Current Biotechnology

Research Progress on Multi-omics Systematic Analysis of Unintended Effects in Biotechnology-derived Breeding Products
Yu GAO, Tianyi HAN, Xiaoyun HE, Nan CHENG, Weichen LIU, Wei FU, Kunlun HUANG, Hong CHEN
2026, 16(4): 732-743. DOI: 10.19586/j.2095-2341.2026.0025
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With the accelerating commercialization of genetically modified （GM） and gene-edited （GE） crops within global agricultural trade， the accurate identification of potential unintended effects （UEs） in biotechnology-derived breeding products has become a central issue in biosafety assessment. For a long duration， the evaluation system has relied largely on the principle of "substantial equivalence"， wherein targeted detection technologies have played a significant role in assessing conventional physicochemical indicators. However， traditional methods struggle to capture complex molecular variations. Moreover， conventional "bulk multi-omics" analysis tends to mask cellular heterogeneity， failing to detect rare variants and their intricate microenvironmental interactions at the microscopic level. This review summarized recent advances in the application of multi-omics technologies for evaluating unintended effects in biotech breeding products. It focused on the potential of emerging technologies such as single-cell omics and spatial omics in deciphering tissue-specific unintended effects. Furthermore， the article provided an in-depth analysis of how non-targeted multi-omics—encompassing genomics， transcriptomics， proteomics， and metabolomics—enable the comprehensive profiling of multi-layered molecular variations through full-spectrum scanning. Finally， the article explored the challenges and prospects of integrated multi-omics data analysis and artificial intelligence （AI） in future biosafety regulatory frameworks. Therefore， a systems biology strategy integrating multi-dimensional omics data， combined with machine learning and artificial intelligence， represents a crucial trend toward achieving comprehensive and precise food safety and unintended effects assessment of biotech breeding products.

Research Progress on Detection Methods of Genetically Modified and Gene-edited Products
Hua ZHANG, Yuteng ZHANG, Xinyao PEI, Litao YANG
2026, 16(4): 744-755. DOI: 10.19586/j.2095-2341.2026.0130
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With the continuous expansion in the commercialization scale of genetically modified （GM） crops and the rapid application of emerging biotechnologies such as gene editing， products derived from these novel biotechnologies have become increasingly complicated and diversified. Establishing sensitive， accurate and traceable detection methodologies and systems for such biotech products serves as an essential foundation for food safety assurance， labeling administration as well as regulatory law enforcement. The article systematically reviewed the research advances in the detection of GM and gene-edited products and other novel biotech-derived products in 2025， focusing on sample pretreatment， nucleic acid amplification assays （including quantitative real-time PCR and digital PCR）， isothermal amplification， clustered regularly interspaced short palindromic repeats-Cas protein （CRISPR/Cas）-assisted detection， biosensing and immunoassays， high-throughput sequencing， alongside databases and regulatory supporting systems. Overall， quantitative real-time PCR （qPCR） remains the core technique for quantitative analysis of genetically modified ingredients； digital PCR （dPCR） exhibits prominent advantages in absolute quantification of low-copy target sequences and value assignment of reference materials. Isothermal amplification and biosensing technologies keep facilitating the development of rapid and on-site detection. High-throughput sequencing provides technical support with superior resolution for the identification of gene-edited products. In the future， relevant detection technologies will further evolve toward the integrated application of multiple techniques， simultaneous identification of multi-target analytes， intelligent data analysis and compatibility with regulatory requirements.

Application and Prospect of CRISPR/Cas System in Genetically Modified Detection
Jiayi ZHOU, Lili ZHU, Gongwen HE, Dagang JIANG
2026, 16(4): 756-764. DOI: 10.19586/j.2095-2341.2026.0049
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With the widespread cultivation of genetically modified crops， there is an urgent need for efficient and accurate detection technologies to protect consumer rights and enable government regulation. Although PCR-based molecular detection techniques have been widely adopted， there remains scope for improvement in on-site rapid screening， simultaneous multi-target detection， and device portability. The clustered regularly interspaced short palindromic repeats-associated protein （CRISPR/Cas） system， with its unique and powerful cleavage activity， offers a breakthrough solution for developing simple， on-site detection technologies. Toward the application of the CRISPR/Cas system in genetically modified detection， this review summarized its recent advances and current implementations. It elaborated on and analyzed the technical principles and practical applications of molecular diagnostic platforms based on Cas9， Cas12， Cas13， and Cas14 proteins， including their advantages in enhancing detection sensitivity， enabling multi-target recognition， simplifying operational procedures， and promoting device portability. The paper also summarized the current limitations of these technologies and discussed future development directions， aiming to advance the progress of rapid， precise， and portable novel detection technologies.

