Biotechnology of the Tree Fern Cyathea smithii (J.D. Hooker; Soft Tree Fern, Katote) II Cell Suspension Culture: Focusing on Structure and Physiology in the Presence of 2,4-D and BAP
Abstract
:1. Introduction
2. Materials and Methods
2.1. Medium Description
2.2. Culture Initiation and Cell Suspension Establishment
2.3. Preparation of Living and Fixed Specimens for Bright-Field Light Microscopy
2.4. Scanning Electron Microscopy (SEM) Preparation
2.5. Semi-Thin Specimen Preparation
2.6. Preparation of Living Specimens for Confocal Microscopy
2.7. Acriflavine Nuclei Staining of Cell Suspension and Determination of Nuclear DNA Content
2.8. Cell Suspension Maintenance
2.9. Fresh, Dry, and Ash Matter Evaluation
2.10. Measurements of the Growth Kinetics of Cell Suspensions Depending on the Type of Energy Source
2.11. Measurements of Quercetin Produced by the Cell Suspension during the 15-Day Subculture in the Post-Culture Medium
2.12. Effectivity of Ethylene Production in Relation to the Fresh Weight of Initial Tissue
2.13. Effectivity of Ethylene Production According to the Length of Time
2.14. Antibacterial Activity of Extracts of Cell Suspension
2.14.1. Antibacterial Character Determined via Agar Disc Diffusion Assay
2.14.2. MIC (Minimum Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) Analyses
3. Results
3.1. Microscopic Analysis of Cell Aggregates
3.2. Morphogenic Potential Evaluation of Cell Suspensions
3.3. Measurement of Cell Suspension Kinetics
3.4. Nuclear DNA Content of Cell Suspension Determined with the Help of DNA-Specific Fluorochrome Acriflavine with Acetic Hydrolysis
3.5. Effectivity of ETH Production in Relation to the Fresh Weight of Initial Tissue of Subculture
3.6. Effectivity of ETH Production According to the Length of Time
3.7. Antibacterial Activity of C. smithii Extracts Tested with a Modified Disc Diffusion Method
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample No. | Time of Cultivation (Days) | Amount of ETH (ppm) | Amount of ETH per mg FW | Amount of ETH per mg DW | Amount ETH per mg of ASH |
---|---|---|---|---|---|
1. | 3 | 5.64 ± 0.00 | 1.77 ± 0.05 a | 17.80 ± 0.40 a | 0.0035 ± 0.0002 a |
2. | 6 | 6.67 ± 0.00 a | 1.34 ± 0.04 b | 17.88 ± 3.12 a,b | 0.0042 ± 0.0001 b |
3. | 9 | 5.34 ± 0.00 | 1.63 ± 0.04 a,b,c | 16.02 ± 4.39 a,b,c | 0.0046 ± 0.0003 c |
4. | 12 | 5.56 ± 0.00 | 0.53 ± 0.02 c | 6.02 ± 0.29 a | 0.0096 ± 0.0007 |
5. | 15 | 6.68 ± 0.00 a | 1.51 ± 0.48 a,b,c | 23.30 ± 6.85 a,b,c | 0.0044 ± 0.0016 a,b,c |
Sample No. | Initial Weight (g) | Amount of ETH (ppm) | Amount of ETH per mg FW | Amount of ETH per mg DW | Amount ETH per mg of ASH |
---|---|---|---|---|---|
1. | 0.5 | 5.25 ± 0.00 | 7.05 ± 0.25 | 58.00 ± 3.05 | 0.0006 ± 0.0001 |
2. | 1.0 | 5.46 ± 0.00 | 3.25 ± 0.04 | 26.05 ± 0.68 | 0.0021 ± 0.0001 |
3. | 1.5 | 5.51 ± 0.00 | 2.20 ± 0.10 | 16.50 ± 0.95 a | 0.0031 ± 0.0001 |
4. | 2.0 | 5.53 ± 0.00 | 1.20 ± 0.04 | 12.50 ± 0.31 a | 0.0045 ± 0.0002 |
5. | 3.0 | 4.55 ± 0.00 | 0.61 ± 0.48 | 10.25 ± 4.68 a | 0.0083 ± 0.0004 |
Sample | Staphylo coccus aureus | S. epidermidis | Escherichia coli | Pseudo monas aeruginosa | S. mutans | Propioni bacterium acnes PCM 2400 | P. acnes PCM 2334 | S. sanguinis PCM 2335 |
---|---|---|---|---|---|---|---|---|
Aerobic Gram-Positive | Aerobic Gram-Negative | Microaerobic Gram-Positive | ||||||
Standards | ||||||||
quercetin | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 0 |
luteolin | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
gallic acid | 15 | 36 | 12 | 10 | 16 | 20 | 22 | 18 |
p-coumaric acid | 0 | 0 | 8 | 6 | 0 | 0 | 0 | 0 |
caffeic acid | 0 | 28 | 4 | 4 | 0 | 0 | 0 | 0 |
vanillic acid | 0 | 0 | 4 | 4 | 0 | 0 | 0 | 0 |
Samples of Cyathea smithii | ||||||||
In vitro * | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Green house ** | 0 | 0 | 0 | 0 | 0 | 6 | 6 | 0 |
Cell suspension *** | 15 | 10 | 40 | 36 | 19 | 18 | 16 | 22 |
Sample | S. aureus ATCC 25923 | MBC/MIC | S. epidermidis ATCC 12228 | MBC/MIC | E. coli ATCC 25992 | MBC/MIC | P. aeruginosa ATCC 27853 | MBC/MIC | S. mutans PCM 2502 | MBC/MIC | P. acnes PCM 2400 | MBC/MIC | P. acnes PCM 2334 | MBC/MIC | S. sanguinis PCM 2335 | MBC/MIC |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Cell * | 62.5 | 8 | 125 | 16 | 7.8 | 16 | 62.5 | 8 | 62.5 | 4 | 62.5 | 4 | 62.5 | 4 | 15.6 | 2 |
Gallic acid | 125 | 16 | 31.25 | 8 | 500 | 8 | 1000 | - | 125 | 8 | 62.5 | 8 | 62.5 | 8 | 62.5 | 8 |
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Rybczyński, J.J.; Kaźmierczak, A.; Dos Santos Szewczyk, K.; Tomaszewicz, W.; Miazga-Karska, M.; Mikuła, A. Biotechnology of the Tree Fern Cyathea smithii (J.D. Hooker; Soft Tree Fern, Katote) II Cell Suspension Culture: Focusing on Structure and Physiology in the Presence of 2,4-D and BAP. Cells 2022, 11, 1396. https://doi.org/10.3390/cells11091396
Rybczyński JJ, Kaźmierczak A, Dos Santos Szewczyk K, Tomaszewicz W, Miazga-Karska M, Mikuła A. Biotechnology of the Tree Fern Cyathea smithii (J.D. Hooker; Soft Tree Fern, Katote) II Cell Suspension Culture: Focusing on Structure and Physiology in the Presence of 2,4-D and BAP. Cells. 2022; 11(9):1396. https://doi.org/10.3390/cells11091396
Chicago/Turabian StyleRybczyński, Jan J., Andrzej Kaźmierczak, Katarzyna Dos Santos Szewczyk, Wojciech Tomaszewicz, Małgorzata Miazga-Karska, and Anna Mikuła. 2022. "Biotechnology of the Tree Fern Cyathea smithii (J.D. Hooker; Soft Tree Fern, Katote) II Cell Suspension Culture: Focusing on Structure and Physiology in the Presence of 2,4-D and BAP" Cells 11, no. 9: 1396. https://doi.org/10.3390/cells11091396