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Ralph Scorza and Harold W. Fogle

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Jean-Michel Hily, Ralph Scorza*, and Michel Ravelonandro

We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.

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Rajeev Arora, Michael Wisniewski, and Ralph Scorza

Deciduous fruit trees undergo endo-dormancy during fall at which time they also attain maximum cold hardiness (CH). Because these two processes occur simultaneously it is difficult to study them independently. We have been able to overcome this limitation with the use of genetically related (sibling) deciduous and evergreen peach trees. Using this system we conducted a time course study to characterize the seasonal fluctuations in CH and proteins in bark and xylem tissues. Cold hardiness (LT50) was assessed using electrolyte leakage method. Polypeptides were separated using SDS-PAGE. The data indicated that 1) CH of bark increased from -5°C (in August) to -49°C (in January) and from -3°C to -22°C for deciduous and evergreen trees, respectively. In January, under favorable conditions, evergreen trees were actively growing. 2) CH of xylem successively increased from -11°C to -36°C in deciduous trees and from -7°C to -16°C (in November) in evergreen trees and then plateaued. 3) LT50 of xylem in both genotypes closely approximated the mid-point of low temperature exotherms determined by differential thermal analysis. 4) As CH increased several qualitative and quantitative differences in polypeptides were noted between two genotypes. These changes during cold acclimation will be compared with those during de-acclimation.

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Ann M. Callahan, Chris Dardick, and Ralph Scorza

The plum (Prunus domestica) cultivar Stoneless was characterized to determine if the lack of stone was the result of reduced endocarp development or a decrease in lignification. Fruit were sampled at several times from 37 days before stone hardening (DBH) until the stone was too hard to cut with a knife and were compared with plum fruit that had normal stones. At all sampling times there was less endocarp tissue and reduced lignin staining in the ‘Stoneless’ plum fruit. The tissue that did stain appeared to be small endocarp remnants present in the ‘Stoneless’ plum, and was concentrated at the suture side and at the blossom end as well as the stem end. The lignin stain was detected in these regions beginning at 19 DBH, while the normal plums had a progression of staining beginning at the blossom end, suture side at 23 DBH and radiating up to the stem end and throughout the presumptive stone tissue at 8 DBH. Comparison of dry weight for dissected tissues agreed with the specific lack of endocarp tissue in the ‘Stoneless’ plum. Gene activity for the lignin pathway was analyzed using four known genes required for lignification. All four genes showed endocarp-specific expression in ‘Stoneless’ similar to that observed for the control. These results support the idea that the phenotype of ‘Stoneless’ plum fruit is due to a decrease in endocarp formation rather than a decrease in endocarp lignification.

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Ralph Scorza, Daniele Bassi, and Alessandro Liverani

A study was conducted to determine genetic control of the columnar or pillar (PI) growth habit, and to evaluate the effects of interactions of various genes that influence peach [Prunus persica (L.) Batsch (Peach Group)] growth habit. The PI habit (brbr) examined in this study was inherited as a monogenic trait expressing incomplete dominance. The heterozygous Brbr derived from crosses between standard (ST) and PI genotypes was recognized as an upright (UP) tree with narrower branch angles than ST trees but wider than PI trees. The combination of brbr and brachytic dwarf (DW) (dwdw) produced dwarf-pillar (DWPI) trees. The effects of the heterozygous Brbr in combination with dw and/or compact (CT) (Ct) could not be recognized by visual observation. Compact pillar (CTPI) trees resulted from the expression of Ct_ brbr. These trees were distinguished from globe-shaped (GL) trees (Ct_Brbr) by the more upright growth habit of the CTPI trees. This genetic study highlights the genetic plasticity of tree growth habit in peach. The investigation of novel growth habits extends our concept of the peach tree. Some growth habits such as PI may have commercial potential for high-density peach production systems. Others, such as DWPI and CTPI may have potential as ornamentals.

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Alexis K. Nagel, Guido Schnabel, Cesar Petri, and Ralph Scorza

The Gastrodia antifungal protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This lectin has previously been shown to provide increased resistance in transgenic Nicotiana tabacum against taxonomically unrelated root pathogens Phytophthora nicotianae, Rhizoctonia solani, and Meloidogyne incognita, but its potential to confer disease resistance in tree species is not known. Agrobacterium tumefaciens-mediated transformation yielded three gafp-1 expressing plum lines (Prunus domestica) designated 4J, 4I, and 5D. These lines possessed one, two, and four copies of the gafp-1 gene, respectively, as demonstrated by DNA blotting. Lines 4J and 4I were not phenotypically different from the nontransformed control line, but line 5D showed significant divergence in leaf morphology and growth habit. Compared with the inoculated control line, lines 4J and 4I exhibited increased tolerance to Phytophthora root rot (PRR) caused by P. cinnamomi. When inoculated with the root-knot nematode, Meloidogyne incognita, the 4J and 4I lines showed a significantly lower degree of root galling than the inoculated control line. Nematode reproduction, as measured by the presence of egg masses and the number of eggs produced per gram fresh root, was significantly reduced in line 4J compared with the inoculated control line. The results of this study suggest that the expression of gafp-1 in the roots of a woody plant may confer some level of resistance to PRR and root-knot nematode. Long-term field trials will be necessary to confirm this hypothesis.

