A Genotype-Independent, Simple, Effective and Efficient in Planta Agrobacterium-Mediated Genetic Transformation Protocol

Kharb, Pushpa; Chaudhary, Rinku; Tuteja, Narendra; Kaushik, Prashant

doi:10.3390/mps5050069

Cited by 6 publications

(5 citation statements)

References 24 publications

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“…In this study, three insertion sites were located in the gene region and four were located in the intergenic region ( Supplementary Table S4 ). Furthermore, transgenic lines with Agrobacterium -mediated integration usually have a relatively small copy number [ 28 ]. In this study, six transgenic lines of PEN were all generated using the Agrobacterium -mediated method, resulting in a total of seven T-DNA insertion sites, all of which were single-copy integrations ( Table 2 ).…”

Section: Discussionmentioning

confidence: 99%

Effect of T-DNA Integration on Growth of Transgenic Populus × euramericana cv. Neva Underlying Field Stands

Zhang,

Huang,

Dong

et al. 2023

IJMS

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Multigene cotransformation has been widely used in the study of genetic improvement in crops and trees. However, little is known about the unintended effects and causes of multigene cotransformation in poplars. To gain insight into the unintended effects of T-DNA integration during multigene cotransformation in field stands, here, three lines (A1–A3) of Populus × euramericana cv. Neva (PEN) carrying Cry1Ac-Cry3A-BADH genes and three lines (B1–B3) of PEN carrying Cry1Ac-Cry3A-NTHK1 genes were used as research objects, with non-transgenic PEN as the control. Experimental stands were established at three common gardens in three locations and next generation sequencing (NGS) was used to identify the insertion sites of exogenous genes in six transgenic lines. We compared the growth data of the transgenic and control lines for four consecutive years. The results demonstrated that the tree height and diameter at breast height (DBH) of transgenic lines were significantly lower than those of the control, and the adaptability of transgenic lines in different locations varied significantly. The genotype and the experimental environment showed an interaction effect. A total of seven insertion sites were detected in the six transgenic lines, with B3 having a double-site insertion and the other lines having single copies. There are four insertion sites in the gene region and three insertion sites in the intergenic region. Analysis of the bases near the insertion sites showed that AT content was higher than the average chromosome content in four of the seven insertion sites within 1000 bp. Transcriptome analysis suggested that the differential expression of genes related to plant hormone transduction and lignin synthesis might be responsible for the slow development of plant height and DBH in transgenic lines. This study provides an integrated analysis of the unintended effects of transgenic poplar, which will benefit the safety assessment and reasonable application of genetically modified trees.

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Section: Discussionmentioning

confidence: 99%

Effect of T-DNA Integration on Growth of Transgenic Populus × euramericana cv. Neva Underlying Field Stands

Zhang,

Huang,

Dong

et al. 2023

IJMS

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“…Following this treatment, the seeds are infected with a solution of Agrobacterium and grown until the T 1 generation for selection. (B) Agro-imbibition [124]. In this approach, seeds are imbibed with a solution of Agrobacterium instead of sterile water and further selected in the T1 generation.…”

Section: Means Of In Planta Transformationmentioning

confidence: 99%

A comprehensive review of in planta stable transformation strategies

Bélanger,

Copley,

Hoyos-Villegas

et al. 2024

Plant Methods

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Plant transformation remains a major bottleneck to the improvement of plant science, both on fundamental and practical levels. The recalcitrant nature of most commercial and minor crops to genetic transformation slows scientific progress for a large range of crops that are essential for food security on a global scale. Over the years, novel stable transformation strategies loosely grouped under the term “in planta” have been proposed and validated in a large number of model (e.g. Arabidopsis and rice), major (e.g. wheat and soybean) and minor (e.g. chickpea and lablab bean) species. The in planta approach is revolutionary as it is considered genotype-independent, technically simple (i.e. devoid of or with minimal tissue culture steps), affordable, and easy to implement in a broad range of experimental settings. In this article, we reviewed and categorized over 300 research articles, patents, theses, and videos demonstrating the applicability of different in planta transformation strategies in 105 different genera across 139 plant species. To support this review process, we propose a classification system for the in planta techniques based on five categories and a new nomenclature for more than 30 different in planta techniques. In complement to this, we clarified some grey areas regarding the in planta conceptual framework and provided insights regarding the past, current, and future scientific impacts of these techniques. To support the diffusion of this concept across the community, this review article will serve as an introductory point for an online compendium about in planta transformation strategies that will be available to all scientists. By expanding our knowledge about in planta transformation, we can find innovative approaches to unlock the full potential of plants, support the growth of scientific knowledge, and stimulate an equitable development of plant research in all countries and institutions.

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“…The Agrobacterium -mediated delivery of CRISPR-Cas9 has been shifted to other field crops, such as Hemp [ 37 ]. These modifications are either in CRISPR vector systems, explant selection, media composition, or the addition of some regulators [ 35 , 36 , 38 , 39 ]. Recently, to enhance the efficiency of Agrobacterium -mediated transformation for CRSIPR-Cas9, scientists have targeted the Agrobacterium genome [ 40 , 41 ] however, the technology faces multiple challenges that need to be improved further.…”

Section: Ways To Deliver Crispr/cas Reagents To Plantsmentioning

confidence: 99%

Plant Virus-Derived Vectors for Plant Genome Engineering

Mahmood

Naqvi

Rahman

et al. 2023

Viruses

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Advances in genome engineering (GE) tools based on sequence-specific programmable nucleases have revolutionized precise genome editing in plants. However, only the traditional approaches are used to deliver these GE reagents, which mostly rely on Agrobacterium-mediated transformation or particle bombardment. These techniques have been successfully used for the past decades for the genetic engineering of plants with some limitations relating to lengthy time-taking protocols and transgenes integration-related regulatory concerns. Nevertheless, in the era of climate change, we require certain faster protocols for developing climate-smart resilient crops through GE to deal with global food security. Therefore, some alternative approaches are needed to robustly deliver the GE reagents. In this case, the plant viral vectors could be an excellent option for the delivery of GE reagents because they are efficient, effective, and precise. Additionally, these are autonomously replicating and considered as natural specialists for transient delivery. In the present review, we have discussed the potential use of these plant viral vectors for the efficient delivery of GE reagents. We have further described the different plant viral vectors, such as DNA and RNA viruses, which have been used as efficient gene targeting systems in model plants, and in other important crops including potato, tomato, wheat, and rice. The achievements gained so far in the use of viral vectors as a carrier for GE reagent delivery are depicted along with the benefits and limitations of each viral vector. Moreover, recent advances have been explored in employing viral vectors for GE and adapting this technology for future research.

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A Genotype-Independent, Simple, Effective and Efficient in Planta Agrobacterium-Mediated Genetic Transformation Protocol

Cited by 6 publications

References 24 publications

Effect of T-DNA Integration on Growth of Transgenic Populus × euramericana cv. Neva Underlying Field Stands

Effect of T-DNA Integration on Growth of Transgenic Populus × euramericana cv. Neva Underlying Field Stands

A comprehensive review of in planta stable transformation strategies

Plant Virus-Derived Vectors for Plant Genome Engineering

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