This document discusses haploid plants, diploidization, and the bulbosum technique. It begins by defining haploid and diploid plants and describing methods of haploid production, including anther culture. It then covers diploidization methods like colchicine treatment and endomitosis. The bulbosum technique is described as using interspecific crosses between Hordeum vulgare and H. bulbosum to produce haploid H. vulgare plants through chromosome elimination. Haploids have various applications including early variety release, hybrid sorting, and developing disease resistant varieties. However, limitations include low yields and albinism in cereals.

1. SIGNIFICANCE OF HAPLOIDS,DIPLOIDIZATION
AND BULBOSUM TECHNIQUE

2. CONTENT
• Introduction
• Methods of haploid production
• Diploidization
• Bulbosum technique
• Significance
• Limitations
• Conclusion
• Reference

3. INTRODUCTION
Haploids are sporophytic plants that are autonomous and possess only
a single set of chromosomes. These are in contrast of diploids containing
two sets of chromosomes. Haploids may be originated from a gametic cell
in the embryo sac or in the pollen grain. Haploids may be divided into two
categories :

4. 1. Monoploids (monohapioids):
These are the haploids that possess half the number of chromosomes
from a diploid species e.g. Maize, Barley.
2. Polyhaploids:
The haploids possessing half the number of chromosomes from a
polyploid species are regarded as polyhaploids e.g. Wheat, Potato. It may be
noted that when the term haploid is generally used it applies to any plant
originating from a sporophyte (2n) and containing half the number (n) of
chromosomes.

5. • The existence of haploids was discovered by Bergner in Datura
stramonium. Plant breeders have been conducting extensive research to
develop haploids. The Indian scientists Guha and Maheshwari (1964)
reported the direct development of haploid embryos and plantlets from
microspores of Datura innoxia by the cultures of excised anthers.
• Subsequently, Bourgin and Hitsch (1967) obtained the first fully-
pledged haploid plants from Nicotiana tabacum. Thereafter, much
progress has been made in the anther cultures of Wheat, Rice, Maize,
Pepper and a wide range of economically important species.

6. METHODS OF HAPLOID
PRODUCTION
There are two way for the production of haploid plants. They are:
(1) In Vivo and (2) In Vitro. The process of apomixis or parthenogenesis
is responsible for producing spontaneous natural haploids. Many
techniques are followed both by in vivo and in vitro methods for haploid
production. In vitro techniques are more applicable for much haploid
production.

7. IN VIVO APPROACH
1. Androgenesis: In the process of androgenesis, haploid plants are
mainly derived from male gamete. Development of a haploid plant from
an egg cell containing male nucleus is referred to as
androgenesis. Before fertilization this egg nucleus has to be inactivated
or eliminated for successful haploid production in vivo techniques.
2. Gynogenesis:
A haploid plant can be produced from an unfertilized egg cell as a result of
delayed pollination. e.g. cross between Solanum tuberosum and Solanum
phureja produced diploid potatoes.

8. 3. Distant hybridization: Haploid hybrids can be produced by elimination. In
this process, one of the parental genomes have to be eliminated to produce
haploid hybrids. Distant hybridization may be continued by inter-specific or
inter-generic crosses. Gersenson (1928) obtained 7 haploids out of 35
offspring by the crossing between Solanum nigrum and Solanum luteum.
4. Parthenogenesis : In plants, parthenogenesis is a component process of
apomixis. Haploids may be developed from an unfertilized egg cell. In this
case, no fertilization is needed.
5. Apogamy: In apogamy, haploids may be developed from other cells of the
mega-gametophyte.

9. 6. Shocks with high or low temperatures :
Haploid plants may also be produced by applying high or low
temperature. Blakesle et al (1927) got haploid plant by the application of high
and low temperature.
7. Irradiation with X-rays or UV light:
For producing haploids, x-rays or ultra violate rays may be used to
induce chromosomal breakage and their subsequent elimination. Arenberger
(1948) got haploid Antirrhinum majus .
8. Chromosome elimination :
Chromosome elimination occurs in some intergeneric and interspecific
crosses to produce haploids. e.g. Cross between Hordeum vulgare and H.
bulbosum.

10. IN VITRO APPROACH
1. Anther culture:
Most research has been carried out on isolated anthers which has been
isolated on to solid or on liquid nutrient media
2. Pollen grain culture:
This is less used technique due to technical problems
3. Inflorescences:
Useful with grasses and other plant species which have small flowers,
e.g. Hordeum

11. 4. Embryo culture:
Hordeum vulgare is crossed with H. bulbosum. Soon after fertilization,
the chromosome of H. bulbosum are eliminated. Haploid embryo of H.
vulgare is produced with no endosperm and without embryo culture this
embryo would abort. Another example is crossing between Triticum aestivum
and H. bulbosum which resulted in the production of a haploid Triticum
aestivum embryo.
5. Pseudo-fertilization:
Hess and Wagner (1974) pollinated Mimulus luteus in vitro with alien
pollen of Torenia fournieri by prickle pollination a haploid cell of Mimulus
developed into a haploid plant.

