Improving the efficiency of wheat microspore culture methodology: evaluation of pretreatments, gradients, and epigenetic chemicals
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The objective of the study was to develop efficient widely adaptable microspore culture methodology for spring and winter wheat genotypes. Current wheat microspore culture methods are still inadequate for routine application, especially for winter genotypes. The present study involved increasing the frequency of microspore embryogenesis and improving the conversion of microspore-derived embryos to fertile green plants. A systematic evaluation was conducted testing numerous factors at all phases of the microspore embryogenesis process. Results from pretreatments, maltose gradients, and histone deacetylase inhibitors are presented. A 28 days cold pretreatment of spikes was optimal for winter wheat genotypes whereas a 21 days cold pretreatment was best for spring genotypes. Microspore suspensions containing a high frequency of embryogenic microspores were collected by resuspension in a 23% maltose gradient with subsequent pelleting with a mannitol containing solution. The application of trichostatin A (TSA) enhanced embryogenesis and/or green plant regeneration. Other epigenetic chemicals like scriptaid, BIX-01294, and sodium butyrate were also evaluated. Scriptaid enhanced embryogenesis but regenerated plantlets showed abnormal shoot and root development. The improved methodology was validated using a range of spring and winter wheat genotypes.
Enhanced microspore embryogenesis and green plant regeneration in spring and winter wheat was observed with 21 or 28 days cold pretreatments, a 23% maltose gradient, and the application of trichostatin A (TSA).
KeywordsDoubled haploidy Histone deacetylase inhibitors Microspore culture Microspore embryogenesis Wheat
Ploidy analyses were conducted at NRC as well as the Global Institute for Food Security, Saskatoon. We thank Rick D. Goertzen for his invaluable guidance and assistance as well as Dr. Timothy Sharbel for helpful advice on our analyses. The authors also thank NRC Canadian Wheat Improvement Program for funding.
HMW, JLE, KLN, JMB, and TDO contributed by performing experiments and compiling data. HMW, JLE, and AMRF participated in drafting the manuscript. AMRF provided the concept for the study, data interpretation, and critical revisions. All authors reviewed the final manuscript.
Compliance with ethical stanadards
Conflict of interest
The authors declare that they have no conflict of interest.
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