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Construction and application of a virus-induced gene silencing system in taro

  • # Authors contributed equally: Yanling Gui, Bicong Li

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  • Received: 25 March 2024
    Revised: 01 June 2024
    Accepted: 07 June 2024
    Published online: 11 September 2024
    Tropical Plants  3 Article number: e030 (2024)  |  Cite this article
  • CePDS was used as an indicator gene to construct a VIGS system in taro.

    The silencing plant rate of VIGS system increased significantly by optimized bacterium concentration.

    CeTCP14 was selected to further verify the robustness of the constructed system.

  • Virus-induced gene silencing (VIGS) technique is an important means for rapid identification of plant gene function, and a stable VIGS system in taro can provide technical support for rapid and efficient gene function verification. A VIGS system was constructed with a tobacco rattle virus (TRV)-based vector, with phytoene desaturase (CePDS) as the indicator gene, then CeTCP14 was silenced to further verify the robustness of this system. Ganyu No.1 was used as the material to construct the taro VIGS system using the leaf injection method with CePDS at OD600 = 0.6, and the silencing plant rate was 12.23%, the expression level of CePDS was about 59.34%−77.18% compared to the control, and the chlorophyll content decreased 37.80%−56.11% in CePDS silencing plants. The silencing plant rate increased significantly by leaf injection at OD600 = 1.0, and reached 27.77%, but there was no significant difference in silencing plant rate between the leaf injection method and bulb vacuum treatment. CeTCP14 was further silenced with bacteria solution OD600 = 1.0 and bulb vacuum treatment in Ganyu No.2, the silencing plant rate was 20%, and the expression level of CeTCP14 was 43.94%−63.34% of the control. Meanwhile, the starch content in the corms decreased significantly to 70.88%−80.61% compared to the control. In conclusion, the results indicated that a TRV-based VIGS system is effective in taro. The concentration of bacterial solution is a key factor affecting the VIGS system, CeTCP14 can affect starch accumulation in taro bulbs. A robust VIGS system establishment in taro could lay a good foundation for the subsequent rapid gene function verification.
    Graphical Abstract
  • Barnyard millet (Echinochloa esculenta) is garnering attention for its potential to contribute significantly for food and nutritional security, particularly in Asia[1]. It ranks as the fourth most produced minor millet and India leads globally in barnyard millet production, covering the largest area (0.146 Mha) and yielding the highest output (0.147 Mha) with an average productivity of 1,034 kg·ha−1 over the past three years[2]. E. esculenta, known as Japanese barnyard millet, is valued for its fast growth, adaptability to various soil and water conditions, and nutritional grain profile, making it an excellent choice for forage and food[3]. Moreover, E. frumentacea, or Indian barnyard millet, shares similar traits but is often preferred in traditional farming systems in South Asia due to its hardiness and ease of cultivation[4]. In crop improvement, E. esculenta's rapid growth and resilience are leveraged to enhance stress tolerance and yield in other cereal crops[5,6]. Barnyard millet emerges as a beacon of hope for regions grappling with unpredictable weather patterns, and its secret lies in its remarkably short life cycle, maturing in just 60−80 days[7]. This swiftness allows farmers to double-crop, squeezing in two harvests within a single season[8].

    Even more crucially, the short cycle enables them to avoid droughts by planting just before the rains and harvesting before potential dry spells[9]. Additionally, the nutritional benefits of barnyard millet, including being rich in protein, carbohydrate and dietary fiber, low glycemic index, gluten-free, abundance of micronutrients like iron and zinc, good source of vitamin B, and rich in antioxidants, position it as a valuable crop for addressing nutritional deficiencies, particularly in hilly and tribal communities[10].

    Moreover, Fig. 1 categorizes different biological data types and resources, along with the number of records available for each in NCBI (National Center for Biotechnology Information) database as of 20th July 2024 for Japanese barnyard millet (E. esculenta) (Taxonomy ID: 121770)[11]. For literature, sources include Bookshelf, PubMed, and PubMed Central, totaling 168 records[11]. Genes are covered by the Gene and PopSet databases with 142 records, while Proteins are split into Identical Protein Groups and Protein databases with 91 and 203 records respectively[11]. Genomes are sourced from BioProject and Taxonomy with five records, BioSample with 165 records, Nucleotide with 69 records and the Sequence Read Archive with 164 records. Lastly, the PubChem database includes 21 BioAssay records[11].

    Figure 1.  Distribution and abundance of different types of biological data of Japanese barnyard millet (E. esculenta) across various databases as of 20th July 2024 [11].

    Moreover, the genetic improvement of barnyard millet holds significant potential but requires concerted research efforts to overcome existing challenges[12]. However, genome research in barnyard millet is still in its early stages and lags significantly behind other minor millets, and this is primarily due to the complex nature of its genome (2n = 6x = 54, hexaploid)[6]. Enhanced genetic characterization, effective utilization of germplasm resources, and advanced breeding techniques, including interspecific hybridization, are essential for unlocking the full potential of this underutilized crop[5]. The limited genetic diversity observed within cultivated varieties, compared to their wild relatives, further complicates breeding efforts aimed at enhancing desirable traits like yield, disease resistance, and abiotic stress tolerance[13].

    On the positive side, recent advancements in genomics have opened new avenues for the genetic enhancement of barnyard millet[5]. The release of genome and transcriptome sequences for both wild and cultivated Echinochloa sp. has facilitated a better understanding of the genetic basis of important agronomic traits[9]. The identification of quantitative trait loci (QTL) and specific genes associated with these traits is crucial for the development of improved varieties[9]. Furthermore, interspecific hybridization between E. esculenta and E. frumentacea has shown promise, despite challenges related to seed sterility in hybrid plants[14]. This hybridization can potentially introduce desirable traits from one species to the other, thereby broadening the genetic base and enhancing the crop's overall performance[15].

    Despite nutritional advantages, barnyard millet remains underutilized[9]. The genetic improvement of barnyard millet faces several challenges and presents numerous prospects. One of the main challenges is the limited genetic and genomic resources available for this crop[7]. Although recent efforts have led to the development of core collections and the identification of single-nucleotide polymorphisms (SNPs) within these collections, there is still a significant gap in understanding the full genetic architecture of barnyard millet[16]. Moreover, integrating advanced molecular and biotechnological tools with conventional breeding approaches can significantly enhance the agronomic traits of E. esculenta[17].

