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2. Introduction
One of the goals in plant breeding program is the production of F1 hybrids. The main limitation of
this scope is the long period required to produce inbred lines. The most time-consuming and
labor-intensive aspect of developing these hybrids is the traditional inbreeding process that
requires manual self pollination necessary to generate homozygous parental lines. This process
requires six or more generations of inbreeding to establish adequately stable lines that can be
used in hybrid combinations. In many species in vitro methods have allowed to speed up the
production of homozygous lines, as alternative to the slower inbreeding process. Haploid plants
can be obtained by anther, non-fertilized ovule, ovaries or whole flowers buds culture.

3. The haploid chromosome number means that meiotic recombinations and recessive gene
effects are manifested at plant level. Then, spontaneous or induced chromosome doubling
consent the regeneration of doubled haploids (DHs) homozygous materials, with restored
fertility, that can be used in different breeding strategies. Because all the alleles of DHs lines
are fixed, selection e.g. for quantitative characters is often more reliable than in conventional
populations.

4. Haploids
 Haploids are plants (sporophytes) that contain a gametic chromosome number (n).
 Blakeslee first described this phenomenon in Datura stramonium in 1922.
 This was subsequently followed by similar reports in tobacco (Nicotiana tabacum),
wheat (Triticum aestivum) and several other species (Forster et al., 2007).
 The potential of haploidy for plant breeding arose in 1964 with the achievement of haploid embryo
formation from in vitro culture of Datura anthers ( Guha and Maheshwari, 1964, 1966)

5. Production of haploids and doubled haploids
Haploids produced from diploid species (2n=2x), known as monoploids, contain
only one set of chromosomes in the sporophytic phase (2n=x).
They are smaller and exhibit a lower plant vigour compared to donor plants and
are sterile due to the inability of their chromosomes to pair during meiosis.
To propagate them through seed and to include them in breeding programs,
their fertility has to be restored with spontaneous or induced chromosome
doubling.
The obtained DHs are homozygous at all loci and can represent a new variety
(self- pollinated crops) or parental inbred line for the production of hybrid
varieties (cross- pollinated crops).

6. The main factors affecting haploid induction and subsequent
regeneration of embryos are:
genotype of the donor plants,
physiological condition of donor plants (i. e. growth at lower temperature and high illumination),
developmental stage of gametes, microspores and ovules,
pre-treatment (i.e. cold treatment of inflorescences prior to culture, hot treatment of cultured
microspores),
composition of the culture medium (including culture on “starvation” medium low with carbohydrates
and/or macro elements followed by transfer to normal regeneration medium specific to the species),
physical factors during tissue culture (light, temperature).

7. Haploid Techniques
Induction of maternal haploids
In situ induction of maternal haploids:-In situ induction of maternal
haploids can be initiated by pollination with pollen of the same species
(e.g., maize), pollination with irradiated pollen, pollination with pollen of a
wild relative (e.g., barley, potato) or unrelated species (e.g., wheat).
Wide hybridization:- Wide crossing between species has been shown to
be a very effective method for haploid induction and has been used
successfully in several cultivated species.
In vitro induction of maternal haploids – gynogenesis:- In vitro
induction of maternal haploids, so-called gynogenesis, is another pathway
to the production of haploid embryos exclusively from a female
gametophyte.
It can be achieved with the in vitro culture of various un-pollinated flower
parts, such as ovules, placenta attached ovules, ovaries or whole flower
buds.
Androgenesis can be induced with in vitro culture of immature anthers, a
technically simple method consisting of surface sterilization of pre-treated
flower buds and subsequent excision of anthers under aseptic conditions.

8. a) Induction of maternal haploids
In situ induction of maternal haploids:-
In situ induction of maternal haploids
can be initiated by pollination with pollen
of the same species (e.g., maize),
pollination with irradiated pollen,
pollination with pollen of a wild relative
(e.g., barley, potato) or unrelated
species (e.g., wheat).
Pollination can be followed by
fertilization of the egg cell and
development of a hybrid embryo, in
which paternal chromosome elimination
occurs in early embryogenesis or
fertilization of the egg cell does not
occur, and the development of the
haploid embryo is triggered by
pollination of polar nuclei and the
development of endosperm.

9. 2. Wide hybridization:-
Wide crossing between species
has been shown to be a very
effective method for haploid
induction and has been used
successfully in several cultivated
species.
It exploits haploidy from the
female gametic line and involves
both inter- specific and inter-
generic pollinations.
The fertilization of polar nuclei
and production of functional
endosperm can trigger the
parthenogenetic development of
haploid embryos, which mature
normally and are propagated
through seeds (e.g., potato).
In other cases, fertilization of
ovules is followed by paternal
chromosome elimination in hybrid
embryos.
The endosperms are absent or
poorly developed, so embryo
rescue and further in vitro culture
of embryos are needed (e.g.,
barley).
The ‘bulbosum’ method was the
first haploid induction method to
produce large numbers of
haploids across most genotypes
and quickly entered into breeding
programs.

11. 3. In vitro induction of
maternal haploids –
gynogenesis:-
In vitro induction of maternal
haploids, so-called
gynogenesis, is another
pathway to the production of
haploid embryos exclusively
from a female gametophyte.
It can be achieved with the in
vitro culture of various un-
pollinated flower parts, such
as ovules, placenta attached
ovules, ovaries or whole
flower buds.
Although gynogenetic
regenerants show higher
genetic stability and a lower
rate of albino plants
compared to androgenetic
ones, gynogenesis is used
mainly in plants in which other
induction techniques, such as
androgenesis and the
pollination methods above
described, have failed.
Gynogenic induction using
un-pollinated flower parts has
been successful in several
species, such as onion, sugar
beet, cucumber, squash,
gerbera, sunflower, wheat,
barley etc. but its application
in breeding is mainly
restricted to onion and sugar
beet.

