Abstract
BackgroundSomatic embryogenesis in grapevines is a complex process that depends on many physiological and genetic factors. One of its main limitations is the process of precocious germination of the somatic embryos in differentiation medium. This process lowers plant conversion rates from the somatic embryos, and it is probably caused by a low endogenous abscisic acid (ABA) content.ResultsPrecocious germination of the somatic embryos was successfully avoided by culturing grapevine cv. Mencía embryogenic aggregates over a semipermeable membrane extended on top of the differentiation medium. The weekly analysis of the endogenous ABA and ABA-glucosyl ester (ABA-GE) contents in the aggregates showed their rapid accumulation. The expression profiles of 9-cis-epoxycarotenoid dioxygenase (VvNCED1), 8′-hydroxylase (VvHyd2), UDP-glucosyltransferase (VvUGT) and β-glucosidase (VvBG2) genes in grapevine revealed that the occurrence of a first accumulation peak of endogenous ABA in the second week of culture over the semipermeable membrane was mainly dependent on the expression of the VvNCED1 gene. A second increase in the endogenous ABA content was observed in the fourth week of culture. At this point in the culture, our results suggest that of those genes involved in ABA accumulation, one (VvNCED1) was repressed, while another (VvBG2) was activated. Similarly, of those genes related to a reduction in ABA levels, one (VvUGT) was repressed while another (VvHyd2) was activated. The relative expression level of the VvNCED1 gene in embryogenic aggregates cultured under the same conditions and treated with exogenous ABA revealed the significant downregulation of this gene.ConclusionsOur results demonstrated the involvement of ABA metabolism in the control of the maturation of grapevine somatic embryos cultured over a semipermeable membrane and two important control points for their endogenous ABA levels. Thus, subtle differences in the expression of the antagonistic genes that control ABA synthesis and degradation could be responsible for the final level of ABA during the maturation of grapevine somatic embryos in vitro. In addition, the treatment of somatic embryos with exogenous ABA suggested the feedback-based control of the expression of the VvNCED1 gene by ABA during the maturation of grapevine somatic embryos.
Highlights
Somatic embryogenesis in grapevines is a complex process that depends on many physiological and genetic factors
Our results demonstrated the involvement of abscisic acid (ABA) metabolism in the control of the maturation of grapevine somatic embryos cultured over a semipermeable membrane and two important control points for their endogenous ABA levels
The treatment of somatic embryos with exogenous ABA suggested the feedback-based control of the expression of the VvNCED1 gene by ABA during the maturation of grapevine somatic embryos
Summary
Somatic embryogenesis in grapevines is a complex process that depends on many physiological and genetic factors. One of its main limitations is the process of precocious germination of the somatic embryos in differentiation medium. This process lowers plant conversion rates from the somatic embryos, and it is probably caused by a low endogenous abscisic acid (ABA) content. It has been shown that the application of exogenous ABA enhanced the tolerance of alfalfa somatic embryos to desiccation, improving the quality of the plants converted from these embryos [2, 3]. During somatic embryogenesis in cotton [5], the transcriptional activation of stress responses was observed, with enhanced expression levels of stress-related genes, including several that are involved in ABA biosynthesis and signaling. The maturation of tulip somatic embryos requires high ABA levels [6]
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