In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis

Embryo development in vivo has been studied in four Citrus aurantium L. polyembryonic genotypes. Seeds were collected 65, 85, 105, 125 and 220 days after pollination (DAP). None of the immature seeds harvested 65 and 85 DAP contained visible embryos. A single embryo at a more advanced developmental stage was observed in the central position at the micropylar apex of the embryo sac in about 74% of seeds harvested at 105 DAP, while at 125 and 220 DAP the majority of seeds had two or more embryos at the same developmental stage crowded together. Restriction fragment length polymorphism (RFLP) analysis of low- and high-copy-number nuclear DNA was used to distinguish zygotic from nucellar seedlings. Analysis of plantlets derived from in vitro culture of the bigger embryos, located in the central position at the micropylar apex of the embryo sac of seeds harvested at 105 DAP, established that the frequencies of zygotic embryos ranged from 82 to 88%. Media for immature embryo germination in vitro were based on the nutrients and vitamins of Murashige and Skoog (MS) and Murashige and Tucker (MT) media supplemented with various concentrations of sucrose and growth regulators. A total of 76% of globular stage embryos (<0.3 mm) germinated on MT medium containing 150 mM sucrose and 14.4 mu M gibberellic acid. Heart stage embryos (0.3-0.8 mm) germinated at 95% on MT medium supplemented with 150 mM sucrose and 2.9 mu M gibberellic acid. The addition of 500 mg/l malt extract to MS medium increased the germination of early cotyledon stage (0.8-2.0 mm) embryos to 98%. The optimum sucrose concentration for embryo rescue was 150 mM for the three embryo developmental stages. The ability to form plants in vitro strongly increased with increasing embryo developmental stage.