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D. Michael Glenn, Ralph Scorza, and William R. Okie

Two unpruned willow leaf and two unpruned standard leaf peach [Prunuspersica(L.) Batsch.] selections were evaluated for physiological components related to water use efficiency (WUE). The purpose of the study was to assess the value of willow leaf phenotypes to improve water use efficiency in peach and separate the environmental from the genetic components. The willow leaf characteristic itself did not confer improved water use efficiency. Light interception was a key determinant of WUE in these genotypes and the relationship of WUE with intercepted photosynthetically active radiation (PAR) by the entire canopy indicated a significant negative correlation. Internal shading of the tree by excessive leaf area reduced WUE and canopies that intercept more than 60% of the PAR have reduced WUE. While WUE is improved by reducing the amount of PAR interception of the canopy, productivity is reduced. Neither of the willow leaf genotypes had a significant correlation of WUE with yield (leaf and fruit weight); however, the standard leaf type cultivars, `Bounty' and `Redhaven', had significantly different regressions that indicate greater productivity in `Bounty' for a given level of WUE. `Redhaven' was the least productive cultivar; `Bounty' was the most productive, and the two willow leaf genotypes were intermediate in the relationship of intercepted PAR with yield. Therefore, genetic differences in peach growth types can be selected for both increased WUE as well as increased productivity. Future work in peach breeding to improve WUE and productivity must take into consideration light interception, productivity, and WUE in an integrated manner to make progress in the efficient use of water and light.

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Ann Callahan, Ralph Scorza, Peter Morgens, Seth Mante, John Cordts, and Reuben Cohen

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Ann M. Callahan, Peter H. Morgens, Reuben A. Cohen, Ken E. Nichols Jr., and Ralph Scorza

We are interested in identifying and isolating genes which affect the rate of softening in peach fruit. It may be possible through the engineering of these genes to delay or extend the softening. This could ultimately allow for the harvest and transport of more mature, higher quality fruit. The clone, pch313, was isolated from a ripe peach fruit cDNA library. RNA homologous to this clone is detected at a low abundance in fruit until softening when a >100 fold increase in abundance of the RNA occurs. Pch313 RNA is also detected 30 min after wounding leaf or fruit tissue and peaks in accumulation within 2-8 hours. Wound ethylene was measured from the same tissue and its rate of evolution paralleled the accumulation of the RNA. The cDNA was sequenced and found to have 78% sequence identity with pTom13, a tomato gene that is expressed during fruit ripening and wounding (Holdsworth et al., NAR 15:731-739, 1987). To determine the universality of pch313 related gene expression, RNA accumulation was measured in other fruits during softening, and in leaf tissue upon wounding.

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Ann M. Callahan, Peter H. Morgens, Reuben A. Cohen, Ken E. Nichols Jr., and Ralph Scorza

We are interested in identifying and isolating genes which affect the rate of softening in peach fruit. It may be possible through the engineering of these genes to delay or extend the softening. This could ultimately allow for the harvest and transport of more mature, higher quality fruit. The clone, pch313, was isolated from a ripe peach fruit cDNA library. RNA homologous to this clone is detected at a low abundance in fruit until softening when a >100 fold increase in abundance of the RNA occurs. Pch313 RNA is also detected 30 min after wounding leaf or fruit tissue and peaks in accumulation within 2-8 hours. Wound ethylene was measured from the same tissue and its rate of evolution paralleled the accumulation of the RNA. The cDNA was sequenced and found to have 78% sequence identity with pTom13, a tomato gene that is expressed during fruit ripening and wounding (Holdsworth et al., NAR 15:731-739, 1987). To determine the universality of pch313 related gene expression, RNA accumulation was measured in other fruits during softening, and in leaf tissue upon wounding.

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Zongrang Liu, Ralph Scorza, Jean-Michel Hily, Simon W. Scott, and Delano James

Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

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Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza

Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.

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Ying Wang, Laura L. George, Gregory L. Reighard, Ralph Scorza, and Albert G. Abbott

Evergreen genotypes of peach [Prunus persica (L.) Batsch] have been identified in Mexico, where terminal growth on evergreen trees is continuous under favorable environmental conditions. This evergreen trait in peach is controlled by one single gene (evg), and this evergreen condition is homozygous recessive. Four dominant AFLP markers, EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC, were found to be tightly linked to the evg locus at 1 cM, 4.6 cM, 5.8 cM, and 11 cM, respectively. All four markers were sequenced and identified. A peach BAC library was constructed by using the pBeloBAC11 vector for building the physical map for the evg gene. This library represents four times the coverage of the peach genome with the average insert size of 50 to 70 kb. The EAT/MCAC AFLP marker fragment was used for screening the peach BAC library. A single BAC clone, 18F12, was confirmed to contain this fragment. The final BAC contig for this evg gene region and the potential homology between peach and Arabidopsis thaliana will be presented and discussed.

