A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables
Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of function...
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MDPI AG
2023-11-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/6/6/113 |
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| author | Pragya Yadav V. V. Santosh Kumar Jyoti Priya Shashank Kumar Yadav Shivani Nagar Meenu Singh Viswanathan Chinnusamy |
| author_facet | Pragya Yadav V. V. Santosh Kumar Jyoti Priya Shashank Kumar Yadav Shivani Nagar Meenu Singh Viswanathan Chinnusamy |
| author_sort | Pragya Yadav |
| collection | DOAJ |
| description | Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for <i>indica</i> cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega <i>indica</i> rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of <i>Agrobacterium</i> culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of <i>indica</i> rice cultivars for functional genomics studies and crop improvement. |
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| spelling | doaj.art-b76962524e4142859034ae185cdeb0ad2023-12-22T14:28:08ZengMDPI AGMethods and Protocols2409-92792023-11-016611310.3390/mps6060113A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal VariablesPragya Yadav0V. V. Santosh Kumar1Jyoti Priya2Shashank Kumar Yadav3Shivani Nagar4Meenu Singh5Viswanathan Chinnusamy6Division of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 110012, IndiaDivision of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 110012, IndiaDivision of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 110012, IndiaDivision of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 110012, IndiaDivision of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 110012, IndiaDepartment of Biotechnology, College of Engineering and Technology, IILM University, Greater Noida 201310, IndiaDivision of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 110012, IndiaRice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for <i>indica</i> cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega <i>indica</i> rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of <i>Agrobacterium</i> culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of <i>indica</i> rice cultivars for functional genomics studies and crop improvement.https://www.mdpi.com/2409-9279/6/6/113in vitro culturecallus induction efficiencytransformation efficiencyregeneration efficiencyBAPkinetin |
| spellingShingle | Pragya Yadav V. V. Santosh Kumar Jyoti Priya Shashank Kumar Yadav Shivani Nagar Meenu Singh Viswanathan Chinnusamy A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables Methods and Protocols in vitro culture callus induction efficiency transformation efficiency regeneration efficiency BAP kinetin |
| title | A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables |
| title_full | A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables |
| title_fullStr | A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables |
| title_full_unstemmed | A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables |
| title_short | A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables |
| title_sort | versatile protocol for efficient transformation and regeneration in mega i indica i rice cultivar mtu1010 optimization through hormonal variables |
| topic | in vitro culture callus induction efficiency transformation efficiency regeneration efficiency BAP kinetin |
| url | https://www.mdpi.com/2409-9279/6/6/113 |
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