Molecular Microbiology Research 2025, Vol.15 http://microbescipublisher.com/index.php/mmr © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher.
Molecular Microbiology Research 2025, Vol.15 http://microbescipublisher.com/index.php/mmr © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Publisher MicroSci Publisher Edited by Editorial Team of Molecular Microbiology Research Email: edit@mmr.microbescipublisher.com Website: http://microbescipublisher.com/index.php/mmr Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Molecular Microbiology Research (ISSN 1927-5595) is an open access, peer reviewed journal published online by MicroSci Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all areas of molecular microbiology, including original articles, reviews and brief reports in microbiology, bacteriology, mycology, molecular and cellular biology and virology at the level of gene expression and regulation, genetic transfer, cell biology and subcellular organization, pathogenicity and virulence, physiology and metabolism, cell-cell communication and signalling pathways as well as the interactions between the various cell systems of microorganisms including the interrelationship of DNA, RNA and protein biosynthesis. All the articles published in Molecular Pathogens are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MicroSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights. MicroSci Publisher is an international Open Access publisher specializing in microbiology, bacteriology, mycology, molecular and cellular biology and virology registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada.
Molecular Microbiology Research (online), 2025, Vol. 15, No. 1 ISSN 1927-5595 http://microbescipublisher.com/index.php/mmr © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content Agrobacterium-Mediated Genetic Transformation Techniques in Cucumis Chunxia Wu Molecular Microbiology Research, 2025, Vol. 15, No. 1, 1-9 Observation of Immune Gene Expression in Goats Under FMD Virus Infection Wenzhong Huang, Zhongmei Hong Molecular Microbiology Research, 2025, Vol. 15, No. 1, 10-17 E. coli Pathogenesis: From Commensal to Pathogenic Strains Chunyang Zhan Molecular Microbiology Research, 2025, Vol. 15, No. 1, 18-27 Review of Disease Resistance Mechanisms in Sweet Potato: Pathogen Response Pathways Jianquan Li Molecular Microbiology Research, 2025, Vol. 15, No. 1, 28-36 Improving Nitrogen Fixation in Soybean: Insights into Rhizobium Interactions Dan Luo Molecular Microbiology Research, 2025, Vol. 15, No. 1, 37-44
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 1 Review Report Open Access Agrobacterium-Mediated Genetic Transformation Techniques in Cucumis Chunxia Wu Modern Agricultural Research Center of Cuixi Academy of Biotechology, Zhuji, 311800, Zhejiang, China Corresponding email: chunxia.wu@cuixi.org Molecular Microbiology Research, 2025, Vol.15, No.1 doi: 10.5376/mmr.2025.15.0001 Received: 02 Nov., 2024 Accepted: 30 Dec, 2024 Published: 15 Jan, 2025 Copyright © 2025 Wu, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wu C.X., 2025, Agrobacterium-mediated genetic transformation techniques in Cucumis, Molecular Microbiology Research, 15(1): 1-9 (doi: 10.5376/mmr.2025.15.0001) Abstract Cucumis plants play an important role in global agricultural production, including common crops such as cucumbers and melons. To meet the demand for high yield, disease-resistant and environmentally friendly crop varieties, Agrobacterium-Mediated Transformation is widely used to improve the traits of these crops. This study systematically reviews the application of this technology in cucumber plants, and explores the mechanism of T-DNA metastasis, factors that affect transformation efficiency, and how to improve transformation efficiency by optimizing Agrobacterium rhizogeness strains, culture conditions, selection markers and reporter genes. In addition, the study also introduced the specific plans for the transformation of cucumber and melon genes in detail, analyzed the current challenges faced in the transformation process, such as resilience, low conversion rate and genetic stability, and proposed solutions. Through case studies and practical field applications, this study summarizes the successful practice of Agrobacterium rhizogenes mediated transformation technology in improving disease resistance, abiotic stress tolerance and nutritional quality improvement. Looking ahead, this technology will provide important support for sustainable breeding and agricultural production of cucumber plants. Keywords Cucumis; Agrobacterium; Genetic transformation; Transformation efficiency; Sustainable agriculture 1 Introduction Crops like cucumbers and melons are usually common on our dining table, but in fact they are also very important in agricultural production. They are not only able to sell money, but are also nutritious, rich in vitamins, minerals and dietary fiber. They have long been one of the protagonists in the agricultural product supply chain (Chai et al., 2020). However, these alone are not enough. Now people pay more attention to new varieties that are high-yield, disease-resistant, and able to withstand environmental changes, while traditional breeding methods are increasingly unable to keep up with this demand. It’s not that traditional methods are useless, but that they are often too slow and too limited. At this time, gene conversion technology has become a new breakthrough. Among them, Agrobacterium rhizogenes mediated transformation technology is considered to be a relatively common method in plant genetic engineering and has good results. It essentially uses Agrobacterium rhizos to “suffle” specific genes into the plant genome, thereby changing certain traits of crops (Wang et al., 2015; Liu et al., 2023). Of course, not all plants are so easy to "treat". Cucumber is quite picky. Problems such as low conversion efficiency and large differences in different genotypes have always been a hurdle for the implementation of technology (Chai et al., 2020a; Liu et al., 2023). Therefore, it is still necessary to improve this method (Fan et al., 2020). This study mainly revolves around this aspect. It not only talks about the technology itself, but also analyzes in detail some key factors that will affect the efficiency of transformation, such as which type of material is used, how to match the culture conditions, how to perform different genotypes, etc. We also sorted out some optimization methods. In addition to the technical level, this study also summarizes the practical potential of this method in improving disease resistance, stress resistance and nutritional quality. For scientific researchers or breeding experts, these contents can provide many operational ideas and lay the foundation for sustainable breeding and improvement of cucumber plants in the future.
