A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material
2.2. Procedure
2.2.1. Surface Sterilization of Mature Rice Seeds
2.2.2. Optimization of Efficient Callus Induction and Regeneration in Rice cv. MTU1010
2.2.3. Gene Constructs and Agrobacterium Strain Used for Rice Transformation
2.2.4. Preparation of Primary and Secondary Agrobacterium Culture
2.2.5. Transformation and Co-Cultivation
2.2.6. Washing of Calli and Resting Medium
2.2.7. Selection of Transformed Calli
2.2.8. Regeneration of Transformed Calli
2.2.9. Rooting Medium and Root Hardening
2.2.10. Molecular Confirmation of Putative Transgenic Plants
2.2.11. Southern Blotting
2.2.12. β-Glucuronidase Staining (GUS) Assay
2.2.13. Statistical Analysis
3. Results and Discussion
3.1. Optimization of In Vitro Embryogenic Callus Induction in Rice cv. MTU1010
3.2. Downscaling Salt Concentration during Infection and Co-Cultivation Can Enhance the T-DNA Delivery and High Transient Expression Efficiency in Rice cv. MTU1010
3.3. Effect of Pre-Incubation on Transformation Efficiency
3.4. Optimization of Regeneration Medium and Measurement of Regeneration Frequency
3.5. Molecular Confirmation
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviation
References
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Name of the Media | Composition |
---|---|
Callus induction media (CIM) | MS Salts with B5 vitamins, 300 mg/L Casein hydrolysate, 560 mg/L L-Proline, 36 g/L Maltose monohydrate, pH-5.8, 4 g/L Phytagel. Autoclave the media and allow them to cool to room temperature and then add 2.5 mg/L 2,4-D and 0.25 mg/L 6-BAP |
Yeast extract Mannitol medium (YEM) | Yeast extract 1 g/L, Mannitol 10 g/L, NaCl 1 g/L, MgSO4.7H2O 0.2 g/L, K2HPO4 0.5 g/L, pH-6.8–7. Autoclave and store the media at room temperature |
Yeast extract Mannitol Agar medium (YEMA) | To YEM medium, add 1.5% Agar |
Resuspension medium (RSM-1) | MS salts with 1.5% sucrose and adjust the pH to 5.4 autoclave and store at room temperature |
Resuspension medium (RSM-2) | ½ MS or ¼ MS salts with 1.5% sucrose and adjust the pH to 5.4 autoclave and store at room temperature |
Co-cultivation medium (CCM-1) | MS Salts with B5 vitamins, 300 mg/L Casein hydrolysate, 560 mg/L L-Proline, 36 g/L Maltose monohydrate, pH-5.8, 4 g/L Phytagel. Autoclave and then add 2.5 mg/L 2,4-D, 0.25 mg/L 6-BAP and 150 µM acetosyringone (freshly prepared) |
Co-cultivation medium (CCM-2) | ½ MS Salts with B5 vitamins, 300 mg/L Casein hydrolysate, 560 mg/L L-Proline, 36 g/L Maltose monohydrate, pH-5.8, 4 g/L Phytagel. Autoclave and then add 2.5 mg/L 2,4-D, 0.25 mg/L 6-BAP and 150 µM acetosyringone (freshly prepared) |
Resting Media | To the CIM medium, add 300 mg/L Cefotaxime, 200 mg/L Timentin. |
Selection medium-1 (SM-1) | To the CIM medium, add 300 mg/L Cefotaxime, 200 mg/L Timentin, 50 mg/L Hygromycin |
Selection medium-2 (SM-2) | To the CIM medium, add 250 mg/L Cefotaxime, 50 mg/L Hygromycin |
Regeneration medium (RM-1) | MS salts, 30 g/L Maltose monohydrate, pH-5.8, 1% Agarose. Autoclave the media and then add 2.5 mg/L 6-BAP, 1 mg/L Kinetin, 0.5 mg/L NAA, 250 mg/L cefotaxime and 35 mg/L Hygromycin |
Regeneration medium (RM-2) | MS salts, 30 g/L Maltose monohydrate, pH-5.8, 0.8% Agarose. Autoclave the media and add 2.5 mg/L 6-BAP, 1 mg/L Kinetin and 0.5 mg/L NAA, 30 mg/L Hygromycin |
Rooting medium | ½ MS salts, 20 g/L Sucrose, 10 g/L Glucose, pH-5.8 then add 2.5 g/L Phytagel. Autoclave the media and then add 0.05 mg/L NAA |
Root hardening | ½ strength Yoshida Medium or ½ strength Hoagland medium |
Acclimatization | Soil rite mixture |
Transplanting | Soil |
Media | Composition | [19] | [18] | [41] | [42] | [43] | [44] |
