Abstract
BackgroundPhytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.ResultsWe pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated.ConclusionsWe developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.
Highlights
Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding
Transformation and functional expression of single late blight R genes in potato varieties The resistance spectra of three potato varieties were tested with five P. infestans isolates with variable virulence spectra and aggressiveness
In order to make Atlantic and Bintje resistant to late blight and to broaden the resistance spectrum of Potae9, these three varieties were transformed with two constructs, each containing a single late blight R gene
Summary
Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Genetic disease resistance is an effective tool for sustainable management of late blight, caused by Phytophthora infestans, which is economically the most important disease of potato. Breeding at the beginning of the twentieth century concentrated on major dominant late blight resistance (R) genes from the Mexican wild species Solanum demissum and eleven of these R genes were introgressed of an entire harvest [7]. Sarpo Mira is an example of a durably late blight resistant potato variety which contains a stack of at least four R genes [8,9]. The variety has not acquired a large market share yet because established varieties are preferred by farmers, processors and consumers
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