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 2 2 Agrobacterium-Mediated Transformation: Basic Principles 2.1 Mechanism of T-DNA transfer Agrobacterium rhizogenes are considered an old acquaintance when sending foreign genes into plants. This transformation method is widely used mainly by a bacteria called Agrobacterium tumefaciens. It is not a high-tech product, but a bacteria in nature that can "secretly" stuff a piece of DNA (called T-DNA) into the plant genome. This T-DNA stays on its Ti plasmid, usually quiet, but once the bacteria hit the wound part of the plant, things start to work (Lacroix and Citovsky, 2013). How did T-DNA be transferred ? There are still many steps. At the beginning, plant wounds will release some phenolic compounds, such as ferulic acid ketone, which trigger the vir gene activation in the bacteria. Then, the bacteria cut the T-DNA off the plasmid and then wrapped it with proteins such as VirD2 and VirE2 to form a structure called the T complex (Gelvin, 2010). This complex will then enter the plant cells and then enter the nucleus. However, entering the nucleus is not the end point. Next, the T complex will be untied, and T-DNA will then be inserted into its genome through the plant's own DNA repair mechanism. The insertion location is usually random, but the target gene can be expressed stably as long as the integration is successful (Gelvin, 2010). Of course, this whole process does not necessarily mean success. Whether bacterial strains are selected well and whether the plant cell status is good will affect the final transformation efficiency (Lacroix and Citovsky, 2013). 2.2 Factors influencing transformation efficiency When using Agrobacterium rhizogenes to make the cucumber (Cucumis sativus L.) gene transformation, efficiency is not simple, it is affected by many factors. Sometimes even if the steps are the same, the results may be completely different. Such as the genotype of cucumber, the age of the explant, the type of strain used, and even the details of the infection will determine the success or failure. There is a big difference between genotypes, and this cannot be ignored. Cucumbers in northern China perform much better in bud regeneration than pickled or sliced varieties in the United States (Figure 1) (Liu et al., 2023). Explants are more popular at younger ages, such as cotyledons or hypocotyls near the apex, and their regeneration ability is generally higher (Liu et al., 2023). Let’s talk about the strain. Not all strains are common, for example, the positive frequency of AGL1 is higher than that of GV3101, while EHA105 is more suitable for use on cucumber variety Poinsett 76 (Wang et al., 2015). So how to choose a strain is also a science. Of course, the handling conditions are also critical. What is the concentration of bacterial solution you use? Is ferulic acid ketone added? How long does infection and co-culture last? Once these variables change, the result may be different. Some people also found that precultivating explants in advance, or wiping the ingredients of the culture medium can help the formation of transgenic buds (Thiruvengadam et al., 2013). The screening stage should not be taken lightly. The use of screening agents such as kanamycin is actually quite effective. They can screen out the truly transformed cells without wasting time (Thiruvengadam et al., 2013; Liu et al., 2023). 3 Techniques for Enhancing Transformation Efficiency 3.1 Optimization of agrobacterium strains 3.1.1 Strain selection and genetic modifications Not all Agrobacterium rhizogenes are suitable for use on cucumber plants, which has become apparent in the experiment. Some strains show better than others. For example, the conversion efficiency of AGL1 is much higher than that of GV3101 (Liu et al., 2023). Although GV3101 may be OK on other crops, it is not very useful when it comes to the genus Cucumber. In addition to these two common strains, the transformation results of EHA105 and LBA4404 in some other plants are also quite good, which also shows a problem-the selection of strains cannot be casually (Thiruvengadam et al., 2013). Moreover, if the effect of ready-made strains is not satisfied, some studies simply use hands-on modification, such as introducing plasmids such as pCAMBIA2300 into the strain to improve their transformation ability (Bhatt et al., 2021). From this perspective, not only do you have to choose the right strain, but sometimes you have to "train" it.
