Modification of lignin content of wood of sengon (Paraserianthes falcataria (L.) Nielsen) by engineered gene of 4-Coumarate CoA Ligase (4CL)

Abstract

Separation of lignin from cellulose requires high input of chemicals and energy resulted in high cost production and high ecological risks. The presence of lignin in plant cells is a factor limiting the efficiency of processing lignocellulosic materials of wood-based industries including pulp and paper industry either chemically or biologically (biopulping) process using white rot fungi. Lignin content and composition affect the efficiency of pulping process. Low-lignin wood or wood containing more reactive lignin which means syringyl content is higher than guaiacyl are more easily separated from cellulose. Therefore, it would be advantage to improve the efficiency process of pulp and paper industry. Sengon (P. falcataria) as a fast growth species is useful as a raw material for pulp because its characteristic which possess good fiber dimensions, high quality of physical and mechanical properties of its paper sheets. This study was aimed at (1) isolating the cDNA encoding 4-coumarate CoA ligase (4CL) from sengon, (2) constructing a binary vector of 4CL gene, with antisense orientation sengon transformation, (3) producing transgenic sengon plants which have characteristics low-lignin or higher syringyl ratio by introducing binary vector construct containing 4CL gene fragment with antisense orientation. Modification of the content or composition of lignin through recombinant DNA technology (genetic engineering) has been carried out in several stages: (1) quantitative analysis and histochemical lignin of sengon (P. falcataria), (2) embryogenesis shoots induction of sengon (3) isolation and cloning of cDNA fragments of genes encoding 4-coumarate: Coenzyme A ligase from sengon, and (4) genetic transformation on sengon by using gene fragment of antisense 4-coumarate CoA ligase (4CL) via A. tumefaciens. Lignin content determination of sengon collected from several areas in Indonesia indicated that lignin content of sengon ranged low to moderate category (16.58-35.59%). Lignin histochemical assay of transverse section of stems by using phloroglucinol-HCl staining showed that the initiation of lignin deposition was noted in 2 weeks old seedling. Quantitative and qualitative assessment through histochemical assay showed that lignin content in trees was varied depending on heights and location. Shoots induction from cotyledon nodes was selected as the most appropriate regeneration system for genetic transformation to improve sengon wood quality for pulp and paper industry. The cDNA fragment of 342 bp in size was obtained using degenerate primer designed using CODEHOP technique. Blast analysis result showed that deduction amino acid sequences of one out of two RT-PCR products nucleotide was highly homologous with the 4CL conserved region from Rubbus ideaus, Oryza sativa, Populus tomentosa, Populus balsamifera, Betulla platyphilla, Nicotiana tabacum, and Arabidopsis thaliana with identity ranging from 78-90%. The 4CL fragment has been successfully constructed and cloned for antisense orientation in pCAMBIA 2301. Recombinant expression vector of antisense 4CL has been successfully introduced into sengon via Agrobacterium tumefaciens. Gene integration test by PCR method showed that 19 out of 112 transformed sengon seedlings which resistant to kanamycine were transgenic seedlings. Lignin content determination of transgenic seedlings stem including histochemical assay, Klason lignin content and FTIR analysis to determine the S/G ratio was indicated that two transgenic sengon seedlings namely 4CLAS-4 and 4CLAS-1 possessed lower lignin content (15:53%) and higher S/G ratio than the control. These plants also showed the best growth characteristic and normal morphological appearance.