Bioscience Method 2024, Vol.15 http://bioscipublisher.com/index.php/bm © 2024 BioSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Bioscience Method 2024, Vol.15 http://bioscipublisher.com/index.php/bm © 2024 BioSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. BioSci Publisher is an international Open Access publisher specializing in bioscience methods, including technology, lab tool, statistical software and relative fields registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher BioSci Publisher Editedby Editorial Team of Bioscience Methods Email: edit@bm.bioscipublisher.com Website: http://bioscipublisher.com/index.php/bm Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Bioscience Methods (ISSN 1925-1920) is an open access, peer reviewed journal published online by BioSci Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all areas of bioscience, the range of topics including (but are not limited to) technology review, technique know-how, lab tool, statistical software and known technology modification. Case studies on technologies for gene discovery and function validation as well as genetic transformation. All the articles published in Bioscience Methods 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. BioSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Bioscience Methods (online), 2024, Vol.15, No.5 ISSN 1925-1920 https://bioscipublisher.com/index.php/bm © 2024 BioSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Study of Post-Harvest Preservation Techniques for Loquat and its Application in Reducing Post-Harvest Losses Xiuying Zhao Bioscience Methods, 2024, Vol.15, No.5, 207-215 CRISPR-based Gene Editing in Bt for Improved Insecticidal Properties Wenfei Zhang Bioscience Methods, 2024, Vol.15, No.5, 216-225 The Integration of Genetic Markers in Maize Breeding Programs Qiong Chen, Qiaohong Ying, Kaozu Lei, Junmei Zhang, Huazhou Liu Bioscience Methods, 2024, Vol.15, No.5, 226-236 Probiotics from Tea Fermentation: Potential Applications in Health Products Yali Deng, Haomin Chen Bioscience Methods, 2024, Vol.15, No.5, 237-243 Comprehensive Genomic Analysis of Atractylodes macrocephala: Unveiling Its Medicinal Functions and Genetic Secrets Xichen Wang, Huixian Li, Chuchu Liu Bioscience Methods, 2024, Vol.15, No.5, 244-254
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 207 Research Insight Open Access Study of Post-Harvest Preservation Techniques for Loquat and its Application in Reducing Post-Harvest Losses Xiuying Zhao Traditional Chinese Medicine Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, China Corresponding author: xiuying.zhao@cuixi.org Bioscience Methods, 2024, Vol.15, No.5 doi: 10.5376/bm.2024.15.0021 Received: 01 Jul., 2024 Accepted: 11 Aug., 2024 Published: 01 Sep., 2024 Copyright © 2024 Zhao, 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: Zhao X.Y., 2024, Study of post-harvest preservation techniques for loquat and its application in reducing post-harvest losses, Bioscience Methods, 15(5): 207-215 (doi: 10.5376/bm.2024.15.0021) Abstract Loquat (Eriobotrya japonica) holds significant importance in agriculture due to its nutritional value and economic relevance. However, post-harvest losses remain a major challenge due to its rapid ripening and sensitivity to environmental factors such as temperature and humidity. This study provides a comprehensive analysis of the post-harvest physiology of loquat, including key factors contributing to spoilage. Traditional preservation techniques, such as cold storage, modified atmosphere packaging (MAP), chemical treatments, and dehydration methods, are discussed, along with emerging technologies like nano-coating, natural antimicrobials, and smart packaging systems. Through a comparative analysis of these methods' efficiency, economic viability, and environmental impact, this study also evaluates the integration of preservation techniques within supply chains and their role in reducing post-harvest losses through a regional case study. Lastly, this study explores future directions for loquat preservation, focusing on technological advancements, regulatory frameworks, and sustainability. The findings are expected to provide insights into improving post-harvest management and reducing loquat losses. Keywords Loquat; Post-harvest preservation; Shelf-life extension; Storage techniques; Food sustainability 1 Introduction Loquat (Eriobotrya japonica Lindl.) is a subtropical evergreen tree that holds significant agricultural value due to its diverse uses and