Sinergistički uticaj giberelina i svetlosti na indukciju somatske embriogeneze iz lateralnih korenova spanaća (Spinacia oleracea L.) in vitro
Synergistic effect of gibberellin and light on the induction of somatic embryogenesis from lateral roots of spinach (Spinacia oleracea l.) in vitro
Abstract:
In this work, the impact of gibberellins (GA) and light, on somatic embryo initiation in vitro from root apices of spinach seedlings (cv. Matador) was studied. The explants were cultivated on induction medium supplemented with 20μM α-naphthaleneacetic acid (NAA) and 5 μM gibberellic acid (GA3). A high variability in embryogenic capacity of spinach, especially in the cultivar "Matador", impedes studying the effects of other factors on the induction of somatic embryogenesis. Analysis of embryogenic potential of randomly chosen seedlings, obtained from seeds of cv. “Matador” purchased from nine European seed companies, showed significant variation in the regeneration frequency (0-100%) among populations and individuals within the population, as well as in the mean SE number per explant (0-14.4 and 0-36 per population and per individual seedlings' explants, respectively). The results strongly evidenced the importance of the sampling of plant material, because it may significantly affect the interpretation of the data. With the aim to overcome such high variability of plant material due to genetic and other impacts, a sampling method was developed and successfully applied in the present work. The sampling method implies the exposure of equal number of root explants taken from each seedling to all treatments. In order to investigate the mechanism of GA3 action on somatic embryogenesis induction, the influence of paclobutrazole (PAC), an inhibitor of GA biosynthesis, and trichostatin A (TSA), a histone deacetylase inhibitor, on this process was studied. Unexpectedly, PAC acted synergistically with GA3 and NAA in promoting somatic embryogenesis. In combination with NAA+GA3, 1–10 μM PAC increased embryogenic capacity of the explants, with maximum effect at 2.5 μM PAC. In combination with NAA, which alone is not sufficient to induce somatic embryogenesis, higher concentrations of PAC were required for satisfactory embryogenic response—10 μM PAC for the explants taken from randomly chosen seedlings and 5 μM PAC for the explants derived from the seedlings genetically predisposed for somatic embryogenesis. TSA (0.1–5 μM applied over 1, 7 or 14 days) could not substitute for either NAA or GA3 in somatic embryogenesis induction, although it significantly increased embryogenic capacity of the explants in combination with 10 μM NAA+5 μM GA3 or 20 μM NAA+5 μM GA3. The positive impact of TSA on somatic embryogenesis induction mostly depended on the treatment duration. Longer TSA treatments were required with lower level of NAA and vice versa. In addition, a significant positive impact of dimethyl sulfoxide, used to dissolve TSA, on embryogenic capacity of the explants was observed. The quality of the light also significantly affected the embryogenic potential of the explants. Explants cultured under blue LED light exhibited the highest embryogenic potential. Explants cultured under white LED and fluorescent light had significantly lower embryogenic potential, while somatic embryo regeneration was completely inhibited in explants cultured under red LED light or in the dark. To elucidate the mechanism of action of GA and light on somatic embryogenesis induction, a detailed analysis of GA metabolism in the explants during induction of somatic embryogenesis was performed by analyzing the expression of genes encoding key enzymes in GA biosynthesis (SoGA20-ox1 and SoGA3-ox1) and inactivation (SoGA2-ox1, SoGA2-ox2 and SoGA2-ox3), as well as the endogenous GA content. The most dramatic differences between embryogenic explants (cultured on medium with NAA+GA3), and non-embryogenic explants (cultured on medium without plant growth regulators or on medium with GA3 or NAA) were detected in the expression of SoGA20-ox1 and SoGA2-ox2 genes. In the non-embryogenic explants, the SoGA20-ox1 expression was lower during 1-14 days compared to the control (roots of intact seedlings), while in the embryogenic explants the expression of this gene was significantly lower than the control during the entire period of SE induction. In contrast, the expression of SoGA2-ox2 was significantly higher in the embryogenic than in non-embryogenic explants. The expression profile of the genes encoding key enzymes of GA metabolism was similar in the explants cultured on medium supplemented with NAA+PAC, NAA+GA3 and NAA+GA3+PAC. Similarly, no significant differences in the expression of these genes were observed in the explants cultured under fluorescent, blue or white LED light. This indicates that PAC and light quality enhance somatic embryogenesis in spinach by some other mechanism that does not include alterations in the expression level of genes encoding the key enzymes involved in GA metabolism. In the non-embryogenic explants, cultured on 20 μM NAA-supplemented medium, only a transient increase in endogenous GA3 was detected, without significant alterations in the levels of other bioactive GAs. By contrast, in the embryogenic explants, cultured on medium supplemented with 20 μM NAA+5 μM GA3, a significantly higher and longer-lasting increase in GA3 was detected, followed by a significant increase in the levels of the majority of endogenous bioactive GAs (GA1, GA4 and GA7). The obtained results evidence that the drastic increase in the levels of bioactive GAs in the explants correlated with the induction of somatic embryogenesis in spinach.