In this study, the various concentrations of casein hydrolysate (25, 50, 75, 100 mg/L) and L-phenylalanine (50, 100, 150, 200 µM/l) were incorporated in MS containing 15 µM BA plus 5 µM 2,4-D for enhancement of secondary metabolites in cell culture of Spilanthes acmella. The presence of casein hydrolysate in the nutrient medium improved the growth of cell biomass and the production of scopoletin. The addition of casein hydrolysate up to 75 mg/L stimulated the accumulation of scopoletin, but increasing excess 75 mg/L the level of casein hydrolysate reduced the production of scopoletin. The addition of L-phenylalanine in the nutrient medium was found to be more effective for production of secondary metabolite in S. acmella. The addition of 50 µM/L of L-phenylalanine in the medium increased scopoletin content to 27.12 ± 0.58 µg/g dry weight, compared to the scopoletin content of control at 7.89 ± 0.61 µg/g dry weight. The highest accumulation of scopoletin was observed in the 100 µM/L L-phenylalanine in cell suspension, which was 4.51 times more than the control. As a result, using moderate concentration of L-phenylalanine was ideal for the production of scopoletin. In general, casein hydrolysate was more effective than L-phenylalanine for production of scopoletin and growth of cell biomass in the cell culture of S. acmella

Cold-acclimated and non-acclimated contrasting Camelina (Camelina sativa L.) biotypes were investigated for changes in stress-associated biomarkers, including antioxidant enzyme activity, lipid peroxidation, protein, and proline content. In addition, a well-known freezing tolerance pathway participant known as C-repeat/DRE-binding factors (CBFs), an inducer of CBF expression (ICE1), and a cold-regulated (COR6.6) genes of the ICE-CBF-COR pathway were studied at the transcriptional level on the doubled-haploid (DH) lines. Freezing stress had significant effects on all studied parameters. The cold-acclimated DH34 (a freezing-tolerant line) showed an overall better performance under freezing stress than non-acclimated plants. The non-cold-acclimated DH08 (a frost-sensitive line) showed the highest electrolyte leakage after freezing stress. The highest activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) was also detected in non-acclimated plants, whereas the cold-acclimated plants showed lower enzyme activities upon stress treatment. Cold acclimation had a significantly positive effect on the total protein and proline content of stressed plants. The qRT-PCR analysis revealed significant differences in the expression and cold-inducibility of CsCBF1-3, CsICE1, and CsCOR6.6 genes among the samples of different treatments. The highest expression of all CBF genes was recorded in the non-acclimated frost-tolerant biotype after freezing stress. Interestingly a significantly higher expression of COR6.6 was detected in cold-acclimated samples of both frost-sensitive and -tolerant biotypes after freezing stress. The presented results provide more insights into freezing tolerance mechanisms in the Camelina plant from both a biochemical point of view and the expression of the associated genes.

Freezing stress is an important abiotic stress that limiting the yield and the spatial distribution of many important crops. This study was undertaken to screen 136 doubled haploid (DH) lines of camelina (Camelina sativa L.) along with four canola (Brassica napus) cultivars (Hyola 401, Lord, Roska and Cascade) as experimental control under freezing stress conditions (-14?C for 6 h) to identify lines with high or low level of tolerance to freezing stress for further studies. First, a protocol was developed for large scale screening of camelina germplasm under freezing stress conditions. For this purpose, an experiment with different freezing temperatures (-5, -10, -15 and -20?C) was conducted to find an appropriate temperature that discriminated best between genotypes (i.e. the LT50 temperature). The LT50 values for camelina lines were varied between -10.2 and -17.1?C with an average of -13.94?C for all of the camelina lines. Therefore, we selected the -14?C exposure for 6 h as an appropriate temperature to screening of camelina lines. The principal components of measured parameters (LT50, survival percentage, relative conductivity and scoring) was using principal component analysis that determine freezing-tolerant and freezing-sensitive lines. Among 136 doubled haploid lines, some lines (58, 62 and 101) had higher level of freezing tolerance and some of them (8, 16, 32, 91 and 107) were freezing sensitive. The selected lines in a preliminary freezing screening are useful for further evaluations.