Indirect Organogenesis and Plant Regeneration in Common Sage ( Salvia officinalis L . ) : An Important Medicinal Plant of Iran

Salvia species are an important resource for medicinal industry. This research was conducted to develop an indirect organogenesis regeneration protocol for Salvia officinalis L. via which callus was obtained from leaf and internode explants, among these explants internode explant gave best result on MS medium supplemented with 0.5 mg/l 6-Benzylaminopurine (BAP), 2.0 mg/l 1-Naphthaleneacetic acid (NAA). The maximum percentage (70%) of regeneration was obtained with 0.5 mg thidiazuron (TDZ) from internode explants. Shootlets were highly rooted on MS/2 medium added with 1.0 mg/l indole-3-butyric acid (IBA). In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facilitate further development of reliable procedures for this genus.


Introduction
Salvia officinalis L., Common sage, (family: Lamiaceae) one among the important medicinal plant species, is a perennial woody shrub native to the Mediterranean region, which is currently cultivated in several countries mostly to obtain the dried leaves to be used as raw material in medicine and perfumery industries 28 .Recent research has shown that sage essential oil can recover the memory and has shown promise in the treatment of Alzheimer's disease 24 .Application of plant extracts prepared from S. officinalis has a long tradition in human society, as this extract manifest remarkable biological effects (fungistatic, antibacterial, antioxidant, virustatic, analgesic) and have preventive and therapeutic activity against several diseases (e.g., bronchial asthma, inflammatory affection, atherosclerosis, cataracts, ischaemic heart disease, cancer, hepatotoxicity, insufficient sperm mobility).The antioxidant properties of S. officinalis reside typically in its phenolic nature 2 and are designated in several researches and reviews.Lima et al. showed significant increase of the liver antioxidant enzyme glutathione-S-transferase activity in rats and mice of sage drinking clusters.Therefore, this herb is also a potential candidate to devise novel medicines to be applied in industry.
Conventionally, S. officinalis is propagated through seeds, however, in nature, seeds germinate slowly and remain dormant for a long time.Alternatively, cutting can be used but low population size hampers the process.Therefore, in vitro methods for large scale multiplication would be a viable option and has been reported for several medicinal herbs, which is considered a powerful tool to multi-ply difficult to propagate, rare or endangered and useful species for commercial cultivation as well conservation.Although, in vitro propagation of S. officinalis has been reported earlier, however, the clonal fidelity of the plants produced through these studies was not ascertained.Therefore, it is not clear whether the plants produced through these studies are genetically stable.The present study reports development of an efficient in vitro propagation protocol using internode explant and assessing the genetic stability of the regenerated plants.

Seed Germination and Explant Preparation
Seeds of Salvia officinalis L. were collected on December 2013 in Iran.The sage seeds were sterilized with 70% ethyl alcohol for 30 seconds and after that solution of 5% sodium hypochlorite solution for 5 minutes.Then the seeds were washed 5 times by sterile distilled water.Disinfected seeds were placed in 15 cm tube containing 5 ml solidified (agar) medium.The seeds were grown under optimal culture condition.45 days old seedlings were used as explants and cut them into 10-15 mm fragment.

Media and Culture Condition
Basic culture was Murashige and Skoog medium.MS medium was supplemented with 30 g/l sucrose (Sigma-Aldrich, USA) and solid with 8 g/l agar (Sigma-Aldrich, USA), and the pH of the medium was adjusted to 5.7 ±0.2 with 0.1 N NaOH or 0.1 N HCl after adding of the plant growth regulators.The medium was dispensed in culture tube and autoclaved at 121°C, for 30 min.All the cultures were kept in a sterilized culture chamber at 25 ±2°C, with 60-65 % relative humidity and 16 h photoperiods provided by cool white fluorescent light (55 µmolm -2 s -1 ).The cultures were subcultured on the fresh medium after 20 days.

Callus Initiation
Explants including leaf (0.5 × 0.5 cm) and internode (1 cm in length), excised from 45 day old in vitro germinated plants, were placed horizontally on MS medium.In this test, the effects of auxins and cytokinin, both separately and in combination were investigated on callus initiation.Auxins such as 1-Naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) (1.0, 2.0 and 3.0 mg/l), and cytokinin 6-Benzylaminopurine (BAP) (5.0 mg/l) were used.These culture conditions were used in all the tests stated below.Data of frequency (%) of callus formation were noted after 45 days of culture.

Shoot Organogenesis from Callus
Well-established compact and hard callus, about 0.5 g fresh weight, was cultured on MS medium suplemented with BAP (0.5 mg/l) +NAA (2.0 mg/l) for shoot organogenesis.Calli were moved to shoot organogenesis media, containing of MS basal media added with TDZ (0.1, 0.5 and 1.0 mg/l) and BAP (5, 10 and 25 mg/l), in combinations of IBA at 0.5 mg/l.Cultures were subcultured on fresh media after 25 th day of inoculation.The average number of shoots per inoculum and average length of shoot organogenesis from callus were noted on the 40 th day after moving the callus on shoot organogenesis media.

Root Formation
Regenerated shoots, with 2-4 cm long, were cultured on MS/2 basal medium supplemented with either of IBA or NAA (0.1, 0.5 and 1.0 mg/l) for adventitious rooting.Data were noted on percentage of rooting, length and number of the roots after 25 days of transmission onto the rooting media.

Acclimatization of plantlets
Rooted plantlets were gotten out from the medium and rinsed in sterile distilled water to remove all the traces of basal callus and agar.The plants were then transferring to plastic pots (7 cm diameter) containing soil mixed of sand and vermiculite (2:1:1).The pots were enclosed with polyethylene bags to maintain the 70-80% relative humidity.These plant were maintained at 25 ± 2° C with light intensity of 25 µmolm -2 s -1 and 16-h photoperiod for 15 days in the culture chamber, and the plants were then transferred to a shade (50 µmolm -2 s -1 ) in the third week.Plants were then transferred to glass house in the 30 days.

Statistical Analyses
The experimental design was a randomized complete with 4 replications.A replicate consisted of 25 explants.Data was statistically analyzed by using two-way ANOVA using IBM SPSS Statistics 22. Mean comparisons were made by least significant difference at the 5% probability level with Duncans's multiple range test.

Effect Plant Growth Regulator Concentration and Types of Explant On Callus Initiation
First experiment was set up to find out the most appropriate plant growth regulator concentration on callus formation (Fig. 1) and in vitro shoot organogenesis of S. officinalis L. were investigated in internode and leaf explants cultured on MS-basal media.The ratio of callus formation varied significantly (P <0.001), depending on the concentration of plant growth regulators present in the medium.Also results showed that a significant interaction between the two factors (P <0.001), indicating that the effects of explant types on in vitro callus formation percentage are dependent on plant growth regulator concentration.There was significant difference among the different explants types in terms of the callus formation percentage.Based on the results of this study, when NAA was added to the medium in combination with BAP, the percentage of callus formation was significantly increased (Fig. 1; Fig. 6, a).Likewise Rajeswari and Paliwal in Albizia odoratissima among the epicotyls, petiole, cotyledon segments tested, maximum of shoot regeneration percentage was obtained from epicotyls cultured on MS medium fortified with 1.5 mg/l BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 0.7 mg/l BAP and 0.1 mg/l NAA.Similarly Gikloo et

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Figure 2. S al.