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COMPARATIVE STUDY OF DIFFERENT NUTRIENT SUPPLEMENTS ON IN VITRO REGENERATION AND BOERAVINONE B PRODUCTION IN BOERHAAVIA DIFFUSA L.

Byline: S. Sharma, A. Koul, J. Sharma, V. Sharma and S. Mallubhotla

Key-words: Carbon sources, CNP ratio, HPTLC profiling, Punarnava.

INTRODUCTION

In recent years, advancement in utilization of secondary metabolites for medicinal purpose has significantly aroused and keen interest has been devoted to the enhancement of production of these metabolites especially by utilizing various media manipulation techniques under in vitro conditions(Ramachandra and Ravishankar 2002).

In context to this, the present study focuses upon the medicinally important herb Boerhaavia diffusa(Punarnava) that belongs to family Nyctaginaceae and is widely prevalent in the tropical and sub-tropical regions of the world. It is a perennial herb which is used in various traditional medicinal systems due to the presence of medicinally important phytochemicals like alkaloids(punarnavine), flavonoids(boeravinone A-F), saponins, carbohydrates, tannins and phenols in them(Chaudhary and Dantu 2011). It has been demonstrated that the plant extracts of B. diffusa possess anticonvulsant(Kaur and Kumar 2009), anti-inflammatory(Agrawal et al. 2011), diuretic(Desai et al. 2008), antifibrinolytic(Sengul et al. 2009) and antibacterial properties(Umamaheswari et al. 2010). Recently drug formulation of B. diffusa has been used as an adjuvant for suppressing secondary tumor formation in anticancer therapy(Milic 2008).

Due to its slow vegetative propagation, less seed viability and low seed germination rate, this species is on the verge of extinction and has become vulnerable. Under such circumstances, plant tissue culture holds a great prominence for plant conservationists and pharmaceutical industries as a suitable method for obtaining large quantity of genetically homogenous and healthy plant material. Such in vitro propagation techniques have been used efficiently for the conservation of endangered plant species(Fay 1992).

Reports on phytochemical and pharmacological aspects of B. diffusa are available; however, reports on successful in vitro regeneration are few and insufficient(Bhansali et al. 1978; Shrivastava and Padhya 1995; Nagaranjan et al. 2005). An attempt has been made to optimize the protocol for in vitro propagation of this valuable herb by investigating the effect of different plant growth regulators, carbon sources(table sugar, sucrose, fructose, maltose, dextrose) and varied ratio of carbon, nitrogen and phosphate on biomass regeneration and production of one of the active phytoconstituents of the herb - boeravinone B. This metabolite is naturally present in different parts of the plant species and has been reported to be used for its anti-inflammatory, anti-apoptotic and anti-ageing properties(Bairwa et al. 2013; Bairwa and Jachak 2015; Biradar et al. 2018).

The present study also aims at enhancing boeravinone B production under in vitro conditions through media manipulation mechanisms. Further, interest was in comparing varied content of boeravinone B in the different culture treatments. The qualitative and quantitative analysis of boeravinone B was done through HPTLC(High performance thin layer chromatography), since it is simple, accurate, rapid and a cost effective method in comparison to HPLC(High performance liquid chromatography). From literature review, it has been observed that only limited studies have been performed and reported on identification and quantification of boeravinone B in whole plant parts(Gomes et al. 2013; Bairwa et al. 2014; Gomes et al. 2014; Vaidya et al. 2014). No reports on analysis and quantification including enhancement of boeravinone B production using in vitro regenerated tissues have been attempted.

MATERIALS AND METHODS

Plant material identification and authentication: Plant material of Boerhaavia diffusa was collected from vegetatively propagated 3-month-old plants habituated at Trikuta Hills Herbal Garden, Shri Mata Vaishno Devi University(SMVDU), Katra, Jammu and Kashmir(Latitude 28Adeg66' North, Longitude 77Adeg21' East and Altitude 754 m). After examining its habit, vegetative structure, flower morphology followed by microscopic examination, the plant was identified as Boerhaavia diffusa(L.) by Dr. Bikarma Singh, Scientist, Regional Research Laboratory Herbarium, Indian Institute of Integrative Medicine(CSIR-IIIM), Jammu and the identified and authenticated plant material under accession number - RRLH23492 has been deposited at Janaki Ammal Herbarium of CSIR-IIIM, Jammu for future reference.

Explant surface sterilization: Actively growing healthy young shoot tips(1.5 - 2 cm) of Boerhavia diffusa chosen as explants, were thoroughly washed under tap water to remove superficial contaminants, sterilized with 1%(v/v) Tween 20(HiCare Solutions, Delhi) for 2 min. Further, the explants were treated with 1%(v/v) sodium hypochlorite(Himedia Chemicals, Mumbai) for 2 min. Secondary treatment was given with 0.2%(w/v) mercuric chloride(Himedia Chemicals, Mumbai) for 3 min under laminar air flow cabinet(Khera Instruments Pvt. Ltd., Delhi) and finally washed with sterile distilled water 3-4 times for removing traces of sterilants.