nutritional benefits. The fruit is not only consumed fresh but is also processed into various products such as jams, jellies, juices, wines, and canned fruits. Additionally, loquat leaves and seeds are utilized in the development of food products and for extracting valuable compounds like starch and oil (Shah et al., 2023). The fruit is rich in essential nutrients, including vitamin A, ascorbic acid, calcium, iron, manganese, and potassium, and contains pharmacologically active constituents such as kaempferol, ursolic acid, and quercetin, which contribute to its anti-inflammatory, antitumor, antioxidative, and antidiabetic properties (Dhiman et al., 2021). Despite its nutritional and economic importance, loquat faces significant post-harvest challenges that limit its shelf life and marketability (Fu et al., 2020). The fruit is highly perishable and susceptible to various physiological disorders and decay after harvesting. Common post-harvest issues include physical and mechanical damage, moisture and nutrient loss, and decay. Additionally, loquat is prone to chilling injury and flesh browning during low-temperature storage, which further complicates its preservation (Jing et al., 2022). Techniques such as modified atmosphere packaging (MAP), controlled atmosphere (CA) storage, and treatments with compounds like 1-methylcyclopropene (1-MCP) and methyl jasmonate (MeJA) have been explored to extend the shelf life and maintain the quality of loquat fruit (Pareek et al., 2014). This study comprehensively evaluates the various post-harvest preservation techniques for loquat and their effectiveness in reducing post-harvest losses; covers physical, chemical, and biological methods of preservation, highlights recent advancements and innovations in the field. By synthesizing information from past and current research, this study provides a clear understanding of the best practices for loquat preservation, thereby aiding researchers, farmers, and industry stakeholders in improving the post-harvest management of this valuable fruit.
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 208 2 Post-Harvest Physiology of Loquat 2.1 Ripening process and physiological changes Loquat (Eriobotrya japonica Lindl.) is a non-climacteric fruit, meaning it does not continue to ripen significantly after being harvested. However, some cultivars exhibit ripening patterns similar to climacteric fruits, which complicates post-harvest management (Zhang et al., 2020). The ripening process in loquat involves several physiological changes, including a decrease in fruit firmness and an increase in sweetness and acidity balance, which are critical for consumer acceptance. During post-harvest storage, loquat fruit undergoes lignification, a process where lignin accumulates in the flesh, leading to increased firmness and reduced quality (Liu et al., 2019). This lignification is associated with the activities of enzymes such as phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) (Cañete et al., 2015). 2.2 Key factors contributing to post-harvest losses Post-harvest losses in loquat are primarily due to mechanical damage, moisture loss, and decay caused by pathogens. Mechanical damage during harvesting and handling can lead to bruising, which accelerates deterioration. Moisture loss results in wilting, shriveling, and a decline in fruit texture and flavor, significantly reducing marketability. Pathogens such as anthracnose, canker, and purple spot are prevalent post-harvest diseases that contribute to decay and spoilage. Additionally, chilling injury (CI) during cold storage can cause browning and other disorders, further reducing the fruit's quality and shelf life (Lufu et al., 2020). 2.3 Sensitivity to external factors (temperature, humidity, etc.) Loquat fruit is highly sensitive to external factors such as temperature and humidity, which play crucial roles in its post-harvest physiology. Low-temperature storage is commonly used to extend shelf life, but it can also lead to chilling injury, characterized by lignification and browning of the flesh. Controlled atmosphere storage, hypobaric storage, and modified atmosphere packaging are some techniques used to mitigate these effects. High humidity levels can exacerbate moisture loss and decay, while optimal humidity conditions can help maintain fruit quality. The rate of respiration and ethylene production in loquat fruit is significantly influenced by storage temperature, with higher temperatures accelerating deterioration (Cai et al. 2006). Therefore, maintaining appropriate temperature and humidity levels is essential for minimizing post-harvest losses and preserving the quality of loquat fruit (Ding et al., 1988). 