George Acquaah - Principles of Plant Genetics and Breeding

Poor storage of synthetic seeds due to lack of dormancy and stress tolerance in the somatic embryos.

Mechanical damage, lack of oxygen supply, invasion by microbes, and lack of nutrients all contribute to poor germination of synthetic seeds.

Strategies are available for addressing some of these challenges. They vary among species and includes desiccation, a process that can damage the embryo.

Viral infections are systemic, being pervasive in the entire affected plant. Heat therapy is a procedure that is used for ridding infected plants of viral infections. After heat treatment, subsequent new growth may be free of viruses. More precisely, meristems dissected from leaf and shoot primordia are often free of viruses even when the plant is infected. Tissue culture technology is used to nurture the excised meristematic tissue into full plants that are free from viruses.

The process starts with detection (e.g. by ELISA) of the presence of a viral infection in the plant. Once confirmed, the meristems on the shoots are aseptically removed and sterilized (dipped in 75–99% ethanol or 0.1–0.5% sodium hypochlorite or household bleach for a few seconds or minutes). The explant is submitted to tissue culture as 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 virus‐free crop. It should be pointed out that virus elimination from plants do not make them virus resistant. The producer should adopt appropriate measures to protect the crop from infection.

Wide cross production is discussed in Chapter 7.

Sometimes, especially in crosses between different plant species, the embryo formed after fertilization in wide crosses fails to develop any further. The breeder may dissect 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, e.g. 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 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 was discussed in Chapter 6.

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 resulting plantlets will have a haploid genotype. Haploid plantlets may arise directly from embryos or indirectly via callus. To have maximum genetic variability in the plantlets, breeders usually use anthers from F 1or F 2plants. Usually, the haploid plant is not the goal of anther culture. Rather, the plantlets are diploidized (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:

Anther culture to induce androgenesis;

Ovary culture to induce gynogenesis;

Embryo rescue from wide crosses.

Flower buds are picked from healthy plants. After surface sterilization, the anthers are excised from the buds and cultured unto an appropriate tissue culture medium. The pollen grains at this stage would be 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. High nitrogen content of the donor plant and exposure to low temperature at meiosis reduces albinos and enhances the chance of green plant regeneration. Pre‐treatment (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% colchicines, or through somatic callus culture).

Development of new cultivarsThrough 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 is needed, versus several 1000s of F2 for selecting desirable genotypes.

Selection of mutantsAndrogenic haploids have been used for selecting especially recessive mutants. In species such as tobacco, mutants that are resistant to methionine analogue (methionine sulfoxide) of the toxin produced by Pseudomonas tabaci have been selected.

Development of supermales in asparagusHaploids of Asparagus officinalis may be diplodized to produce homozygous males or females.

The full range of genetic segregation of interest to the plant breeder is observed because only a small fraction of androgenic grains develops into full sporophytes.

High rates of albinos occur in cereal haploids (no agronomic value).

Chromosomal aberrations often occur, resulting in plants with higher ploidy levels, requiring several cycles of screening to identify the haploids.

Use of haploids for genetic studies is hampered by the high incidence of nuclear instability of haploid cells in culture.

Gynogenesis, using ovules or ovaries, has been achieved in species such as barley, wheat, rice, maize, tobacco, sugar beet, and onion. The method is less efficient than androgenesis because only one embryo sac exists per ovary as compared to thousands of microspores in each anther. Ovaries ranging in developmental stages from uninucleate to mature embryo sac stages are used. However, it is possible for callus and embryos to develop simultaneously from gametophytic and sporophytic cells, making it a challenge to distinguish haploids from those of somatic origin. Generally, gynogenesis is selected when androgenesis is problematic (as in sugar beet and onion).