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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.

Walnut has a large number of chromosomes (2n=32), and there have been very limited cytological studies in this species to date. Since haploid plants have half the number of the chromosomes of the parents, they will be useful to develop the karyotype. In our study, haploid plants of 'Hartley' obtained via parthenogenesis of irradiated pollen with 600 Gy were selected. Chromosome number was assessed in root tip cells of in vitro haploid plants, using the standard Feulgen technique. Micro Measure v3.3 software was used for chromosomal analyses. Length of the 16 chromosomes varied from 1.55 to 3.53 µm. Centromere position of chromosomes were measured according to Levan et al. (1964). The results showed that among the 16 chromosomes, 10 of them were metacentric, 5 submetacentric and 1 subtelocentric.

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