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Latex synthesized in latex cells is expelled from the bark of rubber trees after tapping. Latex production is stimulated by applying ethephon on the tapping panel, which enhances latex flow and regeneration between tapping operations. This exploitation sometimes causes the flow of latex to stop. This tapping panel dryness syndrome (TPD) is related to endogenous ethylene production and oxidative stress.
A reverse genetic analysis was conducted to gain insight into the molecular mechanisms underlying the tolerance to tapping stresses and rubber production. This involved the production of transgenic rubber plants over- or underexpressing a candidate gene. In these studies, the candidate genes were involved in ethylene and jasmonate biosynthesis and signalling and in the cell response (ie rubber biosynthesis, detoxification of reactive oxygen species, etc.). They were characterized with respect to the stress response in three Hevea clones with contrasting metabolisms.

Genetic transformation— a gene function assessment tool

Modified transgenic plants are a source of essential information on functional mechanisms involved in crop production. CIRAD, in collaboration with the Universities of Mahidol and Kasetsart (Thailand) and the Institut français du caoutchouc, has been developing a Hevea genetic transformation procedure for several years. This procedure has been used to carry out an in-depth study on controlling natural rubber production. The process is based on an efficient Agrobacterium tumefaciens-mediated gene transfer method for use with high quality embryogenic callus lines. Friable callus lines with a high plant regeneration capacity via somatic embryogenesis were used for transformation of clone PB 260. Several thousands of transformation events per gramme of calli can be obtained by culturing these embryogenic calli with bacterial strain EHA 105 at low temperature. The transgenic callus lines are then isolated, cryopreserved and the number of transferred DNA copies is analysed by Southern molecular hybridization. Hundreds of plants have already been regenerated via this process—they are derived from dozens of lines bearing the reporter gene encoding ß-glucuronidase (GUS) under control of the 35S promotor of the cauliflower mosaic virus. The transgenic cell selection phase was thus shortened from 6 to 3 months and its efficacy was increased twofold by using the green fluorescent protein (GFP) as visual marker combined with antibiotic selection. More recently, transgenic lines were visually selected with GFP without any antibiotic input.

Directing transgene expression

Transgene expression was targeted in latex cells using the promoter of the HEV2.1 gene encoding hevein, an agglutinin protein involved in the coagulation of rubber particles. Functional analysis of this promoter showed that the HEV2.1 gene, which was assumed to be specifically expressed in latex, was actually only expressed in non-photosynthetic tissues. In leaves, its expression was located in all tissues by in situ hybridization, which is related to light induction of this promoter. Other promoters will be studied to target transgene expression in suitable tissues.

Transgenic plant development is the basis of studies on hormonal stress signalling and oxidative stress tolerance. For instance, overexpression of the gene coding for superoxide dismutase, an enzyme involved in the detoxification of reactive oxygen species, can lead to high phenotypic variability under characterization. Research is under way to develop a gene silencing technique through RNA interference. Moreover, studies will be conducted in partnership with CATAS (China) to characterize transcription factors that might coordinate a network of genes involved in tapping and cold stress tolerance and, in liaison with European and Asian teams, to investigate latex allergy and abiotic stress resistance.

Contact

Pascal Montoro
Plant Development and Genetic Improvement (UMR DAP)
E-mail

Partners

• Universités de Mahidol et de Kasetsart (Thailand)
• Institut français du caoutchouc (France)
• Chinese Academy for Tropical Agricultural Sciences (CATAS, China)

For further information

• Lardet L., Bénistant E., Leclercq J., Martin F., Oliver G., Montoro P., 2008. Comparison of GUS activity in self-rooting and budded primary transformant plants in Hevea brasiliensis. In : IRRDB natural rubber conference. Kuala Lumpur : MRB.
• Leclercq J., Gébelin V., Martin F., Lardet L., Rio M., Chabaud M., Ayar A., Montoro P., 2008. Etude de l'expression de gènes impliqués dans la régulation du stress oxydatif chez Hevea brasiliensis. In : Biologie moléculaire des ligneux. Nancy : Inra.
• Montoro P., Lagier S., Baptiste C., Marteaux B., Pujade-Renaud V., Leclercq J., Alemanno L., 2008. Expression of the HEV2.1 gene promoter in transgenic Hevea brasiliensis. Plant Cell, Tissue and Organ Culture, 94 : 55-63.