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