CaMV 35S promoter directs ß-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree).

Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the ß-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. ß-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex.

Gene insertion into Hevea brasiliensis.

A transformation system has been developed for Hevea brasiliensis using the particle gun method. Anther derived calluses were transformed with vectors harbouring the ß-glucuronidase (gus) gene, the neomycin phosphotransferase (nptII) gene, and the chloramphenicol acetyl transferase (cat) gene. Gene transfer was determined by histochemical staining and fluorometric assay for ß-glucuronidase activity, enzyme linked immunosorbent assay for detecting neomycin phosphotransferase II gene and direct enzyme assay for detection of expression of the cat gene. These independent assays all showed a several-fold increase, compared to control values, in gene product level and enzyme activity in extracts from transformed callus and embryoids of Hevea. These results were confirmed using polymerase chain reaction with primers designed to amplify an internal gus fragment. Together, the results show the feasibility of the particle gun method for the introduction of foreign genes into Hevea.