Cell Biology 2015; 3(4): 50-57 Published online December 22, 2015 (http://www.sciencepublishinggroup.com/j/cb) doi: 10.11648/j.cb.20150304.11 ISSN: 2330-0175 (Print); ISSN: 2330-0183 (Online) Physical, Thermal, and Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media Mahendra Kumar Trivedi1, Alice Branton1, Dahryn Trivedi1, Gopal Nayak1, Khemraj Bairwa2, Snehasis Jana2, * 1Trivedi Global Inc., Henderson, USA 2Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, Madhya Pradesh, India Email address: [email protected] (S. Jana) To cite this article: Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Khemraj Bairwa, Snehasis Jana. Physical, Thermal, and Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media. Cell Biology. Vol. 3, No. 4, 2015, pp. 50-57. doi: 10.11648/j.cb.20150304.11 Abstract: The Murashige and Skoog medium (MS media) is a chemically defined and widely used as a growth medium for plant tissue culture techniques. The present study was attempted to evaluate the impact of biofield energy treatment on the physical, thermal, and spectral properties of MS media. The study was performed in two groups; one was kept as control while another was subjected to Mr. Trivedi’s biofield energy treatment and coded as treated group. Afterward, both the control and treated samples were analyzed using various analytical techniques. The X-ray diffraction (XRD) analysis showed 19.92% decrease in the crystallite size of treated sample with respect to the control. The thermogravimetric analysis (TGA) showed the increase in onset temperature of thermal degradation (T ) by 9.41% and 10.69% in first and second steps of thermal onset degradation, respectively after the biofield energy treatment as compared to the control. Likewise, T (maximum thermal max degradation temperature) was increased by 17.43% and 28.61% correspondingly in the first and second step of thermal degradation in the treated sample as compared to the control. The differential scanning calorimetry (DSC) analysis indicated the 143.51% increase in the latent heat of fusion of the treated sample with respect to the control sample. The Fourier transform infrared spectroscopy (FT-IR) spectrum of treated MS media showed the alteration in the frequency such as 3165→3130 cm-1 (aromatic C-H stretching); 2813→2775 cm-1 (aliphatic C-H stretching); 1145→1137 cm-1 (C-N stretching), 995→1001 cm-1 (S=O stretching), etc. in the treated sample with respect to the control. The UV spectra of control and treated MS media showed the similar absorbance maxima (λ ) i.e. at 201 and 198 nm, respectively. The XRD, TGA-DTG, DSC, and max FT-IR results suggested that Mr. Trivedi’s biofield energy treatment has the impact on physical, thermal, and spectral properties of the MS media. As a result, the treated MS media could be more stable than the control, and might be used as better media in the plant tissue culture technique. Keywords: Biofield Energy Treatment, Murashige and Skoog Medium, X-ray Diffraction, Fourier Transform Infrared Spectroscopy Development of culture medium was a consequence of 1. Introduction several systematic experimentation and trials [2, 3]. An ideal plant tissue culture media should contain the constituents The optimum growth and development of plant tissues are such as micronutrients (<0.5 mM/L), macronutrients (>0.5 vary among the plants according to their nutritive mM/L), amino acids, nitrogen supplements, vitamins, carbon requirements [1]. The tissues from different plant parts may source, undefined organic supplements, plant growth also have different requirements for appropriate growth. regulators and solidifying agents such as agar [4, 5]. The Furthermore, the plant species are sensitive to growth Murashige and Skoog media (MS media) is a chemically regulators and high salts [2]. Therefore, the tissue culture defined and most suitable classic plant cell culture medium, medium was selected based on the species to be cultured. consisting vitamins, carbohydrates, and inorganic salts [6]. 