12. 6. Development of unfertilized ovules without pseudo-pollination:
The main advantage of ovule culture appears to be that the majority of
regenerants are green and are more genetically stable, e.g. rice, wheat,
maize, Hordeum vulgare, etc.
7. Gametophytic irradiation and ovary culture:
In Petunia, the in vitro ovary culture of plants pollinated with
irradiated pollen is so far the most efficient techniques for haploid
production. The irradiated pollen acts not only in stimulating gynogenesis
but also takes part in fertilization.

14. DIPLOIDIZATION
Haploids may grow normally up to flowering stage but
in the absence of homologous chromosomes meiosis is
abnormal and, consequently viable gametes are not formed.
To obtain fertile, homozygous diploids for analyzing the
progenies and the breeding behavior of the pollen plants
the chromosome complement of the diploids must be
duplicated. Spontaneous duplication of the chromosomes
in pollen derived plants has been observed but its
frequency fairly very low.

15. In wheat and barley 20-50% and 20- 70% of the
pollen plants respectively, exhibited spontaneous
diploidization. However, generally, the rate of
spontaneous duplication of chromosomes may be very
low. This can be significantly enhanced by using artificial
means. For Nicotiana tabacum only 0.4% solution of
colchicine is recommended to diploidize the pollen plants.
In practice, the young pollen-derived plants are immersed
in a filter- sterlized solution of colchicine for about 96
hours and then transfer to a culture medium to allow their
further growth.

16. Alternatively the treatment is given in form of a
lanolin paste. It is applied to the axils of the upper leaves
and the main axis is duplicated to stimulate the axillary
buds to grow into diploid and fertile branches. Lichter et
al. (1989) induce chromosome doubling in B. napus by
injecting 2% solution of colchicine into secondary buds.
Besides bringing about chromosome duplication,
colchicine treatment may also result in chromosome and
gene instabilities.

17. Endomitosis: Haploids cells are unstable in culture and
have tendency to undergo Endomitosis. i.e chromosome
duplication without nuclear division. This property can be
used for obtaining homozygous diploid plants. In this
process, a small explant of stem from a haploid plant is
cultured on auxin-cytokinin added medium where the
segment forms the callus tissue. During callus growth,
diploid homozygous cells are produced by endomitosis.
Now large number of isogenic diploid plants can be
obtained by organogenesis..

18. iii) Fusion of Pollen Nuclei: Homozygous diploid callus or
embryos may form by the spontaneous fusion of two
similar nuclei of the cultured pollen after first division. In
Brassica, the frequency of spontaneous nuclear fusion in
microspore is high in culture

20. BULBOSUM TECHNIQUE
The production of haploid plants was not the only
mechanism obtained by pollen or anther culture. There
are other alternatives to haploid production. Selective
elimination of chromosome of one species during
interspecific crosses of two different species of the same
plant and retaining one chromosome of one species is
being utilised to obtain haploids.

21. The tetraploids of Hordeum vulgare and Hordeum
bulbosum were crossed. The products of nearly all the
plants were dihaploids. These dihaploids were again
crossed, the progeny comprised of haploids and
morphologically it is like hordeum vulgare. kasha
et.al.(1978) treated fertilised florets with low
concentration of GA3 for a couple of days considerably
enhanced the frequency of seed set and the recovery of
haploid plants.

22. Detailed cytological studies have revealed that in
these crosses double fertilization occurs normally but
elimination of bulbosum chromosomes during the early
stages of development of the hybrid embryo leads to the
formation of embryos with only vulgare chromosomes.
The chromosome elimination is controlled by genes
present on chromosome 2 and 3 of H.vulgare. In these
crosses the embryo normally aborts 10 days after
pollination. For raising full plants it is essential to excise
the immature embroys and rear them on an artificial
nutrient medium.

24. SIGNIFICANCE
• In haploid plants, as a chromosome possesses dominant and recessive
genes, only one set of homologous chromosomes present derived from
pollen grains. All the receissive genes will be expressed phenotypically in
the progeny as there is no masking effect of recessive gene by dominanat
genes. So in the haploidS, it is easy to identify recessive characters.
• Haploid plants are sterile as there is no zygotene pairing in the first
meiotic division and all the chromosome appear as univalent.

