Elsevier

Plant Science

Volume 168, Issue 2, February 2005, Pages 431-437
Plant Science

Liquid culture system for shoot multiplication and secoiridoid production in micropropagated plants of Centaurium erythraea Rafn

https://doi.org/10.1016/j.plantsci.2004.08.013Get rights and content

Abstract

An efficient shoot multiplication method from shoot tips of Centaurium erythraea using liquid Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (0.1 mg l−1) and 6-benzylaminopurine (BAP) (1.0 mg l−1) was developed. Under these conditions, almost 60 microshoots per explant were produced within 4 weeks. This is three times more as compared to shoots obtained on agar-solidified medium. Shoots taken from liquid culture were rooted with frequency 85% and 77% on hormone-free full-strength and half-strength MS medium, respectively within 6 and 4 weeks. The plantlets were transferred into soil and they survived acclimatization with 90% success, producing healthy plants, morphologically similar to plants derived from shoots grown on solid culture during their multiplication stage. The shoots of 10-week-old micropropagated plants of C. erythraea accumulated secoiridoid glucosides (gentiopicroside, swertiamarin and sweroside) up to 149 mg g−1 dry weight. The value was significantly higher than those achieved for other tested plant materials, such as shoot cultures and aerial parts of wild-grown plants. It is hoped, that the system using liquid shoot culture could be useful for large-scale micropropagation of C. erythraea plants with high production of pharmacologically important products.

Introduction

Centaurium erythraea (Gentianaceae) is an important medicinal plant officially listed in pharmacopoeias of many European and American countries [1]. Extract of the aerial parts of the plant is used principally as stomachic and bitter tonic and to treat heartburn [1], [2]. “Centaurii herba” is also used in the traditional medicine to cleanse blood and kidneys, relieve indigestion, heal wounds and sores [3]. These therapeutic effects are thought to correlate with the content of secoiridoid glucosides. Among them, the most important are gentiopicroside, swertiamarin and sweroside. The secoiridoids show various biological activities, such as fungitoxic, antibacterial, choleretic, pancreatic and hepatoprotective [4]. Van der Beek et al. [5] have reported on the antiamoebic effect in vitro sweroside against Entamoeba histolytica. Because of their bitterness, these glucosides are also used in preparation of some commercial beverages [6].

Today, for medicinal purpose Centaurium erythraea is collected mostly from wild population, which is becoming critical on account of over-exploitation of the plants and the progressive clearance of their habitats. The in vitro culture techniques can be used as an alternative method of propagation of this endangered species. In vitro regeneration of C. erythraea plants has been reported either directly on leaf explants or via callus that developed on the explants cultured on agar-solidified medium [7]. In this paper we report for the first time the micropropagation of C. erythraea from shoot tips grown in liquid culture. This system could be useful for increasing the scale of production and cost reduction. For comparison, results of experiments designed for C. erythraea cultures grown on agar-solidified medium are also presented. Finally, we analyzed by HPLC method contents of secoiridoid glucosides in in vitro and in vivo shoots of C. erythraea under diverse growth conditions. There are only few reports on the production of these active compounds using C. erythraea tissue cultures [6], [8].

Section snippets

Establishment of shoot cultures

Shoot cultures of C. erythraea were initiated from shoot tips (3–5 mm in length) excised from 30-day-old sterile seedlings derived from seeds. Seeds of C. erythraea were obtained from the Botanical Gargen in Powsin, and the same seeds were used to obtain intact C. erythraea plants for comparison reason. The shoot cultures grew on multiplication basal MS medium [9] containing 30 g l−1 of sucrose, 7 g l−1 of agar (SIGMA), vitamins in standard concentration as in MS medium and growth regulators (0.1 mg l

Shoot multiplication in liquid and solid media

Shoot tips of C. erythraea were incubated in liquid and solid (0.7% agar) MS media for a period of 4 weeks to compare effect of these media on shoot proliferation. The media were supplemented with 0.1 mg l−1 IAA and 1 mg l−1 BAP. The combination of growth regulators was based on earlier experiments in our laboratory [10]. Irrespective of the type of culture (solid/liquid), shoot multiplication occurred as a result of the release of axillary buds from apical dominance as well as through callus

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