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 3 3.1.2 Impact of virulence factors When it comes to gene transformation, virulence factors are not optional. They can directly affect the results, especially when you want to increase conversion efficiency. Many people have tried adding ferulic ketone into the culture medium, and the effect is indeed quite obvious. Plants like Mangji and cucumber, with ferulic acid ketone when infected or co-cultured, the conversion rate is significantly higher (Chai et al., 2020; Bhatt et al., 2021). But then again, you can't do anything randomly if you add it. It is useless to have too low concentration, and it may be suppressed if it is too high. Studies have found that the transformation effect is best with 100 µM ferulic acid ketone, especially in the pre-culture and co-culture stages (Udayabhanu et al., 2022). Therefore, it is not only a question of whether to add or not, but also a fine control of how much to add and how much to add. Figure 1 Agrobacterium stain AGL1 exhibited higher transformation efficiency than GV3101 (Adopted from Liu et al., 2023) Image caption: Transgenic plants were developed with RNAi constructs for the CsAPRR2 gene in a PS76 background. Plantlets in the jars were generated from GV3101-mediated (A) and AGL1-mediated (D) transformation, respectively. T0 transgenic plants grown in soil were mediated by GV3101 (B) and AGL1 (E), respectively. PCR verification identified positive T0 transgenic plants from GV3101-mediated (C) and AGL1-mediated (F) transformation, respectively. In (C,F), the first lane is a size marker, and lanes 1-4 are the positive control and three regenerated plantlets from independent transformation events. Scale bar = 1.0 cm (Adopted from Liu et al., 2023) 3.2 Pre-culture and co-cultivation conditions The conversion into unsuccessful results are sometimes not a problem with bacteria, but a lack of good grasp of the operation details. The two stages of pre-cultivation and co-cultivation are simple and simple, and the details are quite particular. Environmental conditions, processing time, whatever is wrong, the conversion efficiency may be reduced in the end. For example, the experiment of Brazilian rubber tree found that the T-DNA transfer effect was much better when controlling the co-culture temperature at 22 °C and allowing the explant to stay in the dark for 84 hours (Udayabhanu et al., 2022). The research on cucumbers is similar. Pre-culture plus dark conditions, combined with a little ferulic acid ketone can also significantly improve the conversion effect (Chai et al., 2020). However, once the conditions are done, the materials themselves cannot be ignored. Some explants are older, but they are not very "obedient". Generally speaking, the younger the tissue, the higher the conversion rate, which has also been verified in cucumber experiments (Liu et al., 2023). 3.3 Selection markers and reporter genes How to determine whether the conversion has been successful, You have to choose the right tool. Marker genes and reporter genes come in handy at this time. Kanamycin is a commonly used selective antibiotic, but the amount
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 4 used is not static, it depends on which step the transformation is carried out. Taking cucumber as an example, the concentration during the early screening is generally 25 mg/L. When it comes to the rooting stage, it is necessary to add 100 mg/L to be sufficient (Chai et al., 2020). If the concentration is set too low, it may not be clean, and if it is too high, it may easily cause the regenerated tissue to be "inadvertently injured". In terms of reporter genes, GUS (β-glucuronidase) should be the most common type. It's good that it can make people intuitively see whether the transformation has been successful. It is intuitive to determine whether the gene is expressed through tissue staining, whether it is transient or stable (Thiruvengadam et al., 2013; Bhatt et al., 2021). Therefore, screening is not just a technical step, but also a "quality barrier" that must be maintained. 4 Protocols for Genetic Transformation in Cucumis 4.1 Transformation in Cucumis sativus (cucumber) Agrobacterium rhizogenes has been used in the study of gene transformation in cucumbers, especially in the production of complex plants with transgenic root systems. However, traditional methods often have many steps and are complicated to operate, so they also choose the experimental conditions. In order to save trouble, Fan et al. (2020) proposed a more direct solution. They did not use the conventional two-step method and did not set up any complicated culture medium. The method is actually quite simple: inoculate Agrobacterium rhizobium with the target gene directly into the oblique incision of the hypocotyl of the 5-day-old cucumber seedlings, and then plant the seedlings into moist sterile vermiculite, and all other steps are saved (Figure 2) (Fan et al., 2020). It sounds a bit risky, but the success rate is unexpectedly high-more than 90% of seedlings have grown roots with genetically modified ones. The efficiency and simplicity of this "one-step method" have opened up new ideas for the verification of promoter functions in cucumbers and root system research. While saving time and effort, it also avoids the damage problems caused by mid-way transplantation, which is a fast and stable alternative. Figure 2 Agrobacterium rhizogenes-mediated root transformation of cucumber achieved by one-step (Adopted from Fan et al., 2020) Image caption: a: Five days old healthy seedlings; b: The apical portion of the hypocotyl was cut diagonally (0.5 cm cut) in the liquid of K599 harboring the desired gene construct; c: A slant cut of the residual hypocotyl was scraped and inoculated on the plate grown A. rhizogenes K599 harboring the desired gene construct. Note the bacterial mass coated in the slant cut; d: Explant inoculated was directly planted into a pot with wet sterile vermiculite; e: An example of composite plant induced at 21 dpi. Bars = 1 cm (Adopted from Fan et al., 2020) 4.2 Transformation in Cucumis melo (melon) The conversion technology of Cucumis melo was not smooth from the beginning. In the early days, problems such as tetraploidy, chimera, and escape often made it difficult to advance the experiment. However, in the early 1990s, the situation began to change. Akasaka-Kenedy et al. found a breakthrough through the liquid cultured somatic