Innovative Applications of Artificial Intelligence in Safety Assessment of Biological Breeding Products
Tianyi HAN, Yu GAO, Nan CHENG, Xiaoyun HE, Weichen LIU, Wei FU, Kunlun HUANG, Hong CHEN
2026, 16(4): 765-773. DOI: 10.19586/j.2095-2341.2026.0032
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Edible safety assessment of biologically breeding products is critical for preventing potential toxic and allergenic risks and protecting consumer safety. It is also indispensable for building consumer confidence and advancing the sustainable development of the industry. The rapid development of artificial intelligence （AI） has provided a brand-new technical path and method of support for the safety evaluation of biological breeding products. It not only enables non-destructive food testing but also significantly improves the accuracy and efficiency of the evaluation process. This article systematically reviewed the application progress of artificial intelligence in the edible safety evaluation of biological breeding products， such as genetically modified and gene-edited foods， from four aspects： multi-modal data sources， allergenicity and toxicological evaluation， nutritional evaluation， and adverse effect evaluation. The aim was to provide theoretical support for the more comprehensive application of artificial intelligence in edible safety evaluation.

Research Progress and Environmental Safety Assessment of Transgenic Cotton in China
Mengyao JIA, Deqin HU, Yuan LI, Dongmei WANG, Wei LU, Hongsheng PAN
2026, 16(4): 774-785. DOI: 10.19586/j.2095-2341.2026.0112
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Transgenic cotton is one of the most successfully commercialized transgenic crops worldwide. In 2023， its planting area accounted for 80.4% of the global total cotton acreage， and by 2025， the adoption rate in China had reached 95%. While transgenic cotton has effectively controlled Helicoverpa armigera infestation and significantly reduced the application of chemical pesticides， the evolution of target pest resistance， ecological risks associated with changing cropping structures， and persistent public concerns regarding the environmental safety of transgenic crops have emerged as core issues constraining the high-quality development of this industry. Against this background， this paper systematically reviewed the progress of transgenic cotton breeding， industrial application， and environmental safety assessment in China over the past three decades. It summarized the diversified breeding technology systems for insect resistance， herbicide tolerance， disease resistance， stress tolerance， and quality improvement， analyzed the mechanisms of Bt resistance evolution in H. armigera and the region-specific prevention and control strategies， and evaluated the ecological risks of transgenic cotton to non-target organisms， soil microecology， and gene flow. Furthermore， in view of the current layout of cotton-growing regions and frontiers in biotechnology， this paper prospected the future directions of gene-editing breeding， long-term ecological monitoring， and refined biosafety management， aiming to provide a scientific reference for the sustainable application of transgenic cotton and the high-quality development of China's cotton industry.

Research Progress on Ecological Risk Assessment of Genetically Modified Plants Based on Plant-associated Microbial Systems
Xiang REN, Mingchao YANG, Yin ZHANG, Yifan CHEN, Shengqian CHAO, Sheng CHEN, Yuping JIANG, Beibei LYU
2026, 16(4): 786-796. DOI: 10.19586/j.2095-2341.2026.0126
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Plant-associated microbial systems represent an important component of ecological risk assessment for genetically modified plants. However， the ecological effects and underlying mechanisms of genetically modified plants on microbial systems remain insufficiently understood. This review summarized recent advances in the impacts of genetically modified plants on rhizosphere microbiota， phyllosphere microbiota and endophytes. Focusing on rhizosphere microbiota， the effects of Bt-transgenic and herbicide-tolerant crops on microbial communities were analyzed， with emphasis on the regulatory roles of root exudates， soil environmental factors， and rhizosphere functional processes in microbial responses. Furthermore， the influences of host characteristics， environmental factors， and interactions among different ecological niches on microbial responses in phyllosphere microbiota and endophytes were summarized. The differences in microbial response patterns among various genetically modified traits were compared， and current research challenges were discussed. This review was expected to provide a theoretical basis for applying microbial indicators in ecological risk assessment and risk analysis of genetically modified plants.