3 Traditional Post-Harvest Preservation Techniques 3.1 Cooling and cold storage Cold storage is a widely used method for preserving the quality of loquat fruit post-harvest. This technique helps in reducing the metabolic rate of the fruit, thereby slowing down the processes that lead to spoilage. However, loquat fruit is susceptible to chilling injury (CI), which can cause browning and other quality issues. Studies have shown that while cold storage can extend the shelf life of loquat, it can also lead to lignification of the flesh tissue, reducing the fruit's quality and economic value (Figure 1) (Zhang et al., 2022). To mitigate these effects, low-temperature conditioning (LTC) and heat treatments have been explored. These methods have been found to alleviate lignification and maintain the fruit's quality during storage (Su et al., 2023). Zhang et al. (2022) investigated the role of the antioxidant system in controlling reactive oxygen species (ROS) during the cold storage of loquat. The study highlighted the importance of enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), among others, in mitigating ROS-induced chilling injuries. These enzymes work in concert to maintain redox balance and protect cellular structures by neutralizing ROS. Additionally, the involvement of the ascorbic acid-glutathione (AsA-GSH) cycle was noted, with components such as reduced and oxidized glutathione (GSH and GSSG) playing crucial roles. The interaction of these antioxidants helps prevent cellular damage, thus extending the shelf life of loquats under cold storage. The findings emphasize the significance of both enzymatic and non-enzymatic antioxidants in enhancing fruit resistance to cold-induced oxidative stress.
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 209 Figure 1 The antioxidant system involved in the control of ROS during cold storage of loquat (Adopted from Zhang et al., 2022) Image caption: SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GPX, glutathione peroxi-dase; PRX, peroxiredoxin; TRX, thioredoxin; MDHA, monodehydroascorbate reductase; MDHAR, dehydroascorbate reductase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; AsA, ascorbic acid (Adapted from Zhang et al., 2022) 3.2 Modified atmosphere packaging (MAP) Modified Atmosphere Packaging (MAP) is another effective technique for extending the shelf life of loquat fruit. MAP involves altering the atmospheric composition around the fruit to slow down respiration and delay spoilage. Research has demonstrated that MAP can significantly reduce water loss and maintain the organic acid levels in loquat fruit, although it may not significantly affect total sugars. Different gas compositions in MAP, such as high nitrogen or low oxygen environments, have been tested. For instance, packaging loquat in 100% nitrogen (MAPN2) has been shown to limit browning and microbial growth, thereby preserving the fruit's sensory and nutritional qualities. However, the storage temperature is crucial, as higher temperatures can lead to increased decay even under MAP conditions (Palumbo et al., 2022). 3.3 Chemical treatments and their role Chemical treatments are often employed to control microbial decay and physiological disorders in loquat fruit. These treatments can include the application of fungicides, antioxidants, and other chemical agents. For example, konjac glucomannan coatings have been found to significantly reduce decay rates and maintain higher levels of total soluble solids, titratable acidity, and ascorbic acid in loquat fruit (Liu et al., 2019). Additionally, chemical treatments can be combined with other preservation methods, such as cold storage and MAP, to enhance their effectiveness. The use of chemical treatments, however, must be carefully managed to avoid residues that could affect consumer health and safety (Ding et al., 2006). 3.4 Drying and dehydration techniques Drying and dehydration are traditional methods used to extend the shelf life of loquat fruit by reducing its moisture content, which inhibits microbial growth and enzymatic activity. Convective tray drying, for instance,