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 5 embryo system and successfully solved these difficult problems (Nonaka and Ezura, 2015). Later, a research team tried to use the entire germinated seed as an explant, and regenerate the cotyledon nodule area and stem junction as targets. The effect is not bad, the conversion frequency of melon reaches 13%, and plants with exogenous genes can be detected by waiting for about a month (Zhang et al., 2014). More importantly, these transgenic plants are almost exactly the same as wild type in appearance and growth, and there are no obstacles to commercialization. Of course, the increase in regeneration rate has never stopped. Researchers have successively tested microbrushing, sonication, vacuum infiltration and other operations, with the purpose of improving infection efficiency. Some people simply start from the molecular level, such as overexpressing developmental regulatory genes such as AtGRF5 and AtPLT5, which does bring about higher conversion success rates and makes conversion less dependent varieties (Wan et al., 2023). Speaking of Oriental Melo (Cucumis melo var. makuwa), there is also a specialized method. Cotyledons were used as explants, benzyl adenine and indoleacetic acid were added to MS medium, and then GUS staining, PCR and Southern hybridization were used to confirm whether the transgene was integrated (Choi et al., 2012). When using kanamycin and genetic dyskinin during screening, the conversion rates were 2.9% and 7.1%, respectively. Although it is not too high, it has practical value. And when it comes to gene editing, researchers are not idle either. Strategies like "optimal infiltration intensity" are specially customized for cucurbita plants and optimize the transformation process. Using this method, CRISPR/Cas9 has successfully knocked out some genes from the ERECTA family and obtained mutants with shorter internodes and more compact plants (Xin et al., 2022). 5 Challenges and Solutions in Cucumis Transformation 5.1 Recalcitrance and low transformation rates When using Agrobacterium rhizogenes for gene transformation of Cucumis, an old problem has not been completely solved - it is not easy to "serve" and is not easy to transform. Especially cucumber (Cucumis sativus L.), the conversion efficiency has been low. Although technology has made a lot of progress in recent years, it is still not easy to make it stable "receive" foreign genes. There are many factors that affect efficiency, and we cannot just blame the technology for not being able to do it. Such as the genotype of cucumber, the origin and age of explants, the selection of strains, etc., they may all influence the experimental results. For example, the regeneration rate of cucumber buds in northern China is much stronger than that in the United States, which shows that the genotype will indeed widen the gap (Liu et al., 2023). There are also some considerations in explants. Organs that are too old are not very "obedient", and young tissues are generally better used. Faced with these difficulties, the researchers did not fail to do anything. These methods have been tried, such as adjusting infection and co-culture conditions, and using screening agents such as kanamycin, and have certain effects. In terms of strains, AGL1 has a higher conversion efficiency than GV3101 (Liu et al., 2023). Some people have tried to add ferulic acid ketone to the culture medium at different stages, and the results show that the conversion efficiency has also been improved (Chai et al., 2020a; 2020b). Overall, the conversion rate can reach about 0.2% to 1.7%. It is not high, but it has been a significant improvement compared to the situation that was almost impossible to achieve in the early years (Liu et al., 2023). 5.2 Somaclonal variation and genetic stability Somatic mutation is also a headache, especially during the transformation process, which may cause genetic instability in the plants. Sometimes, even though the target gene is introduced, the grown plants do not perform as expected, and the problem may lie here. This phenomenon is actually not uncommon in the tissue culture of cucumbers (Pawełkowicz et al., 2021). The mechanism of somatic cell mutation has not been fully understood yet, and it is indeed complicated to say that it is complicated. However, judging from the analysis of the transcriptome, some clues can still be found. For example, polymorphisms in gene regions may have some interaction with molecular networks, which will activate specific signaling pathways, which in turn affect gene expression (Pawełkowicz et al., 2021). Although this kind of mutation brings trouble, it is not entirely worthless - sometimes, it can provide some useful genetic differences that may come in handy in breeding.
