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Background Hairy root (HR) transformation assays mediated by Agrobacterium rhizogenes , both in vitro and ex vitro , are essential tools in plant biotechnology and functional genomics. These assays can be significantly influenced by various factors, which ultimately can enhance the efficiency. In this study, we optimized a two-step ex vitro HR transformation method using the actual mother plant combined with the RUBY system and compared with existing methods. Results The two-step ex vitro method proved more efficient than both the one-step ex vitro and in vitro methods, with the highest transformation efficiency of 90% observed in the actual plant. This technique also demonstrated a faster and less complicated approach, reducing time to achieve massive transgenic HR formation by 9–29 days compared to other methods. Conclusions A novel, quicker, less complicated, and more efficient two-step transformation method for cannabis has been established, presenting a significantly lower risk of contamination. This protocol is particularly interesting to produce secondary metabolites using the CRISPR/Cas system in cannabis. We anticipate that this method will facilitate substantial time savings by rapidly producing hundreds of transformed samples.

Background Today, salinity stress is one of the most important abiotic stresses in the world, because it causes damage to many agricultural products and reduces their yields. Oxidative stress causes tissue damages in plants, which occurs with the production of reactive oxygen species (ROS) when plants are exposed to environmental stresses such as salinity. Today, it is recommended to use compounds that increase the resistance of plants to environmental stresses and improve plant metabolic activities. Salicylic acid (SA), as an intracellular and extracellular regulator of the plant response, is known as one of these effective compounds. Damask rose ( Rosa damascena Mill.) is a medicinal plant from the Rosaceae, and its essential oils and aromatic compounds are used widely in the cosmetic and food industries in the world. Therefore, considering the importance of this plant from both medicinal and ornamental aspects, for the first time, we investigated one of the native cultivars of Iran (Kashan). Since one of the most important problems in Damask rose cultivation is the occurrence of salinity stress, for the first time, we investigated the interaction of several levels of NaCl salinity (0, 4, 8, and 12 ds m − 1 ) with SA (0, 0.5, 1, and 2 mM) as a stress reducer. Results Since salinity stress reduces plant growth and yield, in this experiment, the results showed that the increase in NaCl concentration caused a gradual decrease in photosynthetic and morphological parameters and an increase in ion leakage. Also, increasing the level of salinity stress up to 12 ds m − 1 affected the amount of chlorophyll, root length and leaf total area, all of which reduced significantly compared to plants under no stress. However, many studies have highlighted the application of compounds that reduce the negative effects of stress and increase plant resistance and tolerance against stresses. In this study, the application of SA even at low concentration (0.5 mM) could neutralize the negative effects of salinity stress in the Rosa damascena . In this regard, the results showed that salinity increases the activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) and the concentration of proline, protein and glycine betaine (GB). Overexpression of antioxidant genes (Ascorbate Peroxidase (APX), CAT, Peroxidase (POD), Fe-SOD and Cu-SOD) showed an important role in salt tolerance in Damascus rose. In addition, 0.5 mm SA increased the activity of enzymatic and non-enzymatic systems and increased salinity tolerance. Conclusions The change in weather conditions due to global warming and increased dryness contributes to the salinization of the earth’s surface soils. Therefore, it is of particular importance to measure the threshold of tolerance of roses to salinity stress and the effect of stress-reducing substances in plants. In this context, SA has various roles such as increasing the content of pigments, preventing ethylene biosynthesis, increasing growth, and activating genes involved in stress, which modifies the negative effects of salinity stress. Also, according to the results of this research, even in the concentration of low values, positive results can be obtained from SA, so it can be recommended as a relatively cheap and available material to improve production in saline lands.

Vermicompost (VC) is a rich source of HA that improves plant growth and yield indices such as fresh and dry weights, plant height, stem diameter, leaf area, and chlorophyll index value. In this study, the effect of foliar application of HA extracted from different types of VC enriched with bacteria and/or fertilizers, commercial HA (CHA) and indole acetic acid (IAA) on the growth characteristics of canola ( Brassica napus ) in greenhouse conditions were compared. According to the results, the foliar application of HA extracted from VC had complete superiority over CHA and IAA in most traits except for the leaf number. Furthermore, the highest level of foliar application of HA (600 mg L −1 ) enriched with Azotobacter chroococcum (21Az) +  Pseudomonas fluorescens (Ps 59) (HA-AS) generated the highest height, diameter, leaf area, and chlorophyll index value. Also, the highest stomatal conductance and photosynthesis rate were observed with the application of 600 mg L −1 HA extracted from VC enriched with nitrogen, sulfur, and phosphorus (HA-NSP) compared to the other treatments. Besides, dry and fresh weights and seed yield under HA-NSP and HA-AS treatments were at their highest rate. Among the extracted HAs, the one extracted from the nitrogen enriched VC had the lowest efficiency. Based on the present study, the HA extracted from VC enriched with Azotobacter , Pseudomonas and NSP is recommended to increase canola growth and production.

