Evaluation of gamma irradiated mutant population of sugarcane (Saccharum officinarum) for lignin content and its characterization for COMT gene
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Date
2020
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Punjab Agricultural University, Ludhiana
Abstract
Sugarcane is a C4 plant which is grown for sugar and biofuel in many parts of world. The bagasse left after extracting juice from sugarcane stalk is rich in cellulose, hemicellulose and lignin. This makes it an ideal substrate for bioethanol production. Lignin is the single key factor which makes this lignocellulosic biomass recalcitrant for ethanol extraction. Around 200 γ-irradiated mutant plants of sugarcane variety CoJ 85 were used in present study for evaluating variation for lignin content using acetyl bromide method. The mutant plants showed variation in lignin content ranging from 13-28%. The normal plant (control) showed 22.95% lignin content. To know this variation at molecular level the caffeic acid-o- methyl transferase (COMT) gene was amplified from five low lignin mutant plants and control. Two overlapping primers of the reference gene retrieved from sugarcane genome database (https://sugarcane-genome.cirad.fr) were designed using perl primer software for COMT gene for amplification using high fidelity polymerase chain reaction. The eluted PCR products from mutants as well as control were then separately ligated in pGEM-T vector and cloned in E.coli (strain DH5-ɑ). The plasmids were then isolated and Sanger sequencing was done for all these samples. After removing bad sequences from ABI files the gene lengths were predicted in control as well as mutants in comparison to reference gene. The predicted sequence of control was 2566 bp while those of mutants ranged from 2560-2568 bp in length. Sequencing results showed variations such as SNPs and indels in various regions of COMT gene for all the screened mutants and control in comparison to reference gene. All the CDS, TSS, intron and exonic regions were also identified by using FGENESH program in COMT gene isolated from control as well as in field grown γ-irradiated sugarcane mutants.