Abstract
Objective: Centella asiatica (L.) Urb from Umbelliferae is a potential source of secondary metabolites having immense medicinal value. Asiaticoside is the major therapeutic compound. In the present study, Identification of a possible relationship between concentration/transcript level expression of asiaticoside and concentrations of growth hormones at different growth stages was observed. The current study includes molecular and biochemical evaluation of stress generated in C. asiatica at different time intervals in vitro.
 Methods: The enhancement in auxin, cytokinin and final asiaticoside content were determined using immunoassay kits for auxin, cytokinin and HPLC analysis respectively. Transcript level expression at different growth phases was carried out using real-time RT-PCR. For isolation of stress-related miRNAs, reverse transcription of total RNA using miScript II RT Kit PCR System was carried out as per instructions. The differential expression of five selected miRNAs was done by Real-Time RT-PCR. The analysis of stress in vitro was done by quantification of Hydrogen Peroxide (H2O2), total phenolics and total antioxidants by H2O2 assay kit, total antioxidant assay kit and Folin Ciocalteau reagent respectively. The final asiaticoside content was determined by HPLC.
 Results: Differential expression of key genes involved in asiaticoside pathway showed significantly higher transcript expression, which is in correlation with the final asiaticoside content. The enhanced expression of miRNAs and the analysis of H2O2, total antioxidant capacity and total phenolics are suggestive of generation of oxidative stress under controlled conditions.
 Conclusion: The present study shows a direct correlation between oxidative stress and transcript/phytochemical estimation of asiaticoside content under in vitro conditions.
Highlights
Plants are normally subjected to different types of stresses
The enhanced expression of miRNAs and the analysis of H2O2, total antioxidant capacity and total phenolics are suggestive of generation of oxidative stress under controlled conditions
The present study shows a direct correlation between oxidative stress and transcript/phytochemical estimation of asiaticoside content under in vitro conditions
Summary
Plants are normally subjected to different types of stresses. Stress in plants can be originated from the abiotic factor like surrounding environment or from biotic factors like living organisms. Various plant regulators protect against these stresses through different signalling pathways to nullify the adverse effects. It is reported that miRNAs in plants act as critical post-transcriptional regulators of gene expression which helps to react to various abiotic stresses during different growth phases [3]. MiRNAs, that direct post-transcriptional repression of messenger RNAs, with diverse functions [4] They are generally involved in the modulation of stability and/or translational potential of their mRNA targets [5]. Differential expressions of miRNAs implicated in plant growth and development have been observed in many plant species which were prone to abiotic stress conditions such as drought, salinity etc [6]. Differential expressions of miRNAs implicated in plant growth and development have been observed in many plant species which were prone to abiotic stress conditions such as drought, salinity etc [6]. miRNAs may hold the key as potential targets for genetic manipulations to engineer abiotic stress tolerance in crop plants
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