Humic acid (HA) is a specific and stable component of humus materials that behaves similarly to growth stimulants, esp. auxin hormones, contributing to improving growth indices and performance of plants. As a rich source of HA, vermicompost (VC) is also a plant growth stimulating bio-fertilizer that can enhance growth indices and performance in plants. The purpose of the present study is to compare the influence of VC enriched with bacterial and/or fertilizer, commercial humic acid (CHA) extract, and indole-3-acetic acid (IAA) on improving growth characteristics and performance of rapeseed under greenhouse conditions. The results showed the complete superiority of VC over the CHA and IAA (approximately 8% increase in the dry weights of root and aerial organ and nearly three times increase in seed weight). The highest values of these indices were obtained with VC enriched with Nitrogen, Sulfur, and Phosphorus, Azotobacter chroococcum and Pseudomonas fluorescens; the lowest value was obtained with VC enriched with urea. Additionally, the application of 3% VC and the control involved the highest and lowest values in all traits, respectively. The SPAD (chlorophyll index) value and stem diameter were not significantly affected by different application levels of VC. Overall, the applications of IAA and the CHA were not found to be suitable and therefore not recommended.

The levels of antioxidant activity and vitamins can change with varying ratios of different wavelengths. This study was conducted as a pot experiment under soilless culture conditions in order to investigate the interactive effects of light supplementation and nutrition (calfomyth solution) on some qualitative traits of tomato fruit. The research was carried out as a split-plot experiment based on a completely randomized design with three light treatments including without supplementary light (control), 60% red light+40% blue light and 90% red light + 10% blue light. There were two nutritional treatments including no spraying with calfomyth as commercial fertilizer and foliar application with a concentration of 2 mg/L in three replicates. According to the results, the amounts of vitamin C (16.1 mg/g FW fruit), soluble solids (12.33), and lycopene (2.95 mg/g FW fruit) were all uppermost by the effect of a higher percentage of red light treatment. Higher percentage of t blue light resulted in the highest leaf chlorophyll content (38.4 mg/g FW leaf), but supplementary light treatments had no significant effects on the titratable acidity. Nutrition (calfomyth foliar application) showed positive impacts on all treatment traits compared to control. Beta-carotene content was affected by none of the treatments with no significant differences. According to this research, it can be expected that the use of complementary light treatments and calfomyth foliar spray may have positive effects on most of the qualitative traits in tomato fruit (cv. 240).

Using synthetic biology techniques to engineer secondary metabolic pathways through hairy root transformation is one of the most advanced approaches used in research. In this study, we optimized an ex-vitro one-step hairy root transformation of the RUBY system in both drug- and hemp-type cannabis, shedding light on its potential applications in secondary metabolite production. Three different strains of A. rhizogenes including (A4, ARqual, and K599) were used. Significant variation in HR induction and transformation efficiency (TE) was observed based on A. rhizogenes strains and seed types. Drug-type seedlings exhibited the highest hairy root induction, increasing by 58.8% compared to hemp-type seedlings. Also, the A4 strain consistently demonstrated the highest transformation efficiency (75%) irrespective of genotype, while the ARqual strain yielded the lowest one (8.33%). In conclusion, our study is the first to present an ex-vitro one-step transformation of both hemp- and drug-type cannabis. In comparison to the in vitro method, our ex-vitro method is simpler, faster, and has a lower risk of contamination, making it an excellent choice for the efficient production of secondary metabolites in cannabis using the CRISPR/Cas system. Keywords: Cannabis, Transformation, Hairy root transformation, Hairy root culture, Agrobacterium rhizogenes, Transformation efficiency.Competing Interest StatementThe authors have declared no competing interest.