Articles
INCREASING THE RESISTANCE OF APPLE ROOTSTOCKS TO FIRE BLIGHT BY GENETIC ENGINEERING: A PROGRESS REPORT
These include 51 transgenic lines containing the attacin E gene, 98 transgenic lines containing the cecropin SB-37 gene, 18 transgenic lines containing the cecropin Shiva-1 gene, and 5 transgenic lines containing the hen eggwhite lysozyme gene.
In addition, 15 M.7 lines transformed with pBl121 vector plasmid not containing lytic protein genes were selected.
Based on a GUS histochemical assay, many of these transgenic lines (ca. 75%) are apparently chimeric mixtures of transformed and non-transformed cells.
Solid lines (non-chimeric) were successfully selected from approximately 30% of the chimeric lines by placing leaf pieces on regeneration medium containing 250 μg/ml of paromomycin or 100 μg/ml of kanamycin and selecting regenerated shoots.
Work in progress indicates that, in many cases, a negative result in a GUS histochemical assay is due to a failure of the GUS gene to be expressed rather than an absence of the gene.
T1 is a transgenic M.7 apple rootstock that has been transformed with the gene encoding the lytic protein, attacin E. A 1995 field trial of T1 indicated a significant increase in fire blight resistance when compared with nontransformed M.7 rootstock or M.7 transformed with the pBl121 vector plasmid not containing the attacin E gene (transgenic T791). Ungrafted plants of T1, T791, and M.7 were planted in a field plot in May 1993. Tips of vigorously growing shoots of 3-yr-old plants were inoculated by hypodermic syringe with 5 X 109 cfu/ml of E. amylovora strain Ea273.
Resulting areas under the disease progress curve were T791:1201, M.7:637, and T1:395; and respective Ymax values (maximum proportion of the current season’s shoot length blighted) were 0.55, 0.30, and 0.19. A stoolbed trial indicated that T1 was not significantly different from M.7 in the number or quality of liners it produced.
An orchard trial comparing the performance of grafted fruiting trees on M.7 and T1 rootstocks has been initiated.
NOTE: At the time of the workshop and in previous published reports on this project, the parent cultivar of T1 and other transgenic rootstocks was thought to be M.26. Recent DNA RAPD and isozyme testing has indicated that the parent cultivar is M.7 and not M.26. In addition, plant material referred to as M.26 at the workshop and in previous reports was in fact M.7. These M.7 transgenics will still be used to evaluate the effect of the lytic protein genes on fire blight resistance, but will likely be of little commercial value.
Currently, we are working on producing lytic protein transgenics of the M.26 and M.9 rootstocks.