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| ABSTRACT Purpose: to identify whether seed immersion in 13 homeopathically potentised 15X essential mineral nutrient solutions increases Corn (Zea mays) seed germination when compared with a water control. Method: This was a Randomised Blind Control Pilot Research study. Fourteen (14) interventions including a control of distilled water were each tested using 100 seeds. 60mls of each intervention solution was added to 4 Petri dishes each containing 25 seeds and sequentially numbered 1 to 4. Petris were distributed evenly in a preheated darkened incubator with an average 20°C kept throughout the trial. Germination count was recorded at 8 hour intervals until the 72nd hour. Chi2 analysis was performed to find statistical significance at p < 0.05 when compared with the water control. The Null Hypothesis states that Homeopathically potentised 15X essential mineral nutrient solutions will not increase germination of corn seed when compared to distilled water. Results: No homeopathically potentised 15X essential mineral nutrient solution statistically increased the rate of corn seed germination when compared to water. The three interventions with statistical significance; Calcarea carbonicum, Cuprum metallicum, and the Combination Intervention all produced inhibiting effects on seed germination. Discussion: It is believed that this research shows no significance due to seeds needing only water and oxygen to germinate. The essential mineral nutrients are needed for further plant development only once germination has occurred. (Knox et al 1994) Conclusion: Although most interventions produced similar results to the water control, none showed significance, and therefore the Null Hypothesis is accepted. INTRODUCTION Food security (United Nations 2008) and soil nutrient depletion (WRI 1998-99) are a major problem facing today’s global community. This research was undertaken to identify whether seed immersion in homeopathically potentised 15X essential mineral nutrient solutions would increase corn (Zea mays) seed germination. This could potentially enable the production of a crop with increased grain yield, increased produce bioavailability in soil deficient areas and monetary returns. (Harris et al 2007) Tulip (2008) summarises research showing the effects of homeopathically potentised preparations on plant growth. Scherr et al (2007) discusses potency issues and found enhancing effects; Brizzi et al (2000) found inhibitory effects; Betti et al (2003) found differences between the Decimal and Centesimal potencies; and Hamman et al (2003) found differences in root and shoot lengths. In standard agricultural industry practice, research has been undertaken by Foti et al (2008) which resulted in increased seed emergence of maize germination when primed in a 0.1% osmotica of copper sulphate. To date, there has been no research which identifies the effect of homeopathically potentised essential mineral nutrient solutions on seed germination. The 13 essential mineral nutrients (see Appendix 1) were chosen due to their requirement for plant life, survival and functioning; (Australian Academy of Science, 1990) and the findings of Scherr et al (2007) that homoepathically potentised (21X, 25X & 29X) Phosphorus enhanced duckweed growth. Pacific Seeds (2008/09) state that Nitrogen, Phosphorus and Potassium are the three primary nutrients required for a corn crop representing 83% of total nutrients. Sulphur, Calcium and Magnesium are secondary requirements providing 16% of nutrients, with the micronutrients (trace elements) only contributing 1% of total nutrient uptake. Seed immersion in homeopathically potentised preparations was chosen to mimic the process of seed priming used by standard agricultural industry practice. As stated by Gallardo et al (2001, p835) “priming treatments are used to synchronize the germination of individual seeds… Seed priming generally causes faster germination and faster field emergence.” This research procedure differs from normal seed priming practice by keeping the seeds immersed in homeopathic preparations up to and following seed radicle emergence or germination. Normal procedure is to soak the seed for a set period of time and halt the process before seed radicle emergence or germination. The seed is then dried and stored for future sale. (Harris et al, 1999) The scale of this operation was beyond the abilities of this research project and therefore the immersion techniques applied by Brizzi et al (2000) and Hamman (2003) were referred to as a guide. 45X was the potency which Brizzi et al (2000) found to have the greatest stimulating effect on wheat germination. Due to financial constraints, this research procedure used 15X potency. The Null Hypothesis will be utilized for this research. The Null Hypothesis states: Homeopathically potentised 15X essential mineral nutrient solutions will not increase germination of corn seed when compared with distilled water. Should the Null Hypothesis be rejected, the Alternative Hypothesis states: Homeopathically potentised 15X essential mineral nutrient solutions increase germination of corn seed when compared with distilled water. METHODOLOGY This was a Randomised Blind Control Pilot Research study. As research was undertaken on corn seeds, no approval by an Ethics Committee was required. The research was undertaken by the author, including lab work, result tallying, statistical analysis and reporting. Trial duration: 72 hours Trial commencement: 29 August 2008 at 24:00 Trial conclusion: 1 September 2008 at 24:00 Design Fourteen (14) masked