Plant Transformation and Genome Editing

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

Deadline for manuscript submissions: 30 June 2024 | Viewed by 12450

Special Issue Editor


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Guest Editor
Innovative Genomics Institute, University of California, Berkeley, CA 94704, USA
Interests: plant transformation; genome editing; biotechnology

Special Issue Information

Dear Colleagues,

Efficient and appropriate transformation technologies are essential for genome editing, which will enable major advances in commercial crop product development. In recent decades, tremendous progress has been made in genetic transformation using plant morphogenic genes and plant growth transcription factors, as well as the improvement of tissue culture and DNA delivery efficiency protocols. In addition, the recent discovery of the CRISPR/Cas9 gene-editing system revolutionized the field of plant genetics and breeding. This technology has attracted global attention and has been employed in basic and applied research in numerous crop species.

This Special Issue of Plants will highlight the improved/advanced plant transformation technologies, genome-editing efficiency improvement, and novel genome-editing approaches for crop improvement.

Dr. Myeong-Je Cho
Guest Editor

Manuscript Submission Information

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Keywords

  • plant transformation
  • genome editing
  • DNA (transgene)-free editing
  • base/prime editing
  • HDR-based editing
  • CRISPR/dCas9 system
  • off-target effect

Published Papers (6 papers)

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Research

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16 pages, 3224 KiB  
Article
Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis ‘Purpurea’
by Yun Xiao, Wanli Tuo, Xuexuan Wang, Baomin Feng, Xinyu Xu, Sagheer Ahmad, Junwen Zhai, Donghui Peng and Shasha Wu
Plants 2023, 12(24), 4130; https://doi.org/10.3390/plants12244130 - 11 Dec 2023
Viewed by 1015
Abstract
Oxalis triangularis ‘Purpurea’ has significant ornamental value in landscaping. There is a critical necessity to elucidate the gene functions of O. triangularis ‘Purpurea’ and dissect the molecular mechanisms governing key ornamental traits. However, a reliable genetic transformation method remains elusive. In this study, [...] Read more.
Oxalis triangularis ‘Purpurea’ has significant ornamental value in landscaping. There is a critical necessity to elucidate the gene functions of O. triangularis ‘Purpurea’ and dissect the molecular mechanisms governing key ornamental traits. However, a reliable genetic transformation method remains elusive. In this study, our investigation revealed that various transformation parameters, including recipient material (petioles), pre-culture time (2–5 days), acetosyringone (AS) concentration (100–400 μM), Agrobacterium concentrations (OD600 = 0.4–1.0), infection time (5–20 min), and co-culture time (2–5 days), significantly impacted the stable genetic transformation in O. triangular ‘Purpurea’. Notably, the highest genetic transformation rate was achieved from the leaf discs pre-cultured for 3 days, treated with 200 μM AS infected with Agrobacterium for 11 min at OD600 of 0.6, and subsequently co-cultured for 3 days. This treatment resulted in a genetic transformation efficiency of 9.88%, and it only took 79 days to produce transgenic plants. Our transformation protocol offers advantages of speed, efficiency, and simplicity, which will greatly facilitate genetic transformation for O. triangular ‘Purpurea’ and gene function studies. Full article
(This article belongs to the Special Issue Plant Transformation and Genome Editing)
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9 pages, 966 KiB  
Communication
Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector
by Minjeong Kang, Keunsub Lee, Qing Ji, Sehiza Grosic and Kan Wang
Plants 2023, 12(15), 2799; https://doi.org/10.3390/plants12152799 - 28 Jul 2023
Cited by 3 | Viewed by 2141
Abstract
Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated [...] Read more.
Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing. Full article
(This article belongs to the Special Issue Plant Transformation and Genome Editing)
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14 pages, 2218 KiB  
Article
eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’
by Guoliang Yuan, Yang Liu, Tao Yao, Wellington Muchero, Jin-Gui Chen, Gerald A. Tuskan and Xiaohan Yang
Plants 2023, 12(8), 1657; https://doi.org/10.3390/plants12081657 - 14 Apr 2023
Cited by 1 | Viewed by 1934
Abstract
Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood [...] Read more.
Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone ‘52-225’ (P. trichocarpa × P. deltoides clone ‘52-225’). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides. Full article
(This article belongs to the Special Issue Plant Transformation and Genome Editing)
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16 pages, 3299 KiB  
Article
Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce (Lactuca sativa L.) Using the Endogenous U6 Promoter
by Young-Sun Riu, Gwang Hoon Kim, Ki Wha Chung and Sam-Geun Kong
Plants 2023, 12(4), 878; https://doi.org/10.3390/plants12040878 - 15 Feb 2023
Cited by 3 | Viewed by 2734
Abstract
The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter [...] Read more.
The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that the PHOT2 gene is a versatile target gene. We isolated the LsU6-10 promoter from 10 U6 snRNA genes identified from the lettuce genome database for comparison with the AtU6-26 promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the LsU6-10 and AtU6-26 promoters to drive sgRNA361 to target the PHOT2 gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted PHOT2 gene, as in the Arabidopsis phot2 mutant. The PHOT2 gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent phot2 mutants in the R2 generation. Our results suggest that the LsU6-10 promoter is more effective than the AtU6-26 promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that PHOT2 is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes. Full article
(This article belongs to the Special Issue Plant Transformation and Genome Editing)
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Review

