Abstract
Plant in vitro regeneration systems, such as somatic embryogenesis, are essential in breeding; they permit propagation of elite genotypes, production of doubled-haploids, and regeneration of whole plants from gene editing or transformation events. 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, a 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 a master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1, and BZR1 were up-regulated and the BAS1 catabolic gene was repressed, indicating activation of the 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 up the way for new strategies using GSK-3β inhibitors that could be extended to other species.
Highlights
Agriculture in the 21st century faces significant pressure societal biosafety demands
The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening up the way for new strategies using glycogen synthase kinase 3β (GSK-3β) inhibitors that could be extended to other species
When cells from other tissues are reprogrammed to embryogenesis, somatic embryogenesis has proved to be very useful for propagation of species with long reproductive cycles or low seed set in a large variety of crop and forest species (Loyola-Vargas and Ochoa-Alejo, 2016; DíazSala, 2018; Pais, 2019), due to its great potential for large-scale clonal propagation and cryopreservation of elite genotypes, as well as for production of genetically modified and, more recently, gene-edited plants with improved traits
Summary
Agriculture in the 21st century faces significant pressure societal biosafety demands. Responses of crop plants to environmental stress, as well as to develop efficient and rapid methods to obtain new crop varieties, with increasing yield and better adapted to new environmental conditions. In this context, efficient technologies to exploit plant cell in vitro reprogramming potential for production of doubled-haploids (DHs), for regeneration and propagation of selected plants, and for conducting gene editing and transformation techniques (which require more efficient plant regeneration methods), are needed in plant breeding and biotechnology, as well as in applied and basic plant research. When cells from other tissues are reprogrammed to embryogenesis, somatic embryogenesis has proved to be very useful for propagation of species with long reproductive cycles or low seed set in a large variety of crop and forest species (Loyola-Vargas and Ochoa-Alejo, 2016; DíazSala, 2018; Pais, 2019), due to its great potential for large-scale clonal propagation and cryopreservation of elite genotypes, as well as for production of genetically modified and, more recently, gene-edited plants with improved traits
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