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 210 has been shown to be effective in preserving loquat slices. This method involves drying the fruit at 70 °C for 12 hours, which has been found to maintain good drying efficiency and preserve the fruit's physico-chemical and sensory properties. When combined with MAP, particularly with high nitrogen environments, the dried loquat slices exhibited limited browning and maintained higher concentrations of certain minerals and vitamins (Tinebra et al., 2022). These techniques are not only useful on a small scale but can also be industrialized for larger-scale applications. In summary, traditional post-harvest preservation techniques such as cooling and cold storage, MAP, chemical treatments, and drying and dehydration play crucial roles in reducing post-harvest losses of loquat fruit. Each method has its advantages and limitations, and often, a combination of these techniques is employed to achieve optimal results in preserving the quality and extending the shelf life of loquat fruit. 4 Emerging Preservation Techniques 4.1 Nano-coating and edible films Nano-coating and edible films have emerged as promising techniques for extending the shelf life of loquat fruits. These coatings, often made from biopolymers, provide a biodegradable alternative to traditional preservation methods. Nanoemulsion coatings, in particular, have shown significant potential due to their enhanced mechanical and barrier properties. They offer protection against moisture loss, respiration, and microbial spoilage, thereby maintaining the physicochemical quality of loquats during storage and transportation (Flores-Lopez et al., 2016). Additionally, incorporating antioxidants and antimicrobials into these coatings can further enhance their effectiveness by providing controlled release of these compounds, thus extending the shelf life and improving the nutritional quality of the fruits. 4.2 Use of natural antimicrobials and antioxidants Natural antimicrobials and antioxidants are increasingly being used to preserve the quality of loquat fruits post-harvest. Chitosan, a natural biopolymer, has been particularly effective in enhancing the activities of antioxidant enzymes such as superoxide dismutase and catalase, while inhibiting enzymes responsible for oxidative stress and membrane damage. This treatment not only extends the storage life of loquats but also preserves their membrane integrity and reduces enzymatic browning. Other natural compounds, such as konjac glucomannan, have also been shown to reduce decay rates and maintain higher levels of total soluble solids, titratable acidity, and ascorbic acid in loquats (Cvanić et al., 2023). 4.3 Innovative storage systems (smart packaging, sensors, etc.) Innovative storage systems, including smart packaging and sensors, are being developed to monitor and maintain the quality of loquat fruits during storage and transportation. These systems utilize advanced technologies such as image analysis, electronic noses, and near-infrared spectroscopy to provide real-time, non-destructive monitoring of fruit quality. Smart packaging materials, often enhanced with nanotechnology, offer improved gas and mechanical properties, which help in maintaining the freshness and extending the shelf life of loquats (Pace and Cefola, 2021). These technologies are particularly useful for long-distance transportation, ensuring that the fruits reach consumers in optimal condition. 4.4 Impact of technological advancements on shelf-life extension Technological advancements in post-harvest preservation have significantly impacted the shelf life of loquat fruits. The integration of nanotechnology in packaging and coating materials has provided new insights and solutions for extending the storage period and improving the post-harvest quality of loquats. These advancements have addressed the limitations of conventional methods, such as high costs and residue issues, by offering more efficient and sustainable alternatives. The use of natural antimicrobials and antioxidants, along with innovative storage systems, has further contributed to reducing post-harvest losses and maintaining the nutritional and sensory quality of loquats (Palumbo et al., 2022). Overall, these emerging preservation techniques hold great promise for enhancing the shelf life and marketability of loquat fruits. Palumbo et al. (2022) demonstrated that certain yeast strains produce lytic enzymes, such as protease, glucanase, and chitinase, that can effectively degrade the cell wall of phytopathogenic fungi. These enzymes target key structural components of the fungal cell wall, including chitin, mannoproteins, and β-glucans, breaking down the
