Mestinšek Mubi Špela, Kunej Urban, Vogrinčič Valentin, Jakše Jernej, Murovec Jana
Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
Front Plant Sci. 2024 Feb 15;15:1336519. doi: 10.3389/fpls.2024.1336519. eCollection 2024.
Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity and . The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape ( L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). , , and gene expression analysis revealed dynamic patterns, with displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.
小孢子胚胎发生（ME）是植物育种和分子生物学研究中创建纯合系最有效的工具。它仍然主要基于通过温度、渗透压和/或营养胁迫对小孢子进行重编程。人们正在寻找能够提高小孢子胚胎发生效率，甚至能诱导顽固基因型形成单倍体胚胎的新化合物。其中，有丝分裂因子植物磺肽素α（PSK-α）因其广泛的活性谱而颇具前景。我们研究的目的是探究PSK-α对油菜（L.，DH4079）小孢子单倍体胚胎发生的影响，油菜是最重要的油料作物之一，也是研究控制胚胎形成分子机制的模式植物。我们测试了该肽的不同浓度（0、0.01、0.1和1μM），并在培养四周后评估其对小孢子活力和胚胎再生的影响。我们的结果表明，添加PSK-α与培养的小孢子活力之间呈正相关，并且对发育胚胎的数量也有积极影响。对添加或不添加PSK-α（共15个RNA文库）的三个时间点（第0、2和4天）的转录组进行分析，揭示了在细胞分裂、小孢子胚胎发生及随后再生中起关键作用的差异表达基因。主成分分析（PCA）按RNA采样时间对转录组进行分组，前两个主成分解释了56.8%的变异性。在添加PSK的第2天，添加PSK-α时45个基因（15个上调和30个下调）差异表达，到第4天其数量增加到了304个（30个上调和274个下调）。基因表达分析揭示了动态模式，在第2天和第4天的小孢子培养过程中显示出最高的增加和整体表达。尽管存在一些差异，但只有在添加PSK-α时显示出显著的差异表达。在16个与ME相关的分子标记中，分别有3个和15个在添加PSK的培养物中于第2天和第4天表现出显著的差异表达。胚胎特异性标记在培养4天后主要表达，在不含PSK的培养基中表达更高，而在第0天，许多孢子体特异性标记高度表达。