Establishment of On-site Rapid Identification System for Transgenic Ingredients Based on Microfluidic Platform
Fangqi FU, Xinhao JIN, Wei FU, Pengchong JIANG, Jing GAO, Li CUI, Yimei CAI, Lufeng REN, Hong CHEN
2026, 16(4): 797-804. DOI: 10.19586/j.2095-2341.2026.0037
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Accurate identification and supervision of genetically modified （GM） crops are critical for biosafety assurance and seed industry development. Most existing detection methods rely on laboratory environments and fail to meet the practical requirements for rapid， on-site， and multi-target parallel identification. This study aimed to establish an integrated on-site rapid detection system that enables one-stop nucleic acid extraction， amplification and detection. Based on a handheld integrated GM on-site rapid detection device equipped with a high-monochromatic excitation light source， miniature spectrometer and semiconductor refrigeration module， supporting multi-target nucleic acid detection chips and sample processing tubes were developed via microfluidic technology and precision injection molding. The system achieves fully automated “sample-in， result-out” detection for plant seeds， leaves and other samples within 30 minutes， supporting simultaneous detection of up to seven targets with less than 1 minute of manual operation. The built-in battery allows eight independent detections on one full charge. In simulated samples， the limit of detection （LOD） for the MON810 event reached 0.1% （mass fraction） with no non-specific amplification. Reproducibility tests showed that the coefficient of variation （CV） of C_t values across different operators， instruments and test time points was less than 1.5%， and the results were highly consistent with those of conventional laboratory real-time PCR. This developed method enables accurate， rapid and portable on-site identification of GM ingredients， providing an efficient integrated solution for grassroots supervision and seed industry quality control.

A Nucleic Acid Mass Spectrometry-based Multi-target Detection Method for Genetically Modified Rice
Zehui ZHOU, Chen ZHANG, Rongyan SHEN, Ke WU, Na LIU, Ruihuan LI, Yasi WANG, Cheng WANG, Chao XU, Xin ZHAO, Qingkuo LAN, Yong WANG, Xin QI, Zhongyou PEI
2026, 16(4): 805-821. DOI: 10.19586/j.2095-2341.2026.0059
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Current techniques for detecting genetically modified （GM）rice primarily rely on conventional PCR， with no high-throughput， rapid screening methods available. This study used matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS） to create a multiplex primer panel for detecting multiple targets in GM rice. This method covers five target elements： the TT51 rice transformant-specific sequence， the Cry1A gene， the bar gene， theCaMV 35S promoter and the NOS terminator. The minimum detection limit for target genes was 0.5%. The established detection method offers high-throughput， multi-target capability， strong specificity and high sensitivity， offerring robust technical support for safety regulation， identity authentication and traceability management of genetically modified rice.

Preparation of a Monoclonal Antibody Against SpCas9 Protein and Application of a Sandwich ELISA for Screening Gene-edited Rice
Zhaozhao SI, Zaobing ZHU, Dongdong LIU, Yongping WANG, Litao YANG
2026, 16(4): 822-832. DOI: 10.19586/j.2095-2341.2026.0042
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Rapid and accurate detection of SpCas9 protein in transformed plants is essential for positive plant selection and transgene elimination during gene-edited crop development， yet reliable detection methods validated for plant matrices remain limited. In this study， mouse monoclonal antibodies （mAbs） and rabbit polyclonal antibodies （pAbs） were generated against prokaryotically expressed recombinant SpCas9 protein. A sandwich enzyme-linked immunosorbent assay （ELISA） was established by systematic pairing of 12 mAbs with pAbs， using mAb #10 as the capture antibody and pAbs as the detection antibody， followed by comprehensive analytical performance validation. The results showed that all antibody titers exceeded 1∶729 000. The sandwich ELISA showed excellent linearity over 1.50~48.00 ng·mL^-1 （R2=0.998 4） with a limit of detection （LOD） of 0.56 ng·mL^-1. Intra-assay and inter-assay coefficients of variation were 0.98%~3.90% and 0.70%~7.34%， respectively. Spike recoveries ranged from 97.0% to 109.1%， and dilution linearity recoveries from 96.8% to 114.1%. No cross-reactivity was observed with SaCas9， FnCas12a， BrCas12b or LwaCas13a. The specificity of the sandwich ELISA was validated by Western blotting， and the assay was applied to T₂-generation gene-edited rice leaves. Among 16 SpCas9-positive rice lines， SpCas9 protein contents ranged from 10.30±0.13 to 150.26±0.25 ng·g^-1 fresh weight， with a 14.6-fold variation among the lines. No detectable SpCas9 protein was detected in wild-type rice samples （below the detection limit）. The established sandwich ELISA exhibited high sensitivity， specificity， and reproducibility， and was suitable for analysis in complex plant matrices. This method provides a rapid and cost-effective tool for screening positive gene-edited plants， evaluating Cas9 expression levels， and identifying transgene-free homozygous lines during gene-edited crop development.

