Agrobacterium-mediated barley transformation.

More than ten years have passed since the first successful Agrobacterium-mediated barley transformation experiment, however it is still quite challenging to establish a stably functioning agroinfiltration protocol. Efficiency of the method depends mainly on the transformation and co-cultivation conditions, and also the components of the tissue-culture media. With the use of an optimized media we have been able to set up a reliable, properly functioning transformation protocol. The first generation of transgenic barley plants, transformed with a transformation cassette carrying an aldo-keto-reductase gene from Arabidopsis thaliana and the hpt marker gene, were analyzed at nucleic acid (both DNA and RNA) and at protein levels. The key factors of success proved to be the use of Silwet L-77 (surfactant) in transformation inoculum, the Cu-content of regenerating media and the continuous visual monitoring of the transformed callus during the somatic embryogenesi

Optical microscopy on agrobacterium-mediated transient transformated arabidopsis nahg plants

Agrobacterium tumefaciens-mediated transient transformation has demonstrated to be an invaluable tool in plant cell biology. However, low efficiency and inconsistency of this method in Arabidopsis has forced the implementation of Nicotiana benthamiana as a surrogate system, limiting applicability. Previous results suggested that hormone-mediated defence responses against bacteria might be responsible for the low efficiency of Agrobacterium-mediated transient transformation in Arabidopsis. In this work, we evaluate the efficiency of Agrobacterium-mediated transient transformation in Arabidopsis genotypes affected in JA perception or signalling (coi1, jin1), or with low SA or JA content (sid2, NahG, aos). We demonstrate that expression of the NahG transgene dramatically improves this process. Arabidopsis NahG plants can be efficiently used for transient expression-based optical microscopy assays routinely performed in N. benthamiana, such as determination of subcellular localization of GFP-fused proteins or analysis of protein-protein interactions by Bimolecular Fluorescent Complementation. Considering the wide-spread use of Agrobacterium-mediated transient transformation, this system can enormously facilitate research in the model plant Arabidopsis, allowing for an efficient use of the full potential of the numerous tools and resources currently available to the community.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Agrobacterium-Mediated Transformation

Agrobacterium-mediated transformation (AMT) heavily relies on the capability of bacterial pathogen Agrobacterium tumefaciens in transferring foreign genes into a wide variety of host plants. Currently, AMT is the most commonly used method for generating transgenic plants. On the other hand, A. tumefaciens was very useful for plant breeding. It also accelerated the technology of plant breeding to obtain specific characters. Gene transfer from bacteria to plants is a complex mechanism that involves several functional steps. This chapter will give brief information related to AMT mechanism, including the history of crown gall disease, the natural pathogenesis of A. tumefaciens, and the general protocol of AMT

Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem’s Delight Orchid with Higher Efficiency

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem’s Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusAgene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD’s PLBs. Based on the results, 3-4mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit a

Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation

Plant recalcitrance is the major barrier in developing Agrobacterium-mediated transformation protocols for several important plant species. Despite the substantial knowledge of T-DNA transfer process, very little is known about the factors leading to the plant recalcitrance. Here, we analyzed the basis of Hypericum perforatum L. (HP) recalcitrance to Agrobacterium-mediated transformation using cell suspension culture. When challenged with Agrobacterium, HP cells swiftly produced an intense oxidative burst, a typical reaction of plant defense. Agrobacterium viability started to decline and reached 99% mortality within 12 h, while the plant cells did not suffer apoptotic process. This is the first evidence showing that the reduction of Agrobacterium viability during co-cultivation with recalcitrant plant cells can affect transformation

U.S. Patent Literature Survey of Agrobacterium-Mediated Transformation of Sweet Potato (Ipomoea Batatas)

A team of researchers and patent information scientists at Franklin Pierce Law Center were asked to evaluate the patent and literature landscape related to the Agrobacterium-mediated transformation in sweet potato with respect to the U.S. patents and patent applications. This report provides a patent landscape of the Agrobacterium-mediated transformation of sweet potato. The report includes the applicable methods of transformation and has also included certain patents and patent applications which claim a transformed plant by virtue of these methods. In certain cases, the claim structure covers Agrobacterium-mediated transformation technology via system and composition of matter claims and not the more prevalent method claims. Sweet potato plant (Ipomoea batatas) is adaptable to a broad range of agroecological conditions and fits in low input agriculture. It is highly productive even under adverse farming conditions. Sweet potato is grown in more than 100 countries as a valuable source of food, animal feed and industrial raw material. It is a staple crop in many South East Asian and African countries. Traditional plant breeding has contributed to the improvement of sweet potato, especially in developed countries such as the U.S.A. and Japan. Because of the biological complexities of sweet potato, sexual hybridization strategies have not been very effective in developing improved cultivars. Confidential Therefore, biotechnological tools, such as gene transfer, are very attractive in sweet potato improvement, as they enable direct introduction of desirable genes from other sources into preadapted cultivars

Effect of Agrobacterium Induced Necrosis, Antibiotic Induced Phytotoxicity and Other Factors in Successful Plant Transformation

Agrobacterium tumefaciens infection and antibiotic wash are the critical steps of Agrobacterium mediated plant transformation procedure, most time responsible for lower transformation efficiency due to necrosis and phytotoxicity caused by biotic stress of Agrobacterium and abiotic stress by antibiotics respectively. Ammi majus Egyptian origin medicinal plant and Pearl millet cereal grain crop were studied for their stress responses to Agrobacterium mediated transformation (AMT). Agrobacterium strains LBA4404 (O.D.=0.6-0.8) and EHA105 (O.D.=0.2-0.4) were used for transformation experiments to infect calli of Ammi majus and embryogenic calli of Pearl millet respectively. Incase of antibiotic wash, Cefotaxime 500 mg L-1 was used for LBA4404 infected Ammi majus calli and Timentin 300 mg L-1 was used for EHA105 infected embryogenic calli of Pearl millet. Effects of Agrobacterium infection, antibiotic and NaOCl washes on Agrobacterium removal and both explants physiological changes during transformation experimental procedures were studied. At the end of the experiments explants survival efficiency of Ammi majus and pearl millet were 8% and 5% respectively. Biotic and abiotic stress factors responsible for lower efficiency were investigated with various other factors and strategies were discussed which are need to be considered for higher transformation events and target tissue survival

AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

Background: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer

Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus

Molecular resources and transgenic studies in red algae are lagging behind those for green algae. The Agrobacterium-mediated gene-transfer method routinely used in plant transformation has not been fully utilised in the red algae, which, as an important source of phycocolloids, warrant more studies. In this regard, a stepwise methodology was developed for Agrobacterium-mediated transformation of the carrageenophyte Chondrus crispus using pCAMBIA 1301 and a construct featuring a codon-optimized beta-glucuronidase (GUS) reporter gene driven by the endogenous Chondrus actin promoter. The effects of several factors on transformation efficiency were investigated. An intimate association of Chondrus and bacterial cells was observed using scanning electron microscopy. GUS transient expression within Chondrus cortical and medullary cells with both expression cassettes testified to the amenability of Chondrus to Agrobacterium-mediated transformation. Darker staining, indicative of higher GUS activity, was observed with the Chondrus-specific construct, suggesting its superiority over the pCAMBIA 1301. Presence of acetosyringone, the wounding method and the type of co-cultivation medium significantly affected the transformation outcome and efficiency. The Agrobacterium-mediated transient expression presented here constitutes a first step towards tailoring a transformation strategy for Chondrus, which can serve to facilitate further transgenic studies in this important red alga