51 Mahendra Kumar Trivedi et al.: Physical, Thermal, and Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media Ammonium nitrate and potassium nitrate serves as the (Table 1). The MS media was divided into two groups; one nitrogen sources, while sucrose was used as the source of was kept as control while another was handed over to Mr. carbohydrate. The MS medium was first invented by Toshio Trivedi to render the biofield energy treatment under standard Murashige and Folke Skoog to support the tobacco callus and laboratory conditions. Mr. Trivedi provided the biofield regeneration of shoots and plantlets from the explants [6, 7]. energy treatment to the treated group via his unique energy Autoclaving or heat treatment is the standard method of harnessing process without touching the sample. culture media sterilization [8]. However, the extensive heat Subsequently, both the control and treated samples were treatment of complex culture media may result in nutrient analyzed with respect to physicochemical and spectroscopic destruction via the direct thermal degradation or chemical properties using various analytical techniques such as XRD, reactions between the components [9]. Therefore, it is TGA-DTG, DSC, FT-IR, and UV-vis spectroscopy. advantageous to find out an alternative approach that can enhance the thermal stability of culture media. Table 1. Chemical composition of Murashige and Skoog media. Recently, the energy therapies have been reported for S. No. Ingredients mg/L several beneficial effects throughout the world. Biofield 1 Ammonium nitrate 1650.000 energy treatment is the part of energy therapy that has been 2 Boric acid 6.200 3 Cobalt chloride.6HO 0.025 reported to alter the numerous physicochemical and spectral 2 4 Copper sulphate.5HO 0.025 properties of organic compounds [10] and organic products 2 5 EDTA disodium salt.2HO 37.300 2 [11]. The National Institute of Health/National Center for 6 Ferrous sulphate.7HO 27.800 2 Complementary and Alternative Medicine (NIH/NCCAM) 7 Glycine (Free base) 2.000 considered the healing energy or the putative energy fields 8 Magnesium sulphate 180.690 9 Manganese sulphate.HO 16.900 treatment under the subcategory of energy therapies [12]. The 2 10 Molybdic acid (sodium salt).2HO 0.250 energy therapies such as magnet therapy, bio-electromagnetic 2 11 myo-Inositol 100.000 therapy, healing touch, etc. comprise low-level of energy 12 Nicotinic acid (Free acid) 0.500 field interactions [13]. Numerous mechanism and 13 Potassium iodide 0.830 explanations are proposed in support of the biofield energy 14 Potassium nitrate 1900.000 15 Potassium phosphate monobasic 170.000 therapies including the consciousness. It is described as 16 Pyridoxine hydrochloride 0.500 healer’s intent to heal, which may interact with the physical 17 Sucrose 30000.000 realm [14]. Similarly, physical resonance is another concept 18 Thiamine hydrochloride 0.100 that includes subtle energies. According to this, the energy 19 Zinc sulphate.7HO 8.600 2 might exchange between the energy fields of patient and 2.2. XRD Study therapist [15]. Thus, the human can harness the energy from Universe and transmit it to the objects (living or non-living). The XRD analysis of control and treated samples of MS The objects receive this energy and respond in a useful way. media was done on PW 1710 Phillips Holland X-ray This process is called as biofield energy treatment. Mr. diffractometer with copper anode and nickel filter. The Trivedi is well known for his unique biofield energy wavelength of XRD system was set to 1.54056Å. The treatment (The Trivedi Effect®) that has been studied in the percent change in crystallite size (G) was calculated using field of biotechnology [16], agricultural science [17], following equation: microbiology [18], etc. Therefore, after considering the significant impact of G = [(G -G )/G ] × 100 T C C biofield energy treatment in different areas, this study was Here, G and G are the crystallite size of treated and attempted to evaluate its impact on the plant cell culture T C control samples, respectively. media such as MS media. The biofield energy treated MS media, and the respective control sample were analyzed 2.3. TGA-DTG Analysis using several analytical techniques such as X-ray diffractometry (XRD), thermogravimetric analysis-derivative The TGA-DTG analysis of control and treated samples thermogravimetry (TGA-DTG), Differential scanning was carried out on Mettler Toledo simultaneous TGA/DTG calorimetry (DSC), Fourier transform infrared (FT-IR) STARe SW 8.10 thermal analyzer. The analytes were heated spectroscopy, and ultraviolet-visible (UV-vis) spectroscopy. up to 400°C from room temperature at the heating rate of 5°C /min under air atmosphere. The onset temperature of 2. Materials and Methods thermal degradation (T ) and the temperature at which onset maximum weight loss occur (T ) were obtained from TGA- max 2.1. Study Design DTG thermograms. The MS media was