Peach plants were repeatedly regenerated from immature embryos but not from callus derived from mature embryos. A white, nodular, highly regenerative callus was obtained when friable, primary callus from immature embryos was transferred from medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 0.44 μM benzyladenine (BA) to media containing 0.27 μM α-naphthaleneacetic acid (NAA) and 2.2 μM BA. This callus retained its morphogenetic potential for a minimum of three subcultures. Green nodular callus, that lacked regenerative capacity, was produced from primary callus derived from mature embryos. Maximum regeneration of shoots occurred when highly regenerative callus was transferred to a medium in which the NAA concentration was reduced five times and the BA concentration was increased two times. Regenerated shoots were rooted in the dark on a medium containing 28.5 μM indoleacetic acid. Cytogenetic analysis of regenerated plants indicated that all plants were diploid, 2n = 2x = 16. Phenotypic evaluation of regenerated plants, grown under field conditions, is now in progress.

Success at propagating peach (Prunus persica (L.) Batsch) scion cultivars in vitro has been limited. This study describes factors influencing in vitro multiplication and rooting of 8 peach scion cultivars and one rootstock, as well as acclimatization and genetic stability of these cultivars. Shoot multiplication was best when 8.8/~M 6-benzylamino purine (BA) was added to the shoot proliferation medium. Maximum rooting occurred when shoots were placed on I/2-strength Murashige and Skoog (MS) medium, stored in the dark at 4 °C for 35 to 40 days and then incubated on rooting medium in the dark at 26 °C for 14 days. All cultivars exposed to l/2-strength MS medium supplemented with 28.5/~M of either indoleacetic acid (IAA), indolebutyric acid (IBA) or ct-napthaleneacetic acid (NAA) rooted best on NAA medium. A 5-fold reduction in NAA concentration to 5.8/~M, the use of IAA plus phenolic rooting cofactors, and length of time shoots were in vitro prior to rooting each increased the percentage of rooting for most cultivars. No plant loss occurred during acclimatization. Cytogenetic analysis of micropropagated plants indicated that all plants were diploid, 2n = 2x = 16. Examination of the performance of in vitro propagated plants under field conditions is now in progress.

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high efficiency shoot regeneration from leaf explants of shoots propagated in vitro. The effects on shoot organogenesis of age of explant source, length of dark treatment, the addition of either thidiazuron (TDZ) at 1 or 5 μm, or zeatin riboside at 20 μm to the regeneration medium, and a photosynthetic photon flux (PPF) of either 18 ± 5 or 55 ± 5 μmol·m–2·s–1 were investigated. A maximum of 13.0, 13.0, 12.6, and 4.6 shoots regenerating per explant for cultivars Duke, Georgiagem, Sierra, and Jersey, respectively, occurred on regeneration medium with zeatin riboside and under a PPF of 55 ± 5 μmol·m–2·s–1. `Duke' regenerated equally well on medium with either zeatin riboside or 1 μm TDZ, whereas the number of shoots per explant for `Georgiagem' and `Sierra' was significantly higher on zeatin riboside. Regeneration of `Duke', `Jersey', and `Sierra' on zeatin riboside was significantly better under a PPF of 55 ± 5 μmol·m–2·s–1 than under 18 ± 5 μmol·m–2·s–1, but the higher PPF inhibited regeneration of `Duke' on 5 μm TDZ. There were no significant differences in percentage of regeneration or the number of shoots per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures, or when either 1 week or 2 weeks of darkness preceded light treatments. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(-β-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

Individual callus cultures were initiated from 400 immature embryos of bacterial leaf spot-susceptible 'Sunhigh' peach. Each was subjected to several selection cycles of a toxic culture filtrate produced by Xanthomonas campestris pv. pruni, the causal agent of leaf spot of peach. Progressively higher concentrations of the filtrate were used in each cycle. Two calli survived, and two plants were regenerated from each of the surviving calli. Each of the four clones was propagated in vitro and tested for whole plant resistance to X. c. pv. pruni. Results from bioassays on greenhouse-grown plants indicated that two out of the four selected clones were significantly more resistant to X. c. pv. pruni than the parental cv 'Sunhigh'. In addition, one clone was significantly more resistant than the moderately resistant cv 'Redhaven'.

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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.

A detached-leaf bioassay was used to evaluate peach [Prunus persica (L.) Batsch] regenerants derived from zygotic embryo callus cultures of cultivars Sunhigh (susceptible to leaf spot) and Redhaven (moderately resistant to leaf spot) for resistance to Xanthomonas campestris pv. pruni [(E.F. Sm.) Dews], the causal agent of bacterial leaf spot. Regenerants obtained from calli produced on two `Sunhigh' embryos, #61 and #156, and on three `Redhaven' embryos were evaluated. Sixty-four percent of the regenerants derived from `Sunhigh' embryo #156 and 13% of the regenerants derived from `Sunhigh' embryo #61 demonstrated significantly greater spot resistance than `Sunhigh'. Regenerants with resistance greater than `Redhaven' were also obtained from both `Sunhigh' embryos. The frequency of variation in the `Sunhigh' seedling population, with respect to the response to bacterial leaf spot, was not so great as that exhibited by the regenerants derived from `Sunhigh' embryo #156. None of the `Redhaven' seedlings or any of the regenerants derived from `Redhaven' embryos were more resistant than `Redhaven'. These studies suggest that the frequency of somaclonal variation is genetically determined and that screening for somaclonal variation may be a feasible approach to obtaining leaf spot-resistant peach plants.