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 6 In order to reduce this uncertainty, researchers have also put a lot of effort into cultivating conditions. For example, which explant is used, it is very important. Experiments have found that cotyledon explants are more stable than other types and have a lower incidence of somatic cell mutation (Pawełkowicz et al., 2021). In addition, the combination and timing of antibiotics are not casually set. Some studies have pointed out that drugs such as cefotaxime sodium and temetin can not only inhibit Agrobacterium rhizogenes, but also reduce the occurrence of somatic mutations to a certain extent (Chai et al., 2020a; 2020b). 6 Applications of Genetic Transformation in Cucumis 6.1 Development of disease-resistant varieties To improve the disease resistance of cucumber plants, traditional breeding does not always keep up with the pace, especially when facing complex diseases, progress is often slow. At this time, the gene transformation technology mediated by Agrobacterium rhizogenes becomes particularly useful. It does not rely on hybridization, but directly "send" disease-resistant genes into the plant body to enhance resistance from the source. Some experiments have proved that this path works. For example, after introducing specific disease resistance genes into cucumbers (Cucumis sativus), the disease resistance of plants is significantly improved, and can be topped off when some traditional methods are not effective (Chai et al., 2020a; 2020b). More importantly, this transformation is not just the kind that "try it" - using Agrobacterium rhizogenes with pCAMBIA2300s plasmid, which not only can be transformed, but can also stably integrate the target gene and subsequent expression is also reliable. 6.2 Enhancing abiotic stress tolerance Whether the cucumber genus grows well in the fields sometimes depends not only on how much fertilizer is applied and how fine the pipe is, but also on abiotic stresses such as drought, saline and extreme temperatures may greatly reduce both yield and quality. To deal with this kind of problem, conventional breeding alone is not very useful. Researchers have also begun to use gene transformation to find breakthroughs in recent years. Agrobacterium nodule mediated transformation technology has already been used in this regard. There is a very representative example: researchers transferred the reversible gene in peas (Pisum sativum) to citrus plants, and the results showed that both drought resistance and salt resistance have been significantly improved (Hasan et al., 2019). Although this experiment is not done for cucumbers, it provides a way of thinking - a similar method can be used entirely on cucumber plants. And it’s not just a talk, but there are already related attempts. Chai et al. (2020a; 2020b) have introduced stress-resistant genes into cucumbers, and it turns out that this technology can indeed play a role in improving abiotic stress tolerance. 6.3 Improvement of nutritional and quality traits When it comes to cucumber plants, many people may think of yield or disease resistance, but in fact their nutrition and quality are also a part of what they pay great attention to when breeding. How to improve these indicators, traditional breeding efficiency is too slow, and conversion technology becomes an important tool. Agrobacterium rhizogenes-mediated gene transformation has been used to introduce some nutrition-related genes, such as those that can make the fruit contain more vitamins or minerals. In addition to nutrition, some studies have also tried to improve flavor, fruit size, and even extend freshness (Ziemienowicz et al., 2014; Chai et al., 2020a; 2020b). Although it is not easy to improve either, technical attempts are being made slowly. Of course, importing genes alone is not enough. To improve success rates, researchers also worked on transformation programs, such as the use of certain antibiotics or the addition of ferulic acid ketone, which showed that these detailed operations can indeed make the improvement process more efficient (Chai et al., 2020a; 2020b). 7 Case Studies and Field Applications 7.1 Successful examples in commercial cultivation Can Agrobacterium rhizogenes be used in commercial breeding of cucumbers? Many experiments have given answers. Examples of this variety like Poinsett 76 are quite typical. The researchers used the EHA105 strain. After optimizing cotyledon explants and screening systems, the conversion efficiency suddenly reached 23% (Wang et al., 2015). This is relatively high in cucumber transformation, and it also makes this method a genetic
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 7 improvement tool worth referring to. In addition to cucumber itself, it may actually be used for the production of vaccines or certain biological materials, and there is a lot of room for expansion. Of course, this is not the only success story. The cucumber variety "Xintai Mischi" in northern China has also made progress. This time it was replaced by the GV3101 strain, and the researchers mainly optimized the use of antibiotics and ferulic acid ketone, and finally achieved an 8.1% conversion efficiency (Chai et al., 2020a; 2020b). Although not as high as Poinsett 76, this is already one of the best levels that can be found in the "New Tai Misty" variety. More importantly, this method also shows good application potential in improving stress resistance and improving nutritional quality, and is easier to implement in actual cultivation. 