anther/pollen culture

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

• 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

• 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

Haploid Plant Formation

• History

– 1964, 1966 Datura innoxia (Guha and Maheshwari)

– 1967 Nicotiana tabacum (Nitsch)

• Critical factor - change in developmental pattern from mature pollen to embryogenesis.

Androgenesis

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

• 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

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)

• 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)

Other Factors Influencing Androgenesis

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

Pollen mother cell

Tetrad

Pollen forming

Pollen Development

Pathways to Androgenesis

Normal pollen

development

•Endoreduplication

“ colchicine”

Colchicine

• 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

MicrosporesFilter paperAnthers

Pollen in hanging drops

Isolated microspore 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

Ovule Culture

• 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

Production of Doubled Haploids

• 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

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