---|---|---|---|---|---|---|---|
Genotypes | IR64 | CSR10, IR64, PB1, Swarna | ADT 43 | AC39020 | MTU1010 | CO39 | |
CIM (Callus Induction Media) |
| 4.4 g/L 0.03 g/L 0.065 g/L --- 30 g/L --- 4 g/L 2 g/L 2.5 mg/L 0.15 mg/L 5.8 | 4.4 g/L 0.3 g/L 0.60 g/L 30 g/L --- 10 g/L 3 g/L --- 3 mg/L 0.25 mg/L 5.2 | --- --- --- --- --- --- 3 g/L --- 2.5 mg/L --- 5.8 | --- --- --- --- --- --- --- --- 2 mg/L --- 5.8 | 4.4 g/L 0.4 g/L 0.7 g/L 30 g/L --- --- 4 g/L --- 2.5 mg/L --- 5.8 | 4.3 g/L 0.3 g/L 2.8 g/L 30 g/L --- --- 3 g/L --- 3 mg/L --- 5.8 |
Resuspension medium |
| 4.4 g/L 68 g/L 36 g/L 3 g/L 4 g/L 150 µM 5.2 | 4.4 g/L 68 g/L 36 g/L 3 g/L 4 g/L 150 μM 5.2 | Not Mentioned | Not Mentioned | Not Mentioned | 4.3 g/L 68 g/L 36 g/L 3 g/L 4 g/L 150 µM 5.2 |
CO-C (Co-Cultivation Media) |
| 4.4 g/L 0.03 g/L 0.065 g/L --- 30 g/L --- 4 g/L 2 g/L 2.5 mg/L 0.15 mg/L 150 µM 5.8 | 4.4 g/L 0.3 g/L 0.60 g/L 30 g/L --- 10 g/L 3 g/L --- 3 mg/L 0.25 mg/L 150 µM 5.2 | --- --- --- --- --- --- 3 g/L --- 2.5 mg/L --- 100 µM 5.8 | Not Mentioned | 4.4 g/L --- 0.5 g/L --- 20 g/L 10 g/L 4 g/L --- 1.5 mg/L --- 200 µM 5.2 | 4.3 g/L 0.3 g/L 0.60 g/L --- --- 10 g/L 4 g/L --- 3 mg/L --- 150 µM 5.8 |
Selection Media |
| 4.4 g/L 0.03 g/L 0.065 g/L --- 30 g/L --- --- 4 g/L 2 g/L 2.5 mg/L 0.15 mg/L 50 mg/L 300 mg/L --- --- 5.8 | 4.4 g/L 0.3 g/L 0.60 g/L 30 g/L --- 10 g/L --- 3 g/L --- 3 mg/L 0.25 mg/L 50 mg/L 250 mg/L --- --- 5.2 | --- --- --- --- --- --- --- 3 g/L --- 2.5 mg/L --- --- --- --- --- 5.8 | Not Mentioned | 4.4 g/L 0.4 g/L 0.7 g/L --- 30 g/L --- 1 g/L 4 g/L --- 3 mg/L --- 50 mg/L --- --- 600 mg/L 5.8 | 4.3 g/L 0.3 g/L 2.8 g/L --- 30 g/L --- --- 3 g/L --- 3 mg/L --- 50 mg/L --- 250 mg/L --- 5.8 |
Regeneration Media (I) |
| 4.4 g/L --- 30 g/L 3 mg/L 0.5 mg/L 1 mg/L --- --- 40 mg/L 10 g/L 5.8 | 4.4 g/L 30 g/L --- --- 0.2 mg/L 2 mg/L 250 mg/L --- 30 mg/L 10 g/L 5.8 | 4.4 g/L 30 g/L --- 1 mg/L 1.5 mg/L 1.2 mg/L --- --- 30 mg/L 10 g/L 5.8 | 4.4 g/L 30 g/L --- 4 mg/L 0.5 mg/L --- --- --- 30 mg/L 10 g/L 5.8 | 4.4 g/L --- 30 g/L 2 mg/L 0.5 mg/L 1 mg/L --- 600 mg/L 30 mg/L 4 g/L 5.8 | 4.3 g/L 30 g/L --- --- 0.2 mg/L 2 mg/L --- --- --- 10 g/L 5.8 |
Regeneration Media (II) |
| 4.4 g/L --- 30 g/L 3 mg/L 0.5 mg/L 1 mg/L --- 40 mg/L 8 g/L 5.8 | 4.4 g/L 30 g/L --- 2.7 mg/L 0.5 mg/L 1.2 mg/L 250 mg/L 30 mg/L 8 g/L 5.8 | Not Mentioned | Not Mentioned | Not Mentioned | 4.3 g/L 30 g/L --- --- 0.2 mg/L 2 mg/L --- 30 mg/L 8 g/L 5.8 |
Rooting Media |
| 4.4 g/L 20 g/L 10 g/L --- --- --- 4 g/L | 2.2 g/L 30 g/L --- --- 250 mg/L 30 g/L 3 g/L | Not Mentioned | Not Mentioned | 2.2 g/L 15 g/L --- --- --- --- 4 g/L | 2.15 g/L 30 g/L --- --- --- 30 mg/L 3 g/L |
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Yadav, P.; Santosh Kumar, V.V.; Priya, J.; Yadav, S.K.; Nagar, S.; Singh, M.; Chinnusamy, V. A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables. Methods Protoc. 2023, 6, 113. https://doi.org/10.3390/mps6060113
Yadav P, Santosh Kumar VV, Priya J, Yadav SK, Nagar S, Singh M, Chinnusamy V. A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables. Methods and Protocols. 2023; 6(6):113. https://doi.org/10.3390/mps6060113
Chicago/Turabian StyleYadav, Pragya, V. V. Santosh Kumar, Jyoti Priya, Shashank Kumar Yadav, Shivani Nagar, Meenu Singh, and Viswanathan Chinnusamy. 2023. "A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables" Methods and Protocols 6, no. 6: 113. https://doi.org/10.3390/mps6060113
APA StyleYadav, P., Santosh Kumar, V. V., Priya, J., Yadav, S. K., Nagar, S., Singh, M., & Chinnusamy, V. (2023). A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables. Methods and Protocols, 6(6), 113. https://doi.org/10.3390/mps6060113