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Methods Mol Biol ; 2287: 227-244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270033


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.

Pão/análise , Flores/crescimento & desenvolvimento , Flores/genética , Melhoramento Vegetal/métodos , Técnicas de Cultura de Tecidos/métodos , Triticum/crescimento & desenvolvimento , Triticum/genética , Haploidia , Pólen/genética , Pólen/crescimento & desenvolvimento
Plants (Basel) ; 9(11)2020 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-33114625


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.