Plant Genetic Engineering and Biotechnology

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 (20 September 2023) | Viewed by 27001

Special Issue Editors


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Guest Editor
1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China
2. College of Advanced Agriculture Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
Interests: plant genetics; bioengineering and biotechnology; crop improvement
Center for Excellence in Molecular Plant Science, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
Interests: plant biotechnology; hormone signaling; fruit development
National Watermelon and Melon Improvement Center, Beijing Academy of Agricultural and Forestry Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing 100097, China
Interests: biomass accumulation; sugar transporter; expression regulation

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Guest Editor
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China
Interests: crop architecture improvement; crop oil metabolism; Interaction of plant and microbial

Special Issue Information

Dear Colleagues,

In the past few decades, genetic engineering and biotechnology has been widely used in the field of plants to improve nutritional quality, increase production, improve genetic characteristics, carry out important trait gene screening, and reduce biological and abiotic stress; transgenic plants can even be used as a biological reactor, producing organic compounds, drug proteins, plant secondary metabolites, and so on. This Special Issue of Plants aims to analyze economic crop genomics and biotechnology research, focusing on research on the establishment of new plant expression systems, exploration of genetic transformation methods, gene cloning, the regularity of gene expression in plants, and important economic crop genetic engineering improvement and research achievements. We welcome all research articles and review articles covering the above subject to submit in this special issue.

Prof. Dr. Zanmin Hu
Dr. Han Xiao
Dr. Yi Ren
Dr. Chengming Fan
Guest Editors

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Keywords

  • modified plants
  • plant improvement
  • genome engineering
  • biotechnology
  • plant transformation

Published Papers (14 papers)