A Qualitative Detection Method for Genetically Modified Maize KJ1003 by Real-time PCR
Tongtong ZHAO, Ziyan CHEN, Hua ZHANG, Xinyao PEI, Chenyao WANG, Jiake LI, Dan WANG, Yueqiu ZHANG, Wei FU, Hong CHEN
2026, 16(4): 833-842. DOI: 10.19586/j.2095-2341.2026.0027
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The genetically modified maize line KJ1003 is a transformation event developed by Kejidalong （Beijing） Biotechnology Co.， Ltd. It exhibits excellent insect resistance and herbicide tolerance， has obtained a genetically modified organism safety certificate， and possesses promising industrialization prospects. In this study， a real-time fluorescent PCR qualitative detection method for the KJ1003 transformation event was established， targeting the specific sequence of KJ1003 through primer and probe design and screening， reaction system optimization， as well as specificity， sensitivity， limit of detection， and robustness tests， together with inter-laboratory validation. The results showed that this method could effectively detect KJ1003 transgenic components and amplify a 158 bp specific fragment， with a detection sensitivity and limit of detection both reaching 0.05% and favorable robustness. The verification results of the method′s specificity， limit of detection and reproducibility conducted by 8 laboratories all complied with relevant standard requirements. This study provides a scientific basis and technical support for the detection and safety supervision of genetically modified crops in China.

PCR Detection Method for Transgenic Maize KJ1003
Fangrui GAO, Ziyan CHEN, Tongtong ZHAO, Hongye JIANG, Hua ZHANG, Yichen LIAO, Xiaodan JIANG, Fangqi FU, Yahui LI, Haoqian WANG, Hong CHEN
2026, 16(4): 843-852. DOI: 10.19586/j.2095-2341.2025.0179
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The genetically modified maize KJ1003 is a strain developed by Kejidalong （Beijing） Biotechnology Co.， Ltd. It shows insect resistance and herbicide tolerance has obtained a genetically modified organism safety certificate and has a good industrialization prospect. This study established a qualitative PCR detection method for maize KJ1003， through primer design， primer screening， specificity test， sensitivity test， detection limit test， stability test， and laboratory validation， with the specific sequence of KJ1003 as the target. Results showed that a 165 bp specific fragment could be amplified， with a detection sensitivity of 0.05% and a limit of detection of 0.1%， and had a good stability. Verification results of specificity， detection limit， repeatability and reproducibility for this method from 8 laboratories all met the national standard requirements. This study provides a scientific basis and technical support for the detection and safety supervision of genetically modified crops in China.

Establishment of a Real-time Quantitative PCR Detection Method for Insect-resistant and Herbicide-tolerant Maize BBL2-2
Mei DONG, Lixia MENG, Tingting WEN, Chaohua MIAO, Wujun JIN
2026, 16(4): 853-862. DOI: 10.19586/j.2095-2341.2026.0041
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The adoption of genetically modified （GM） maize has expanded rapidly in China， and it has become the crop with the highest number of safety certificates issued for genetically modified organisms （GMOs）. Therefore， it is essential to establish a specific， sensitive， and operable detection method for GM maize lines. This study designed specific primer-probe sets based on the flanking sequences of the insect-resistant and herbicide-tolerant maize event BBL2-2， establishing a real-time quantitative PCR detection method for this event. The method exhibited high specificity. Its limit of detection （LOD） reached 0.05% （equivalent to 20 copies）， and the limit of quantification （LOQ） reached 0.1% （equivalent to 40 copies）. The standard curve showed excellent linearity， and both the trueness and precision of the method were ≤25%， complying with relevant standard requirements. Inter-laboratory validation results were consistent with internal test results， demonstrating good repeatability and reproducibility. The established uncertainty assessment method was highly operable， ensuring the reliability of quantitative results and providing a basis for labeling threshold management. The developed real-time quantitative PCR detection method for insect-resistant and herbicide-tolerant maize BBL2-2 offers technical support for detection and regulatory oversight， facilitating the commercial deployment of this GM maize event.

