Cell culture and conventional breeding studies to establish cytoplasmic male sterility in the cultivated tomato (Lycopersicon esculentum)

Cytoplasmic male sterility is a mitochondrial trait which has great economic importance in hybrid seed production. The research reported in this dissertation was undertaken to produce cytoplasmic male sterility in the cultivated tomato (Lycopersicon esculentum).;Somatic hybrid plants were produced following polyethylene glycol-mediated fusion of L. esculentum mesophyll protoplasts with cell suspension-derived protoplasts of L. peruvianum. A partial selection system was established as L. peruvianum protoplasts did not divide in the culture medium used and unfused L. esculentum protoplasts were sensitive to the fusion treatment. Nuclear hybridity was confirmed through analysis of the isozymes phophoglucomutase and aspartate amino transferase. EcoRI digests of chloroplast DNA revealed that all somatic hybrid plants contained L. esculentum chloroplasts. Total DNA probed with the maize mitochondrial genes cytochrome oxidase II and 18S/5S ribosomal DNA and the carrot mitochondrial DNA probe PMNS 178 revealed that all somatic hybrids contained mitochondria from L. peruvianum and that no mitochondrial recombination had occurred. Methods were developed for analysis of mitochondrial DNA from frozen leaf tissue. The somatic hybrid plants were determined to be primarily hexaploid by comparing the number of chloroplasts in stomatal guard cells with parental lines. This method was shown to be a reliable, statistically significant way to determine relative ploidy of several species, but not for determining chromosome number. Somatic hybrid plants were self-sterile and could not be backcrossed by either parent.;It was determined that the low regeneration response of L. esculentum protoplasts was responsible for the failure to recover cybrids from the fusion experiments. Increased plant regeneration from leaf explants was obtained by intercrossing L. pennellii chromosome substitution lines. Hybrids were constructed that were self-fertile and sexually compatible with the cultivated tomato. Several of these hybrids produced 6.0 to 6.2 shoots per leaf explant whereas L. esculentum averaged only 1.0 shoot per explant. An alternative method of establishing cytoplasmic male sterility involved crossing L. pimpinellifolium and L. cheesmanii with L. esculentum. Repeated backcrosses to the cultivated tomato resulted in recovery of male sterile plants in the BC...