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IN VITRO REGENERATION OF VALERIAN (VALERIANA OFFICINALIS L.) INDUCED BY DIFFERENT CONCENTRATIONS OF NAA AND BAP
Homa Mahmoodzadeh, Hida Fatehi
Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran
In this research, regeneration of Valeriana officinalis via
shoot apex was studied. Shoot apex explants were cultured on MS medium supplemented
with different concentrations and combinations of NAA and BAP. The highest callus
induction percentage was obtained in the medium containing 0.5 mg/l BAP and 0.5
mg/l NAA. Multiple shoot were induced from the shoot apex explant of V. officinalis by
culturing them in MS medium supplemented with 0.5 mg/l of 6-benzylaminopurine
(BAP). The explants also induced root formation in MS medium supplemented with
1 mg/l of NAA. The highest fresh and dry weight of callus was observed when 0.5
mg/l BAP was combined with 0.5 mg/l NAA. The addition of BAP up to 1 mg/l to
the medium containing different concentrations of NAA lead to the production
of callus which is compact rather than friable. This result suggests that this
methodology can be applied for rapid propagation of this species.
In this research, regeneration of Valeriana officinalis via shoot apex was studied. Shoot apex explants were cultured on MS medium supplemented with different concentrations and combinations of NAA and BAP. The highest callus induction percentage was obtained in the medium containing 0.5 mg/l BAP and 0.5 mg/l NAA. Multiple shoot were induced from the shoot apex explant of V. officinalis by culturing them in MS medium supplemented with 0.5 mg/l of 6-benzylaminopurine (BAP). The explants also induced root formation in MS medium supplemented with 1 mg/l of NAA. The highest fresh and dry weight of callus was observed when 0.5 mg/l BAP was combined with 0.5 mg/l NAA. The addition of BAP up to 1 mg/l to the medium containing different concentrations of NAA lead to the production of callus which is compact rather than friable. This result suggests that this methodology can be applied for rapid propagation of this species.
Key words: Propagation, MS medium, Explant, Culture.
Valeriana officinalis, commonly called valerian, is a perennial herb of the Valerianaceae. Its habitats include marshy thickets, and borders of ditches and rivers of Europe and North America. Valerian can be identified by its unpleasant odor and pinkish-colored flowers that grow from a rhizome . Historically called the wild nard, valenan was originally used as a stimulant, and valued for its odor and food flavoring charactenstics . During the 200-year period from 1733–1936, valerian was one of the six most prescribed medicines in European and American medicine. It was used as a antispasmodic to treat hysteria and nervous afflictions, an emmenagogue, a carminative, and a diuretic, among other uses. In the 20th century, valerian is well known for its sedative and restorative affects on the nervous system, and is widely used in herbal and allopathic medicines. The root of the Valeriana officinalis has been used safely and effectively as a sedative and sleep aid for several thousand years, and is widely supported by modern research as a mild sedative for the central nervous system . The petition argues that unlike prescription drugs that reduce REM sleep and cause drowsiness, valerian is not a hypnotic agent or psychotropic tranquilizer and has been proven to be effective as a mild sedative and sleep-aid without side effects and limitations. The rhizome of valerian contains a vanety of compounds including valepotriates, valeric acid, and volatile oils. These compounds affect brain receptors for the neurotransmitter gamma-aminobutyric acid (GABA) . Extracts from the rhizome of valerian have been found to inhibit the uptake and stimulate the release of GABA using the [3H] muscimol binding technique on synaptic membranes isolated from rat brain cortices. The release of [3H] GABA is caused by the reversal of the GABA carrier, independent of Na(+)-K(+)-ATPase activity and the membrane potential of the brain cortex [2, 6]. The use of Valeriana officinalis on the central nervous system of mice has been shown to produce sedative activity at high dosage, anxiolytic activity at low dosage, and weak anticonvulsive properties [8, 18].
Clinical studies of valerian have included studies utilizing the Semliki Forest virus (SFV) expression system, which concluded that Valeriana officinalis does not inhibit in vitro [3H]naloxone binding to the mu-opioid receptors, which may be a possible mechanism for its anti-anxiety effects . The neurotropic activity of valerian has been confirmed through pharmacological assessment during central nervous system ischemia in frogs, without ethanol driving-off, which is associated with volatilization of ethanol oil, the active factor in tincture of valerian . Other studies have shown that valerian may possess some level of antifungal properties . In addition, a variety of valerian known as Valeriana officinalis var latifolia, which relieves smooth muscle spasms and vasodilatation, has been indicated to be effective in the remission of angina symptoms, decreasing frequency of attacks and shortening duration of angina, restoring blood supply to ischemic myocardium, and lowering plasma lipids without toxic action .
