Natural rubber production in Hevea brasiliensis is determined by both tapping and ethephon frequencies. It is affected by a complex physiological disorder called tapping panel dryness. This syndrome is likely to be induced by environmental and latex harvesting stresses. Defence responses, including rubber biosynthesis, are dramatically mediated by wounding, jasmonate and ethylene (ET), among other factors. Using real-time RT-PCR, the effects of wounding, methyl jasmonate (MeJA) and ET on the relative transcript abundance of a set of 25 genes involved in their signalling and metabolic pathways were studied in the bark of 3-month-old epicormic shoots. Temporal regulation was found for 9 out of 25 genes. Wounding treatment regulated the transcript abundance of 10 genes. Wounding-specific regulation was noted for the HbMAPK, HbBTF3b, HbCAS1, HbLTPP and HbPLD genes. MeJA treatment regulated the transcript abundance of nine genes. Of these, the HbMYB, HbCAS2, HbCIPK and HbChi genes were shown to be specifically MeJA inducible. ET response was accompanied by regulation of the transcript abundance of eight genes, and six genes, HbETR2, HbEIN2, HbEIN3, HbCaM, HbPIP1 and HbQM, were specifically regulated by ET treatment. Additionally, the transcript level of the HbGP and HbACR genes was enhanced by all three treatments simultaneously. Overall, a large number of genes were found to be regulated 4 h after the treatments were applied. This study nevertheless revealed some jasmonic acid-independent wound signalling pathways in H. brasiliensis, provided a general characterization of signalling pathways and will serve as a new base from which to launch advanced studies of the network of pathways operating in H. brasiliensis.

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An efficient genetic transformation procedure using a recombinant green fluorescent protein (GFP) has been developed in Hevea brasiliensis clone PB260. Transformation experiments have been performed using an Agrobacterium tumefaciens binary vector harbouring both uidA and S65T-GFP reporter genes in order to compare selection methods using glucuronidase assay (GUS activity) and paromomycin resistance, GFP activity and paromomycin resistance, or GFP activity only. At transient level, the number of spots showing GUS or GFP activities was similar for 4 and 5 days after coculture. After selection, stable transformation events were observed and led to the establishment of transgenic callus lines. A higher number of lines were generated with GFP selection compared to the GUS one. GFP selection is less time-consuming in terms of callus subculturing, and offers the possibility of producing antibiotic resistance marker-free transgenic plants.

An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20 degrees C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged.

The South American Leaf Blight (SALB), caused by the fungus Microcyclus ulei, is the major rubber tree disease in all Central and South America. A population of 192 progeny individuals derived from a cross between a resistant clone and a susceptible cultivated clone was planted in a field trial in French Guiana in order to evaluate the resistance parameters under real infestation conditions. The resistance type (RT), the presence of stromata (ST) and the level of attack (AT) were observed 20-times on a 22-months period, and semi-quantitative evaluation of stromata was registered only once. The search for QTLs was performed using the Kruskal-Wallis test, Interval Mapping and the Composite Interval Mapping method. One major QTL located on linkage group g13 was detected on the RO 38 map, responsible for 36 to 89% of the phenotypic variance of resistance. This resistance QTL corresponds to one that had previously been detected under controlled conditions of infestation and we called it M13-1bn. Surprisingly, the effect of this QTL was larger under natural conditions of infestation than under controlled inoculation. Other minor QTLs (four on the RO38 map and one on the PB 260 map) were also detected. The type of resistance brought by M13-1bn, as well as its durability, are discussed. Applications for rubber tree breeding programs are considered.

Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.

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Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression in budded plants should be verified for functional analysis and further development.