In situ Parthenogenetic Doubled Haploid Production in Melon “Piel de Sapo” for Breeding Purposes

Abstract

Doubled haploids in cucurbit species are produced through in situ parthenogenesis via pollination with irradiated pollen for further use as parental lines for hybrid F1 production. In this study, seven genotypes of melon "Piel de Sapo" were appraised for agronomic traits and pathogen resistances to evaluate its commercial value and used as donor plant material for the parthenogenetic process. Then, in situ parthenogenetic capacity of melon "Piel de Sapo" germplasm was evaluated and optimized. Several steps of the parthenogenetic process were assessed in this study such as melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of parthenogenetic embryos and plantlets, in vitro and in vivo chromosome doubling with colchicine or oryzalin and fruit set of doubled haploid lines. Parthenogenetic efficiencies of "Piel de Sapo" genotypes showed a high genotypic dependency during the whole process. Three different methods were assayed for parthenogenetic embryo detection: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was four times faster than one-by-one method and jeopardized eight times less parthenogenetic embryo obtention than liquid medium. One third of melon fruits set after pollination with irradiated pollen contained at least one parthenogenetic embryo. The 50.94% of the embryos rescued did not develop into plantlets because failed to germinate or plantlet died at the first stages of development because of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of the 26 parthenogenetic plantlets obtained was: 73.08% haploid, 23.08% spontaneous doubled haploid and 3.84% mixoploid. Two in vitro chromosome doubling methods, with colchicine or oryzalin, were compared with a third in vivo colchicine method. In vivo immersion of apical meristems in a colchicine solution for 2 h showed the highest results of plant survival, 57.33%, and chromosome doubling, 9.30% mixoploids and 20.93% doubled haploids. Fruit set and seed recovery of doubled haploids lines was achieved. In this study, doubled haploid lines were produced from seven donor genotypes of melon "Piel de Sapo," however, further improvements are need in order to increase the parthenogenetic efficiency.

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

“…The haploid and mixoploid plantlets need to undergo in vitro or in vivo chromosome doubling, usually using colchicine as antimitotic compound. Once the plantlets have doubled their chromosomes, they are cultured in the greenhouse together with the spontaneously-doubled haploid lines to recover DH seed [3,6,7]. During the DH process, a high genotypic dependency and other factors continuously hamper each step causing a loss of efficiency that might be critical.…”

“…However, this pollen is genetically inactive and unable to fertilize the egg-cell and the polar nuclei. Therefore, irradiated pollen stimulates egg-cell division and parthenogenetic embryo induction [3]. Overall, the dose of ionizing radiation can range from 25 to 500 Gy, depending on the species and can yield less parthenogenetic haploid embryos at higher or more diploid embryos at lower dosages.…”

“…The parameters that define the success or failure of pollination with irradiated pollen are the number of fruits set and the ratio of parthenogenetic embryos per fruit. 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].…”

“…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.…”

“…Parthenogenic DH methodology in cucurbits consists of two basic steps, the initial production of haploid material through in situ parthenogenesis via pollination with irradiated pollen and the subsequent chromosome doubling of haploid plants to restore the diploid chromosome content and to allow the generation of DH seed. Nevertheless, parthenogenesis in cucurbits has several bottlenecks and limitations that may jeopardize the final production of DH lines, such as high genotypic dependency; low fruit set when pollinated with irradiated pollen; difficulties in detecting parthenogenetic embryos; low production of parthenogenetic embryos; recalcitrant culture in vitro; high mortality of parthenogenetic plants during in vitro culture and after the chromosome doubling treatment; low rates of chromosome doubling; and low fruit set of DH lines once chromosomes are doubled [3].…”