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Agrobacterium-Mediated Plant Transformation during the Genome Engineering Era
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Agrobacterium-Mediated Plant Transformation during the Genome Engineering Era

A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Genetics, Genomics and Biotechnology".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 12602

Special Issue Editor

Dr. Keunsub Lee

E-Mail Website
Guest Editor
Department of Agronomy, Iowa State University, Ames, IA 50011, USA
Interests: plant genome editing; agrobacterium-mediated transformation; small noncoding RNA; plant-microbe interactions

Special Issue Information

Dear Colleagues,

The rapid emergence of genome editing technologies utilizing the famous CRISPR-Cas systems has revolutionized every single aspect of plant biological research, and only the power of imagination seems the limit of their applications. For most plant biologists, however, the delivery systems for such powerful tools into plant cells still suffer from low efficiencies, and thus are considered as bottlenecks for many plant species. “Nature’s genetic engineer,” the Agrobacterium species have served as essential gene delivery systems for diverse plant species, including crops. Enhancing Agrobacterium-mediated transformation efficiencies can boost both the fundamental gene functional research and crop breeding applications. This Special Issue of Plants will highlight recent advancements in applications of Agrobacterium-mediated genome editing tools delivery into various plant cells and provide useful guidance for expanding their future applications.

Dr. Keunsub Lee
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Plants is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • agrobacterium-mediated transformation
  • genome engineering
  • gene editing
  • CRISPR

Published Papers (4 papers)

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Research

14 pages, 3275 KiB  
Article
Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis
by Jinu Udayabhanu, Tiandai Huang, Shichao Xin, Jing Cheng, Yuwei Hua and Huasun Huang
Plants 2022, 11(8), 1067; https://doi.org/10.3390/plants11081067 - 14 Apr 2022
Cited by 5 | Viewed by 2579
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Agrobacterium-Mediated Plant Transformation during the Genome Engineering Era)
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18 pages, 2486 KiB  
Article
Genetic Transformation of Quercus ilex Somatic Embryos with a Gnk2-like Protein That Reveals a Putative Anti-Oomycete Action
by Susana Serrazina, Mª Teresa Martínez, Vanesa Cano, Rui Malhó, Rita Lourenço Costa and Elena Corredoira
Plants 2022, 11(3), 304; https://doi.org/10.3390/plants11030304 - 24 Jan 2022
Cited by 4 | Viewed by 3176
Abstract
Holm oak is a key tree species in Mediterranean ecosystems, whose populations have been increasingly threatened by oak decline syndrome, a disease caused by the combined action of Phytophthora cinnamomi and abiotic stresses. The aim of the present study was to produce holm [...] Read more.
Holm oak is a key tree species in Mediterranean ecosystems, whose populations have been increasingly threatened by oak decline syndrome, a disease caused by the combined action of Phytophthora cinnamomi and abiotic stresses. The aim of the present study was to produce holm oak plants that overexpress the Ginkbilobin-2 homologous domain gene (Cast_Gnk2-like) that it is known to possess antifungal properties. Proembryogenic masses (PEMs) isolated from four embryogenic lines (Q8, E2, Q10-16 and E00) were used as target explants. PEMs were co-cultured for 5 days with Agrobacterium EHA105pGnk2 and then cultured on selective medium containing kanamycin (kan) and carbenicillin. After 14 weeks on selective medium, the transformation events were observed in somatic embryos of lines Q8 and E2 and a total of 4 transgenic lines were achieved. The presence of the Cast_Gnk2-like gene on transgenic embryos was verified by PCR, and the number of transgene copies and gene expression was estimated by qPCR. Transgenic plants were obtained from all transgenic lines after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In an in vitro tolerance assay with the pathogen P. cinnamomi, we observed that transgenic plants were able to survive longer than wild type. Full article
(This article belongs to the Special Issue Agrobacterium-Mediated Plant Transformation during the Genome Engineering Era)
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8 pages, 1627 KiB  
Communication
Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback
by Anna Hebda, Aleksandra Liszka, Piotr Zgłobicki, Katarzyna Nawrot-Chorabik and Jan J. Lyczakowski
Plants 2021, 10(11), 2524; https://doi.org/10.3390/plants10112524 - 20 Nov 2021
Cited by 3 | Viewed by 1994
Abstract
The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design [...] Read more.
The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity. Full article
(This article belongs to the Special Issue Agrobacterium-Mediated Plant Transformation during the Genome Engineering Era)
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21 pages, 4741 KiB  
Article
A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example
by Giovana Acha, Ricardo Vergara, Marisol Muñoz, Roxana Mora, Carlos Aguirre, Manuel Muñoz, Julio Kalazich and Humberto Prieto
Plants 2021, 10(9), 1882; https://doi.org/10.3390/plants10091882 - 10 Sep 2021
Cited by 2 | Viewed by 3729
Abstract
In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained [...] Read more.
In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated. Full article
(This article belongs to the Special Issue Agrobacterium-Mediated Plant Transformation during the Genome Engineering Era)
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