V. officinalis preparations are considered safe despite the known in vitro cytotoxic activity of valepotriates  and no acute side effects have been reported. Biotechnological tools are important for multiplication and genetic improvement of the medicinal plants by adopting techniques such as in vitro regeneration In this context, many studies have been carried out in association with in vitro organogenesis of different species of Valeriana genus [10, 20]. Among the constituents of valerian, valerenic acids (valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid) and valepotriates are often regarded as active principles that display tranquilizing effects. Production of these resources is high in the root and scarce in the aboveground parts of V. officinalis plants . Plant cell and tissue culture may offer an alternative method for a controlled production procedure of these natural products. One of the major bottlenecks in regeneration procedures is the production of primary calli. Exogenous auxins and cytokinins are the main plant growth regulators (PGRs) involved in the control of cell division and differentiation . The role of these PGRs in the regeneration performance of valerian has been previously described. It is therefore of importance to optimise the tissue culture conditions in order to extend the regeneration protocols to valerian propagation.
The objective of the present study was to study the effect of various PGRs, particularly of the auxins (naphthalene acetic acid (NAA), and of the cytokines (benzyl adenine (BA) on the callogenesis of valerian.
MATERIALS AND METHODS
Seeds of valerian were obtained from Pakan Bazr company of Isfahan, Iran. The seeds surface sterilized for ten minutes in a 20% (v/v) sodium hypochlorite solution containing 0.1% (v/v) Tween 20 as a wetting agent. The seeds were then rinsed three times for five minutes with sterile distilled water. The sterilized seeds were then germinated on MS  medium. Five seeds were placed in each petri dish; the petri dishes were incubated at 25 ± 2°C with 16/8 hour photoperiod under cool-white fluorescent light at 40 μmol/m2/s.
Callus induction using shoot apex explants
After one week of germination shoot apices explants were excised and cultivated in Petri dishes on MS basal medium supplemented with plant growth regulators in five replications. Plant growth regulators were NAA (0, 0.1, 0.5 and 1.0 mg/l) and BAP (0, 0.1, 0.5 and 1 mg/l). The MS medium without plant growth regulators (PGRs) was used as control. Callus induction, fresh and dry weight of callus, shoot and root regeneration and length of shoot and root and was analyzed after 45 days. All culture media used were adjusted to pH 5.7 with NaOH and autoclaved at 121°C and 1.2 Kg/cm2 for 20 minutes before use. The cultures were incubated at 25°C, with a light to dark period of 16/8 hours.
Experiments were conducted using a randomized completely block design. Data were subjected to variance analysis (ANOVA) and the differences between treatment means established with Duncan's test. Statistical analysis was done using SPSS program.
In this study, the effect of different concentrations of BAP and NAA on micropropagation of Valeriana officinalis, an medicinal plant, through organogenesis was evaluated. Studied characteristics were callus induction, fresh and dry weight of callus, shoot and root length and shoot and root number. The results are summarized in Tables 1–3. Our data revealed that there are differences in the effect of the different concentrations of BAP and NAA on these characters. Maximum callus induction per explants was obtained when 0.5 mg/l NAA and 0.5 mg/l BAP was used in the media. Up to 73% callus induction was observed when BAP was used at 0.5 mg/l and accompanied by 0.5 mg/l NAA. This percentage was not under the same level of significance as that obtained in media supplied with 0.5 mg/l BAP followed by 0.5 mg/l and 1 mg/l NAA (Tab. 1). Callus induction was highly reduced when BAP used alone. NAA by itself led to the production of callus and the BAP did not have any effect in this respect. MS medium supplemented with 0.5 mg/l BAP resulted in the highest shoot length (4.37 cm) and shoot number (3.5). Lowest root length (0.34 cm) was seen in medium containing 1 mg/l BAP along with 0.5 mg/l NAA (Tab. 1). Lowest number of shoot (1.1) were obtained in control medium (without any plant growth regulators) (Tab. 1). The highest callus fresh weight was obtained from explants cultured onto medium containing 0.5 mg/l NAA without BAP, although the same significant level was observed in media containing 0.5 mg/l NAA and 0.5 mg/l BAP, also 1 mg/l NAA and 0.5 mg/l BAP. The results of the study also showed that the callus dry weight was significantly reduced when BAP included in the media (Tab. 1). The addition of BAP up to 1 mg/l to the medium containing different concentrations of NAA lead to the production of callus which is compact rather than friable (Tab. 2 and Fig. 1A). Friable callus induced MS basal medium containing 0.5 mg/l NAA and 0.1 mg/l BAP (Tab. 2 and Fig. 1B). Also analysis of variance (ANOVA) showed that interaction effect of BAP and NAA was significant on callus induction, shoot number and shoot length (Tab. 3).