    Techniques such as genome editing with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) - Cas9 (CRISPR-associated protein 9) can precisely target and modify genes associated with important agronomic traits in barnyard millet[18]. Marker-assisted selection (MAS) allows for the efficient identification and incorporation of desirable traits by using molecular markers linked to key agronomic characteristics in barnyard millet[19]. Combining these molecular tools with traditional breeding ensures the development of superior E. esculenta varieties with enhanced performance and resilience[2022].

    Therefore, the main focus of this review is to explore how these advanced tools can be effectively integrated with conventional methods to improve the agronomic traits of E. esculenta, emphasising the potential for developing high-yield, stress-resistant barnyard millet varieties to enhance food security and agricultural sustainability.

    Barnyard millet, despite its name, is a grain belonging to the grass family Poaceae[22]. While its exact origin remains unclear, it's believed to have been domesticated in India or Southeast Asia. Classified within the genus Echinochloa, it shares some relatives with other millets but possesses a unique genetic makeup[23]. Moreover, barnyard millet is a significant crop primarily cultivated in semi-arid tropical regions of Asia and Africa appreciated for its drought tolerance, rapid maturation, and nutritional benefits[24]. The genus Echinochloa comprises multiple species, with the two major cultivated types being E. frumentacea (Indian barnyard millet) and E. esculenta (Japanese barnyard millet)[25]. The origins, classification, and genomic relationships of barnyard millet can be understood through a synthesis of genetic and genomic research[24].

    Barnyard millet has a complex origin involving multiple species and subspecies[13,22]. The genomic relationship of barnyard millet species has been explored using various molecular markers[16]. A study utilizing whole-genome genotyping-by-sequencing on a core collection of 89 barnyard millet accessions identified several thousand SNPs segregating within the collection[16]. This study revealed four population clusters within E. colona and three within E. crus-galli, suggesting that the genetic diversity within these species is substantial and likely influenced by geographic factors[16]. Additionally, the use of RAPD (Random Amplified Polymorphic DNA) primers confirmed the genetic diversity among 21 accessions of the Echinochloa spp. complex, classifying them into two morphological races, E. frumentacea and E. colona, with a high degree of molecular diversity[26].

    Moreover, interspecific hybridization between E. esculenta and E. frumentacea offers potential for genetic enhancement[26]. A successful hybrid between E. esculenta cultivar PRJ 1 and E. frumentacea cultivar ER 72 is also reported, confirmed through rice SSR markers[15]. Although the hybrid plants exhibited vigor and disease resistance, they were sterile, highlighting challenges in utilizing interspecific hybrids for breeding programs[15]. Therefore, the genetic characterization of barnyard millet is crucial for breeding and improvement programs. For instance, association analysis in germplasm and F2 populations have shown positive direct effects of traits like stem girth, ear head weight, and plant height on grain yield[27]. Moreover, understanding its genomic relationships with other Echinochloa species is crucial for breeding programs to develop improved barnyard millet varieties with enhanced yields and desirable traits[23].

    The phylogeny of barnyard millet has been the subject of multiple studies focusing on genetic diversity, population structure, and evolutionary relationships among different species and accessions[16]. However, these studies identify thousands of SNPs and revealed distinct population clusters, and interestingly, these genetic clusters correlated more with geographic origin rather than morphological classification, highlighting the influence of geographic isolation on genetic diversity[28]. Another significant contribution to understand the phylogeny of Echinochloa species comes from the analysis of chloroplast DNA (cpDNA) sequences from non-coding regions (trnT-L-F)[29], and the findings suggest that domestication and adaptation to different environments occurred after the species diverged in Asia[28].

    Further phylogenetic insights are provided by the complete chloroplast genome sequencing of Indian barnyard millet (E. frumentacea), and this study revealed that E. frumentacea diverged from its close relatives E. oryzicola and E. crus-galli approximately 1.9–2.7 million years ago[30]. However, morpho-agronomic trait analysis has contributed to our understanding of barnyard millet phylogeny[31]. Lastly, the assessment of genetic diversity using molecular markers further elucidates the phylogenetic relationships within and between E. crus-galli and E. frumentacea[32]. However, the phylogeny of barnyard millet is complex and influenced by geographic, genetic, and morphological factors[33]. Moreover, the phylogeny of barnyard millet is characterized by significant genetic diversity and complex evolutionary relationships, and studies utilizing whole-genome sequencing, interspecific hybridization, and molecular markers have provided a deeper understanding of these relationships, offering valuable insights for crop improvement[34].

    Germplasm resources in barnyard millet hold significant potential for crop improvement, particularly in terms of enhancing traits such as drought tolerance, nutritional quality, salinity tolerance, and disease resistance[35]. Several studies have characterized the genetic variability and diversity within barnyard millet germplasm, providing valuable insights for breeders and researchers[35]. This genetic diversity is crucial for the development of improved cultivars with desirable traits[26]. The formation of a core set in barnyard millet germplasm using data on 24 morpho-agronomic traits has been instrumental in managing and utilizing the vast genetic resources available[16]. This core set captures most of the available diversity and serves as a representative sample for detailed evaluation and breeding programs[31].

    Similarly, another study characterized 494 barnyard millet germplasm for quantitative traits, revealing significant variation for most traits and highlighting traits like days to 50% flowering, plant height, and days to maturity as key contributors to total variance[36]. Moreover, barnyard millet germplasm has been screened for resistance to grain smut with accessions showing varying levels of susceptibility, and the study suggests that smut resistance and grain yield can be improved simultaneously through proper breeding strategies[37]. Additionally, the crop's tolerance to salinity has been investigated, with certain genotypes demonstrating the ability to withstand up to 200 mM salt concentration during germination, and it is found that, this tolerance is associated with traits such as higher relative water content and enzyme activity under salt stress[38]. The genetic variability and association studies under sodic soil conditions have identified traits like days to 50% flowering, days to maturity, ear width, and thousand-grain weight as important selection indices for enhancing grain yield[39].

    The characterization of genetic diversity and the formation of core collections facilitate the efficient utilization of barnyard millet germplasm in breeding programs aimed at developing high-yielding, stress-tolerant, and disease-resistant cultivars[40]. While the exact number of barnyard millet germplasm accessions globally remains around 8,000 conserved in various gene banks, such as Vivekananda Parvatiya Krishi Anusandhan Sansthan (VPKAS), India, Indian Institute of Millets Research (IIMR), India, National Institute of Agrobiological Sciences (NIAS), Japan, and Consultative Group on International Agricultural Research (CGIAR), including the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India, recent efforts focus on optimizing their use and maintenance[9]. The extensive genetic resources available in barnyard millet germplasm provide a solid foundation for crop improvement[4,41].