Development of Multi-target Plasmid DNA Reference Materials for Screening Transgenic Soybeans
Pengfei LIU, Na LIU, Ruihuan LI, Wei FU, Chao XU, Qingkuo LAN, Yong WANG, Xin ZHAO
2026, 16(4): 863-877. DOI: 10.19586/j.2095-2341.2026.0029
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Soybeans are rich in plant proteins and edible oils， and also provide beneficial nutrients such as isoflavones and lecithin， making them important crops for food， feed， and oil production. China's demand for and import volume of soybeans have increased year by year， with import dependence at times exceeding 80%. Five transgenic soybean events （DBN9004， DBN8002， SHZD32-1， ZH6106， and CAL16）， which were independently developed in China and have obtained biosafety certificates for genetically modified organisms， were used as research objects. On the basis of the standard qualitative and quantitative detection methods for each transgenic event and its derivative varieties， the five transgenic soybean events and the endogenous standard gene Lectin were assembled into a binary vector. A series of experiments， including plasmid molecular verification， screening of plasmid preservation medium， method specificity validation， and detection applicability and interchangeability testing using genomic DNA， was then carried out to develop the plasmid DNA reference molecule pUC-soy-identification for transgenic screening. The results showed that the sequence of the prepared plasmid reference molecule was completely consistent with the target sequence， and both restriction enzyme digestion and qPCR amplification produced the expected results. The addition of salmon sperm DNA helped bring the copy number ratio closer to the theoretical value. The plasmid preparation met the specified criteria for use as a positive control in specificity， applicability， and interchangeability tests targeting DBN9004， DBN8002， SHZD32-1， ZH6106， and CAL16. Therefore， the resulting plasmid reference molecule can be applied to the quantitative detection of the aforementioned transgenic soybeans and their processed products， and provides a robust technical tool for the biosafety management and labeling compliance of genetically modified organisms in China.

Study on Rapid Detection Method for CRISPR/Cas9-mediated Gene-edited High-oleic Soybean
Xiaoya YUAN, Zhen ZHEN
2026, 16(4): 878-884. DOI: 10.19586/j.2095-2341.2025.0077
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Gene editing， as an important and emerging cutting-edge biotechnology， has been widely applied in fields， such as life science research， species improvement， and human health. Currently， more than ten species of animals and plants have been approved for commercialization. The detection technology of gene-edited plants is the core support for controlling biosafety and regulating the seed industry market. Due to different regulatory requirements， detection methods and a standardized and complete monitoring system are crucial. Based on this， for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 （CRISPR/Cas9）-mediated gene-edited high-oleic soybean， multiple sets of specific primer probe sequences were designed by comparing their gene sequences with wild-type soybean. A set of primer probe sequences that met the requirements was screened using fluorescence PCR method， with a detection sensitivity of up to 0.01%， good stability， and specificity in the digital PCR detection system. The establishment of detection methods not only provides technical support for biosafety supervision， but also lays the foundation for the formulation of relevant detection standards.

DNA Extraction Techniques for Genetically Modified Organism Testing of Soybean Products
Yingying WANG, Wenlong LIU, Juntian ZENG, Fulu CHEN, Li LIU, Jindong FU, Longguo JIN
2026, 16(4): 885-897. DOI: 10.19586/j.2095-2341.2026.0030
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Establishing an effective DNA extraction technique for soybean products to meet the increasingly complex and diverse demands for transgenic detection helps verify product label authenticity， safeguard food safety， and protect consumers' rights. This method improved the standard SDS-PVP-based approach by precipitating proteins with an ammonium acetate salt solution， detecting genomic characteristics of soybean products， and validating the method's effectiveness through the detection of the Lectin gene. The results showed that the DNA concentration extracted by this method was relatively high， ranging from 30 to 5 683 ng·μL^-1. Based on absorbance ratios and agarose gel electrophoresis results， most samples were relatively pure with minimal protein contamination. Electrophoretic analysis showed that the main DNA bands were concentrated around 100 bp， with increased genomic degradation and shorter fragments observed as processing depth increases. The average quantification cycle （Cq） values for Lectin gene detection range from 19.99 to 33.63， meet the requirements for subsequent transgenic detection. This method has broad applicability and yields high-quality DNA， which would contribute to advancing transgenic detection in soybean processed foods and the formulation of related standards.

Research on Fourth-generation Sequencing Based on Biological Nanopores
Xinxin ZHANG, Tianli GUO, Hua GUAN
2026, 16(4): 898-908. DOI: 10.19586/j.2095-2341.2025.0148
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As a crucial pillar of molecular biology， sequencing technology is experiencing rapid development worldwide. Nanopore sequencing technology has emerged as the leading fourth-generation sequencing technology， addressing the limitations of previous-generation technologies in terms of throughput， read length， portability and cost. By comparing the key parameters of traditional sequencing and nanopore sequencing， this study elaborated on the principles of biological nanopore sequencing and the composition of sequencing instruments， analyzed the structural characteristics and functional adaptability of biological nanopore proteins， expounded on the application scenarios of nanopore sequencing in genomics， transcriptomics， epigenetics and other fields， and prospected for the development of sequencing technology. This paper aims to provide a comprehensive reference for researchers to deeply understand nanopore sequencing technology， and facilitates the further promotion， optimization and upgrading of this technology in life science research and clinical practice.

