Abstract
BackgroundMicrospore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.ResultsWe performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants.ConclusionsProplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.
Highlights
Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars
Based on the analysis of expression profiles of genes involved in transcription and translation occurring in plastids and genes related to chloroplast differentiation, together with determining the plastome copy number and analysis of plastid morphology and ultrastructure, we indicated the mechanisms that underlie the alterations in chloroplast development leading to the formation of albino plants
Rapture of the exine wall and further cell divisions led to the formation of structures that could be observed on the 21st day of culture (21dC) as globular
Summary
Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. The regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Isolated microspore culture via microspore embryogenesis is the most effective of haploid/doubled haploid (DH) production methods that are used in plant breeding programmes to shorten the time required for development of new varieties [1,2,3]. Stress treatment or pre-treatment reprograms the gametophytic pathway of microspore development into the sporophytic one (androgenesis), which results in induction of embryo formation and regeneration of androgenic plants [7, 8]. Albinism occurring in androgenic cultures is a phenomenon that is distinctive for cereals and grasses, and occurs in a wide frequency range in most agronomically important monocot species, including wheat [9, 10], rice [11], barley [12, 13] and triticale [14]
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