New Epigenetic Modifier Inhibitors Enhance Microspore Embryogenesis in Bread Wheat.

The use of doubled haploid (DH) technology enables the development of new varieties of plants in less time than traditional breeding methods. In microspore embryogenesis (ME), stress treatment triggers microspores towards an embryogenic pathway, resulting in the production of DH plants. Epigenetic modifiers have been successfully used to increase ME efficiency in a number of crops. In wheat, only the histone deacetylase inhibitor trichostatin A (TSA) has been shown to be effective. In this study, inhibitors of epigenetic modifiers acting on histone methylation (chaetocin and CARM1 inhibitor) and histone phosphorylation (aurora kinase inhibitor II (AUKI-II) and hesperadin) were screened to determine their potential in ME induction in high- and mid-low-responding cultivars. The use of chaetocin and AUKI-II resulted in a higher percentage of embryogenic structures than controls in both cultivars, but only AUKI-II was superior to TSA. In order to evaluate the potential of AUKI-II in terms of increasing the number of green DH plants, short and long application strategies were tested during the mannitol stress treatment. The application of 0.8 µM AUKI-II during a long stress treatment resulted in a higher percentage of chromosome doubling compared to control DMSO in both cultivars. This concentration produced 33% more green DH plants than the control in the mid-low-responding cultivar, but did not affect the final ME efficiency in a high-responding cultivar. This study has identified new epigenetic modifiers whose use could be promising for increasing the efficiency of other systems that require cellular reprogramming.

Barley Isolated Microspore Culture.

The production of doubled haploids (DHs) has proved to be a highly valuable tool to obtain new cultivars. Among the cereals, barley (Hordeum vulgare L.) is the most successful species in large-scale haploid production. Techniques employed for this purpose are based on either the gynogenetic or the androgenetic pathway. Interspecific cross with Hordeum bulbosum L., haploid gene inducer (the hap gene), ovary culture, anther culture (AC), and isolated microspore culture (IMC) are the most used methods. Among all of them, IMC is regarded as a particularly effective system owing to the great increase in green plant numbers per spike and also the higher induction of chromosome doubling when compared with other methods. Thus, IMC provides the best way to mass scale production of new varieties.

Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci.

KEY MESSAGE: The genetic substitution of transformation amenability alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars. Barley (Hordeum vulgare) cv. 'Golden Promise' is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from 'Haruna Nijo' × 'Golden Promise,' as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross 'Golden Promise' × 'Full Pint.' Based on SNP genotype, we selected lines having 'Golden Promise' alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to 'Golden Promise.' The results validate that the genetic substitution of TFA alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars.