Protoplasts may
be isolated by either mechanical or enzymatic procedures,
as discussed in Chapter 10.
The most common methods of fusion are by chemical
agents or electrical manipulation. Fusogenic agents
include salt solutions (e.g., KCl, NaCl). However, the
most commonly used agent is polyethylene glycol
(PEG). The protoplasts are agglutinated by the application
of PEG to facilitate the fusion. Addition of the
compound called concanavalin A to PEG enhances
the fusion. Protoplast fusion can also be accomplished
by an electrical process (electrofusion). Protoplasts are
agglutinated by the technique of dielectrophoresis, in
which they are subjected to a non-uniform AC field of
low intensity (500–1,000 V/cm) for a very short time.
This is followed by an application of high voltage AC
pulse to destabilize the cell membrane at specific sites
to facilitate the fusion. Maintenance of proper osmotic
potential is critical to the success of fusion. Chemicals
(e.g., manitol, sorbitol) are added to the tissue culture
medium for this purpose.
Fusion of protoplast does not necessarily guarantee
fusion of nuclei. For a stable hybrid to form, the two
nuclei must fuse within a single cell, followed by mitosis
involving the two genomes. Somatic hybrids are difficult
to identify. A selection system is used to verify hybridity
since fusion is non-specific and therefore allows the
formation of various products – multiple fusions,
homokaryons (fusion of protoplasts from the same parent),
heterokaryons (fusion of protoplasts from different
parents), and unfused protoplasts. Some of the methods
used to authenticate hybridity include genetic complementation
of non-allelic mutants, use of selective media,
isozyme analysis, and microisolation. The mechanical
methods are most precise but tedious. A microscope is
used to examine the products to identify fused products.
After fusion, the tissue culture environment is modified
to induce cell wall formation.
Sexual hybridization and somatic hybridization have
some differences. Sexual hybridization involves fusing of
two haploid nuclei and one maternal cytoplasm; somatic
hybridization combines diploid nuclear genomes and
two maternal cytoplasmic genomes (symmetric hybrid).
Whereas sexual hybrids are uniform, somatic hybrids
produce significant variability in the population, resulting
from genetic instability, mitotic recombination,
somaclonal variation, and cytoplasmic segregation.