Interventions including the Control, were tested on 100 seeds split between four (4) sequentially numbered Petri dishes. Each dish contained 25 seeds and was labeled with the intervention code (A-N) and a sequential number (1-4) on the base. 15mls of Intervention was added to each of the four corresponding Petri dishes, totaling 60mls per Intervention. Each dish was enclosed with a lid to prevent cross-Intervention contamination. Dishes were placed in a preheated darkened incubator with an average 20°C temperature. These conditions were maintained throughout the 72 hours of the trial. (Hamman B et al 2003) To allow for internal variance of temperature to similarly affect each intervention, Petri dishes labeled number 1 were placed on the top shelf, number 2 were placed on the second shelf, number 3 were placed on the third shelf and number 4 were placed on the lowest shelf. Germination per Petri dish was counted at 8 hour intervals until the 72nd hour. This resulted in 9 lots of data being collected. Total seed germination per intervention was calculated at the 72nd hour. Chi2 analysis was performed to statistically ascertain germination differences between Interventions and Control based on comparisons between observed frequencies to expected frequencies. A one degree of freedom with significance at p < 0.05 was used to evaluate the thirteen (13) individual interventions against the water control. (Polgar & Thomas 2008, p325) Data was stored and manipulated using Microsoft Office Excel 2003. CRITERIA Blinding was performed by a person (Blinder) external to the trial study. Fourteen (14) interventions including the control were randomly assigned a letter from A – N. (See Table 2) All interventions were stored in 200ml amber bottles. The Blinder documented the code, labeled all interventions, and took the code sheet away from the research site. All Interventions remained masked until data collection, observations and statistical analysis were recorded and calculated. Measurement of germination was specified according to the scale of Raynes & Bowey (2008, p4). This scale lists: Germination 05: Radicle (root) emerged from Caryopsia (seedcoat). For this research, radicle length was not included as a factor and therefore germination was deemed complete once the author saw that the radicle had broken through the caryopsia. All seeds were included in the germination count regardless of the appearance of mould. It was deemed that mould was due to research study technique, rather than the germination ability of the seed. Criteria for concluding this pilot research study was based on a pre-trial where it was found that excessive mould formed on the seeds after 72 hours. PRODUCT The Corn (Zea mays) seed was donated by Pacific Seeds. This variety is research grade Hycorn 424 which is untreated with pesticides. This is a hybrid corn which tolerates cold and dry conditions and has a high stress tolerance. It produces a starchy silage crop for animal fodder. (Pacific Seeds 2008-09) 1400 seeds were sorted from the batch to include those with: a strong yellow colour; average size compared to batch; and no visible defects. Seeds were excluded if they appeared: mouldy, seemed defective in shape, were showing little colour, or were smaller than the average size. INTERVENTION The Homeopathic preparations were sourced from Simillimum Homeopathic Pharmacy; Registration number 2008/495. Contact details are: 20 Panama Street, Wellington, New Zealand; Phone +64 4 4999242. All preparations excluding the Control, were prepared in 15X medicating potency with 90% alcohol content. The Decimal potency was chosen based on the stimulatory germination effect described by Brizzi et al (2000) and the ability of the Decimal rather than the Centesimal potency to stimulate a plant to reach its maximum potential, as described by Betti et al (2003). As mentioned previously, 15X rather than 45 X was chosen due to financial limitations. Individual homeopathic preparations (Table 1) were chosen based on similarity to the essential mineral nutrients. (Boericke, Reprint 2004; Simillimum 2008) Potassium was not available as a singular preparation, so Potassium chloride was substituted for the singular elements of Potassium and Chlorine. Table 1 – Homeopathic preparations chosen for essential Mineral Nutrient 15X potentisation
In total 13 homeopathic solutions and 1 Control were made at the research site. The homeopathic solutions included the above 12 essential mineral nutrient solutions and one Combination solution, being a mixture of all 12 homeopathic preparations. Solution ratio for each individual preparation was one drop of an homeopathic preparation to 15ml of “Projecta” distilled water. The pH of the distilled water was 5.5 being a semi-acidic reading. 75ml was prepared for each solution resulting in 5 drops per solution. The solution was then hand succussed 10 times before use. The Combination solution contained 5 drops of each of the 12 listed homeopathic essential mineral nutrient preparations and was hand succussed 10 times before use. The Control was made with 75ml of distilled water and was not hand succussed before use. All 13 solutions plus the non-succussed distilled water control were stored in separate 200ml amber bottles. The solution was added at the beginning of the experiment and was not changed throughout the 72 hour trial period. RESULTS Figure 1 shows Total Germination per Intervention recorded at the 72nd hour. Seven of the interventions produced at least 90 germinated seeds which is similar to the Control. Six interventions produced at least 80 germinated seeds.