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12 pages, 827 KiB  
Review
Improving the Traits of Perilla frutescens (L.) Britt Using Gene Editing Technology
by Sivabalan Karthik, Jia Chae, Seong Ju Han, Jee Hye Kim, Hye Jeong Kim, Young-Soo Chung, Hyun Uk Kim and Jae Bok Heo
Plants 2024, 13(11), 1466; https://doi.org/10.3390/plants13111466 (registering DOI) - 25 May 2024
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Abstract
Plant breeding has evolved significantly over time with the development of transformation and genome editing techniques. These new strategies help to improve desirable traits in plants. Perilla is a native oil crop grown in Korea. The leaves contain many secondary metabolites related to [...] Read more.
Plant breeding has evolved significantly over time with the development of transformation and genome editing techniques. These new strategies help to improve desirable traits in plants. Perilla is a native oil crop grown in Korea. The leaves contain many secondary metabolites related to whitening, aging, antioxidants, and immunity, including rosmarinic acid, vitamin E, luteolin, anthocyanins, and beta-carotene. They are used as healthy and functional food ingredients. It is an industrially valuable cosmetics crop. In addition, perilla seeds are rich in polyunsaturated fatty acids, such as α-linolenic acid and linoleic acid. They are known to be effective in improving neutral lipids in the blood, improving blood circulation, and preventing dementia and cardiovascular diseases, making them excellent crops whose value can be increased through improved traits. This research will also benefit perilla seeds, which can increase their stock through various methods, such as the increased production of functional substances and improved productivity. Recently, significant attention has been paid to trait improvement research involving gene-editing technology. Among these strategies, CRISPR/Cas9 is highly adaptable, enabling accurate and efficient genome editing, targeted mutagenesis, gene knockouts, and the regulation of gene transcription. CRISPR/Cas9-based genome editing has enormous potential for improving perilla; however, the regulation of genome editing is still at an early stage. Therefore, this review summarizes the enhancement of perilla traits using genome editing technology and outlines future directions. Full article
(This article belongs to the Special Issue Plant Transformation and Genome Editing)
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22 pages, 3460 KiB  
Review
Unintended Genomic Outcomes in Current and Next Generation GM Techniques: A Systematic Review
by Philomena Chu and Sarah Zanon Agapito-Tenfen
Plants 2022, 11(21), 2997; https://doi.org/10.3390/plants11212997 - 7 Nov 2022
Cited by 12 | Viewed by 3115
Abstract
Classical genetic engineering and new genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities for modifying the genetic material in organisms. These technologies have the potential to provide novel agricultural traits, including modified microorganisms and environmental applications. However, legitimate safety concerns arise [...] Read more.
Classical genetic engineering and new genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities for modifying the genetic material in organisms. These technologies have the potential to provide novel agricultural traits, including modified microorganisms and environmental applications. However, legitimate safety concerns arise from the unintended genetic modifications (GM) that have been reported as side-effects of such techniques. Here, we systematically review the scientific literature for studies that have investigated unintended genomic alterations in plants modified by the following GM techniques: Agrobacterium tumefaciens-mediated gene transfer, biolistic bombardment, and CRISPR-Cas9 delivered via Agrobacterium-mediated gene transfer (DNA-based), biolistic bombardment (DNA-based) and as ribonucleoprotein complexes (RNPs). The results of our literature review show that the impact of such techniques in host genomes varies from small nucleotide polymorphisms to large genomic variation, such as segmental duplication, chromosome truncation, trisomy, chromothripsis, breakage fusion bridge, including large rearrangements of DNA vector-backbone sequences. We have also reviewed the type of analytical method applied to investigate the genomic alterations and found that only five articles used whole genome sequencing in their analysis methods. In addition, larger structural variations detected in some studies would not be possible without long-read sequencing strategies, which shows a potential underestimation of such effects in the literature. As new technologies are constantly evolving, a more thorough examination of prospective analytical methods should be conducted in the future. This will provide regulators working in the field of genetically modified and gene-edited organisms with valuable information on the ability to detect and identify genomic interventions. Full article
(This article belongs to the Special Issue Plant Transformation and Genome Editing)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: eYGFPuv-Assisted Plant Transformation and Multiplex Genome Editing in Populus deltoides
Authors: Guoliang Yuan; Gerald A. Tuskan; Xiaohan Yang
Affiliation: 1Biosciences Division, Oak Ridge National Laboratory, Oak Ridge TN 37831, USA 2The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
Abstract: Poplar (Populus spp.) is an important woody crop for biofuels and carbon sequestration. Agrobacterium-mediated stable transformation is a major approach for genetic engineering in poplar. High-quality poplar genome sequences have been generated for multiple poplar species, laying a solid foundation for genetic engineering in poplar. However, some of these poplar species are not easily transformed by A. tumefaciens. Eastern cottonwood (P. deltoides) is an important model for poplar genomics and biotechnology research due to 1) availability of high-quality genome sequence, 2) amenability to plant transformation, 3) relatively high stress tolerance, and 4) fast growth. Although P. deltoides WV94 can be transformed by A. tumefaciens, several challenges need to be addressed, including the relatively low efficiency of transformation and relatively high rate of false positives from selection based on antibiotic resistance. Here, using a UV-visible reporter eYGFPuv, we optimize the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94. The transgenic events can be easily recognized and counted in a non-invasive assay. On the other hand, CRISPR-Cas based genome editing has been widely used for plant genetic engineering because of its simplicity, high efficiency, low cost, and the possibility of targeting multiple genes. In this study, we examine the efficacy of multiplex CRISPR genome editing in P. deltoides WV94 protoplasts. Furthermore, we integrate multiplex CRISPR genome editing and eYGFPuv-assisted plant transformation to accelerate muti-gene engineering in P. deltoides WV94.

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