RESUMEN
For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.
Asunto(s)
Genes de Plantas , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Plastidios/genética , Solanum tuberosum/genética , Transformación Genética , Productos Agrícolas , Regulación de la Expresión Génica de las Plantas , Fitomejoramiento , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Solanum tuberosum/crecimiento & desarrolloRESUMEN
The PII protein in plants has been associated to many different tissue specialized roles concerning the Nitrogen assimilation pathways. We report here the further characterization of L. japonicus transgenic lines overexpressing the PII protein encoded by the LjGLB1 gene that is strongly expressed in the guard cells of Lotus plants. Consistently with a putative role played by PII in that specific cellular context we have observed an alteration of the patterns of stomatal movement in the overexpressing plants. An increased stomatal closure is measured in epidermal peels from detached leaves of normally watered overexpressing plants when compared to wild type plants and this effect was by-passed by Abscisic Acid application. The biochemical characterization of the transgenic lines indicates an increased rate of the Nitric Oxide biosynthetic route, associated to an induced Nitrate Reductase activity. The phenotypic characterization is completed by measures of the photosynthetic potential in plants grown under greenhouse conditions, which reveal a higher stress index of the PII overexpressing plants.
RESUMEN
Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental concerns. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum). By optimizing the tissue culture system and using transformation vectors carrying homologous potato flanking sequences, we obtained up to one transplastomic shoot per bombardment. Such efficiency is comparable to that usually achieved in tobacco. The method described in this chapter can be used to regenerate potato transplastomic plants expressing recombinant proteins in chloroplasts as well as in amyloplasts.
Asunto(s)
Biolística/métodos , Cloroplastos/genética , Solanum tuberosum/genética , Transformación Genética , Resistencia a Medicamentos , Regulación de la Expresión Génica de las Plantas , Vectores Genéticos , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/genética , Espectinomicina/farmacología , Transgenes/genéticaRESUMEN
Low transformation frequencies limit the use of plastid transformation in potato and other crops. Hence, a breakthrough in chloroplast genetic engineering of agronomically important species is a highly desirable goal. We succeeded in achieving potato transformation efficiency up to one shoot every bombardment using a modified regeneration procedure and novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. Such efficiency corresponds to 15-18-fold improvement compared to previous results obtained in potato with a progenitor vector of that used in our study, and is comparable to that usually achieved with tobacco. The results obtained represent a significant advancement towards the implementation of the plastid transformation technology in potato breeding and biotechnology.
Asunto(s)
Vectores Genéticos/genética , Plantas Modificadas Genéticamente/genética , Plastidios/genética , Solanum tuberosum/genética , Transformación Genética/genética , Ingeniería Genética/métodosRESUMEN
Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5'-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5'-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5'-UTR construct, described above, and another containing the same terminator, but with the promoter and 5'-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Plastidios/genética , Solanum tuberosum/genética , Transformación Genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Biotecnología/métodos , Cloroplastos/genética , Cloroplastos/metabolismo , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hojas de la Planta/genética , Raíces de Plantas/genética , Plastidios/metabolismo , Recombinación Genética , Secuencias Reguladoras de Ácidos Nucleicos , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , TransgenesRESUMEN
Gene expression in nongreen plastids is largely uncharacterized. To compare gene expression in potato (Solanum tuberosum) tuber amyloplasts and leaf chloroplasts, amounts of transcripts of all plastid genes were determined by hybridization to plastome arrays. Except for a few genes, transcript accumulation was much lower in tubers compared with leaves. Transcripts of photosynthesis-related genes showed a greater reduction in tubers compared with leaves than transcripts of genes for the genetic system. Plastid genome copy number in tubers was 2- to 3-fold lower than in leaves and thus cannot account for the observed reduction of transcript accumulation in amyloplasts. Both the plastid-encoded and the nucleus-encoded RNA polymerases were active in potato amyloplasts. Transcription initiation sites were identical in chloroplasts and amyloplasts, although some differences in promoter utilization between the two organelles were evident. For some intron-containing genes, RNA splicing was less efficient in tubers than in leaves. Furthermore, tissue-specific differences in editing of ndh transcripts were detected. Hybridization of the plastome arrays with RNA extracted from polysomes indicated that, in tubers, ribosome association of transcripts was generally low. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association. Selected nuclear genes involved in plastid gene expression were generally significantly less expressed in tubers than in leaves. Hence, compared with leaf chloroplasts, gene expression in tuber amyloplasts is much lower, with control occurring at the transcriptional, posttranscriptional, and translational levels. Candidate regulatory sequences that potentially can improve plastid (trans)gene expression in amyloplasts have been identified.