Effect of histone deacetylation in the regulation of somatic embryogenesis related genes in coconut (Cocos nucifera L.)

Abstract

The study entitled “Effect of histone deacetylation in the regulation of somatic embryogenesis-related genes in coconut (Cocos nucifera L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, during 2023-2024. The objective of this study was to know the effect of histone deacetylation in the regulation of somatic embryogenesis related genes (SERK, BBM, WUS) and histone deacetylation gene (HDAC) in coconut (Cocos nucifera L.) in presence of HDAC inhibitor Trichostatin. Coconut somatic embryogenesis holds significant promise for cultivating superior coconut plants. Currently, no repeatable and efficient protocol exists for inducing somatic embryogenesis in this crop. Epigenetic regulators have been found to enhance cell differentiation and their role in promoting embryogenic induction has been observed in various recalcitrant crops (Abrahamsson et al., 2017). In many such species, histone acetylation modification of genes that control somatic embryogenesis has been reported to increase gene expression and subsequently improve the rate of somatic embryogenesis (Martinez et al., 2021). The acetylation of genes can be enhanced by using histone deacetylase inhibitors (HDAs). Trichostatin A (TSA), the most used histone deacetylase inhibitor (Görisch et al., 2005), blocks HDAC activity in cultured cells, leading to a significant increase in embryogenic growth (Wójcikowska et al., 2018). By specifically inhibiting HDAs, TSA causes an accumulation of acetylated histones, a corresponding reduction in DNA methylation, and an increase in gene activity (Wójcikowska et al., 2018). However, how HDAC inhibitors affect the acetylation of genes related to somatic embryogenesis in coconut is still not well explored. Keeping this in view, the present study was planned to find out the effect of Trichostatin in embryogenic callus induction. For analysing the effect of Trichostatin on callus induction, plumules from 11-month-old West Coast Tall (WCT) coconuts were scooped out, surface sterilized, and pre-cultured in Y3 basal medium for one month. The pre-cultured

plumules were then inoculated into Callus Induction Medium (Y3 + 2,4-D (74.6μM), TDZ (4.5μM), spermine (50μM)) with varying concentrations of TSA (0.5μM to 2μM). The results showed no change in the rate of callus induction with Trichostatin treatment. In the control, a 20% embryogenic callus induction was achieved in 50 days. However, in the treatments with TSA, instead of callus development, enlargement of plumules was observed at 50 days of inoculation, with no change in status even after that period. The percentage of enlargement of plumules varied with TSA concentration and maximum was observed in 0.5μM TSA. Following the Trichostatin treatment, an analysis of the expression of somatic embryogenesis (SE) related genes and HDAC gene in control and treated samples was conducted. For gene expression analysis, RNA was isolated from plumules inoculated in 0.5μM TSA and control cultures using Trizol. Complementary DNA (cDNA) was synthesized by reverse transcription mix and subjected to quantitative real-time PCR (RT-qPCR) to analyze the expression levels of somatic embryogenesis marker genes (SERK, WUS, BBM) and histone deacetylase genes (HDAC). The results showed no significant change in the expression of the HDAC gene in both the control and the CIM with 0.5μM TSA. Additionally, the results revealed no expression of somatic embryogenesis-related genes in Trichostatin-treated samples. This finding suggests that Trichostatin treatment does not influence embryogenic callus induction, indicating a need to fine-tune the concentration and conditions of the treatment to optimize its effects. Conclusively, further optimization is required to achieve desirable results in somatic embryogenesis induction in coconut.