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David Mills and Freddi A. Hammerschlag

Pathogenic bacteria, such as Xanthomonas campestris pv. pruni, cause diseases of significant economical implications in the Prunus genus. Cecropins are naturally occurring bactericidal peptides found in the hemolymph of insects. Cecropins cause channel formation in membranes and lysis of bacterial cells. We are interested in engineering the gene for cecropin into peach (Prunus persica) and other fruit tree species. The objective of this study was to determine the effect of cecropin B on viability, using fluorescein diacetate staining, and on changes in transmembrane electrical potential (PD) using the fluorescing probe merocyanine-540. Protoplasts were isolated from shoot-tip cultures in a CPW13M (salts + 0.71M mannitol) solution containing 2% cellulase and 0.5% macerase, while cells were isolated in CPW15.4S (salts + 0.45M sucrose) containing 0.5% cellulase and 0.5% macerase. Cecropin B (1μM) had no effect on viability and changes in PD, while 10μM had a slight effect, and 100μM cecropin B caused significant depolarization and lysis of peach protoplasts. No effect on viability and change in PD were observed in cells when treated with 1-100μM cecropin B. These results suggest that cells and protoplasts of peach can resist cecropin B in the concentration range that causes lysis of plant pathogenic bacteria. The implication of using cecropin to increase microbial disease resistance will be discussed.

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Xiaoling Cao, Freddi A. Hammerschlag, and Larry Douglass

As part of a program to improve highbush blueberry (Vaccinium corymbosum L.) cultivars via tissue culture and genetic engineering, studies were conducted to determine optimum conditions for organogenesis from leaf explants of the previously recalcitrant cv. Bluecrop. The effects of a pretreatment, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% explants regenerated shoots with a mean of 11 shoots per leaf explant after 62 days when explants of 2-week-old shoot cultures were incubated on the following regime: pretreatment medium #1 containing 5 μm TDZ and 2.6 μm NAA for 4 days, pretreatment medium #2 containing 7 μm zeatin riboside and 2.6 μm NAA for 3 days, regeneration medium containing 1 μm TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatment prior to incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on regeneration medium containing 1 μm TDZ was about three times that on 0.5 μm TDZ or 20 μm zeatin riboside, and nine times that on 5 μm TDZ. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(β-D-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

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Ann C. Smigocki and Freddi A. Hammerschlag

Immature `Redhaven' peach [Prunus persica (L.) Batsch] embryos were infected with a shooty mutant strain of Agrobacterium tumefaciens, tms328::Tn5, which carries an octopine-type Ti plasmid with a functional cytokinin gene and a mutated auxin gene. Shoots were regenerated from embryo-derived callus that was initiated on MS medium lacking phytohormones. Shoots exhibited increased frequency of branching and were more difficult to root than the noninfected. Transcripts of the tms328::Tn5-cytokinin gene were detected using northern analyses of total plant RNA. Polymerase chain reaction of genomic DNA and cDNA resulted in amplification of DNA fragments specific for the cytokinin gene, as determined by restriction enzyme and Southern analyses. The concentrations of the cytokinins zeatin and zeatin riboside in the leaves of regenerated plants were on the average 51-fold higher than in leaves taken from nontransformed plants. None of the shoots or callus tissues were postive for octopine. The expression of the T-DNA encoded cytokinin gene promotes growth of peach cells in the absence of phytohormones, thus serving as a marker for transformation. In addition, this gene appears to promote morphogenesis without an auxin inductive step.

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Wayne A. Mackay, Timothy J Ng, and Freddi A. Hammerschlag

Studies examining exposure methods and callus type were conducted to develop an in vitro selection system using roridin E as a selection agent. Vacuum infiltration of callus with the toxin solution was the only successful selection method at the concentrations tested. Primary callus (callus originating directly from the explant) was not sensitive to roridin A or E at the concentrations used. Secondary callus (callus produced from primary callus) exhibited a differential response to roridins A and E similar to that of detached-leaf assays. Electrolyte leakage studies of callus were not conclusive in establishing the membrane as the site of toxin action or useful for screening tolerance in vitro. A small percentage of callus from tolerant and susceptible cultivars survived repeated exposure to roridin E at 50 μg·ml-1.

