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GEMINI
Genetic determinism of maritime pine pulp and paper properties

Natural populations of maritime pine trees show large, in part genetically determined, variations in the properties of lignocellulosic fibres and in the derived pulp and paper products. So far, tree breeding programmes have failed to exploit this variation due to the costly and destructive traditional chemical and technological assays, and the requirement for mature trees to evaluate such properties. In this multi-disciplinary project, it is proposed to solve this problem by taking it from two angles. Firstly, a reliable, fast, cheap and non-destructive selection criteria for pulp and paper properties will be used and further developed. Secondly, a combination of QTL mapping, global gene expression analysis, Arabidopsis genomics and linkage disequilibrium mapping will be used to identify the genes controlling the traits, which are underlying the natural variation in pulp and paper properties. This should yield valuable criteria for the selection of elite trees.

Objectives
There are two main objectives of this project. Firstly, to develop/optimise reliable and cheap methods for measuring lignocellulosic, pulp and paper properties, to use these methods to obtain accurate estimates of genetic parameters (variability, heritability, genetic correlation between traits) and select elite trees with high pulping quality. Secondly, to identify, characterise and validate the genes involved in the genetic control of wood biosynthesis (lignin, cellulose and hemicellulose content, lignin composition, cellulose fibre morphology) that could be used as an efficient early selection criteria in breeding programmes. The combination of rapid methods for the chemical/physical characterisation of lignocellulosic fibre, pulp and paper characteristics, and genomic sciences to find the genes controlling these traits, should increase the efficiency of selection by shortening the time and reducing the costs associated with measuring such properties. This project is specifically directed by market requirements of the pulp and paper industry, and to a lesser extent by the wood panel industry. It focuses on a single tree species, the maritime pine (Pinus pinaster Ait.), used as a model softwood, but results are expected to have broader applications for other conifers, which will be of considerable interest for the EU (for example, the Scots pine and Norway spruce). The primary goal is to provide new strategic options for forest tree breeding by obtaining phenotypic and/or molecular selection criteria for genetic improvement of lignocellulosic fibre quality and, as a result, improving the quality of pulp and paper products. This project will lead to the selection of elite trees for improved variety deployment.

Progress to Date
The following results and milestones were achieved during the first three years of the project.

1. Screening methods for measuring lignocellulosic, pulp and paper properties
A database is being constructed to relate fast chemical (FTIR, NIRS, analytical pyrolysis) and physical assessments of wood (X-ray densitometry), pulp and paper, to conventional pulping tests carried out on an industrial scale. Various methodologies were also explored for chemical characterisation of wood samples by FTIR and solid state NMR, and to develop UV microscopy. These investigations should lead to the proposal of a non-destructive technique for estimating the papermaking potential of the maritime pine.

2. Estimates of genetic parameters for traits related to pulp and paper
Several genetic tests continue to be collected and analysed for X-ray analysis, chemical measurement, fibre morphology assessment and the Kraft pulping test. Several thousand samples are under analysis.

3. Identification of genes/proteins whose expression varies in developing xylem associated with different types of wood
Approximately 10 000 xylem ESTs were generated. At the transcriptome level, several genes were distinctive in differentiating xylem associated with different types of wood (early vs. late wood, opposite vs. compression wood, juvenile vs. mature wood) and were identified using high-density filters of cDNAs. At the proteome level, interesting proteins were also identified. Bioinformatic tools are being developed to manage the vast quantity of data that is being produced.

4. Identification of genes whose genetic map positions colocalise with wood quality QTLs
Candidate genes were mapped and colocalisation with wood quality QTL identified. Comparative mapping with Pinus taeda allowed the validation of some QTLs.

5. Characterisation of novel mutants in Arabidopsis
The screening of 25 000 T-DNA lines, as well as EMS mutagenised lines for short hypocotyl mutants, has been completed, and over 400 dwarf mutants are now available. An innovative classification procedure for cell wall mutants, based on FTIR microscopy, has been applied and clusters containing cellulose-deficient mutants and mutants with defects in the cortical microtubule cytoskeleton have been identified. A second screening of 3 500 EMS-mutagenised lines for temperature-sensitive mutants with altered FTIR spectra of the petiole has yielded 75 mutants with heritable phenotypes. The cellulose-deficiency for several mutants has been confirmed by FTIR-microspectroscopy and chemical analysis. Gene sequences have been identified and pine orthologs were identified. Five extragenic suppressors for a cellulose deficient mutant (prc1), covering at least three loci have also been identified. In a minimum of two suppressors, hypocotyl growth has been restored without restoring cellulose synthesis, suggesting that the genes are involved in the coordination between cell expansion and cellulose synthesis. The positional cloning of two loci is underway.

6. Regeneration and genetic transformation procedures for maritime pine
An improved protocol for somatic embryogenesis in maritime pine was established by P1-o. Maturation allowed the regeneration of mature somatic embryos of high quality that assured high germination and plantlet frequencies. Development of a transformation procedure for maritime pine, via Agrobacterium tumefaciens, was carried out. Transgenic EMs were obtained for the first time. By comparing these with previous results, it has been possible to develop a new transformation method.

7. Selection of elite trees based on phenotypic and/or molecular criteria
It will be possible to select elite trees with a high pulping quality by the end of the project.

Classified in FORESTRY, NON-FOOD PRODUCTS, GENOMICS

Scientist responsible for the project

Dr CHRISTOPHE PLOMION
UMR BIOGECO 1202
33610 Cestas
France - FR

Phone: +33 5 57 12 28 38
Fax: +33 5 57 12 28 81
E-mail: plomion@pierroton.inra.fr

References

Project ID QLRT-1999-00942
Organisation Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers
Area 5.3.2
Start date 01 February 2000
Duration (months) 48
Total cost 4 001 172 €
Total EC contribution   2 199 999 €
Status Ongoing
Web address of the project   http://www.pierroton.inra.fr/Gemini/

The partners

  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
  • Institut National de la Recherche Agronomique-Equipe de Génétique et Amélioration des Arbres Forestiers, France - FR
    plomion@pierroton.inra.fr
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