Reproducibility of effects of the homeopathic dilutions 14x - 30x of gibberellic acid on growth of Lemna gibba L.

Background: Reproducibility of investigations in homeopathy is still challenging. Duckweed (Lemna gibba L.), a monocotyledonous waterplant which mostly reproduces vegetatively and therefore builds genetically identical clones, may be a suitable test system for standardised trials. Aims: This study investigated if formerly observed effects of gibberellic acid 14x – 30x on growth of Lemna gibba were reproducible. Methododology: Duckweed was grown in dilutions of gibberellic acid (14x–30x) as well as once succussed (c1) and unsuccussed (c0) water control. Area-related growth rate for day 0–7 was determined by a computerised image analysis system. Three series including five independent blinded and randomised experiments each were carried out in the same way as in the original study. Only time and conductor of experiments were modified. System stability was controled by three series of systematic negative control (SNC) experiments with the same set-up, but distilled and autoclaved water was used as the only test substance. According to the series with gibberellic acid, each serie of SNC experiments included five experiments. Full two-way ANOVA (α = 5%) was used for statistical analysis. Independent variables were treatment and experiment number, dependent variable was r(area) for day 0–7. Data of each experiment was normalised to its mean value to allow a better comparison between experiments. Only if the global ANOVA F-test was significant (p < 0.05) we compared the investigated groups with Fisher`s LSD test (protected Fisher`s LSD). Results: No specific effects of agitated dilutions of gibberellic acid were found in the first two replication series (p=0.263 and p=0.062). In the third serie with gibbous Lemna gibba L. we observed a significant effect (p=0.009) of the homeopathic treatment, however growth was increased in contrast to decreasing in the former study. Variability in experiments with gibberellic acid 14x – 30x was lower than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. Conclusions: When designing new studies to investigate reproducibility, different physiological states of the test organism must be considered. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

New Insights into Interspecific Hybridization in Lemna L. Sect. Lemna (Lemnaceae Martinov)

Duckweeds have been increasingly studied in recent years, both as model plants and in view of their potential applications as a new crop in a circular bioeconomy perspective. In order to select species and clones with the desired attributes, the correct identification of the species is fundamental. Molecular methods have recently provided a more solid base for taxonomy and yielded a consensus phylogenetic tree, although some points remain to be elucidated. The duckweed genus Lemna L. comprises twelve species, grouped in four sections, which include very similar sister species. The least taxonomically resolved is sect. Lemna, presenting difficulties in species delimitation using morphological and even barcoding molecular markers. Ambiguous species boundaries between Lemna minor L. and Lemna japonica Landolt have been clarified by Tubulin Based Polymorphism (TBP), with the discovery of interspecific hybrids. In the present work, we extended TBP profiling to a larger number of clones in sect. Lemna, previously classified using only morphological features, in order to test that classification, and to investigate the possible existence of other hybrids in this section. The analysis revealed several misidentifications of clones, in particular among the species L. minor, L. japonica and Lemna gibba L., and identified six putative ‘L. gibba’ clones as interspecific hybrids between L. minor and L. gibba.

Morphological Characterization and DNA Barcoding of Duckweed Species in Saudi Arabia

Duckweeds, or Lemnaceae, are widespread aquatic plants. Morphology-based identification of duckweed species is difficult because of their structural complexity. Hence, molecular tools provide significant advantages for characterizing and selecting species or clones for sustainable commercial use. In this study, we collected and characterized ten duckweed isolates from nine different regions in Saudi Arabia (SA). Based on the morphological characterization and phylogenetic analysis of intergenic spacer sequences of chloroplast DNA using six barcoding markers, the clones were classified into three genera, represented by seven species: Lemna gibba L., Lemna minor L., Lemna japonica Landolt, Lemna aequinoctialis Welw., Lemna perpusilla Torr., Spirodela polyryiza (L.) Schleid., and Landoltia punctate G. Mey. Lemna gibba was revealed to be a distinct dominant duckweed species in many regions of SA. Five barcoding markers showed that L. gibba, L. minor, and L. punctata were the most widely distributed species in the country. However, L. punctata, L. perpusilla, and S. polyryiza were the dominant species in the Al-Qassim, Madinah-1, and Madinah-2 regions, respectively. Moreover, the morphological traits revealed variations for these clones, relative to other studied duckweed clones. According to the results obtained in this study, three out of six plastid markers (trnH-psbA, matK, and atpF-atpH) helped to identify the dominant duckweed species in Saudi Arabia. Further evaluation based on adaptability, molecular genetic studies, and functional genomics is needed for these species to be used at the commercial level in Saudi Arabia.

Reproducibility of effects of homeopathically potentised Argentum nitricum on the growth of Lemna gibba L. in a randomized and blinded bioassay

