|Padilla,, Isabel - MALAGA, SPAIN|
|Golis,, Agnieszka - SKIERNIEWICE, POLAND|
|Gentile,, Adele - ISF, ROME, ITALY|
|Damiano,, Carmine - ISF, ROME, ITALY|
Submitted to: Plant Cell Tissue And Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 16, 2005
Publication Date: March 15, 2006
Citation: Padilla,, I., Golis,, A., Gentile,, A., Damiano,, C., Scorza, R. 2006. Evaluation of transformation in peach [Prunus persica (l.) Bortsch] and plum (Prunus domestica l.) explants using green fluorescence protein (gfp) and beta-glucoronidase (gus) reporter genes. Plant Cell Tissue And Organ Culture. 84:309-314. Interpretive Summary: Genetic engineering of peach trees can provide new cultivars that have resistance to diseases such as plum pox virus, that cannot otherwise be obtained through traditional breeding. Genetic engineering approaches can also provide consumers with a higher nutritive value, improved sweetness and flavor, and novel fruit types. The limitation to the use of genetic engineering of peach is the difficulty in producing peach plants through regeneration in tissue culture. The production of new peach plants through tissue culture allows for the insertion of genes that can improve peach. We tested several common gene transfer vectors and genes that would indicate gene transfer in both plum (which can be genetically engineered) and in peach. Our results show that while the major block to peach genetic engineering is the inability to regenerate peach plants through tissue culture, the gene transfer step is also limiting. Our work points the way to additional research approaches that may overcome these difficulties and allow for the genetic engineering of peach in the future.
Technical Abstract: Peach and plum explants, including cotyledons, embryonic axes (peach), and hypocotyls slices (peach and plum) from non-germinated seeds and epicotyl slices from germinating seeds (peach), were exposed to Agrobacterium-mediated transformation treatments and regeneration protocols. The objective of the current study was to determine the optimum conditions for gene transfer to peach explants. GFP and GUS marker genes were tested using six different A. tumefaciens strains, 2 plasmids, and three promoters (CaMV35s, doubleCaMV35s, and chlorophyll a/b binding protein (CAB-B). Both GUS and GFP were highly expressed in plum hypocotyls slices. However, GUS and GFP expression were significantly lower in peach explants. The best result for both plum and peach explants was obtained with EHA105/pBINI9nptII-GUS/CaMV35s. Interactions between Agrobacterium strain, plasmid, and promoter were recorded. In some, the single CaMV35s promoter appeared more effective than the double CaMV35s. In general, the CaMV35s promoters produced more transformation event expression than the CAB-B promoter. Moreover, A. tumefaciens strains EHA105 and LBA4404 differed in their effects on GUS expression but not GFP expression suggesting an interaction between Agrobacterium strain and Ti plasmid. For peach, in general, internodes (56.8%), cotyledons (52.7%), and embryonic axes (46.7%) had the highest transformation rates. While no putative transgenic peach shoots were produced, two GFP-transgenic plum lines were confirmed through PCR. While GFP expression in these lines was visible in the early stages of regeneration, fluorescence subsided rapidly with shoot growth. While GFP presents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, the optimization of its use for plum and especially for peach is necessary.