Table 1. Callus induction, Fresh and dry weight of callus, number and length of shoot and root formed from V.officinalis shoot tip explants on MS basal medium supplemented with different levels of NAA and BAP supplied after 4 weeks of culture at 25°C under light condition
In each column, means with the similar letters are not significantly different at 5% level of probability using Duncan’s test
Table 2. Morphology of callus formed from V. officinalis shoot tip explants on MS basal medium supplemented with different levels of NAA and BAP
PG – indicats pale green color, PY – indicats pale yellow color, WY – indicats white yellow color
Fig. 1. A) Compact callus produced on 0.5 mg/l NAA and 1 mg/l BAP, B) Friable callus produced on 0.5 mg/l NAA and 0.5 mg/l BA
Table 3. Analysis of variance (ANOVA) for the effect of different concentrations of BAP and NAA on some characters of V. officinalis
Significant at α = 5%, ns – not significant
Now, shoot tip explant is being routinely used for the micropropagation and rooting of ornamental and medicinal plants [4, 7].
The effect of BAP and NAA on the callus induction, root and shoot regeneration ability and other characters of valerine shoot apex explants is shown in Table 1. Maximum callus induction per explants was obtained when 0.5 mg/l NAA and 0.5 mg/l BAP were used in the media.These results are in agreement with those reported by Hatzilazarou et al.  who found that using a high concentration of auxin (NAA) led to the production of callus only and the BA and Kinetin did not have any role in producing callus in Ebenus cretica L. meristem culture. On the other hand, the addition of NAA to the basal medium within concentrations, which ranged from 0–1 mg/l, significantly increased the caulogenesis efficiency in the treatment of callus tissue. Using cytokinin alone at the tested concentration produced shoots without callus . It was also noticed that using moderate concentrations of auxin and cytokinin produced a high amount of callus. The results revealed that 1 mg/l NAA induced the formation of rootlets obtained from in vitro plantlets (Fig. 2 A,B). The findings are in agreement with those reported by other researchers . Some plants are known to produce high levels of auxin. This was evident in the rooting ability of valerine cultures in the present study. Producing roots from callus caused problems in the production of adventitious shoots. The more production of roots from callus, the fewer the productions of adventitious shoots. As a result, whenever callus leads to regeneration, the culture medium is used to produce the least number of roots on callus .
|Fig. 2. Rootlets induction of the in vitro plantlet derived from V. officinalis shoot tip explants cultured in the MS basal medium supplemented with A) 1 mg/l NAA and B) 0.1 mg/l BAP+0.1 mg/l NAA|
In the present study, in valerine MS media alone was sufficient to regenerate the plantlet from the nodal explant. In MS medium supplemented with BAP (0.5 mg/l) multiple shoots were developed. In this condition highest number of multiple shoots were produced. When the concentration of BAP was increased to 0.5 and 1 mg/l, shoots were formed earlier. Hence, increase in concentration of BAP has positive effect on the earlier shoot formation, i.e. higher the concentration of BAP, less time taken for the formation of shoot. Sripichitt et al.  observed that BAP was more effective than kinetin in inducing shoot formation on MS medium. In the present investigation, in MS + BAP (0.5 mg/l), the explant initiated the shoot formation followed by callus formation. Similar result was obtained when the concentration of BAP was increased to 1 mg/l. Joshi et al.  also reported that MS medium supplemented with BAP (1 mg/l) + NAA (0.5 mg/l) was the ideal condition for the induction of micro shoots from the nodal segment of Foeniculum vulgare, a member of Umbelliferae.
Most of the work on tissue culture established that auxin like IAA, IBA, NAA induced root formation while cytokinin like BAP, Kinetin induce shoots and bud formation. Similarly in the present study, use of BAP alone and combined with NAA showed initiation of multiple shoots from the shoot apex explants of valerine.
By this experiment, it can be concluded that shoot apex explants are the appropriate explants for the formation of multiple shoots in valerine. MS medium supplemented with BAP (0.5 mg/l) and BAP (0.5 mg/l) + NAA (0.5 mg/l) were the ideal condition for the formation of multiple shoots from the shoot apex explants.
Results of the current study indicated that the highest callus induction percentage was obtained in the medium containing 0.5 mg/l BAP and 0.5 mg/l NAA. Multiple shoot were induced from the shoot apex explant of V. officinalis by culturing them in MS medium supplemented with 0.5 mg/l of 6-benzylaminopurine (BAP). The explants also induced root formation in MS medium supplemented with 1 mg/l of NAA. The highest fresh and dry weight of callus was observed when 0.5 mg/l BAP was combined with 0.5 mg/l NAA. The addition of BAP up to 1 mg/l to the medium containing different concentrations of NAA lead to the production of callus which is compact rather than friable. In general, our results indicated that optimum concentration of regulatory hormones is important for increasing the efficiency of in vitro production of V. officinalis.
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Accepted for print: 29.12.2015
Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran
Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran
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