AGRE-0021 Optimisation of Lignin in Crop and Industrial Plants Through Genetic Engineering

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Biotechnology : ECLAIR Cluster II - Lignocellulose : Paper/Pulp : Plant Genetics : Pulping : Straw : Wood (Lignocellulose)

This shows the pulp obtained from tobacco plants agter simulated alkaline kraft pulping experiments on control and antisense tobacco stems. The antisense plant lignin is more accessible to chemical attack and the corresponding pulp contains lower residual lignin

SUMMARY

Wood, agricultural residues and many other plant materials which can be used for feed or paper production consist largely of lignocellulose. Of the various components of such materials, the carbohydrates cellulose and hemicellulose are of most value. The third component, lignin, has a negative effect on the digestibility of forage crops, contributes to the effluents generated by the paper/pulp industry and limits the attack on cellulose by enzymes and microorganisms during saccharification. Hence, it is of interest to design plants with a modified lignin content, adapted to specific agricultural and industrial uses. Recent advances in plant genetic engineering, particularly the use of what is termed antisense RNA, make this possible in theory. However, at the time this work was started no lignification genes had been characterised. The activities carried out under this ECLAIR project have changed the situation and shown that down regulation of certain lignification genes can result in marked changes in lignin composition and reactivity. These induced changes would appear particularly advantageous in terms of improved pulping characteristics and animal feed utilisation. The commercial exploitation of these results is already envisaged.

INTRODUCTION

Lignin is a complex phenolic heteropolymer, resulting from the hydrogenative polymerisation of three different cinnamyl alcohols, which is deposited in the walls of specific cells during the final stages of their differentiation. Lignin reinforces and waterproofs the walls of plant cells and plays a fundamental role in the strategies of mechanical support, solute conductance and also disease protection of higher plants. Lignins represent more than 25% of the weight of terrestrial biomass, but their presence in plants has a negative impact on biomass utilisation, particularly in forage digestibility, as well as papermaking where they have to be removed from wood to obtain the chemical pulp required. In Europe, millions. of tonnes of soluble lignin derivatives are produced every year. They are poorly utilised substances and their extraction, which is the cause of a lot of pollution, is an extremely expensive process. The content and chemical diversity of lignin in plants varies according to the species, the environmental conditions and the specific cultivar. For a long time, plant breeders have tried, through conventional genetics, to design plants with a reduced and modified lignin content more adapted to specific agricultural and industrial uses.

OBJECTIVES AND ACTIVITIES

The work carried out under this project used a plant genetic engineering approach to achieve the objective of modifying lignin content. It employed what is termed antisense RNA technology to interfere with the regulation of three genes coding for enzymes involved in the lignification process. These genes encode the following enzymes:
O-methyltransferase (OMT) which controls the degree of methylation of lignins, cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD). The latter two are both specific enzymes of lignin monomer synthesis which control the flux of lignin production. A prerequisite was the obtention of the three corresponding genes which had never been characterised prior to this project. An important research effort was thus devoted to the purification and molecular characterisation of the lignification enzymes. From the data obtained, specific oligonucleotides were used to screen cDNA libraries and several cDNAs corresponding to lignification enzymes were characterised for the first time in plants. Transformation experiments were conducted on both model plants (tobacco) and target plants of economic interest (poplar and alfalfa). In addition genetic transformation procedures have been designed for other potential target plants (tall fescue, eucalyptus) with the aim to extend the first results to a wider range of plant species. The chemical and phenotypic characteristics of the transformed plants were investigated as was digestibility and pulping quality.

COMMERCIALISATION

The results of these studies show that manipulation of the lignin content of plants using genetic engineering techniques is possible. This offers both an experimental tool which can be used to study the basic mechanisms of lignin structure and synthesis and, from an applied point of view, a way of improving annual crops and trees. Results to date suggest that such manipulation does not induce significant changes in growth, structure or morphology of the plants. This suggests that a degree of variability in the lignin polymer has no repercussions on the physiology of the plant and hence this technique can be used to improve commercial varieties or lines. For example transformed forest trees with more easily extractable lignins will be of value to the paper pulp industry, since less chemicals will have to be used. While longer field trials of these plants are required, for instance to determine their resistance to disease and climate variations, possibilities of commercial production are under consideration by the industrial partner.

PARTICIPANTS

Centre de Biologie et Physiologie Vegetales URA CNRS (Toulouse, France), ZENECA (Bracknell, UK), Laboratorium Voor Genetica, Universiteit Gent (Gent, Belgium), INRA Laboratoire de Biologie Cellulaire (Versailles, France), Ste RAG T (Rodez, France), INRA-SRNH (St Genes-Champanelle, France), Plant Genetic Systems NV (Gent, Belgium), IBMP-CNRS (Strasbourg, France), Universitat Hohenheim Institut fur Pflanzenphysiologie (Stuttgart, Germany), Laboratoire de Chimie Biologique Institut National Agronomique (Thiverval-Grignon, France), Centre Technique du Papier, Domaine Universitaire (Grenoble, France), Empresa Nacional de Celulosas SA, (Pontevedra-Spain).