    Genome sequencing in barnyard millet has made significant strides in recent years, providing valuable insights into the genetic makeup and potential for crop improvement in this under-researched species[15,16]. A groundbreaking study on the genetic characterization of a global barnyard millet germplasm collection utilized whole-genome genotyping-by-sequencing to identify several thousand SNPs in 89 accessions, providing a robust framework for breeders to enhance barnyard millet for smallholder farmers[16]. However, researchers in China successfully released and annotated the whole genome sequence of weedy E. crus-galli, and the genome, sequenced at a depth of 171x, was estimated to be 1.27 Gb, covering approximately 90.7% of the predicted size[9]. Moreover, the study identified 4,534 contigs ranging from 1 kb to 11.7 Mb, with gene annotations revealing 108,771 protein-coding genes, 785 miRNAs, 514 Mb of repetitive elements, and various non-coding RNAs[9]. NCBI resources for Echinochloa species included 1,246 nucleotide sequences, 822 gene sequences, 2,468 protein sequences, and other genomic data, with E. crus-galli leading in sequence availability[9]. Despite this, cultivated barnyard millet species E. frumentacea and E. esculenta had significantly fewer sequences, representing only 4% of the total[9].

    Further insights were gained from the complete chloroplast genome sequence of Indian barnyard millet, E. frumentacea[30]. The chloroplast genome, which is 139,593 bp in length with a typical quadripartite structure, includes 112 individual genes, and these genes consist of 77 protein-coding genes, 30 tRNA genes, four rRNA genes, and one conserved open reading frame, with an overall GC content of 38.6%[30]. Phylogenetic analysis indicated that E. frumentacea diverged from its close relatives, E. oryzicola and E. crus-galli, approximately 1.9–2.7 million years ago, highlighting the evolutionary history and genetic uniqueness of this species[30]. Moreover, genomics and genetic studies in barnyard millet are rapidly advancing, providing a wealth of data and opportunities for crop improvement[14]. However, in a recent study, the transcriptome profiling of barnyard millet was performed during grain development to reveal the genomic insights into iron accumulation, which has an insight towards genome sequencing[5,8].

    Molecular markers, specifically nucleotide sequences, play a crucial role in genetic diversity studies, linkage map construction, and marker-assisted selection in crop plants[9]. Early research utilized RAPD markers to study genetic diversity and phylogeny among Echinochloa species, effectively distinguishing between cultivated and wild progenitors[42,43]. Although RAPD markers provided insights into genetic diversity, their low polymorphism levels were noted in various studies[44]. Moreover, Amplified Fragment Length Polymorphism (AFLP) markers later showed greater efficacy in revealing genetic diversity due to their higher allele production per primer[5]. Advancements in sequencing technologies introduced sequence-based markers like SRAP (Sequence-Related Amplified Polymorphism); SSRs (Simple Sequence Repeats); EST-SSRs: (Expressed Sequence Tag-Simple Sequence Repeats); and SNPs, which are more desirable due to co-dominant, reproducible, and highly polymorphic nature[18]. Studies utilizing SSR markers in Echinochloa species have shown the formation of distinct clusters, highlighting the genetic diversity[4548].

    EST markers have proven effective in analyzing genetic diversity, with in silico mining and validation of EST-SSR primers identifying frequent repeat motifs in barnyard millet[4951]. Additionally, the RAD (Restriction-site associated) approach combined with Illumina DNA sequencing in E. phyllopogon facilitated the rapid discovery of SSR and SNP markers, demonstrating the usefulness of these markers in studying genetic diversity and aiding molecular breeding[52]. Further developments in whole-genome genotyping-by-sequencing (GBS) have identified a significant number of SNPs across various biotypes of Echinochloa species[53]. Moreover, population structure analysis using these SNPs has provided clear genetic differentiation among species, forming distinct clusters[35]. This detailed SNP data enhances our understanding of genetic diversity, origins, and distribution, particularly in herbicide-resistant populations, and supports molecular breeding efforts[54]. Recent studies have continued to use EST-SSR markers to analyze genetic diversity in Indian barnyard millet germplasm, reinforcing the importance of advanced molecular markers in crop genetic research and breeding programs[55]. However, Table 1 summarizes the availability of molecular resources in barnyard millet.

    Table 1.  Availability of molecular markers in barnyard millet.
    Experiment Type of marker Results Ref.
    Proximate and molecular study 20 Echinochloa frumentacea L. 10 RAPD primers generated 50 bands All 50 bands were polymorphic (100%) having 45 shared and five unique bands; fragment size ranged 212 bp [42]
    Proximate and molecular study 20 Echinochloa frumentacea L. 10 ISSR primers generated 42 bands 40 bands were polymorphic (90%) with 39 shared one unique bands; fragment size ranged 250 bp [42]
    Genetic diversity analysis of millet crop Four RAPD primers OPC06, OPC18, OPD13, and OPW04 Two primers OPC06 and OPD13 showed the highest polymorphism (83%) [44]
    Assessment of genetic diversity in Echinochloa
    crus-galli (L.)
    Eight specific SSR markers 48 alleles were identified; genetic variation among populations (37.01%) [45]
    Assessment of molecular diversity and proximate composition of barnyard millet, pearl millet and sorghum (five varieties of each) 10 SRAP primer 65 polymorphic bands having 56 shared and nine unique bands with an average of 6.5 bands per primer and 98.75% polymorphism per primer [47]
    Comparative analysis of whole chloroplast genomes of Echinochloa crus-galli var. crus-galli, E. crus-galli var. zelayensis, and E. glabrescens 139 SSRs Phylogenetic tree between 10 barnyard grass species and other common Gramineae plants, showing new genetic relationships of the genus Echinochloa [46]
    Validation of ssr markers for barnyard millet obtained by partial genome assembly 46,157 SSRs identified from 11,39,481 contigs 15 SSR markers validated among the 30 barnyard millet accessions [48]
    Genetic diversity in the barnyard millet 51 EST-SSR markers used 14 primers found polymorphic with 29 alleles, and average PIC of 0.43 [49]
    Transcriptomic profiling of Echinochloa frumentacea and barnyard grass
    30 EST-SSR primer pairs 10 EST-SSR primers were found polymorphic; revealing putative genes involved in drought adaptation and micronutrient accumulation by [50]
    Inheritance and identification of EST-SSR marker(s) associated with the anthocyanin pigments in barnyard millet 51 EST-SSR markers used Marker BMESSR 39 found associated with the anthocyanin pigment [51]
    Barnyard millet: EST, SNP markers identified 41 EST sequences 22 microsatellite markers and SNPs identified; one potential SNP and one reliable SNP and two haplotypes obtained [35]
    Molecular diversity analysis of 48 Echinochloa frumentacea genotypes 182 RAPD marker loci 170 RAPD marker loci (93.40%) found to be polymorphic [55]
     | Show Table
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    The genetic improvement of barnyard millet is essential for enhancing its productivity, resilience, and nutritional value, making it a key player in sustainable agriculture[4]. This process involves utilizing advanced breeding techniques such as genomic selection, and biotechnological tools to develop superior cultivars with improved traits including higher yield, enhanced resistance to biotic and abiotic stresses, and better nutritional profiles[56]. Despite its potential, the genetic improvement of barnyard millet faces challenges like limited genetic diversity, insufficient genomic resources, and inadequate funding for research[5,8]. Addressing these issues through comprehensive research initiatives and international collaboration can pave the way for the development of robust barnyard millet varieties[15]. However, Fig. 2 illustrates the process of improving barnyard millet cultivars by integrating genetic and omics resources. Genetic resources such as core germplasm, recombinant inbred lines and mutants are utilized alongside genome sequencing to facilitate the identification of molecular markers and genetic improvement techniques like genomic selection and genome editing. Omics resources, including genomics, transcriptomics, and proteomics play a crucial role in phenotyping, genotyping, and ultimately developing enhanced barnyard millet varieties.