Research Advances and Clinical Application Prospects of in Vivo CAR-T Therapy
Mingdi ZHANG, Qigai LI, Lu WANG, Hui OUYANG, Jinghua LIAN, Fei XIE
2026, 16(4): 909-918. DOI: 10.19586/j.2095-2341.2025.0159
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Chimeric antigen receptor T-cell （CAR-T） therapy， as a revolutionary immunotherapy method， has demonstrated remarkable clinical efficacy in the treatment of hematological malignancies. However， the traditional in vitro CAR-T therapy is limited in wide application due to numerous problems. In recent years， the research on in vivo CAR-T therapy， which directly modifies T cells in the body， holds the promise of overcoming the limitations of traditional therapies. This article reviewed the technical principles，latest research progress， clinical application potential， safety issues， and future development directions of CAR-T therapy in vivo， aiming to provide comprehensive references for clinical research on malignant tumors and promote the further optimization and transformation of innovative therapy.

Analysis of Subcellular Localization and Expression Patterns of Soybean Transcription Factor GmMYB109
Hongqi ZHANG, Yuehua YU, Zhiyong NI
2026, 16(4): 919-925. DOI: 10.19586/j.2095-2341.2026.0057
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To investigate the function of the GmMYB109 transcription factor in soybean， we performed bioinformatics analysis on its sequence， examined its subcellular localization using transient expression in tobacco， and determined its expression levels in different tissues and at different time points under salt stress by qRT?PCR. Bioinformatics prediction showed that GmMYB109 contains two SANT domains and is localized in the nucleus. Transient expression assays confirmed its nuclear localization in tobacco epidermal cells， indicating that GmMYB109 is a nuclear?localized transcription factor. qRT?PCR results revealed that GmMYB109 was most highly expressed in leaves， followed by stems， cotyledons and roots. Its expression gradually increased under salt stress， suggesting that GmMYB109 is induced by salt stress. These results provide theoretical basis for further exploring the biological function and regulatory mechanism of the GmMYB109 transcription factor.

Screening of Salt-tolerant, Low-Cr-accumulating Hulless Barley Varieties and Their Growth Response Characteristics
Xinping ZHANG, Ma MI, Chongshuang PAN, Lu LI, Qinghai LIU, Cheng QIU, Bin WU
2026, 16(4): 926-933. DOI: 10.19586/j.2095-2341.2026.0073
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To address the need for safe hulless barley production and varietal selection in Xizang under the combined pressures of escalating soil salinization and chromium （Cr） environmental risk， this study evaluated eight barley varieties. Under NaCl stress conditions， germination rate， germination index， vigor index and early seedling growth parameters were measured. Based on these results， five varieties were selected for subsequent screening under Cr stress， where root and shoot biomass， Cr content， bioconcentration factor （BCF）， and translocation factor （TF） were determined. The results indicated that NaCl stress significantly inhibited barley germination and seedling growth， with the inhibitory effect intensifying as salt concentration increased. The varietal differences were most pronounced under 200 mmol·L^-1 NaCl. Comprehensive analysis identified Zangqing 148， Zangqing 320， Zangqing 2000， Shanqing 9， and Xila 19 as salt-tolerant varieties suitable for further Cr stress testing. Under Cr stress， the biomass of the five varieties generally decreased with increasing concentration. Cr was significantly enriched in the roots， with all TFs being less than 1. Among the varieties， Shanqing 9 exhibited the lowest Cr content in shoots， as well as the lowest BCF and TF， establishing it as a superior salt-tolerant and low-Cr-accumulating variety. This study provides data support for selecting barley varieties suited to environments with combined salinization and chromium contamination risks.

Optimization of Rapid Propagation System of Chinese Herbal Medicine Reynoutria japonica Houtt. in Vitro
Ming LIU, Junxin ZHANG, Zhimin HE, Liping YU, Tiran HUANG, Lanqing MA, Liusheng DUAN
2026, 16(4): 934-940. DOI: 10.19586/j.2095-2341.2023.0105
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To rapid propagate high-quality Reynoutria japonica Houtt. seedlings and optimize a rapid propagation system of Reynoutria japonica Houtt. in vitro tissue， the effects of different disinfection times on the stem segments of Reynoutria japonica Houtt. and different hormone concentrations in the culture medium on the proliferation induction and adventitious bud rooting of the stem segments of Reynoutria japonica Houtt. were investigated. The results showed that the best sterilization method for the stem segment with axillary buds of Reynoutria japonica Houtt. was 75% alcohol 15 s+（0.1% Tween-20+2% sodium hypochlorite） for 12 min， and the acquisition rate of sterile explants reached 90.89%. The optimal medium for inducing axillary bud sprouting in stem segments was MS+30 g·L^-1 sucrose+7 g·L^-1 agar+1.5 mg·L^-1 kinetin+0.2 mg·L^-1 naphthalene acetic acid， with a proliferation rate of 113.3%. The optimal medium for rooting culture of adventitious buds was 1/2 MS+30 g·L^-1 sucrose+5.5 g·L^-1 agar+3 mg·L^-1 green plant growth regulator （GGR6）. The rooting rate could reach 70.37% and adventitious roots could be differentiated after about 3 days， and complete plants could be obtained after about 15 days. The survival rate of transplanted tissue cultured seedlings after rooting reached 100%. This study optimized a rapid culture system for stem segments of Reynoutria japonica Houtt.， with a sterile explant acquisition rate， proliferation rate， and transplant survival rate of over 90%. Moreover， the rooting time is fast， which can significantly shorten the transplant time and reduce reproduction costs.

