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Improving the efficiency of isolated microspore culture in six-row spring barley: I-optimization of key physical factors

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An improved isolated microspore culture protocol alleviating the recalcitrance typically observed in six-row spring barley was developed by optimizing four key physical factors to increase embryogenesis and reduce albinism.

Abstract

Doubled haploid (DH) plants are completely homozygous individuals that can be generated in just a few months via androgenesis in vitro. DHs are useful tools in genetic research and in plant breeding. Isolated microspore culture (IMC) is the most efficient way to produce DHs, but a strong genotype dependency imposes limitations to its wide application. Six-row, spring barley genotypes are considered as particularly recalcitrant due to a low frequency of embryogenesis and a high rate of albinism. Seeking to develop an efficient IMC protocol for this type of barley, we explored four important factors: (1) the harvest stage of immature spikes, (2) the type of pretreatment applied, (3) the osmotic potential in the induction medium, and (4) the plating density of microspores. This work was first performed using four barley genotypes: two typical six-row spring cultivars (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) cultivar. First, by optimizing the harvest stage for each genotype we obtained a twofold to fourfold increase in the yield of embryogenic microspores. Second, two pretreatments (0.3 M mannitol for 2 days, or a combination of cold and heat over 15 days) both performed significantly better than the commonly used cold pretreatment (28 days at 4 °C). Third, an induction medium-containing mannitol (32 g/l) doubled green plant regeneration. Fourth, a plating density of 106 microspores/ml yielded the highest number of green regenerated plants. Our most important findings were then confirmed using sets of F1s from a six-row, spring-type breeding program.

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Acknowledgments

The financial supports of the Natural Sciences and Engineering Research Council of Canada (NSERC) and of La Coop Fédérée are acknowledged. The authors wish to thank Isabelle Clermont, Martine Jean and Suzanne Marchand for their assistance and constructive comments, as well as B. Legge (AAFC Brandon) for providing seeds of some of the cultivars used here.

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  1. Département de phytologie, Faculté des sciences de l’agriculture et de l’alimentation, Pavillon Paul-Comtois, Université Laval, 2425, rue de l’Agriculture, Local 3236, Quebec, QC, G1V 0A6, Canada

    Patricio Esteves & François Belzile

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  1. Patricio Esteves
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  2. François Belzile
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Correspondence to François Belzile.

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Communicated by X. S. Zhang.

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Esteves, P., Belzile, F. Improving the efficiency of isolated microspore culture in six-row spring barley: I-optimization of key physical factors. Plant Cell Rep 33, 993–1001 (2014). https://doi.org/10.1007/s00299-014-1583-x

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