    Figure 2.  Process of improving barnyard millet cultivars by integrating genetic and omics resources.

    The identification of QTLs and the application of MAS in barnyard millet have been facilitated by the development of molecular markers such as SSRs[45,46] and SNPs[52]. Although many SSR and SNP markers have been created to aid in linkage map construction and QTL mapping, progress has been slower compared to other millets like foxtail and finger millet[55]. Notable studies that identified SNP markers for waxy traits controlled by three loci (EeWx1, EeWx2, EeWx3)[57], and linked the SSR marker BMESSR 39 with anthocyanin pigments using bulk segregant analysis using F2 individuals of ACM 331 × MA 10[58], provide opportunities to facilitate the construction of linkage maps and QTL mapping in barnyard millet followed by MAS[5]. Despite these advancements, barnyard millet genome mapping remains in its initial stages, requiring further investigation to apply these findings to MAS for improved breeding[8].

    Barnyard millet's breeding is poised for a breakthrough with the combined power of genomic selection and speed breeding[45]. Genomic selection, unlike traditional methods, analyzes a plant's entire genetic code to predict valuable traits like yield and disease resistance[46]. This allows breeders to identify promising candidates[55]. Speed breeding, on the other hand accelerates the breeding cycle by manipulating the growing conditions to produce multiple generations per year[5,8]. By identifying superior genetics early and rapidly testing them through multiple generations, this combined approach can significantly speed up the development of improved barnyard millet varieties with enhanced yields, disease resistance, and adaptation to local environments[59,60].

    Barnyard millet, despite its potential as a nutritious and climate-smart crop faces limitations in breeding due to the lack of a complete genome sequence[61]. While traditional breeding methods are ongoing, genome editing offers a promising but nascent approach[62]. CRISPR-Cas, a revolutionary gene editing technique could be a game-changer[63]. However, its application in barnyard millet is still in its early stages[61]. Moreover, chloroplast genomic data can offer significant advantages when implementing the CRISPR/Cas9-based strategy for genetic improvement in barnyard millet. Firstly, chloroplasts possess unique genetic characteristics that can be exploited for genetic engineering[64]. Unlike nuclear DNA, chloroplast DNA is maternally inherited in most plants, which can help contain the spread of transgenes via pollen[63]. This characteristic is advantageous for minimizing gene flow to non-target species and ensuring biosafety in genetically modified crops[65]. Secondly, the efficiency of chloroplast transformation has traditionally been lower than that of nuclear transformation due to the low rate of homologous recombination in chloroplasts[63].

    However, recent advancements in CRISPR/Cas9 technology have shown that this can be improved[65]. For instance, using CRISPR/Cas9 to introduce double-strand breaks at specific sites within the chloroplast genome can activate the DNA damage repair mechanism, thereby enhancing the efficiency of homologous recombination. Studies have demonstrated that incorporating CRISPR/Cas9 into the chloroplast transformation system can increase transformation efficiency 6−10 times, which is a substantial improvement[65]. Moreover, CRISPR/Cas9 technology allows for precise genetic modifications, including insertions, deletions, and substitutions, which are crucial for targeted genetic improvement[46].

    In the context of barnyard millet, precise editing of the chloroplast genome could be used to introduce traits that enhance photosynthetic efficiency, nutritional value, or stress tolerance, directly impacting crop yield and resilience[64,66]. Additionally, the use of chloroplast genomic data can facilitate multiplex genome engineering, where multiple genes can be targeted simultaneously[63]. This is particularly beneficial for complex traits that are controlled by multiple genes[46]. For example, in cotton, CRISPR/Cas9 has been used to generate mutations in multiple genes concurrently, demonstrating the potential for similar approaches in barnyard millet[46]. Furthermore, chloroplast-targeted CRISPR/Cas9 applications can improve the nutritional quality of barnyard millet by modifying pathways involved in the synthesis of essential nutrients[65]. Given that chloroplasts are the site of important biosynthetic pathways, such as those for amino acids and fatty acids, editing the chloroplast genome provides a direct route to enhancing the nutritional profile of the crop[66]. By successfully utilizing CRISPR-Cas, scientists could directly target genes responsible for desirable traits like higher yield, improved drought tolerance, or resistance to specific diseases, and this would significantly accelerate the development of enhanced barnyard millet varieties, but further research is needed to unlock the full potential of genome editing for this promising crop[61].

    Functional genomics is a powerful approach to understanding the roles of genes in complex biological systems and can be highly beneficial for improving crops such as barnyard millet[67,68]. Applying RNAi techniques to barnyard millet could similarly facilitate the identification of key genes involved in stress responses, growth, and yield[69,70]. Another critical tool in functional genomics is insertional mutagenesis, such as transfer DNA (T-DNA) tagging, and adapting T-DNA tagging to barnyard millet would enable the systematic characterization of genes, potentially leading to the discovery of traits beneficial for crop improvement[71]. Functional genomics databases creation for barnyard millet could provide comprehensive resources for storing, querying, and analyzing large-scale data sets and these databases integrate various types of data, including metabolite profiles and small RNA (sRNA) data, and offer tools for identifying co-expressed genes and significant biological processes[68,72].