Chi2 (x2) analysis to compare frequencies for each Intervention against the water Control was used to ascertain germination differences. This shows the likelihood of the result not being due to chance (p<0.05). There are four components to each Frequency Observed summation: Germination totals for each Intervention and Control; and non-germination totals for each Intervention and Control. The Frequency Expected was calculated by the ratio of the combined Intervention and Control total seed germination / or non-germination, divided by the total number of seeds used for the comparison (200) and multiplied by the total number of seeds used per Intervention (100). The equation to calculate Chi2 (x2) for each Intervention is: (Guilford 1956 p232) x2 = Σ (Freq Obs – Freq Exp)2 Freq Exp To prove significance (p < 0.05) and reject the null hypothesis, individual Intervention x2 values need to be greater than 3.84 (Polgar & Thomas 2008, p325) These findings are shown in Table 2. Table 2 - Chi2 and Significance per Intervention
(Expected Frequency G: Germinated; Expected Frequency NG: Non-germinated) The water control produced 94 of 100 germinated seeds but even though Ferrum metallicum 97/100 (x2 1.046), Magnesium metallicum 96/100 (x2 0.42) and Manganum metallicum 95/100 (x2 0.094) produced higher germination rates than water, these results were not found to be statistically significant at p<0.05. The three (3) interventions which showed a significant effect (Calcarea carbonicum x2 6.8 with p<0.01; Cuprum metallicum x2 6.8 with p<0.01; and the Combination Intervention x2 6.8 with p<0.01) all produced inhibiting effects on seed germination. DISCUSSION No homeopathic essential mineral nutrient solution at 15X potency promoted the germination of corn seeds. Figure 2, shows cumulative germination results from four (4) extreme Interventions mapped against the Control over Time, as per Hossain (2005). Figure 2 – Cumulative Germination over Time: Results from 4 extreme Interventions compared to Water Control
Figure 2 displays a minimal rate of germination difference between the 4 extreme Interventions and Control. The rate of increase was greatest between 32-56 hours and plateaued after the 56th hour. It is believed that this research shows no significance due to seeds needing only water and oxygen to germinate. The essential mineral nutrients are needed for further plant development and absorbed through the leaf or root systems, only once germination has occurred. (Knox et al 1994, pp321, 374-380) CONCLUSION The Null Hypothesis is accepted and it is found that Homeopathically potentised 15X essential mineral nutrient solutions do not increase the seed germination of corn (Zea mays) when compared to distilled water. Future research will focus on the technique of priming seeds in homeopathically potentised essential mineral nutrients with analysis of the ensuing crop production. (Harris et al 2007) ACKNOWLEDGEMENTS Many thanks to John & Noela Eddington; Pacific Seeds; Department of Primary Industries; Endeavour College of Natural Health and Dr David Tulip. REFERENCES Australian Academy of Science, 1990, Biology – The Common Threads, Part 1, Australian Academy of Science, Australian Capital Territory, Australia Betti, L., Lazzarato, L., Trebbi, G., Brizzi, M., Calzoni, GL., Borghini, F., & Nani, D. 2003, ‘Effects of homeopathic arsenic on tobacco plant resistance to tobacco mosaic virus. Theoretical suggestions about system variability, based on a large experimental data set’, Homeopathy, Vol 92, pp195-202, viewed 23 April 2008, Science Direct database Boericke, W, 2004 Reprint, Pocket manual of Homeopathic Materia Medica with Indian Medicine & Repertory, 9th Edn, Indian Books & Periodicals Publishers, New Delhi, India Brizzi, M., Nani, D., Peruzzi, M., & Betti, L. 2000, ‘Statistical analysis of the effect of high dilutions of arsenic in a large dataset from a wheat germination model’, British Homeopathic Journal, Vol 89, pp63-67, viewed 23 April 