Optimization of Cucumber Doubled Haploid Line Production Using In Vitro Rescue of In Vivo Induced Parthenogenic Embryos

Abstract

ADDITIONAL INDEX WORDS. Cucumis sativus, Cucurbitaceae, parthenogenesis, haploid, DHL, pure line, hybrid ABSTRACT. Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co 60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identifi ed by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by fl ow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfi ng of selected lines. Codominant markers and fl ow cytometry confi rmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μM colchicine. Rescue of diploid or chimeric plants was shown by fl ow cytometry, prior to their acclimation and planting in the greenhouse. Selfi ng of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.

THE EFFECTS OF ANTI-MITOTIC AGENTS ON DIHAPLOIDIZATION AND FERTILITY IN WINTER SQUASH (Cucurbita maxima Duch.) AND PUMPKIN (Cucurbita moschata Duch.) ANDROGENIC HAPLOIDS

“…In accordance with our results, in melon, Solmaz et al [2011] reported the SR in the range of 23% and 53%, and findings of Lim and Earle [2008] also introduced the 39-83% SR; besides, it varied from 23% to 81% in Iranian melons [Nasertorabi et al 2012]. Correlatively, 20-60% of regenerants were only survived in cucumber lines [Claveria et al 2005].…”

“…Mean SR was about 40% in CL treatments, while it was up to 60.0% in OR. The phytotoxicity and lower SR of in vitro CL treatments was also reported in melon [Koksal et al 2002, Yashiro et al 2002, Lotfi et al 2003, Yetisir and Sari 2003, Lim and Earle 2008, Solmaz 2011, Nasertorabi et al 2012, or in cucumber [Caglar and Abak 1997, Claveria et al 2005, Galazka et al 2015. In accordance with our results, in melon, Solmaz et al [2011] reported the SR in the range of 23% and 53%, and findings of Lim and Earle [2008] also introduced the 39-83% SR; besides, it varied from 23% to 81% in Iranian melons [Nasertorabi et al 2012].…”

Effect of in vitro and in vivo colchicine treatments on pollen production and fruit set of melon plants obtained by pollination with irradiated pollen

“…Our best duplication rate was 22% for 3 cm tips treated for 3 h with 500 mg/l colchicine. Claveria et al (2005) reported the survival rate of cucumber treated in vitro with 500 lM (*200 mg/l) colchicine for 48 h was 20-60%, and the duplication rate was 30%. Koksal et al (2002) and Yetisir and Sari (2003) tested several different in vivo treatments for chromosome duplication in muskmelon.…”

Induction and development of anther-derived gametic embryos in cucumber (Cucumis sativus L.) by optimizing the macronutrient and agar concentrations in culture medium

“…(Kurtar and Balkaya, 2010), parthenogenesis method using gamma-irradiated pollen in snap melon (Cucumis melo L.) (Godbole and Murthy, 2012), and anther culture method in watermelon (Citrullus lanatus L.) (Abdollahi et al, 2015). Several methods including spontaneous parthenogenesis (Aalders, 1958), in vitro gynogenesis (Diao et al, 2009;Li et al, 2013), pollination with irradiated pollen (Claveria et al, 2005;Lotfi and Salehi, 2008), and in vitro androgenesis (Ashok Kumar et al, 2003; Ashok Song et al, 2007;Chen et al, 2008) were also used to obtain haploid and double haploid (DH) plants in cucumber. Anther culture was reported as an efficient method for haploid production in many plants species (Ferrie et al, 1995).…”