procured from HiMedia Laboratories, 2.4. DSC Study India. It contained several inorganic salts such as ammonium The melting temperature and latent heat of fusion of nitrate, EDTA, ferrous sulphate, magnesium sulphate, myo- control and treated MS media were determined using the inositol, potassium nitrate, potassium phosphate, sucrose, etc. Pyris-6 Perkin Elmer differential scanning calorimeter. The Cell Biology 2015; 3(4): 50-57 52 samples were heated up to 300°C at the heating rate of XRD diffractogram of control MS media showed the peaks at 10°C/min under air atmosphere with air flow rate of 5 Bragg’s angle (2θ) equal to 23.35º, 29.29º, 32.25º, 33.67º, mL/min. and 40.92º. Similarly, the XRD diffractogram of treated MS media exhibited the XRD peaks at 2θ equal to 23.39º, 23.69º, 2.5. FT-IR Spectroscopic Characterization 29.28º, 33.67º, and 46.64º. The result showed that positions of XRD peaks in biofield energy treated sample were slightly The FT-IR spectroscopic analysis of control and treated altered with respect to the control sample peaks. It is reported samples of MS media was carried out on Shimadzu’s Fourier that due to the presence of internal strain within the transform infrared spectrometer (Japan) in the frequency region of 500-4000 cm-1. The samples for FT-IR analysis molecules the positions and shape of X-ray diffraction peaks can alter [19]. Based on this, it is hypothesized that the were prepared by mixing of MS media sample (1%) with biofield energy treatment possibly produced some internal KBr powder and then pressed to a disc or pellets. strain within the treated sample. Furthermore, the average 2.6. UV-Vis Spectroscopic Analysis crystallite sizes of both the samples were determined using Scherrer equation [20]. The UV-vis spectra of control and treated MS media were The result exhibited the crystallite size of the control and recorded on Shimadzu’s UV spectrometer (2400 PC). The treated samples as 95.53 and 76.25 nm, respectively. The instrument was equipped with quartz cell of 1 cm with a slit result showed 19.92% decrease in the crystallite size of width of 2.0 nm. The samples were analyzed at the UV treated sample as compared to the control (Fig. 2). It was wavelength region of 200-400 nm. reported that increase in lattice strain may reduce the crystallite size of the sample [21, 22]. Therefore, it presumed 3. Results and Discussion that biofield energy had induced some lattice strain within the treated molecules of MS media. As a result, the grains 3.1. XRD Analysis were fractured into sub grains that could lead to decrease the crystallite size of the treated sample as compared to the control. This decrease in crystallite size of the treated sample might enhance its solubility as compared to the corresponding control during the preparation of culture media for plant tissue culture [23]. Fig. 2. Crystallite size of control and treated Murashige and Skoog media. 3.2. TGA-DTG Analysis The TGA/DTG thermograms of control and treated MS media are shown in Fig. 3. The TGA thermogram of control sample showed the three steps of thermal degradation process. The first step was started at 85°C (T ) and onset terminated at 148°C (T ) with 3.33% weight loss. During endset this phase, the maximum thermal degradation (T ) was max observed at 120.5°C. The second step was started from Fig. 1. XRD diffractograms of control and treated Murashige and Skoog media. 159°C and terminated at 202°C with a percent weight loss of 12.24% and T of 180°C. Similarly, the third step was max The XRD diffractograms of control and treated MS media initiated at 210°C and terminated at 258°C with T of max are shown in Fig. 1. The XRD diffractograms showed the 233°C and 12.6% of sample loss The results exhibited a . sharp and intense peaks in both the samples. This suggested cumulative 28.17% weight loss during first, second and third the crystalline nature of control and treated MS media. The steps of thermal degradation. On the other hand, the treated 53 Mahendra Kumar Trivedi et al.: Physical, Thermal, and Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media sample showed the two steps of thermal degradation. The Furthermore, the T was increased by 17.43% and 28.61% max first step was initiated at 93°C, which was ended at 170°C in first and second steps of thermal degradation in treated with 9.05% weight loss and T of 141.5°C. The second step sample with respect to the control. The increase in T and max onset was initiated at 176°C and terminated at 245°C with T of T of both the steps in treated sample suggested the max max 231.5°C, and 7.78% sample loss. This showed the cumulative increased thermal stability as compared to the control [24]. 