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.

Microspore embryogenesis induction by mannitol and TSA results in a complex regulation of epigenetic dynamics and gene expression in bread wheat.

Reprogramming of microspores development towards embryogenesis mediated by stress treatment constitutes the basis of doubled haploid production. Recently, compounds that alter histone post-translational modifications (PTMs) have been reported to enhance microspore embryogenesis (ME), by altering histones acetylation or methylation. However, epigenetic mechanisms underlying ME induction efficiency are poorly understood. In this study, the epigenetic dynamics and the expression of genes associated with histone PTMs and ME induction were studied in two bread wheat cultivars with different ME response. Microspores isolated at 0, 3 and 5 days, treated with 0.7M mannitol (MAN) and 0.7M mannitol plus 0.4µM trichostatin A (TSA), which induced ME more efficiently, were analyzed. An additional control of gametophytic development was included. Microspores epigenetic state at the onset of ME induction was distinctive between cultivars by the ratio of H3 variants and their acetylated forms, the localization and percentage of labeled microspores with H3K9ac, H4K5ac, H4K16ac, H3K9me2 and H3K27me3, and the expression of genes related to pollen development. These results indicated that microspores of the high responding cultivar could be at a less advanced stage in pollen development. MAN and TSA resulted in a hyperacetylation of H3.2, with a greater effect of TSA. Histone PTMs were differentially affected by both treatments, with acetylation being most concerned. The effect of TSA was observed in the H4K5ac localization pattern at 3dT in the mid-low responding cultivar. Three gene networks linked to ME response were identified. TaHDT1, TaHAG2, TaYAO, TaNFD6-A, TabZIPF1 and TaAGO802-B, associated with pollen development, were down-regulated. TaHDA15, TaHAG3, TaHAM, TaYUC11D, Ta-2B-LBD16 TaMS1 and TaDRM3 constituted a network implicated in morphological changes by auxin signaling and cell wall modification up-regulated at 3dT. The last network included TaHDA18, TaHAC1, TaHAC4, TaABI5, TaATG18fD, TaSDG1a-7A and was related to ABA and ethylene hormone signaling pathways, DNA methylation and autophagy processes, reaching the highest expression at 5dT. The results indicated that TSA mainly modified the regulation of genes related to pollen and auxin signaling. This study represents a breakthrough in identifying the epigenetic dynamics and the molecular mechanisms governing ME induction efficiency, with relevance to recalcitrant wheat genotypes and other crops.

Bread Wheat Doubled Haploid Production by Anther Culture.

The use of doubled haploid (DH) plants in plant breeding programmes is the fastest route to release new varieties (4-6 years), allowing for a rapid response to end-user needs. Microspore embryogenesis is one of the most efficient methods for DH plant production in bread wheat. In this process, microspores triggered by a stress treatment or by application of bioactive compounds are reprogrammed to follow an embryogenic pathway that leads to the production of haploid or DH plants. In this chapter, we describe a protocol for anther culture of bread wheat. This protocol is based on an osmotic and starvation treatment of the anthers followed by the application of a microtubule disrupting agent. Anthers are cultured in an ovary pre-conditioned medium with mature ovaries from cv. Caramba. This protocol has been applied to a wide range of genotypes and F1s from bread and spelt wheat.

Trichostatin A Affects Developmental Reprogramming of Bread Wheat Microspores towards an Embryogenic Route.

Microspores can be developmentally reprogrammed by the application of different stress treatments to initiate an embryogenic pathway leading to the production of doubled haploid (DH) plants. Epigenetic modifications are involved in cell reprogramming and totipotency in response to stress. To increase microspore embryogenesis (ME) efficiency in bread wheat, the effect of the histone deacetylase inhibitor trichostatin A (TSA) has been examined in two cultivars of wheat with different microspore embryogenesis response. Diverse strategies were assayed using 0-0.4 µM TSA as a single induction treatment and after or simultaneously with cold or mannitol stresses. The highest efficiency was achieved when 0.4 µM TSA was applied to anthers for 5 days simultaneously with a 0.7 M mannitol treatment, producing a four times greater number of green DH plants than mannitol. Ultrastructural studies by transmission electron microscopy indicated that mannitol with TSA and mannitol treatments induced similar morphological changes in early stages of microspore reprogramming, although TSA increased the number of microspores with 'star-like' morphology and symmetric divisions. The effect of TSA on the transcript level of four ME marker genes indicated that the early signaling pathways in ME, involving the TaTDP1 and TAA1b genes, may be mediated by changes in acetylation patterns of histones and/or other proteins.