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In vitro production of Haploids
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Historical
Natural haploids
Blakeslee (1922): Datura stramonium
Clausen and Mann (1924): Tobacco
Gaines and Aase (1926): wheat
Haploids through anther culture
Guha & Maheshwari (1964): D. innoxia
Niizeki and Oono (1968): Rice
Today in vitro haploids reported in > 200 species
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Haploid production : Methods
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Advantages
Instant homozygosity
Recessive phenotypes revealed
Simplified genetic analysis Homozygous inbreds in
self-incompatible
species
In vitroselection for seed/embryo traits
Basic studies: Molecular maps, tagging
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Origin of microspore embryos
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Factors influencing anther culture
Temperature
Tobacco - 5C,72h response 21 to 58%
Rice: 13C, 10-14 d
Rye: 6C, 6-10 d
Brassica microspore: 32C, 24 h
Light
Initial dark period in most cases
Vitis, potato, D. innoxia: light during first 24 h
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Anther wall factors
Fate of exine bursting and embryogenesis
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Nutrition
Tobacco & Datura: Pollen divn. in anthersmaintained in humid
condn. agar + sucrose (2-4%)
sufficient for embryogenesis
Higher sucrose favours anther embryogenesis
Potato, wheat, rice ~ 6% sucrose
Brassica microspore culture: 13% sucroseMaltose superior to
sucrose in many species
Sucrose starvation: Tobacco
NitrogenLow NH4
+ (1-2 mM) - promotive
Glutamine: Highly promotive, can substitute foranther-wall
factor
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Gelling agent
AgarLess callus compared to liquid medium
but liquid medium leads to more albinosGenotype effect
Stage of microspore
Early-late uninucleate stage: most responsive
Early bi-nucleate stage: Atropa, N. sylvestris,
absolutely necessary for N. knightiana
Arabidopsis & tomato: Meiosis I
Physiological condition of donor plant
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Problems in anther culture
Albino plants
Chromosome doubling
Regeneration from non-haploid tissue Somaclonal variation
Distorted segregation due to genetic selection
B. napus x B. carinata : 93% haploids from 2ngametes (Nelson et
al. 2009)
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Gene expression studies BABYBOOM gene B. napusmicrospore
culture
16 genes specific markers for embryogenesis; also
expressedduring zygotic embryogenesis
Micropsores at 0, 3, 5 & 7 d of culture
A- light micropscope,B- DAPI-stained, C- scanning micrograph
D- 7d preglobular embryo, E- 7d zygotic embryo
Malik et al. 2007, Plant Physiol
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Doubled haploids versus selfing
Limited recombination
Maize:
Av. 10 recomb. per genome in DH v/s 15 in RI % lines with 4 or
more intact chromosomes:
37% in DH v/s 13% in RI
When to produce haploids?DHF2 best compromise (Bernardo, 2009,
TAG)
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Varieties developed
Barley: Nearly 50% vars in Europe from doubled
haploid breeding
B. napus: routinely used
Tomato, capsicum, B. oleracea
Rice :
China - Huafeng #10, Zhong-hua 8, Hua Ju #2, Hua-03;
JapanJoiku #394 (first DH cultivar in Japan);
Korea - Joryeongbyeo
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Doubled haploids & MAS
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GE approach to haploid production
Ravi & Chan: Nature 2010
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Other applications
Microspore transformation and DH production
Microspore transformation and in vitro culture
to obtain mature pollen
Microspore mutagenesis and DH production