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Freddi A. Hammerschlag, Ghazala Hashmi, Robin Huettel, Dennis Werner, and David Ritchie

One approach for obtaining useful genetic variation is to select for somaclonal variants generated by tissue culture techniques. Increased levels of resistance to bacterial leaf spot (Xanthomonas campestris pv. pruni) have been observed in toxin-selected and unselected peach regenerants in vitro, in the greenhouse and under field conditions. Peach regenerants have also demonstrated increased levels of bacterial canker (Pseudomonas syringae pv. syringae) and root-knot nematode (Meloidogyne incognita) resistance. Random amplified polymorphic DNA (RAPD) primers have been used to study genetic variation at the DNA level among the somaclonal variants. Sixty RAPD primers (10-mers) were screened and 10 proved useful as markers to detect polymorphisms, thus establishing a genetic basis for somaclonal variation. These studies demonstrate the feasibility of using tissue culture techniques to generate fruit trees with increased levels of disease resistance.

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Freddi A. Hammerschlag, Richard H. Zimmerman, Umedi L. Yadava, Sally Hunsucker, and Petya Gercheva

A range of antibiotics and short-term exposure to an acidified (pH 3.0) medium were evaluated for their effects on eliminating Agrobacterium tumefaciens, supervirulent strain EHA101 (pEHA101/pGT100), from leaf explants of `Royal Gala' apple (Malus ×domestica Borkh.) and on shoot regeneration. Exposure of leaf explants to regeneration and elongation media containing 100 μg·mL-1 concentrations of the antibiotics carbenicillin (crb), cefotaxime (cef), and cefoxitin [=mefoxin (mef)], singly or in combination for 52 days did not eliminate A. tumefaciens from the explants. The percentage of regeneration on crb, cef, and mef was 97%, 11%, and 50%, respectively, compared to 67% for the controls. Short-term (1- to 18-hour) vacuum infiltration with 500 μg·mL-1 of any of the above antibiotics did not inhibit regeneration and failed to eliminate A. tumefaciens from leaf explants. Cef (2000 μg·mL-1) did not inhibit the percentage of regeneration and was more effective than crb or mef in preventing growth of A. tumefaciens when vacuum infiltrated into apple leaf explants for 30 minutes. Further experiments demonstrated that the incidence of A. tumefaciens contamination could be reduced to 28% without negatively impacting shoot regeneration by using a 1-hour vacuum infiltration with an acidified medium, an 18-hour vacuum infiltration with cef (5000 μg·mL-1), and a 52-day incubation on regeneration and elongation media containing 100 μg·mL-1 each of mef and crb. Kan resistant, GUS (β-glucuronidase) positive, putative transformants without A. tumefaciens were generated by adding kan (10 μg·mL-1) to the regeneration and elongation media.

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Kathleen Heuss-LaRosa, Rosemarie Hammond, James M. Crosslin, Christine Hazel', and Freddi A. Hammerschlag

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] shoot cultures with Prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. Infected shoots served as graft bases and source of the virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a (DIG)-labeled cRNA probe derived from PNRSV RNA 3. Rates of successful grafting ranged from 55% to 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in source shoots, suggesting that in vitro micrografting should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV. Chemical name used: digoxigenin (DIG).

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Freddi A. Hammerschlag*, Sandra Garces, Margery Koch-Dean, Stephanie Ray, Kim S. Lewers, John L. Maas, and Barbara J. Smith

Diseases affecting strawberries have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose caused by the fungus Colletotrichum acutatum is one of the most serious disases of strawberry worldwide. Although chemical controls are being used to treat anthracnose, generating disease resistant plants is a more attractive solution to the problem because chemicals can pose a health hazard, have a negative impact on the environment and may only be moderately effective. Tissue culture-induced (somaclonal) variation provides us with one strategy for generating disease-resistant genotypes. An in vitro screening system was used to evaluate several commercially important cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie, and shoots regenerated from leaf explants of these cultivars for resistance to C. acutatum isolate Goff (highly virulent). Somaclones with increased levels of anthracnose resistance were identified for all the cultivars. The greatest increases in disease resistance were observed for somaclones of cultivars Chandler, Pelican and Sweet Charlie that exhibited 6.8-, 12-, and 4.2-fold increases in resistance, respectively. These studies provide evidence that: 1) in vitro screening can be used to evaluate strawberry germplasm for anthracnose resistance, 2) soma-clonal variation is influenced by stawberry genotype, and 3) generating somaclonal variants may be a feasible approach to obtaining strawberry plants with increased levels of anthracnose resistance.