    In addition to these specific techniques, by comparing the functional genomics data of barnyard millet across different species and model organisms, researchers can gain insights into conserved genetic mechanisms and evolutionary adaptations[73,74]. Moreover, the integration of bioinformatics and systems biology in barnyard millet research could advance the understanding of its genetic and molecular underpinnings, facilitating the development of improved crop varieties[59,75]. Bioinformatics tools could be used to analyze the barnyard millet genome, identify genes, and uncover genetic variations, while systems biology approaches map out complex interactions between genes, proteins, and metabolites[60,76]. This integration allows for comprehensive analysis of gene expression, regulatory networks, and metabolic pathways[61,77]. By combining these approaches, researchers can better understand the biological processes underlying key traits, such as stress tolerance and nutrient utilization, and apply this knowledge to optimize breeding strategies and develop resilient, high-performing varieties[8,60,74,78].

    Barnyard millet's potential as a nutritious and climate-adaptable crop is undeniable, but genetic improvement hinges on advancements in breeding techniques. QTL identification and Marker-Assisted Selection offer promising avenues to target desirable traits like yield and disease resistance. Additionally, CRISPR-Cas gene editing holds immense promise for precise improvement, although challenges like incomplete genome knowledge and transformation protocols need to be addressed. By overcoming these limitations and actively pursuing these genetic improvement strategies, researchers can unlock barnyard millet's full potential, significantly contributing to global food security and sustainable agriculture.

    The authors confirm contribution to the paper as follows: study conception and design: Sahoo JP; data collection: Pradhan PP, Bhuyan P, Nag G; analysis and interpretation of results: Sahoo JP; draft manuscript preparation: Sahoo JP; review & editing: Pradhan PP, Bhuyan P, Nag G; Sahoo JP. All authors reviewed and approved the final version of the manuscript.

    Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

  • The authors declare that they have no conflict of interest.

  • Supplemental File 1 Gene IDs and sequences for CePDS and CeTCP14.
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  • Cite this article

    Gui Y, Li B, Zhu Q, He Y, Zhang Y, et al. 2024. Construction and application of a virus-induced gene silencing system in taro. Tropical Plants 3: e030 doi: 10.48130/tp-0024-0025
    Gui Y, Li B, Zhu Q, He Y, Zhang Y, et al. 2024. Construction and application of a virus-induced gene silencing system in taro. Tropical Plants 3: e030 doi: 10.48130/tp-0024-0025

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ARTICLE   Open Access    

Construction and application of a virus-induced gene silencing system in taro

Tropical Plants  3 Article number: e030  (2024)  |  Cite this article

Abstract: Virus-induced gene silencing (VIGS) technique is an important means for rapid identification of plant gene function, and a stable VIGS system in taro can provide technical support for rapid and efficient gene function verification. A VIGS system was constructed with a tobacco rattle virus (TRV)-based vector, with phytoene desaturase (CePDS) as the indicator gene, then CeTCP14 was silenced to further verify the robustness of this system. Ganyu No.1 was used as the material to construct the taro VIGS system using the leaf injection method with CePDS at OD600 = 0.6, and the silencing plant rate was 12.23%, the expression level of CePDS was about 59.34%−77.18% compared to the control, and the chlorophyll content decreased 37.80%−56.11% in CePDS silencing plants. The silencing plant rate increased significantly by leaf injection at OD600 = 1.0, and reached 27.77%, but there was no significant difference in silencing plant rate between the leaf injection method and bulb vacuum treatment. CeTCP14 was further silenced with bacteria solution OD600 = 1.0 and bulb vacuum treatment in Ganyu No.2, the silencing plant rate was 20%, and the expression level of CeTCP14 was 43.94%−63.34% of the control. Meanwhile, the starch content in the corms decreased significantly to 70.88%−80.61% compared to the control. In conclusion, the results indicated that a TRV-based VIGS system is effective in taro. The concentration of bacterial solution is a key factor affecting the VIGS system, CeTCP14 can affect starch accumulation in taro bulbs. A robust VIGS system establishment in taro could lay a good foundation for the subsequent rapid gene function verification.

    • Taro (Colocasia esculenta (L.) Schott), from the Araceae family, is a tropical and subtropical crop, which is commonly used as food and for the industrial processing of raw material with high economic benefits. It ranks 5th among root crops and 14th among vegetable crops worldwide[1], and world annual production of taro in 2022 was estimated at 19.67 million tons (FAOSTAT 2024). Taro is typically propagated from corms with a long life cycle (harvested usually around 6 to 12 months after planting) that lead scientists to largely overlook it.

      Taro is rich in mucus protein, polysaccharides, vitamins, mineral elements, and many other physiologically active substances, which have been applied to alleviate a variety of human health symptoms[2]. It can be predicted that taro will be a promising crop. In recent years, taro and its products are favored by consumers because of their unique nutritional value and healthcare functions, higher requirements are also put forward for new varieties, and clear gene function will lay the foundation for high-quality new variety breeding to further meet consumer needs.

      As research continues, the elucidation of gene function is urgent, but the genetic transformation system is still unstable in taro, which hinders the gene functional verification and greatly limits the utilization of excellent taro germplasm resources. An effective gene function verification system is urgently required. Due to virus-induced gene silencing (VIGS) advantages of stability, high efficiency, simplicity, low cost, high throughput and short cycle, it has been widely applied in the gene function research of vegetable crops[3]. The robust VIGS system establishment in taro can not only verify gene function, but also greatly promote the utilization of germplasm resources, which has an important role in promoting the improvement and breeding of taro.

      At present, VIGS technology has been widely applied in the study of gene function related to growth and development, stress, substance synthesis, and metabolic regulation of vegetable crops[35]. However, the silencing efficiency induced by viral vectors is host-depended. Based on previous studies, several viral vectors have been successfully applied in VIGS, including tobacco mosaic virus (TMV), potato virus X (PVX), tobacco rattle virus (TRV), tomato golden mosaic virus (TGMV), and cabbage leaf curl virus (CbLCV)[6]. The TRV virus vector has been widely used in gene silencing experiments because of its high silencing efficiency, long silencing duration, and mild virus symptoms[7,8]. During the VIGS system establishment process, phytoene desaturase (PDS) is often selected as the target gene to indicate the success of gene silencing.

      TCP TFs are named after the first known members (TB1, CYC, and PCFs) that share a highly conserved TCP domain, which harbors a non-canonical basic-helix-loop-helix (bHLH) structural motif[9,10]. They are classified into two divergent groups; TCP class I and TCP class II, the latter is further divided into two clades, CIN and CYC/TB1[9,11]. As a plant-specific transcription factor, they play a crucial role in plant growth and development, including regulating flower organ development, leaf morphology and lateral branch growth[11]. Currently available evidence has shown that most TCP class I genes are activators of plant development, whereas TCP class II members often function as a repressor of various growth and development pathways[1214]. The function of TCP TFs in taro have however not yet been reported.

