Analysis of goblet and LeGAI spatial-temporal expression in tomato introgression lines with high regeneration capacity

Abstract

The ability of certain plant tissues to regenerate new organs has been known for a long time and was used primarily in the domestication of plants that are propagated vegetatively. Later on, efforts to extend this regenerative capacity to in vitro propagation enabled a better understanding of the mechanisms controlling the post-embryonic plant organogenesis. Recent studies on cell signaling that initiates the regeneration process have allowed the detection of genes and signaling molecules related mainly to the specific phase of "induction" of the differentiation process to form new organs. However, the molecular mechanisms involved in the phase of "acquisition of competence" for regeneration are still poorly understood. The CUC2 gene from Arabidopsis, which homologue in tomato is GOBLET, is a marker of both root and shoot formation sites. The mutant procera contains a point mutation in the DELLA gene LeGAI and has reduced in vitro shoot and root formation that is rescued by the allele REGENERATION1 (Rg1), which confers enhanced regeneration capacity. This indicates that GOBLET and LeGAI may be involved in cell competence. Using a set of introgression lines (IL) containing Solanum pennellii LA716 chromosomal segments into the Solanum lycopersicum cv. M82 genetic background, three introgression lines with enhanced in vitro root and shoot regeneration where identified: the ILs 3-2, 8-3 and 7-1. The high in vitro regeneration capacity of both root and shoot of these ILs indicates that these chromosomal segments may contain genes controlling the competence to undertake distinct cell fates. The goal of this work is to perform a detailed characterization of GOBLET and LeGAI gene expression in the ILs 3-2, 8-3 and 7-1 during shoot regeneration. To accomplish this, the GOBLET promoter will be fused to the coding region of the reporter genes GFP-GUS, and the LeGAI promoter and coding region will be fused to the coding region of the reporter gene GFP. The fused genes will be further cloned into a binary vector, and the selected ILs will be used for transformation. The gene expression in the explants will be visualized with fluorescence microscopy (GFP) and stereomicroscopy (GUS). In addition we propose to analyze the expression of genes markers of in vitro shoot induction using RT-PCR. This characterization will further contribute to better understand the general principles involved in the regeneration process. (AU)