Growth medium and aseptic culture conditions: The most commonly utilized basal Murashige and Skoog [(MS) Himedia Chemicals, Mumbai] medium(Murashige and Skoog 1962) was used. Throughout the experiment, the medium pH was maintained at 5.8 with 0.1 N sodium hydroxide(NaOH)/1 N hydrochloric acid(HCl)(Himedia Chemicals, Mumbai) and 0.7% agar [(w/v)(Himedia Chemicals, Mumbai)] was used as the gelling agent. The medium was dispensed in culture tubes and autoclaved at 121 AdegC for 15 min at 15 psi. The cultures were incubated in a growth room maintained at 25 +- 2 AdegC temperature with 16/8 hr day/night photoperiod provided by cool white fluorescent light(3000 - 3500 lux)(Phillips, India).

Influence of medium strength and different plant growth regulators(PGR's): MS medium of varying strengths(quarter, half, full, double) were utilized to test their impact on shoot proliferation and boeravinone B production. Reproducible and effective protocol of in vitro regeneration depends upon the type of plant growth regulators(PGR's) used. Therefore, shoot tip explants were inoculated vertically on MS medium fortified with different concentrations and combinations of PGR's; auxin [1-Napthaleneacetic acid(NAA)] and cytokinins [Thiadiazuron(TDZ); Kinetin(Kn); 6 - Benzyl aminopurine(BAP); Zeatin(Zn)(Himedia Chemicals, Mumbai)](Table 1) along with 3% sucrose [(w/v),(Himedia Chemicals, Mumbai)]. Explants inoculated on MS medium without the supplementation of PGR's was considered as the control. After one month, cultures were sub-cultured onto medium of original composition and all data were recorded on weekly basis.

In vitro rooting: To find out the effective strength of auxin for in vitro rooting of this plant, in vitro shoots were isolated and inoculated in MS medium strengthened with various concentrations of auxins(Indole Butyric acid(IBA), Indole Acetic acid and 1-Napthylacetic acid [(NAA):(0.5; 1.0; 1.5, 2 mg/L),(Himedia Chemicals, Mumbai)(Table 2).

Hardening and acclimatization: For acclimatization, the well developed in vitro plantlets having atleast 2 to 4 cm roots were carefully removed from culture tubes and washed with distilled water in order to remove the remnants of agar adhered to the roots of the plantlets. They were then transplanted into perforated plastic cups containing autoclaved river sand and garden soil(1:1). In order to prevent desiccation and to avoid rapid changes in environment, cups were wrapped in plastic bags and incubated in a plant growth chamber(Spire Automation and Innovation, India) under 25 +- 2 AdegC temperature, 70 - 80% humidity, 16/8-hr photoperiod and irrigated twice a week. The bags were gradually perforated after 1 week and finally removed. Hardened plantlets were then transferred to earthen pots and successfully acclimatized to field conditions.

Survival rate and growth parameters(shoot length, mean number of leaves, etc.) were recorded and statistically evaluated after three weeks of incubation in growth chamber and also after transfer to the field conditions.

Effect of carbon, nitrogen and phosphate(CNP) ratio and different carbon sources on biomass regeneration and boeravinone B Production: In vitro regenerated shoot tips regenerated in best combination and concentration of PGR's of appropriate size(1.5-2 cm) were used as source material for further experiments. Carbon, nitrogen and phosphate(CNP) are the essential constituents of MS medium. In this study, the ratio of these three elements was changed to find out their effect on proliferation and production of secondary metabolite in B. diffusa cultures. 3%(w/v) sucrose(carbon source), nitrogen(NH4NO3 + KNO3 equivalent to 840 mg/L nitrogen) and phosphate(KH2PO4 equivalent to 170 mg/L phosphate) at a concentration of 1:1:1 was used as control.

In various treatments, the concentration of each component was first decreased to one half and then increased to two folds with respect to standard concentration(1:1:1) present in MS medium by keeping constant the concentration of other components to investigate their effect on culture development(Supplementary Table S2). Further, for evaluating the effect of carbon sources on biomass regeneration and boeravinone B production, different types(sucrose, fructose, table sugar, maltose, dextrose) at varied concentrations(1%, 3%, 5%)(Supplementary Table S3) along with the best combination and concentration of auxin and cytokinin were utilized. Cultures were maintained under controlled conditions and MS medium with 3% sucrose was regarded as control.

