An experiment was conducted in a persian walnut (Juglans regia) orchard in the north of Iran to evaluate the effects of zinc (Zn) as zinc sulfate and/or boron (B) as boric acid in foliar spray with different concentrations and combinations. Three B and three Zn concentrations (0, 174, and 348 mg·L−1 for B and 0, 1050, and 1750 mg·L−1 for Zn) were applied either independently or in combination. Leaf nutrient concentrations, pollen germination, fruit set, leaf chlorophyll index, nut and kernel characteristics, vegetative growth, nut weight, and nut yield were measured to assess the effects of treatments. The results showed that all B and Zn applications and combinations had a significant effect on all traits except nut and kernel diameter, shell percent, husk thickness, and pistillate flower abscission (PFA). Pollen germination, fruit set, vegetative growth, nut weight, kernel percent, nut and kernel length, and chlorophyll index were highest when B and Zn were applied simultaneously at 174 and 1050 mg·L−1 concentrations, respectively.
Karim Keshavarz, Kourosh Vahdati, Mahmoud Samar, Behzad Azadegan, and Patrick H. Brown
Azadeh Behrooz, Kourosh Vahdati, Farhad Rejali, Mahmoud Lotfi, Saadat Sarikhani, and Charles Leslie
Drought stress is one of the main constraints limiting worldwide crop production. Arbuscular mycorrhizae (AM) and plant growth-promoting bacteria (PGPB) such as Azotobacter chroococcum and Azospirillium lipofrum have been shown to alleviate drought stress effects. Therefore, the interaction effect of AM fungi [Glomus mosseae, G. etunicatum, and a mix of these (G. mix), and PGPB bacteria (Azotobacter chroococcum + Azospirillium lipofrum)] was investigated in 1-year-old walnut seedlings (cv. Chandler) under normal and drought stress conditions. Drought stress reduced growth (plant height, root length, number of leaves, and fresh weight) and leaf nutrient content (N, P, and Zn) significantly of walnut plants. In contrast, proline, total soluble sugar, starch peroxidase enzyme activity, and total phenolic content of walnut leaves increased under this stress. Application of fungi or bacteria, and especially their simultaneous use, alleviated the negative effects of drought stress on walnut seedlings. AM fungi and PGPB increased significantly the content of some metabolites, including total phenolic content, proline level, peroxidase activity, total soluble sugar, and starch content as well as peroxidase enzyme activity. This led to an increase in walnut plant growth under the drought stress condition. Among AM fungi, G. etunicatum was more effective in reducing drought stress symptoms than either G. mosseae or the G. mix of fungi. In conclusion, use of G. etunicatum, along with PGPB, can reduce negative effects of drought stress on walnut seedlings.
Mohammad Sadat Hosseini Grouh, Kourosh Vahdati, Mahmoud Lotfi, Darab Hassani, and Nejat Pirvali Biranvand
We report the first successful regeneration of haploid lines in persian walnut (Juglans regia) developed by in situ parthenogenesis followed by embryo rescue. Female flowers of cultivars Hartley and Pedro and two native Iranian selections (Z63 and Z67) were pollinated using pollen of selections Z53 and Z30 that had been irradiated with gamma rays at five doses (50, 150, 300, 600, and 900 Gy). Gamma-irradiated pollen induced fruit set and development of some parthenogenetic embryos. The immature embryos were excised 30 and 45 days after pollination, cultured in vitro, and then stratified for 30 days at 4 °C to overcome dormancy. Ploidy level of the resulting plantlets was determined by chromosome counting and flow cytometry. Haploid plants were obtained from ‘Hartley’, ‘Pedro’, Z63, and Z67 after pollination using pollen irradiated at 300 and 600 Gy. Plants obtained from pollen irradiated at 50 and 150 Gy were all diploid. Molecular marker analysis using four simple sequence repeat (SSR) markers also showed that all the diploid plants recovered were zygotic and no spontaneous double haploid plants were obtained in this work. Also, the haploid plantlets presented only one allele of their female parents. These profiles confirmed the parthenogenetic origin of the obtained haploid plants. The techniques used to induce haploid walnut plants by irradiated pollen were successful and could be used in breeding programs and accelerate genome analysis in this plant in which the genome size is approximately three times the size of the human genome.
Darab Hassani, Mohammad Reza Mozaffari, Asghar Soleimani, Raana Dastjerdi, Reza Rezaee, Mansureh Keshavarzi, Kourosh Vahdati, Ahmad Fahadan, and Jamal Atefi
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.
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.