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Desmond R. Layne, Guido Schnabel, Kerik Cox, Ralph Scorza, and Karen Bussey

Armillaria root rot (ARR) of peach caused by the soil-borne basidiomycete fungus Armillaria tabescens is causing premature decline and mortality of peach trees on most southeastern U.S. peach farms. Soil inoculum may be present both in former peach orchard sites and on sites that were once in hardwood forest. The fungus is protected under the bark of dead root pieces and may survive up to 100 years at various depths in the soil profile. No commercially available rootstocks are resistant to ARR. Since 2002, we have embarked on a multipronged strategy to develop control options to combat ARR. First, we have two replicated trials on commercial grower replant sites with a history of ARR. Trial 1 compares four preplant fumigation treatments (none, Telone II, methyl bromide, and Enzone), three rootstocks (Lovell, Halford, and Guardian) and preplant root dips with endomycorrhizal fungi. Trial 2 compares the use of raised beds, root collar excavation and preplant root dips. Both trials examine long-term productivity and tree survival. Second, we are examining the use of systemic fungicide injection into infected trees to protect trees around infection foci. Third, we are trying to develop a genetically modified ARR-resistant rootstock. We have inserted the gene encoding the gastrodia antifungal protein (GAFP—a low molecular weight lectin that binds mannose and chitin) from a Chinese orchid into tobacco (model herbaceous system) and plum (model Prunus system). GAFP has antifungal activity against several basidiomycete root rot pathogens. Pathogenicity tests with transformed tobacco plants show enhanced tolerance to several root rot pathogens when compared to nontransformed plants. Transformed plums are being multiplied for pathogenicity tests.

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Xiaojian Ye, Susan K. Brown, Ralph Scorza, John Cordts, and John C. Sanford

Physical and biological parameters affecting the efficiency of biolistic transformation of peach were optimized using ß-glucuronidase (GUS) as a reporter gene, such that efficiency of transient GUS expression in peach embryo-derived callus was increased markedly. Transient expression was also obtained in embryonic axes, immature embryos, cotyledons, shoot tips, and leaves of peach. Stable expression of a fusion gene combining neomycin phosphotransferase (NPTII) and ß-glucuronidase activities has been achieved in peach embryo calli. Sixty-five kanamycin-resistant callus lines were obtained from 114 pieces of bombarded calli after 4 months of selection. Nineteen of the 65 putative transformant lines produced shoot-like structures. Seven lines were examined to confirm stable transformation using the colorimetric GUS assay and PCR analysis. All seven lines showed GUS activity. PCR analysis confirmed that, in most of the putative transformants, the chimeric GUS/NPTII gene had been incorporated into the peach genome. The transgenic callus lines were very weakly morphogenic, presumably because the callus was 5 years old and no transgenic shoots developed from this callus. Results of this research demonstrate the feasibility of obtaining stable transgenic peach tissue by biolistic transformation.

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John C. Beaulieu, Karen L. Bett, Elaine T. Champagne, Daphne A. Ingram, James A. Miller, and Ralph Scorza

Many consumers do not buy peaches due to the fuzzy skin and seed stone and because out-of-season peaches do not possess optimum tree-ripe flavor. The feasibility of using a non-browning freestone peach to deliver high-quality fresh-cut products was investigated. Changes in fresh-cut flavor, texture, and postharvest attributes of commercial-ripe (CR) vs. tree-ripe (TR) harvested and shipped `Bounty' peach was assessed. Fresh-cut CR wedges had an initial firmness of 20.9 N, whereas TR wedges had 11.2 N. On day 2, firmness decreased roughly 3% to 12% and 35% to 45% for CR and TR wedges held at 1 °C, respectively. By day 5, CR wedges hardened (24.5 N) whereas TR did not return to their initial firmness; increasing marginally through day 7. Sensory panel hardness for CR did not change through storage, but with TR wedges, hardness decreased through day 2 then increased until day 7. Little variation was noted in the initial soluble solids for CR vs. TR wedges (11.7, vs. 11.4 °Brix, respectively). After 7 days storage, °Brix decreased 7.5% to 12% in CR and 4.5% to 12% in TR wedges. Yellow flesh color (b*) decreased in all CR and TR treatments through storage. Flavor compounds in expressed juice were analyzed by solid phase microextraction with GC-MS. Several peaks were identified that may be associated with flavor-related changes that occurred during storage. For example, low molecular weight acetates and 6C compounds almost disappeared during storage, whereas short chain fatty acids, lactones, and palmitic acid increased markedly through storage. In TR, the “fruity” descriptor decreased throughout storage and “sweet aromatic” increased slightly (day 2) then decreased through day 7.