Principles of Plant Genetics and Breeding. George Acquaah

the embryo sac), and semigamy (sperm nucleus and egg nucleus develop independently without uniting, leading to one haploid embryo). The resulting haploid plants contain sectors of material from both maternal and paternal origin, and is therefore chimeric.

7.16 Other tissue culture applications

      There are other tissue culture‐based applications of interest to plant breeders besides micropropagation.

      7.16.1 Synthetic seed

      Somatic embryogenesis has potential commercial applications, one of which is in the synthetic seed technology (production of artificial seeds). A synthetic seed consists of somatic embryos enclosed in protective coating. There are two types currently being developed:

      1 Hydrated synthetic seed – This kind of seed is encased in hydrated gel (e.g. calcium alginate).

      2 Desiccated synthetic seed – This kind of seed is coated with water soluble resin (e.g. polyoxethylene).

      To develop synthetic seed, it is critical to achieve a quiescent phase, which is typically lacking in somatic embryogenesis (i.e. without quiescence, there is continuous growth, germination, and eventually death, but no stationary stage as in embryos in mature seeds). The application will depend on the crop. Lucerne (Medicago sativa) and orchardgrass (Dactylis glomerata) are among the species that have received much attention in artificial seed development. Potential application of artificial seed is in species that are highly heterozygous and in which conventional breeding is time‐consuming. Trees can be cloned more readily by this method. In some tropical species that are seed propagated but in which seeds have short duration of viability, artificial seed production could be economical, because of the high economic value of these crops (e.g. cacao, coconut, oil palm, coffee). Also, hybrid synthetic seed could be produced in species in which commercial hybrid production is problematic (e.g. cotton, soybean).

      7.16.2 Limitations to commercialization of synthetic seed technology

      Whereas the prospect of commercial synthetic seed is appealing, several factors make this impractical at this time. Problems may occur at maturation, germination, rooting, shot apex formation, or acclimatization.

       Large‐scale production of high quality viable propagules remains a key challenge.

       A major limitation is the poor conversion of apparently normal propagules into normal plantlets.

       Improper development and maturation of somatic embryos causes poor germination and conversion problems.

       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.

      7.16.3 Production of virus‐free plants

      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.

      7.16.4 Applications in wide crosses

      Wide cross production is discussed in Chapter 7.

       Embryo rescue

      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

      Somatic hybridization was discussed in Chapter 6.

7.17 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 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 F₁ or F₂ plants. 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.

      7.17.1 Anther culture

      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