12. b) Induction of
paternal haploids –
Androgenesis:-
Androgenesis is the process of induction and
regeneration of haploids and double haploids
originating from male gametic cells.
Due to its high effectiveness and applicability in
numerous plant species, it has outstanding
potential for plant breeding and commercial
exploitation of double haploids.
Its major drawbacks are high genotype dependency
within species and the recalcitrance of some
important agricultural species, such as woody
plants, leguminous plants and the model plant
Arabidopsis thaliana.

13. Androgenesis can be induced with in vitro
culture of immature anthers, a technically
simple method consisting of
surface sterilization of pre-treated flower
buds and subsequent excision of anthers
under aseptic conditions. The anthers are
inoculated and cultured in vitro on solid,
semi-solid or liquid mediums or two-
phase systems (liquid medium overlaying
an agar-solidified medium).
The method relies on the ability of
microspores and immature pollen grains
to convert their developmental pathway
from gametophytic (leading to
mature pollen grain) to sporophytic,
resulting in cell division at a haploid level
followed by formation of calluses or
embryos.

15. Applications of gynogenesis
In practice, production of haploid plants by gynogenesis is not used as frequently as
androgenesis in crop improvement programs because:
 The dissection of unfertilized ovaries and ovules is quite difficult; and
 The presence of small numbers of ovaries per flower compared to the large number of
pollen grains in an anther.
Now, if there is androgenesis which is highly successful, why should you use gynogenesis?
Gynogenesis is mainly used to produce haploids of male-sterile plants or of dioecious plant
species (each individual has only female or male flowers), such as mulberry and cannabis.

16. The advantage this method offers you over androgenesis is the
low occurrence of albinos (white plants without chlorophyll) in
cereals like wheat, maize and rice. In crop plants like onion, sugar
beet, and melon, gynogenesis is the only way to produce haploid
plants. Also, in some plant species such as rice, gynogenesis is
more efficient than androgenesis.

17. Haploid Plants from Tissue
Culture
Specialized plant tissue culture methods have enabled the production of
completely homozygous breeding lines from gametic cells in a shortened time
frame compared to conventional plant breeding. Plants from gametic cells of an
F1 hybrid represent a gametic array each having a different genetic contribution
from the parents. Lines exhibiting the desired characteristics are chosen for large-
scale field trials as a prelude to commercialization. Although the number of new
plant varieties developed via this method has been limited, refinement of tissue
culture techniques has extended the range of crop species from which haploid
plants have been produced as well as the efficiency resulting in large-scale
haploid plant production. Several varieties developed via this method are grown
on considerable acreage while others are being tested as candidates to replace
varieties developed by conventional methods.

18. Haploid Plants from Tissue Culture
Application in Crop Improvement
Haploid plants are of great interest to geneticists and plant breeders. Haploids
offer geneticists the opportunity to examine genes in the hemizygous condition
and facilitate identification of new mutations. Plant breeders value haploids as a
source of homozygosity following chromosome doubling from which efficient
selection of both quantitative and qualitative traits is accomplished (Griffing, 1975).
As a tool in crop improvement strategies, it is imperative that haploids are
produced from individual heterozygous genotypes in sufficiently large numbers to
compensate for undesirable gene combinations that result from linkage and
random assortment via meiosis. Just as plant breeders advance large populations
through several generations (to maximize variability in later generations),
application of haploidy in plant breeding is dependent on the ability to produce a
haploid population of sufficient size to accommodate selection of desired gene
combinations.

19. What is a doubled haploid (DH)
plant?
In a DH plant, the chromosome set of a haploid plant has been
doubled spontaneously or artifi cially. Chromosome doubling is
necessary since haploid plants are generally frail, have reduced
organ size and are not fertile. The most commonly used chemical
agent to render haploid plantlets diploid is colchicine, which
blocks cell division without blocking chromosome . Quick guide
duplication. This treatment acts like a ‘copy–paste’ of the haploid
genome into a diploid genome. Consequently, in DH plants all loci
are homozygous. Chromosome doubling creates ‘pure’
homozygotes or fully inbred lines.

20. Why is doubled haploid
technology impactful for agriculture?
Doubled haploid technology comprises both the production of
haploid plants and the chromosome doubling process. It has
become an important tool in plant breeding, since it shortens the
time needed to create pure homozygous lines, which can either
be released directly to farmers as cultivars or used as genitors
(inbred lines) for the production of hybrid seeds. The primary
advantage of DH plants is to possess a phenotypic stability due to
the fact that all alleles are in a homozygous state. In short, DH
technology increases the efficiency of plant breeding.

22. What future improvements are
needed for DH technology?
In plant breeding, and apart from maize, the production of DH plants requires
at least an in vitro-based process, the success of which remins highly
species- and genotype-dependent, as well as labor-intensive and time-
consuming. Moreover, the DH technology has not yet been applied to all
breeding programs, and there are still some major crop species (e.g.,
soybean, tomato, sunflower) that are recalcitrant the currently available
procedures, or for which present protocols are not efficient enough. The
understanding of the maize in situ system should help to extend the DH
technology to more species or breeding programs by either directly knocking
out the functional ortholog of the maize gene, or if needed by transferring the
cellular and molecular knowledge acquired on maize.