Optimisation of In Vitro Cultural Conditions of Some Officinal Species

In this work in vitro culture conditions for three species belonging to the Labiatae family were studied. In particular, a micropropagation protocol of Lavandino cv Grosso (Lavandula officinalis Chaix × Lavandula latifolia Villars) was developed. As regards this species it was verified that the hyperhydricity symptoms of axillary shoots were related to the agar concentration in the culture medium. Moreover, a reproducible in vitro propagation procedure was established for Mentha piperita L.. The results demonstrated that the method of supplying cytokinins affected the proliferation percentage. Moreover, studies concerning the influence of gravity on shoot formation showed that the perturbation of the explant orientation modified BA absorption inducing shoot hyperhydricity. Explants from wild plants of Ajuga reptans L. were micropropagated giving the source of material to start callus production: different callus proliferation and qualities were observed in dark and light conditions. The results from the experiments on the three species suggest that the interaction among physicalchemical factors may affect tissue cultured plants and it might often mask or give unexpected results generating mistakes in data evaluation. INTRODUCTION Many reports and patents concerning optimisation of in vitro cultural conditions have been focused on the chemical components of culture media. However, physical properties of the medium, temperature, aeration, light, etc. may affect growth rates of plant tissues and consequently they may improve the yield of active compounds in species of medicinal interest. In this work, in vitro culture conditions for three species belonging to the Labiatae family were studied: Ajuga reptans L., Mentha piperita L. and Lavandula officinalis Chaix × Lavandula latifolia Villars. Tissue cultures from Ajuga reptans L. and A. pyramidalis have been recognized as donors of flavonoids and other valuable phytochemicals including anthocyanin pigments with higher stability than the phytochemicals extracted from in vivo plants. The growth and pigment production of this species are so predictable that this system is frequently used as a model to test how the chemical or physical factors affect the micropropagation and the callus culture pattern (Smith and Rogers, 2000) In this work explants from wild plants of A. reptans were micropropagated giving the source of material to start callus production. Furthermore, the influence of explant source, growth regulators and physical micro-environmental treatments (light and dark conditions) was examined on callus induction and growth. As regards the micropropagation protocol of mint, the present research stressed two particular aspects related with the observed tendency of this species to develop hyperhydricity as observed in many other species (Ziv, 1991). In particular two different methods of BA supplying were compared and at the same time the explants were displaced from their normal upright position modifying the direction of the gravitational vector by means of a horizontal clinostat. Furthermore, during the in vitro culture of lavandin a significant reduction of shoot quality was observed in connection with the occurrence of visible symptoms of hyperhydricity in the new formed shoots (Panizza et al. 1993). Methods for in vitro propagation of lavandin cultivar “Grosso” (Lavandula officinalis Chaix × Lavandula 303 Proc. I IC on Labiatae Eds.: B. Ruffoni et al. Acta Hort. 723, ISHS 2006 latifolia Villars) were established as an alternative to the use of cuttings (Panizza and Tognoni, 1988). The aim of the present work was to verify the possibility to overcome this problem modifying gel strength by changes of the agar content in the culture medium. MATERIALS AND METHODS Ajuga reptans L. The composition of the basal medium used to establish micropropagated plantlets and callus was: LS (Linsmaier and Skoog, 1965) mineral and vitamins medium, sucrose (30 g L) and Difco Bacto agar (7 g L). The pH was adjusted to 5.8 before autoclaving. Cultures were grown at of 23 ± 1 °C, and at a 16-h photoperiod with irradiance of 70 μmol m s. All cultures were subcultured every four weeks. Micropropagation was performed with basal medium supplemented with 0.5 mg L indol-3-acetic acid (IAA) and 1.0 mg L 6-benzylaminopurine (BA). One-node explants and shoot tips were excised from vigorously growing runners of wild plants and surface sterilized in diluted (15 % v/v) sodium hypochloride (8% active chlorine w/v) for 15 min followed by three rinses in sterile water. Callus cultures were started from young leaves of wild plants disinfected as above described and placed on Petri dishes containing basal medium supplemented by 1 mgL 2,4-dichlorophenoxyacetic acid (2,4 D) or 2,4 D (1 mg L) plus BA (0.5 mg L) under light and dark condition. Furthermore, callus formation was induced in leaves, flowers and bracts excised from in vitro propagated plantlets and cultured on 2,4 D (1 mg l) plus BA (0.5 mg l) under light condition. To quantify callus increments over time (four subcultures) a rating scale was adopted with reference to the initial inoculum (0.5 cm diameter): 1= 1/3, 2= 2/3, 3= 3/3. Mentha piperita L. Stem node explants, excised from greenhouse grown mint plants were washed in current water and then surface sterilized with 0.1% sodium hypochloride (8% active chlorine w/v) solution, adding a drop of Tween 20 as a wetting agent. After sterilization, the plant material was rinsed four times in sterile distilled water. Nodes were cultured on solidified LS medium (7 ml/each vial) supplemented with 30 g L sucrose and 7 g L Difco Bacto Agar. The pH was adjusted to 5.8 before autoclaving at 120°C for 20 minutes. Before autoclaving BA, 2 mg L was added to the culture medium whilst in half of the cultures filter sterilized aqueous solutions of BA (2 mg L) were dispensed in drops above the explants (125 μL/explant). The explants were placed in a growth chamber at the same culture conditions described for Ajuga. Vials with vertically oriented explants were mounted on the clinostat with the apical portion oriented to the centripetal direction (Ccp). The rotation rate of the clinostat (Drum Roller, PBI International) was 3.75 rpm and the resulting average acceleration was in the order of 10 g. Lavandula officinalis Chaix × Lavandula latifolia Villars Lavandin micropropagation was performed by induction of axillary shoots from single-node stem segments. The explants, excised from actively growing vegetative plants, were pre-washed in water and Tween (0.1% v/v) for 30 min. Plant material was surface-sterilized in 10% aqueous solution of sodium hypochlorite (8% active chlorine w/v) for 5 min followed by 3 rinses in sterile distilled water. The basal nutrient medium was LS supplemented with 30 g L sucrose and 7.5 g l Difco Bacto agar and 0.2 mg L BA. To evaluate the effects on hyperhydricity media were solidified also with 5, 2.5 e 0 g l Difco bacto Agar. Rooting was induced on the same basal medium added with 1 mg L naphtaleneacetic acid (NAA). The pH was adjusted to 5.8 before autoclaving. Cultures were placed in a growth chamber at the same culture conditions described for Ajuga. Acclimatization was achieved by transferring plantlets into pots in the greenhouse. Each pot was placed in a plastic bag for 1 week to prevent desiccation. The plants were transferred outdoors 30 days later.