In vitro rooting of three commercial cultivars of Persian walnut (Juglans regia L.), `Sunland', `Chandler', and `Vina', was examined using a two-phase rooting procedure: root induction in the dark on Murashige and Skoog (MS) medium with 15 μm IBA followed by root development in the light on a mixture of one-quarter strength Driver Kuniyuki Walnut (DKW) basal medium and vermiculite (1:1.25, v/v). Rooting percentages were: `Sunland' (94%), `Chandler (55%), and `Vina' (27%). A positive relationship was observed between the vigor of cultivars and rooting ability, but shoot length did not affect rooting success. Rooting was optimum when shoots were cultured on root induction media for 6 to 8 days. Increasing the sucrose level in the root induction medium to 40 g·L-1 improved rooting, and shoots induced to root at 22 °C rooted more readily than those induced at 30 °C. Either increasing or decreasing the nitrogen level in the multiplication medium had a negative effect on rooting. Rooted walnut shoots often cease growth during acclimatization, resulting in shoot rosetting. Spray application of Promalin® (25 mL·L-1) caused buds to break and induced elongation of shoots. Chemical name used: indole-3-butyric acid (IBA).
Kourosh Vahdati, Charles Leslie, Zabihollah Zamani, and Gale McGranahan
Gale H. McGranahan, David E. Ramos, Harold I. Forde, and Ronald G. Snyder
Gale H. McGranahan, Demetrios G. Voyiatzis, Peter B. Catlin, and Vito S. Polito
The role of pollen in abscission of pistillate flowers of Persian walnut (Juglans regia L.) cv. Serr was investigated over a 4-year period by controlled pollinations and pollen counts. Self-pollen, pollen from other walnut selections or cultivars, or dead pollen was applied at high and low doses to pistillate flowers enclosed in pollination bags. Unbagged, open-pollinated flowers and bagged, nonpollinated flowers served as controls. In all cases, presence of pollen significantly increased the probability of pistillate flower abscission (PFA). Dead pollen resulted in as much PFA as live pollen. Counts of pollen grains confirmed that PFA-type flowers had significantly more pollen than normal flowers. In the fourth year `Serr' pollen was applied to unbagged flowers of `Serr' and ten other Persian walnut cultivars, and the amount of PFA on the artificially pollinated flowers was significantly higher than on the open-pollinated flowers, while the control flowers dusted with talc or pine pollen had almost no PFA. These results clearly indicate that excess pollen is involved in pistillate flower abscission in `Serr' walnut and suggests that other cultivars may also be sensitive to pollen load. This phenomenon may have implications in the biology of selfing and evolution.
Mohammed A.M. Aly, Robert G. Fjellstrom, Gale H. McGranahan, and Dan E. Parfitt
Somatic embryos derived from walnut (Juglans regia L.) ovule tissues were evaluated to determine whether they were of zygotic or maternal origin. Molecular markers were used to permit evaluation at an early stage, before whole plant development. Somatic embryos developed from potentially apomictic `Sunland' and `Cisco' ovule tissue isolated from bagged putatively unpollinated flowers. Phosphoglucomutase (PGM) isozyme analysis showed that all of these embryos, except one from each cultivar, carry the same zymotype as the maternal tissue. However, restriction fragment length polymorphism (RPLP) analysis combined with isozyme evaluation demonstrated that the tested embryos originated from zygotic rather than maternal tissues. This study demonstrates the application of molecular marker analyses, particularly RFLPs, evaluation of somatic embryo origin.
L. Carl Greve, Gale McGranahan, Janine Hasey, Ronald Snyder, Kathy Kelly, David Goldhamer, and John M. Labavitch
The variation in polyunsaturated fatty acid content of walnut (Juglans regia L.) oils was determined by analysis of samples isolated from specimens growing in four germplasm collections [California (55 cultivars), Washington (64 seedlings), China (12 cultivars), and France (20 cultivars)]. In addition, the impact of within-state geographic differences on oil composition was examined by comparing samples from three California cultivars (`Ashley', `Hartley', and `Franquette') grown in three locations. Local environmental effects on oil composition of `Chico' were also examined by comparing 1) samples collected from shaded and sun-exposed locations of the same trees and 2) samples collected from trees subjected to three irrigation regimes. Polyunsaturated fatty acid content, as a percentage of total fatty acids, ranged from 47.2% in nuts from PI 142323 from France to 81.0% in `Ashley' from California. However, our data indicate that environment, genotype, nut maturity, and their interactions all contribute significantly to variation in the degree of unsaturation of walnut oil.
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.
Abhava M. Dandekar, Gale H McGranahan, Sandra L. Uratsu, Charles Leslie, J. Steven Tebbets, and Patrick V. Vail
Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis (Bt) encoded by cryIA(c) gene were shown in diet incorporation studies to be lethal to codling moth (CM; Cydia pomonella) the key insect pest for walnut. However transformed walnut tissues expressing cryIA(c) with Bt codon usage patterns and native DNA sequence revealed very low levels of expression in planta. To correct this problem synthetic versions of one of these genes, cryIA(c) was used to transform walnut tissue. A total of 61 individual transgenic embryo lines were obtained. 34% of these lines (21/61) were high expressors (“class A”) demonstrating 80 to 100% mortality of first in star CM larvae and displaying no further larval development. Twelve clones (20%) were designated “class B” and these showed a marked retardation of larval development and a mortality between 40 to 79%. Embryos from the remaining 28 lines designated “class C” (46%). although transformed, were indistinguishable from the control (untransformed embryos) and showed a mortality of 0 to 39%.
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.
Gale H. McGranahan, Harold I. Forde, Ronald G. Snyder, G. Steven Sibbett, Wilbur Reil, Janine Hasey, and David E. Ramos
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.