Sato Yuka, Minamikawa Mai F, Pratama Berbudi Bintang, Koyama Shohei, Kojima Mikiko, Takebayashi Yumiko, Sakakibara Hitoshi, Igawa Tomoko
Plant Cell Technology Laboratory, Graduate School of Horticulture, Chiba University, Matsudo, Japan.
Institute for Advanced Academic Research (IAAR), Chiba University, Chiba, Japan.
Front Plant Sci. 2024 Apr 3;15:1308417. doi: 10.3389/fpls.2024.1308417. eCollection 2024.
The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of () and () on the differentiation of tobacco transgenic cells was examined. We found that the fusion to , when co-expressed with the - fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.
据报道，发育调节因子（DR）基因的异位过表达可促进细胞培养过程中的细胞分化，从而改善难转化植物物种的转化。换句话说，在利用DR基因进行植物再生的情况下，组织和细胞培养过程中仍需要施加外源植物生长调节剂（PGR）。在此，研究了（）和（）对烟草转基因细胞分化的影响。我们发现，与-融合基因共表达时，与的融合显著影响无PGR培养条件下自主分化的诱导，在其他一些植物物种中也有类似效果。此外，为了了解细胞分化向再生的内源性背景，我们使用转基因细胞正在自主分化的烟草叶片外植体进行了植物激素和RNA测序分析。随着细胞分化向器官发生发展，活性生长素、细胞分裂素、脱落酸和非活性赤霉素的水平升高。除了与多糖和硝酸盐代谢相关的基因外，与植物激素和器官发生相关的基因本体术语也被鉴定为差异表达基因。对四个选定的作为差异表达基因的基因进行的qRT-PCR验证了RNA测序数据。这种分化诱导系统以及所报道的植物激素和转录谱为开发各种植物物种的无PGR组织培养奠定了基础，有助于未来的生物技术育种。
