Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption
Pigeonpea is an important legume valued for its high nutritional, agricultural, and economic significance in the Asian subcontinent. Despite its potential for high yield, productivity remains stagnant due to several abiotic and biotic stresses. To mitigate these challenges, biotechnological interven...
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Elsevier
2025-03-01
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| Series: | Journal of Genetic Engineering and Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S1687157X25000095 |
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| author | Kameshwaran Senthil Maniraj Rathinam Manisha Parashar Narasimham Dokka Shaily Tyagi Vandana Mathur Sandhya Sharma Kishor Gaikwad Ramcharan Bhattacharya Rohini Sreevathsa |
| author_facet | Kameshwaran Senthil Maniraj Rathinam Manisha Parashar Narasimham Dokka Shaily Tyagi Vandana Mathur Sandhya Sharma Kishor Gaikwad Ramcharan Bhattacharya Rohini Sreevathsa |
| author_sort | Kameshwaran Senthil |
| collection | DOAJ |
| description | Pigeonpea is an important legume valued for its high nutritional, agricultural, and economic significance in the Asian subcontinent. Despite its potential for high yield, productivity remains stagnant due to several abiotic and biotic stresses. To mitigate these challenges, biotechnological interventions like genome editing offer promising solutions. Towards this, developing a species-specific editing toolkit is crucial for recalcitrant species like pigeonpea. In this study, we established a CRISPR/Cas9 genome editing system targeting the phytoene desaturase (PDS) gene. We developed pigeonpea-compatible vector components, including the CcU6_7.1 promoter and an amenable Cas9 gene driven by the potato ubiquitin promoter, creating a pigeonpea-specific CRISPR/Cas9 binary vector (PP_CRISPR_pCAMBIA2301). The system was validated by Agrobacterium tumefaciens-mediated apical meristem-targeted in planta and in vitro embryonic axis explant transformations, with gene knockout confirmed by albino/bleached phenotypes. Editing efficiencies were 8.80% and 9.16% in the in planta and in vitro transformations respectively. While PCR analysis confirmed T-DNA integration, sequence analysis identified PDS gene mutations. Stability of the phenotype was demonstrated in T1 generation plants of in planta transformation-developed mutants. This system may support functional genomics studies and trait improvement in pigeonpea and other legumes. |
| format | Article |
| id | doaj-art-2434ab32a87344c3b0466d66871d7368 |
| institution | Kabale University |
| issn | 1687-157X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Genetic Engineering and Biotechnology |
| spelling | doaj-art-2434ab32a87344c3b0466d66871d73682025-02-07T04:47:21ZengElsevierJournal of Genetic Engineering and Biotechnology1687-157X2025-03-01231100465Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruptionKameshwaran Senthil0Maniraj Rathinam1Manisha Parashar2Narasimham Dokka3Shaily Tyagi4Vandana Mathur5Sandhya Sharma6Kishor Gaikwad7Ramcharan Bhattacharya8Rohini Sreevathsa9ICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaCorresponding authors.; ICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaCorresponding authors.; ICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012 IndiaPigeonpea is an important legume valued for its high nutritional, agricultural, and economic significance in the Asian subcontinent. Despite its potential for high yield, productivity remains stagnant due to several abiotic and biotic stresses. To mitigate these challenges, biotechnological interventions like genome editing offer promising solutions. Towards this, developing a species-specific editing toolkit is crucial for recalcitrant species like pigeonpea. In this study, we established a CRISPR/Cas9 genome editing system targeting the phytoene desaturase (PDS) gene. We developed pigeonpea-compatible vector components, including the CcU6_7.1 promoter and an amenable Cas9 gene driven by the potato ubiquitin promoter, creating a pigeonpea-specific CRISPR/Cas9 binary vector (PP_CRISPR_pCAMBIA2301). The system was validated by Agrobacterium tumefaciens-mediated apical meristem-targeted in planta and in vitro embryonic axis explant transformations, with gene knockout confirmed by albino/bleached phenotypes. Editing efficiencies were 8.80% and 9.16% in the in planta and in vitro transformations respectively. While PCR analysis confirmed T-DNA integration, sequence analysis identified PDS gene mutations. Stability of the phenotype was demonstrated in T1 generation plants of in planta transformation-developed mutants. This system may support functional genomics studies and trait improvement in pigeonpea and other legumes.http://www.sciencedirect.com/science/article/pii/S1687157X25000095CRISPR/Cas9PDS knockoutPigeonpeaIn planta transformationRegenerationGenome editing |
| spellingShingle | Kameshwaran Senthil Maniraj Rathinam Manisha Parashar Narasimham Dokka Shaily Tyagi Vandana Mathur Sandhya Sharma Kishor Gaikwad Ramcharan Bhattacharya Rohini Sreevathsa Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption Journal of Genetic Engineering and Biotechnology CRISPR/Cas9 PDS knockout Pigeonpea In planta transformation Regeneration Genome editing |
| title | Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption |
| title_full | Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption |
| title_fullStr | Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption |
| title_full_unstemmed | Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption |
| title_short | Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption |
| title_sort | establishing a crispr cas9 genome editing framework in pigeonpea cajanus cajan l by targeting phytoene desaturase pds gene disruption |
| topic | CRISPR/Cas9 PDS knockout Pigeonpea In planta transformation Regeneration Genome editing |
| url | http://www.sciencedirect.com/science/article/pii/S1687157X25000095 |
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