Background: Reproducibility of basic research investigations in homeopathy is still a challenging issue. A former study reported a significant interaction between date of experiment and treatment effect of Argentum nitricum 14x–30x on the growth rate of duckweed (Lemna gibba L.). In a reproduction trial with duckweed (Lemna gibba L.) and potencies of gibberellic acid duckweed appeared only then sensitive to treatment with homeopathic potencies of gibberellic acid potencies when the plants were in the growth state of gibbosity. Aims: This study investigated if formerly observed effects of Argentum nitricum 14x – 30x on growth of Lemna gibba were reproducible and if sensitivity to homeopathic treatment was influences by the growth state of duckweed. Methods: Duckweed was grown in potencies of Argentum nitricum (14x–30x) and one time succussed and unsuccussed water control (c0 and c1). Area-related growth rate for day 0–7 was determined by a computerised image analysis system. The original study including six independent blinded and randomised experiments each were repeated one time completely and a second time with 3 experiments. Every repetition experiment was carried out in the same way as in the original study, only time and conductor of experiments were modified. System stability was controled by systematic negative control (SNC) experiments with the same set-up, but water as the only test substance. All water used in potency and SNC experiments was distilled and autoclaved before. Statistical analysis was evaluated with full two-way ANOVA (α = 5%), using treatment and experiment number as independent variables and r(area) for day 0–7 as dependent variable. Data of each experiment was normalised to its mean value to allow a better comparison between experiments. Only if the global ANOVA F-test was significant (p < 0.05) investigated groups were compared with Fisher`s LSD test (protected Fisher`s LSD). Results: The original study observed a significant interaction between treatment and experiment number compared to cpool (c0+c1).Caused to the fact that in the first repetition series a significant treatment difference between c0 and c1 was found, additionally a significant interaction between experiment number and c0 and c1, it was not possible to pool c0 and c1. All data (original study, as well as repetition series 1 and 2) was evaluated comparing potencies to one time succussed control c1. This new statistical evaluation confirmed the significant interaction between treatment and experiment number for data of the original study (p = 0.0055). In the first repetition series a significant treatment effect (p = 0.016) was observed, in the second series no effect.Variability in experiments with Argentum nitricum 14x – 30x was higher than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. An a posteriori subgroup analysis suggests that using a specific growth state of Lemna gibba (gibbosity) seems to be crucial for successful reproduction of the significant interaction of the original study. Conclusions: With the original study design chosen (disregarding gibbosity of Lemna gibba), results of the original study could not be reproduced. Bioassays for use in homeopathic basic research must carefully consider different physiological states of the test organisms. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

Tolerance and decolorization potential of duckweed (Lemna gibba) to C.I. Basic Green 4

With growing human culture and industrialization, many pollutants are being introduced into aquatic ecosystems. In recent years, dyes have become a major water pollutant used in the manufacture of paints and other production purposes. In this research, the potential of duckweed (Lemna gibba) plant was investigated spectrophotometrically as an obvious bioagent for the biological decolorization of the organic dye C.I. Basic Green 4 (Malachite Green, BG4). Photosynthetic efficiency analysis showed that the photosynthetic apparatus of L. gibba is very tolerant to BG4. Significant induction of reactive oxygen species (ROS) scavenging enzymes was observed after 24h of biodecolorization process in L. gibba treated with 15 and 30 mg/l BG4. The experimental results showed that L. gibba has a strong ability to extract BG4 from contaminated water and the best results were obtained at 25–30°C and pH 8.0. We conclude that duckweed L. gibba can be used as a potent decolorization organism for BG4.

Effects of Homeopathic Preparations of Mercurius corrosivus on the Growth Rate of Moderately Mercury-Stressed Duckweed Lemna gibba L

Background A bioassay with severely mercury-stressed duckweed (Lemna gibba L.) had revealed growth-inhibiting effects of homeopathically potentised mercury(II) chloride (Mercurius corrosivus, Merc-c.). We hypothesised that effects of potentised preparations are dependent on the stress level of the organisms used in the bioassay. The aim of the present investigation was to examine the response of duckweed to potentised Merc-c. at a lower stress level. Methods Duckweed was moderately stressed with 2.5 mg/L mercury(II) chloride for 48 hours. Afterwards plants grew in either Merc-c. (seven different potency levels, 24x–30x) or water controls (unsuccussed or succussed water) for 7 days. Growth rates of the frond (leaf) area were determined using a computerised image-analysis system for day 0–3 and 3–7. Three independent experiments with potentised Merc-c. and three systematic negative control experiments were performed. All experiments were randomised and blinded. Results Unsuccussed and succussed water did not significantly differ in their effects on duckweed growth rate. The systematic negative control experiments did not yield any significant effects, thus providing evidence for the stability of the experimental system. Data from the two control groups and the seven treatment groups (Merc-c. 24x–30x) were each pooled to increase statistical power. Duckweed growth rates for day 3–7 were enhanced (p < 0.05) after application of Merc-c. compared with the controls. Growth rates for day 0–3 were not influenced by the homeopathic preparations. Conclusions Moderately mercury-stressed Lemna gibba L. yielded evidence of growth-enhancing specific effects of Merc-c. 24x–30x in the second observation period (day 3–7). This observation is complementary to previous experiments with severely mercury-stressed duckweed, in which a decrease in growth was observed in the first observation period (day 0–3). We hypothesise that the differing results are associated with the level of stress intensity (moderate vs. severe).

Bioremediation Potential of Duckweed (Lemna Gibba) in The Decolorization of C.I. Basic Green 4 and Chlorophyll a Fluorescence Analysis: Understanding Plant Performance

In this research, the duckweed (Lemna gibba) potential has been investigated spectrophotometrically as an obvious bioagent for the biological decolorization of organic dye Basic Green 4 (BG4). Experiment result showed that L. gibba has a potent ability to extract BG4 from contaminated water. The study showed that for better results the temperature at 25-30 oC and pH 8.0 is considered to be optimum. A significant induction in SOD, GPOD and CAT activity was observed in L. gibba treated with 15 and 30 mg / l BG4, respectively, after 24 hours of biodecolorization process. It was observed during five repeated batch run that L. gibba showed comparable efficiency in dye decolorization. The chlorophyll fluorescence analysis also shows that photosynthetic apparatus of L. gibba is highly tolerant of BG4. The overall results of the observations here demonstrate the duckweed L. gibba can be used as a potent biodegrading organism for BG4.