RESUMEN
Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.
Asunto(s)
Brassica napus/embriología , Diferenciación Celular , Polen/fisiología , Semillas/crecimiento & desarrollo , Solanum melongena/embriología , Brassica napus/genética , Genotipo , Solanum melongena/genéticaRESUMEN
Eggplant is an economically important vegetable crop in Asia and Africa, and although it is grown in Europe and the United States, it does not account for a significant percentage of agricultural production. It is susceptible to a number of pathogens and insects, with bacterial and fungal wilts being the most devastating. Attempts to improve resistance through introgression of traits from wild relatives have had limited success owing to sexual incompatibilities. Therefore, a crop improvement approach that combines both conventional breeding and biotechnological techniques would be beneficial. This chapter describes an Agrobacterium-mediated transformation protocol for eggplant based on inoculation of seedling explants (cotyledons and hypocotyls) and leaves. We have used this protocol to recover transformants from two different types of eggplant, a Solanum melongena L. breeding line, and S. melongena L. var. Black Eggplant. The selectable marker gene used was neomycin phosphotransferase II (nptII) and the selection agent was kanamycin. In vitro grown transformants acclimated readily to greenhouse conditions.
Asunto(s)
Agrobacterium tumefaciens/genética , Técnicas de Transferencia de Gen , Plantas Modificadas Genéticamente/genética , Solanum melongena/genética , Transformación Genética , Agrobacterium tumefaciens/crecimiento & desarrollo , Cotiledón/citología , Cotiledón/embriología , Cotiledón/genética , Cotiledón/microbiología , Productos Agrícolas/citología , Productos Agrícolas/embriología , Productos Agrícolas/genética , Productos Agrícolas/microbiología , Resistencia a Medicamentos/genética , Marcadores Genéticos , Infertilidad Vegetal/genética , Hojas de la Planta/citología , Hojas de la Planta/embriología , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/embriología , Plantas Modificadas Genéticamente/microbiología , Solanum melongena/citología , Solanum melongena/embriología , Solanum melongena/microbiología , Especificidad de la EspecieRESUMEN
An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.