Anther/Pollen culture

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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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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.

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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

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Androgenesis

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

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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

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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.

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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)

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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)

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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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Tetrad
Pollen mother cell
Pollen forming
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Pollen Development
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Pathways to Androgenesis
Normal pollen development
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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

80 pollen grains/drop
Isolated microspore culture
Pollen in hanging drops
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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

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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

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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

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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