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 26 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 410 oC for 210 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,

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 thousands of F2 for selecting desirable genotypes.
Selection of mutants. Androgenic haploids have been used for selecting especially
recessive mutants. In species such as tobacco, mutants resistant to methionine analogue
(methionine sulfoxide) of the toxin produced by Pseudomonas tabaci have been selected.
Development of supermales in asparagus. Haploids 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 develop 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.

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).
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 (2n2x14,
VV) X Hordeum bulbosum (2n2x14, BB), commonly called the bulbosum method, and also
in wheat X maize crosses.
However, during the tissue culture of the embryo, the bulbosum chromosomes are eliminated,
leaving a haploid (2nx7V). This is then doubled by colchicines treatment to obtain
2n2x14 VV.
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 sources. Haploids 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 1pp (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 sources. Haploid 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, 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 (23) generations.
o It is easier and more efficient to select among homogeneous progeny (versus
heterogeneous progeny in conventional breeding).
o The cultivar released is homogeneous.
Disadvantages
o The procedure requires special skills and equipment in some cases.
o Additional technology for doubling may increase the cost of a breeding program.
o Frequency of haploids generated is not predictable.
o There is a lack of opportunity to observe line performance in early generations
prior to homozygosity.
Genetic issues. Unlike the conventional methods of inbreeding, it is possible to achieve
completely homozygous individuals. 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. It is advantageous if the female also has agronomically
desirable traits. F1 hybrids are suitable because their female gametes will be segregating.