Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations—2nd Edition

A Special Issue of Plants (ISSN 2223-7747) belonging to the section "Plant Genetics, Genomics and Biotechnology".

Deadline for manuscript submissions: 10 April 2027 | Viewed by 2258

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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
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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 genomics data to understand gene function, together with novel gene cloning and up-to-date tissue culture methods, is further accelerating crop improvement and trait development.

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

These innovations have included improving procedures in Agrobacterium-mediated gene transfer, protoplasts 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 continuous 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 regeneration procedures of species of relevance in breeding. We welcome manuscripts dealing with the following:

  • Transformation of recalcitrant species;
  • 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;
  • CRISPR-mediated genome editing;
  • Other new approaches to genetic transformation (for instance, the use of genes affecting plant regeneration).

Dr. Humberto Prieto
Guest Editor

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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-anonymized 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.

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Keywords

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

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Related Special Issue

Published Papers (4 papers)

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Research

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19 pages, 13962 KB  
Article
A RUBY-Based Visual Hairy Root Transformation and CRISPR-PTG Genome-Editing System in Eucommia ulmoides
by Linqing Xu, Yani Zhou, Yongfeng Sun, Yating Dong, Rong Kang, Nan Yao and Xia Cai
Plants 2026, 15(18), 2795; https://doi.org/10.3390/plants15182795 - 11 Sep 2026
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Abstract
Eucommia ulmoides Oliv. is a woody plant with significant medicinal and industrial value. However, the lack of an efficient genetic transformation and gene-editing system has seriously hindered the research on its functional genes and the progress of molecular breeding. To break through this [...] Read more.
Eucommia ulmoides Oliv. is a woody plant with significant medicinal and industrial value. However, the lack of an efficient genetic transformation and gene-editing system has seriously hindered the research on its functional genes and the progress of molecular breeding. To break through this technical bottleneck, this study successfully established an efficient Agrobacterium-mediated genetic transformation system for E. ulmoides hairy roots based on the RUBY visual reporter gene. Through systematic optimization, the optimal transformation conditions were identified as infecting hypocotyl explants with Agrobacterium strain K599 at an OD600 of 0.6 for 20 min. Based on this optimized system, a RUBY-based CRISPR-PTG (polycistronic tRNA-gRNA) construct was further generated. With squalene synthase (EuSQS) as the target gene, Sanger sequencing and ICE (Inference of CRISPR Edits) analysis provided preliminary evidence for CRISPR-PTG-mediated editing at the EuSQS locus in transgenic hairy roots, although the potentially chimeric nature of hairy roots may contribute to variation in the observed editing profiles. In this study, a RUBY-based visual hairy root genetic transformation system was established, which provided preliminary evidence that CRISPR-PTG-mediated targeted mutagenesis can occur in E. ulmoides hairy roots. These results provide a useful basis for further optimization and application of genome editing in subsequent gene function analysis and metabolic engineering research of E. ulmoides, and also offer a reference for genetic transformation studies of other woody plants. Full article
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18 pages, 2972 KB  
Article
Culture Medium Composition and Light Intensity Shape Alternative Morphogenic Pathways from the Mature Embryo Shoot Apex of Eragrostis curvula
by Eduardo Daniel Souza Canada, Ingrid Garbus, Juan Pablo Selva, Hugo Raúl Permingeat and Viviana Echenique
Plants 2026, 15(18), 2793; https://doi.org/10.3390/plants15182793 - 11 Sep 2026
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Abstract
Eragrostis curvula (weeping lovegrass) is a perennial C4 forage grass and a model for apomixis whose genetic transformation and genome editing remain constrained by the lack of efficient regeneration systems. Here we assessed the effect of culture medium composition and light intensity [...] Read more.
Eragrostis curvula (weeping lovegrass) is a perennial C4 forage grass and a model for apomixis whose genetic transformation and genome editing remain constrained by the lack of efficient regeneration systems. Here we assessed the effect of culture medium composition and light intensity during the induction phase on morphogenic responses from explants of mature embryos of cv. Tanganyika INTA. Eleven media were tested: nine designed to induce somatic embryo-like structures (SELSs; Group 1) and two to promote multiple shoot proliferation (Group 2). Four light regimes—low, medium, high, and darkness—were applied during an 8-week induction period. Responsive and non-responsive explants were recorded at the Petri dish level and analyzed separately within each functional group using binomial generalized linear models (GLMs) in R 4.5.0. Morphogenic responses originated from the apical region of the embryonic axis, encompassing the shoot apical meristem. Medium composition influenced the predominant morphogenic response, whereas light intensity affected the efficiency of the response (significant medium × light interaction). Medium I (WPBS + 2,4-D + low BAP) under medium light resulted in the highest morphogenic response within Group 1, corresponding to an estimated 60.6% of explants exhibiting SELSs and/or associated shoot-forming meristematic tissue, although this estimate was not statistically distinguishable from those obtained for the same medium under low and high light intensity. Medium J (WPBS + high BAP:2,4-D ratio) under medium light resulted in a 74.0% multiple shoot proliferation response. Darkness reduced morphogenic responses in both groups. These protocols provide a reproducible regeneration platform for future genetic transformation and genome-editing studies in E. curvula. Full article
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16 pages, 6458 KB  
Communication
Dual-gRNA CRISPR/Cas9 Deletion of CsDMR6 in Sweet Orange Supported by Improved In Vitro Regeneration
by Sandra Sopalda, Ricardo Vergara, Marisol Muñoz, Carlos Aguirre, Carlos Muñoz and Humberto Prieto
Plants 2026, 15(17), 2664; https://doi.org/10.3390/plants15172664 - 31 Aug 2026
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Abstract
Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6 [...] Read more.
Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of ‘Valencia’ and hybrid genotypes (CsH1–CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L−1 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in ‘Valencia’ and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure. Full article
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13 pages, 1823 KB  
Brief Report
Enhancing Agrobacterium-Mediated Hairy-Root Transformation Efficiency in Peanut Through the Application of GRF, GIF and WOX Genes
by Qianqian Zhang, Yuanyuan Cui, Fangjun Chen and Xiaoqin Liu
Plants 2026, 15(12), 1889; https://doi.org/10.3390/plants15121889 - 18 Jun 2026
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Abstract
Peanut (Arachis hypogaea L.) is a major oil and economic crop, yet genetic transformation remains inefficient and time-consuming, hindering functional genomics and molecular breeding. In this study, we found that the use of GRF, GIF and WOX genes improved the efficiency [...] Read more.
Peanut (Arachis hypogaea L.) is a major oil and economic crop, yet genetic transformation remains inefficient and time-consuming, hindering functional genomics and molecular breeding. In this study, we found that the use of GRF, GIF and WOX genes improved the efficiency of Agrobacterium-mediated peanut hairy-root transformation. Here, we identified multiple peanut Growth-Regulating Factor (GRF) genes, GRF-Interacting Factor (GRF-GIF) fusion genes and WUSCHEL-related homeobox (WOX) genes, constructed high-expression vectors, and delivered them into A. rhizogenes to infect 3–5 cm peanut stem segments cut from 30-day-old seedlings. Statistical analysis of the data showed that, relative to the empty-vector control, expression of these developmental regulators markedly enhanced hairy-root growth: the number of roots per explant increased by 1.3–2.4-fold. Observations using reporter constructs showed that growth factors (besides 2S-PL-GUS and GRF-2A-T-GUS) improved the transformation efficiency of hairy roots, among which the highest transformation efficiency of GRF-2A (396)-GIF-GUS was 85.14 ± 2.94%. Collectively, these findings provide an efficient and rapid platform for the study of peanut gene function. Full article
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