      Although VIGS technology has been widely used to verify plant gene function, there are no reports of VIGS technology in taro. The establishment of a taro VIGS technology system would lay the foundation for rapid verification of gene function, and clarify the gene function that controls important agronomic traits in taro, which would promote germplasm resource utilization and taro breeding improvement.

    • Ganyu No. 1 and Ganyu No. 2 were used for VIGS system construction, corms with 1 cm diameter were used as test materials. pTRV1 and pTRV2 were selected as VIGS vectors and stored in the laboratory (Fig. 1a & b). Escherichia coli DH5α and Agrobacterium GV3101 receptor cells were purchased from Huayueyang Biotechnology Co., Ltd (Beijing, China).

      Figure 1. 

      Vectors used and infection flow chart in this study. The plasmid profile of (a) pTRV1, (b) pTRV2, (c) pTRV2::PDS410, (d) pTRV2::CeTCP14. Color change of leaves by (e) leaf injection and (f) bulb vacuum injection methods.

    • First, the protein sequence of SlPDS (Solyc03g123760) from tomato and AtPDS (AT4G14210) from Arabidopsis were used to blast in the taro reference genome Taro_JAAS_v1.0 (BioProject: PRJNA587719)[15]. Then, the obtained sequences were validated in the transcriptome of Ganyu No. 1, which has been published previously[16]. Finally, the CePDS (EVM0008568.1) sequence with 1,743 bp was obtained and verified, CeTCP14 (EVM0000825.2) was screened out based on RNA-seq analysis, the gene sequences are shown in the Supplemental File 1. The mRNA from young leaves was reverse transcribed into cDNA, and then used as a template, the primers used in this study are listed in Table 1, pTRV2-CePDS was constructed by restriction enzymes (EcoRI and BamHI), pTRV2-CeTCP14 was constructed by homologous recombination, and the vector construction was verified by sanger sequencing (Fig. 1c & d).

      Table 1.  Primers used in this study

      Primer name Primer sequence (5'−3') Length (bp) Purpose
      CePDS-EcoRI-F GGAATTCATGGGCTTTACCAGTTCTCTTTCGG 410 Used for fragments amplified of CePDS and CeTCP14 inserted in TRV2 vector
      CePDS-BamHI-R CGGGATCCTCCAGCAATATAGGCTTATGACCTG
      TRV2-TCP14_F GTGAGTAAGGTTACCGAATTCATGGGGGAGAGCCACCAG 300
      TRV2-TCP14_R CGTGAGCTCGGTACCGGATCCATCGACGGCCTTGCTGGG
      qCePDS-F GGTCGTTGGGGAGGAAGC 140 Used for the qRT-PCR of CePDS
      qCePDS-R TCTAGTCGGGCGTGGTGA
      qCeTCP14_F CCACACCGCCATCCAGTT 110 Used for the qRT-PCR of CeTCP14
      qP_TCP14_R CGAGCTCGTCTATGGCGG
      CeActinF CTAGTGGTCGCACAACAGGT 191 Used for the qRT-PCR of reference genes
      CeActinR TTCACGCTCAGCAGTGGTAG
      TRV1F1 CGTGTTGCATTTCGATGAA 525 Used for the detection of RdRp in TRV1
      TRV1R1 GACAACGCCACGATTAAGT
      TRV2F1 GTTGAAGAAGTTACACAGCA 407 Used for the detection of coat protein in TRV2
      TRV2R1 TCTTCAACTCCATGTTCTCT
      pTRV2_F TGTCAACAAAGATGGACATTGTTAC 198 / 480 Used for the detection of TRV2 and TRV2-CeTCP14 expression
      pTRV2_R ACACGGATCTACTTAAAGAA
      TRV2_F TGTTACTCAAGGAAGCACGATGAGCT Used for vector construction sequencing and colony PCR detection
      TRV2_R GTACAGACGGGCGTAATAACGCTTA
      Note: underline indicates cleavage sites, bold indicates protective bases.
    • Plasmids pTRV1, pTRV2, TRV2-CePDS and pTRV2-CeTCP14 were transferred into agrobacteria GV3101 using the freeze-thaw method[17]. First, 5 ml YEB solution containing rifampicin (25 mg/L) and kanamycin (50 mg/L) was used to culture bacteria by shaking, and colony PCR detection was performed with specific primers to obtain positive monoclones (Table 1). Then 500 μL of bacteria solution was transferred to 50 mL of YEB solution containing MES (10 mM), acetosyringone (20 μM) and corresponding antibiotics for overnight culture. The bacteria were centrifuged at 3000× when the OD reached 1.2, and collected with MS containing MES (10 mM), acetyl syringone (200 μM) and MgCl2 (10 mM) at pH = 5.6 and then stood at room temperature for 1−2 h. Afterwards, pTRV1 was mixed with pTRV2, TRV2-CePDS and PTRV2-CeTCP14 resuspension in equal volume respectively, the mixed solution was used for infection in subsequent research.

    • Leaf injection[18] and bulb vacuum infiltration[8] were applied in agrobacterium infection. Infection solution containing these four combinations: pTRV1 + pTRV2, pTRV1 + pTRV2-PDS, pTRV1 + pTRV2-CeTCP14, negative control (suspensions). In the leaf injection method, 1 mL syringe without needle was used, and plant material with 2−3 fully unfolded leaves were selected for infection material, about 1/3−1/2 of the leaf area were injected, and then cultured in a seedling room with 16 h of light and 8 h of darkness at 22−24 °C (Fig. 1e). For the bulb vacuum method, the cleaned corms were punctured by needles that facilitate infection liquid entry, the pressure was 750 mm Hg for 30 min, then the surface of the taro was washed after infection. The plants were planted in pots (10 cm × 10 cm) and moved to the seedling room (Fig. 1f). The temperature and light conditions were the same as those for the leaf injection method. Leaf color change was continuously observed.

    • After 20 d of infection, the photobleaching phenotype appeared, the leaves/corms sample were collected, the RNA was extracted using the polysaccharide polyphenol plant RNA extraction kit (0416-50), Huayueyang Biotechnology Co. Ltd (Beijing, China), and the expression levels of CePDS and CeTCP14 were detected by RT-qPCR, actin with stable expression level was selected as the internal reference, and the relative expression level of genes was normalized by the 2ΔΔCᴛ method with three biological repeats[19]. The primers are listed in Table 1.

