The effects of osmotic stress induced by polyethylene glycol on the seed germination of 16 walnut genotypes (‘Z ₃₀ ’, ‘Z ₅₃ ’, ‘Z ₆₇ ’, ‘Z ₆₀ ’, ‘Z ₆₃ ’, ‘K ₇₂ ’, ‘B ₂₁ ’, ‘V ₃₀ ’, ‘Panegine ₂₀ ’, ‘Hartley’, ‘Pedro’, ‘Vina’, ‘Lara’, ‘Serr’, ‘Ronde de Montignac’, and ‘Chandler’) of Juglans regia L. were studied. Potted seeds were kept under controlled conditions (12/12-h light/dark photoperiod and 25 ± 1 °C) during the experiments. The objective was to screen genotypes and determine the critical range of osmotic potential (ψ _S ) for walnut seeds during germination. Decreasing the ψ _S of the germination solutions markedly reduced germination percentage in all genotypes, but there were variations in degree among the genotypes. The Z genotypes were the most sensitive to osmotic stress, and their germination rates were the lowest at ψ _S more negative than –0.75 MPa. Cluster analysis produced a dendrogram with four groups differing in their tolerance to osmotic stress. Based on factor analysis, four factors explained 90.45% of data total variance. Factor analysis showed that tissue fresh and dry weight, tissue water content, and thickness were the most important traits under drought condition. Regression analysis failed to show a significant relationship between percent germination and either seed weight ( r ² = 0.0601) or kernel weight ( r ² = 0.0258).

The objective of this work was to determine the chilling and heat requirements of Persian walnut cultivars and genotypes using excised twigs. The experiment was carried out from Nov. 2006 and 2007 to Mar. 2007 and 2008. One-year-old twigs were prepared from four cultivars and four domestic genotypes of Juglans regia L. After leaf fall, the twigs were taken and placed in plastic bags and kept at 4 ± 1 °C to stimulate 400 to 1500 chilling hours. After chilling, the excised twigs were transferred to the greenhouse with a natural photoperiod and a temperature from 18 to 27 °C. The evaluation of budbreak was made three times a week and the number of accumulated growing degree hours (°C) was determined until the buds reached the balloon or green tip stage. The chilling requirements were lowest (400 h) for catkins and highest (1000 h) for lateral buds. The Serr cultivar and ‘Z ₃₀ ’ genotype had the lowest chilling requirements (650 and 650 h). ‘Lara’, ‘Z ₆₃ ’, ‘Z ₅₃ ’, ‘Pedro’, and ‘Z ₆₇ ’ showed intermediate chilling requirements with values of 900, 900, 800, 750, and 750 h, respectively. Finally, ‘Hartley’ completed its dormancy after an accumulation of 1000 h, being the walnut cultivar with the highest chilling requirement in our study. As the final result, the cultivars and genotypes were classified into three groups based on their heat requirements: low requirement (‘Z ₃₀ ’ and ‘Serr’), medium requirement (‘Z ₅₃ ’, ‘Z ₆₇ ’, ‘Lara’, and ‘Pedro’), and high requirement (‘Hartley’ and ‘Z ₆₃ ’).

Iran is a center of origin and diversity for walnuts (Juglans regia L.) with very good potential for breeding purposes. The rich germplasm available, creates an opportunity for study and selection of the diverse walnut genotypes. In this study, the population structure of 104 Persian walnut accessions was assessed using AFLP markers in combination with phenotypic variability of 17 and 18 qualitative and quantitative traits respetively. The primers E-TG/M-CAG, with high values of number of polymorphic bands, polymorphic information content, marker index and Shannon’s diversity index, were the most effective in detecting genetic variation within the walnut germplasm. Multivariate analysis of variance indicated 93.98% of the genetic variability was between individuals, while 6.32% of variation was among populations. A relatively new technique, an advanced maximization strategy with a heuristic approach, was deployed to develop the core collection. Initially, three independent core collections (CC1–CC3) were created using phenotypic data and molecular markers. The three core collections (CC1–CC3) were then merged to generate a composite core collection (CC4). The mean difference percentage, variance difference percentage, variable rate of coefficient of variance percentage, coincidence rate of range percentage, Shannon’s diversity index, and Nei’s gene diversity were employed for comparative analysis. The CC4 with 46 accessions represented the complete range of phenotypic and genetic variability. This study is the first report describing development of a core collection in walnut using molecular marker data in combination with phenotypic values. The construction of core collection could facilitate the work for identification of genetic determinants of trait variability and aid effective utilization of diversity caused by outcrossing, in walnut breeding programs.

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 (Z ₆₃ and Z ₆₇ ) were pollinated using pollen of selections Z ₅₃ and Z ₃₀ 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’, Z ₆₃ , and Z ₆₇ 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.