The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells.

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ-tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral's effect on microtubules was both dose- and time-dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ-tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP-Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral's effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules.

Development-specific association of amyloplasts with microtubules in scale cells of Narcissus tazetta.

Narcissus tazetta is one of the major geophyte crops worldwide, but little is known about its cell biology. The narcissus storage organ was studied by monitoring scale cell biology during the growth stage and dormancy, and it was found that amyloplasts gradually increased in size and reached a maximum at dormancy. In parallel, microtubules changed their organisation: during the growth phase (February to March) they were oblique; during April and May, microtubules formed a network with round "holes"; by late June and the beginning of July, when dormancy started, they were organised in parallel arrays. The holes formed in the microtubule array corresponded to amyloplasts. A closer look showed that during a short time window, while the plants were preparing for dormancy, the microtubules surrounded the amyloplasts. In vitro reconfirmation of this phenomenon was obtained when fluorescent bovine brain microtubules enwrapped isolated amyloplasts that had been purified between April and July but not those purified between January and March. Interestingly, protease treatment of amyloplasts did not completely prevent binding of microtubules, which suggests the existence of a protease-resistant factor that docks microtubules to the outer membrane of amyloplasts.

Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions.

Very short exposures of embryogenic calli of Vitis vinifera cv. Superior Seedless grape plants to diluted cultures of Agrobacterium resulted in plant tissue necrosis and subsequent cell death. Antibiotics used for Agrobacterium elimination or as plant selectable markers were not responsible for this necrotic response. Rather, cell death seemed to be oxygen-dependent and correlated with elevated levels of peroxides. Therefore, we studied the effects on necrosis of various combinations of antioxidants during and after grape-Agrobacterium cocultivation. The combination of polyvinylpolypyrrolidone and dithiothreitol was found to improve plant viability. Tissue necrosis was completely inhibited by these antioxidants while Agrobacterium virulence was not effected. These treatments enabled the recovery of stable transgenic grape plants resistant to hygromycin.

Lycopersicon esculentum lines containing small overlapping introgressions from L. pennellii.

The objective of this project was to introgress small overlapping chromosome segments which cover the genome of L. pennellii into Lycopersicon esculentum lines. The interspecific hybrid was backcrossed to L. esculentum, and a map of 981 cM, based on 146 molecular markers covering the entire genome, was produced. A similar backcross 1 population was selfed for six generations, under strong selection for cultivated tomato phenotypes, to produce 120 introgression lines. The introgression lines were assayed for the above-mentioned molecular markers, and 21 lines covering 936 cM of L. pennellii, with an average introgression of 86 cM, were selected to provide a resource for the mapping of new DNA clones. The rest of the lines have shorter introgressions consisting of specific regions with an average size of 38 cM. The proportion of the L. pennellii genome in the introgression lines was lower than expected (252 cM) because of strong selection against the wild-parent phenotype. The mean introgression rate for ends of linkage groups in the 120 lines was 3 times higher than for other regions of the genome. The introgression lines can assist in RFLP-based gene cloning by allowing the rapid selection of DNA markers that map to specific chromosome segments. The introgression lines also provide a base population for the mapping and breeding for quantitative traits such as salt and drought tolerance that characterize the wild species L. pennellii.

RFLP mapping of I1, a new locus in tomato conferring resistance against Fusarium oxysporum f. sp. lycopersici race 1.

The inheritance and linkage relationships of a gene for resistance to Fusarium oxysporum f. sp. lycopersici race 1 were analyzed. An interspecific hybrid between a resistant Lycopersicon pennellii and a susceptible L. esculentum was backcrossed to L. esculentum. The genotype of each backcross-1 (BC1) plant with respect to its Fusarium response was determined by means of backcross-2 progeny tests. Resistance was controlled by a single dominant gene, I1, which was not allelic to I, the traditional gene for resistance against the same fungal pathogen that was derived from L. pimpinellifolium. Linkage analysis of 154 molecular markers that segregated in the BC1 population placed I1 between the RFLP markers TG20 and TG128 on chromosome 7. The flanking markers were used to verify the assignment of the I1 genotype in the segregating population. The results are discussed with reference to the possibility of cloning Fusarium resistance genes in tomato.

Genetic mapping of tomato cDNA clones encoding the chloroplastic and the cytosolic isozymes of superoxide dismutase.

The isozyme pattern of superoxide dismutase (SOD) in tomato consists of two Cu,Zn isozymes located, respectively, in the chloroplast and in the cytosol, as well as additional isozymes of the Mn or Fe SOD type. We have shown that SOD-1 is the chloroplastic Cu,Zn SOD and is related to cDNA clone T10. Restriction fragment length polymorphism (RFLP) analysis was performed with two cDNA clones representing tomato Cu,Zn-superoxide dismutases. T10, coding for the chloroplast isozyme, was thus mapped to chromosome 11, between marker TG46 and TG108, while clone P31, coding for the cytosolic Cu,Zn SOD isozyme, was mapped to chromosome 1 between TG24 and TG81. SOD is associated with the response of plants to various environmental stresses; the mapping information presented here would permit the demonstration of this association by genetic analysis.