An Improved Approach to Protoplast Regeneration and Transfection in Banana (Musa acuminata AAA cv. Williams)
Abstract
1. Introduction
2. Materials and Methods
2.1. Buffers and Media
2.2. Protoplast Isolation and Purification
2.3. Preparation of Conditioned Media
2.4. Protoplast Culture and Plant Regeneration
2.5. Protoplast Transfection
2.6. Post Transfection and Culture Maintenance
2.7. Statistical Analysis
3. Results
3.1. Protoplast Isolation
3.2. Protoplast Culture and Plant Regeneration
3.3. Transfection
3.4. Culture of Transfected Protoplasts
4. Discussion
4.1. Protoplast Isolation
4.2. Protoplast Regeneration
4.3. Protoplast Transfection
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Buffers and Media | Components * |
---|---|
Banana Cell Culture Media (BL) | MS macro and micronutrients (1×), MS-Fe-EDTA (1×), sucrose (20 g/L), Bluggoe vitamins (1×), ascorbic acid (50 mg/L), 2,4-D (1.1 mg/L), zeatin (0.25 mg/L), pH 5.7 |
Protoplast Isolation Buffer (PIB) | CaCl2.2H2O (7 mM), NaH2PO4.2H2O (0.7 mM), mannitol (0.55 M), MES (3 mM), pH 5.7 |
Protoplast Wash Buffer (PWB) | KCl (0.4 M), CaCl2.2H2O (0.034 M), mannitol (0.55 M), MES (3.5 mM), pH 5.7 |
Antioxidant Mix (AOM) | Ascorbic acid (50 mg/L), citric acid (5 mg/L), L-cysteine (50 mg/L) |
Protoplast Culture Media-8G (PCM-8G) | N6 salts (1×), KM vitamins (1×), KM sugars (1×), KM organic acids (1×), KH2PO4 (0.25 g/L), glucose (80 g/L), MES (0.5 mM), pH 5.7 |
Protoplast Culture Media-2G (PCM-2G) | N6 salts (1×), KM vitamins (1×), KM sugars (1×), KM organic acids (1×), KH2PO4 (0.25 g/L), glucose (20 g/L), sucrose (1 g/L), MES (0.5 mM), pH 5.7 |
Protoplast Culture Medium for Embryogenesis (PCM-E) | SH macro and micro salts (½×), MS micronutrients (½×), KM vitamins (½×), sucrose (20 g/L), glucose (2 g/L), MES (0.5 mM), pH 5.7 |
Regeneration Medium-I (RM-I) | MS macro and micronutrients (1×), MS-Fe-EDTA (1×), MW vitamins (1×), sucrose (30 g/L), IAA (0.1 mg/L), BAP (0.5 mg/L), MI (100 mg/L), pH 5.7 |
Regeneration Medium-II (RM-II) | MS macro and micronutrients (1×), MS-Fe-EDTA (1×), MW vitamins (1×), sucrose (30 g/L), BAP (0.05 mg/L), MI (100 mg/L), pH 5.7 |
MS | MS macro and micronutrients (1×), MS-Fe-EDTA (1×), MS vitamins (1×), sucrose (30 g/L), pH 5.7 |
MaMg | MgCl2.6H2O (15 mM), mannitol (0.5 M), MES (4 mM), pH 5.7 |
PEG-MaMg | MgCl2.6H2O (15 mM), mannitol (0.55 M), MES (5.12 mM), pH 5.7, PEG-3350 (20%) |
Stop Buffer | Mannitol (0.5 M), KCl (5 mM), MES (4 mM), pH 5.7 |
Enzyme Combinations | Enzymes (%) | |||
---|---|---|---|---|
Cellulase | Macerozyme | Driselase | Pectinase | |
E1 | 1 | 0.5 | 0 | 0 |
E2 | 1 | 1 | 0 | 0 |
E3 | 1 | 1 | 0.5 | 0 |
E4 | 1 | 1 | 0 | 0.5 |
E5 | 1 | 1 | 0.5 | 0.5 |
Tested Variable | Comparative Results * | |
---|---|---|
Protoplast and DNA incubation (min) | 1 | +++ |
5 | - | |
PEG size (ave. molecular weight) | 3350 | +++ |
4000 | - | |
PEG-3350 concentration (%) | 10 | +++ |
20 | + | |
PEG-3350 incubation time (min) | 5 | +++ |
10 | - | |
PEG-3350 transfection buffer | MaMg a | +++ |
CaCl2 b | - | |
PEG-3350 transfection buffer pH | 5.6 | +++ |
7 | - | |
PEG-3350 dilution regime | 20× | - |
40× | +++ | |
Transfection culture vessel | Falcon tubes | - |
Microfuge tubes | +++ | |
ECS subculture regime (days) | 7 | +++ |
10 | + | |
Protoplast recovery | Ice (30 min) | - |
RT (60 min) | +++ | |
Temperature shock (5 min) | Ice | +++ |
45 °C | + | |
Protoplast culture post-transfection | Liquid medium | +++ |
Solid medium | +++ |
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Deo, P.C.; Paul, J.-Y.; James, A.; Harding, R.; Dale, J. An Improved Approach to Protoplast Regeneration and Transfection in Banana (Musa acuminata AAA cv. Williams). Appl. Biosci. 2025, 4, 42. https://doi.org/10.3390/applbiosci4030042
Deo PC, Paul J-Y, James A, Harding R, Dale J. An Improved Approach to Protoplast Regeneration and Transfection in Banana (Musa acuminata AAA cv. Williams). Applied Biosciences. 2025; 4(3):42. https://doi.org/10.3390/applbiosci4030042
Chicago/Turabian StyleDeo, Pradeep Chand, Jean-Yves Paul, Anthony James, Rob Harding, and James Dale. 2025. "An Improved Approach to Protoplast Regeneration and Transfection in Banana (Musa acuminata AAA cv. Williams)" Applied Biosciences 4, no. 3: 42. https://doi.org/10.3390/applbiosci4030042
APA StyleDeo, P. C., Paul, J.-Y., James, A., Harding, R., & Dale, J. (2025). An Improved Approach to Protoplast Regeneration and Transfection in Banana (Musa acuminata AAA cv. Williams). Applied Biosciences, 4(3), 42. https://doi.org/10.3390/applbiosci4030042