Wheat germination and highly diluted agitated gibberellic acid (10e-30) - a multi researcher study

Grains of winter wheat (Triticum aestivum L., Capo variety) were observed under the influence of highly diluted gibberellic acid (10-30) prepared by stepwise dilution and agitation according to a protocol derived from homeopathy (“G30x”). Adequate control was used (water prepared according to the homeopathic protocol “W30x” and/or untreated water “W0”). Two sets of multicenter experiments were performed, 4 in 2009-2010 and 4 in 2011, involving altogether 6 researchers, 6 laboratories and 4,000 grains per treatment group. Data were found to be homogeneous within the control groups as well as within the verum groups. When the 2009-2010 experiments were pooled, mean germination rates after 24 hours were (85.9 + 2.6) for the control group and (82.1 + 5.7) for G30x (mean + SD at the level of experiments in %) (N = 2,000 per group). Verum germination rate was 4.4% lower than (i.e. equal to 96.6% of) (4.4 + 96.6 = 101) the control germination rate (100%). The difference is statistically significant (p < 0.001) and the effect size (d) is large (> 0.8). Observations at other points in time between 0 and 40 hours of germination yielded similar results. Practically no difference was found between W30x and W0 groups (p > 0.05). When the 2011 experiments were pooled, the mean germination rates after 24 hours were (73 + 12) for the control group and (73 + 14) for G30x (N = 2,000 per group), i.e. there was practically no difference between the groups (p > 0.05). We interpret the data from 2009-2010 on wheat germination within 40 hours as being in line with our previous findings on wheat stalk growth after one week, i.e. as confirmation that gibberellic acid 30x can influence, i.e. slow down, wheat development. Various possible reasons for the absence of any difference between groups in the 2011 experiments, including seasonal variance, are discussed and it is suggested to perform wheat germination experiments in the very beginning of autumn season only.

Poisson distribution and process as a well-fitting pattern for counting variables in biologic models

One of the major criticisms directed to basic research on high dilution effects is the lack of a steady statistical approach; therefore, it seems crucial to fix some milestones in statistical analysis of this kind of experimentation. Since plant research in homeopathy has been recently developed and one of the mostly used models is based on in vitro seed germination, here we propose a statistical approach focused on the Poisson distribution, that satisfactorily fits the number of non-germinated seeds. Poisson distribution is a discrete-valued model often used in statistics when representing the number X of specific events (telephone calls, industrial machine failures, genetic mutations etc.) that occur in a fixed period of time, supposing that instant probability of occurrence of such events is constant. If we denote with λ the average number of events that occur within the fixed period, the probability of observing exactly k events is: P(k) = e-λ λk /k! , k = 0, 1,2,… This distribution is commonly used when dealing with rare effects, in the sense that it has to be almost impossible to have two events at the same time. Poisson distribution is the basic model of the socalled Poisson process, which is a counting process N(t), where t is a time parameter, having these properties: - The process starts with zero: N(0) = 0; - The increments are independent; - The number of events that occur in a period of time d(t) follows a Poisson distribution with parameter proportional to d(t); - The waiting time, i.e. the time between an event and another one, follows and exponential distribution. In a series of experiments performed by our research group ([1], [2]., [3], [4]) we tried to apply this distribution to the number X of non-germinated seeds out of a fixed number N* of seeds in a Petri dish (usually N* = 33 or N* = 36). The goodness-of-fit was checked by different tests (Kolmogorov distance and chi-squared), as well as with the Poissonness plot proposed by Hoaglin [5]. The goodness-of-fit of Poisson distribution allows to use specific tests, like the global Poisson test (based on a chi-squared statistics) and the comparison of two Poisson parameters, based on the statistic z = X1–X2 / (X1+X2)1/2 which is, for large samples (at least 20 observations) approximately standard normally distributed. A very clear review of these tests based on Poisson distribution is given in [6]. This good fit of Poisson distribution suggests that the whole process of germination of wheat seeds may be considered as a non-homogeneous Poisson process, where the germination rate is not constant but changes over time. Keywords: Poisson process, counting variable, goodness-of-fit, wheat germination References [1] L.Betti, M.Brizzi, D.Nani, M.Peruzzi. A pilot statistical study with homeopathic potencies of Arsenicum Album in wheat germination as a simple model. British Homeopathic Journal; 83: 195-201. [2] M.Brizzi, L.Betti (1999), Using statistics for evaluating the effectiveness of homeopathy. Analysis of a large collection of data from simple plant models. III Congresso Nazionale della SIB (SocietàItaliana di Biometria) di Roma, Abstract Book, 74-76. [3] M.Brizzi, D.Nani, L.Betti, M.Peruzzi. Statistical analysis of the effect of high dilutions of Arsenic in a large dataset from a wheat germination model. British Homeopathic Journal, 2000;, 89, 63-67. [4] M.Brizzi, L.Betti (2010), Statistical tools for alternative research in plant experiments. “Metodološki Zvezki – Advances in Methodology and Statistics”, 7, 59-71. [5] D.C.Hoaglin (1980), A Poissonness plot. “The American Statistician”, 34, 146-149. [6] L.Sachs (1984) Applied statistics. A handbook of techniques. Springer Verlag, 186-189.

