Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture
Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isola...
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MDPI AG
2022-09-01
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| Series: | International Journal of Molecular Sciences |
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| author | Jong-Eun Han Han-Sol Lee Hyoshin Lee Hyunwoo Cho So-Young Park |
| author_facet | Jong-Eun Han Han-Sol Lee Hyoshin Lee Hyunwoo Cho So-Young Park |
| author_sort | Jong-Eun Han |
| collection | DOAJ |
| description | Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes <i>SERK1, WUS, BBM, LEC1,</i> and <i>DRN</i> was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 10<sup>6</sup> protoplasts·g<sup>−1</sup> FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 10<sup>4</sup> protoplasts·g<sup>−1</sup> FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, <i>WUS</i> and <i>SERK1</i> expression increased 12-fold, and <i>LEC1</i> and <i>BBM</i> expression increased 3.6–6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing. |
| first_indexed | 2024-03-09T21:39:33Z |
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| spelling | doaj.art-c4bab50d11c440abb9c42f52fa4375112023-11-23T20:35:17ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-09-0123191155610.3390/ijms231911556Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast CultureJong-Eun Han0Han-Sol Lee1Hyoshin Lee2Hyunwoo Cho3So-Young Park4Department of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28644, KoreaDepartment of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28644, KoreaDepartment of Forest Genetic Resources, National Institute of Forest Science, 39 Onjeong-ro, Suwon 16631, KoreaDepartment of Industrial Plant Science and Technology, College of Agricultural, Life and Environmental Sciences, Chungbuk National University, Cheongju 28644, KoreaDepartment of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28644, KoreaProtoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes <i>SERK1, WUS, BBM, LEC1,</i> and <i>DRN</i> was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 10<sup>6</sup> protoplasts·g<sup>−1</sup> FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 10<sup>4</sup> protoplasts·g<sup>−1</sup> FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, <i>WUS</i> and <i>SERK1</i> expression increased 12-fold, and <i>LEC1</i> and <i>BBM</i> expression increased 3.6–6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.https://www.mdpi.com/1422-0067/23/19/11556embryogenic callus-derived protoplastgene expressionplant regeneration |
| spellingShingle | Jong-Eun Han Han-Sol Lee Hyoshin Lee Hyunwoo Cho So-Young Park Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture International Journal of Molecular Sciences embryogenic callus-derived protoplast gene expression plant regeneration |
| title | Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture |
| title_full | Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture |
| title_fullStr | Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture |
| title_full_unstemmed | Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture |
| title_short | Embryogenic Stem Cell Identity after Protoplast Isolation from <i>Daucus carota</i> and Recovery of Regeneration Ability through Protoplast Culture |
| title_sort | embryogenic stem cell identity after protoplast isolation from i daucus carota i and recovery of regeneration ability through protoplast culture |
| topic | embryogenic callus-derived protoplast gene expression plant regeneration |
| url | https://www.mdpi.com/1422-0067/23/19/11556 |
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