Qualitative and quantitative analysis of boeravinone B content through HPTLC: Field grown donor plant and all the in vitro regenerated plant samples were shade dried, powdered and extracted(200mg/20ml) with methanol for 5 hours using soxhlet extraction method. Finally, the extracts were filtered using a 0.45 um PVDF syringe filter, air dried and further dissolved in HPLC grade methanol(Sigma Aldrich Co., USA) prior to HPTLC study. Quantification of boeravinone B in different extracts was done by using CAMAG HPTLC analysis. Activated and pre-washed TLC aluminium plates pre-coated with silica gel 60F - 254(E. Merck) of 20 x 10 cm size were used as stationary phase and toluene: ethyl acetate: methanol(7:1:2)(v/v) was used as mobile phase for the analysis. Deuterium and tungsten lamp was used as source of radiation. Development of CAMAG twin trough chamber was done by its pre-saturation with mobile phase for 20 mins. Slit dimension was kept as 5.00 X 0.45 mm with 100 um/step data resolution.

Detection was carried out at 254 nm wavelength at a speed of 20 mm/s by using denistometric scanner(CAMAG TLC scanner operated by Win CATS software). Samples were automatically injected in the injector and 15 ul of each sample was spotted on the plate by automated sample applicator or ul syringe(Hamilton, Switzerland) in the form of bands of width 6 mm. Temperature was maintained between 23-27 AdegC and 9.4 mm distance was kept between each track.

Standard stock solution of boeravinone B and calibration curve preparation: Standard stock solution of boeravinone B(Natural Remedies, Bangalore) was prepared by dissolving 10 mg of standard drug powder in 10 ml(1 mg/ml) of HPLC grade methanol and working solution was prepared by diluting 1 ml of stock solution in 10 ml of methanol to get a final 100 ug ml/L concentration. Eight point calibration curves were utilized ranging from 100 - 800 ng and curves were established by plotting areas of peaks against various concentration of standard solution. Quantification of boeravinone B content in different samples was done by utilizing linear regression equation of calibration curves and each sample was quantified in triplicates in order to maintain consistency.

Experiment design and statistics: Cultures were regularly sub-cultured after four weeks and all the experiments were repeated thrice and six replicates per treatment were used. Data was scored after every seven days of inoculation for leaf number, shoot length and shoot number in each experiment. Growth expressed as growth index(GI) was calculated for the all treatments on the basis of fresh weight(FW) and dry weight(DW)(GI= final weight-initial weight/initial weight) in order to assess the increase in biomass regeneration. ANOVA(SPSS version 17.0)(SPSS Inc., Chicago, USA) was used to analyze the differences in means of data recorded and Duncan's multiple range test(DMRT) was used to compared means of different treatments at p value a$? 0.05.

RESULTS AND DISCUSSION

Medium composition is a determining factor for growth and successful plant regeneration depends on the right choice of nutrient medium. As nutrient requirement of plants vary from species to species, therefore standardization of a particular medium on the basis of concentration of different mineral nutrients is very difficult. Also, concentration of nutrient salts in growth medium significantly influences the shoot growth and relevant studies so far show that half and full strength of MS medium is more suitable for in vitro regeneration of many plant species(Mustafa et al. 2013). Shoot tip explants cultured on different concentrations of MS medium have been frequently used for micropropagation in large number of plants for in vitro multiplication and callus induction of Drosera genus(Perica and Berljak 1996), micropropagation of Coptis teeta and Terminalia belerica(Rathore et al. 2008).

In the present study also it was observed that half and full strength(control) MS medium showed similar results for biomass production, however full strength medium had the best results when compared to the other treatments.Whereas a two fold salt strength was found to be detrimental leading to weak shoot growth with fewer shoot and leaf number(Supplementary Table S1).

Reduction in biomass production expressed as growth index(GI) was in the order of full > half > quarter > double(Figure 1 A). Moreover, when different strengths treatment were analysed for the presence of boeravinone B, it was observed in all the treatments as compared to standard boeravinone B(Figure 1 B, C). In case of quantitative analysis, maximum yield of this specific secondary metabolite was obtained from shoots cultured on full strength MS medium(8.2%)(Figure 1 A, D).

Shoot multiplication: Multiple shoots(with an average of 13.0 +- 0.33) from shoot tip explants were produced within 3-4 weeks in MS medium fortified with 1 mg/L Zn and 0.5 mg/L NAA and 3% sucrose. Among the different treatments used, this treatment was found to be optimal for growth and production of boeravinone B and significantly resulted in a nearly two fold increase(14.13%) in boeravinone B content as compared to control(MS medium devoid of PGR's)(7.53%) and threefold increase as compared to 3 month old field grown donor plant(4.83%)(Table 1, Figure 2).

