Transcriptional profiling of the AFL subfamily of B3-type transcription factors during the in vitro induction of somatic embryogenesis in the model legume Medicago truncatula

Abstract

In many legume species, somatic embryogenesis is a limiting step of in vitro regeneration, with profound implications to genetic engineering and plant breeding. In Arabidopsis thaliana, the small AFL subfamily of B3-type transcriptional factors is composed by LEC2 (LEAFY COTYLEDON 2), FUSCA (FUSCA3), and ABI3 (ABSCISIC ACID INSENSITIVE 3), which are directly involved in both zygotic and somatic embryogenesis. This study aimed to identify and analyze the expression profile of AFL subfamily genes during in vitro induction of the somatic embryogenesis in the model legume Medicago using near-isogenic genotypes contrasting for their capability of regenerating in vitro. Three AFL genes were identified in the Medicago truncatula genome: MtLEC2, MtFUSCA3 and MtABI3. RT-qPCR was used to compare the expression of these genes during the induction of somatic embryogenesis in the embryogenic genotype M9-10a and the non-embryogenic genotype M9. The AFL genes identified were highly expressed 10 days after introducing leaflet explants in vitro in the embryogenic genotype (M9-10a), whereas absence or low expression was observed in explants of the non-embryogenic genotype (M9). During zygotic embryogenesis, the Medicago Gene Atlas revealed that expression of MtFUSCA3 and MtABI3 occurred specifically during seed development, starting at 10 days after pollination. This study allowed for the identification and transcriptional characterization of MtLEC2, MtFUSCA3, and MtABI3. The expression pattern of these genes in the M9-10a genotype suggests they are involved in the Medicago truncatula somatic embryogenesis. The transcriptional profile of AFL transcription factors highlights the evolutionary conservation of this developmental pathway between the Arabidopsis and Medicago lineages and opens avenues to improve the efficiency of in vitro embryogenesis in legume crops. Expression of MtLEC2, MtFUSCA3, and MtABI3 genes was induced during somatic embryogenesis and these genes to be used as in vitro regeneration biomarkers as well as to improving somatic embryogenic rates in legumes and other crops recalcitrant to in vitro regeneration.