Anther/Pollen culture

• Method to produce haploid

plants
• Spontaneous occurrence in
low frequency
• Induction by physical
and/or chemical treatment
• Chromosome elimination
following interspecific
hybridization
 Value of Haploids in Breeding
• 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.
 Haploid Plant Formation
• Formation in vivo
– 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.
• 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
 Androgenesis
• History
– 1964, 1966 Datura innoxia (Guha and
Maheshwari)
– 1967 Nicotiana tabacum (Nitsch)
• Critical factor - change in developmental
pattern from mature pollen to embryogenesis.
Factors influencing androgenesis

– Genotype of donor plants

– Anther wall factors
– Culture medium and culture density
– Stage of microspore or pollen development
– Effect of temperature and/or light
– Physiological status of donor plant
 Factors Influencing Androgenesis
• 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.
 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.
• Medium particularly important in cereals and rice to be
able to produce green plants. A major difficulty was large
number of albino plants that resulted.
– Sucrose - ranges from 2% (Nicotiana) to 10%
(Brassica)
 Other Factors Influencing Androgenesis
• Density
• Atmospheric volume of the vessel
• For embryos 15 ml/anther
• For producing plants 5.5 ml/anther
• Effect may be ethylene
– 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 gametic 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)
 Normal pollen development

• Pollen mother cells are in anther primordia

• First phase - meiosis - pollen mother cell (PMC)
• A tetrad froms from each PMC
• Second phase - microspores released from tetrads
• Third phase - microspores mature into pollen grains -
first pollen mitosis
• Second pollen mitosis, maybe after germination
• Generative and vegetative cells formed
 Tetrad
Pollen mother
cell

Pollen forming
Pollen Development
Pathways to Androgenesis

Normal
pollen
developmen
t
•Endoreduplication

Colchicin
“ colchicine” e
 Isolated Microspore Culture
• 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
80 pollen grains/drop Isolated microspore culture
– Brassica oleracea
Pollen in hanging drops Microspores
Anthers Filter pap

Medium
 Ovule Culture
• 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
 Production of Doubled Haploids

• Use solution of colchicine which interferes

with cell division, but DNA is doubled
• For polygenic traits, use two anther-derived
plants
– Shortens the breeding cycle considerably by
reducing number of generations required in
noarmal breeding programs
 Associated Problems with Anther Culture

• 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
Haploid production by the bulbosum
method in barley

• Pollen is collected from plants of

Hordeum bulbosum, a wild relative of
cultivated barley (H. vulgare).
• The H. bulbosum pollen is brushed onto
emasculated barley florets.
• 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.
The haploid embryos must be germinated
in vitro.
The haploid plants can be treated with
colchicine to obtain doubled haploids.
 Uses of haploids and
doubled haploids

• Completely homozygous plants

• Inbred lines
• Mutation studies
• Breeding (equal ploidy levels)
• Mapping