Table 1:Effect of plant growth regulators on multiple shoot proliferation of Boerhaavia diffusa after 30 days of culture under in vitro conditions

*MS medium+###Mean leaf###Mean Shoot###Mean shoot###Boeravinone-B

###PGRs(mg/L)###Number+-SE###Number+-SE###Length+-SE###content +- SE(%)

###Field plant###-###-###-###4.83 +- 0.09

###Control###5.4 +- 0.27f###1.0 +- 0e###4.3 +- 0.20b###7.53 +- 0.13

###BAP

###0.5###8.5 +- 0.17c###2.1 +- 0.11c,d###2.6 +- 0.10d

###1.0###9.0 +- 0.19c###2.0 +- 0.19c,d###2.7 +- 0.13d###6.13 +- 0.12

###1.5###8.0 +- 0.18c###2.0 +- 0.14c,d###2.7 +- 0.16d

###Kn

###0.5###8.1 +- 0.25a###1.6 +- 0.17d,e###3.6 +- 0.11b

###1.0###8.0 +- 0.19d###1.5 +- 0.18d,e###3.8 +- 0.12b###8.20 +- 0.21

###1.5###8.1 +- 0.20a###1.0 +- 0.17d,e###3.7 +- 0.16b

###TDZ

###0.5###4.3 +- 0.10g###1.3 +- 0.17e###1.3 +- 0.11f

###1.0###4.0 +- 0.12g###1.3 +- 0.19e###1.2 +- 0.1f###4.33 +- 0.19

###1.5###4.9 +- 0.22g###1.4 +- 0.20e###1.1 +- 0.1f

###Zn

###0.5###10.4 +- 0.12b###3.6 +- 0.21b###3.0 +- 0.21c

###1.0###10.5 +- 0.17b###3.8 +- 0.22b###3.1 +- 0.25c###11.54 +- 0.03

###1.5###10.0 +- 0.11b###3.7 +- 0.20b###3.1 +- 0.20c

BAP + NAA

###1.0 + 0.5###7.8 +- 0.18d###2.3 +- 0.25c###2.0 +- 0.14e

###1.0 + 1.0###7.6 +- 0.13d###2.2 +- 0.19c###2.1 +- 0.13e###7.15 +- 0.14

###1.5 + 1.5###7.6 +- 0.11d###2.2 +- 0.19c###2.1 +- 0.14e

Kn + NAA

###1.0 + 0.5###7.0 +- 0.18e###1.3 +- 0.11e###1.2 +- 0.07f

###1.0 + 1.0###7.1 +- 0.14e###1.2 +- 0.12e###1.1 +- 0.1f###9.60 +- 0.23

###1.5 + 1.5###7.0 +- 0.15e###1.2 +- 0.22e###1.0 +- 0.11f

TDZ + NAA

###1.0 + 0.5###3.0 +- 0.19h###1.1 +- 0.09e###1.2 +- 0.1f

###1.0 + 1.0###3.1 +- 0.14h###0.9 +- 0.12e###1.3 +- 0.1f###3.88 +- 0.36

###1.5 + 1.5###3.0 +- 0.16h###1.0 +- 0.22e###1.1 +- 0.1f

Zn + NAA

###1.0 + 0.5###13.0 +- 0.33a###5.1 +- 0.25a###5.3 +- 0.18a

###1.0 + 1.0###12.4 +- 0.30a###4.9 +- 0.21a###5.0 +- 0.16a###14.13 +- 0.11

###1.5 + 1.5###12.0 +- 0.36a###4.5 +- 0.22a###4.7 +- 0.11a

In vitro production of boeravinone B from shoot cultures of B. diffusa suggested that the pathway for its biosynthesis is not affected by lab conditions. Shoot multiplication was further enhanced by subsequent subcultures. There are few other reports(Roy 2008; Kumar et al. 2013) which also suggest that shoot tips were the best source of explants for the induction of multiple shoots in B. diffusa. In medium combination containing both cytokinin and auxin, explants produced a greater number of shoots as comparison to medium fortified with only cytokinin(Table 1). Under in vitro conditions PGR's are highly significant as they control apical dormancy as well as help in maintaining a proper balance between organic and inorganic nutrients required for growing tissues. Synergetic effect of cytokinins and auxins in shoot proliferation has also been reported by various scientists in different plant species(Roy 1998; Madhulatha et al. 2004).