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Daniela Giovannini, D. Michael Glenn, Ralph Scorza, and W.V. Welker

Our objective was to evaluate the dry-matter partitioning between the roots and shoots of two genetically size-controlled peach [Prunus persica (L.) Batsch] types, dwarf and pillar, compared to a full-sized standard peach type. Compared to the pillar and standard types, the dwarf type had a reduced leaf: root ratio, less allocation of dry matter to woody tissue and more to leaf tissue. Genetically size-controlled peach trees have a smaller root system, but a lower leaf: root ratio and may require modified soil and water management techniques to ensure high productivity.

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Richard L. Bell, Ralph Scorza, Chinnathambi Srinivasan, and Kevin Webb

`Beurre Bosc' pear (Pyrus communis L.) was transformed with Agrobacterium tumefaciens (E.F. Smith & Townsend) Conn strain EHA101 containing the binary vector pGA-GUSGF into which the rolC gene had been inserted. Leaf explants from in vitro shoot tip cultures were wounded, Agrobacterium-inoculated, and cultured on kanamycin selection medium. Regenerating shoots were transferred to proliferation medium without antibiotics. Three clones tested positive for GUS and nptII enzyme activity. Transformation with the rolC gene was confirmed by DNA, RNA, and protein blot analyses. The number of copies of the rolC transgene varied from one to three. Plantlets of the three transgenic clones were acclimated and transferred to the greenhouse. Preliminary observations of phenotype indicate that the rolC gene reduced height, number of nodes, and leaf area of transgenic `Beurre Bosc'.

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Ann Callahan, Chris Dardick, Roberta Tosetti, Donna Lalli, and Ralph Scorza

The theme running through many of Luther Burbank’s breeding programs was to make plants more tailored to human uses. Mr. Burbank thought that the stone in plum fruits was unessential to a tree that was propagated vegetatively, so he chose stoneless plums as a breeding goal. He made two releases, ‘Miracle’ in 1903 and his final and almost perfect ‘Conquest’ in 1916, which he considered one of his best accomplishments in plum breeding. ‘Conquest’ had only a grain of stone and flavor and size comparable to the best French types of the time but was not commercially successful. In view of the current desire for convenience food such as seedless fruit (citrus, grapes, watermelon) and advanced knowledge of genetics and breeding technologies, we have taken up where Mr. Burbank left off in the production of a better than “almost perfect” stoneless plum. We began by locating what were most likely remnants from Mr. Burbank’s breeding program and we are now using 21st century technology to achieve a completely stoneless, high-quality plum fruit. These technologies include molecular markers, genetic engineering, and accelerated breeding cycles (FasTrack). Initial experiments had characterized the stoneless trait as a decrease in the number of endocarp cells that form the stone. We defined the time critical to the formation of endocarp by analyzing gene expression of a number of transcription factors involved with determining endocarp cells. We identified genes that were expressed differently during this period between normal stone cultivars and one of the stoneless cultivars. In addition, we targeted genes for genetic engineering to reduce the lignification in endocarp and to reduce or convert endocarp cells to non-lignifying cells. A system, FasTrack, using a flowering gene from poplar, has been incorporated to reduce the juvenility period and eliminate the seasonal aspect of fruiting to see the results of the breeding as well as the genetic engineering approach much faster. The combination of these approaches is now in place to attempt to improve on Mr. Burbank’s stoneless plum.

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Hetal M. Kalariya, Guido Schnabel, Cesar Petri, and Ralph Scorza

The Gastrodia antifungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously generated, transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were created from seeds derived from open pollination of the cultivar Bluebyrd (BB-OP) with gafp-1 under the control of the polyubiquitin promoter bul409 and evaluated for Phytophthora root rot (PRR) and Root knot nematode (RKN) susceptibility. One of nine transgenic lines synthesizing GAFP-1 exhibited increased tolerance to PRR caused by P. cinnamomi. The same line (BB-OP-1) was also significantly more tolerant to RKN infection caused by Meloidogyne incognita. BB-OP-1 was more resistant to PRR and equally resistant to RKN compared with the cultivar Stanley-derived 4J line, which expresses gafp-1 under the control of the 35SCaMV promoter. GAFP-1 synthesis in BB-OP-1 was not elevated by pathogen infection, suggesting that the bul409 promoter is not inducible in the plum/GAFP-1 system. This study confirms the usefulness of the gafp-1 gene in various cultivars of transgenic plum and establishes that the bul409 promoter is at least equal in effectiveness to the 35SCaMV promoter for gafp-1 expression in transgenic lines of woody plants.