Allelopathic Effects of Essential Oils on Seed Germination of Barley and Wheat

In this study, we evaluated the allelopathic effects of essential oils (EOs) from six different plant species, namely, lavender (Lavandula angustifolia), hyssop (Hyssopus officinalis), English thyme (Thymus vulgaris), lovage (Levisticum officinale), costmary (Chrysanthemum balsamita), and cumin (Cuminum cyminum), on seed germination and seedling growth of barley (Hordeum vulgare) and wheat (Triticum aestivum). The main constituents of the EOs of L. angustifolia were 47.0% linalool acetate and 28.4% linalool; H. officinalis’ main constituents were 39.8% cis-pinocamphone, 9.8% trans-pinocamphone, 11.4% β-pinene, and 7.5% β-phellandrene; T. vulgaris’ were 38.2% para-cymene, 25.6% thymol, and 13.6% γ-terpinene; L. officinale’s were 64.8% α-terpinyl acetate and 14.7% β-phellandrene; C. balsamita’s were 43.7% camphor, 32.4% trans-thujone, and 11.6% camphene; C. cyminum’s were 49.6% cumin aldehyde, 10.4% para-cymene, 11.6% α-terpinen-7-al, and 9.1% β-pinene. All six EOs exhibited an allelopathic effect and suppressed the seed germination and seedling development of wheat and barley; however, the concentrations that exhibited a suppressing effect were different among the plants. C. cyminum EO completely suppressed both barley and wheat germination at 10-, 30-, and 90-µL application rates, making it the most effective treatment among the tested EOs. C. balsamita’s and H. officinalis’ EOs at 30 and 90 µL application rates completely suppressed barley and wheat radicles per seed, radicle length (mm), seedling height (mm), and germination (%). L. angustifolia’s EOs at 30- and 90-µL and T. vulgaris’ EO at 90 µL application rates also completely suppressed barley and wheat radicles per seed, radicle length (mm), seedling height (mm), and germination (%). C. balsamita’s, H. officinalis’, L. angustifolia’s, and T. vulgaris’ EOs at a 10 µL application rate reduced barley radicle length, seedling height, and % germination relative to the control. Wheat seed germination % was completely suppressed by the application of L. angustifolia’s and T. vulgaris’ EOs at 30 and 90 µL, while T. vulgaris’ EO at 10 µL rate reduced the germination relative to the control. Interestingly, C. balsamita and H. officinalis at 10 µL did not reduce wheat germination; however, they did reduce the number of radicles per seed, radicle length (mm), seedling height (mm), germination (%), and vigor index. Furthermore, L. officinale’s EO reduced the measured indices (radicles per seed, radicle length, seedling height, and vigor index) at the 10, 30, and 90 µL application rates relative to the non-treated control; however, none of the application rates of L. officinale’s EO had a suppression effect on wheat germination. This study demonstrated the allelopathic effects of the EOs of six different herbal plant species on seed germination of barley and winter wheat. The results can be utilized in the development of commercial products for controlling pre-harvest sprouting of wheat and barley. Further research is needed to verify the results under field conditions.

Peat as a promising raw material for the creation of bacterial preparations in solid form

The possibility of using peat as a solid base for a microbial preparation was investigated. The basis of the microbial consortium of the drug was made up of Pseudomonas extremorientalis and Aeromonas media isolated from coprolites of the earthworms Eisenia fetida, Savigny. Among the studied peat species, eutrophic woody peat (pH = 5.7, degree of decomposition 48%) was chosen, which stimulated the increase in the aboveground mass (+ 11%) of wheat seedlings in preliminary tests. We assessed the viability of bacteria within 2 months after their adsorption on peat. On the 56th day of the experiment, the number of A. media in the variant with a peat carrier remained at the highest possible level - 107 CFU / ml. In a vegetation experiment, it was found that the use of two microbial cultures (in liquid form and in solid after adsorption of bacteria on peat) for presowing seed treatment led to an increase in wheat germination by 9% (83% for the liquid form and 92% for the solid form). Plant biomass increased in variants using bacteria on peat compared to using bacteria in liquid form. The presence of peat and P. extremorientalis increased the aboveground weight of wheat by 5%, while peat with a mixture of bacteria increased this figure by 15%. In variants with the use of P. extremorientalis on peat, the rate of development of plant diseases decreased in comparison with the liquid form by more than 3 times. With the combined use of bacteria, peat provides high rates of germination of seeds and aboveground mass. The research results indicate a greater efficiency of using a solid preparative form of a microbial preparation in comparison with a liquid one.

