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Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations
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Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations

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

Deadline for manuscript submissions: 31 July 2025 | Viewed by 10674

Special Issue Editor

Dr. Humberto Prieto

E-Mail Website1 Website2
Guest Editor
Biotechnology Laboratory, Instituto de Investigaciones Agropecuarias, Santiago de Chile, 8831314, Chile
Interests: Plant molecular biology; Fruit crops; Gene editing; Gene silencing; In vitro culture

Special Issue Information

Dear Colleagues,

The intersection of genomics and gene editing has ushered in a transformative era in plant biology and biotechnology. The rapidly increasing number of sequenced plant genomes and information from functional genomic data to understand gene function, together with novel gene cloning and up-to-date tissue culture methods, is accelerating crop improvement and trait development.

Genetic transformation is key for these advances to contribute to breeding and research in plants, aiding the understanding of complex biological phenomena like pathogenesis, genome organization, light reception, signal transduction, epigenetic modulation, and others. Efforts for improving transformation techniques have contributed to speeding up precision breeding; newly created phenotypes have been the result of modulating the activity of the host’s gene by RNA interference mechanisms and, more recently, the powerful CRISPR-Cas gene-editing technology, which today aims to hold immense promise for global agriculture, food security, and scientific advancements.

These innovations have included improving procedures in Agrobacterium-mediated gene transfer, protoplast protocols, direct organogenesis, somatic embryogenesis, and in planta techniques. The use of morphogenic genes has also been explored in different plant systems. In addition, there is also a continued search for progress in screening and selection procedures, including the design and application of new vectors and delivery systems.

Currently, while transgenic technology is beneficial for breeding, it faces challenges like low transformation efficiency and the complexity of molecular genetic mechanisms involved in the gene transfer process. Several aspects of genetic transformation achieved in model species have not been reached for relevant crops, including both monocots and dicots.

This Special Issue aims to cover new developments in the field of gene transfer and the regeneration procedures of species of relevance in breeding. We welcome manuscripts dealing with the following:

  • The transformation of recalcitrant species;
  • The employment of Agrobacterium strains with different transformation capabilities;
  • In planta transformation procedures;
  • Protoplast techniques;
  • New vectors for transformation, including cisgenic and viral vectors;
  • Gene silencing through the application of siRNA, miRNA, or artificial miRNA methodology;
  • CRISPRmediated genome editing;
  • Other new approaches to genetic transformation (for instance, the use of genes affecting plant regeneration).

Dr. Humberto Prieto
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

  • gene transfer procedures
  • Agrobacterium
  • regeneration
  • in planta genetic transformation
  • RNA interference
  • gene expression
  • gene editing

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Published Papers (8 papers)