In vitro root induction: Successful rooting of in vitro regenerated shoots is important for their establishment in soil. For rooting, elongated shoots were inoculated on MS basal medium containing various concentrations of auxins along with 3% sucrose and 0.7% agar. Among the various auxins tested, maximum in vitro rooting of individual shoots was observed in MS medium supplemented with 0.5 mg/L NAA(Table 2, Figure 3 A, D) wherein maximum root number(11.3 +- 0.39) and the longest roots(7.8 +- 0.25) were recorded after three weeks of culture. Moreover, rooting was also observed when NAA was used along with Zn but it took longer time period of eight weeks of culture. Results obtained for rooting are in correspondence with previous findings(Sudarshana et al. 2008), wherein maximum rooting was observed on MS medium fortified with 0.5 mg/L NAA. The rooted plantlets were transferred to plastic pots and kept inside the growth chamber for hardening and acclimatization.

After 21 days of culture, well rooted hardened plants were acclimatized under green house conditions with a 95% survival rate. Growth parameters like shoot length, mean number of leaves and number of nodes were determined on zero day and after 21 days of transfer and there was an increase in the growth parameters recorded for the transferred plants. The regenerated plantlets showed normal plant morphology when compared to field plants(Figure 3 E, H).

Effect of CNP ratio on proliferation and boeravinone B production in Boerhaavia diffusa: When the effect of different ratio of media major nutrients was examined in comparison to control i.e. 1:1:1 ratio of carbon, nitrogen and phosphate; culture morphology was significantly influenced but no major influence on production of boeravinone B was observed. One half reduction in concentration of nitrogen(CNP3) and phosphate(CNP4) leads to reduction in culture growth whereas when concentration of nitrogen(CNP6) and phosphate(CNP7) were increased to its two fold, culture showed normal morphology with that of control(CNP1). Conversely, when concentration of carbon(CNP2) was reduced to half, large internodal distance was observed and when the concentration(CNP5) was increased to two fold, plantlets showed stunted morphology(Supplementary Table S2, Figure 4).

Existing corpus of study confirmed that an appropriate concentration of nitrogen and phosphorus is required for growth and proliferation of plant species under in vitro conditions(Vidal and Guitierrez 2008). Moreover, when concentration of phosphate salt was reduced to half of its normal concentration, there was a decrease in boeravinone B production(9.43%) as compared to that of control(13.78%) and when concentration of carbon source was increased to its two fold, highest boeravinone B content was recorded(16.78%)(Figure 4 A). In case of culture morphology of CNP5(two fold increase in carbon source), it was observed that increase in concentration of carbon source is not suited for culture growth and it leads to stress condition in plantlets.

As a result, the plantlet showed stunted morphology. But from higher yield of boeravinone B under these culture conditions, it may be concluded that stress conditions are stimulatory for production of this secondary metabolite and further studies can be attempted to ascertain the fact. From previous finding it was also reported that type and concentration of carbon source in nutrient significantly influenced the metabolite production(Ramachandra and Ravishanker 2002).

Table 2: Effect of auxins(IBA, NAA, IAA) on in vitro root induction in Boerhaavia diffusa after 30 days of culture

###PGR concentration mg/L###Root number +- SE###Root length +- SE(cm)

###Control###No root induction###No root induction

###IBA

###0.5###4.3 +- 0.27b###3.0 +- 0.19e,f

###1.0###3.8 +- 0.18b###2.5 +- 0.12f

###1.5###3.3 +- 0.21b###2.6 +- 0.18f

###2.0###2.7 +- 0.12b###2.5 +- 0.12f

###NAA

###0.5###11.3 +- 0.39a###7.8 +- 0.25a

###1.0###5.0 +- 0.19b###5.0 +- 0.19b

###1.5###5.0 +- 0.27b###4.8 +- 0.19b

###2.0###5.1 +- 0.25b###3.8 +- 0.16c,d

###IAA

###0.5###2.2 +- 0.12b###2.6 +- 0.11f

###1.0###3.9 +- 0.18b###3.5 +- 0.12d,e

###1.5###4.8 +- 0.26b###4.3 +- 0.26c

###2.0###3.8 +- 0.20b###4.0 +- 0.19c,d

Effect of different type and concentration of carbon sources on regeneration and boeravinone B production in Boerhaavia diffusa: Exogenous carbon source added to the culture medium highly influenced culture growth and acted as an osmotic agent and energy source(Haque et al. 2013). In most of the plant species like Centella asiatica(Hossain et al. 2005) Prunus persica(Ahmed et al. 2007), Pogostemon cablin(Kuamara et al. 2010) normally 3% sucrose is used as carbon source. Variance in shoot response is a consequence of the differential ability of plant species within the plant kingdom to metabolize different types of carbohydrates. In the present study, 3% sucrose(control) and 3% table sugar showed similar results(Figure 5 A, B) and had the highest response rate. Therefore, the finding indicates a possible way of using table sugar(a cheaper source of carbon) in place of sucrose for in vitro regeneration of B. diffusa to reduce the production cost without significant loss in plant quality and growth.