Can Wheat Germination Test Be Used to Predict Pregnancy in Ewes?

Several invasive diagnosis methods to predict pregnancy in humans and animals were used in the past. Since Antiquity, grains germination test was used to predict more safely the pregnancy in women in order to avoid the possibility of miscarriage. The abscisic acid is a component of pregnant females’ urine that prevents germination of grains. Considering this principle, 20 samples of urine were collected from 10 pregnant ewes and 10 from non-pregnant animals. After urinalysis, 15 ml urine sample was added to 20 grains of wheat in a Petri dish, followed by a dilution of 1:4 with distilled water. Only water has been added in the control sample. There were performed duplicates for each animal. For 10 days, all plates were kept in a constant environmental temperature (22°C) and humidity (42%). Statistical analysis revealed that after 5 and 10 days, wheat grains belonging to pregnant ewes were less germinated, and values regarding shoot length were lower than those from the control group. Even if the number of samples is small, it can be concluded that wheat germination test can be used to predict pregnancy in ewes.

Application of High Voltage Electrical Discharge Treatment to Improve Wheat Germination and Early Growth under Drought and Salinity Conditions

The environmentally friendly, physical method of high voltage electrical discharge (HVED) was developed to improve the drought and salinity tolerance of two wheat genotypes. Unlike other plasma technologies, HVED treatment involves the discharge of electricity in water. In this study, the effect of HVED pretreatment on wheat germination and early vegetative growth under drought (0%, 15%, 20% and 30% PEG) and salinity (0, 90, 160 and 230 mM NaCl) stress conditions was investigated. HVED-exposed seeds showed altered seed surfaces and became more permeable to water uptake, resulting in higher germination percentages, germination index values, and shoot and root growth under the control and all drought and salinity concentrations. Moreover, the electrical conductivity of the water medium increased significantly, indicating HVED-induced reactions of ionization and dissociations of water molecules occurred. In addition, HVED pretreatment in the salt experiment improved the tolerance index values of the shoots and roots. The most pronounced genotypic variations occurred under the highest stress levels (30% PEG or 230 mM NaCl) and varied with the stress intensity and growth stage. The study results indicate that HVED pretreatment has the potential to improve drought and salt tolerance in wheat.

Nanoscale cluster structure of O2 gas-filled nanobubbles in O2 gas-supersaturated alkaline solution

There exists a close parallelism between gas-filled nanobubble solutions and ultra-high dilutions. Both these solutions contain Brownian nano-sized bubbles. We have studied aggregation kinetics of O2 gas-filled Brownian nanobubbles and their nanoscale cluster structure in 0.1M NaCO3 with NanoSight Particle Tracking Ananlysis (NTA) and transmission electron microscopy (TEM). A number of discrete peaks in a NTA size distribution of O2 nanobubbles in 0.1M NaCO3 eight days later after production are closely related to the existence of stable nanobubble clusters composed of 6-9nm nanobubbles that are inferred from the internal void size observed with TEM. Recent reports on 1HNMR of nanobubble water and its biological effects of wheat germination by Ohshita’s group may correlate with Demangeat`s NMR studies and germination experiments performed by many groups since the first publication by Lily Kolisco in 1923. Demangeat’s has recently published a review on the essential role of succussion that can cause nanobubble formation.

Intoxication of wheat seedlings with 50 mM NaCl and follow-up attempt to “cure” by extremely diluted NaCl (30cH)

Background: “High level of salinity deteriorates seed germination, growth and yield of crops in cultivated lands all over the world. There is no effective remedy to mitigate this global problem”, Mondal, Sukul et al.1 summed up the background of their study that led to the conclusion that homeopathically prepared, extremely diluted Natrum muriaticum (NaCl) 200cH promotes seed germination in seedlings of cowpea under salt stress.1 Aim: To investigate if the wheat germination model standardized at the Interuniversity College2,3 can be useful in investigating the NaCl issue; i.e. the study was not designed as a replication of the study by Mondal, Sukul et al.1 Methods: Grains of winter wheat (Triticum aestivum) were observed under the influence of extremely diluted NaCl (10-60) prepared by stepwise dilution and agitation according to a protocol derived from homeopathy (“N30c”). Analogously prepared water was used for control (“W30c”). Grains (500 per group) were pretreated (intoxicated) with (non-agitated) NaCl 50mM (“N”) or with water (“W”) prior to treatment with N30c or W30c. Seedlings were allowed to develop under standardized conditions for 32 h; germination of stalks and of roots were monitored at intervals of 4 h. Results: With regard to intoxication, the groups treated with N exhibited less growth than the W control groups. With regard to the attempt to “cure”, the N+N30c group was not statistically different from the N+W30c group, and the W+N30c group was not statistically different from the W+W30c group. Conclusion: The hypothesis that treatment of wheat seedlings with the extreme dilution NaCl 30cH will “cure” the effect of previous intoxication with NaCl 50 mM on seed germination could not be accepted. If further experiments are to be performed on the wheat model, these should include different intoxication solutions (e.g. 100mM1), different treatment dilutions (e.g. 200cH1) or different durations of the experiment3. The use of cowpea1 instead of wheat should also be considered.