2008, Science Direct database Foti, R., Abureni, K., Tigere, A., Gotosa, J. & Gere, J. 2008, (Abstract) ‘The efficacy of different seed priming osmotica on the establishment of maize (Zea mays L.) caryopses’, Journal of Arid Environments, Vol 72, Issue 6, pp1127-1130, viewed 12 May 2008, Science Direct database Gallardo, K., Job, C., Groot, P.C., Puype, M., Demol, H., Vandekerckhove, J., & Job, D. 2001, ‘Proeomic Analysis of Arabidopsis Seed Germination and Priming, Plant Physiology, Vol 126, pp835-848, viewed 7 May 2008, Google Scholar Guilford J, 1956, Fundamental Statistics in Psychology and Education, 3rd Edn, McGraw-Hill Book Company, London, United Kingdom Hamman, B., Koning, G., and Him Lok, K., 2003, ‘Homeopathically prepared gibberellic acid and barley seed germination’, Homeopathy, (2003) 92, pp140-144, viewed 23 April 2008, Science Direct database, DOI: 10.1016/S1475-4916(03)00045-6 Harris, D., Joshi, A., Khan, P.A., Gothkar, P., & Sodhi, P.S. 1999, (Abstract) ‘On-Farm Seed Priming in Semi-Arid Agriculture: Development and Evaluation in Maize, Rice and Chickpea in India Using Participatory Methods’, Experimental Agriculture, Cambridge Journals, Feb 1999, Vol 35, pp15-29, viewed 7 May 2008, Google Scholar, http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=2999 Harris, D., Rashid, A.b, Miraj, G.c, Arif,b M. & Shah, H. 2007 (Abstract) ‘On-farm seed priming with zinc sulphate solution – A cost-effective way to increase the maize yields of resource-poor farmers’, Field Crops Research, Vol 102, Issue 2, pp119-127, viewed 12 May 2008, Science Direct database, DOI: 10.1016/j.fer.2007.03.005 Hossain M, Arefin M, Khan B & Rahman M, 2005, “Effects of Seed Treatments on Germination and Seedling Growth Attributes of Horitaki (Terminalia chebula Retz.) in the nursery”, Research Journal of Agriculture and Biological Sciences 1(2): 135-141, INSInet Publication, viewed 5 October 2008, http://library.georgetown.edu/newjour/r/msg02863.html Knox B, Ladiges P & Evans B, 1994, Biology, McGraw-Hill Book Company, Sydney, Australia Pacific Seeds, 2008-09, Yearbook – Hybrid Corn, viewed 14 August 2008, http://www.pacificseeds.com/products/corn/documents/cornproduct08.pdf Polgar S & Thomas S, 2008, Introduction to Research in the Health Sciences, 5 Edn, Churchill Livingstone Elsevier, Sydney, Australia Raynes, M. and Bowey, G., 2008, The Decimal Growth Scale for Cereals, State of Victoria, Department of Primary Industries, AG0013, ISSN 1329-8062, viewed 12 March 2008, http://www.dpi.vic.gov.au/DPI/nreninf.nsf/v/859B65A8E6368D27CA2574100000B98B1/$file/The_Decimal_Growth_Scale_for_Cereals.pdf Scherr, C., Simon, M., Spranger, J. & Baumgartner, S. 2007 ‘Duckweed (Lemna gibba L.) as a Test Organism for Homeopathic Potencies, The Journal of Alternative and Complementary Medicine, Volume 13, Number 9, pp931-937, viewed 27 March 2008, Ebsco database, DOI: 10.1089/acm.2007.0506 Simillimum, 2008, Simillimum remedy list June 2008, viewed 10 June 2008, http://www.simillimum.co.nz Tulip J, 2008, Literature Review For Research Proposal, unpublished United Nations, 2008, The Secretary
General's High Level Task Force on the (WRI) World Resources Institute, 1989-99, Environmental change and human health – Soil-friendly farming practices are coming into wider use, viewed 20 October 2008, http://www.wri.org/publication/content/8427 APPENDIX 1 Essential Mineral Nutrients and Significance to Plant Function Information concerning Significance is taken from Sengusch (2003) and the Australian Academy of Science (1990, p178). These references will be denoted by an (S) and an (A) respectively.
--------------------------------------------- Australian Academy of Science, 1990, Biology – The Common Threads, Part 1, Australian Academy of Science, Australian Capital Territory, Australia Sengusch, P. 2003, Mineral Nutrients, viewed 8 May 2008, http://www.biologie.uni-hamburg.de/b-online/e16/16a.htm, Google Scholar |
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