Double Haploids in Plant Breeding

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 plantlets resulting 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 diplodized (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.

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. A high nitrogen content of the donor plant and exposure to low
temperature at meiosis reduces albinos and enhances the chance of green plant regeneration. Pretreatment (e.g., storing buds at 4–10 oC 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 articifial doubling
with 0.2% colchicines, or through somatic callus culture).
Applications

Development of new cultivars. Through 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,

This is then doubled by colchicines treatment to obtain 2n¼2x¼14 VV. and also in wheat X maize crosses. commonly called the bulbosum method. resulting in plants with higher ploidy levels. However. Consequently. Haploids of Asparagus officinalis may be diplodized to produce homozygous males or females. VV) X Hordeum bulbosum (2n¼2x¼14. In species such as tobacco.  enabling fixation to occur in only one cycle of selection. making it a challenge to distinguish haploids from those of somatic origin. Ovule/ovary culture Gynogenesis. tobacco. Chromosomal aberrations often occur. Doubled haploids . gynogenesis is selected when androgenesis is problematic (as in sugar beet and onion). Androgenic haploids have been used for selecting especially recessive mutants. wheat. Ovaries ranging in developmental stages from uninucleate to mature embryo sac stages are used. BB). The method is less efficient than androgenesis because only one embryo sac exists per ovary as compared to thousands of microspores in each anther. Heritability is high because dominance is eliminated. mutants resistant to methionine analogue (methionine sulfoxide) of the toxin produced by Pseudomonas tabaci have been selected. Development of supermales in asparagus. High rates of albinos occur in cereal haploids (no agronomic value). versus several thousands of F2 for selecting desirable genotypes. Well established systems include the interspecific crosses between Hordeum vulgare (2n¼2x¼14. Selection of mutants. Haploids from wide crosses Certain specific crosses between cultivated and wild species are known to produce haploids. and onion. maize. Generally. it is possible for callus and embryos to develop simultaneously from gametophytic and sporophytic cells. rice. the bulbosum chromosomes are eliminated. However. leaving a haploid (2n¼x¼7V). Limitations    The full range of genetic segregation of interest to the plant breeder is observed because only a small fraction of androgenic grains develop into full sporophytes. Use of haploids for genetic studies is hampered by the high incidence of nuclear instability of haploid cells in culture. sugar beet. using ovules or ovaries has been achieved in species such as barley. during the tissue culture of the embryo. requiring several cycles of screening to identify the haploids. only a small number of doubled haploid plants in the F1 is needed.

Spontaneous doubling occurs in corn at the rate of 10% of haploids developed. Procedure The first step in using doubled haploids in breeding is identifying the source of haploids. 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. Another marker used is the purple aleurone color. The haploid plants are retained and grown in the greenhouse or field. It is estimated that haploids occur in corn at the rate of 1 in 1000 diploids. cytological evaluation of plants with the recessive female marker (by root tip squash) is conducted. 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 1pp (green haploid). Chase based on seedling color. or in vitro. and wheat. corn. Haploid production through interspecific and intergeneric crosses is in use.   Natural sources. discarding seedlings with the dominant male marker. pleiotropy. The key is distinguishing between haploid and diploid plants. and detection of gene linkages. Such doubling may be accomplished in vivo naturally or through crossing of appropriate parents. These include canola.Researchers exploit haploidy generally by doubling the chromosome number to create a cell with the double dose of each allele (homozygous). The haploids may be maternal or paternal in origin. barley. calculating combining abilities. 99% of which arise from parthenogenesis of maternal origin. and chromosome locations. To use this marker system. The seed from those with dominant endosperm marker of the male is saved and planted. Haploids originate in nature through the phenomenon of parthenogenesis (gamete formation without fertilization). Additionally. estimating the number of genetic variances. A marker system for this purpose was first developed by S. Haploids are used in mutation studies (recessive mutants are observable instantly) and in selecting against undesirable recessive alleles. Next. and self-pollinated to produce diploids. the breeder should cross a heterozygous female to a male with marker genes. doubled haploids are used to generate general genetic information that can be applied to facilitate the breeding process. 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. After . Key features Inbred lines are homozygous genotypes produced by repeated selfing with selection over several generations.S. Such information includes gene action and interaction. Applications Doubled haploids have been successfully used in breeding species in which efficient haploid generation and doubling systems have been developed. through the use of colchicine. one of the most well known being the barley system (previously discussed). Artificial sources.

F1 hybrids are suitable because their female gametes will be segregating. there is no need for growing segregating generations as obtains in conventional programs. o The cultivar released is homogeneous. . Advantages and disadvantages. Disadvantages o The procedure requires special skills and equipment in some cases. Using an F1 hybrid or a segregating population as female parent in the production of maternally derived haploids increases genetic diversity in the doubled haploid line. the diploid plants are grown to maturity. it is possible to achieve completely homozygous individuals. Genetic issues. o Frequency of haploids generated is not predictable. It is advantageous if the female also has agronomically desirable traits. Unlike the conventional methods of inbreeding. o Additional technology for doubling may increase the cost of a breeding program. The technique of doubled haploids has certain advantages and disadvantages. Seeds are harvested for planting plant rows. the key ones including: Advantages o Complete homozygosity is attainable in a shorter period o Duration of the breeding program can be reduced by several (2–3) generations. Because diploids produced by this method are normally completely homozygous. o There is a lack of opportunity to observe line performance in early generations prior to homozygosity. o It is easier and more efficient to select among homogeneous progeny (versus heterogeneous progeny in conventional breeding).    doubling the chromosome.