Department Of Botany

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    Transgene-free genome editing supports the role of carotenoid cleavage dioxygenase 4 as a negative regulator of ?-carotene in banana
    (Oxford University Press, 2022-02-01T00:00:00) Awasthi, Praveen; Khan, Shahirina; Lakhani, Hiralben; Chaturvedi, Siddhant; Shivani, S.; Kaur, Navneet; Singh, Jagdeep; Kesarwani, Atul Kumar; Tiwari, Siddharth
    Carotenoid cleavage dioxygenases (CCDs) belong to a small gene family and have an important role in the intricate metabolism of carotenoids in plants. In this study we aimed to understand the regulatory mechanism of ?-carotene homeostasis by establishing transgene-free genome editing in banana. We found that the expression patterns of multiple CCDs were correlated with the concentrations of carotenoids in two cultivars with contrasting contents of ?-carotene, Nendran (high) and Rasthali (low). Higher expression of CCD4 in Rasthali (RAS-CCD4) was negatively correlated with ?-carotene accumulation in the fruit-pulp. Docking analysis and enzyme assays of purified RAS-CCD4 suggested that ?-carotene and 10-Apo-?-carotenal were the preferred substrates of RAS-CCD4. Bacterial complementation assays demonstrated the role of RAS-CCD4 in ?-carotene degradation, and this was further confirmed by in vivo overexpression of RAS-CCD4 in Arabidopsis, which resulted in significant reduction in ?-carotene concentration. CRISPR/Cas9-mediated editing of CCD4 was conducted in protoplasts and embryogenic cell lines of Rasthali, and carotenoid profiling in the resulting stable mutant lines showed greater increases in ?-carotene accumulation in the roots than in the leaves compared with unedited controls. The differences in expression of carotenoid pathway genes were correlated with differences in metabolites in the edited lines. Overall, our study suggests that carotenoid catabolism is regulated by CCD4 in ways that are tissue-and cultivar-specific, and it also demonstrates the successful use of the genome-editing tool in developing transgene-free biofortified lines of banana. � 2022 The Author(s) 2022.
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    Genetically modified crop regulations: scope and opportunity using the CRISPR-Cas9 genome editing approach
    (Springer Science and Business Media B.V., 2021-06-10T00:00:00) Gupta, Shweta; Kumar, Adarsh; Patel, Rupali; Kumar, Vinay
    Global demand for food is increasing day by day due to an increase in population and shrinkage of the arable land area. To meet this increasing demand, there is a need to develop high-yielding varieties that are nutritionally enriched and tolerant against environmental stresses. Various techniques are developed for improving crop quality such as mutagenesis, intergeneric crosses, and translocation breeding. Later, with the development of genetic engineering, genetically modified crops came up with the transgene insertion approach which helps to withstand adverse conditions. The process or product-focused approaches are used for regulating genetically modified crops with their risk analysis on the environment and public health. However, recent advances in gene-editing technologies have led to a new era of plant breeding by developing techniques including site-directed nucleases, zinc finger nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) that involve precise gene editing without the transfer of foreign genes. But these techniques always remain in debate for their regulation status and public acceptance. The European countries and New Zealand, consider the gene-edited plants under the category of genetically modified organism (GMO) regulation while the USA frees the gene-edited plants from such type of regulations. Considering them under the category of GMO makes a long and complicated approval process to use them, which would decrease their immediate commercial value. There is a need to develop strong regulatory approaches for emerging technologies that expedite crop research and attract people to adopt these new varieties without hesitation. � 2021, The Author(s), under exclusive licence to Springer Nature B.V.
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    Transgene-free genome editing supports the role of carotenoid cleavage dioxygenase 4 as a negative regulator of ?-carotene in banana
    (Oxford University Press, 2022-02-01T00:00:00) Awasthi, Praveen; Khan, Shahirina; Lakhani, Hiralben; Chaturvedi, Siddhant; Shivani, S.; Kaur, Navneet; Singh, Jagdeep; Kesarwani, Atul Kumar; Tiwari, Siddharth
    Carotenoid cleavage dioxygenases (CCDs) belong to a small gene family and have an important role in the intricate metabolism of carotenoids in plants. In this study we aimed to understand the regulatory mechanism of ?-carotene homeostasis by establishing transgene-free genome editing in banana. We found that the expression patterns of multiple CCDs were correlated with the concentrations of carotenoids in two cultivars with contrasting contents of ?-carotene, Nendran (high) and Rasthali (low). Higher expression of CCD4 in Rasthali (RAS-CCD4) was negatively correlated with ?-carotene accumulation in the fruit-pulp. Docking analysis and enzyme assays of purified RAS-CCD4 suggested that ?-carotene and 10-Apo-?-carotenal were the preferred substrates of RAS-CCD4. Bacterial complementation assays demonstrated the role of RAS-CCD4 in ?-carotene degradation, and this was further confirmed by in vivo overexpression of RAS-CCD4 in Arabidopsis, which resulted in significant reduction in ?-carotene concentration. CRISPR/Cas9-mediated editing of CCD4 was conducted in protoplasts and embryogenic cell lines of Rasthali, and carotenoid profiling in the resulting stable mutant lines showed greater increases in ?-carotene accumulation in the roots than in the leaves compared with unedited controls. The differences in expression of carotenoid pathway genes were correlated with differences in metabolites in the edited lines. Overall, our study suggests that carotenoid catabolism is regulated by CCD4 in ways that are tissue-and cultivar-specific, and it also demonstrates the successful use of the genome-editing tool in developing transgene-free biofortified lines of banana. � 2022 The Author(s) 2022.
  • No Thumbnail Available
    Item
    Genetically modified crop regulations: scope and opportunity using the CRISPR-Cas9 genome editing approach
    (Springer Science and Business Media B.V., 2021-06-10T00:00:00) Gupta, Shweta; Kumar, Adarsh; Patel, Rupali; Kumar, Vinay
    Global demand for food is increasing day by day due to an increase in population and shrinkage of the arable land area. To meet this increasing demand, there is a need to develop high-yielding varieties that are nutritionally enriched and tolerant against environmental stresses. Various techniques are developed for improving crop quality such as mutagenesis, intergeneric crosses, and translocation breeding. Later, with the development of genetic engineering, genetically modified crops came up with the transgene insertion approach which helps to withstand adverse conditions. The process or product-focused approaches are used for regulating genetically modified crops with their risk analysis on the environment and public health. However, recent advances in gene-editing technologies have led to a new era of plant breeding by developing techniques including site-directed nucleases, zinc finger nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) that involve precise gene editing without the transfer of foreign genes. But these techniques always remain in debate for their regulation status and public acceptance. The European countries and New Zealand, consider the gene-edited plants under the category of genetically modified organism (GMO) regulation while the USA frees the gene-edited plants from such type of regulations. Considering them under the category of GMO makes a long and complicated approval process to use them, which would decrease their immediate commercial value. There is a need to develop strong regulatory approaches for emerging technologies that expedite crop research and attract people to adopt these new varieties without hesitation. � 2021, The Author(s), under exclusive licence to Springer Nature B.V.