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

Abstract

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 lM in B 5 g 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.

The Role of Endogenous Ethylene in Carbohydrate Metabolism of Medicago sativa L. Somatic Embryos in Relation to Their Regenerative Ability

“…Rangelander due to the presence of two nonspecific ethylene biosynthesis inhibitors SA and AVG suggests that the process requires ethylene biosynthesis. As demonstrated earlier, application of those inhibitors during the proliferation phase of somatic embryogenesis not only brings about a decline in ethylene production and embryogenic suspension proliferation, it also inhibits production of embryos and their development, and, moreover, results in a loss of ability of the cotyledonary embryos kept on the differentiation and maturation media to germinate and convert when kept on the regeneration medium (Kępczyńska and Zielińska 2011). Thus, the results obtained previously and those reported in this article clearly indicate that endogenous ethylene, dependent on ACO and ACS activities, is required for germination and conversion of the somatic embryos of M. sativa L. cv.…”

“…Moreover, residual amounts of ABA present in somatic embryos after they have been treated with 20 lM ABA during the maturation phase can affect the activity of the enzyme because retardation of starch hydrolysis in the presence of ABA in the germination medium was correlated with ABA's inhibitory effect on a-amylase activity (Kępczyńska and Zielińska 2006). There is a lack information about the role of ethylene in embryo germination and conversion, although the part played by ethylene in somatic embryogenesis (SE) of M. sativa L. has already been widely documented in different phases and regeneration protocols (Meijer and Brown 1988;Meijer 1989;Kępczyński and others 1992;Huang and others 2001;Kępczyński and others 2009a, b;Kępczyńska and Zielińska 2011). It has been unequivocally demonstrated that endogenous ethylene is necessary during differentiation (Meijer and Brown 1988;Meijer 1989;Kępczyński and others 1992) and also in proliferation of alfalfa somatic embryos (Kępczyńska and others 2009a, b).…”

“…It has been unequivocally demonstrated that endogenous ethylene is necessary during differentiation (Meijer and Brown 1988;Meijer 1989;Kępczyński and others 1992) and also in proliferation of alfalfa somatic embryos (Kępczyńska and others 2009a, b). Moreover, disturbance of ethylene biosynthesis and action during these phases of SE adversely affects the vigor of the somatic embryos obtained (Kępczyńska and Zielińska 2011).…”

AGAMOUS-Like15 Promotes Somatic Embryogenesis in Arabidopsis and Soybean in Part by the Control of Ethylene Biosynthesis and Response

“…An ACO was previously reported as up-regulated during SE, although transcript accumulation was higher on the abaxial side than the adaxial side that forms embryos (Thibaud-Nissen et al, 2003). In other systems, ethylene has been shown to have a role in cotyledon development of B. napus microspore embryos (Hays et al, 2000) and in the development and conversion of alfalfa somatic embryos (Kepczynska et al, 2009;Kepczynska and Zielinska, 2011). In some situations, a suppression of embryogenesis with ethylene is observed (for review, see Jiménez and Thomas, 2006), and a number of systems show enhanced SE by the addition of AgNO 3 to medium (for review, see Kumar et al, 2009).…”

Plant Somatic Embryogenesis: Some Useful Considerations

“…Giridhar et al (2004) reported that by adding silver nitrate at different stages of the plant regeneration through somatic embryogenesis of Coffea arabica L. and Coffea canephora was good for the production of somatic embryos. Recent research conducted by Kępczyńska & Zielińska (2011) focused on the effects ethylene inhibitors binding to receptors at different phases of somatic embryogenesis in Medicago sativa L.; the findings showed that ethylene biosynthesis and its action influenced individual phases of somatic embryogenesis. Moreover, alterations of these processes affected adversely the activity of the production of somatic embryos.…”