Haploid plant formation can occur through several methods, including anther/pollen culture, ovule culture, and distant hybridization. Anther/pollen culture involves culturing anthers or isolated microspores to induce microspores or pollen to undergo embryogenesis rather than gametogenesis. Success requires triggering a shift from the gametophytic to sporophytic development pathway. Temperature shock, culture medium composition, and donor plant conditions influence response. Haploids can be doubled to create homozygous diploid lines using colchicine, valuable for shortening plant breeding cycles.

3. Pollen mother cell
Tetrad
Pollen formation

5. A.D Bergner discovered haploid plant of
Datura stamoniun in 1921.

6. Formation in vivo
1. Genome elimination by distant hybridization:
In intergeneric and interspecfic crosses due to selective elimination of
one parental genomes during the process haploid are generated.
2. Symigamy: Egg cell and generative nuclei of pollen divide
independently
3. Chemical treatment: Some chemicals such as chloramphenical and
paraflurophenyalanine may induce chromosome elimination in
somatic cells.
4. Temperature shock: High or low temperature treatments may play
a role in induction of haploids
5. Irradiation effects. X-rays or UV light reportedly induce breakage
in chromosomes.
Haploid Plant Formation

7. • in vivo methods:
Ovule androgenesis - production of haploid
plants by development of egg containing male
nucleus only. Inactivation of egg takes place
before fertilization by microspores.
Gynogenesis - production of haploid plants
from unfertilized egg cell as result of
delayed fertilization
In vivo method yield low frequency of haploids, therefore
pollen / anther culture (anther androgenesis) is preferred.

8. • History
– 1964, 1966 Datura innoxia (Guha and
Maheshwari)
– 1967 Nicotiana tabacum (Nitsch)
• Critical factor - change in developmental
pattern from mature pollen to embryogenesis.
Androgenesis (Pollen embryogenesis)
• Anther culture for production of haploids reported in
about 250 species
• Solanaceae, Cruciferae, Gramineae, Ranunculaceae
most common

9. Requirements to trigger androgenesis
a.Healthy plants grown under controlled conditions
b.Knowledge of pollen ontogenesis
c.Temperature treatment to arrest existing metabolism in order to
shift pollen from gametophytic stage (gamete formation to form
mature pollen) to sporophytic phage (induce embryo)

10. Factors influencing androgenesis
1. Genotype of donor plants
2. Anther wall factors
3. Culture medium and culture density
4. Stage of microspore or pollen development
5. Effect of temperature and/or light
6. Physiological status of donor plant.

11. • Genotype
– Response is genotypically determined depending on
the species. In cereals, there is a major genetic
component controlled by many genes.
– In plants such as tobacco, genotype is less important.
• Anther wall factors
– The specific compounds are not known. Addition of
anther wall extracts, however was promotive in
tobacco.
– In some plants, glutamine alone in in combination
with serine and myoinositol replaced the wall factors.
Factors Influencing Androgenesis

12. Effect of culture medium
Two hormone groups
• Without hormones - mostly dicots. Most success with solanaceous
species. Do not want the anther wall to form callus.
• With hormones - most non-solanaceous species. Many monocots.
Require hormones or complex organics such as coconut milk.
– Sucrose - ranges from 2% (Nicotiana) to 10% (Brassica)

13. • Density
– Density of pollen or anthers
• In Brassica napus minimum density required is 3000 pollen/ml of
culture medium
• Stage of development of microspore or pollen
development
– Microspore or pollen must shift from gametophytic to
sporophytic pattern of development
– Best time to induce such a shift is either just prior to division of
the microspore or after microspore mitosis (forms generative
and vegetative cells)
Other Factors Influencing Androgenesis

14. Effect of temperature and/or light Temperature
shock helps in androgenesis. Bud treated with
cold temp. 3ºC or 5ºC for 72hrs induce haploid
of pollen embryos in Datura, tobacco.
Physiological status of donor plant. Anthers from
plants grown in short day (8hrs) and high light
shows better response

15. Pathways to Androgenesis
Normal pollen
development

16. Pathways1:
Microspores divide by equal division and two daughter cells contribute
to sporophyte. Vegetative and generative cells are not distinct.
Pathway 2:
Unequal division of uninucleate microscopre resulting in formation off
vegetative and generative cell. Sporophye rise due to division of
vegetative cell. Generative cells dives slow or does not divide before
degenarates.
Pathway 3:
The uninucleate microspore undergoes a normal unequal division but
pollen embryo re predominantly formed from generative cell.
Pathway IV:
The division of microspore in asymmetrical as in pathway II. Both
generative and vegetative cell divide and contribute to sporophytye

18. •Endoreduplication
(Chromosome doubling)
“ colchicine”
Haploid can be made
Double haploid (DH) by
Colchicine treatment

19. • 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
cells are produced
• Doubled haploids sometimes are not homozygous
– Segregation may be seen in progency
Associated Problems with Anther Culture

20. • Of interest because formation of embryo is
known to be from one cell only and thus no
chimeras are formed
• Much more difficult than anther culture
• Cultured either isolated microspores or pollen
– Brassica oleracea
Isolated Microspore Culture
80 pollen grains/drop
Medium
Microspores
Filter papeAnthers
Pollen in hanging drops
Isolated microspore culture

21. • Haploids can be induced from ovules
• The number of ovules is less and thus is used
less than anther culture
• May be by organogenesis or embryogenesis
• Used in plant families that do not respond to
androgenesis
– Liliaceae
– Compositae
Ovule Culture

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

23. Hordeum bulbosum,
a wild relative cultivated
barley
H. vulgare
cultivated barley
Elimination of bulbosum chromosome during zygote formation
Haploid plants having only H. vulgare chromosome

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

26. • A hybrid zygote forms, but during the
first few cell divisions the H. bulbosum
chromosomes are eliminated.
• The seeds that develop contain haploid
embryos with one set of H. vulgare
chromosomes.

27. The haploid embryos must be germinated
in vitro.

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

29. • 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
– Shorten the time for inbreeding for production
of superior hybrids genotypes.
Value of Haploids in Breeding

30. Alleles are alternate form of a gene
A
A a
Haploid
A
A
A
a
Dominant
Recessive
A
A
A
a
Dominant
Recessive
A
a
Cochicine treatment