25. • Significance in Early Release of Varieties:
As time taken for getting the homozygous line is less so ultimately
many new hybrid varieties can be obtained in less time applying this
technique.
• Hybrid Sorting in Haploid Breeding:
Haploid breeding involves the hybrid sorting and it is considered
superior to pedigree and bulk method as the superior haploid plants are
selected and hybridized.

26. • Induction of Mutagenesis:
Haploid cell cultures are useful material for induction of mutations and
to study the effect of mutation. This method can overcome the masking
effect of presence of dominant gene. The screening method for detection of
mutational effect is also easier in this technique.
Ex: the anther derived haploids of Hyoscymus niger when treated with
x ray achieved mutant plants which show higher alkaloid contents.

27. • Induction of Genetic Variability:
The pollen/microspore are easy explant for production of
genetically variable types by introducing the different foreign
genes through different transformation procedure. These
transformed or transgenic haploids can be used further in
breeding programme.

28. • Generation of Exclusively Male Plants:
By haploid production of some dioecious male plant, the
dihaploid plant containing both Y chromosomes can produce supermale. In
case of Asparagus officinalis, male plants give more yield than female
plants, so if haploids are produced from anthers of male (XY) plants and by
chromosome doubling the Y chromosome containing plant will produce
super male plant (YY) which can be subsequently vegetatively propagated.

29. • Development of Disease Resistant/Insect Resistant Varieties:
Haploid production can be used for the introduction of disease resistance
genes into cultivars. An established cultivar is crossed with a donor disease
resistant species and either F1 or F2 anthers are cultured. Haploids are
developed and screened for resistance and then diploidized.
New hybrids are produced in tobacco which exhibited resistance to
bacterial wilt.

30. • Development of Salt Tolerant Varieties:
Salt tolerant breeding lines have been developed in different crop
species which have been further integrated in conventional breeding
procedure.
Ex: Datura innoxia from haploid culture derived from anther culture
In raising isogenic pure lines which takes 7to8 years by conventional
methods in cross pollinated crops, haploid culture of f1 plants, this may be
achieved in single generation.

31. • Development of Aneuploids:
Haploids have been used in the production of aneuploids like
monosomies in wheat, trisomies in potato. In tobacco nullisomics were
derived from haploids obtained from monosomies which could not produce
nullisomics on selfing.
• Pollen culture allows a greater survival rate of embryos. This technique
is effective in induction frequency of the economically important plants.

32. LIMITATIONS
The practical applications of androgenic haploids are still limited as
the desired success with anther and pollen culture has been largely
restricted to only a few families, such as Brassicaceae, Poaceae and
Solananceae.
Low yields
Generally only 5 to 8 % of the total pollen grains in a responding
anther under go androgenic development, and a very small proportion of
these androgenic grains develop into full sporophytes. This turns out to be a
serious limitation in obtaining full range of gametic segregants of interest
of plant breeders.

33. Conversion of pollen embryos into plants
One of the major problems in anther/pollen culture of brassica
species is the structural, physical and biochemical abnormalities of pollen
embryos so that 70 to 80% of the embryos are incapable of normal
germination.
Albinism in cereals
The degree of albino formation may vary with the variety. The
different variety of rice shows different rate of formation albino.
The albino plants are morphologically normal except for the
absence of chlorophyll. Since the albino plants cannot survive in nature there
is no nagronomic value.

34. Genetic changes
Instabilioty of genetic material during
androgenesis could be an hindrance in the use of
haploids. In tobacco the androgenic plants exhibited
growth depression, reduced agronomic performanced
and abnormalities such as reduced leaf and flower size
and tumerization of leaf cells, which could be correlated
to amplification of DNA.

35. CONCLUSION
Haploids have been produced for a very large number of angiosperms,
and the technique of anther/pollen culture for haploid production has
become an integral part of plant breeding programs. The main factors that
hinder the application of anther and pollen culture to cereals are low rates
of androgenesis and high frequency of albinism(upto to 80%).

36. REFERENCE
• Bhojwani,S.S.,& Razdan,M.K.,(2013). Plant tissue culture: theory and
practice. Elsevier pvt.ltd. Netherland pp-167-214
• Singh,s.k., & Srivastava,S.,(2012). Plant tissue culture. Campus Books
International.New delhi. pp-46-56
• Narayaswamy,S,.(2017). Plant cell and tissue culture. Tata McGraw Hill
Education Private Limited.New delhi pp- 340-347
https://www.biologydiscussion.com/plants/haploid-plants/top-10-
applications-of-haploid-plants-biotechnology/61373
https://www.slideshare.net/jannatiftikhar/haploid-culture-final
https://plantlet.org/haploid-breeding-development-of-pure-homozygous-line/

37. THANK YOU
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