      After the photobleaching phenotype occurred in the VIGS-CePDS group, leaves without coarse leaf veins were collected. The samples were cut into small strips, and about 0.2 g of fresh samples were placed into 50 mL centrifuge tubes with three biological repeats, 25 mL of 95% ethanol was added, and left to stand for 36 h until the leaves become white under dark conditions. The well-mixed supernatant was taken for spectrophotometer determination at wavelengths of 649 and 665 nm respectively, OD values, A649 and A665 were recorded. chlorophyll a and chlorophyll b were calculated as follows: Ca = 13.95 × A665 − 6.88 × A649, Cb = 24.96 × A649−7.32 × A665, total chlorophyll content = Ca + Cb[19].

      After 20 d of VIGS-CeTCP14 suspension infection, 0.5 g fresh corms flesh samples at the same sites were collected to determine starch content with three biological repeats. The methods were as those reported by Gao[20].

    • To confirm whether the TRV-based vector could induce gene silencing in taro, Ganyu No. 1 was selected as material for preliminary testing with CePDS as the indicator by leaf injection at OD600 = 0.6. After 20 d, the leaves began to show photobleaching phenomenon (Fig. 2b), about 10 d later, almost the entire leaf turned white (Fig. 2c), there was no colour change in the negative control group and the empty-vector group (Fig. 2a, d). Then the total number of plants showing the photobleaching phenomenon were counted, and the average value was 3.67 (30 plants each time with three biological replicates). Therefore, the TRV-based vector could induce gene silencing in taro, but the proportion with photobleaching phenotype was low (12.23%).

      Figure 2. 

      VIGS system constructing in taro. (a) Empty vector phenotype at 20 d of leaf infection with OD600 = 0.6, (b) VIGS-PDS410 phenotype at 20d of leaf infection OD600 = 0.6, (c) VIGS-PDS410 phenotype at 30 d of leaf infection with OD600 = 0.6, (d) mock phenotype after 20 d infection, (e) RdRp gene expression in TRV1 and CP gene expression in TRV2 were detected by RT-PCR with actin as the internal reference, (f) detection of CePDS expression level for photobleaching phenotype plants, (g) chlorophyll content detection for photobleaching phenotype plants. Scale bars = 3 cm.

    • To verify that the phenotypic changes were caused by endogenous CePDS silencing through RNA expression derived from pTRV1 and pTRV2-PDS, RT-PCR was applied for detection. Transcriptional analysis of pTRV1 coat protein (CP) and RNA-dependent RNA polymerase (RdRp) from pTRV2 confirmed the successful expression of pTRV1 and pTRV2 vectors in taro leaves (Fig 2e). The expression level of CePDS were about 59.34%−77.18% in VIGS-CePDS plants compared to the control through further detection (Fig. 2f), and the chlorophyll content decreased by 37.80%−56.11% in VIGS-CePDS plants (Fig. 2g). Phenotypic and molecular results of VIGS-CePDS seedlings clearly showed that CePDS expression in taro was targeted and silenced after VIGS, but the number of silenced lines was low and needed to be further improved.

    • Studies have shown that factors such as infecting solution concentration and infecting method would influence the silencing effect[8,18,21]. The VIGS system was therefore optimized based on these two factors. Based on photobleaching phenotype, the results showed that when OD600 = 1.0, the number of silent lines was the largest compared to OD600 = 0.6 and OD600 = 0.8, the proportion reached 27.77%, but there was no significant difference compared with D600 = 1.2 at 30 d after infection (Table 2). Subsequently, the bulb vacuum infection and leaf injection infection were compared, the number of photobleached lines showed no significant difference (Table 3). The above results indicate that the concentration of infective bacterial solution was a key factor affecting gene silencing in taro.

      Table 2.  Bacterial concentrations optimization for the VIGS system.

      OD600 Total number of inoculation plants Number of photobleaching phenotype The percentage of
      photobleaching phenotype
      0 10 0 d 0
      0.6 30 3.00 ± 1.00 c 10%
      0.8 30 5.33 ± 0.58 b 17.77%
      1.0 30 8.33 ± 0.58 a 27.77%
      1.2 30 7.67 ± 0.58 a 25.57%

      Table 3.  Comparison of the VIGS system based on infection type.

      Infection type Total number of inoculation plants Number of photobleaching phenotype The percentage of
      photobleaching phenotype
      Leaf injection 30 8.33 ± 0.58 a 27.77%
      Bulb evacuation 30 8.00 ± 0.58 a 26.67%
    • In our previous work, it was found that CeTCP14 presented obvious upregulated trend during the early stage of corm expansion, this gene was further selected to verify the robustness of the constructed VIGS system. CeTCP14 was silenced with OD600 = 1.0 and the bulb vacuum infiltration method to identify its function. RT-PCR results confirmed the successful expression of pTRV1 and pTRV2 vectors in taro corms (Fig. 3a), the expression level of CeTCP14 in VIGS-CeTCP14 plants decreased by 36.66%~56.06% (Fig. 3b), this resulted in significant starch content decrease, only 70.88%~80.61% of the control (Fig. 3c). In maize, ZmTCP7 regulated the starch content through binding to the ZmBt2 promoter[22], CeTCP14 and ZmTCP7 belong to the same TCP class II, the factors interacting with CeTCP14 in taro to regulate starch content need further study.

      Figure 3. 

      Detection of taro VIGS system for CeTCP14 in Ganyu No. 2. (a) RdRp gene expression in pTRV1 and fragement expression in pTRV2 were detected by RT-PCR. (b) Detection of CeTCP14 expression at 20 d after bulb vacuum infiltration. (c) Starch content detection for VIGS-CeTCP14 plants.

    • The genome of taro has been previously reported[23], which facilitates the gene mining that controls important agronomic traits. CePDS was cloned as an indicator gene from taro based on homologous alignment and a VIGS system was constructed, for color changes are the most intuitive way after gene silencing or gene editing[19,2426].

