Disturbance of ethylene biosynthesis and perception during somatic embryogenesis in Medicago sativa L. reduces embryos’ ability to regenerate

Effects of non-specific ethylene biosynthesis inhibitors: salicylic acid (SA) and aminoethoxyvinylglycine (AVG), and of specific inhibitors of ethylene binding to receptors: 1-methylcyclopropene (1-MCP) and 2,5-norbornadiene (NBD) applied during proliferation and differentiation phases of indirect somatic embryogenesis (SE) of Medicago sativa L. cv. Rangelander on embryogenic suspension growth, embryo production, development, and ability to germinate and convert were studied. Application of SA and AVG alone or together at concentrations from 1 to 500 μM in B5g liquid medium during the proliferation phase had an inhibitory effect on ethylene production and embryogenic suspension growth. Additionally, it caused a drastic reduction in production of embryos and their development on BOi2Y solid differentiation medium. The inhibitory effect of SA was more visible than that of AVG. In addition, disturbance of ethylene biosynthesis during the proliferation phase of SE resulted in diminished lateral germination and conversion of cotyledonary embryos on MS solid medium. Moreover, blocking of ethylene receptors by 1-MCP during the proliferation phase also inhibited ethylene production and embryogenic suspension growth and reduced embryo production during differentiation. MCP almost completely inhibited development of cotyledonary embryos. At the same time, development of more embryos was arrested at the globular stage, and the number of abnormal embryos almost doubled. Similarly, addition of 1-MCP or NBD to the ambient atmosphere during the differentiation phase evidently arrested the development of embryos and, consequently, their ability to germinate and convert on MS regeneration medium. All the results presented above demonstrated that not only ethylene biosynthesis, but also ethylene action is involved in the control of individual phases of SE in Medicago sativa L. cv. Rangelander. And what is more, disturbance of these processes during distinct phases of SE adversely affects vigor of the somatic embryos obtained.