Production of Haploids in Persian Walnut through Parthenogenesis Induced by Gamma-irradiated Pollen

Abstract

<jats:p>We report the first successful regeneration of haploid lines in persian walnut (<jats:italic>Juglans regia</jats:italic>) developed by in situ parthenogenesis followed by embryo rescue. Female flowers of cultivars Hartley and Pedro and two native Iranian selections (Z<jats:sub>63</jats:sub> and Z<jats:sub>67</jats:sub>) were pollinated using pollen of selections Z<jats:sub>53</jats:sub> and Z<jats:sub>30</jats:sub> 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<jats:sub>63</jats:sub>, and Z<jats:sub>67</jats:sub> 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.</jats:p>

Implications of ionizing radiation on pollen performance in comparison with diverse models of polar cell growth

“…All here presented data, which derive from the surveyed studies, were reconsidered and collectively standardized, and thus they are here comparatively indicated as percentage of their respective untreated controls (Tables 1, 2, & 3). In summary, IR caused a suppression of pollen germination (Table 1) and tube growth (Table 2) in a dose‐dependent manner (Cuny et al, 1993; Kumar & Rai, 2006; Peixe et al, 2000; Pfahler, 1971; Sadat Hosseini Grouh et al, 2011; Todorova et al, 2004; Zhang & Lespinasse, 1991; Zottini et al, 1997). Compared to the extensively performed rather low dose research on human and animal cells, the artificially applied radiation doses on pollen were very high and the results clearly demonstrate that pollen performance withstands much higher radiation levels.…”

“…Most experiments studying the effects of ionizing radiation on pollen germination and tube growth were performed in breeding programs or classical genetic studies utilizing acute high doses of gamma irradiation on mature pollen of culture plants to induce haploidy by artificial parthenogenesis (Aleza et al, 2009; Blasco, Badenes, & Del Mar Naval, 2016; Bouvier, Zhang, & Lespinasse, 1993; Lecuyer, Zhang, Tellier, & Lespinasse, 1991; Peixe, Campos, Cavaleiro, Barroso, & Pais, 2000; Todorova, Ivanov, Nenova, & Encheva, 2004; Zhang & Lespinasse, 1991). Since Blakeslee et al, (1922) first reported that pollination with irradiated pollen induces in situ haploid plants of Jimson Weed, this has become a widely used technique which has been applied to produce parthenogenetic seeds of several crop plants, such as apple (Zhang & Lespinasse, 1991), citrus (Aleza et al, 2009), kiwifruit (Pandey, Przywara, & Sanders, 1999), loquat (Blasco et al, 2016), muskmelon (Cuny, Grotte, Dumas de Vaulx, & Rieu, 1993), pear (Bouvier et al, 1993), rose (Meynet, Barrade, Duclos, & Siadous, 1994), winter squash (Kurtar, 2009; Kurtar & Balkaya, 2010), pumpkin (Kurtar, 2009; Kurtar, Balkaya, Ozbakir, & Ofluoglu, 2009), sun flower (Todorova et al, 2004), sweet cherry (Höfer & Grafe, 2003), walnut (Sadat Hosseini Grouh, Vahdati, Lotfi, Hassani, & Biranvand, 2011) and watermelon (Sari, Abak, Pitrat, Rode, & Dumas de Vaulx, 1994).…”

Genome Size: A Novel Predictor of Nut Weight and Nut Size of Walnut Trees

“…Furthermore, analytical tools such as flow cytometry are a reliable and convenient method to estimate genome size of plant communities such as ornamental plants (Abedi et al, 2015), medicinal plants (Javadian et al, 2017;Mahdavi and Karimzadeh, 2010;Majdi et al, 2010;Sadat Noori et al, 2017;Tarkesh Esfahani et al, 2016;Tavan et al, 2015) and fruit trees such as peach (Baird et al, 1994), olive (Brito et al, 2008;Loureiro et al, 2007b), coconut (Freitas et al, 2015), and Malus species (Hofer and Meister, 2010;Korban et al, 2009;Podwyszy nska et al, 2016). Unlike other fruit trees and plant species, there has been no study on the genome size of different walnut genotypes and cultivars, only flow cytometry is used to determine the ploidy level of walnut samples in in vitro conditions with the aim of identifying haploid plants (Sadat Hosseini Grouh et al, 2011).…”

Karyotype Analysis of Haploid Plants of Walnut (Juglans Regia L.)

“…Fixed root tips were placed in aceto-ironhematoxylin dye for staining chromosomes (Lu and Raju, 1970). The fixed root tips were hydrolyzed in 1 N HCl for 12 min at 60°C and squashed in a drop of 45% (v/v) acetic acid as outlined in our previous studies (Sadat Hosseini Grouh et al, 2011). Arm ratios, average lengths and relative lengths were calculated using Excel 2007 (Microsoft) using a sample size of five cells/observation.…”