Small molecule inhibitors of mammalian GSK-3β promote in vitro plant cell reprogramming and somatic embryogenesis in crop and forest species

Abstract

Plant in vitro regeneration systems, like somatic embryogenesis, are essential in breeding; they permit to propagate elite genotypes, to produce doubled-haploids, and to convert gene editing or transformation events into plants. However, in many crop and forest species somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3β (GSK-3β), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis- genes FUS3, LEC2 and AGL15. Plant GSK-3 kinase BIN2 is master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1 and BZR1 were upregulated and BAS1 catabolic gene was repressed, indicating activation of BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening the way for new strategies using GSK-3β inhibitors that could be extended to other species.