7.2 Comparative analysis of transformation techniques There have been many "versions" of cucumber gene transformation technology in recent years. Once there are many methods, the effects will naturally be high and low. The most commonly used old method is to use the EHA105 strain combined with cotyledon explants. This combination is most widely used because of its stable transformation efficiency (Wang et al., 2015). But that doesn't mean other methods are not worth a try. Such as vacuum penetration combined with filter paper core co-culture, it sounds not traditional, but it really improves the conversion efficiency. On the one hand, it can enhance the infection effect of Agrobacterium rhizogenes, and on the other hand, it also reduces explant necrosis, with an average conversion rate of 11.9% (Nanasato et al., 2012). This idea obviously solves the infection bottleneck more "treat" with it. There is also a more "quick knife" approach, which is to use Agrobacterium rhizogenes to generate a complex plant with a genetically modified root, a stem or a wild type. This single-step method saves a lot of trouble. The success rate of transgenic roots can exceed 90%, which is particularly convenient for functional gene analysis or studying root traits (Fan et al., 2020). As for CRISPR/Cas9, it is still in the exploratory stage on cucumbers. Its efficiency is not as good as the traditional method for the time being, but its advantage is that it is more directional. For example, by regulating the penetration intensity, researchers have made plants with shorter internodes and more compact structures, which shows that this technology does have some potential in the cucurbitae (Xin et al., 2022). 8 Concluding Remarks In plant genetic engineering, the transformation system of Agrobacterium rhizogenes can be said to be unavoidable, especially when studying cucumber plants, it is used very much. But its advantages are not outstanding from the beginning. Many improvements have actually been accumulated slowly in recent years. For example, the transient transformation method was not favored by many people in the early stage, but later because it was fast, step-saving, and easy to scale, it has now been widely used in the research of functional genomes and recombinant proteins. In terms of efficiency, traditional transformation has not been improved, especially after choosing the right genotype, explant and strain, some solutions can even achieve a conversion rate of 23% - on crops like cucumbers, this achievement is not low. In addition, there is a one-step approach that has also attracted attention: directly using the root strain bacteria to generate a composite plant of genetically modified roots and wild-type stems, which does not require staged processing, the process is simple, and the effect is quite stable. In recent years, technical details like this have been continuously optimized, making Agrobacterium rhizogenes more and more like a "standard configuration in the toolbox" in crop improvement and genetic research. Although transformation technology has made a lot in recent years, the problem has not been solved. Some old problems are stuck there. For example, some cucumber varieties cannot improve their efficiency no matter how they are done - this is directly related to the genotype. In other words, it is not a general solution that can handle all the materials, and targeted optimization still needs to be done. Another direction that is attracting great attention now is combining CRISPR/Cas9 with Agrobacterium rhizogenes transformation technology. Although traditional methods are stable, their accuracy is limited; while CRISPR can be edited in a fixed location. If used in conjunction, it may better control the conversion effect and reduce some biosafety concerns. But then again, whether the gene can be inserted stably is not enough. It also needs to figure out where it lands in the genome, whether it causes mutations or large-scale rearrangements. If you don’t understand this kind of
Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 8 problem, it will be difficult to improve the reliability and predictability of genetically modified plants. How to go in the future? In addition to improving conversion efficiency and editing accuracy, a better screening system is also a point worth working on. Looking further away, the potential of cucumber in vaccine carriers and biomaterial production may also be a new way to explore. The concept of sustainable agriculture is quite big, but often it starts with a little technological breakthrough. Agrobacterium rhizogenes mediated gene transformation is one of the directions that make people see the changes. It doesn’t solve all problems out of thin air, but it does play an increasingly role in improving crop adaptability, nutrition and disease resistance. Genetically modified cucumbers, which are resistant to insects and diseases, are a very typical example. After making it, the number of pesticides used can be significantly reduced, and the burden on the environment is also much lighter. This approach is indeed more sustainable than relying on chemical control alone. In addition, some people may have overlooked that it is not just a matter of "farming". Research is using plants to produce recombinant proteins and other biologically active substances, using this transformation system. This alternative production method is not only energy-saving, but also more environmentally friendly than traditional industrial processes. Although it is not a perfect solution, it is certain that as long as this technology continues to move forward, there is still a lot of room for it to play in dealing with major issues such as food security and environmental pressure. Acknowledgments I express my sincere thanks to the Biotechnology Research Center of Cuixi Academy of Biotechnology for their provision of essential resources for this research. I also appreciate the reviewers for their valuable recommendations, which helped in improving the manuscript. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Reference Bhatt R., Asopa P.P., Jain R., Kothari-Chajer A., Kothari S.L., and Kachhwaha S., 2021, Optimization of Agrobacterium mediated genetic transformation in Paspalum scrobiculatum L. (Kodo Millet), Agronomy, 11(6): 1104. https://doi.org/10.3390/AGRONOMY11061104 Chai L.A., Du C.X., Fan H.F., Liu C., and Si Y.Y., 2020a, Improved Agrobacterium tumefaciens-mediated transformation of cucumber via modified use of antibiotics and acetosyringone, Research Square, 2020: 1-28. https://doi.org/10.21203/rs.3.rs-38940/v1 Chai L., Fan H., Liu C., and Du C., 2020b, The progress of transgenic cucumber mediated by Agrobacterium tumefaciens, Trends in Horticulture, 3(1): 93-101. https://doi.org/10.24294/th.v3i1.1791 Choi J., Shin J., Chung Y., and Hyung N., 2012, An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa), Plant Cell Tissue and Organ Culture (PCTOC), 110: 133-140. https://doi.org/10.1007/s11240-012-0137-6 Fan Y., Xu F., Zhou H., Liu X., Yang X., Weng K., Sun X., and