Development of a Feasible and Efficient In Vitro Rescue Protocol for Immature Prunus spp. Embryos

The major factors affecting the in vitro immature embryo rescue efficiencies from Prunus persica or P. armeniaca accessions have been identified, along with improving the feasibility. Variations in the woody plant medium (WPM) were used depending on the embryo size. Embryos less than 5 mm long were cultured in WPM supplemented with 1 μM BAP and 1 μM GA₃, while embryos bigger than 5 mm long were cultured in hormone-free medium, with or without vermiculite. The environmental in vitro culture conditions consisted of three phases: a (I) stratification at 4 °C during a 3- to 5-month-long period in the dark, followed by (II) growth of germinated embryos at 14 °C for a 4-week-long period, with 12 h light a day, which favors plantlet development, and finally, (III) growth at 24 °C, with 16 h light a day, until the plantlets were acclimatized in the greenhouse. The germination of smaller embryos, at the end of phase I, ranged from 82.2% to 22.1% for apricots and flat peaches, respectively, whereas for bigger embryos, the germination varied from 97.3% to 53.2% for the same species. The embryo germination for peaches and nectarines ranged from 40.1% to 30.3% for smaller embryos, and from 91.9% to 63.0% for bigger embryos. Endo- and epiphytic contamination, affecting from 7.4% to 52.9% of cultured embryos, depending on the fruit type and conservation conditions, and the capacity to acclimate to soil conditions, ranging from 50.4% to 93.2%, were the two most important factors influencing the protocol's efficiency and feasibility. Considering the overall efficiencies, expressed as hardened plants transferred to field plots over clean uncontaminated embryo, the values ranged from 55.8% for nectarines, 54.0% for peaches, 45.6% for apricots, and 23.3% for flat fruits. The addition of vermiculite to the culture medium significantly improved the plantlet development, avoiding subculture to fresh medium when an extension of phase III was required before acclimatization. Compared to laboratory glassware, the use of food glass containers with air-permeable sealing film, along with vermiculite-containing medium, significantly reduced the costs when handling the large number of embryos required for breeding programs.