Plant Biotechnology Reports最新文献

Pak-choi (Brassica rapa ssp. chinensis) is a popular vegetative crop in southern China, East Asia, and Southeast Asia. Owing to the threat of climate change, rapid breeding strategies for vegetable cultivars that are tolerant to abiotic and biotic stresses are required. Thus, the rapid fixation of useful agronomic traits using doubled haploid technology is urgent. The haploid-inducer gene is key to doubled haploidization. Two known CENH3 and pPLAIIγ genes, in which altered or partially deleted forms lead to haploid induction, were selected, and direct editing of Pak-choi CENH3 and pPLAIIγ genes (BcCENH3 and BcpPLAIIγ) was conducted using an Agrobacterium-mediated CRISPR/Cas9 system. First, BcCENH3 and BcpPLAIIγ genes were characterized by analyzing the spatial expression patterns and subcellular localization. The CENH3 expression levels in carpels and pPLAIIγ in various parts of Pak-choi flowers were higher than those of other parts. BcCENH3 and BcpPLAIIγ proteins targeted in the nucleus and plasma membrane, respectively. Whole plants were successfully regenerated from the shoot apical meristem (SAM) regions of Pak-choi seedlings using the optimized procedure and culture conditions. The regeneration results of SAM explants after Agrobacterium-mediated transformation of constructs expressing CRISPR/Cas9 and BcCENH3 or BcpPLAIIγ sgRNAs confirmed four independent BcCENH3-targeted transgenic lines with 2.1%, 1.8%, 1.8%, and 1.7% INDEL frequencies, and three independent BcpPLAIIγ-targeted transgenic lines with 24.5%, 33.7%, and 33.0% INDEL frequencies. Thus, our results suggested the possibility of developing transgenic Pak-choi lines by applying the CRISPR/Cas9 genome editing technology to BcCENH3 and BcpPLAIIγ as two haploid-inducer genes.

Plants are among the many creatures that have benefited from the widespread application of the CRISPR-associated Cas system as a genome-editing tool for investigating gene function, identifying disease, and enhancing agricultural yields. Although the CRISPR/Cas systems for DNA editing are widely employed, post-transcriptional manipulation of RNA remains difficult despite the prevalence of Cas9. Type VI CRISPR/Cas systems, which were recently found, allow for precise RNA editing without permanently affecting the genome. Cas13d has been put to good use in RNA-related studies across a wide range of RNA knock-down, and RNA detection without affecting DNA. Regulation of cas13d specificity and activity helps to avoid the off-target effects and immune responses in plants. Cas13d as highly efficient RNA-targeting tools for the virus resistance, gene function studies, disease diagnostics, and crop improvement in plants. However, CRISPR/Cas13d applications in plant RNA biology are just getting started. This article discusses how RNA editing tools derived from the CRISPR/Cas13d system are currently being used and where they may be used in the future for plant research.

Abstract

Tomato (Solanum lycopersicum L.) crop is well-known for its versatility worldwide and is also recognized as model species used extensively for various genetic studies. The aim of this research was to investigate both inter and intra-genetic diversity present among various tomato genotypes. This investigation was carried out through a comprehensive analysis encompassing morphological observations, biochemical assessments, and the utilization of SSR markers. A total of 15 discrete agro-morphological traits and six biochemical traits were undertaken in the current study for evaluating the analysis of variance, genetic parameters and correlation. The analysis of variance indicated significant differences across genotypes for all 15 agro-morphological traits and 6 biochemical traits tested, indicating that the experimental material included considerable variability. Morphological clustering divided the genotypes into 2 clusters and the genotype wise distance matrix was obtained to identify the most diverse genotypes. PCA analysis was conducted to understand the directive relation of traits and magnitude of variability contributed by them. SSR profiling with 24 primers identified 44 alleles with 1.83 as mean number of alleles/SSR with an average PIC value of 0.31. Structure analysis revealed two sub-populations (K = 2). The AMOVA indicated that 98% of the total variation was present within populations. This study presents a roadmap for composing future breeding strategies for integrating desirable traits in novel tomato lines that combine robustness and nutritive value.

