DEVELOPMENT OF LOW LIGNIN EUCALYPTUS CAMALDULENSIS THROUGH DOWN-REGULATION OF ENDOGENOUS CINNAMOYL COA REDUCTASE (CCR)

Abstract

Renewable fuel resources can provide an alternative to meet the energy demands and compensate the diminishing resources of fossil fuels. Lignocellulosic biomass from non-crop plants can provide an alternative for the production of biofuels. However, presence of lignin in lignocellulosic biomass is a limiting factor to achieve industrial objectives. Industrial methods for the removal of lignin from lignocellulosic biomass are cumbersome, inefficient and expensive. The objective of this study was to produce lignocellulosic biomass with low lignin contents by down regulation of endogenous Cinnamoyl CoA Reductase (CCR) in Eucalyptus camaldulensis. Lignin downregulation was accomplished through RNAi. Putative transgenic plants were screened for the presence of transgene through polymerase chain reaction (PCR). CCR expression profiling, lignin estimation and effects of lignin down regulation leading to morphological changes were studied in transgenic and control E. camaldulensis plants. Real time Reverse transcriptase PCR for expression profiling of endogenous CCR gene revealed down regulation of CCR RNA transcripts from 72% to 80% in transgenic as compared to control and wild type plants. CCR enzyme activity was significantly reduced in transgenic plant as compared to control plant. Lignocellulosic biomass from stem of transgenic plants showed reduction in Klason lignin to 25.5% in transgenic plants as compared to wild type E. camaldulensis. Transgenic plants showed ~28% height reduction and ~53% stem girth reduction in comparison to untransformed control plants. Transformation efficiency of 0.13% was observed after successful transformation, regeneration, selection, molecular screening and expression profiling of the transgenic plants. This study confirmed role of CCR downregulation leading to lignin reduction in E. camaldulensis through RNAi for development of lignocellulosic biomass with low lignin contents.