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Research

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14 pages, 3489 KiB  
Article
Validation of Novel Reference Genes in Different Rice Plant Tissues through Mining RNA-Seq Datasets
by Xin Liu, Yingbo Gao, Xinyi Zhao, Xiaoxiang Zhang, Linli Ben, Zongliang Li, Guichun Dong, Juan Zhou, Jianye Huang and Youli Yao
Plants 2023, 12(23), 3946; https://doi.org/10.3390/plants12233946 - 23 Nov 2023
Cited by 1 | Viewed by 957
Abstract
Reverse transcription quantitative real-time PCR (RT-qPCR) is arguably the most prevalent and accurate quantitative gene expression analysis. However, selection of reliable reference genes for RT-qPCR in rice (Oryza sativa) is still limited, especially for a specific tissue type or growth condition. [...] Read more.
Reverse transcription quantitative real-time PCR (RT-qPCR) is arguably the most prevalent and accurate quantitative gene expression analysis. However, selection of reliable reference genes for RT-qPCR in rice (Oryza sativa) is still limited, especially for a specific tissue type or growth condition. In this study, we took the advantage of our RNA-seq datasets encompassing data from five rice varieties with diverse treatment conditions, identified 12 novel candidate reference genes, and conducted rigorous evaluations of their suitability across typical rice tissues. Comprehensive analysis of the leaves, shoots, and roots of two rice seedlings subjected to salt (30 mmol/L NaCl) and drought (air-dry) stresses have revealed that OsMED7, OsACT1, and OsOS-9 were the robust reference genes for leaf samples, while OsACT1, OsZOS3-23, and OsGDCP were recommended for shoots and OsMED7, OsOS-9, and OsGDCP were the most reliable reference genes for roots. Comparison results produced by different sets of reference genes revealed that all these newly recommended reference genes displayed less variation than previous commonly used references genes under the experiment conditions. Thus, selecting appropriate reference genes from RNA-seq datasets leads to identification of reference genes suitable for respective rice tissues under drought and salt stress. The findings offer valuable insights for refining the screening of candidate reference genes under diverse conditions through the RNA-seq database. This refinement serves to improve the accuracy of gene expression in rice under similar conditions. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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14 pages, 5969 KiB  
Article
A SUPERMAN-like Gene Controls the Locule Number of Tomato Fruit
by Mi Zhang, Enbai Zhou, Meng Li, Shenglan Tian and Han Xiao
Plants 2023, 12(18), 3341; https://doi.org/10.3390/plants12183341 - 21 Sep 2023
Viewed by 1146
Abstract
Tomato (Solanum lycopersicum) fruits are derived from fertilized ovaries formed during flower development. Thus, fruit morphology is tightly linked to carpel number and identity. The SUPERMAN (SUP) gene is a key transcription repressor to define the stamen–carpel boundary and [...] Read more.
Tomato (Solanum lycopersicum) fruits are derived from fertilized ovaries formed during flower development. Thus, fruit morphology is tightly linked to carpel number and identity. The SUPERMAN (SUP) gene is a key transcription repressor to define the stamen–carpel boundary and to control floral meristem determinacy. Despite SUP functions having been characterized in a few plant species, its functions have not yet been explored in tomato. In this study, we identified and characterized a fascinated and multi-locule fruit (fmf) mutant in Solanum pimpinellifolium background harboring a nonsense mutation in the coding sequence of a zinc finger gene orthologous to SUP. The fmf mutant produces supersex flowers containing increased numbers of stamens and carpels and sets malformed seedless fruits with complete flowers frequently formed on the distal end. fmf alleles in cultivated tomato background created by CRISPR-Cas9 showed similar floral and fruit phenotypes. Our results provide insight into the functional conservation and diversification of SUP members in different species. We also speculate the FMF gene may be a potential target for yield improvement in tomato by genetic engineering. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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17 pages, 3431 KiB  
Article
Integrated Full-Length Transcriptome and Metabolome Profiling Reveals Flavonoid Regulation in Response to Freezing Stress in Potato
by Zhiguo Zhu, Lingling Wei, Lei Guo, Huihui Bao, Xuemei Wang, Philip Kear, Zhen Wang and Guangtao Zhu
Plants 2023, 12(10), 2054; https://doi.org/10.3390/plants12102054 - 22 May 2023
Viewed by 1241
Abstract
Cold stress impairs plant growth and development, resulting in crop failure. Cultivated potato (Solanum tuberosum L.) is sensitive to freezing, while its wild relative, S. commersonii, has a strong freezing tolerance. To decipher the anti-freezing mechanism of CM, we carried out [...] Read more.
