Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry
Abstract Background Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-03-01
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| Series: | Journal of Genetic Engineering and Biotechnology |
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| Online Access: | https://doi.org/10.1186/s43141-022-00323-6 |
| _version_ | 1797204944558751744 |
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| author | Paulo Cesar Alves de Sousa Stênio Steferson Silva e Souza Gabriela Ferreira Nogueira Inaê Mariê de Araújo Silva-Cardoso Jonny Everson Scherwinski-Pereira |
| author_facet | Paulo Cesar Alves de Sousa Stênio Steferson Silva e Souza Gabriela Ferreira Nogueira Inaê Mariê de Araújo Silva-Cardoso Jonny Everson Scherwinski-Pereira |
| author_sort | Paulo Cesar Alves de Sousa |
| collection | DOAJ |
| description | Abstract Background Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L−1 NAA + 2.5 mg L−1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L−1 NAA + 2.5 mg L−1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. Results The treatments 2.5 mg L−1 BAP and 5 mg L−1 NAA + 2.5 mg L−1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. Conclusion This protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum. |
| first_indexed | 2024-04-24T08:43:16Z |
| format | Article |
| id | doaj.art-76bd2a0601db44988dac697d6b9f86ce |
| institution | Directory Open Access Journal |
| issn | 2090-5920 |
| language | English |
| last_indexed | 2024-04-24T08:43:16Z |
| publishDate | 2022-03-01 |
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| series | Journal of Genetic Engineering and Biotechnology |
| spelling | doaj.art-76bd2a0601db44988dac697d6b9f86ce2024-04-16T14:44:29ZengElsevierJournal of Genetic Engineering and Biotechnology2090-59202022-03-0120111410.1186/s43141-022-00323-6Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometryPaulo Cesar Alves de Sousa0Stênio Steferson Silva e Souza1Gabriela Ferreira Nogueira2Inaê Mariê de Araújo Silva-Cardoso3Jonny Everson Scherwinski-Pereira4Universidade de Brasília, Campus Universitário Darcy RibeiroUniversidade de Brasília, Campus Universitário Darcy RibeiroPosdoctoral Fellows from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil)/Embrapa Recursos Genéticos e BiotecnologiaPosdoctoral Fellows from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil)/Embrapa Recursos Genéticos e BiotecnologiaEmbrapa Recursos Genéticos e BiotecnologiaAbstract Background Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L−1 NAA + 2.5 mg L−1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L−1 NAA + 2.5 mg L−1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. Results The treatments 2.5 mg L−1 BAP and 5 mg L−1 NAA + 2.5 mg L−1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. Conclusion This protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum.https://doi.org/10.1186/s43141-022-00323-6PiperaceaeMorphogenesisSomatic embryosPlant regenerationCellular DNA content |
| spellingShingle | Paulo Cesar Alves de Sousa Stênio Steferson Silva e Souza Gabriela Ferreira Nogueira Inaê Mariê de Araújo Silva-Cardoso Jonny Everson Scherwinski-Pereira Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry Journal of Genetic Engineering and Biotechnology Piperaceae Morphogenesis Somatic embryos Plant regeneration Cellular DNA content |
| title | Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry |
| title_full | Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry |
| title_fullStr | Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry |
| title_full_unstemmed | Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry |
| title_short | Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry |
| title_sort | indirect somatic embryogenesis of piper hispidinervum l and evaluation of the regenerated plants by flow cytometry |
| topic | Piperaceae Morphogenesis Somatic embryos Plant regeneration Cellular DNA content |
| url | https://doi.org/10.1186/s43141-022-00323-6 |
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