High efficiency in vitro plant regeneration from epicotyl explants of Ponkan Mandarin (Citrus reticulata Blanco)

Abstract

Ponkan mandarin (Citrus reticulata Blanco) is one of the most important commercial cultivars of mandarin orange in China. This study reports an improved and efficient protocol for in vitro plant regeneration of Ponkan mandarin. Epicotyl segments, which were cut longitudinally into two halves, were used as explants. The shoot regeneration frequency was significantly increased by longitudinal cutting. A 100% shoot regeneration frequency and 13.2 shoots per explant were obtained when cultures were maintained in darkness for 20 d before being transferred to light conditions, with bud induction by indirect organogenesis. A 72.5% shoot regeneration frequency and 7.8 shoots per explant were obtained when explants were incubated under a 16-h light photoperiod continuously with buds differentiating directly from the cutting wound surface. The optimal medium for shoot formation was Murashige and Tucker basal medium supplemented with 2 mgL−1 BA and 30 gL−1 sucrose both under light conditions. The addition of the auxin NAA reduced the frequency of regeneration. A “filter-paper bridge” technique was used for rooting in this study. The basal portion of regenerated shoots was dipped into 1,000 mgL−1 IBA solution for 15 min before placement on a filter-paper bridge that was maintained in 1/2 MS liquid medium supplemented with 10 gL−1 sucrose. Eighty percent of the shoots rooted, and an average of 2.0 roots per shoot were achieved. Survival rate through acclimatization was 100%.