Cold stress impairs plant growth and development, resulting in crop failure. Cultivated potato (Solanum tuberosum L.) is sensitive to freezing, while its wild relative, S. commersonii, has a strong freezing tolerance. To decipher the anti-freezing mechanism of CM, we carried out a transcriptomic and metabolomic analysis of an anti-freezing variety of CM (a type of S. commersonii) and a freeze-sensitive variety of DM (a type of Solanum tuberosum L.). A total of 49,232 high-quality transcripts from 12,811 gene loci, including 46,772 coding sequences and 2018 non-coding RNAs, were identified. KEEG enrichment analysis of differentially expressed genes (DEGs) between the two varieties showed that the flavonoid biosynthesis pathway was strongly induced by freezing stress, which was proven by flavonoid metabolome analysis. Consistent with the accumulation of more flavonoids, nearly all the pathway genes were significantly upregulated in CM than those in DM. The transcript levels of two chalcone synthase (CHS-1) isoforms and four isoforms of flavonoid 3′-hydroxylase (F3′H-1) were confirmed by qRT-PCR. Co-expression analysis identified one Myb-related and three UGTs (UDP-glycosyltransferase) that were significantly upregulated in CM during freezing stress. Our findings support that the flavonoid pathway was significantly enhanced by freezing stress and the greater accumulation ofglycosylatedflavonoids in resistant types than that of sensitive types, maybe accounting for the increased freezing tolerance of freeze-resistant potato varieties. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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7 pages, 787 KiB  
Communication
A Method for Electroporation of Cre Recombinase Protein into Intact Nicotiana tabacum Cells
by Yuichi Furuhata, Emiko Egi, Tomi Murakami and Yoshio Kato
Plants 2023, 12(8), 1631; https://doi.org/10.3390/plants12081631 - 12 Apr 2023
Viewed by 1463
Abstract
The Cre/lox recombination system has become a powerful technology for gene function analysis in a broad spectrum of cell types and organisms. In our previous report, Cre protein had been successfully delivered into intact Arabidopsis thaliana cells using electroporation. To expand the [...] Read more.
The Cre/lox recombination system has become a powerful technology for gene function analysis in a broad spectrum of cell types and organisms. In our previous report, Cre protein had been successfully delivered into intact Arabidopsis thaliana cells using electroporation. To expand the feasibility of the method of protein electroporation to other plant cells, here we attempt the protein electroporation into tobacco-derived BY-2 cells, one of the most frequently used plant cell lines for industrial production. In this study, we successfully deliver Cre protein into BY-2 cells with intact cell walls by electroporation with low toxicity. Targeted loxP sequences in the BY-2 genome are recombined significantly. These results provide useful information for genome engineering in diverse plant cells possessing various types of cell walls. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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10 pages, 1849 KiB  
Article
A Set of Molecular Markers to Accelerate Breeding and Determine Seed Purity of CMS Three-Line Hybrids in Brassica napus
by Yanfeng Zhang, Ran An, Min Song, Changgen Xie, Shihao Wei, Daojie Wang, Yuhong Dong, Qingli Jia, Shuhua Huang and Jianxin Mu
Plants 2023, 12(7), 1514; https://doi.org/10.3390/plants12071514 - 30 Mar 2023
Cited by 2 | Viewed by 1612
Abstract
Cytoplasmic male sterility (CMS) is the main mechanism employed to utilize the heterosis of Brassica napus. CMS three-line rapeseed hybrids have dramatically enhanced yield and brought about the global revolution of hybrid varieties, replacing conventional crop varieties. Over the last half century, [...] Read more.
Cytoplasmic male sterility (CMS) is the main mechanism employed to utilize the heterosis of Brassica napus. CMS three-line rapeseed hybrids have dramatically enhanced yield and brought about the global revolution of hybrid varieties, replacing conventional crop varieties. Over the last half century, China has led the development of hybrid Brassica napus varieties. Two sterile lines, polima (pol) and shaan 2A, were of particular importance for the establishment of three-line hybrid systems in rapeseed, which has opened up a new era of heterosis utilization. However, in current breeding practices, it takes up to three years to identify the restorer or maintainer relationship and the cytoplasmic type of any inbred material. This greatly affects the breeding speed of new varieties and inhibits the rapid development of the rapeseed industry. To address this problem, we developed a set of molecular markers for the identification of fertile cytoplasmic gene N and sterile cytoplasmic gene S, as well as for the fertile nucleus gene R and sterile nucleus gene r, based on differences in the gene sequences between the CMS line, maintainer line and restorer line of Brassica napus. Combining these markers can accurately identify the CMS line, maintainer and restorer of both the pol and shaan systems, as well as their hybrids. These markers can not only be used to identify of the maintainer and restorer relationship of inbred materials; they can also be used as general molecular markers to identify the CMS-type hybrid purity of pol and shaan systems. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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14 pages, 5636 KiB  