Maltose was found to be a poor source of carbon for plant regeneration and MS medium supplemented with dextrose 1% produced maximum leaf number(14.1 +- 0.32) followed by incorporation of dextrose 3%(12.1+-0.25). Proliferative shoots were produced in dextrose supplemented medium; however the regenerants were weak and light green in color. Additionally, the formation of callus in dextrose containing medium reflects that table sugar is a better carbon source for in vitro growth of this plant species. Negative effect of fructose on plant growth was also observed, probably due to its inefficient metabolization by the cells of plant. The rate of culture growth expressed as GI was highly influenced by different sources of carbon at different concentrations and highest biomass regeneration was observed in dextrose containing medium(6.32 DW) at different concentrations. 3% sucrose(5.58 DW) and 3% table sugar(5.63 DW) showed almost similar results for biomass increment(Fig 5 b).

Most remarkable observation in this experiment was a significant increase(~3.9 fold) in boeravinone B production(19.15%) in medium containing table sugar(5%) as compared to field grown mother plant(4.83%) and yield was 1.3 fold more than that of control treatment(14.18%), wherein 3% sucrose was used as carbon source. It is also remarkable that in both the media manipulation experiments, increase in the concentration of carbon source highly influences the production of boeravinone B. Likewise, fructose and maltose sugar which showed least response for biomass regeneration at different concentrations also contributed to an increase in production of boeravinone B and dextrose sugar showed least results for its production as compared to control(Supplementary Table S3, Figure5).The study explicitly hinges upon development of a cost effective and reproducible method for conservation of Boerhaavia diffusa by utilizing a minimal quantity of cytokinin in combination with auxin along with 3% table sugar.

It also establishes an efficient protocol for enhancement of boeravinone B(medicinally valuable metabolite) production by simply manipulating the media components. Enhanced boeravinone B production by the regenerated tissues emphasises on the fact that the activity of enzymes related to its biosynthesis are significantly increased by altering the media components. Moreover, its production is not bound to any specific season and is not dependent upon the age of the plant material as is usually the case in field grown plants. In cue with the above, the study serves as a basis for pharmaceutical companies to utilize this medicinally important pharmaceutical component of B. diffusa without posing threats to its natural population. Further, it assists in selection of high metabolite yielding tissues which can be utilized for enhanced metabolite production in cultured tissues.

Acknowledgement: The authors wish to express their sincere gratitude to Shri Mata Vaishno Devi University, Katra for providing the necessary facilities. The authors gratefully acknowledge the kind support from Dr. Deepika Singh(Senior Scientist - Quality Control, Quality Assurance and CMC Division, CSIR-IIIM, Jammu) for interpretation of HPTLC results. Supplementary Material

REFERENCES

Agrawal B, Das S. and Pandey A: 2011. Boerhaavia diffusa Linn: A review on its phytochemical and pharmacological profile. Asian Journal of Applied Sciences 4: 663-684.

Ahmed T, Abbasi NA, Hafiz IA. and Ali A: 2007. Comparison of sucrose and sarbitol as main carbon energy sources in micropropagation of peach root stock GF-677. Pakistan Journal of Botany 39: 1269-1275.

Bairwa K, Singh IN, Roy SK, Grover J, Srivastava A. and Jachak SM: 2013. Rotenoids from Boerhaavia diffusa as potential anti-inflammatory agents. Journal of Natural Products 76: 1393-1398.

Bairwa K, Srivastava A. and Jachak SM: 2014. Quantitative analysis of boeravinones in the roots of Boerhaavia diffusa by UPLC/PDA. Phytochemical Analysis 25: 415-420.

Bairwa K. and Jachak SM: 2015. Anti-inflammatory potential of a lipid based formulation of a rotenoid rich fraction prepared from Boerhaaviadiffusa. Pharmaceutical Biology 53: 1231-1238.

Bhansali RR, Kumar A. and Arya HC: 1978. In vitro induction of adventitious shoots on stem explants of Boerhaaviadiffusa. Current Science 47: 551-552.

Biradar SP, Tamboli AS, Khandare RV. and Pawar PK: 2018. Chebulinic acid and boeravinone B act as anti-ageing and anti-apoptosis phyto-molecules during oxidative stress. Mitochondrion 1: 110-114.

Chaudhary G. and Dantu PK: 2011. Morphological, phytochemical and pharmacological studies on BoerhaaviadiffusaJournal of Medicinal Plants Research 5: 2125-2130.

Desai SK, Gawali VS, Naik AB. and D'souza LL: 2008. Potentiating effect of piperine on hepatoprotective activity of Boerhaaviadiffusa to combat oxidation stress. International Journal of Pharmacology 4: 393-397.

