Improved and Highly Efficient Agrobacterium rhizogenes-Mediated Genetic Transformation Protocol: Efficient Tools for Functional Analysis of Root-Specific Resistance Genes for Solanum lycopersicum cv. Micro-Tom
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material
2.2. Agrobacterium Strain and Vector
2.3. Agrobacterium Culture Preparation and Infection Medium
2.4. Preparation of Explants, Agrobacterium Infection and Co-Cultivation
2.5. Tissue Culture and Transplantation
2.6. Fluorescence Observation and Imaging
2.7. DNA Extraction and PCR Verification
2.8. Determination of Agrobacterium Infection and Transformation Efficiency
2.9. Nematode Susceptibility Test
3. Results
3.1. Rhizogenes Inoculation Method
3.2. Elimination of A. rhizogenes
3.3. Agrobacterium Transformation of Explants
3.4. DsRed Allows for Quick and Easy Identification of Positive Plants
3.5. Confirmation of Genetic Co-Transformation of the Roots by PCR
3.6. Long-Term Evaluation of Transformed Roots after Nematode Infection and Analysis of the Presence of Transgenes
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Organ Explant | Inoculation Method | Total No. of Transformed Plant | No. of Explants Surviving | No. of DsRed-Positive Roots | Mortality Rate (%) |
---|---|---|---|---|---|
Radicles | Coating—method (1) | 40 | 11 | 11 | 73 |
Hypocotyl | Coating—method (2) | 40 | 0 | 0 | 100 |
Hypocotyl | Puncturing—method (3) | 40 | 1 | 1 | 98 |
Inoculation with A. rhizogenes Strain ARqua1 (pKWGFS7-RR) | Mock Inoculation—A. rhizogenes Strain ARqua1 (pKWGFS7) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Organ Explant | Inoculation Method | No. of Experiments | Total No. of Transformed Plant | No. of Explants That Showed Hairy Roots | No. of DsRed-Positive Roots | Total No. of Transformed Plant | No. of Explants That Showed Hairy Roots | No. of DsRed-Positive Roots | Transformation Efficiency ¹ (%) |
Radicles | Coating—method (1) | 2× | 60 | 57 | 55 | 20 | 18 | 0 | 91 |
Hypocotyl | Coating—method (2) | 2× | 60 | 58 | 56 | 20 | 18 | 0 | 92 |
Hypocotyl | Puncturing—method (3) | 2× | 60 | 58 | 57 | 20 | 19 | 0 | 95 |
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Tóth, M.; Tóth, Z.G.; Fekete, S.; Szabó, Z.; Tóth, Z. Improved and Highly Efficient Agrobacterium rhizogenes-Mediated Genetic Transformation Protocol: Efficient Tools for Functional Analysis of Root-Specific Resistance Genes for Solanum lycopersicum cv. Micro-Tom. Sustainability 2022, 14, 6525. https://doi.org/10.3390/su14116525
Tóth M, Tóth ZG, Fekete S, Szabó Z, Tóth Z. Improved and Highly Efficient Agrobacterium rhizogenes-Mediated Genetic Transformation Protocol: Efficient Tools for Functional Analysis of Root-Specific Resistance Genes for Solanum lycopersicum cv. Micro-Tom. Sustainability. 2022; 14(11):6525. https://doi.org/10.3390/su14116525
Chicago/Turabian StyleTóth, Máté, Zoltán Gábor Tóth, Sándor Fekete, Zoltán Szabó, and Zoltán Tóth. 2022. "Improved and Highly Efficient Agrobacterium rhizogenes-Mediated Genetic Transformation Protocol: Efficient Tools for Functional Analysis of Root-Specific Resistance Genes for Solanum lycopersicum cv. Micro-Tom" Sustainability 14, no. 11: 6525. https://doi.org/10.3390/su14116525
APA StyleTóth, M., Tóth, Z. G., Fekete, S., Szabó, Z., & Tóth, Z. (2022). Improved and Highly Efficient Agrobacterium rhizogenes-Mediated Genetic Transformation Protocol: Efficient Tools for Functional Analysis of Root-Specific Resistance Genes for Solanum lycopersicum cv. Micro-Tom. Sustainability, 14(11), 6525. https://doi.org/10.3390/su14116525