An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation
Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of trans...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-01-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2020.562056/full |
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| author | Karthik Kesiraju Shaily Tyagi Soumyadeep Mukherjee Rhitu Rai Nagendra K. Singh Rohini Sreevathsa Prasanta K. Dash |
| author_facet | Karthik Kesiraju Shaily Tyagi Soumyadeep Mukherjee Rhitu Rai Nagendra K. Singh Rohini Sreevathsa Prasanta K. Dash |
| author_sort | Karthik Kesiraju |
| collection | DOAJ |
| description | Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax. |
| first_indexed | 2024-12-20T01:17:54Z |
| format | Article |
| id | doaj.art-46dae012a92b4064a17f1ad097214483 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-20T01:17:54Z |
| publishDate | 2021-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-46dae012a92b4064a17f1ad0972144832022-12-21T19:58:31ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2021-01-011110.3389/fpls.2020.562056562056An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and ValidationKarthik KesirajuShaily TyagiSoumyadeep MukherjeeRhitu RaiNagendra K. SinghRohini SreevathsaPrasanta K. DashEfficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.https://www.frontiersin.org/articles/10.3389/fpls.2020.562056/fulltransgenic flax/linseedin planta transformationapical meristemGUSnptIIGM crops |
| spellingShingle | Karthik Kesiraju Shaily Tyagi Soumyadeep Mukherjee Rhitu Rai Nagendra K. Singh Rohini Sreevathsa Prasanta K. Dash An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation Frontiers in Plant Science transgenic flax/linseed in planta transformation apical meristem GUS nptII GM crops |
| title | An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation |
| title_full | An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation |
| title_fullStr | An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation |
| title_full_unstemmed | An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation |
| title_short | An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation |
| title_sort | apical meristem targeted in planta transformation method for the development of transgenics in flax linum usitatissimum optimization and validation |
| topic | transgenic flax/linseed in planta transformation apical meristem GUS nptII GM crops |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2020.562056/full |
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