Article
Characterization of the NAC Transcription Factor in Passion Fruit (Passiflora edulis) and Functional Identification of PeNAC-19 in Cold Stress
by Yi Xu, Pengfei Li, Funing Ma, Dongmei Huang, Wenting Xing, Bin Wu, Peiguang Sun, Binqiang Xu and Shun Song
Plants 2023, 12(6), 1393; https://doi.org/10.3390/plants12061393 - 21 Mar 2023
Cited by 2 | Viewed by 1486
Abstract
The NAC (NAM, ATAF and CUC) gene family plays an important role in plant development and abiotic stress response. However, up to now, the identification and research of the NAC (PeNAC) family members of passion fruit are still lacking. In this [...] Read more.
The NAC (NAM, ATAF and CUC) gene family plays an important role in plant development and abiotic stress response. However, up to now, the identification and research of the NAC (PeNAC) family members of passion fruit are still lacking. In this study, 25 PeNACs were identified from the passion fruit genome, and their functions under abiotic stress and at different fruit-ripening stages were analyzed. Furthermore, we analyzed the transcriptome sequencing results of PeNACs under four various abiotic stresses (drought, salt, cold and high temperature) and three different fruit-ripening stages, and verified the expression results of some genes by qRT-PCR. Additionally, tissue-specific analysis showed that most PeNACs were mainly expressed in flowers. In particular, PeNAC-19 was induced by four various abiotic stresses. At present, low temperatures have seriously endangered the development of passion fruit cultivation. Therefore, PeNAC-19 was transformed into tobacco, yeast and Arabidopsis to study their function of resisting low temperature. The results show that PeNAC-19 responded to cold stress significantly in tobacco and Arabidopsis, and could improve the low temperature tolerance of yeast. This study not only improved the understanding of the PeNAC gene family characteristics and evolution, but also provided new insights into the regulation of the PeNAC gene at different stages of fruit maturation and abiotic stresses. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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13 pages, 1171 KiB  
Article
In Vitro Mass Propagation of Coffee Plants (Coffea arabica L. var. Colombia) through Indirect Somatic Embryogenesis
by Consuelo Margarita Avila-Victor, Víctor Manuel Ordaz-Chaparro, Enrique de Jesús Arjona-Suárez, Leobardo Iracheta-Donjuan, Fernando Carlos Gómez-Merino and Alejandrina Robledo-Paz
Plants 2023, 12(6), 1237; https://doi.org/10.3390/plants12061237 - 08 Mar 2023
Cited by 4 | Viewed by 4527
Abstract
Coffea arabica is one of the two most consumed coffee species in the world. Micropropagation through somatic embryogenesis has allowed the large-scale propagation of different coffee varieties. However, the regeneration of plants using this technique depends on the genotype. This study aimed to [...] Read more.
Coffea arabica is one of the two most consumed coffee species in the world. Micropropagation through somatic embryogenesis has allowed the large-scale propagation of different coffee varieties. However, the regeneration of plants using this technique depends on the genotype. This study aimed to develop a protocol for the regeneration of C. arabica L. var. Colombia by somatic embryogenesis for its mass propagation. Foliar explants were cultured on Murashige and Skoog (MS) supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and phytagel for inducing somatic embryogenesis. In total, 90% of the explants formed embryogenic calli with a culture medium containing 2 mg L−1 of 2,4-D, 0.2 mg L−1 BAP, and 2.3 g L−1 phytagel. The highest number of embryos per gram of callus (118.74) was obtained in a culture medium containing 0.5 mg L−1 2,4-D, 1.1 mg L−1 BAP, and 5.0 g L−1 phytagel. In total, 51% of the globular embryos reached the cotyledonary stage when they were cultured on the growth medium. This medium contained 0.25 mg L−1 BAP, 0.25 mg L−1 indoleacetic acid (IAA), and 5.0 g L−1 of phytagel. The mixture of vermiculite:perlite (3:1) allowed 21% of embryos to become plants. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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15 pages, 2512 KiB  
Article
An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus
by Daniela Cordeiro, Ana Alves, Ricardo Ferraz, Bruno Casimiro, Jorge Canhoto and Sandra Correia
Plants 2023, 12(5), 1202; https://doi.org/10.3390/plants12051202 - 06 Mar 2023
Cited by 2 | Viewed by 3272
Abstract
Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic [...] Read more.
Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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19 pages, 6845 KiB  
Article
Genome-Wide Analysis of MYB Transcription Factor Gene Superfamily Reveals BjPHL2a Involved in Modulating the Expression of BjCHI1 in Brassica juncea
by Chang Gen Xie, Ping Jin, Jiamin Xu, Shangze Li, Tiantian Shi, Rui Wang, Shuangwei Jia, Zixuan Zhang, Weike Guo, Wenfang Hao, Xiaona Zhou, Jun Liu and Ying Gao
