Haploid plants have half the normal number of chromosomes due to developing from an unfertilized egg or synergid cell. Haploids can be produced in vitro through anther or ovule culture, or in vivo through chromosome elimination after interspecific hybridization in some plants like barley. Anther culture involves excising anthers and exposing them to stress before culture. Microspores then develop into haploid embryos or calli. Ovule culture is less common. Haploids are valuable for breeding as recessive traits are expressed, but are sterile. Chromosome doubling through colchicine produces fertile doubled haploids that are useful for breeding programs. Factors like genotype, culture medium composition

2. Haploid Production
• Haploid plants are characterized genetically
by the presence of gametic number of
chromosomes in their cells (i.e., half the
number of chromosomes as in somatic
cells).
• In nature, haploids arise as an abnormality
when the egg or a synergid cell develops
into an embryo without fertilization

3. • The natural haploids of Datura stramonium
were first discovered in 1922 by Blakeslee
et al.
• Since then spontaneous occurrence of
haploids has been reported in about 100
species (Vasil 1997).

4. • Haploids are sexually sterile and require
doubling of chromosomes to produce fertile
doubled haploids (DH) or homozygous
diploids.
• Haploids and doubled haploids are of
considerable importance in genetics and
plant breeding programs.

7. Pollen mother cell
Tetrad
Pollen forming

8. Pollen Development

9. • Haploids are very valuable in plant breeding
for several reasons
– Since they carry only one allele of each gene,
mutations and recessive characteristics are
expressed in the plant.
– Plants with lethal genes are eliminated from the
gene pool.
– Can produce homozygous diploid or polyploid
plants - valuable in breeding
Value of Haploids in Breeding

10. • In vitro methods:
– Anther culture (androgenesis) - production of
haploid plants from microspores
• Anther culture for production of haploids reported in about
250 species
• Solanaceae, Cruciferae, Gramineae, Ranunculaceae most
common
– Ovule culture (gynogenesis) - production of haploid
plants from unfertilized egg cell
• in vivo method:
– Spontaneous occurrence in low frequency
– Induction by physical and/or chemical treatment
– Chromosome elimination following interspecific hybridization.
Specific for some plants such as barley. Not widespread.
Haploid Plant Formation

11. Androgenesis
• Androgenesis refers to the development of
plants (sporophytes) from microspores or
immature pollen (male gametophyte).
• History
– 1964, 1966 Datura innoxia (Guha and
Maheshwari)
– 1967 Nicotiana tabacum (Nitsch)
Two techniques are used to produce androgenic
haploids, viz. anther culture and isolated pollen
culture

12. • Anther culture is technique by which the
developing anthers at a precise and critical stage
are excised aseptically from unopened flower
bud and are cultured on a nutrient medium
• The cultures are exposed to a suitable stress
treatment before incubation under normal
culture conditions in dark.
• Microspore within the cultured anther develop
into callus tissue or embryoids that give rise to
haploid plantlets.
Anther Culture

14. Pollen Culture:
Pollen or microspore culture is an in vitro
technique by which the pollen grains, preferably
at the uninucleated stage, are squeezed out
aseptically from the intact anther and then
cultured on nutrient medium, develop into
haploid embryoids or callus tissues that give rise
to haploid plantlets.

16. Ovule Culture:
Used in plant families that do not respond to
androgenesis eg. Liliaceae, Compositae
Ovule culture is an elegant experimental system by
which ovules are aseptically isolated from the
ovary and are grown aseptically on chemically
defined nutrient medium under controlled
conditions.
• Haploids can be induced from ovules
• The number of ovules is less and thus is used less than
anther/pollen culture

17. • Use solution of colchicine which interferes
with cell division, but DNA is doubled
• Shortens the breeding cycle considerably by
reducing number of generations required in
normal breeding programs
Production of Doubled Haploids

18. • Anthers fail to grow, embryos fail to continue growth
• Developing tissue or callus may be diploid or
polyploid
– Chimera of different ploidy may result
• Formation of albinos in cereals (especially rice)
• Low success rate - not commercially viable
• Use of growth regulators for callus production
usually detrimental for haploid production since
diploid and polyploid callus is produced
• Doubled haploids sometimes are not homozygous
– Segregation may be seen in progeny
Associated Problems with Anther Culture

19. In vivo - Haploid production by the
bulbosum method in barley
• Pollen is collected from plants of
Hordeum bulbosum, a wild relative of
cultivated barley (H. vulgare).

21. • The H. bulbosum pollen is brushed onto
emasculated barley florets.

22. • A hybrid zygote forms, but during the
first few cell divisions the H. bulbosum
chromosomes are eliminated.
• The seeds or the haploid embryo
developed rescue contain haploid
embryos with one set of H. vulgare
chromosomes.

23. The haploid embryos must be germinated
in vitro.

24. The haploid plants can be treated with
colchicine to obtain doubled haploids.

25. Uses of haploids and
doubled haploids
• Completely homozygous plants
• Helps in plant breeding (equal ploidy levels)
• Time taken is shorter
• Mutation studies are possible

26. • Genotype
– Difficult in cereals, as genetic component controlled
by multiple genes.
– In plants such as tobacco little success is reported,
genotype is less important.
• Anther wall factors (only in androgenesis)
– The specific compounds initially were not known.
However addition of anther wall extracts was found
to promote anther culture in tobacco.
– Glutamine alone in in combination with serine and
myoinositol replaced the wall factors.
Factors Influencing Andro/gyno genesi

27. Effect of culture medium
HORMONE EFFECT in dicot and monocots.
• In dicots most success with solanaceous species Without
hormones
• In most non-solanaceous species and many monocots it
require hormones or complex organics such as coconut
milk.
SUCROSE EFFECT (High sucrose required)- The higher
levels of sucrose may fulfill an osmotic rather than a nutritional
requirement. It ranges from 2% (Nicotiana) to 10% (Brassica).

28. Application of a variety of stresses – Applying stress such as
temperature shock, osmotic stress, and sugar starvation, at the initial
stage of anther or pollen culture has proved promontory or essential
for the induction of androgenesis.
CHILLING PETREATMENT
Yields of tobacco haploids are often increased by storing excised buds
at 7 to 8° C for 12 days prior to anther excision and culture
Density
Density of pollen or anthers
In Brassica napus minimum density required is 3000 pollen/ml of culture
medium.
Other Factors Influencing Andro/gyno genesis