Effects of adenine sulphate, glutamine and casein hydrolysate on <i>in vitro</i> shoot multiplication and rooting of Kinnow mandarin (<i>Citrus reticulata</i> Blanco)

Abstract

In vitro shoot regeneration was carried out for Kinnow mandarin (Citrus reticulata Blanco) through shoot tip explants obtained from in vitro germinated seedling. The medium supplemented with 2.5 mg/L benzylaminopurine (BAP) supported maximum shoot proliferation (2.45 shoots/explant). This rate was further enhanced (7.23 shoots/explant) by adding 25 mg/L glutamine, 50 mg/L adenine sulphate and 100 mg/L casein hydrolysate. From the in vitro proliferated shoots, the shoot apices and nodes were excised and sub-cultured on four different medium namely: SCM-1, SCM-2, SCM-3 and SCM-4 for six consecutive months. Problems like defoliation, chlorosis and loss of vigour in shoots were observed at different stages of subculture. These problems were successfully controlled by SCM-4 having 1.5 mg/L of BAP, 0.5% charcoal, 25 mg/L glutamine, 50 mg/L adenine sulphate and 100 mg/L casein hydrolysate. The SCM-4 also maintained the high proliferation rate throughout the subculturing process. Rooting was best induced on 2 mg/L indole-butyric acid (IBA) in combination with 0.1 mg/L indole-3-acetic acid (IAA). Rooting was significantly enhanced by 100 mg/L casein hydrolysate. The in vitro raised plantlets were acclimatized under culture room conditions in different potting mixture, of which the combination of garden soil, sand and vermiculite mixture in 1:1:2 ratio was found most supportive. After 30 days of acclimatization, plantlets were transferred to soil, where established plants  showed more than 90% survival.

Key words: In vitro shoot regeneration, micro-shoots, enhanced shoot multiplication, subculture, acclimatization.