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Mohammad Sadat-Hosseini, Kourosh Vahdati, and Charles A. Leslie

Somatic embryos (SEs) can play important roles in genetic manipulation and breeding. They can be used as targets for induced mutagenesis, as material for cryopreservation and germplasm conservation, and for transformation or gene editing in support of plant improvement and proof of gene function. However, germination rates of walnut (Juglans regia) SEs are low, and the genetic stability of plantlets regenerated from them has not been explored. Here, we studied first the effects of gibberellic acid (GA3) and low temperature storage (LTS) on germination of walnut somatic embryos. Second, we assessed the genetic fidelity of plantlets regenerated from these SEs by comparing them to each other and to their cultivar of origin. Results showed that GA3 and LTS increased the walnut SE germination rate. The best rate was observed when SEs were subjected to LTS for 60 d followed by culture on a medium with either 1 or 3 mg·L−1 GA3 (56.6% and 46.6% germination respectively). Genetic stability was evaluated, using flow cytometry and 15 sets of ISSR primers. Flow cytometry indicated that all samples (i.e., regenerated and parental counterpart) showed the same peak. Amplified fragments of inter simple sequence repeats (ISSR) primers ranged in size from ≈200 to 1800 bp. All ISSR profiles of regenerants were monomorphic. Results did not show any genetic differences among plantlets regenerated from SEs or from their parental counterpart. Due to this apparent genetic stability, walnut SEs can be useful for genetic transformation and germplasm conservation.

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Greg T. Browne, Joseph A. Grant, Leigh S. Schmidt, Charles A. Leslie, and Gale H. McGranahan

Seedlings from seven open-pollinated selections of Chinese wingnut (Pterocarya stenoptera) (WN) representing collections of the USDA-ARS National Clonal Germplasm Repository at Davis, CA, and the University of California at Davis were evaluated as rootstocks for resistance to Phytophthora cinnamomi and P. citricola and graft compatibility with scions of five cultivars of Persian walnut (Juglans regia). Seedlings of Northern California black walnut (NCB) (J. hindsii) and Paradox hybrid (PH) (typically J. hindsii × J. regia) were used as standards. In greenhouse experiments, potted plants of the rootstocks were subjected to intermittent flooding in soil artificially infested with the pathogens. All WN seedlings were relatively resistant to the pathogens (means of 0% to 36% of root and crown length rotted) compared with NCB (44% to 100%) and PH seedlings (11% to 100%). Negligible disease occurred in flooded control soil without the pathogens. In 9-year graft compatibility trials in an orchard, NCB and PH rootstocks supported relatively good survival and growth of all tested scion cultivars (‘Chandler’, ‘Hartley’, ‘Serr’, ‘Tulare’, and ‘Vina’; final scion survival 80% to 100%, mean scion circumference increase 292 to 541 mm), whereas results with WN were mixed. Wingnut rootstocks from all sources were incompatible with ‘Chandler’ (final scion survival 20% to 60%, scion circumference increase 17 to 168 mm). Conversely, all WN rootstocks from all sources were compatible with ‘Tulare’ and ‘Vina’ (final scion survival 80% to 100%, scion circumference increase 274 to 556 mm). Use of the WN rootstocks produced variable results in ‘Hartley’ and ‘Serr’ (final scion survival 10% to 100%, mean scion circumference increase 69 to 542 mm). There was a tendency for more rootstock sprouts on WN selections than on NCB or PH. In a commercial walnut orchard infested with P. cinnamomi, ‘Hartley’ survived and grew markedly better on WN selections than on PH. High resistance to P. cinnamomi and P. citricola was common to all of the WN selections. The results indicate that WN selections may be useful rootstocks for cultivars Tulare and Vina in soils infested with P. cinnamomi or P. citricola and that WN selections may contribute valuable resistance to these pathogens in walnut rootstock breeding efforts.

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Fatemeh Khodadadi, Masoud Tohidfar, Mehdi Mohayeji, Abhaya M. Dandekar, Charles A. Leslie, Daniel A. Kluepfel, Timothy Butterfield, and Kourosh Vahdati

Plants respond to pathogens with both active and passive defense mechanisms. These defense responses include the induction of defense or defense-related genes such as polyphenol oxidase (PPO) and pathogenesis-related (PR) proteins. The role of PPO in the interaction between bacterial blight [Xanthomonas arboricola pv. juglandis (Xaj)] and walnut (Juglans regia) was studied. JrPPO-1 and P14a genes were identified in two walnut cultivars, Chandler and Serr, using standard polymerase chain reaction (PCR) to understand their inducible ability in response to Xaj. ‘Serr’ and ‘Chandler’ were inoculated with Xaj strain 417. PPO activity in leaves was assayed at 0, 24, 72, 96, 120, and 144 hours after inoculation. Results showed a steady increase in activity commencing within 24 hours of inoculation. Increase in PPO activity was close to 2-fold greater in ‘Chandler’ than in ‘Serr’ at all time points examined. Real-time PCR analysis showed differences between cultivars in PPO gene expression. The JrPPO-1 gene was highly expressed in both cultivars 24 hours after inoculation but expression in ‘Serr’ was much greater than in ‘Chandler’. Significant expression of P14a gene was observed in both cultivars within 24 hours. Expression in ‘Serr’ was strong and maximized with a significant increase at 96 hours. Expression in ‘Chandler’ was far weaker than ‘Serr’ at 24 hours and did not increase further. Our results imply that the walnut–bacterial blight interaction induces the expression of JrPPO-1 and P14a as well as the activity of PPO.

