Principles of Plant Genetics and Breeding. George Acquaah

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).

       Applications

       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.

       Limitations

       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.

      7.17.2 Ovule/Ovary 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).

      7.17.3 Haploids from wide crosses

Certain specific crosses between cultivated and wild species are known to produce haploids. Well‐established systems include the interspecific crosses between Hordeum vulgare (2n = 2x = 14, VV) × Hordeum bulbosum (2n = 2x = 14, BB), commonly called the bulbosum method, and also wheat × maize crosses. The bulbosum method is illustrated in Figure 7.1. The F₁ zygote has 2n = 2x = 14 (7 V + 7B). However, during the tissue culture of the embryo, the bulbosum chromosomes are eliminated, leaving a haploid (2n = x = 7 V). This is then doubled by colchicine treatment to obtain 2n = 2x = 14 VV.

Figure 7.1 Generating haploids in barley by the bulbosom method.

      7.17.4 Doubled haploids

      Researchers exploit haploidy generally by doubling the chromosome number to create a cell with the double dose of each allele (homozygous).

       Key features

      Inbred lines are homozygous genotypes produced by repeated selfing with selection over several generations. The technique of doubled haploids may be used to produce complete homozygous diploid lines in just one year (versus more than four years in conventional breeding) by doubling the chromosome complement of haploid cells. Such doubling may be accomplished in vivo naturally or through crossing of appropriate parents, or in vitro, through the use of colchicine. The success of doubled haploids as a breeding technique depends on the availability of a reliable and efficient system for generating haploids and doubling them in the species.

       Applications

      Doubled haploids have been successfully used in breeding species in which efficient haploid generation and doubling systems have been developed. These include canola, barley, corn, and wheat. Additionally, doubled haploids are used to generate general genetic information that can be applied to facilitate the breeding process. Such information includes gene action and interaction, estimating the number of genetic variances, calculating combining abilities, and detection of gene linkages, pleiotropy, and chromosome locations. Haploids are used in mutation studies (recessive mutants are observable instantly) and in selecting against undesirable recessive alleles.

       Procedure

      The first step in using doubled haploids in breeding is identifying the source of haploids.

       Natural SourcesHaploids originate in nature through the phenomenon of parthenogenesis (gamete formation without fertilization). The haploids may be maternal or paternal in origin. It is estimated that haploids occur in corn at the rate of 1 in 1000 diploids, 99% of which arise from parthenogenesis of maternal origin. Spontaneous doubling occurs in corn at the rate of 10% of haploids developed. The key is distinguishing between haploid and diploid plants. A marker system for this purpose was first developed by S.S. Chase based on seedling color, purple plants being encoded by the dominant gene (P) while normal green plants are recessive (p). A cross of F1(pp) × PP would yield 999Pp (purple diploids) and 1 pp (green haploid). Another marker used is the purple aleurone color.To use this marker system, the breeder should cross a heterozygous female to a male with marker genes. The seed from those with dominant endosperm marker of the male is saved and planted, discarding seedlings with the dominant male marker. Next, cytological evaluation of plants with the recessive female marker (by root tip squash) is conducted. The haploid plants are retained and grown in the greenhouse or field, and self‐pollinated to produce diploids.

       Artificial sourcesHaploid production through interspecific and intergeneric crosses is in use, one of the most well‐known being the barley system (previously discussed). After doubling the chromosome, the diploid plants are grown to maturity. Seeds are harvested for planting plant rows. Because diploids produced by this method are normally completely homozygous, there is no need for growing segregating generations as obtains in conventional programs.

       Advantages and disadvantages

      The technique of doubled haploids has certain advantages and disadvantages, including the following:

       AdvantagesComplete homozygosity is attainable in a shorter period.Duration of the breeding program can be reduced by several (two to three) generations.It is easier and more efficient to select among homogeneous progeny (versus heterogeneous progeny in conventional breeding).The cultivar released is homogeneous.

       DisadvantagesThe procedure requires special skills and equipment in some cases.Additional technology for