THE SULFUR-RICH BRAZIL NUT 2S ALBUMIN IS SPECIFICALLY FORMED IN TRANSGENIC SEEDS OF THE GRAIN LEGUME VICIA-NARBONENSIS

Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EH A101 to transfer a chimeric 2S albumin gene construct carried in the b inary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vici a narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of tither the CaMV 35S promoter which permits ge ne expression in all organs, or the Vicia faba legumin B4 promoter whi ch elicits seed-specific gene expression. After callus formation and s election for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained b y grafting transgenic shoots on the appropriate seedlings. R(1) and R( 2) generations were raised and analysed for GUS as well as 2S albumin gene expression. Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyl edons of seeds, whereas seed-specific gene expression was found in tra nsformants with the legumin promoter/2S albumin gene fusion. The 2S al bumin accumulated in the 2S protein fraction of transgenic seeds and i ts primary translation product was processed into the 9 and 3 kDa poly peptide chains. The foreign protein was localised in the protein bodie s of the grain legume. Analysis of the R(2) plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plan ts the amounts of 2S albumin were twice that present in the correspond ing heterozygous plants. Whereas only low level formation of the forei gn protein was achieved if the gene was under the control of the 35S p romoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promo ter. Some of these transformants exhibited a three-fold increase in th e methionine content of the salt-soluble protein fraction extracted fr om seeds.