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.

Efficient regeneration protocol is important for mass propagation, genetic transformation and germplasm alteration of desired plants. Thus, development of an efficient protocol is of the considerable importance for further genetic manipulation. In vitro plant regeneration was studied from two age groups of seedlings (2-11 weeks and 12-21 weeks old) by using their cotyledons, hypocotyls & leaf segments as explants. Shoot organogenesis was evaluated by using different explants on MS medium supplemented with varying concentrations of phtyohormones (1 mg/l BAP; 0.2 mg/l BAP + 1 mg/l Zn ; 0.5 mg/l B AP + 0.1 mg/l NAA; 1.5 mg/l BAP + 0.5 mg/l NAA). Similarly, rooting of regenerated shoots was optimized on different concentrations of IBA (0.1 mg/l; 0.5 mg/l; 1 mg/l). Hypocotyls of two to eleven weeks old seedlings as explants showed the best results of direct organogenesis on BAP (0.5 mg/l) and NAA (0.1 mg/l) in 21 days. However, BAP (0.2 mg/l) and Zn (1 mg/l) showed no growth. Indole-3-butyric acid (1 mg/l) induced rooting within a minimum period of 12 days. Overall, rate of shoot and root formation was found to be 50%, respectively. Successfully, the platelets were acclimatized in net house and further transferred to open field conditions.

The objective of the study was to develop an efficient protocol for Populus deltoides transformation through Agrobacterium tumefaciens LBA4404. Agrobacterium strain harboring binary plasmid pGA482 with Gus (uidA) gene under CamV35S promoter and Neomycin phosphotransferase (nptII) gene under Nos promoter was used for the transformation. Nodal, internodal and leaf explants from 4-5 months In vitro and fieldgrown plants were used for the transformation. Transformation was done under different conditions including, preculture time, optical density, acetosyringone concentration, infection time and co-cultivation time. Confirmation of transformation was done through GUS histochemical staining. Highest transformation efficiency was observed in one week precultured leaf explants from field grown source on preculture medium containing 200µM acetosyringone. Precultured explants from In vitro source also gave good results for transformation but the callus formation was found to be slow in leaf explant. Calli from the both sources did not show any transformation when infected with O.D A600nm range from 0.3-0.8. Node and internode though showed less transformation rate but the callogenesis was found to be highest in node and internode explants on CIM 1. Leaf explants from field source also gave high callus induction on CIM 5. A. tumefaciens O.D A600nm 0.3-0.5 was found to be effective. Infection time of 1-2 hour and co-cultivation time of 1day in dark were found to be optimum for the transformation. 200mg/l of timentin was found the best to control the overgrowth of Agrobacterium.100mg/l Kanamycin in growth medium was found to sufficient for selection for transformants. Selected transformants were confirmed through PCR for the presence of transgene. The present protocol for P. deltoides was found to be efficient for genetic transformation and can be used to introduce novel traits in the P. deltoides.