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 211 integrity of the cell wall and leading to fungal cell death. This study suggests that yeast-mediated enzyme production represents a promising biological control strategy for managing fungal plant pathogens, potentially offering an environmentally friendly alternative to chemical fungicides. By disrupting the cell wall's structural components, these enzymes help to weaken the fungus, ultimately preventing its spread and infection in crops. The findings highlight the potential for using yeast in sustainable agricultural practices to reduce crop losses caused by phytopathogenic fungi. 5 Comparative Analysis of Preservation Techniques 5.1 Efficiency in reducing post-harvest losses Various preservation techniques have been studied to reduce post-harvest losses in loquat fruit. Wrapping with sterile non-woven gauze, expanded polyethylene, and polyethylene foam fruit net, as well as coatings with konjac glucomannan, have shown significant efficacy in reducing fruit decay and extending shelf life. These methods also help in maintaining higher levels of total soluble solids, titratable acidity, and ascorbic acid. Additionally, low-temperature storage, although effective in prolonging shelf life, can lead to chilling injury, which reduces the quality and economic value of the fruit. Nanotechnology-based active packaging has emerged as a promising alternative, offering extended storage periods and improved post-harvest quality by removing ethylene and providing antioxidant and antimicrobial properties. 5.2 Economic viability and cost analysis The economic viability of preservation techniques is a crucial factor for their adoption. Traditional methods like cold storage and chemical treatments are often cost-effective but come with drawbacks such as chilling injury and chemical residues. On the other hand, advanced techniques like nanotechnology-based active packaging, while potentially more effective, may involve higher initial costs due to the need for specialized materials and technology. Wrapping methods using sterile non-woven gauze and polyethylene foam fruit net are relatively low-cost and have been suggested as convenient and practical options for extending shelf life. The tray drying method, although useful on a small scale, can be industrialized, making it a viable option for larger operations. 5.3 Environmental impact and sustainability considerations Sustainability is an important consideration in the selection of post-harvest preservation techniques. Traditional chemical methods often leave residues that can be harmful to the environment. In contrast, physical methods like wrapping and low-temperature storage have a lower environmental impact but may still contribute to energy consumption and waste generation. Nanotechnology-based active packaging offers a more sustainable alternative by reducing the need for chemical preservatives and potentially lowering energy consumption through more efficient storage conditions. Additionally, biological control methods and the use of natural coatings like konjac glucomannan are environmentally friendly options that can reduce the reliance on synthetic chemicals. In summary, while traditional methods remain widely used due to their cost-effectiveness, emerging technologies like nanotechnology and natural coatings offer promising alternatives that balance efficiency, economic viability, and environmental sustainability (Lu, 2024). 6 Application of Preservation Techniques in Reducing Post-Harvest Losses 6.1 Integration of techniques in supply chains The integration of various preservation techniques into supply chains is crucial for reducing post-harvest losses of loquat fruit. Several methods have been identified as effective in extending the shelf life and maintaining the quality of loquat. For instance, wrapping loquat fruits in sterile non-woven gauze or polyethylene foam fruit nets, and coating them with konjac glucomannan have shown significant promise in reducing decay rates and preserving sensory quality. Additionally, advanced postharvest treatments such as active packaging, vacuum impregnation, and high hydrostatic pressure have been implemented to maintain the nutritional value and safety of fresh produce during transportation to distant markets. The use of nanotechnology in active packaging is also emerging as a state-of-the-art solution to extend the post-harvest storage period and improve the quality of loquats by removing ethylene and incorporating antioxidants and antimicrobials (Santos et al., 2019).