Plants 2023, 12(5), 1011; https://doi.org/10.3390/plants12051011 - 23 Feb 2023
Viewed by 1318
Abstract
Brassica juncea is an economically important vegetable and oilseed crop. The MYB transcription factor superfamily is one of the largest transcription factor families in plants, and plays crucial roles in regulating the expression of key genes involved in a variety of physiological processes. [...] Read more.
Brassica juncea is an economically important vegetable and oilseed crop. The MYB transcription factor superfamily is one of the largest transcription factor families in plants, and plays crucial roles in regulating the expression of key genes involved in a variety of physiological processes. However, a systematic analysis of the MYB transcription factor genes in Brassica juncea (BjMYB) has not been performed. In this study, a total of 502 BjMYB superfamily transcription factor genes were identified, including 23 1R-MYBs, 388 R2R3-MYBs, 16 3R-MYBs, 4 4R-MYBs, 7 atypical MYBs, and 64 MYB-CCs, which is approximately 2.4-fold larger than that of AtMYBs. Phylogenetic relationship analysis revealed that the MYB-CC subfamily consists of 64 BjMYB-CC genes. The expression pattern of members of PHL2 subclade homologous genes in Brassica juncea (BjPHL2) after Botrytis cinerea infection were determined, and BjPHL2a was isolated from a yeast one-hybrid screen with the promoter of BjCHI1 as bait. BjPHL2a was found to localize mainly in the nucleus of plant cells. An EMSA assay confirmed that BjPHL2a binds to the Wbl-4 element of BjCHI1. Transiently expressed BjPHL2a activates expression of the GUS reporter system driven by a BjCHI1 mini-promoter in tobacco (Nicotiana benthamiana) leaves. Taken together, our data provide a comprehensive evaluation of BjMYBs and show that BjPHL2a, one of the members of BjMYB-CCs, functions as a transcription activator by interacting with the Wbl-4 element in the promoter of BjCHI1 for targeted gene-inducible expression. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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19 pages, 3166 KiB  
Article
Identification and Phylogenetic Analysis of the R2R3-MYB Subfamily in Brassica napus
by Dingfan Luo, Desheng Mei, Wenliang Wei and Jia Liu
Plants 2023, 12(4), 886; https://doi.org/10.3390/plants12040886 - 16 Feb 2023
Cited by 2 | Viewed by 1467
Abstract
The R2R3-MYB sub-family proteins are composed of most members of MYB (v-Myb avian myeloblastosis viral oncogene homolog) protein, a plant-specific transcription factor (TF) that is classified into four classes depending on the number of MYB repeats. R2R3-MYB TFs are involved in physiological and [...] Read more.
The R2R3-MYB sub-family proteins are composed of most members of MYB (v-Myb avian myeloblastosis viral oncogene homolog) protein, a plant-specific transcription factor (TF) that is classified into four classes depending on the number of MYB repeats. R2R3-MYB TFs are involved in physiological and biochemical processes. However, the functions of the Brassica napus R2R3-MYB genes are still mainly unknown. In this study, 35 Brassica napus MYB (BnaMYB) genes were screened in the genome of Brassica napus, and details about their physical and chemical characteristics, evolutionary relationships, chromosome locations, gene structures, three-dimensional protein structures, cis-acting promoter elements, and gene duplications were uncovered. The BnaMYB genes have undergone segmental duplications and positive selection pressure, according to evolutionary studies. The same subfamilies have similar intron–exon patterns and motifs, according to the genes’ structure and conserved motifs. Additionally, through cis-element analysis, many drought-responsive and other stress-responsive cis-elements have been found in the promoter regions of the BnaMYB genes. The expression of the BnaMYB gene displays a variety of tissue-specific patterns. Ten lignin-related genes were chosen for drought treatment. Our research screened four genes that showed significant upregulation under drought stress, and thus may be important drought-responsive genes. The findings lay a new foundation for understanding the complex mechanisms of BnaMYB in multiple developmental stages and pathways related to drought stress in rapeseed. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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19 pages, 7226 KiB  
Article
Genome-Wide Identification of the Cytochrome P450 Superfamily Genes and Targeted Editing of BnCYP704B1 Confers Male Sterility in Rapeseed
by Zhilai Wang, Yanfeng Zhang, Min Song, Xiuhua Tang, Shuhua Huang, Bin Linhu, Ping Jin, Weike Guo, Fang Li, Liwen Xing, Ran An, Xiaona Zhou, Wenfang Hao, Jianxin Mu and Changgen Xie
Plants 2023, 12(2), 365; https://doi.org/10.3390/plants12020365 - 12 Jan 2023
Cited by 1 | Viewed by 1822
Abstract
The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance [...] Read more.
The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar “Zhongshuang 11” (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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Review

