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Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis

The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation pro...

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Autores principales: Udayabhanu, Jinu, Huang, Tiandai, Xin, Shichao, Cheng, Jing, Hua, Yuwei, Huang, Huasun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9024888/
https://www.ncbi.nlm.nih.gov/pubmed/35448795
http://dx.doi.org/10.3390/plants11081067
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author Udayabhanu, Jinu
Huang, Tiandai
Xin, Shichao
Cheng, Jing
Hua, Yuwei
Huang, Huasun
author_facet Udayabhanu, Jinu
Huang, Tiandai
Xin, Shichao
Cheng, Jing
Hua, Yuwei
Huang, Huasun
author_sort Udayabhanu, Jinu
collection PubMed
description The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 μM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.
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spelling pubmed-90248882022-04-23 Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis Udayabhanu, Jinu Huang, Tiandai Xin, Shichao Cheng, Jing Hua, Yuwei Huang, Huasun Plants (Basel) Article The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 μM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future. MDPI 2022-04-14 /pmc/articles/PMC9024888/ /pubmed/35448795 http://dx.doi.org/10.3390/plants11081067 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Udayabhanu, Jinu
Huang, Tiandai
Xin, Shichao
Cheng, Jing
Hua, Yuwei
Huang, Huasun
Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
title Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
title_full Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
title_fullStr Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
title_full_unstemmed Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
title_short Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
title_sort optimization of the transformation protocol for increased efficiency of genetic transformation in hevea brasiliensis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9024888/
https://www.ncbi.nlm.nih.gov/pubmed/35448795
http://dx.doi.org/10.3390/plants11081067
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