Effects of adenine sulphate, glutamine and casein hydrolysate on in vitro shoot multiplication and rooting of Kinnow mandarin (Citrus reticulata 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.