Anther/Pollen culture

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3 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

4 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.

5 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

6 Androgenesis History 1964, 1966 Datura innoxia (Guha and Maheshwari) 1967 Nicotiana tabacum (Nitsch) Critical factor - change in developmental pattern from mature pollen to embryogenesis.

7 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

8 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.

9 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)

10 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)

11 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

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13 Pollen Development

14 Pathways to Androgenesis Normal pollen development

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17 Endoreduplication colchicine Colchicine

18 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 Brassica oleracea Pollen in hanging drops 80 pollen grains/drop Anthers Isolated microspore culture Microspores Filter paper Medium

19 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

20 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

21 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

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).

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24 The H. bulbosum pollen is brushed onto emasculated barley florets.

25 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.

26 The haploid embryos must be germinated in vitro

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

28 Uses of haploids and doubled haploids Completely homozygous plants Inbred lines Mutation studies Breeding (equal ploidy levels) Mapping