Stress treatment of microspore embryogenesis leads to the reprogramming of male gametophyte development pathways, which generates haploid green plants. However, low green plant production efficiency is a major hindrance in the adoption of double haploid (DH) production platform for wheat breeding. The aim of the current study was to induce the expression of microspore embryogenesis associated genes by over expressing the RKD transcription factor using Transcription activator-like effector (TALE) proteins. The TALE protein can modulate the gene expression by recognizing and binding host plant promoter sequences. Taking into consideration, we have cloned and analysed RKD promoter from wheat and triticale and used for the custom synthesis of TaRKD-TALE protein. The isolated and purified protein was conjugated with R9 (Cys (Npys)-(D-Arg)9) cargo peptides and successfully transduced into wheat microspores. The expression of early-culture abundant (ECA1), RWP-RK domain-containing proteins (RKD1) and Tapetum determinant 1 (TPD1) marker genes in wheat linked to microspore embryogenesis was studied. The expression of all three (ECA1, TPD1 and RKD1) embryogenesis related marker genes in the TALE transduced microspore was significantly higher than the control. Microspore culture transduced by R9-TaRKD-TALE protein yielded a significantly higher number of embryo-like structures (ELS) and the total green plants in wheat cultivars AC Fielder. Conversely, the microspore cell death has not been significantly affected by the transduction of R9-TaRKD-TALE, while showing a higher recovery at 120 h of microspore culture. This work will be helpful to exploit the TALE protein to improve our understanding of DH production and other pathways related genes.

小孢子胚胎发生的应激处理导致雄配子体发育途径的重编程，从而产生单倍体绿色植物。但是，绿色植物生产效率低是采用双单倍体（DH）小麦育种平台的主要障碍。本研究的目的是通过使用转录激活因子样效应子（TALE）蛋白过表达RKD转录因子来诱导小孢子胚胎发生相关基因的表达。TALE蛋白可以通过识别并结合宿主植物启动子序列来调节基因表达。考虑到这一点，我们克隆并分析了RKD来自小麦和黑小麦的启动子，用于定制合成TaRKD-TALE蛋白。分离和纯化的蛋白质与R9（Cys（Npys）-（D-Arg）9）货物肽缀合，并成功地转导到小麦小孢子中。早期培养丰富（表达ECA1），RWP-RK含有结构域的蛋白（RKD 1）和绒毡层行列式1（TPD1在小麦链接到孢子胚胎）标记基因进行了研究。所有这三种（ECA1，TPD1和RKD1TALE转导的小孢子中与胚胎发生有关的标记基因显着高于对照。通过R9-TaRKD-TALE蛋白转导的小孢子培养物在AC Fielder小麦品种中产生的胚样结构（ELS）和绿色植物总数明显增加。相反，R9-TaRKD-TALE的转导并没有显着影响小孢子细胞的死亡，尽管在120h的小孢子培养中显示出更高的恢复率。这项工作将有助于开发TALE蛋白，以增进我们对DH产生和其他途径相关基因的了解。