RESUMEN
Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.
Asunto(s)
Abies/genética , Agrobacterium tumefaciens/genética , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente , Semillas/genética , Abies/embriología , Toxinas de Bacillus thuringiensis , Vectores Genéticos , Técnicas de Embriogénesis Somática de Plantas , Regiones Promotoras Genéticas , Semillas/crecimiento & desarrollo , Transcripción Genética , Transformación GenéticaRESUMEN
A positive selection system using phosphomannose isomerase was employed for Agrobacterium tumefaciens mediated transformation of lettuce (Lactuca sativa L. var. 'Achát'). It was shown that the mannose-based selection system works very well with the lettuce genotype used, reaching up to 25% transformation efficiency on the medium with 20 g/L mannose and 20 g/L sucrose. The best transformation efficacy with the commonly-used kanamycin at 100 mg/L as a selection agent was 21%. Southern blot analyses of thirteen chosen mannose-resistant regenerants revealed that some of them have clonal origin, about one-half harbour a single T-DNA copy and one plant contains an incomplete T-DNA segment with only the left part of T-DNA with the pmi gene present in the genomic DNA. The following Northern analysis showed transcriptional activity of the introduced pmi gene in all plants analysed with very high differences in the level of pmi specific mRNA. The results demonstrate that both mannose and kanamycin provide comparable transformation efficiencies in our lettuce genotype. An alternative selection method with mannose as a selection agent is now available for lettuce transgenosis.