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. 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. We 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. Proplastids 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.

中文翻译：

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质体生物发生的变化导致大麦小孢子培养物中白化再生体的形成

小孢子胚胎发生可能是获得双单倍体 (DH) 的最有效方法，双单倍体可用于育种计划以加速新品种的生产。然而，白化植物的再生严重限制了利用雄激素生成谷物中的DH。尽管做出了许多努力，但导致白化再生体发育的确切机制尚未阐明。本研究的目的是揭示叶绿体分化中基因型依赖性分子差异，这些差异导致大麦小孢子培养物中绿色和白化再生体的形成。我们对两个大麦品种“Jersey”和“Mercada”雄激素发生连续阶段的质体分化进行了详细分析，这两个品种产生绿色再生体的能力不同。我们证明了 cv 质体中缺乏从 NEP 依赖性转录到 PEP 依赖性转录的转变。“Mercada”在小孢子培养物中主要产生白化再生体。NEP 到 PEP 转变的失败与 Sig2 基因缺乏活性有关，该基因编码质体 rRNA 基因转录所必需的 sigma 因子。非常低水平的 16S 和 23S rRNA 转录本以及受损的质体翻译机制导致再生胚胎和白化再生体的光形态发生受到抑制。此外，分化的“Mercada”胚胎中存在的质体含有少量质体拷贝，其复制并不总是完成。与“Mercada”相反，cv。产生 90% 绿色再生体的“Jersey”显示出 PEP 聚合酶的高活性，Sig2、质体 rRNA 和 tRNAGlu 的表达高度增加，这表明 NEP 受到抑制。在“泽西”再生体中还观察到编码诱导光形态发生所需转录因子的 GLK 基因表达增加。白化基因型的小孢子衍生胚胎中存在的原生质体没有通过其发育的早期检查点，而这些检查点是诱导叶绿体进一步光依赖性分化所需的。胚胎分化过程中质体编码的RNA聚合酶激活失败与大麦小孢子培养物中基因型依赖性的无法再生绿色植物有关。更好地了解白化再生体形成的分子机制可能有助于克服谷物雄激素发生中的白化病问题。