      Virus silence-inducing vector is another important factor affecting the success of VIGS system construction. Although a variety of viral vectors have been used in VIGS, including RNA viruses, DNA viruses, and satellite viruses, the silencing efficiency induced by each viral vector is host-dependent. For example, the potato virus X (PVX) is an RNA virus, studies in tomatoes have found that in vitro transcription is required before RNA infection[27]. DNA viral vectors are simple to construct, easy to operate, and do not require in vitro transcription, such as beet curl top virus (BCTV) and tomato curl leaf virus (ToLCV) can be effectively used for gene silencing[28,29]. Satellite viruses have a small genome, replicate quickly in the host and are easy to inherit. Chinese tomato yellow leaf curl virus (TYLCV) and tobacco stalk curl virus (TbCSV) of these virus vectors have been successfully used to verify gene function[30,31]. The virus-silencing induction vector selected in this study is TRV, which belongs to the RNA virus and is widely used because of its long silencing time, mild virus symptoms and high silencing efficiency[7,8]. In this study, the TRV-based vector could produce silencing effects in Ganyu No. 1 and Ganyu No. 2 which further verified that the TRV vector is suitable for gene silencing in taro, but the percentage of photobleaching phenotype is relatively low compared to banana. In banana VIGS research, another RNA virus, cucumber mosaic virus (CMV) performed better gene silencing effect with a 95% infection rate and reduced target gene transcripts to 10%−18% of the control, the success of developing a banana VIGS system could be partially credited to the choice of utilizing a CMV isolate naturally infecting bananas during its construction[32], while in taro, TRV is not a serious taro-hosting virus, and greatly affected the infection rate, one way that can be used to improve the silencing effect is to increase the concentration of bacterial solution. It is also reported that CMV could infect taro[16], both banana and taro are monocots and exhibit similar growth habits, it might be worth testing CMV in taro as well as in the subsequent optimization experiments. The infecting bacterial solution concentration and infection method were also important factors affecting the silencing effect in VIGS. In the present study, the best infection effect was achieved with OD600 = 1.0, and this concentration was also applied in other plants used in VIGS system construction[18,21]. It may be related to bacterial vitality and infection ability at this concentration. But recent research of engineered TRV vectors on tobacco resulted in a OD600 value of bacterial suspensions of 1 × 10−5 still achieving good silencing effect through a one-strain/two-vector approach[33], these JoinTRV vectors that used pLX as back-bones have been tested in a range of agrobacterium strains, are notably more compact than any other TRV vector system currently documented, except for TRV are tobacco-hosting viruses, perhaps compact structure is another key factor to high efficiency at low concentration. The main VIGS inoculation methods are vacuum penetration[34,35], friction inoculation[36], root-filling method[37], and leaf injection[38]. In corm crops, it usually turns out that vacuum penetration is superior to leaf injection[8,38]. However, the results in taro bulbs showed no significant difference between these two methods (Table 3). The possible reason is that taro bulbs are firm and full of starch with multiple buds. Buds with less infection would display obvious growth advantages, this may be an important factor resulting in no significant differences in the photobleaching phenomenon.

      Currently available evidence has shown that most TCP class I genes are activators of plant development, whereas TCP class II members often function as a repressor of various growth and development pathways[1214]. In maize, ZmTCP7 affects the accumulation of starch by targeting the promoter of ZmBt2[22], besides, ZmTCP7 lacks transactivation activity in yeast systems[10,12], this means that ZmTCP7 may dimerize with other regulatory protein(s) or TF(s) to regulate ZmBt2 to control the accumulation of starch. In this research, it was found that after CeTCP14 expression level was downregulated by VIGS, the starch content decreased above 20% (Fig. 3), this means CeTCP14 also contributes to the accumulation of starch. In VIGS-CeTCP14 plants, the expression level of CeTCP13, CeTCP10, and starch biosynthesis gene (ADP-glucose pyrophosphrylase (AGPL), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), starch branching enzyme (SBE), isoamylase (ISA1) were also detected in taro corms, they all presented a significant down-regulated trend (data not shown), these results indicate that CeTCP14 is an upstream regulatory gene in the starch biosynthesis pathway, but whether it interacts with CeTCP13 and CeTCP10 to regulate the starch pathway remains to be further investigated.

      VIGS is a powerful tool used in plant molecular biology to study gene function. VIGS allows for high-throughput functional analysis of plant genes. By silencing individual genes and observing the resulting phenotypic changes, researchers can elucidate the roles of specific genes in various biological processes such as development, stress response, and disease resistance.

    • In this study, we constructed a VIGS system initially in taro with TRV as vector and CePDS as indicator gene, combined with phenotype change, gene expression, and chlorophyll content determination. Through the optimization of bacterial solution concentration and infection mode, it was found that the silencing effect was the best at OD600 = 1.0, and the silencing plants rate reached 27.77%. There was no significant difference in the silencing plant numbers between leaf injection method and bulb vacuum infiltration method. After silencing the CeTCP14 based on the established system, the starch content in the bulb is reduced by more than 20%, preliminarily verified CeTCP14 could promote starch accumulation in taro, which laid a foundation for rapid gene function verification in taro.

    • The authors confirm contribution to the paper as follows: study conception and design, writing - review & editing: Zhou Q, Xiao Y; experiment design: Cui J, Pan R, Zhu Q, Huang Y, Zhou Q (directing), Xiao Y (directing); experiment execution, writing - draft manuscript: Gui Y, Li B, He Y, Zhang Y, Zhou Q, Xiao Y; data analysis: Gui Y, Li B, He Y, Zhang Y, Zhou Q, Xiao Y; analysis and interpretation of results: Cui J, Pan R. All authors have read and approved the final manuscript.

    • The CePDS and CeTCP14 sequence used in this study are available in the taro reference genome Taro_JAAS_v1.0 (BioProject: PRJNA587719), and the gene numbers are EVM0008568.1 and EVM0000825.2, respectively.

      • This research was supported by Jiangxi Provincial Key Research and Development Project of China (Grant No. 20212BBF61001), the National Natural Science Foundation of China (32060683), the Natural Science Youth Foundation of Jiangxi Province (20224BAB215025),and the Youth Foundation of Jiangxi Provincial Department of Education (GJJ210442).

      • The authors declare that they have no conflict of interest.

      • Received 25 March 2024; Accepted 7 June 2024; Published online 11 September 2024

      • CePDS was used as an indicator gene to construct a VIGS system in taro.

        The silencing plant rate of VIGS system increased significantly by optimized bacterium concentration.

        CeTCP14 was selected to further verify the robustness of the constructed system.

      • # Authors contributed equally: Yanling Gui, Bicong Li

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press on behalf of Hainan University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (3)  Table (3) References (38)
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    Gui Y, Li B, Zhu Q, He Y, Zhang Y, et al. 2024. Construction and application of a virus-induced gene silencing system in taro. Tropical Plants 3: e030 doi: 10.48130/tp-0024-0025
    Gui Y, Li B, Zhu Q, He Y, Zhang Y, et al. 2024. Construction and application of a virus-induced gene silencing system in taro. Tropical Plants 3: e030 doi: 10.48130/tp-0024-0025

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