Production of three new grapefruit cybrids with potential for improved citrus canker resistance

Abstract

Cybrid production, combining the nucleus of one species with alien cytoplasmic organelles of another, is a potentially valuable method used for improvement of various crops including Citrus species. Furthermore, this technology is considered a non-GMO biotechnology strategy. In citrus, cybrid plants can be produced as a by-product of somatic fusion. Host resistance is the most desirable strategy for control of citrus canker. By using a cybridization approach, several putative cybrids were created by protoplast fusion of embryogenic suspension culture-derived protoplasts of canker resistant ‘Meiwa’ kumquat ( Thunb ), with mesophyll-derived protoplasts of three grapefruit (Citrus paradisi Macfad.) cultivars ‘Marsh,’ ‘Flame,’ and ‘N11-11’ somaclone of ‘Ruby Red.’ In an effort to generate new grapefruit cultivars with enhanced canker resistance, putative cybrid grapefruit plants morphologically equivalent to standard grapefruit from all three combinations were produced. Four mitochondrial (mt) introns, a mt ribosomal RNA spacer region, and four chloroplast (cp) DNA regions previously shown to have polymorphism among different Citrus species were tested. Four molecular markers, two mt DNA regions (intron nad7i2 and a rRNA spacer), and two cp DNA regions (NADH dehydrogenase subunit K (ndhk) gene and a trnG-trnR intergenic spacer) revealed polymorphism between kumquat and grapefruit and were used to validate the cybrids. All the cybrids had the mt genome of kumquat, and most had the cp genome of kumquat with a few exceptions. EST-SSR marker analysis confirmed that the nuclear genome in all the generated cybrids came from the grapefruit parent. All the cybrid clones have been propagated and are undergoing extensive canker assays to identify any clones that have improved canker tolerance/resistance. These cybrid populations provide a valuable tool for investigating the contribution of cytoplasmic organelles to plant disease resistance.