The degradation of potato virus M (PVM) particles in plant cells

Degradation of potato virus M particles was observed in the cells of <i>Solanum tuberosum</i>, <i>Solanum rostratum</i>, <i>Lycopersicon esculentum</i> and <i>Lycopersicon chilense</i> plants infected with this virus. PVM particles found in the cytoplasm of infected parenchyma cells grouped together in the form of inclusions, often found near the tonoplast. The ends of the virus particles and the tonoplast came into close contact. Cytoplasmic protrusions containing PVM particles, reaching into vacuoles were formed in those places. In addition to a large central vacuole, small vacuoles were observed in cells containing PVM particles. Various stages of degradation of cytoplasmic protrusions were observed both in the large and small vacuoles.

(260) Development of SCAR and CAPS Markers Linked to Tomato Begomovirus Resistance Genes Introgressed from Lycopersicon chilense

Resistance to begomoviruses tomato mottle virus (ToMoV) and tomato yellow leaf curl virus (TYLCV) has been introgressed to tomato (Lycopersicon esculentum) from L. chilense accessions LA 1932, LA 2779, and LA 1938. Resistance genes have been mapped to three regions on chromosome 6 using randomly amplified polymorphic DNA (RAPD) markers. We call these regions 1, 2, and 3. To facilitate breeding by marker assisted selection, advanced breeding lines with resistance from the above sources were assayed for the presence of RAPD markers to determine which were most tightly linked to begomovirus resistance. The best RAPD markers were then converted to sequence characterized amplified region (SCAR) markers or cleaved amplified polymorphic sequence (CAPS) markers. In addition, selected restriction fragment length polymorphism (RFLP) markers near the three regions were converted into CAPS markers, which were tested for association with the advanced breeding lines. Only LA 2779 derivatives have the L. chilense introgression in region 1, which is near the location of the Ty-1 gene and spans across CAPS markers 32.5Cla and TG118. Two region 1 RAPD markers UBC197 and UBC621 were converted co-dominant SCAR or CAPS markers, which were present in all 16 resistant breeding lines tested. Derivatives from all three accessions have introgressions in region 2. Further assays with more markers in this region are under way to determine the lengths and locations of the introgressions. No tightly linked RAPD markers have been found for the resistance gene from LA 1932 in region 3. RFLP and CAPS markers are being used to more precisely locate the region 3 gene.

Enhanced Resistance to Verticillium dahliae in Transgenic Strawberry Plants Expressing a Lycopersicon chilense Chitinase Gene

A chitinase gene (pcht28), isolated from Lycopersicon chilense, was transferred into `Joliette' strawberry using a stipule regeneration method and Agrobacterium-mediated gene transfer technique. Stipules showed a high rate of shoot regeneration (>90%) through direct or indirect organogenesis. Stipules were cocultured with a transformation plasmid in which pcht28 was under the control of the CAMV 35S promoter. A high tendency in production of chimaeric shoots was observed. Shoots which did not show any sign of bleaching after several subcultures in kanamycin containing medium were considered as stable transformants. These shoots were successfully rooted in the presence of 50 μg·mL-1 kanamycin. Transgenic nature of the plants was confirmed by PCR as well as Southern blot analysis. Constitutive expression of the chitinase gene was demonstrated by Northern analysis. In growth chamber studies, the transgenic strawberry plants which expressed pcht28 had significantly higher resistance to Verticillium dahliae as compared to nontransgenic controls. These results demonstrate that pcht28 plays a role in defense against this fungal disease in strawberry.

Inheritance and Linkage of Tomato Mottle Virus Resistance Genes Derived from Lycopersicon chilense Accession LA 1932

Tomato mottle virus (ToMoV) is a silverleaf whitefly (Bemisia argentifolii Bellows and Perring n. sp.) transmitted, bipartite geminivirus that infects tomatoes (Lycopersicon esculentum Mill.). Inbred lines resistant to ToMoV were derived from Lycopersicon chilense Dunal accession LA 1932. Inheritance was studied using a family developed from the crossing of a resistant inbred with a susceptible tomato inbred over two seasons. The F1 had resistance intermediate to the parents and generation means analysis of F1 and F2, backcross and parental populations suggested that the action of at least two additive genes with high heritability (h2n.s. = 0.87) controlled ToMoV resistance. When data from the two seasons were combined, an acceptable fit to an additive-dominance genetic model was obtained. Single plant comparisons, bulk comparisons, and tailends of F2 populations segregating for ToMoV resistance derived from LA 1932 identified randomly amplified polymorphic DNA (RAPD) markers using eight hundred 10-mer oligonucleotide primers. The F2 populations used for inheritance studies were screened for polymorphic markers, and 12 RAPD markers associated with the ToMoV resistant line were linked to the morphological markers self-pruning (sp) and potato leaf (c) on chromosome 6. RAPD markers that were associated with ToMoV resistance segregated into two linked regions flanking either side of the sp and c loci. The molecular studies suggested that the action of at least two additive regions controlled ToMoV resistance which supported the inheritance analysis.