Improved Clonal Propagation of <i>Alpinia calcarata</i> Rosc., a Commercially Important Medicinal Plant and Evaluation of Chemical Fidelity through Comparison of Volatile Compounds

Abstract

An efficient and improved clonal propagation of Alpinia calcarata, a commercially important medicinal plant was established on Murashige and Skoog medium. The axillary shoot proliferation was achieved with maximum 5.2 ± 0.7 shoots in 92.8% of rhizome explants in medium with 2.0 mg/L 6-benzylamiopurine (BAP) and 0.2 mg/L indole-3-acetic acid (IAA). Axillary shoot buds (60%) upon subculture for 8 weeks in the same medium produced multiple shoot initials (12.1 ± 0.4) mediated with meristemoids (4.0 ± 0.5) and callus. A gradual reduction in the concentration of BAP or elimination of IAA was required for rapid induction of normal plants devoid of callus from propagules during subsequent subculture. Single clump of 3-4 multiple shoot initials during second subculture on medium with 1.0 mg/L BAP and 0.1 mg/L IAA yielded an average of 21 shoots which was best among different propagules tried. The shoot multiplication rate was further enhanced to 32 shoots when the similar propagules passed to third subculture on medium with 1.0 mg/L BAP alone. Clumps of multiple shoot initials upon subculture on medium with 1.0 mg/L BAP alone exhibited 10 fold multiplication rates. Use of liquid medium in culture bottles with polypropylene caps supported fast growth of the shoots and spontaneous root formation on 50% of the shoots. Shoots transferred to half-strength MS liquid medium with 0.2 mg/L of IAA and IBA was optimum for maximum roots (8.14 ± 1.34) in 100% shoots. The rooted plants were hardened in mist chamber showed 95% survival and well established in the field. The acclimatized plants showed rhizome formation after 4-6 weeks of growth under shade house. Volatile chemicals profile of the leaves, rhizome and root of the in vitro and conventionally propagated plants analyzed by gas chromatography-mass spectrometry were qualitatively and quantitatively similar. The analysis of growth characteristics of 36 month old in vitro and conventionally propagated plants showed a 50% increment of rhizome fresh biomass with prolific root and leaf growth in the former than the latter ones. The protocol described herein will have practical applications for the large scale production of uniform efficient plants for commercial cultivation of A. calcarata.