Lyu S., 2020, A fast simple high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation, Plant Cell Tissue and Organ Culture (PCTOC), 141: 207-216. https://doi.org/10.1007/s11240-020-01781-x Gao D.J., Zhang Q., Xu T.B., Zhou P., Cheng W.J., and Zhang W.W., 2024, Bioinformatics identification and expression profiles of SBP family genes in cucumber (Cucumis sativus L.), Plant Gene and Trait, 15(1): 8-14. https://doi.org/10.5376/pgt.2024.15.0002 Gelvin S., 2010, Plant proteins involved in Agrobacterium-mediated genetic transformation, Annual Review of Phytopathology, 48: 45-68. https://doi.org/10.1146/annurev-phyto-080508-081852 Hasan N., Kamruzzaman M., Islam S., Hoque H., Bhuiyan F.H., and Prodhan S.H., 2019, Development of partial abiotic stress tolerant Citrus reticulata Blanco and (Citrus sinensis L.) osbeck through Agrobacterium-mediated transformation method, Journal of Genetic Engineering and Biotechnology, 17(1): 14. https://doi.org/10.1186/s43141-019-0014-3 Křenek P., Šamajová O., Luptovčiak I., Doskočilová A., Komis G., and Šamaj J., 2015, Transient plant transformation mediated by Agrobacterium tumefaciens: principles methods and applications, Biotechnology Advances, 33(6): 1024-1042. https://doi.org/10.1016/j.biotechadv.2015.03.012
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Molecular Microbiology Research, 2025, Vol.15, No.1, 10-17 http://microbescipublisher.com/index.php/mmr 10 Research Insight Open Access Observation of Immune Gene Expression in Goats Under FMD Virus Infection Wenzhong Huang, Zhongmei Hong Tropical Animal Medicine Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572000, Hainan, China Corresponding author: zhongmei.hong@hitar.org Molecular Microbiology Research, 2025, Vol.15, No.1 doi: 10.5376/mmr.2025.15.0002 Received: 03 Nov., 2024 Accepted: 05 Jan., 2025 Published: 26 Jan., 2025 Copyright © 2025 Huang and Hong, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Huang W.Z., and Hong Z.M., 2025, Observation of immune gene expression in goats under FMD virus infection, Molecular Microbiology Research, 15(1): 10-17 (doi: 10.5376/mmr.2025.15.0002) Abstract This study explores the immune gene expression patterns of goats under foot-and-mouth disease virus (FMDV) infection to reveal the key immune response mechanisms of the host in response to viral infection. Foot-and-mouth disease is a highly infectious disease affecting even-unghou animals and poses a serious threat to global animal husbandry. This study uses high-throughput sequencing technology to conduct transcriptome analysis of goat immune cells infected with FMDV to identify differentially expressed genes (DEGs), and explore the functions and pathways of these genes in the immune response through gene ontology (GO) annotation and KEGG pathway enrichment analysis. This study provides important data for a deep understanding of the host-pathogen interactions of goats under FMDV infection, and provides a theoretical basis for the development of targeted vaccines and gene editing technologies, which has important practical application value. Keywords Foot-and-mouth disease virus; Goat immune response; Differentially expressed genes; Transcriptome analysis; Vaccine development 1 Introduction Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cows, sheep, pigs and other even-unged animals. Goats are one of them. Once infected, not only will the animal health be affected, but it will also cause serious economic losses, especially in developing countries (Lüet al., 2018). Therefore, controlling and preventing FMDV has become a focus of the animal husbandry industry. To do a good job of prevention and control, we must first figure out how animals respond to the virus. Goats are common in animal husbandry, but there is not much research on how they resist FMDV at present. In contrast, there are much more research on cattle and pigs (Li et al., 2015). However, existing studies have pointed out that genes such as immune-related can play a big role in viral infection (Li et al., 2019). This shows that it is necessary to further understand the immune mechanism of goats when infected with FMDV. To this end, we conducted this study. We will collect goat immune cells infected with FMDV and use high-throughput sequencing for transcriptome analysis. We intend to use RNA-Seq technology to find differentially expressed genes (DEGs) and see what role these genes play in the immune response. At the same time, we will conduct gene ontology (GO) annotation and KEGG pathway enrichment analysis to classify these genes to see which immune processes they are involved in. Ultimately, we hope to find some key immune regulation mechanisms to provide data support for the prevention and treatment of foot-and-mouth disease and vaccine development. 2 Overview of Foot-and-Mouth Disease Virus 2.1 Classification and genomic characteristics of FMDV Foot and mouth disease virus (FMDV) is a particularly contagious virus that affects many farmed animals around the world. It mainly infects even-hung species, such as goats, cattle, pigs, sheep, and some wild animals can also be infected (Eschbaumer et al., 2016). This virus belongs to the Picornaviridae family, Picornaviridae, and is a single positive-strand RNA virus. A major feature of FMDV is its fast mutation, which is why it is difficult to completely control or eliminate. Its genome is not complex, it only has one RNA strand. This chain can encode
