Scholarlink

Main conclusion <p>This study establishes a genotype-specific transformation system for <i>indica</i> rice cultivars Ranjit, Mahsuri, and Kon Joha using <i>35S:RUBY</i> and CRISPR/LbCas12a constructs, enabling functional genomics studies and genetic improvement.</p> Abstract <p>The <i>indica</i> rice subspecies generally faces challenges in functional genomics and genetic improvement due to its recalcitrance to tissue culture and <i>Agrobacterium</i>-mediated transformation. Three <i>indica</i> rice varieties, Ranjit, Mahsuri, and Kon Joha, cultivated in Assam (India) were selected to optimize callus induction, regeneration, and genetic transformation. Ranjit and Mahsuri are high-yielding cultivars, whereas Kon Joha is an indigenous aromatic landrace of high commercial value. Initially, key steps, such as callus induction and regeneration, were optimized for Ranjit and Kon Joha using mature seed. Thereafter, for transformation, immature embryos were selected as explants because of their competence for agro-infection. The highest callus induction frequency of 70.33% and 90% was achieved with 3.0 and 3.5&#xa0;mg L<sup>−1</sup> of 2,4-dichlorophenoxyacetic acid (2,4-D) in Ranjit and Kon Joha, respectively. The best regeneration, in Ranjit (77%) and Kon Joha (54.5%), was achieved with 4.0&#xa0;mg L<sup>−1</sup> 6-benzylaminopurine (BAP), 1.0&#xa0;mg L<sup>−1</sup> kinetin, and 0.5&#xa0;mg L<sup>−1</sup> naphthalene acetic acid (NAA). Transformation efficiencies with CRISPR/LbCas12a (OD<sub>660</sub> = 0.8) were 5.6% (Ranjit), 7.5% (Mahsuri), and 15.33% (Kon Joha). The optimal in vitro regeneration conditions previously identified for Ranjit were adopted for Mahsuri transformation. Furthermore, a visual non-invasive reporter, <i>RUBY</i>, was employed to investigate the transformation of immature embryos in Ranjit and Kon Joha. The vivid red coloration in the early callusing stage indicates successful transformation events. Kon Joha plants regenerated from red calli showed a distinct phenotype with pink leaves and red roots. These findings confirm <i>RUBY</i>’s effectiveness as a non-invasive reporter for rapid monitoring of rice transformation and underscore the importance of immature embryos in <i>indica</i> rice transformation.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Genotype-specific optimization of in vitro regeneration and Agrobacterium-mediated transformation in indica rice with 35S:RUBY and CRISPR/LbCas12a system

  • 期刊论文

  • 发表日期：

    2026年4月24日

查看全文
  • Yogita N. Sarki,
  • Ajay Kumar Keot,
  • Riwandahun Marwein,
  • Dhanawantari L. Singha,
  • Dhrubo Jyoti Gogoi,
  • Natarajan Velmurugan,
  • Channakeshavaiah Chikkaputtaiah

摘要

Main conclusion

This study establishes a genotype-specific transformation system for indica rice cultivars Ranjit, Mahsuri, and Kon Joha using 35S:RUBY and CRISPR/LbCas12a constructs, enabling functional genomics studies and genetic improvement.

Abstract

The indica rice subspecies generally faces challenges in functional genomics and genetic improvement due to its recalcitrance to tissue culture and Agrobacterium-mediated transformation. Three indica rice varieties, Ranjit, Mahsuri, and Kon Joha, cultivated in Assam (India) were selected to optimize callus induction, regeneration, and genetic transformation. Ranjit and Mahsuri are high-yielding cultivars, whereas Kon Joha is an indigenous aromatic landrace of high commercial value. Initially, key steps, such as callus induction and regeneration, were optimized for Ranjit and Kon Joha using mature seed. Thereafter, for transformation, immature embryos were selected as explants because of their competence for agro-infection. The highest callus induction frequency of 70.33% and 90% was achieved with 3.0 and 3.5 mg L−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) in Ranjit and Kon Joha, respectively. The best regeneration, in Ranjit (77%) and Kon Joha (54.5%), was achieved with 4.0 mg L−1 6-benzylaminopurine (BAP), 1.0 mg L−1 kinetin, and 0.5 mg L−1 naphthalene acetic acid (NAA). Transformation efficiencies with CRISPR/LbCas12a (OD660 = 0.8) were 5.6% (Ranjit), 7.5% (Mahsuri), and 15.33% (Kon Joha). The optimal in vitro regeneration conditions previously identified for Ranjit were adopted for Mahsuri transformation. Furthermore, a visual non-invasive reporter, RUBY, was employed to investigate the transformation of immature embryos in Ranjit and Kon Joha. The vivid red coloration in the early callusing stage indicates successful transformation events. Kon Joha plants regenerated from red calli showed a distinct phenotype with pink leaves and red roots. These findings confirm RUBY’s effectiveness as a non-invasive reporter for rapid monitoring of rice transformation and underscore the importance of immature embryos in indica rice transformation.