Pollen mother cell

Tetrad

Pollen formation

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

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

• 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.

• 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

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)

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.

• 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.

– Sucrose - ranges from 2% (Nicotiana) to 10% (Brassica)

• 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

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

Pathways to Androgenesis

Normal pollen

development

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

•Endoreduplication (Chromosome doubling)

“ colchicine”

                               

  

Haploid can be made

Double haploid (DH) by

Colchicine treatment

• 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

• 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/dropMedium

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

Haploid production by the bulbosum method in barley

• Pollen is collected from plants of Hordeum bulbosum, a wild relative of cultivated barley (H. vulgare).

Hordeum bulbosum, a wild relative cultivated barley

H. vulgarecultivated barley

Elimination of bulbosum chromosome during zygote formation

Haploid plants having only H. vulgare chromosome

• 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.

aa

• 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

Alleles are alternate form of a gene

AA a

Haploid

AA

Aa

Dominant

Recessive

AA

Aa

Dominant

Recessive

A

a

Cochicine treatment