Agriculture Reference
In-Depth Information
2.1.1 General Steps of Androgenesis Methods
Plant grown in the field or/greenhouse
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Spikes are harvested from plants when microspores are at early to mid uninucleate stage
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Harvested spikes pre-treated under cold condition at 3-4 °C for 3-21 days
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Spikes sterilized with 70 % ethanol or mercuric chloride (0.1 %) for 2-3 minutes
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Anther culture
Anthers removed from the middle zone of spike
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Microspore culture
Microspores are isolated mechanically with the help of a sterile glass rod or blender
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In both cases, cultures are incubated at 28 °C for 3-6 weeks for embryo formation
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Suitable embryos are transferred to regeneration medium
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For regeneration, cultures are incubated at 26 °C under the light condition
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Regenerated plantlets are transferred to suitable medium for root and shoot development
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Ploidy levels are analyzed by flow cytometry/karyotypic methods
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If required colchicine treatment may be applied for chromosome doubling in haploid plants
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Treated plants are transferred to soil
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Plants are grown in field and/or greenhouse and DH seeds are harvested
2.2 Genetic Engineering to Develop Selected Abiotic Stress
Tolerence Homozygous Plants
There are different ways of plant transformation and analysis of transgenes. Gen-
erally, some steps are common for almost all cereal crops which are mentioned
herein. Protocol varies a little bit depending on the use of the crop and the explant.
This chapter highlights mainly the
Agrobacterium -
mediated transformation. The
transformation steps through androgenesis (anther or microspore-derived callus)
