Treatment of licorice seeds with colchicine: changes in seedling DNA levels and anthocyanin and glycyrrhizic acid contents of derived callus cultures

The use of colchicine to induce polyploids increases secondary metabolite production potential and has been used for many years for the production of valuable compounds in plants. This project took advantage of this method to increase the production of secondary metabolites in licorice. For this purpose, seeds of licorice, Glycyrrhiza glabra var. glandulifera, were treated with different concentrations of colchicine for 24 hours and then cultivated in vitro. After a month, the effect of colchicine on the cellular DNA level of cotyledons was analyzed by spectrophotometry and flow cytometry. For callus induction, root explants of one month old plantlets derived from colchicine treated seeds were transferred to MS medium containing growth regulators and the anthocyanin and glycyrrhizic acid levels of the callus tissues were measured after two months of growth. The total DNA content of plantlets derived from seeds treated with 0.05%, 0.08% and 0.1% of colchicine for 24 hours was increased significantly. Treated plants had increased numbers of larger stomata, significantly in those treated with 0.1% of colchicine for 24 hours. After colchicine treatment, the root, shoot and leaf thickness was found to be increased, while their length was decreased. Results of flow cytometry showed changes in ploidy level in plantlets obtained from treatment with 0.08% (mixoploids) and 0.1% (tetraploids) of colchicine. Anthocyanin level was significantly increased in callus obtained from plantlets treated with 0.08% of colchicine. The amount of glycyrrhizic acid in all treatments increased, especially in the 0.1 and 0.03% colchicine treatments and this seems to prove an increased production of metabolites in polyploid licorice tissues

Colchicine effect on the DNA content and stomata size of Glycyrrhiza glabra var.glandulifera and Carthamus tinctorius L. cultured in vitro

In vitro induction of polyploids using colchicine causes an increase in DNA content in plants. This is of high importance especially for plants that have medicinal and commercial values. Seeds of two medicinal plants, licorice Glycyrrhiza glabra L. var.glandulifera and safflower Carthamus tinctorius were treated with different concentrations of colchicine, 0%, 0.03%, 0.05%, 0.08%, 0.1% (W/V) in vitro for 24 and 48 h. Treated seeds then were cultured on solid Murashige and Skoog (MS) media under controlled conditions. After a month, the length of the stomata was measured to study the effect of colchicine on stomata size. Cellular DNA content of the regenerated plants was measured by spectrophotometry. Flow cytometry was used for confirming the results obtained from stomata size measurement and spectrophotometry. Results suggested that treated plants have a fair amount of larger stomata, significantly in licorice plantlets that were treated with 0.1% colchicine for 24 h and safflower plantlets that were treated with 0.03%, 0.05% and 0.1% colchicine. Safflower DNA content in all treatments enhanced significantly, but in licorice only DNA content of plantlets that were treated with 0.05% colchicine for 24 h and 0.1%, 0.03% colchicine for 48 h found to be increased significantly. The morphological features of treated plantlets such as shoot and leaf thickness were found to be increased. Flow cytometry confirmed the previously mentioned results and suggested tetraploids in all treated safflower plantlets and licorice plantlets obtained from treatment with 0.08% of colchicine and mixoploids in licorice plantlets obtained from treatment with 0.1% of colchicine

A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13 min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260 nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RS

Nicotine alkaloid levels, and nicotine to nornicotine conversion, in Australian Nicotiana species used as chewing tobacco

A range of endemic Nicotiana species are chewed as a smokeless tobacco by several Aboriginal populations of Australia. In tobacco research, nicotine to nornicotine conversion is important because nornicotine lowers tobacco quality and is detrimental to health. A diverse group of cytochrome P450 genes with different transcriptional regulations are involved in this conversion. The primary aims of this study were to quantify the pyridine alkaloids and investigate nicotine to nornicotine conversion in laboratory-grown Australian Nicotiana spp. Nicotine, nornicotine, anatabine, anabasine, myosmine and cotinine were quantified in fresh leaves of 24 out of the 26 recognised Australian Nicotiana taxa. Conserved regions of CYP82E related genes were PCR amplified in all studied taxa. The conversion process in fresh leaves was compared with that in leaves that underwent a simulated curing process for species that we identified as being high converters (N. cavicola, N. goodspeedii, N. velutina) and low converters (N. benthamiana, N. excelsior, N. gossei). Agarose gel electrophoretic analysis of CYP82E related genes obtained from the PCR amplification of the cDNA in fresh versus leaves with simulated curing showed about a 3-fold increase in transcript accumulation levels in cured leaves of the high converter species, while the transcript accumulation in N. gossei and N. excelsior maintained a steady basal level and increased by a small amount in N. benthamiana. This suggests the presence of functional loci that are triggered by curing in only high converter species and indicates a potential risk for chewers of high converter species

Corrigendum to “In vitro cytotoxicity of Nicotiana gossei leaves, used in the Australian Aboriginal smokeless tobacco known as pituri or mingkulpa” [Toxicol. Lett. 254 (2016) 45–51]

The authors regret that the list of authors was incomplete and has now been corrected. Additionally, the first sentence of the conclusion should read ‘This is the first chemical analysis of pituri undertaken to consider the potential for negative consequences of use.’ A new second sentence should be inserted into the conclusion to read ‘This article is one component of a larger research project investigating the use of native Nicotiana plants in Australia.’ The last sentence of the acknowledgements commencing with ‘and Angela Ratsch…’ is not required. The authors would like to apologise for any inconvenience caused