Cargando…

Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection

In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner in...

Descripción completa

Detalles Bibliográficos
Autores principales: Luo, Bai-Xue, Zhang, Li, Zheng, Feng, Wu, Kun-Lin, Li, Lin, Zhang, Xin-Hua, Ma, Guo-Hua, Teixeira da Silva, Jaime A., Fang, Lin, Zeng, Song-Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795287/
https://www.ncbi.nlm.nih.gov/pubmed/33374823
http://dx.doi.org/10.3390/ijms22010084
_version_ 1783634409553920000
author Luo, Bai-Xue
Zhang, Li
Zheng, Feng
Wu, Kun-Lin
Li, Lin
Zhang, Xin-Hua
Ma, Guo-Hua
Teixeira da Silva, Jaime A.
Fang, Lin
Zeng, Song-Jun
author_facet Luo, Bai-Xue
Zhang, Li
Zheng, Feng
Wu, Kun-Lin
Li, Lin
Zhang, Xin-Hua
Ma, Guo-Hua
Teixeira da Silva, Jaime A.
Fang, Lin
Zeng, Song-Jun
author_sort Luo, Bai-Xue
collection PubMed
description In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50–54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.
format Online
Article
Text
id pubmed-7795287
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-77952872021-01-10 Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection Luo, Bai-Xue Zhang, Li Zheng, Feng Wu, Kun-Lin Li, Lin Zhang, Xin-Hua Ma, Guo-Hua Teixeira da Silva, Jaime A. Fang, Lin Zeng, Song-Jun Int J Mol Sci Article In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50–54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics. MDPI 2020-12-23 /pmc/articles/PMC7795287/ /pubmed/33374823 http://dx.doi.org/10.3390/ijms22010084 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Luo, Bai-Xue
Zhang, Li
Zheng, Feng
Wu, Kun-Lin
Li, Lin
Zhang, Xin-Hua
Ma, Guo-Hua
Teixeira da Silva, Jaime A.
Fang, Lin
Zeng, Song-Jun
Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection
title Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection
title_full Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection
title_fullStr Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection
title_full_unstemmed Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection
title_short Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection
title_sort ovule development and in planta transformation of paphiopedilum maudiae by agrobacterium-mediated ovary-injection
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795287/
https://www.ncbi.nlm.nih.gov/pubmed/33374823
http://dx.doi.org/10.3390/ijms22010084
work_keys_str_mv AT luobaixue ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT zhangli ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT zhengfeng ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT wukunlin ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT lilin ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT zhangxinhua ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT maguohua ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT teixeiradasilvajaimea ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT fanglin ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection
AT zengsongjun ovuledevelopmentandinplantatransformationofpaphiopedilummaudiaebyagrobacteriummediatedovaryinjection