Biotechnological Optimization of Switchgrass: In Vitro Regeneration and Endoglucanase E1 Transgenic Approaches to Boost Biofuel Efficiency

ABSTRACT: In the fight against climate change and for the conservation of finite fossil fuel resources, biofuels are indispensable. To improve biofuel production, this research aimed to genetically engineer switchgrass (Panicum virgatum L.) cultivars with increased cellulose content by establishing efficient in vitro culture methods and using the Endoglucanase E1 gene from Acidothermus cellulolyticus. After an average of 28 days, the shoot regeneration rate in SG2 (MS+0.3mg/l BAP) was 94%, whereas in SG6 (MS+2mg/l+0.8mg/l NAA), the rate was the slowest at less than 5%. The first signs of callus formation on MS-B5 culture media using in vitro clumps as an explant were noticed on the 30th day. A pCambia 1301 vector was used to clone the Endoglucanase E1 gene. Use of quantitative real-time polymerase chain reaction (QRT-PCR) validated Endoglucanase E1 gene expression in transfected callus samples. An increase in sugar release and protein concentration in transfected in vitro switchgrass cells further validated the biochemical screening for the Endoglucanase E1 gene. Thus, proves that the Endoglucanase E1 gene and its enzyme activity were integrated in switchgrass in vitro cultures, leading to a two-fold increase in the ethanol content that is acceptable for biofuel production.