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Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.

Dehydration-responsive element binding/C-repeat-binding factors (DREB/CBF) control the activity of multiple stress response genes and therefore represent attractive targets for genetic improvement of abiotic stress tolerance. Paper mulberry (Broussonetia papyrifera L. Vent) is well known for its bark fibers and high levels of chalcone and flavonoid derivatives. Transgenic paper mulberry plants expressing a tall fescue (Festuca arundinacea Schreb.) FaDREB1 gene under the control of CaMV 35S were produced to examine the potential utility of FaDREB1 to increase the tolerance of paper mulberry plants to abiotic stress. The overexpressing FaDREB1 plants showed higher salt and drought tolerance than the wild-type plants (WT). After 13 days of withholding water, or 15 days in the presence of 250 mM NaCl, all the WT plants died, while the over-expressing FaDREB1 plants survived. The FaDREB1 plants had higher leaf water and leaf chlorophyll contents, accumulated more proline and soluble sugars, and had less ion leakage (which reflects membrane damage) than the WT plants had under high salt- and water-deficient conditions. The 35S promoter-driven expression of FaDREB1 did not cause growth retardation under normal growth conditions. Therefore, improved tolerance to multiple environmental stresses in paper mulberry might be achieved via genetic engineering through the ectopic expression of an FaDREB1 gene.

The purpose of this study was to produce recombinant xylanase in transgenic plants and to test its potential application for pulp bleaching. The xynII xylanase gene from Trichoderma reesei was inserted into the Arabidopsis genome. Many transgenic plants produced biologically active XYNII and accumulated in leaves at level of 1.4-3.2% of total soluble proteins. The bleaching ability of XYNII on Kraft pulp was demonstrated by a reduction in the kappa number and the residual lignin contents. The bleaching efficiency of transgenic plant produced XYNII was similar to commercial xylanase on unbleached Kraft pulp. The effect of xylanase treatment on Kraft pulp was also investigated by SEM. Clear physical change on the pulp fiber surface was observed and was related to the amount xylan removed and microfibrils were visible on the fiber surface. This report demonstrates the potential application of plant produced recombinant xylanase for pulp and paper bleaching.

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.

Transgenic plants severely suppressed in the activity of cinnamoyl-CoA reductase were produced by introduction of a partial sense CCR transgene into tobacco. Five transgenic lines with CCR activities ranging from 2 to 48% of wild-type values were selected for further study. Some lines showed a range of aberrant phenotypes including reduced growth, and all had changes to lignin structure making the polymer more susceptible to alkali extraction. The most severely CCR-suppressed line also had significantly decreased lignin content and an increased proportion of free phenolic groups in non-condensed lignin. These changes are likely to make the lignin easier to extract during chemical pulping. Direct Kraft pulping trials confirmed this. More lignin could be removed from the transgenic wood than from wild-type wood at the same alkali charge. A similar improvement in pulping efficiency was recently shown for poplar trees expressing an antisense cinnamyl alcohol dehydrogenase gene. Pulping experiments performed here on CAD-antisense tobacco plants produced near-identical results--the modified lignin was more easily removed during pulping without any adverse effects on the quality of the pulp or paper produced. These results suggest that pulping experiments performed in tobacco can be predictive of the results that will be obtained in trees such as poplar, extending the utility of the tobacco model. On the basis of our results on CCR manipulation in tobacco, we predict that CCR-suppressed trees may show pulping benefits. However, it is likely that CCR-suppression will not be the optimal target for genetic manipulation of pulping character due to the potential associated growth defects.

International audience; The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

Significant environmental advantages may be gained by applying biotechnologies further upstream in industrial processes.