Some edible Leguminous are toxic when raw, and the Chinese are particularly fond of beans, so Leguminous poisoning is very common in China. Rapid and accurate identification of poisoned species and determination of their toxic components would better assist physicians in treating patients. However, traditional morphology-based identification methods possess many limitations. DNA barcoding technique is a new species identification technique developed in recent years, which is expected to make up for the shortcomings of traditional morphological identification. In this study, a comprehensive evaluation system based on DNA barcoding and ELISA kits was attempted. A total of 30 Leguminous toxic plants were collected, involving 9 genera and 10 species. We used simulated gastric fluid (SGF) to simulate the human gastric environment. Three markers (rbcL, trnH-psbA, and ITS) were amplified and sequenced for all untreated and 15 mock-digested samples. The validity of DNA barcoding for species identification was assessed using the Basic Local Alignment Search Tool (BLAST) method and the tree construction method. The levels of three toxic components (saponin, phytoagglutin and trasylol) were determined in all samples using ELISA kits. The amplification success rate of all three regions was high (rbcL 96.67%, trnH-psbA 100%, and ITS 100%), but the sequencing of the trnH-psbA region was less satisfactory (66.67%), and SGF had a significant impact on the sequencing of the ITS region (After 40 min of SGF treatment, the sequencing success rate decreased by 46.67%). The samples from different species and origins contained different levels of toxic components, and the levels of all three substances decreased significantly after undergoing SGF digestion. After 1 h of SGF treatment, the saponin content decreased to 0–8.60% in untreated content (PHA decreased to 8.62–36.88%, trasylol decreased to 4.70–47.06%). The current results suggest that DNA barcoding has great potential for rapid identification of Leguminous poisoning in clinical settings. Toxins are probably not detectable in the patient for longer periods of poisoning. We recommend DNA barcoding technology as a first step for rapid screening and combined with toxin analysis for clinical diagnosis.

The WRKY transcription factor family plays a key role in plant growth and development, hormone signaling, and resistance to environmental stress. In this study, we investigated the gene sequence, subcellular localization, and response pattern of a member of the WRKY transcription factor family to reveal its protein structure and involvement in the resistance signaling pathway.The BsWRKY51 gene was cloned by RT-PCR, and the structural characteristics of its encoded protein WRKY51 were analyzed by bioinformatics. The vector was next transiently transformed into tobacco to analyze the subcellular localization, and real-time fluorescence quantitative PCR was performed to analyze the changes in the expression pattern of BsWRKY51. The BsWRKY51 gene has a coding sequence (CDS) length of 987 bp.The respective unstable hydrophilic protein BsWRKY51 is localized in the nucleus. It most closely related to the WRKY protein of Dendrobium catenatum in the Orchidaceae family. Fluorescence quantitative PCR results showed that the BsWRKY51 expression in the leaves was significantly higher than that in the roots, stems, and pseudobulbs of Bletilla striata seedlings. Under the conditions of salt and drought stress, the BsWRKY51 expression gradual increased and then a slightly decreased, and under salicylic acid (SA) treatment, the expression of BsWRKY51 showed an overall decreasing trend.The BsWRKY51 gene of Bletilla striata may play an important regulatory role in its salt and drought stress responses. Our present findings provide the foundation for elucidating the mechanisms of salt and drought tolerance in Bletilla striata and for breeding new varieties.

CRISPR/Cas9-based targeted gene editing has emerged as a versatile tool for deciphering gene function and improving traits in plants. However, this technique has not been applied to Zoysia japonica, a prominent warm-season turfgrass widely used for green spaces. Leaf senescence, a vital process affecting crop quality, occurs in Z. japonica during late growth, diminishing its aesthetic value and performance. In this study, we adeptly employed CRISPR/Cas9-mediated gene editing to create Z. japonica exhibiting delayed leaf senescence by targeting the ZjEIN2 gene, a crucial regulator of ethylene-mediated senescence. Precise gene editing, which generated knockout mutations in ZjEIN2, led to delayed leaf senescence in both dark and ethylene treatment conditions. This provided strong evidence for ZjEIN2’s role in leaf senescence regulation. These findings highlight the potential of CRISPR/Cas9-mediated gene editing as a biotechnological strategy to enhance anti-senescence traits in Z. japonica and potentially other crops. This study carries significant implications for sustainable agriculture and turfgrass management, offering promising avenues for future applications and research.

The development of an effective and eco-friendly silver nanoparticle (AgNPs) to abate the effect of abiotic stress is an important area of nano-biotechnology. This study aimed to study the priming effect of plant-based green-synthesized silver nanoparticles and melatonin on the physiological and biochemical activities of drought-stressed E. uniflora. Sterilized seeds of E. uniflora were primed with 0.06 mg/l of ML-AgNPs, 0.06 mg/l of melatonin, and a nano-silver formulation of melatonin (1:1). Primed seeds were planted and subjected to 7 days under drought stress. The ML-AgNPs enhanced germination percentage, speed and vigor, and shoot elongation and induced the production of APx, CAT, and proline dehydrogenase (100% increases). Melatonin improved the activities of APx and CAT, total protein, accumulation of proline, and proline dehydrogenase (200% increases) and stabilized MDA content. Meanwhile, silver nano-formulation of melatonin increased leaves proliferation of leaves and production of APx, GPx, SOD, and CAT. Accumulation of proline and 100% upregulation of proline dehydrogenase osmo-regulated the effects of the drought, reduced MDA contents, and stabilized the excessive production of H₂O₂ and O²⁻. The ML-AgNO₃ showed an efficient delivery system of melatonin into the plant under drought stress. As a result, our research shows that melatonin in silver nano-formulation (1:1) is a useful biostimulant against drought stress.

