|Santos, Margarida -|
|Sanchez, Ana Maria -|
|Gomes, Joao -|
|Miguel, Celia -|
|Oliviera, Margarida -|
Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 29, 2009
Publication Date: August 11, 2009
Citation: Santos, M., Oliver, M.J., Sanchez, A., Payton, P.R., Gomes, J.P., Miguel, C., Oliviera, M.M. 2009. An Integrated Strategy to Identify Key Genes in Almond Adventitious Shoot Regeneration. Journal of Experimental Botany. 60(14):4519-4173. Interpretive Summary: The development of organs during the regeneration of plants that have been genetically engineered is a crucial step in the development of transgenic crop plants. The regeneration process in almonds offers a good model system for understanding the genetic basis for organ development in plants and for understanding the process of genetic transformation. By studying the cellular architecture of the regenerating tissues we were able to determine that organ development is the major road block to obtaining engineered plants. Our studies were extended by investigating the gene expression profiles associated with the key developmental stages of organ development. In this manner we were able to identify several key genes whose expression can be used to track organ development. These studies will allow us to determine ways in which to improve organ development during regeneration of whole plants from engineered tissues thus increasing the efficiency of our transformation protocols. Although almond was the model system used here these studies have relevance to all crop plants.
Technical Abstract: Plant genetic transformation usually depends on efficient adventitious regeneration systems. In almond (Prunus dulcis Mill.), regeneration of transgenic adventitious shoots was achieved but with low efficiency. Histological studies identified two main stages of organogenesis in almond explants that were induced for adventitious shoot regeneration; a dedifferentiation stage (early) and shoot initiation stage (late). Histological observation revealed that the limitation in the recovery of transformed shoots is primarily a function of the low organogenic competence of the transformed tissues rather than transformation efficiency. To identify key genes involved in organogenesis, we used shoot-induced leaves and suppression-subtractive hybridization, to build a cDNA library from each organogenic stage. cDNA clones from both libraries were randomly picked, PCR-amplified and arrayed on glass slides. For transcript profiling, micro-array hybridization was performed using cDNA pools from early and late stages. Statistically significant differential expression was found for 128 cDNA clones (58-early, and 70-late), representing 92 unique gene functions. Genes encoding proteins related to protein synthesis and processing and nitrogen and carbon metabolism were differentially expressed in the early stage, whilst genes encoding proteins involved in plant cell rescue and defence and interaction with the environment were mostly found in the late stage. We found an LTP/alpha-amylase inhibitor/trypsin gene with stronger expression in early stage, as confirmed by quantitative RT-PCR, while a gibberellic acid stimulated protein gene seems to be a good marker for the late stage. These results are discussed on the basis of the putative roles of the annotated differentially regulated genes in almond organogenesis.