Diluted versus potentitized probes of silver nitrate (10e-2 to 10e-10) and wheat germination

Corinne Kraus, Ute Knobloch, Scherer Waltraud, Peter Christian Endler Interuniversity College for Health and Development Graz / Castle of Seggau, Austria Background In 1926, an influence of a homeopathically prepared high dilution of silver nitrate on the growth of coleoptiles of wheat seedlings was described (Kolisko 1926). Later, in an extensive series of experiments, wheat was observed under the influence of extremely diluted agitated silver nitrate (10e-23, “24x”). Stalk lengths clearly indicate that development is enhanced by the probe silver nitrate 24x as compared to control (Scherer et al. 2015). A preliminary experiment was performed in early autumn 2015 on stalk growth of wheat seedlings treated with (not potentized) dilutions of silver nitrate 10e-6 to 10e-10 (“6e to 10e”), compared to potentized silver nitrate 6x to 10x (N = 100 per group). A clear, albeit not statistically significant trend was observed of 6x-stalks being longer (23.4 + 16.2 mm) than 6e-stalks (13.0 + 10.9 mm). Objective The aim of this study was to investigate the influence of diluted versus potentized low dilutions of silver nitrate (10e-2 to 10e-10) on wheat germination. Method The experiments were performed in late autumn 2015 on wheat grain (Triticum aestivum L., Capo variety). The grains were observed under the influence of aqueous solutions 10-2 to 10-10 part per weight of silver nitrate, either diluted in steps of 1 : 10 in distilled water by mere pipetting (probes “2e – 10e”), or diluted and agitated in steps of 1:10 (to create potentized probes “2x – 10x”). Untreated distilled water (“w”) served as an additional control. All probes were applied blindly. 100 grains were observed per treatment group in each of the groups resulting a total of 2,000 grains. Grains were placed in glass dishes, probes were added and dishes were covered with lids and placed in drawers . The following endpoint criteria were defined: K1 = visible emergence of sprout material, K2 = lifting of the operculum and emergence of the sprout and W1 = development of three roots.   Result Germination rates K1 of seedlings treated with “w”-probes (blue), with “e”-probes ranging from 2e to 10e (black) and with “x”-probes ranging from 2x to 10x (red) at the measuring points 20h, 24h and 28h (from left to right for each of the probes). In K1, K2 and W, there is an obvious increase of germination rates from the high to the lower concentrations of silver nitrate, both in the “e” and in the “x”-groups and observable at 20h, 24h and 28h (p < 0.01). In contrast, germination rates of the two „w“-probes are practically alike (p > 0,05) When “e” and “x”-data are compared, germination rates are higher under the influence of “x” than under the influence of “e” (p < 0.01 for the pooled “x”-values compared to the pooled “e”-values with regard to K1 as well as K2 as well as W). Conclusion: A significant difference was found between wheat grains treated with mere dilutions compared to grains treated with potentised dilutions. References 1. Endler PC, Belavite P, Bonamin L,Jäger T, Mazon S. Replication of fundamental research models in ultra high dilutions 1994 and 2015 – update on a bibliometric study. Special issue Homeopathy London. 2015 a Oktober;104(4):234-45. 2. Endler PC, Schulte J, Stock-Schroeer B, Stephen S. Ultra high Dilution 1994 revisited 2015 – the state of follow-up research. Special issue Homeopathy London. 2015 b Oktober;104(4):223-6. 3. Kolisko L. Physiologischer Nachweis der Wirksamkeit kleinster Entitäten bei 7 Metallen – Wirkung von Licht und Pflanzen auf das Pflanzenwachstum. Dornach Schweiz: Philosophisch-Anthroposophischer Verlag am Goetheanum; 1926. 4. Kraus C, Knobloch U. Diluted versus diluted and agitated probes of silver nitrate (10-2 to 10-10) and wheat germination, Thesis (MSc); branch campus UCN at Interuniversity College Graz / Schloss Seggau; 2016. 5. Scherer-Pongratz W., Endler P.C., Lothaller H., Stephen S. Wheat and ultra high diluted silver nitrate – further experiments and re-analysis of data. Special issue Homeopathy London. 2015;104(4):246-9.