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Research

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15 pages, 2677 KiB  
Article
CRISPR-Based Editing of the Medicago truncatula LEC1 Gene
by Elina A. Potsenkovskaia, Varvara E. Tvorogova, Veronika Y. Simonova, Zakhar S. Konstantinov, Anna S. Kiseleva, Andrew G. Matveenko, Anna V. Brynchikova and Ludmila A. Lutova
Plants 2024, 13(22), 3226; https://doi.org/10.3390/plants13223226 - 16 Nov 2024
Viewed by 1180
Abstract
Arabidopsis thaliana LEAFY COTYLEDON1 (LEC1) gene is shown to have numerous diverse functions in plant development, including the regulation of embryo morphogenesis and maturation, hypocotyl elongation, flowering transition, etc. However, the functions of LEC1 orthologs in different plant species have not been extensively studied. [...] Read more.
Arabidopsis thaliana LEAFY COTYLEDON1 (LEC1) gene is shown to have numerous diverse functions in plant development, including the regulation of embryo morphogenesis and maturation, hypocotyl elongation, flowering transition, etc. However, the functions of LEC1 orthologs in different plant species have not been extensively studied. In this study, we obtained a line of Medicago truncatula, a model leguminous plant, carrying the loss-of-function mutation in the MtLEC1 (MtNF-YB10) gene, orthologous to LEC1, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas9) genome editing system. Edited plants with loss of MtNF-YB10 function did not demonstrate any severe abnormalities during their normal growth and gave viable seeds, but their capability for somatic embryogenesis in vitro was dramatically reduced. The T1 progeny of unedited plants with a Cas9-gRNA cassette insertion was also analyzed based on the suggestion that editing could occur during seed formation. However, no edited plants were found in the T1 generation. These results suggest divergent functions of LEC1 orthologs and make it possible to investigate potential specific MtNF-YB10 functions. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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18 pages, 7616 KiB  
Article
Application of an Efficient Enhancer in Gene Function Research
by Feng-Xian Guo, Rui-Xue Yang, Xia Yang, Jing Liu and Yin-Zheng Wang
Plants 2024, 13(22), 3120; https://doi.org/10.3390/plants13223120 - 6 Nov 2024
Viewed by 831
Abstract
Although great progress has been made in transgenic technology, increasing the expression level and thus promising the expected phenotypes of exogenous genes in transgenic plants is still a crucial task for genetic transformation and crop engineering. Here, we conducted a comparative study of [...] Read more.
Although great progress has been made in transgenic technology, increasing the expression level and thus promising the expected phenotypes of exogenous genes in transgenic plants is still a crucial task for genetic transformation and crop engineering. Here, we conducted a comparative study of the enhancing efficiency of three putative translational enhancers, including Ω (natural leader from a plant virus), OsADH 5′ (natural leader from a plant gene), and ARC (active ribosomal RNA complementary), using the transient gene expression systems of Nicotiana benthamiana and Chirita pumila. We demonstrate that three tandem repeats of ARC (3 × ARC) are more efficient than other enhancers in expression. The enhancing efficiency of 6 × ARC is further increased, up to 130 times the expression level without the insertion of enhancers. We further evaluated the enhancing efficiency of 6 × ARC under agrobacterium-mediated transformation systems. In C. pumila, 6 × ARC significantly amplifies the phenotypic effect of CpCYC1 and CpCYC2 in repressing stamen development and yellow pigmentation. In Arabidopsis thaliana, 6 × ARC and the AtAP1 promoter work together to promote the accumulation of anthocyanin pigments in vegetative and reproductive organs. Most significantly, the fusion of 6 × ARC in a CpCYC1/2 transgenic system in C. pumila fully reveals that these genes have the complete function of repressing the yellow spots, displaying an advantage in manifesting the function of exogenous genes. This study highlights the application potential of the enhancer 6 × ARC in gene function research in plants. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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25 pages, 47764 KiB  
Article
Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes
by Van Giap Do, Seonae Kim, Nay Myo Win, Soon-Il Kwon, Hunjoong Kweon, Sangjin Yang, Juhyeon Park, Gyungran Do and Youngsuk Lee
Plants 2024, 13(19), 2803; https://doi.org/10.3390/plants13192803 - 6 Oct 2024
Viewed by 1907
Abstract
Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as [...] Read more.
Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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19 pages, 21418 KiB  
Article
Genetic Transformation of Triticum dicoccum and Triticum aestivum with Genes of Jasmonate Biosynthesis Pathway Affects Growth and Productivity Characteristics