16.87% weight loss during the first and second steps of Further, the decreases in cumulative percent weight loss thermal degradation. during the thermal degradation process in treated sample also Overall, the TGA/DTG study revealed the increase in T suggested its enhanced thermal stability than the control [25, onset by 9.51% (in first step) and 10.69% (in second step) in the 26]. Based on this, it is assumed that the biofield treated MS biofield energy treated sample as compared to the control. media is thermally more stable than the control sample. Fig. 3. TGA-DTG thermograms of control and treated Murashige and Skoog media. change (i.e. solid into liquid) is termed as the ∆H. The DSC 3.3. DSC Analysis thermograms (Fig. 4) of MS media showed the latent heat of fusion as 28.68 J/g in the control and 69.84 J/g in the treated The DSC analysis was performed to determine the latent sample (Table 2). This showed about 143.51% increase in the heat of fusion (∆H) and the melting temperature of both the latent heat of fusion of treated sample as compared to the control and treated MS media. A considerable amount of control sample. It might be due to increase in intermolecular interaction force present in the chemical bonds of any force after the biofield energy treatment of MS media than substance holds them tightly on their positions. The energy the corresponding control. Consequently, the treated MS required to overcome the interaction force during the phase Cell Biology 2015; 3(4): 50-57 54 media molecules absorbed more heat (∆H) to change the 3.4. FT-IR Spectroscopic Analysis phase from solid to liquid as compared to the control. FT-IR spectra of the control and treated MS media are Previously, our group has reported the biofield energy shown in Fig. 5. The contents like inorganic salt, plant growth treatment induced alteration in ∆H in organic products such regulators, and carbohydrates comprised in MS media may as bile salt and proteose peptone [27]. Therefore, it is contain functional groups such as O-H, N-H, C-H, C=C, N=O, supposed that biofield treatment might increase the C-O, etc. Therefore, the FT-IR spectra of MS media may be intermolecular interaction forces in treated MS media expected with the vibrational frequencies associated with these molecules, which may lead to increase its latent heat of groups. The vibration peak at 3546 cm-1 in control and 3543 fusion. cm-1 in treated sample were assigned to O-H stretching. The Moreover, the DSC thermograms of MS media exhibited vibration peak at 3165 cm-1 in control sample was assigned to the melting temperature at 132.21°C in control and 132.07°C aromatic C-H stretching that was shifted to lower frequency in treated sample (Table 2). The result showed no significant region i.e. at 3130 cm-1 after the biofield energy treatment. The change in the melting temperature of the treated sample with position of IR peak in the FT-IR spectrum depends on the respect to the control. force constant and the dipole moment of respective bond. The Table 2. Thermal analysis of control and treated samples of Murashige and increase in dipole moment or force constant in the same atomic Skoog media. bond cause to increase in the frequency (upstream shift) and vice versa [28, 29]. Based on this, it is hypothesized that the Parameter Control Treated force constant or dipole moment of aromatic C-H might 85.00 93.00 decrease; therefore, the IR peak of aromatic C-H was shifted to Onset temperature (°C) 159.00 176.00 lower frequency region. 210.00 The IR peak at 2895 cm-1 in control sample was assigned to 120.50 141.50 Tmax (°C) 180.00 231.50 aliphatic C-H3 stretching. This peak was shifted to 2902 cm-1 in 233.00 the treated sample. Furthermore, the peak at 2813 cm-1 in Latent heat of fusion (J/g) 28.68 69.84 control sample was assigned to aliphatic C-H, which was 2 Melting point (°C) 132.21 132.07 shifted to lower frequency region i.e. at 2775 cm-1 in the treated sample. This might be due to alteration in force T : temperature at maximum weight loss occurs. max constant or dipole moment of CH and CH group in treated 3 2 sample with respect to the control. The IR peak at 1762 cm-1 in control and treated sample were assigned to N=O stretching due to nitrate salt (ammonium nitrate or potassium nitrate) present in the sample. In addition, the peaks at 1398 cm-1 in control and 1402 cm-1 in treated samples were assigned to asymmetric NO vibration. 