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Gerald S. Dangl, Keith Woeste, Mallikarjuna K. Aradhya, Anne Koehmstedt, Chuck Simon, Daniel Potter, Charles A. Leslie, and Gale McGranahan

One hundred and forty-seven primer pairs originally designed to amplify microsatellites, also known as simple sequence repeats (SSR), in black walnut (Juglans nigra L.) were screened for utility in persian walnut (J. regia L.). Based on scorability and number of informative polymorphisms, the best 14 loci were selected to analyze a diverse group of 47 persian walnut accessions and one J. hindsii (Jepson) Jepson ex R.E. Sm × J. regia hybrid (Paradox) rootstock. Among the 48 accessions, there were 44 unique multi-locus profiles; the accessions with identical profiles appeared to be synonyms. The pairwise genetic distance based on proportion of shared alleles was calculated for all accessions and a UPGMA (unweighted pair group method with arithmetic mean) dendrogram constructed. The results agree well with what is known about the pedigree and/or origins of the genotypes. The SSR markers distinguished pairs of closely related cultivars and should be able to uniquely characterize all walnut cultivars with the exception of budsports. They provide a more powerful and reliable system for the molecular characterization of walnut germplasm than those previously tested. These markers have numerous applications for the walnut industry, including cultivar identification, verification of pedigrees for cultivar and rootstock breeding programs, paternity analysis, and understanding the genetic diversity of germplasm collections.

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Gregory T. Browne, Charles A. Leslie, Joseph A. Grant, Ravindra G. Bhat, Leigh S. Schmidt, Wesley P. Hackett, Daniel A. Kluepfel, Reid Robinson, and Gale H. McGranahan

Species of Phytophthora are serious soilborne pathogens of persian (english) walnut, causing crown and root rot and associated production losses worldwide. To facilitate the development of improved walnut rootstocks, we examined resistance of 48 diverse clones and seedlings of Juglans species to P. cinnamomi and P. citricola. Plants were micropropagated, acclimatized to a greenhouse environment, and then exposed to the pathogens in artificially infested potting soil mix. Inoculated plants, as well as noninoculated controls, were subjected to soil flooding for 48 hours every 2 weeks to facilitate infection by the pathogens. Two to 3 months after inoculation, resistance to the pathogens was assessed according to the severity of crown and root rot. Clonal hybrids of J. californica × J. regia were highly susceptible to the pathogens (means 52% to 76% root crown length rotted), while several clones of J. microcarpa × J. regia were significantly less susceptible (means 8% to 79% crown length rotted). Among clones of other parentages tested, including: J. microcarpa, (J. californica × J. nigra) × J. regia, J. hindsii × J. regia, (J. hindsii × J. regia) × J. regia, [(J. major × J. hindsii) × J. nigra] × J. regia, and J. nigra × J. regia, responses varied, but tended to be intermediate. When ‘Serr’ scions were budded or grafted on J. microcarpa × J. regia clone ‘RX1’ or Paradox (J. hindsii × J. regia) seedling rootstocks in a commercial orchard infested with P. cinnamomi, all trees on ‘RX1’ remained healthy, whereas only 49% of those on Paradox survived. Thus, useful resistance to Phytophthora is available among J. microcarpa × J. regia hybrids and is evident in ‘RX1’ rootstock.

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Kourosh Vahdati, James R. McKenna, Abhaya M. Dandekar, Charles A. Leslie, Sandie L. Uratsu, Wesley P. Hackett, Paola Negri, and Gale H. McGranahan

Walnuts (Juglans spp.) are difficult-to-root woody plants. The rolABC genes (rolA + rolB + rolC), derived from the bacteria Agrobacterium rhizogenes, have been shown to increase the rooting potential of other difficult-to-root woody plants. We inserted the rolABC genes into somatic embryos of a `Paradox' hybrid (J. hindsii × J. regia) clone PX1 using the A. tumefaciens gene transfer system. A transgenic sub-clone, designated PX1 rolABC 2-2 was selected and compared to the untransformed clone for a variety of phenotypic characteristics, including rooting potential. Transformed and untransformed shoots were budded onto seedling J. regia rootstock in the greenhouse and established in the field. Transformed trees displayed reduced internode length, an increase in lateral branching, and wrinkled leaves. In another test, a commercial persian walnut cultivar J. regia `Chandler' was grafted onto rooted cuttings of both the untransformed and transformed plants. The presence of the rolABC genes in the rootstock had no visible effects on the grafted scion. Several of these trees were excavated from the field and the root systems of each genotype were examined for root number, diameter, and biomass. Trees with the rolABC rootstock had significantly more small diameter roots compared to the controls and less recovered biomass. Tests of the rooting potential of leafy semi-hardwood cuttings for two years resulted in 14% to 59% rooting of the transformed cuttings compared to 51% to 81% rooting of the control. Both transformed hardwood cuttings and microshoots in tissue culture also rooted significantly less (52% and 29% respectively) than untransformed hardwood cuttings and tissue cultured shoots (82% and 54% respectively). Thus, although the rolABC genes induced a shorter internode length and a more fibrous root system (typical of rol-tranformed plants), they were not useful for increasing the rooting potential, and as rootstock they did not affect the phenotype of the scion.

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Chad E. Finn and John R. Clark