No abstract

An alternative to the leaf disk system for studies of the metabolism of senescence is described. the only advantage of using leaf disks for the study of the metabolism of senescence is convenience, but this may turn into a disadvantage if corrective measures for the above differences are not taken.An alternative method for the study of senescence is described in this report. This method uses the intact leaf in which the metabolic rates can be estimated with simple precursors, and in which senescence can be deferred by endogenous substances. Senescence can be delayed and physiological vigor restored to leaves by removal of the apical portion of the plant before senescence has progressed too far (5,8,20,27,40). Removal of the epicotyl above a given pair of leaves at regular time intervals permits one to determine the time at which apex removal no longer defers senescence of the leaves in question. Before the time at which apex removal no longer defers senescence, the leaves contain a minimal amount of some substance(s) or structure(s) required for survival. This time shall be referred to as the point of no return. The period of recovery in which the progress of senescence is reversed can be referred to as regeneration. Metabolism to the point of no return and during the regeneration subsequent to apex removal can be studied by the incorporation of labeled C02- MATERIALS AND METHODSSoybean seeds (Glycine mlax L. var. Harosoy) were germinated in vermiculite in a growth chamber under 16-hr day length (29 C day, 25 C night) for 7 days. On the 7th day, seedlings were transplanted into 4-inch plastic pots containing garden soil and irrigated with 120 mg of 20-20-20 fertilizer per pot.Cotyledons were harvested, frozen immediately, and stored at -20 C. Ribonuclease preparations were obtained by homogenizing the cotyledons in 0.3 M sucrose and filtering the homogenate through glass wool. The homogenates were then adjusted to a constant volume (2 or 2.5 ml/cotyledon) with 0.3 M sucrose and centrifuged at 1OOOg for 15 min at 5 C. Aliquots of the supernatant were assayed for RNase activity by the method described by Wilson (39). The pellet was not assayed for activity.For isotope incorporation studies, intact soybean plants (17 days), or those from which epicotyls had been removed on the 16th or 17th day, were held for 24 hr in a Plexiglas chamber, in which 1.25 mc of "'CO2 from barium carbonate were liberated with 50% lactic acid (16-hr day and 8-hr night, 30 C and 26 C, respectively). The initial concentration of CO2 in the chamber was approximately 2.06%.The soluble protein of cotyledons was extracted as described for RNase, but the centrifugation step was omitted. Aliquots (1 ml) of the extract were mixed with an equal volume of 10% (w/v,) trichloroacetic acid and held overnight at 0 C. The

Tumor formation in Vitis species and hybrids, incited by Agrobacterium tumefaciens, was altered by chemical, physical, developmental, and genetic variables. Knowledge of the effect of these variables was used to develop a stringent in vitro assay system to select parents for a study of genetic factors that modulate tumor formation. Tumor formation was reduced by short day preconditioning of assay plants and by inoculation of the morphological apex of isolated stem segments. Pretreatment of plants with auxin or cytokinin altered specificity in various combinations of strains and host genotypes. All Vitis species and hybrids formed tumors in response to strains designated as limited host range, but some displayed a necrotic reaction (cell death at and below site of inoculation) or a null response (same as the response to inoculation with an avirulent strain) to strains designated as wide host range (VC Knauf, CG Panagopoulos, EW Nester [1982] Phytopathology 72: 1545-1549). Screens of F1 progeny, derived from crosses of null, necrotic, and tumor-producing phenotypes, demonstrated that the null and the necrotic phenotypes were modulated by dominant and recessive host genes. The extent of cellular necrosis in the necrotic phenotype was modified by the morphological location of the inoculation site, by the presence of buds on the host stem, and by deletion of the tryptophane monooxygenase locus gene of the Ti-plasmid.The tumor-inducing plasmid ofAgrobacterium tumefaciens contains two regions, termed the virulence (vir) and tumorinducing DNA (T-DNA), that coordinately participate in the excision of DNA from the pathogen and its transfer into the host genome (11,17). The current model for infection of host cells by Agrobacterium predicts that phenolic metabolites produced by wounded plant cells activate plasmid vir genes of the bacterium (22). Once activated, the genes modulate the excision and packaging of T-DNA and facilitate its transfer into the host plant genome (17,22). Expression of T-DNA in host cells alters the endogenous levels of auxin, cytokinin, and ethylene (6,11,17), which results in the loss of cellular totipotency (11,17 3,17,27). Bacterial host range and virulence determinants have been attributed to structural and organizational differences in T-DNA loci (1,17,21,(27)(28)(29)30) and to structural differences in at least two vir loci (30,31). Some investigators suggest that chromosomal loci might also modulate host selectivity (17,25). The role of the host genome in Agrobacterium-host compatibility has not received as much consideration as that of the pathogen. However, intergeneric (3,12,25,26,29,30) and intraspecific and cultivar variations (1, 2, 7-9, 19, 23, 24, 27) in hostresponse to WHR and/or LHR strains have been reported. A preliminary analysis of Vitis hybrids showed that resistance to A. tumefaciens was a heritable trait (23).The primary goal of our research was to develop a system to study the interactions of bacterial virulence genes and host genes so that the molecular and bioch...