Soybean is one of the richest and cheapest proteins and vegetable oil sources. It is adapted to grow in a varied environment; however, yield loss occurs due to multiple abiotic stresses. Abiotic stresses negatively impact plant growth and development, damaging the crop and decreasing productivity. The last two decades have focused tremendously on improving soybean productivity by dissecting physiological and molecular mechanisms for developing abiotic stress-tolerant varieties. Here, we present a review with a comprehensive outlook on the biotechnological approaches to explore the pathways involved in abiotic stress tolerance in soybean. The review focuses on summarizing transgenic and RNA interference-based strategies as well as genome editing tools to validate the function of abiotic stress-associated genes in soybean. We have also highlighted the significant challenges faced in increasing soybean yield against climatic changes using diverse techniques.

In recent years, there has been significant progress in enhancing the genetic foundation underlying important agricultural traits such as resistance to scab and the development of a columnar growth habit. V. inaequalis is a hemibiotrophic fungus widely distributed in temperate regions where apples are grown on commercial scale. The present investigation was undertaken to identify Vf gene and Co gene, which, respectively, confer resistance against apple scab disease and columnar phenotype in apple cultivar ‘Rosalie’ and introgression of both the genes in commercially important cultivar ‘Fuji’. Polymorphism survey was carried out between the two parents using 22 simple sequence repeat (SSR) and sequence-characterized amplified region (SCAR) markers. The observations revealed that almost 50% hybrids fall in resistant category and 50% in susceptible category. The results of marker-assisted screening confirmed 38 F₁s carrying resistance gene for scab while the remaining 32 F₁ plants were found to be lacking the gene. The 38 genotypically scab-resistant hybrids were selected for further characterization as columnar and non-columnar plants. Based on the selection criteria, 21 individuals were categorized as columnar and the remaining 17 were categorized as non-columnar. The phenotypic screening was followed by screening of F₁ s using molecular markers for Co gene. The amplification of Co-specific markers yielded columnar-specific fragments in the population and fitted the expected 1:1 Mendelian ratio. 18 scab-resistant F₁ hybrids were found to carry Co gene and the remaining 20 did not possess the gene for columnar growth habit. Gene-specific primers identified in the present study can be directly used for screening large apple germplasm in a short period of time for developing resistant varieties against apple scab as well as varieties with columnar growth habit. Hybrids with verified scab resistance and columnar growth can be swiftly utilized as scab-resistant columnar cultivars.

Sorghum is considered a fifth major cereal, widely used as a multipurpose crop worldwide. The use of sorghum as a major forage crop is limited due to cyanogenic glycoside dhurrin in the vegetative shoot tissues. This cyanogenic glycoside is harmful to livestock when fed as fodder. The present study selected three sorghum genotypes for estimating hydrogen cyanide potential (HCNp) in vegetative tissues under well-watered (WW) conditions. The HCNp concentration varied from genotype to genotype and ranged from 364 to 512 ppm. The HCNp estimation was observed more in ICSR 14001 with 511 ppm, followed by ICSV 93046 (443 ppm) and CSH 24 MF (364 ppm). A significant difference was noticed between the genotypes. Sequence information of dhurrin biosynthesis pathway genes was retrieved and characterized using different bioinformatic tools. The gene expression analysis of dhurrin biosynthesis pathway genes showed different expression patterns, with the highest in ICSV 93046 and less in ICSR 14001 and CSH 24 MF. Genes CYP79A1, CYP71E1 and UGT85B1 showed a 2.5- to 4 fold increase in ICSV 93046 and no significant expression in ICSR 14001 and CSH 24 MF. The genotype CSH 24 MF observed a 1.5-fold increase in CYP79A1 gene expression, and the other genes observed no significant increase. This study assisted in identifying the contrasting genotypes inducing HCNp and the key genes of the dhurrin pathway producing hydrogen cyanide (HCN) under WW conditions, which can be used as potential candidates for gene editing, providing safe feed for the livestock.