Current Status and Hotspot Analysis on Tobacco Biotechnology Research Based on Bibliometrics
Pan WEI, Peijian CAO, Qiansi CHEN
2026, 16(4): 941-951. DOI: 10.19586/j.2095-2341.2026.0013
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Using the Web of Science （WOS） database and the VOSviewer literature analysis tool， the relevant literature in the field of tobacco biotechnology research from 2005 to 2024 was analyzed to explore the research status and hot spots of tobacco biotechnology. The results showed that the number of tobacco biotechnology research papers published in the United States and China in the past two decades ranked the top two in the world. Especially since 2020， the number of tobacco biotechnology research papers published in China has become the first in the world. The United States has the most frequent cooperation in tobacco biotechnology research with countries around the world， and China temporarily ranks third. The research strength of tobacco biotechnology in China has been greatly improved， showing a good development trend and gradually narrowing the gap with developed countries in Europe and the United States. However， more efforts are still needed in international exchange and cooperation and high-quality research. Biological breeding， functional gene research， microbial resource development and utilization， biosynthesis and other fields are the hotspots of tobacco biotechnology research in recent years. It is necessary to further increase investment in basic scientific research and pay attention to the transformation of scientific research results.

Application of bar Gene Screening Markers in Genetic Transformation of Flammulina filiformis
Axiu MAO, Yeran SHANG, Xiaodong SHANG, Guirong TANG, Zhonglin ZHANG
2026, 16(4): 952-959. DOI: 10.19586/j.2095-2341.2026.0048
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Flammulina filiformis is an economically important edible and medicinal mushroom. However， its genetic transformation has historically relied solely on hygromycin resistance as a selectable marker and this limitation has impeded research into the coordinated regulation of multiple genes. In this study， we established an Agrobacterium tumefaciens-mediated genetic transformation system for F. filiformis using the herbicide resistance gene bar and glufosinate-ammonium as the selectable marker and agent， respectively. Two vectors were used， pAgbar， in which the bar gene is driven by the constitutive gpd promoter from Agaricus bisporus； and pAgbarG， which harbors a bar-egfp translational fusion expression cassette. Both constructs were delivered into the monokaryotic strain Dan3， yielding stable， glufosinate-ammonium-resistant transformants. Transformation efficiency was markedly higher on M-100 medium than on PDA. PCR and Southern blot hybridization confirmed the integration of the exogenous bar gene into the F. filiformis genome. Moreover， the bar-egfp fusion system enabled effective fluorescence signal expression， providing a rapid and visual means of identifying positive transformants. This study establishes a genetic manipulation platform for F. filiformis using bar as a resistance selectable marker， offering reliable technical support for functional gene analysis， molecular-assisted breeding， and mechanistic studies of secondary metabolic pathway regulation.

Molecular Property, Phyletic Evolution and Gene Expression Pattern of Apis mellifera Transcription Factor Capicua
Yuanyuan LIU, Yongsheng LI, Jiarun YANG, Xiongjie WANG, Yuan CHENG, Yazhou LU, He ZANG
2026, 16(4): 960-967. DOI: 10.19586/j.2095-2341.2025.0190
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This study aimed to investigate the function of the RNA transcription factor Capicua （AmCIC） in the Apis mellifera. Bioinformatic analyses were conducted to predict the physicochemical properties， molecular characteristics， conserved motifs， and functional domains of AmCIC， and its relative expression levels were examined by RT-qPCR. The results showed that the predicted molecular formula of AmCIC is C₉₀₅₀H₁₄₃₃₉N₂₆₅₃O₂₉₃₂S₅₉， with a molecular weight of 209.11 kD， an aliphatic index of 62.64， and an isoelectric point of 7.42. The protein exhibits a hydrophilicity index of -0.760 and lacks both transmembrane domains and signal peptides. Subcellular localization analysis indicated that AmCIC is distributed in the nucleus， mitochondria， and cytoplasm， suggesting that it is a hydrophilic， non-transmembrane intracellular protein. Comparative analysis revealed that CIC proteins from seven bee species share two conserved domains and five conserved motifs， with A. mellifera and Apis laboriosa clustering closely in phylogenetic analyses. Expression profiling showed that AmCIC is expressed in all examined tissues of adult worker bees， with the highest expression detected in the brain. During larval development， AmCIC expression was highest at the egg stage， while among adult workers of different ages， peak expression occurred at 12 days post-emergence. Notably， following Ascosphaera apis inoculation， AmCIC expression in the larval midgut was extremely significantly downregulated （P<0.001）， indicating that AmCIC is involved in the host response to pathogen infection. Collectively， these results suggest a potential role for this transcription factor in honeybee development and antifungal immune responses.