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14 pages, 1294 KiB  
Review
Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops
by Nikolaos Tsakirpaloglou, Endang M. Septiningsih and Michael J. Thomson
Plants 2023, 12(20), 3564; https://doi.org/10.3390/plants12203564 - 13 Oct 2023
Viewed by 2192
Abstract
With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of [...] Read more.
With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of gene expression. Likewise, new trait improvement approaches can take advantage of targeted editing to improve stress tolerance, disease resistance, and nutritional traits. However, several key steps in the process can prove tricky for researchers who might be new to plant genome editing. Here, we present step-by-step guidelines and best practices for a crop genome editing pipeline that should help to improve the rate of success. Important factors in the process include proper target sequence analysis and single guide RNA (sgRNA) design, sequencing of the target site in the genotypes of interest, performing an in vitro CRISPR/Cas9 ribonucleoprotein (RNP) assay to validate the designed sgRNAs, preparing the transformation constructs, considering a protoplast editing step as further validation, and, finally, stable plant transformation and mutation detection by Sanger and/or next-generation sequencing. With these detailed guidelines, a new user should be able to quickly set up a genome editing pipeline in their crop of interest and start making progress with the different CRISPR/Cas-based editing variants for gene validation and trait improvement purposes. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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12 pages, 802 KiB  
Review
Recent Advancements and Biotechnological Implications of Carotenoid Metabolism of Brassica
by Lichun Shi, Lin Chang, Yangjun Yu, Deshuang Zhang, Xiuyun Zhao, Weihong Wang, Peirong Li, Xiaoyun Xin, Fenglan Zhang, Shuancang Yu, Tongbing Su, Yang Dong and Fumei Shi
Plants 2023, 12(5), 1117; https://doi.org/10.3390/plants12051117 - 02 Mar 2023
Cited by 1 | Viewed by 1692
Abstract
Carotenoids were synthesized in the plant cells involved in photosynthesis and photo-protection. In humans, carotenoids are essential as dietary antioxidants and vitamin A precursors. Brassica crops are the major sources of nutritionally important dietary carotenoids. Recent studies have unraveled the major genetic components [...] Read more.
Carotenoids were synthesized in the plant cells involved in photosynthesis and photo-protection. In humans, carotenoids are essential as dietary antioxidants and vitamin A precursors. Brassica crops are the major sources of nutritionally important dietary carotenoids. Recent studies have unraveled the major genetic components in the carotenoid metabolic pathway in Brassica, including the identification of key factors that directly participate or regulate carotenoid biosynthesis. However, recent genetic advances and the complexity of the mechanism and regulation of Brassica carotenoid accumulation have not been reviewed. Herein, we reviewed the recent progress regarding Brassica carotenoids from the perspective of forward genetics, discussed biotechnological implications and provided new perspectives on how to transfer the knowledge of carotenoid research in Brassica to the crop breeding process. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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Other

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12 pages, 5154 KiB  
Technical Note
Construction of an Efficient Genetic Transformation System for Watercress (Nasturtium officinale W. T. Aiton)
by Jiajun Ran, Qiang Ding, Yunlou Shen, Zhanyuan Gao, Guangpeng Wang, Yue Gao, Xiaoqing Ma and Xilin Hou
Plants 2023, 12(24), 4149; https://doi.org/10.3390/plants12244149 - 13 Dec 2023
Viewed by 561
Abstract
Based on the established efficient regeneration system for watercress in our laboratory, we optimized the processes of pretreatment, co-culture, and differentiation culture. Through GFP fluorescence and PCR identification, we successfully obtained transgenic watercress with the DR5 gene, which allowed us to investigate the [...] Read more.
Based on the established efficient regeneration system for watercress in our laboratory, we optimized the processes of pretreatment, co-culture, and differentiation culture. Through GFP fluorescence and PCR identification, we successfully obtained transgenic watercress with the DR5 gene, which allowed us to investigate the distribution details of auxin in the growth process of watercress. Our findings provide an effective method for gene function research and lay the foundation for innovative utilization of germplasm resources of watercress. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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