A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing t...

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Autores principales:	Ke Duan, Ying-Jie Zhao, Zi-Yi Li, Xiao-Hua Zou, Jing Yang, Cheng-Lin Guo, Si-Yu Chen, Xiu-Rong Yang, Qing-Hua Gao
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<i>Fragaria</i> × <i>ananassa</i> sugar transporter intragenesis <i>Agrobacterium</i>-mediated transformation droplet digital PCR Botany QK1-989
Acceso en línea:	https://doaj.org/article/daa3cdbdb20043f79060a6428606aa74
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spelling	oai:doaj.org-article:daa3cdbdb20043f79060a6428606aa742021-11-25T18:43:57ZA Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry10.3390/plants101122292223-7747https://doaj.org/article/daa3cdbdb20043f79060a6428606aa742021-10-01T00:00:00Zhttps://www.mdpi.com/2223-7747/10/11/2229https://doaj.org/toc/2223-7747Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from <i>F. vesca,</i> harboring a sugar transporter gene <i>FvSTP8</i> driven by a fruit-specific <i>FvKnox</i> promoter. Transformed strawberry was developed through an <i>Agrobacterium</i>-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.Ke DuanYing-Jie ZhaoZi-Yi LiXiao-Hua ZouJing YangCheng-Lin GuoSi-Yu ChenXiu-Rong YangQing-Hua GaoMDPI AGarticle<i>Fragaria</i> × <i>ananassa</i>sugar transporterintragenesis<i>Agrobacterium</i>-mediated transformationdroplet digital PCRBotanyQK1-989ENPlants, Vol 10, Iss 2229, p 2229 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<i>Fragaria</i> × <i>ananassa</i> sugar transporter intragenesis <i>Agrobacterium</i>-mediated transformation droplet digital PCR Botany QK1-989
spellingShingle	<i>Fragaria</i> × <i>ananassa</i> sugar transporter intragenesis <i>Agrobacterium</i>-mediated transformation droplet digital PCR Botany QK1-989 Ke Duan Ying-Jie Zhao Zi-Yi Li Xiao-Hua Zou Jing Yang Cheng-Lin Guo Si-Yu Chen Xiu-Rong Yang Qing-Hua Gao A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
description	Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from <i>F. vesca,</i> harboring a sugar transporter gene <i>FvSTP8</i> driven by a fruit-specific <i>FvKnox</i> promoter. Transformed strawberry was developed through an <i>Agrobacterium</i>-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.
format	article
author	Ke Duan Ying-Jie Zhao Zi-Yi Li Xiao-Hua Zou Jing Yang Cheng-Lin Guo Si-Yu Chen Xiu-Rong Yang Qing-Hua Gao
author_facet	Ke Duan Ying-Jie Zhao Zi-Yi Li Xiao-Hua Zou Jing Yang Cheng-Lin Guo Si-Yu Chen Xiu-Rong Yang Qing-Hua Gao
author_sort	Ke Duan
title	A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
title_short	A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
title_full	A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
title_fullStr	A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
title_full_unstemmed	A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
title_sort	strategy for the production and molecular validation of agrobacterium-mediated intragenic octoploid strawberry
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/daa3cdbdb20043f79060a6428606aa74
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A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

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