by Dmitry N. Miroshnichenko, Alexey V. Pigolev, Alexander S. Pushin, Valeria V. Alekseeva, Vlada I. Degtyaryova, Evgeny A. Degtyaryov, Irina V. Pronina, Andrej Frolov, Sergey V. Dolgov and Tatyana V. Savchenko
Plants 2024, 13(19), 2781; https://doi.org/10.3390/plants13192781 - 4 Oct 2024
Viewed by 1121
Abstract
The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates [...] Read more.
The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates by overexpressing ALLENE OXIDE SYNTHASE from Arabidopsis thaliana (AtAOS) and bread wheat (TaAOS) and OXOPHYTODIENOATE REDUCTASE 3 from A. thaliana (AtOPR3) under the strong constitutive promoter (ZmUbi1), either individually or both genes simultaneously. The delivery of the expression cassette encoding AOS was found to affect morphogenesis in both wheat species negatively. The effect of transgene expression on the accumulation of individual jasmonates in hexaploid and tetraploid wheat was observed. Among the introduced genes, overexpression of TaAOS was the most successful in increasing stress-inducible phytohormone levels in transgenic plants, resulting in higher accumulations of JA and JA-Ile in emmer wheat and 12-OPDA in bread wheat. In general, overexpression of AOS, alone or together with AtOPR3, negatively affected leaf lamina length and grain numbers per spike in both wheat species. Double (AtAOS + AtOPR3) transgenic wheat plants were characterized by significantly reduced plant height and seed numbers, especially in emmer wheat, where several primary plants failed to produce seeds. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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20 pages, 4529 KiB  
Article
Organogenesis in a Broad Spectrum of Grape Genotypes and Agrobacterium-Mediated Transformation of the Podarok Magaracha Grapevine Cultivar
by Galina Maletich, Alexander Pushin, Evgeniy Rybalkin, Yuri Plugatar, Sergey Dolgov and Pavel Khvatkov
Plants 2024, 13(19), 2779; https://doi.org/10.3390/plants13192779 - 3 Oct 2024
Cited by 1 | Viewed by 1273
Abstract
We present data on the ability for organogenesis in 22 genotypes of grapevine and developed a direct organogenesis protocol for the cultivar Podarok Magaracha and the rootstock Kober 5BB. The protocol does not require replacement of culture media and growth regulators, and the [...] Read more.
We present data on the ability for organogenesis in 22 genotypes of grapevine and developed a direct organogenesis protocol for the cultivar Podarok Magaracha and the rootstock Kober 5BB. The protocol does not require replacement of culture media and growth regulators, and the duration is 11 weeks. The cultivation of explants occurs on modified MS medium with the addition of 2.0 mg L−1 benzyladenine and indole-3-butyric acid (0.15 mg L−1 for the rootstock Kober 5BB or 0.05 mg L−1 for the cultivar Podarok Magaracha). The direct organogenesis protocol consists of three time periods: (1) culturing explants for 2 weeks in dark conditions for meristematic bulk tissue, (2) followed by 4 weeks of cultivation in light conditions for regeneration, and (3) 5 weeks of cultivation in dark conditions for shoot elongation. Based on this protocol, conditions for the Agrobacterium-mediated transformation of the Podarok Magaracha cultivar were developed with an efficiency of 2.0% transgenic plants per 100 explants. Two stably transformed lines with integration into the genome of the pBin35SGFP plasmid construction, confirmed by Southern blotting, were obtained. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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16 pages, 2564 KiB  
Article
An Effective Somatic-Cell Regeneration and Genetic Transformation Method Mediated by Agrobacterium tumefaciens for Portulaca oleracea L.
by Mengyun Xu, Xinyu Zhao, Jiahui Fang, Qinwen Yang, Ping Li and Jian Yan
Plants 2024, 13(17), 2390; https://doi.org/10.3390/plants13172390 - 27 Aug 2024
Viewed by 1331
Abstract
Purslane (Portulaca oleracea L.) is highly valued for its nutritional, medicinal, and ecological significance. Genetic transformation in plants provides a powerful tool for gene manipulation, allowing for the investigation of important phenotypes and agronomic traits at the genetic level. To develop an [...] Read more.
Purslane (Portulaca oleracea L.) is highly valued for its nutritional, medicinal, and ecological significance. Genetic transformation in plants provides a powerful tool for gene manipulation, allowing for the investigation of important phenotypes and agronomic traits at the genetic level. To develop an effective genetic transformation method for purslane, various organ tissues were used as explants for callus induction and shoot regeneration. Leaf tissue exhibited the highest dedifferentiation and regeneration ability, making it the optimal explant for tissue culture. By culturing on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzyleaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA), somatic cells from leaf explants could be developed into calli, shoots, and roots. The shoot