Isolation of Phosphate-solubilizing Bacteria from Alpine Mining Soil and Evaluation of Their Cadmium Immobilization Capacity
Mingzhen HAN, Qinghai LIU, Bin WU, Junqi HU, Cheng QIU, Chongshuang PAN
2026, 16(4): 968-978. DOI: 10.19586/j.2095-2341.2026.0074
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Phosphate-solubilizing bacteria-mediated biomineralization has great potential for heavy metal remediation， yet its application efficiency and underlying mechanisms in fragile alpine ecosystems remain poorly understood. A cold-tolerant phosphate-solubilizing strain Serratia quinivorans SH was isolated from an alpine tailings reservoir， and its phosphorus-solubilizing capacity， cadmium （Cd） tolerance and Cd immobilization mechanism under low-temperature conditions were systematically explored. The results showed that the maximum net soluble phosphorus concentration of strain SH reached 210.6 mg·L^-1 under low temperature， and the strain exhibited prominent tolerance to Cd stress at 5~40 mg·L^-1. Furthermore， the net soluble phosphorus concentration ranged from 187.2 to 210.6 mg at 5~15 ℃， with the optimal pH of 7~8 and optimal C/N ratio of 20∶1. Adsorption-precipitation experiments revealed that the maximum net Cd adsorption rate and net precipitation rate of strain SH were 83.3% and 35%， respectively. Characterizations via scanning electron microscopy-energy dispersive X-ray spectroscopy （SEM-EDS） verified that strain SH transformed Cd into stable phosphate precipitates. Whole-genome sequencing analysis further identified functional genes involved in phosphorus solubilization and heavy metal resistance. The results showed that the cold-resistant phosphate-solubilizing strain Serratia quinivorans SH possesses low-temperature phosphorus-solubilizing ability and Cd biomineralization capacity， which can provide strain resources and theoretical support for the remediation of heavy metal pollution in alpine tailings.

Mechanistic Study on Total Flavonoids from Hedyotis diffusa Willd. Inhibiting Lung Cancer Through the CDCA8-p53-glycolysis Axis
Yue LUO, Xing JIANG, Chao WANG, Yingqiu WU, Hongwei LUO, Lu ZHANG
2026, 16(4): 979-989. DOI: 10.19586/j.2095-2341.2025.0147
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This study aimed to investigate the molecular mechanism by which total flavonoids of Hedyotis diffusa Willd. （HDF） exert anti-lung cancer effects through modulation of the CDCA8-p53-glycolysis axis. A549 cell models with CDCA8 knockdown/overexpression were constructed， and cell proliferation， colony formation， migration， and invasion abilities were assessed. Quantitative real-time PCR（qPCR） and Western blot were employed to detect the expression of CDCA8， p53， and key glycolytic enzymes. Glucose uptake， lactate production， and ATP levels were measured using colorimetric assays. Cells were treated with varying concentrations of HDF， and their effects on the CDCA8-p53-glycolysis axis and malignant cell phenotypes were evaluated. The results showed that CDCA8 knockdown significantly inhibited A549 cell proliferation， migration， and invasion （P<0.01）， upregulated p53 and p-p53 （Ser15） protein expression （P<0.05）， downregulated the expression of key glycolytic enzymes GLUT1， HK2， and PKM2 （P<0.05）， and reduced glucose uptake， lactate production， and ATP levels （P<0.05）； CDCA8 overexpression exhibited opposite effects. The IC₅₀ of HDF against A549 cells was 1.6 mg·mL^-1. HDF dose-dependently downregulated CDCA8， upregulated p53/p-p53， inhibited the expression and metabolism of key glycolytic enzymes （P<0.05）， and significantly suppressed cell proliferation， migration， and invasion abilities （P<0.05）. The results indicated that HDF exerts anti-lung cancer effects by downregulating CDCA8 expression， activating the p53 signaling pathway， and inhibiting glycolysis.

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