RESUMEN
Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.
Asunto(s)
Hevea/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Hevea/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. RESULTS: The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. CONCLUSIONS: This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.
Asunto(s)
Etilenos/biosíntesis , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hevea/genética , Técnicas de Embriogénesis Somática de Plantas , Factores de Transcripción/genética , Arabidopsis/genética , Familia de Multigenes , Filogenia , Proteínas de Plantas/genética , ARN de Planta/genética , TranscriptomaRESUMEN
Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression in budded plants should be verified for functional analysis and further development.
Asunto(s)
Glucuronidasa/metabolismo , Hevea/genética , Plantas Modificadas Genéticamente/genética , Transgenes , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Ingeniería Genética/métodos , Glucuronidasa/genética , Hevea/metabolismo , Técnicas de Embriogénesis Somática de Plantas , Plantas Modificadas Genéticamente/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl(2) concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl(2) before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl(2) concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl(2) concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl(2) concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.
Asunto(s)
Cloruro de Calcio/farmacología , Criopreservación , Desarrollo Embrionario/efectos de los fármacos , Hevea/crecimiento & desarrollo , Hevea/fisiología , Regeneración/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo , Hevea/efectos de los fármacos , Hevea/embriología , Tamaño de los Órganos/efectos de los fármacos , Agua/metabolismoRESUMEN
An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20 degrees C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged.