Influence of Ethylene Inhibitor Silver Nitrate on Direct Shoot Regeneration from in Vitro Raised Shoot Tip Explants of Sphaeranthus indicus Linn.—An Important Antijaundice Medicinal Plant

In the present investigation, an attempt has been made to study the influence of ethylene inhibitor silver nitrate on direct shoot regeneration in Sphaeranthus indicus, an important antijaundice medicinal plant, by using in vitro raised shoot tip explants. The effect of various concentrations of kinetin, BAP (0.5 3.0 mg/l), and NAA (0.1 0.5 mg/l) along with AgNO3 (0.1 1.0 mg/l) was studied. Among the combinations tested MS medium augmented with kinetin (1.0 mg/l), NAA (0.1 mg/l) and AgNO3 (0.4 mg/l) was found to be optimum for production of multiple shoots (34.3 ± 0.36). Addition of AgNO3 to the media not only increases shoot number in all the concentrations tested but also shoot length. AgNO3 at the concentration of 0.4 mg/l produced 35% more number of multiple shoots when compared to multiple shoots (10.8 ± 0.12) produced in control. In the present study by the addition of ethylene inhibitor silver nitrate and growth regulators, more number of multiple shoots (three folds) and shoot length was observed compared to control. These in vitro raised shoots were transferred to the rooting medium containing different concentrations of auxins such as NAA and IAA along with AgNO3 (0.1 0.6 mg/l). Better rooting response (21.6) was observed on NAA (2.0 mg/l) and AgNO3 (0.4 mg/l) containing media. The healthy rooted plantlets were transferred to polybags containing soil and vermiculate in 1:1 ratio for hardening. Finally the hardened plants were transferred to field environment for utmost survivability.


Introduction
Sphaeranthus indicus Linn., belonging to the family Asteraceae, is a herbaceous aromatic medicinal plant distributed in India, Srilanka, Australia, Malaysia, China and Africa.It is commonly known as "Boddasoram" in Telugu and "East Indian globe thistle" in English.The plant is much branched, aromatic and grows up to 30 -60 cm height.All parts of the plant are employed for treating jaundice, diseases of spleen, pain in uterus and vagina, dysentery and hemicranias.The powdered seeds and roots are given as an anthelmentic.The bark powder mixed with whey is a valuable remedy for piles.Flowers are credited with alternative, depurative and tonic properties [1].The natural means of propagation of S. indicus is via seeds.However, it encounters the problem of low seed set, viability and germination.As the plant is exploited for its medicinal use, immediate need is required to propagate the plant for its conservation.Hence, efforts have been made to study the effect of ethylene inhibitor silver nitrate on in vitro propagation of this medicinally important plant.
Ethylene is well-known as an ever-present plant hormone which influences the growth as promoter or inhibitor based on the species used [2].In tissue culture, closed vessels are used with the purpose of avoiding contamination.However, sometimes this may cause abnormal plant growth due to gas accumulation such as ethylene in tissue culture vessels.Ethylene, as a plant growth regulator, produced by tissue, callus and plantlets, is known to influence in vitro morphogenesis [3].In vitro studies have indicated that ethylene can hamper shoot rejuvenation, callus growth and somatic embryogenesis.Addition of ethylene antagonists into the culture media directly affects the level of ACC (1-amino cyclopropane-1-carboxylic acid), thereby affecting the ethylene levels [4].Various views and experimental evidences have been put forth to explain the silver ions capability in blocking ethylene receptors to make plants insensitivity to ethylene [5].Thus, in the present investigation an attempt has been made to study the efficacy of silver nitrate on in vitro direct shoot regeneration from shoot tip explants for the first time through micropropagation.

Source of Plant Material
The plant material of Sphaeranthus indicus (Linn.)for the experiment was collected from the Herbal garden of Dravidian University, Kuppam, Andhra Pradesh, India, and also from the fields in the surroundings of the Dravidian University campus.

Preparation and Sterilization of Plant Material
Actively growing shoots with 2 -3 nodes were excised from 2 -3 months old field grown mature plants raised from the seeds.After leaf excision, the shoot tips were dissected from the shoots and washed in running tap water for 10 -15 mins, then in 5% (v/v) Tween-20, a liquid detergent for 5 mins, followed by continuous washing in distilled water until all the traces of detergent was removed.Then the explants were soaked in 0.4% (w/v) bavistine for 10 mins and finally surface decontamination of the explant was performed by passing through a solution of 0.1% HgCl 2 (w/v) for 2 mins.The optimal exposure time and optimal concentration of the surface sterilent was determined after several initial trials.Surface sterilization was followed by 4 -5 rinses in sterile distilled water.The cut ends of the explants were further trimmed and shoot tips of length (1.0 -1.5 cm) were prepared.Then the explants were blotted on sterile filter paper discs to absorb excess of water before planting them vertically on agar gelled MS media in culture vessels.

Selection of Medium
Surface sterilized explants were inoculated on different media such as MS, B 5 , SH and KS supplemented with Kn (2.0 mg/l), NAA (0.5 mg/l) and AgNO 3 (0.1 -1.0 mg/l).It was found that MS medium showed maximum response for shoot induction, regeneration and proliferation from field grown plants.Further experiments were continued with the MS medium.

Culture Medium and Culture Conditions
The shoots formed in vitro in different media were separated aseptically and shoot tips excised from the regenerated shoots were used as explants as a prerequisite for the present study.The excised shoot tips were inocu-lated on MS medium [6] containing 3% (w/v) sucrose, various concentrations of cytokinins (BAP and Kn), auxins (NAA and IAA) and AgNO 3 .pH of the medium was adjusted to 5.8 with 0.1N Hcl and 0.1N NaOH and it was made to a known volume.Before dispensing the media into the containers (15 ml for 25 × 150 mm test tubes) 0.8% (w/v) agar was added to the media and melted.Culture vessels containing media were sterilized at 15 lbs.Pressure in an autoclave at 121˚C for 15 -20 min.After the completion of sterilization, the tubes were removed from the autoclave and placed in a slanting position to get more surface area to inoculate the explants.
All cultures of Sphaeranthus indicus were maintained in a culture room at temperature of 24˚C ± 2˚C and 55% -65% RH with 16 h/8h photoperiod at a photon flux density of 3000 lux or 50 -70 Em −2 •s −1 provided by cool white fluorescent tubes.Sub culturing was carried out at regular intervals of thirty days.Visual observations of the cultures were taken for every transfer and the effects of different treatments were quantified on the basis of percentage of cultures showing response.

Data Collection and Statistical Analysis
Visual observations were recorded on the frequency in terms of number of cultures responding for axillary shoot proliferation, shoot development, number of shoots per explant, average length of the regenerated shoots, number of roots per shoot and average root length.
Despite scarcity and limitations encountered with the plant material, for most of the treatment a minimum of 10 replicates were used.All the experiments were repeated at least twice/thrice and the cultures were observed at regular intervals.The qualitative data were subjected to statistical analysis by using standard error (SE±) for shoot length, rate of shoot multiplication and then number of roots per shoot.

Effect of Silver Nitrate on Shoot Bud Induction and Multiplication from Shoot Tip Explants
Shoot tip explants derived from in vitro raised shoots of Sphaeranthus indicus were grown on MS medium supplemented with various concentrations of cytokinins (BAP and Kn) in combination with auxins (NAA and IAA) and ethylene inhibitor silver nitrate.Shoot initiation from cultured explants were observed in all the treatments with varied shoot number and shoot length depending up on the type of plant growth regulators and concentration of AgNO 3 used.Shoot buds were emerged from shoot tip explants within 10 -15 days of inoculation (Figure 1(a)).Among the cytokinins and auxins tested, Kn treated explants along with NAA and AgNO 3 achieved the highest regeneration frequency, shoot number and shoot length when compared to BAP.
The data obtained revealed that addition of AgNO 3 (0.1 -1.0 mg/l) along with cytokinins such as BAP, Kn (1.0 -2.0 mg/l) and auxins like NAA, IAA (0.1 mg/l) showed significant enhancement in shoot number and shoot length (Table 1).Shoot number increased with increasing the concentrations of AgNO 3 up to 0.4 mg/l, but thereafter decreased in growth as the concentration increases.Based on the results, maximum regeneration frequency (100%) and shoot number (34.3 ± 0.36) was observed in the media supplemented with Kn (1.0 mg/l), NAA (0.1 mg/l) and AgNO 3 (0.4 mg /l) (Figure 1(c)).This treatment (AgNO 3 at 0.4 mg/l) produced 35% number of shoots when compared to control.Maximum shoot length (10.5 ± 0.18 cm) was observed in Kn (2.0 mg/l), NAA (0.1 mg/l) and AgNO 3 (0.8 mg /l) supplemented media (Figure 1(d)).It was found that, silver nitrate at the concentration of 0.4 mg/l showed maximum number of multiple shoots on cultured shoot tips of S. indicus compared with the control.Hence, these results clearly emphasised that KN-NAA-AgNO 3 supplemented media produced more number of multiple shoots when compared to BAP supplemented media (Table 2).The obtained results are in line with the results of sugarcane [7] and date palm [8].
The formation of multiple shoots was detected after 3 weeks (Figure 1(b)).Between 82% -100% of the explants cultured in the MS medium supplemented with all concentrations of AgNO 3 (0.1 -0.4 mg/l) produced multiple shoots ranging from 16.7 -34.3.There was a significant difference in the number of shoots produced between the control and AgNO 3 supplemented medium.This might be due to the fact that when ethylene is produced through cell division during establishment phase under in vitro conditions, Ag + ions are produced in the medium and an ethylene receptor; ETR1 contains one ethylene binding site per homodimer which is further mediated by a single copper ion (Cu + ) present in the ethylene binding site.The replacement of copper co-factor by silver also serves to lock the receptor into a conformation such that it continuously represses ethylene responses [9].These findings suggest that silver nitrate plays a more significant role in multiple shoot formation in S. indicus.

Effect of Silver Nitrate on in Vitro Rooting
The healthy micro shoots developed in vitro were excised from the shoot clumps and transferred to MS basal medium augmented with different concentrations of auxins such as IAA and NAA along with silver nitrate.Among all the combinations tested, maximum number of roots (29.4 ± 0.12) and root length (12.8 ± 0.23) with highest rooting frequency (93%) was observed in NAA (2.0 mg/l) and AgNO 3 (0.4 mg/l) supplemented media (Table 3).Among the auxins employed for rhizogenesis, NAA facilitates maximum rooting frequency along with AgNO 3 (Figure 1(e)).Whereas, the number of roots produced per shoot was less in media containing auxins alone when compared to those produced in the presence of an auxin and silver nitrate.These results emphasized that addition of silver nitrate to the medium along with an auxin significantly augmented the induction of roots.The above results are similar with the previous studies in Decalepis hamiltonii [10] and Vanilla planifolia [11].

Acclimatization and Hardening of in Vitro Raised Plantlets
The well rooted plantlets were separated from the culture tubes, washed thoroughly and transferred to polybags containing soil and vermiculate in 1:1 ratio for hardening.Finally the hardened plants were transferred to field condition for maximum survivability.

Conclusion
In conclusion, this is the first report in Sphaeranthus indicus with protocol for direct organogenesis from shoot tip explants using ethylene inhibitor silver nitrate.A rapid multiplication rate could be obtained through shoot tip explants by combining the growth regulators with AgNO 3 .In the present study by the addition of ethylene inhibitor silver nitrate and growth regulators, more number of multiple shoots (three folds) and shoot length was observed compared to control.This protocol has great potential for rapid multiplication and conservation of S. indicus.

Table 1 .
Effect of different concentrations of Kn, IAA, and NAA in combination with AgNO 3 on multiple shoot regeneration from in vitro grown shoot tip explants of Sphaeranthus indicus.Observation: After 8 weeks, values are mean ± S.E of 10 replicates.

Table 2 .
Effect of different concentrations of BAP, IAA, and NAA in combination with AgNO 3 on multiple shoot regeneration from in vitro grown shoot tip explants of Sphaeranthus indicus.Observation: After 8 weeks, values are mean ± S.E of 10 replicates.

Table 3 .
Effect of AgNO 3 and different concentrations of NAA and IAA on in vitro rooting of Sphaeranthus indicus using MS medium.Observation: After 8 weeks, values are mean ± S.E. of 10 independent determinants.