3 Likely, the IR peak at 825 cm-1 in both the control and treated sample was attributed to symmetric vibration of NO group, 3 possibly due to nitrate salts [30]. The IR peak at 1626 cm-1 in control was assigned to C=C stretching, which was slightly sifted to 1620 cm-1 in the treated sample. This showed the decrease in force constant of C=C bond of treated sample as compared to the control. The peak at 1145 cm-1 in the control sample was assigned to C-N stretching (might be due to EDTA) that was shifted to 1137 cm-1 in the treated sample. It suggested the reduction in force constant or dipole moment of C-N bond after the biofield energy treatment as compared to the control. The IR peak at 995 cm-1 in control was attributed to S=O stretching possibly due to presence of sulfates salts such as magnesium sulphate and manganese sulphate comprised in MS media [31]. This S=O stretching peak was sifted to 1001 cm-1 in the treated sample. The IR peaks at 603- 663 cm-1 in control and 603-657 cm-1 in the treated sample were assigned to C-S stretching, possibly due to thiamine hydrochloride, present in the MS media [32]. Overall, the FT- IR data suggested that biofield energy treatment acted at the molecular level of treated MS media and altered the dipole moment or force constant with respect to the corresponding Fig. 4. DSC thermograms of control and treated Murashige and Skoog control sample. media. 55 Mahendra Kumar Trivedi et al.: Physical, Thermal, and Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media Fig. 5. FT-IR spectra of control and treated Murashige and Skoog media. 3.5. UV-Vis Spectroscopic Characterization The UV spectra of both the control and treated MS media are shown in Fig. 6. It showed the absorbance maxima (λ ) max at 201 nm in control sample and 198 nm in treated sample. It is reported that the UV absorbance occurred due to excitation of electrons from highest energy occupied molecular orbital (HOMO) to lowest energy unoccupied molecular orbital (LUMO) [33]. However, the UV analysis of MS media showed no change in the λ of biofield treated sample as max compared to the control. It indicated that energy gap between the HOMO and LUMO was not altered in the treated sample as compared to the control. Fig. 6. UV spectra of control and treated MS media. Cell Biology 2015; 3(4): 50-57 56 4. Conclusions [4] De Fossard R (1976) Tissue culture for plant propagation. Armidale: University of New England. The XRD study revealed the crystalline nature of both the [5] George EF, Hall MA, Klerk GD (2007) Plant propagation by control and treated samples. Moreover, the crystallite size of tissue culture. (3rdedn), Springer, Netherland. biofield treated sample was significantly decreased (19.92%) as compared to the control. This might be due to the [6] Murashige T, Skoog F (1962) A Revised medium for rapid fracturing of grains into sub grains caused by biofield growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473-497. induced lattice strain in the treated MS media. The TGA- DTG study revealed the considerable increase of T in [7] Sridhar TM, Aswath CR (2014) Review on medicinal plants onset treated sample by 9.41% (in first step) and 10.69% (in second propagation: A comprehensive study on role of natural organic step) as compared to the control. Similarly, T was extracts in tissue culture medium. 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Ind J Med Microbiol 23: the vibrational frequency of functional groups such as 159-163. aromatic and aliphatic C-H, C=C, C-N, and S=O in the [10] Trivedi MK, Patil S, Shettigar H, Singh R, Jana S (2015) An treated sample with respect to the control. This might be due impact of biofield treatment on spectroscopic characterization to alteration in the force constant or dipole moment of of pharmaceutical compounds. Mod Chem Appl 3: 159. respective groups in treated sample as compared to the [11] Trivedi MK, Nayak G, Patil S, Tallapragada RM, Jana S, et al. control. (2015) Bio-field treatment: An effective strategy to improve Based on these results, it is suggested that Mr. Trivedi’s the quality of beef extract and meat infusion powder. J Nutr biofield energy treatment has the significant impact on the Food Sci 5: 389. physical, thermal and spectral properties of MS media. 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