Haploid Induction in Hull-less Seed Pumpkin through Parthenogenesis Induced by X-ray-irradiated Pollen

Abstract

<jats:p>Production of hull-less seeds of styrian oil pumpkin (<jats:italic>Cucurbita pepo</jats:italic> ssp. <jats:italic>pepo</jats:italic> var. <jats:italic>styriaca</jats:italic>) is increasing as a result of demand for aromatic seed oil and for other uses. Hybrid cultivars have recently been released but a method for haploid induction has not been established. We focused on the development of a parthenogenetic haploid induction protocol based on pollination with pollen irradiated with X-ray radiation at 0, 50, 100, 150, 200, 300, and 350 Gy. Fruit set largely decreased at 200 Gy, whereas a decline in embryo formation was observed at 100 Gy. Various accessions were tested as the female parent or as the pollen donor and large differences were found. The best parthenogenetic response was found in ‘Turkey #2’ (10.0%), ‘Gleisdorfer Ölkürbis’ (4.4%), and ‘Naked Seed’ (3.9%), whereas ‘GL Opal’ and ‘White Acorn’ were efficient as pollen donors. The ploidy level of 3830 putative parthenogenetic embryos was determined using flow cytometry. Four ploidy levels (n, 2n, 3n, and 4n) were found with the majority being diploid. Interestingly, a significant proportion was determined to be tetraploid and this was clearly correlated with increased radiation delivered to pollen grains. Using selected simple sequence repeat markers on diploid embryos, no spontaneous chromosome doubling could be confirmed. In this study, haploid induction in styrian oil pumpkin was elaborated for the first time. We also showed that X-ray pollen irradiation provides an alternative to gamma radiation treatment, yielding a sufficient percentage of haploid plantlets.</jats:p>

In vitro embryo rescue and plant regeneration following self-pollination with irradiated pollen in cassava (Manihot esculenta Crantz)

“…Related studies show that the number of seeds generated by pollination with irradiated pollen is a function of genotype (Nasertorabi et al, 2012), while the frequency of haploids generated using irradiated pollen is a function of the recipient female parent and the pollen donor (Koṧmrlj et al, 2013). Also, haploid production from the same genotype is not consistent across different pollination events with irradiated pollen (Koṧmrlj et al, 2013). Our study consisted of only one cassava genotype, Nase 14.…”

“…These results annul an earlier hypothesis by Viera et al (2012) claiming the presence of a specific substance found in the stigma but not in the medium that induces pollen germination, which opens up opportunities to study the impact of irradiation in cassava. The effects of ionizing radiation on pollen germination have been studied in a number of plants (Visser and Oost, 1981;Pooler and Scorza, 1997;Musial and Pryzwara, 1998;Yue and Zou, 2012;Koṧmrlj et al, 2013). These studies generally concluded that irradiation results in a reduction of pollen germination.…”

“…In kiwifruit (Actinidia deliciosa), the number of abnormal seeds drastically increased in fruits when pollinations with irradiated pollen were performed, that is by 68% for 700 Gy and 74% for 900 Gy (Musial and Pryzwara, 1998). Related studies show that the number of seeds generated by pollination with irradiated pollen is a function of genotype (Nasertorabi et al, 2012), while the frequency of haploids generated using irradiated pollen is a function of the recipient female parent and the pollen donor (Koṧmrlj et al, 2013). Also, haploid production from the same genotype is not consistent across different pollination events with irradiated pollen (Koṧmrlj et al, 2013).…”

“…The use of irradiated pollen in combination with embryo culture is an efficient method for induction of haploids in several plants notably plum (Prunus domestica L; Peixe et al, 2000), oil pumpkin (Curcurbita pepo; Koṧmrlj et al, 2013) and melon (Cucumis melo; Sari et al, 1994;Nasertorabi et al, 2012). In cassava, embryo rescue methods are available (Fregene et al, 1999;Akinbo et al, 2010;Yan et al, 2014), which further motivates the use of the pollen irradiation technology.…”

Opportunities and Challenges in Doubled Haploids and Haploid Inducer-Mediated Genome-Editing Systems in Cucurbits

“…Fruit set is lower when pollination is performed using irradiated pollen [3]. The number of female flowers that develop into a fruit can range between 10-25% in melon [3], 20-25% in pumpkin [8] or 50% in cucumber [9]. The initial number of parthenogenetic embryos is crucial to have enough plant material during the whole process.…”