Shoot Regeneration via Callus derived from Shoot Apex of Apple Cultivar 'Fuji'

Shoot regeneration experiments from growth-point-derived callus were conducted to improve apple genome editing techniques. The plant material studied was in vitro maintained shoots of 'Fuji'. In the first step, a procedure for shoot formation from apple callus was established. After using the two earlier reported cases' experimental procedures and media compositions, we investigated the effect of media variations in callus induction, callus multiplication, and shoot induction from the meristem. The procedure that yielded a higher shoot regeneration rate with meristem-derived callus involved shoot multiplication with 1001 medium followed by the Caboni's callus induction medium, then without callus propagation on liquid medium, and then either Caboni's or Saito-Suzuki's shoot induction medium. In this experiment, axillary buds may remain as the shoot apexes were excised at 1 mm. In the next experiment, shoot apexes were excised at 0.5 mm to completely eliminate the axillary bud. Then treatment in the dark was added to the procedure to further improve shoot regeneration rates for the meristem-derived callus. Shoot multiplication medium and shoot induction medium procedures were conducted under dark conditions. This yielded the following optimal procedure: excising meristems from chlorosis shoots after 2-3 months of dark treatment in shoot multiplication medium on 1001; placing the excised meristems in Carboni's callus induction medium in the dark for 20 days; then transferring the formed callus Saito-Suzuki's shoot induction medium with incubation in the dark for at least 2 weeks. The shoot regeneration rates of calli treated in the dark for 6 weeks with shoot induction medium reached 68%. Relative to previous reports, this value is considered high for shoot regeneration from calli in apple cultivars.