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 212 6.2 Regional practices and their effectiveness Regional practices in post-harvest preservation of loquat vary significantly, reflecting local conditions and available resources. In Sichuan, China, treatments with L-Cysteine and γ-Aminobutyric Acid (GABA) have been effective in reducing weight loss, browning, and decay, while enhancing antioxidant activity and sensory quality during cold storage. In Brazil, the central supply (CEASA) of Salvador has identified key determinants of post-harvest losses, including inadequate transportation, poor hygienic conditions, and lack of refrigeration. Strategies such as price reduction, donation practices, and consumption by sellers have been employed to mitigate these losses. These regional practices highlight the importance of tailored approaches to post-harvest preservation that consider local challenges and resources (Zhang et al., 2023). 6.3 Challenges in practical implementation Despite the advancements in post-harvest preservation techniques, several challenges remain in their practical implementation. One major challenge is the susceptibility of loquat to chilling injury during cold storage, which can lead to browning and other quality issues. Additionally, the high costs and potential residues associated with conventional preservation methods pose significant barriers4. Inadequate infrastructure and poor hygienic practices in supply chains further exacerbate post-harvest losses, as observed in the CEASA of Salvador, Brazil. Addressing these challenges requires a multifaceted approach that includes improving infrastructure, adopting cost-effective and residue-free preservation methods, and enhancing the overall management and hygienic practices in supply chains. By integrating effective preservation techniques, considering regional practices, and addressing practical challenges, it is possible to significantly reduce post-harvest losses of loquat and improve its availability and quality for consumers. 7 Case Study 7.1 Overview of case study: region/company involved This case study focuses on the implementation of post-harvest preservation techniques for loquat fruit in a commercial setting in the Zhejiang province of China. The company involved, Zhejiang Loquat Co., Ltd., is a leading producer and exporter of loquat fruit, facing significant challenges related to post-harvest losses due to the fruit's susceptibility to mechanical damage, moisture loss, and microbial decay (Santos et al., 2019). 7.2 Techniques implemented and results Zhejiang Loquat Co., Ltd. implemented several advanced post-harvest preservation techniques to mitigate these issues. These included the use of konjac glucomannan coatings, polyethylene foam fruit nets, and sterile non-woven gauze wrappings. The konjac glucomannan coatings, in particular, were applied in two concentrations: 0.5% and 1% aqueous solutions. These methods were compared to untreated controls stored at 4±1 °C and 95% relative humidity for up to 42 days. The results demonstrated that all treatments significantly reduced the rate of fruit decay and extended the shelf life of the loquat fruit. Specifically, the konjac glucomannan coatings maintained higher levels of total soluble solids, titratable acidity, and ascorbic acid over the first 21 days. The polyethylene foam fruit net was most effective in preventing fruit decay, while the sterile non-woven gauze was noted for its convenience and significant impact on storage quality. 7.3 Analysis of post-harvest loss reduction The implementation of these preservation techniques led to a substantial reduction in post-harvest losses. The konjac glucomannan coatings and polyethylene foam fruit nets were particularly effective, reducing weight loss and decay rates while preserving the nutritional quality of the fruit. The higher levels of superoxide dismutase (SOD) and catalase (CAT) associated with these treatments also contributed to better sensory quality and extended shelf life (Wang et al., 2023). Additionally, the use of bio-materials such as Nigella sativa oil and propolis extract further enhanced the storage quality by reducing weight loss, maintaining fruit firmness, and preventing browning anddecay. Wang et al. (2023) found that the application of different treatments, such as TaEO and TaEO-ME, significantly influenced the decay index, ascorbic acid content, total phenolic content, and malondialdehyde (MDA) levels in loquat fruit during storage. The study revealed that TaEO-treated loquats exhibited the lowest decay index and the