Molecular Microbiology Research, 2025, Vol.15, No.1, 10-17 http://microbescipublisher.com/index.php/mmr 11 various proteins needed by the virus, including non-structural proteins involved in replication, as well as structural proteins that make up the viral shell (Lazarus et al., 2020). 2.2 Transmission routes and epidemiology FMDV can be spread in many ways. Contact between animals can be contagious, contaminated things can also be poisonous, and short-distance aerosols in the air may also spread. What’s even more troublesome is that the virus can survive in the environment for a while, which makes it less easy to prevent and control. From the perspective of epidemics, FMDV can easily cause large-scale outbreaks, not only affecting domestic animals, but sometimes even wild animals can be infected. Each epidemic will bring considerable economic losses, especially for the livestock industry (Rodríguez-Habibe et al., 2020). 2.3 Clinical manifestations and pathological features of fmd in goats Once a goat is infected with FMDV, it usually has fever and blisters, and common locations include hooves, mouth and nipples. Some goats will start to limp. Compared with other even-unged animals, goats have similar pathological manifestations, and blisters are the most obvious characteristics. These blisters sometimes break and are prone to infection after breaking, and the situation may become serious. However, the virulence of different strains is different, and the immunity of each animal is different, so the severity of the disease will also vary (Mitoma et al., 2021). 3 Immune Genes and Antiviral Response Mechanisms 3.1 Role of the innate immune system in fmdv infection Once FMDV invades, the innate immune system will take action first. This is the first line of defense for the body. It activates defense mechanisms by identifying the "pattern" of the virus, such as receptors such as TLR and RIG-I, which will be activated (Liu and Huang, 2024). In the experiment, the researchers used DAPI staining to check the nucleus and found that the distribution was even; and staining with cytokeratin can also clearly see the infected cells, indicating that these cells are in good condition and are suitable for subsequent analysis. In a group of cells treated with 3D-7414 siRNA, the signal of the FMDV-3D gene was significantly weakened. The control group (using the U6 snRNA probe) has a strong signal, which indicates that the viral RNA is still there. This shows that siRNA can effectively suppress the expression of viral RNA and thus prevent viral replication. These results further demonstrate that in the transgenic model of goats, innate immunity does play a key role when the virus first invades (Figure 1) (Li et al., 2015). Figure 1 Expression analysis of 3D-7414siRNA in epithelium cells (Adopted from Li et al., 2015)
Molecular Microbiology Research, 2025, Vol.15, No.1, 10-17 http://microbescipublisher.com/index.php/mmr 12 3.2 Adaptive immune response and related gene expression 3.2.1 B-cell-mediated humoral immune response and antibody production After adaptive immunity is initiated, B cells are activated and then specific antibodies are started. These antibodies recognize and neutralize FMDV, preventing the virus from entering the cells. In this process, a gene called C1QB also plays a role, which enhances the complement system and thus improves the efficiency of the antibody (Lüet al., 2018). 3.2.2 T-cell-mediated cellular immune response (differentiation and activation of CD4+ and CD8+ T cells) In addition to B cells, T cells are also very important, especially CD4+ and CD8+. They are activated after the antigen is recognized and then are involved in the removal of infected cells. Some studies have pointed out that genes like TIMD4 are related to T cell activation, which shows that T cells play a very critical role in fighting FMDV (Chen et al., 2021). 3.2.3 Establishment of immunological memory and its role in secondary infection The immune system also "remembers" the virus. This is immune memory. After the initial infection, some B and T cells are converted into memory cells. When FMDV appears again, they can react quickly and effectively control the virus. This is important in preventing recurrence (Hussain et al., 2019). 3.2.4 Role of MHC molecules in antigen presentation and gene polymorphism To make T cells work, the first thing to do is to have an antigen "display". This process relies on MHC molecules. They deliver viral antigens to T cells, guiding the direction of the immune response. There are differences in MHC genes, and this diversity will affect their efficiency and will also affect the ability of the entire immune system to respond to FMDV (Nahas et al., 2021). 3.3 Cytokine network function in the antiviral process Cytokines are also very active during FMDV infection. These small molecules, like "messengers", are responsible for transmitting immune signals between cells. For example, TNF, IL-6 and IL-10 are involved in the inflammatory response and the mobilization of immune cells. Studies have found that the pathways related to these cytokines are significantly activated in FMDV infection (Wani et al., 2018). They can help control virus replication and also help the body quickly start defense. 4 Immune Gene Expression Analysis Results 4.1 Differential expression of immune genes in early, middle, and late stages of infection When FMDV infects goats, the expression of immune genes changes at different stages. In the early stages of infection, the expression of cytokines such as IL6, CXCL2, CCL20, CCL4, and transcription factors such as NFKBIA will be increased. These genes and molecules are primarily responsible for initiating inflammation and immune responses (Zhang et al., 2018). In the mid-term, some chemokines have been downregulated, such as CCL23 and CXCL17. This change may affect the arrival of immune cells, making it easier for the virus to stay and continue to infect (Zhu et al., 2020). In the late stages, the situation was more complicated. The study found that some genes associated with Th17 immune response are upregulated, while the classic NF-κB pathway is downregulated. This combination may make the virus easier to stay in the body and less easily cleared (Zhu et al., 2022). 4.2 Functional analysis of upregulated and downregulated genes The study also analyzed what all genes with obvious expression changes did during the infection process. It was found that many upregulated genes are related to immune and inflammatory pathways, such as those involved in the cytokine-receptor interaction pathway or the TNF signaling pathway. These pathways are important to fight viruses. Some downregulated genes are often related to cell apoptosis or cell killing. If these genes are attenuated, the body may not be able to remove the virus smoothly, resulting in the persistence of the virus (Eschbaumer et al., 2016). In addition, some chemokines that attract neutrophils or T cells are also inhibited after infection. This may leave the immune system “cannot find the direction” and thus allow the virus to escape (Wani et al., 2019).
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