induction results of 27 different purslane accessions elucidated the impact of genotype on somatic-cell regeneration capacity and further confirmed the effectiveness of the culture medium in promoting shoot regeneration. On this basis, a total of 17 transgenic plants were obtained utilizing the genetic transformation method mediated by Agrobacterium. The assessment of GUS staining, hygromycin selection, and polymerase chain reaction (PCR) amplification of the transgenic plants as well as their progeny lines indicated that the method established could effectively introduce foreign DNA into the purslane nucleus genome, and that integration was found to be stably inherited by offspring plants. Overall, the present study demonstrates the feasibility and reliability of the Agrobacterium-mediated genetic transformation method for introducing and integrating foreign DNA into the purslane genome, paving the way for further research and applications in purslane genetic modification. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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20 pages, 681 KiB  
Review
Promising Biotechnological Applications of the Artificial Derivatives Designed and Constructed from Plant microRNA Genes
by T. N. Erokhina, Ekaterina V. Ryabukhina, Irina S. Lyapina, Dmitry Y. Ryazantsev, Sergey K. Zavriev and Sergey Y. Morozov
Plants 2025, 14(3), 325; https://doi.org/10.3390/plants14030325 - 22 Jan 2025
Viewed by 701
Abstract
MicroRNAs (miRNAs) are small regulatory RNAs that are expressed in a tissue-specific manner during the development of plants and animals. The genes of miRNAs have been found to produce the following two products: (i) primary transcripts of these genes (pri-miRNA) are processed to [...] Read more.
MicroRNAs (miRNAs) are small regulatory RNAs that are expressed in a tissue-specific manner during the development of plants and animals. The genes of miRNAs have been found to produce the following two products: (i) primary transcripts of these genes (pri-miRNA) are processed to give rise to mature miRNA, and (ii) in some cases, the pri-miRNA molecules can be translated to form small peptides, named as miPEPs. Gene silencing by artificial microRNAs (amiRNAs) is one of the potential crucial methods for the regulation of desired genes to improve horticultural plants. Likewise, external application of chemically synthesized miPEPs may help plants to resist biotic/abiotic stresses and grow faster. These potent and reliable derivatives of miRNA genes can be applied for improving useful traits in crop plants. This review summarizes the progress in research on the artificial gene derivatives involved in regulating plant development, virus and pest diseases, and abiotic stress resistance pathways. We also briefly discuss the molecular mechanisms of relevant target genes for future research on breeding in plants. In general, this review may be useful to researchers who are implementing amiRNA and miPEP for accelerating breeding programs and developmental studies in crop plants. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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14 pages, 1665 KiB  
Perspective
The Global Advance of Genome-Edited Plants to the Market: The Key Role of Chile in Its Development
by Miguel A. Sánchez
Plants 2024, 13(24), 3597; https://doi.org/10.3390/plants13243597 - 23 Dec 2024
Viewed by 1305
Abstract
The global advancement of genome-edited plants toward commercialization has been significantly shaped by the functionality and flexibility of some regulatory frameworks governing plant genome editing. These frameworks vary widely across countries, reflecting diverse approaches to assessing and managing the risks and benefits of [...] Read more.
The global advancement of genome-edited plants toward commercialization has been significantly shaped by the functionality and flexibility of some regulatory frameworks governing plant genome editing. These frameworks vary widely across countries, reflecting diverse approaches to assessing and managing the risks and benefits of genome-editing technologies. While some nations have adopted product-based frameworks that focus on the characteristics of the final plant rather than the technique used, others rely on more restrictive process-based regulations. This variability influences the pace of innovation, the types of products able to enter the market, and their global trade potential. Chile stands out as a leader in this landscape, having implemented a science-driven and flexible regulatory framework. Its system promotes innovation by facilitating genome-edited plant research and development, field testing, and local commercialization. This regulatory adaptability positions Chile as a critical player in supporting the global integration of genome-editing technologies into agriculture, fostering advancements that address food security, sustainability, and climate resilience. Full article
(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)
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