Identification of LEC1, L1L and Polycomb Repressive Complex 2 genes and their expression during the induction phase of Medicago truncatula Gaertn. somatic embryogenesis

Abstract

Although somatic embryogenesis (SE), a multi-step process starting from somatic tissues and ending with somatic embryos, has been applied to numerous plants including Medicago sp., the molecular basis of development reprograming in somatic cells toward the embryogenic pathway is still incompletely known. Though recent analysis of the proteome and transcriptome has led to the identification and characterization of new genes involved in SE, lot of these genes are up-regulated only in the late developmental stages. Consequently, this work was aimed at finding out if and when the genetic program changed during the SE induction phase in both the highly embryogenic line M9-10a of Medicago truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. Based on multipoint (day 0, 2, 7, 14 and 21) gene expression qPCR analysis of two embryogenesis marker genes LEC1 and L1L and selected genes encoding proteins of PRC2 complex (CLF, SWN, FIE, MSI1, VRN2) it was possible to distinguish two periods during the induction phase. The first week was related to dedifferentiation with no visible changes in explant morphology and lack of transcripts of LEC1 and L1L; the expression of PRC2 members, however, increased in the embryogenic line. The next two weeks were regarded as the expression phase involving the beginning of a rapid callus growth and the appearance of products for LEC genes, which was observed in the embryogenic line only. However, the callus formation was observed in the non-embryogenic line as well, but the LEC1 and L1L genes were not expressed and the transcription of PRC2 genes was at stable level. It was only the SWN expression that decreased at the beginning of induction and did not change in the subsequent days in both lines. Our results indicate that LEC1 and L1L, known as marker genes of the late developmental stages, may be associated with the acquisition of embryogenic competency by somatic cells (prime events in the SE induction). The PRC2 complex genes are also expressed during embryogenic callus tissue formation on Medicago tuncatula leaf explants.