JXB Advance Access originally published online on August 11, 2009
Journal of Experimental Botany 2009 60(14):4159-4173; doi:10.1093/jxb/erp250
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RESEARCH PAPER |
An integrated strategy to identify key genes in almond adventitious shoot regeneration
1Instituto de Tecnologia Química e Biológica/ Instituto de Biologia Experimental e Tecnológica, Quinta do Marquês, 2784-505 Oeiras, Portugal
2United States Department of Agriculture-ARS, Plant Genetics Research Unit, 205 Curtis Hall, University of Missouri, Columbia, MO 65211, USA
3United States Department of Agriculture-ARS, 3810 4th St, Lubbock, TX 79415, USA
4Instituto Nacional de Saúde, Avenida Padre Cruz, 1649-016 Lisbon, Portugal
* To whom correspondence should be addressed: E-mail: mmolive{at}itqb.unl.pt
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 a 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, shoot-induced leaves and suppression-subtractive hybridization were used, 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, microarray hybridization was performed using cDNA pools from both the early and the 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. The LTP/
-amylase inhibitor/trypsin gene was more strongly expressed at an 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.
Key words: Adventitious shoot, almond, early stage (ES), late stage (LS), quantitative PCR, suppression subtractive hybridization (SSH), transcript profiling
Received 12 May 2009; Revised 13 July 2009 Accepted 29 July 2009