Sustained Shoot Multiplication and Method for Overcoming In Vitro Browning in Medicinally Important Plant, Piper chaba Hunt

Abstract

The current study evaluated for the first time plant regeneration in P. chaba. Adventitious shoot regeneration was achieved in Murashige and Skoog (MS) medium supplemented with three concentrations of 6-benzyladenine (BA; 0.25, 0.5 and 1 mg/l) and 40 mg/l ascorbic acid. Maximum number of 18 adventitious shoot buds was formed in 0.5 mg/l BA but it was accompanied by severe callusing. Therefore, for subsequent multiplication, shoots were subcultured in MS with 0.25 mg/l BA to prevent excessive callusing, though the number of shoots per culture was reduced to 12. A major problem with cultures of this plant was the excessive browning of the shoots and medium due to phenolic exudation. To control browning of tissue and medium two anti-browning agents, ascorbic acid (AA, 10, 40 and 100 mg/l) and activated charcoal (AC, 5 and 10 mg/l) were used individually or in combination with the multiplication medium. Univariate analysis (Kruskal–Wallis test) and principal component analysis plot were performed to assess impact of anti-browning agents on reducing browning but still maintaining consistent rates of proliferation (16.5 ± 0.42) indicated that AC (5 mg/l) was significantly better than either AA alone or any of the combinations of AC and AA. Single shoots were rooted in 0.25 mg/l indole butyric acid and successfully acclimatized under net-house conditions.