Standardization of protocols for plant regeneration and genome editing CF1 potato cultivar to enhance scab resistance

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Canada produces potato in 138 thousand ha (4.6 million t, farm gate value of $1 billion per annum) and US**produces five times more. More than 200 potato cultivars are currently grown in Canada. The cultivar CF1**(registered name held confidential) is grown both in Canada and also in USA and is very susceptible to scab,**caused by Streptomyces scabies, a bacterial pathogen. The pathogen infects young tubers and causes scabby**lesions, often deforming the tubers. Current management options are not effective, often leading to severe yield**losses; in 2015, the loss only for CF1 cultivar was $800 000. Genetic resistance is the best way to manage this**disease. However, the R gene compositions of several commercial cultivars are rather poor, thus have only low**to moderate levels of resistance. Furthermore, the sexual incompatibility limits potato breeding. Based on**metabolomics and transcriptomics Kushalappa lab has identified several R genes against late blight in potato**and to fusarium head blight in wheat. These R genes biosynthesize resistance related metabolites that are**deposited to reinforce the cell walls to contain the pathogen spread; this mechanism is common to different**plants and pathogens, thus can confer multiple disease resistance. In this proposal, some of these R genes will**be sequenced in CF1, and if polymorphic, the best one will be used in genome editing. The protocol for calli**production and plant regeneration will be established. The R gene segment that is mutated in CF1 will be**replaced with the functional gene segment, based on CRISPR-Cas 9 system using geminivirus vectors. The**replaced genes will be confirmed in the regenerated plants based on amplicon sequencing. The mini-tubers of**cisgenic CF1 and the original CF1 will be produced aeroponically, young tubers will be inoculated with scab**pathogen and disease severity will be assessed to confirm enhanced resistance. After this project period, an**elaborate project will be developed to identify more polymorphic genes and to edit them in CF1 to obtain high**levels of multiple disease resistance, in collaboration with the Cavendish Farms, and submitted to**NSERC-SPG for funding.