Fay MF: 1992. Conservation of rare and endangered plants using in vitroIn vitro cellular and Developmental Biology Plant 28: 1-4.

Gomes AG, Vaidya VV, Bhagat RD. and Gadgil JN: 2013. Separation and quantification of pharmacologically active markers boeravinone B, eupalitin 3-O-[beta]-D-galactopyranoside and [beta]-sitosterol from Boeraviadiffusa and from marketed formulation. International Journal of Pharmaceutical Sciences 5: 953-956.

Gomes AG, Vaidya VV, Patankar S. and Kekare MB: 2014. A rapid bio analytical method for simultaneous quantification of boeravinone B and eupalitin 3-O-[beta]-D-galactopyranoside from Boerhaaviadiffusa using LC-MS/MS. International Journal of Pharmaceutical and Clinical Research 6: 149-154.

Haque MS, Wada T. and Hattori K: 2013. Effects of sucrose, mannitol and KH2PO4 on proliferation of root tip derived shoots and subsequent bulblet formation in garlic. Asian Journal of Plant Sciences 2: 903-908.

Hossain AMD, Hossain TMD, Ali RMD. and Rahman SM: 2005. Effect of different carbon sources on in vitro regeneration of Indian penny wort(Centella asiatica). Pakistan Journal of Biological Sciences 8: 963-965.

Kaur M. and Kumar RG: 2009. Anti convulsant activity of Boerhaaviadiffusa: plausible role of calcium channel antagonism. Evidence Based Complementary and Alternative Medicine 18: 1741-1748.

Kuamara M, Balasubramanya MS. and Anuradha M: 2010. In vitro multiplication of patchouli through direct organogenesis. African Journal of Biotechnology 9: 2069-2075.

Kumar A, Kumari P, Priyadarshni M, Anjali K. and Shukla LN: 2013. Development of protocols for efficient clonal propagation of Boerhaaviadiffusa through in vitro culture of different explants. International Journal of Science and Research 2: 17-19.

Madhulatha P, Anbalagan M, Jayachandran S. and Sakthivel N: 2004. Influence of liquid pulse treatment with growth regulators on in vitro propagation of banana(Musa AAA). Plant Cell Tissue and Organ Culture 76: 189-192.

Milic N: 2008. Biological and phytochemical studies on Boerhaaviadiffusa. PhD, University of Naples, Federico II, Italy.

Murashige T. and Skoog F: 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.

Mustafa NS, Rania A, Taha SAM, Hassan Z. and Nagwa SM: 2013. Effect of medium strength and carbon sources on in vitro shoot multiplication of two FicuscaricaJournal of Applied Sciences Research 9: 3068-3074.

Nagaranjan SM, Suresh T, Rajasekaran S, Kannam TMS. and Kulothungan S: 2005. In vitro micropropagation of BoerhaaviadiffusaGeobios 32: 169-172.

Perica MC. and Berljak J: 1996. In vitro growth and regeneration of DroseraspathulataLabill on various media. Journal of Horticulture Science 31: 1033-1034.

Ramachandra RS. and Ravishanker GA: 2002. Plant cell cultures: chemical factories of secondary metabolites. Biotechnology Advances 20: 101-153.

Rathore P, Suthar R. and Purohit SD: 2008. Micropropagation of Terminalia bellerica from juvenile explants. Indian Journal of Biotechnology 7: 246-249.

Roy PK, Islam MS. and Hadiuzzaman S: 1998. Micropropagation of Elaeocarpus robustusPlant Cell Reports 17: 810-813.

Roy PK: 2008. Rapid multiplication of Boerhaaviadiffusa through in vitro culture of shoot tip and nodal explants. Plant Tissue Culture and Biotechnology 18: 49-56.

Sengul M, Yildiz H, Gungor N, Cetin B, Ecer Z. and Ercilsi S: 2009. Total phenolic content, antioxidant and antimicrobial activity of some medicinal plants. Pakistan Journal of Pharmaceutical Sciences 22: 102-106.

Shrivastava N. and Padhya GC: 1995. Punarnavine profile in the regenerated roots of Boerhaaviadiffusa from leaf segments. Current Science 68: 653-656.

Singh AH, Singh R, Pant P, Srikant N. and Dhiman KS: 2018. Identification and quantification of boeravinone B in whole plant extract of Boerhaaviadiffusa and its phytoherbal formulation. Journal of Natural Remedies 17: 88-95.

Sudarshana MS, Niranjan MH. and Girisha ST: 2008. In vitro flowering, somatic embryogenesis and regeneration in Boerhaaviadiffusa, a medicinal plant. Global Journal of Biochemistry and Biotechnology 3: 83-86.

