Analysing the loss of embryogenic competence in long-term cell lines of Solanum betaceum Cav.: involvement of miR827, phosphate and sugar.

Somatic embryogenesis (SE) is a pivotal model for understanding cellular differentiation and totipotency while also offering practical applications for micropropagation and breeding of many crops. Therefore, there is a great interest in understanding the gene regulatory networks underlying cell reprogramming during this process. Accordingly, SE in Solanum betaceum Cav. (tamarillo) is a reliable system for studying embryogenic competence acquisition, expression, and maintenance. Since sugar signalling mediates phosphate starvation responses and the phosphate-starvation-induced miR827 was found upregulated in S. betaceum calli with no embryogenic potential, it was hypothesised that the long exposure to high sucrose concentrations during subcultures might be responsible for the loss of embryogenic competence, through phosphate-starvation activated mechanisms, such as miRNA upregulation. Indeed, the results obtained showed a higher miR827 expression and lower phosphate levels with decreased embryogenic potential. These results were further confirmed by fluorescent in situ hybridization and virus-based miRNA silencing techniques. However, PHOSPHATE TRANSPORTER 5, a known miR827-target gene involved in phosphate transport, was more expressed in long-term maintained callus (LTC). Although LTC and non-embryogenic callus showed lower phosphate levels when compared to embryogenic callus, changing the phosphate availability on culture media did not result in higher embryogenic competence acquisition or expression. The results obtained so far reinforce the hypothesis that phosphate metabolism is a determinant of embryogenic competence commitment and expression during SE. Overall, this work is a step further in understanding the behaviour of long-term conserved cultures, a common bottleneck in the development of fully optimized SE-based protocols for several woody plant species.