ANTHER AND POLLEN CULTURE

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ANTHER AND POLLEN CULTURE By- Mr. Bhimgonda B. Patil. Regn. No. 07/148

Anther and microspore culture: 

Anther and microspore culture Suggested reading: Vasil ch . 7, Bhojwani ch . 7 Importance of anther/microspore culture completely homozygous inbred lines no segregation in progeny (doubled haploid lines) uniform inbred lines in one generation inbred lines for F 1 hybrid cultivars (e.g., oilseed brassicas ) in polyploids (e.g., Medicago & potato), used for making DHs for classical breeding & for somatic hybridization _ genome mapping (easier ID of markers)

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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

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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

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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

Anther and microspore culture: 

Anther and microspore culture Culturing methods anther culture – easiest and simplest pollen (microspore) culture – advantages less competition among microspores no diploid anther walls greater potential haploid plant production protocol for tobacco anther culture (aseptically) detach anther from tobacco filament float anther on a liquid (MS-type) culture medium

Anther and microspore culture: 

Anther and microspore culture Culturing methods protocol for Brassica microspores (see Bhojwani, fig. 7-4 (done aseptically) squeeze out microspores into liquid medium filter through nylon screen of approp. pore size (e.g., 40 μ m for Brassicas) centrifuge at 50-100g for ca. 5 min. resuspend and load onto a 24%/32%/40% Percoll gradient solution and spin

Anther and microspore culture: 

Anther and microspore culture Culturing methods protocol for Brassica microspores (see Bhojwani, fig. 7-4 (done aseptically) separate upper 2 layers and pipet out into fresh medium spin, resuspend, pellet and adjust plating density of pollen to 2-5 x 10 4 microspores per ml plate suspensions as a thin layer in petri dishes and incubate at 32° C in the dark 3-5 days, then at 25° C

Anther and microspore culture: 

Anther and microspore culture Culturing methods protocol for Brassica microspores (see Bhojwani, fig. 7-4 (done aseptically) transfer embryos to liquid medium in flasks and shake at 60 rpm at 32° C transfer embryos to solid medium w/2% sucrose and incubate at the lower temp.

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

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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.

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The haploid embryos must be germinated in vitro.

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

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

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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

Anther and microspore culture: 

Anther and microspore culture Uses in plant breeding group 1 (asparagus, maize, rubber) DHs produced with low efficiency goals - asparagus all male F 1 hybrids maize DHs are screened like other inbreds group 2 (wheat, melons, peppers) rel. large no. of DHs can be produced goals – genetic maps; separating & IDing disease resistance genes (e.g., CMV resistance in pepper)

Anther and microspore culture: 

Anther and microspore culture Uses in plant breeding group 3 (rapeseed, barley, rice) – DH lines easy rice – lines used to develop varieties, esp. japonica subspecies rapeseed – pedigree selection programs and parental lines for F 1 hybrid varieties barley – winter barley varieties use microspore culture, spring barley varieties use the bulbosum method (to be discussed later)

Advantages of pollen culture over anther culture: 

Advantages of pollen culture over anther culture Overcrowding of pollens eliminated Unwanted growth of diploid cells eliminated Pollen is ideal for uptake, transmission, mutagenic studies Pollen may be directly transformed into an embryo Higher yield

Achievement :: 

Achievement : “PARAG” is the rice variety developed by pollen culture technique.

References :: 

References : Introduction to plant biotechnology -By- H. S. Chawala Embryogenesis in angiosperms : a developmental and experimental study -By Valayyamghat Raghavan

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Thank you…