Umamaheswari A, Nuni A. and Shrevidya R: 2010. Evaluation of antibacterial activity of Boerhaaviadiffusa leaves. International Journal of Green Pharmacy 4: 213-219.

Vaidya VV, Gomes A, Gadgil J. and Patil S: 2014. HPLC analysis of boeravinone B and eupalitin 3-O-[beta]-D-galactopyranoside from plant and formulation of BoerhaaviadiffusaInternational Journal of Research in Pharmaceutical Chemistry 4: 982-986.

Vidal EA. and Guitierrez RA: 2008. A system view of nitrogen nutrient and metabolite responses in Arabidopsis. Current Opinion in Plant Biology 11: 529-531.

Table S1:Effect of different strengths of MS medium on shoot regeneration in Boerhaavia diffusa.

###* Medium###No. of leaves +- SE###No. of Shoots +- SE###Shoot Length +- SE###Culture morphology

###concentration###(cm)

###Quarter###5.8 +- 0.17c###1.5 +- 0.12b###4.3 +- 0.10c###Stunted shoot

###Half###6.5 +- 0.24b###1.6 +- 0.18b###4.7 +- 0.11b###Normal shoot

###Full###9.94 +- 0.25 a###2.1 +- 0.17a###4.9 +- 0.12a###Healthy and

###elongated shoot

###Double###3.0 +- 0.19d###1.6 +- 0.17c###3.3 +- 0.07d###Stunted shoot

Table S2:Effect of different concentration of CNP on biomass production of Boerhaavia diffusa.

* MS + varied ratio###No. of leaves +- SE###No. of Shoots +- SE###Shoot Length +- SE###Culture morphology

###of CNP(w/v)###(cm)

###CNP1###9.1 +- 0.74a###1.5 +- 0.22a###6.5 +- 0.82a###Healthy shoot

###CNP2###7.6 +- 1.11b###1.5 +- 0.34a###5.3 +- 0.69b###Normal shoot

###CNP3###7 +- 1.99bc###1 +- 0.16b###4.1 +- 0.53bc###Large internodal

###distance

###CNP4###7 +- 1.52bc###1 +- 0.16b###4.2 +- 0.29bc###Normal shoot, small

###leaf size

###CNP5###6 +- 1.15cd###1.1 +- 0.16b###2.6 +- 0.21d###Stunted shoot

###CNP6###9 +- 1.36a###1.5 +- 0.34a###5.8 +- 0.57b###Normal shoot

###CNP7###8.3 +- 0.84ab###1.5 +- 0.34a###5.3 +- 0.63b###Normal shoot

Table S3:Effect of different carbon sources at different concentrations on biomass production of Boerhaavia diffusa.

###* MS + carbon###No. of leaves +- SE###No. of Shoots +- SE###Shoot Length +- SE###Culture

###source(w/v)###(cm)###morphology

###C1###7.4 +- 0.20d###3.5 +- 0.23a###6.9 +- 0.21a###Healthy shoot

###C2###6.0 +- 0.19e###1.6 +- 0.18cde###5.3 +- 0.14c###Unhealthy shoot

###with small callus at

###base

###C3###5 +- 0.19f###1.5 +- 0.18def###4.4 +- 0.10e###Unhealthy shoot

###C4###2.4 +- 0.12i###1 +- 0f###1.5 +- 0.1 g###Stunted shoot

###C5###4.0 +- 0.19gh###2.1 +- 0.25c###4.1 +- 0.17e###Large internodal

###distance

###C6###7.5 +- 0.23d###3.6 +- 0.18a###7.0 +- 0.29a###Healthy shoot

###C7###4.3 +- 0.26g###1.9 +- 0.20cd###3.6 +- 0.09f###Normal shoot with

###small callus at base

###C8###3.5 +- 0.23h###1.3 +- 0.11ef###4.9 +- 0.17d###Normal shoot with

###small callus at base

###C9###4.0 +- 0.19gh###1.3 +- 0.12ef###4.2 +- 0.15e###Normal shoot with

###small callus at base

###C10###2.3 +- 0.22i###1 +- 0f###1.5 +- 0.1g###Unhealthy shoot

###C11###14.1 +- 0.32a###2.8 +- 0.16b###5.6 +- 0.13c###Highly proliferated

###shoot with small

###callus at base

###C12###12.1 +- 0.25b###2.6 +- 0.21b###6.1 +- 0.16b###Proliferated shoot

###with small callus at

###base

###C13###9.1 +- 0.25c###1.8 +- 0.16cde###5.7 +- 0.11bc###Proliferated shoot

###with small callus at

###base
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Author:S. Sharma, A. Koul, J. Sharma, V. Sharma and S. Mallubhotla
Publication:Journal of Animal and Plant Sciences
Geographic Code:9INDI
Date:May 7, 2021
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