In planta agro-infiltration system for transient gene expression in grapevine (Vitis spp.)

Ben-Amar, Anis; Cobanov, P.; Buchholz, Günther; Mliki, Ahmed; Reustle, Götz M.

doi:10.1007/s11738-013-1348-0

Cited by 22 publications

(18 citation statements)

References 36 publications

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“…However, leaf culture in vitro is complex and susceptible to the influence of environmental conditions which leads to different expression level of foreign protein and cannot play its maximum advantage of vacuum infection. Moreover, the use of detached leaves (Amar et al 2013) did not provide possible monitoring of target infiltrated tissue during days and weeks of plant development. For this reason, we suggest adopting in planta infiltration system that could give us a better outlook of plant gene expression.…”

Section: Discussionmentioning

confidence: 99%

Large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L.

Wang

Yang

et al. 2016

Plant Biotechnol Rep

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Transient expression of foreign genes byAgrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed.Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factorsingle chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agromediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.

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Section: Discussionmentioning

confidence: 99%

Large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L.

Wang

Yang

et al. 2016

Plant Biotechnol Rep

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“…In grapevines, this technology has been recently applied to analyze the function of several genes such as VviAdh [75], VviPIP2 [76], VviCCD1 [77], and VviWRKY33 [78]. Likewise, this method has been proved to be very useful for transgenic complementation [79][80][81], technical studies [82][83][84][85][86][87], promoter analysis [88][89][90][91], subcellular localization analysis [92], and protein production [69,93] in grapevine. Nonetheless, the genetic background and plant growth conditions are key factors in performing successful Agrobacterium-mediated transformation.…”

Section: Indirect Gene Transfer Methods: Agrobacterium-mediated Transmentioning

confidence: 99%

“…Similarly, the agroinfiltration method consists of plunging detached leaves [69,79] or whole plants [81] into the bacterial suspension. As the flow of bacterial suspensions through stomata and across the epidermis is impeded by the high surface tension of aqueous solutions, transient transformation is obtained through the rapid release of a vacuum to introduce the bacterial suspension into the mesophyll cells.…”

Section: Agroinjection and Agroinfiltrationmentioning

confidence: 99%

“…Bertazzon et al [79] assayed the transient downregulation of a grapevine defense-related gene by the agroinfiltration of the constructs for the expression of dsRNA. Interestingly, Ben-Amar et al [81] established a protocol to agroinfiltrate leaves of greenhouse-grown plants using a vacuum device. In their work, they delivered the first evidence of GFP gene silencing in grapevine achieved for the first time using in planta agroinfiltration method.…”

Section: Agroinjection and Agroinfiltrationmentioning

confidence: 99%

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Development and Use of Biotechnology Tools for Grape Functional Analysis

Chialva¹,

Eichler²,

Muñoz³

et al. 2016

Grape and Wine Biotechnology

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The aim of this chapter is to provide a description of the latest scientific advances in the field of gene functional analysis in grapevine. It provides general information about the studies conducted during the past decade to understand the natural variation of this plant and how this information has been exploited for the understanding of traits of interest. Likewise, it is exposed how the use of biotechnology tools have helped to characterize the mechanisms of gene expression and its regulation, as well as the subcellular localization of proteins and their interactions with other molecules. Finally, an approximation to the new technologies of gene editing and their potential application in the functional study of grapevine has been carried out.

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“…In previous studies, different methods of in planta transformation have been documented in literature such as injection of bacterial culture into meristematic tissues or fruit in tomato (Orzaez et al 2006;Vinoth et al 2013), infection of germinating seeds by Agrobacterium in cotton , dropping of DNA directly onto florets in Arabidopsis thaliana (Tague and Mantis 2006), infection of shoot apical node in alfalfa (Weeks et al 2008) and vaccum infiltration of green leaves (Ben-Amar et al 2013). Among them, infection of germinating seeds have been found as better explants source for efficient in planta transformation strategy in various crops such as Medicago truncatula, radish and brinjal (Trieu et al 2000;Park et al 2005;Subramanyam et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.)

Shah

Ali²,

Jan

et al. 2014

Plant Cell Tiss Organ Cult

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Cold is a major constraint for tomato growth and productivity; as it is a cold sensitive crop. DREB1A plays a key role in generating cold tolerance in tomato by regulating the response of multiple genes under chilling stress. In this study, cold tolerant gene (DREB1A) driven by Lip9 promoter, was transformed in three tomato genotypes through Agrobacterium tumefaciens, employing tissue culture independent transformation strategy. Overnight imbibed seeds of tomato were surface sterilized, 3-day old shoot apical meristem of the developing seedling were pierced and incubated for twenty minutes with A. tumefaciens strain EHA-105 having OD 600 nm of 1.0. The treated seeds were co-cultivated with Agrobacterium for 2 days. The regenerated shoots were subjected to 35 mg/l hygromycin as a selection for a period of 2-3 weeks. The presence of DREB1A and hpt genes in the hygromycin resistant (T 0 ) transgenic plants was evaluated by PCR analysis. The transgene activity was detected in T 1 plants by Reverse Transcriptase Polymerase Chain Reaction and Southern blotting that showed the stable integration of the transgene to the next generation. Physiological analysis of T 2 transgenic plants depicted that increased expression of DREB1A induced only during chilling stress. After various chilling stresses, stomatal conductance, transpiration rate and relative water contents of transgenic lines were significantly higher than those of NT plants. While leaf osmotic potential of transgenic plants was lower as compared to NT plants. The established procedure is novel and can produce stable transgenic tomato plants in efficient manner by saving potential resources in terms of cost and time.

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In planta agro-infiltration system for transient gene expression in grapevine (Vitis spp.)

Cited by 22 publications

References 36 publications

Large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L.

Large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L.

Development and Use of Biotechnology Tools for Grape Functional Analysis

Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.)

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