Optimized conditions for callus induction, plant regeneration and alkaloids accumulation in stem and shoot tip explants of Phyla nodiflora

A.B.A Ahmed,R Pallela,R Mat Taha,A.S Rao,M.V Rao

doi:10.5424/sjar/20110904-453-10

Abstract

The present study describes callus induction and the subsequent plant regeneration with alkaloids accumulation instem and shoot tip explants of Phyla nodiflora. Both explants were cultured on different media (MS, B5, SH and WPM) for callus induction. Stem explants showed better callus biomass (dry weight) than shoot tip explants with green compact callus when cultured on MS medium containing 1.5 mg L–1 α-naphthalene acetic acid. Shoots were regenerated from the callus on MS medium with 1.5 mg L–1 α-naphthalene acetic acid and 1.0 mg L–1 benzyl adenine. The rooting of all regenerated shoots was successfully performed on half-strength MS medium with 1.0 mg L–1 indole-3-butyric acid. The plantlets were acclimatized and established in soil (90%) and exhibited morphological characteristics similar to those of the mother plant. In addition, the alkaloids content was higher in regenerated callus than intact stem and shoot tip explants, which were analyzed by a gravimetric method, TLC (thin layer chromatography) and HPTLC (high performance thin layer chromatography). The proposed method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important species

Highlights

Phyla nodiflora (L.) Greene (= Lippia nodiflora (L.) Mihex) belongs to Verbenaceae family, which is widely distributed in South Africa and Central America (Terblanche and Kornelius, 1996)
Stem and shoot tip explants of P. nodiflora were grown in MS medium (Murashige and Skoog, 1962), SH medium (Schenk and Hildebrandt, 1972), B5 medium (Gamborg et al, 1968) and WPM (Lloyd and McCown, 1980) supplemented with 1.0-2.5 mg L–1 of NAA, 2,4-D (2,4-dicholorophenox acetic acid), IBA and IAA; 1.0-3.0 mg L–1 of BA and KN were used for callus induction
Methanol extract of intact plant and regenerative callus raised from stem and shoot tip explants were analyzed by TLC and HPTLC

Summary

Introduction

Phyla nodiflora (L.) Greene (= Lippia nodiflora (L.) Mihex) belongs to Verbenaceae family, which is widely distributed in South Africa and Central America (Terblanche and Kornelius, 1996) It is a runner plant with scanty roots possessing various ethanobotanical and medical applications in adenopathy, chronic indolent ulcers, etc. P. nodiflora extracts have been used to cure multiple skin diseases and hair afflictions (Abbasi et al, 2010) This plant is over-exploited due to its high medicinal value and propagation of this plant by tissue culture may be mandatory, which offers a greater potential to deliver large quantities of disease-free, true-totype healthy stock within a short span of time (Hussain et al, 2001). The present study is an advancement over the earlier protocol, because it describes the hormonal regulation, callus initiation, regeneration and alkaloid accumulation from in vivo stem and shoot tip explants of P. nodiflora. This study deals with the development of a rapid regeneration system, the quantification of alkaloids from stem and shoots tip callus (indirect organogenesis) and the subsequent transplantation of the plantlets to natural environmental conditions

Material and methods

Results and discussion

MS SH WPM