Bioscience Methods 2024, Vol.15, No.5, 207-215 http://bioscipublisher.com/index.php/bm 213 highest ascorbic acid and total phenolic content throughout the storage period. In contrast, control groups, especially the water control, showed a faster rate of decay and a more rapid decline in ascorbic acid and phenolic content. Additionally, MDA, an indicator of oxidative stress, increased more rapidly in the control groups, signifying higher levels of lipid peroxidation. The results suggest that TaEO and its combination with ME can enhance the shelf life and antioxidant capacity of loquat fruits, reducing oxidative damage and slowing decay during storage. This study supports the potential of natural treatments for fruit preservation. 7.4 Lessons learned and future recommendations The case study highlights the importance of selecting appropriate post-harvest preservation techniques to extend the shelf life and reduce losses of loquat fruit. The success of konjac glucomannan coatings and polyethylene foam fruit nets suggests that these methods are practical and effective for commercial use. However, the study also underscores the need for continuous innovation and optimization of preservation techniques. Future recommendations include exploring the potential of nanotechnology-based active packaging to further enhance the quality and shelf life of loquat fruit. Additionally, integrating advanced monitoring technologies such as electronic noses and near-infrared spectroscopy could provide non-destructive methods for quality assessment, ensuring better management of post-harvest losses (Kahramanoğlu, 2020). 8 Future Perspectives and Recommendations 8.1 Advancements in preservation techniques The future of post-harvest preservation techniques for loquat fruit looks promising with the advent of advanced technologies. Traditional methods such as cold storage, controlled atmosphere storage, and chemical treatments have been widely used but come with limitations such as high costs and potential residues. Emerging technologies like nanotechnology offer a state-of-the-art alternative, enhancing the post-harvest quality and extending the shelf life of loquat by removing ethylene, antioxidants, and antimicrobials. Additionally, advanced physical and chemical treatments such as active packaging, vacuum impregnation, and cold plasma are being explored to preserve the nutritional value and safety of fresh produce. These methods, combined with contactless and non-destructive quality monitoring techniques like image analysis and near-infrared spectroscopy, present numerous advantages over traditional methods. The integration of these advanced technologies could significantly reduce post-harvest losses and improve the overall quality of loquat fruit. 8.2 Policy and regulatory considerations To fully realize the benefits of these advanced preservation techniques, it is crucial to address policy and regulatory considerations. The implementation of novel sustainable strategies is essential for reducing synthetic fungicide residues on fruit surfaces and minimizing environmental impact. Regulatory frameworks should encourage the adoption of advanced technologies by providing guidelines and standards for their safe and effective use. Policies should also promote research and development in the field of post-harvest preservation, ensuring that new methods are both economically viable and environmentally friendly. Collaboration between government agencies, research institutions, and industry stakeholders is necessary to develop and enforce regulations that support the widespread adoption of these innovative techniques (Hong and Huang, 2024). 8.3 Recommendations for further research Further research is needed to optimize and validate the effectiveness of advanced preservation techniques for loquat fruit. Studies should focus on the long-term effects of these methods on fruit quality, nutritional value, and safety. Research should also explore the potential of combining multiple preservation techniques to achieve synergistic effects and further extend the shelf life of loquat. Additionally, there is a need for more comprehensive studies on the economic feasibility and environmental impact of these advanced methods. Investigating the molecular and physiological mechanisms underlying the effectiveness of these techniques can provide valuable insights for improving their application. Finally, research should aim to develop user-friendly and cost-effective technologies that can be easily adopted by small-scale farmers and producers, ensuring that the benefits of advanced preservation methods are accessible to all.
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Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 216 Research Insight Open Access CRISPR-based Gene Editing inBt for Improved Insecticidal Properties Wenfei Zhang College of Life Sciences, Hainan Normal University, Haikou, 571158, Hainan, China Corresponding author: wenfei2007@163.com Bioscience Methods, 2024, Vol.15, No.5 doi: 10.5376/bm.2024.15.0022 Received: 11 Jul., 2024 Accepted: 22 Aug., 2024 Published: 13 Sep., 2024 Copyright © 2024 Zhang, 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: Zhang W.F., 2024, CRISPR-based gene editing in Bt for improved insecticidal properties, Bioscience Methods, 15(5): 216-225 (doi: 10.5376/bm.2024.15.0022) Abstract CRISPR-based gene editing has emerged as a powerful tool for enhancing the insecticidal properties of Bacillus thuringiensis (Bt) by targeting specific genetic components associated with resistance in insect pests. This study explores the application of CRISPR/Cas9 technology in Bt to improve its efficacy against various insect species. Key studies demonstrate the successful knockout of ATP-binding cassette (ABC) transporter genes, such as ABCC2 and ABCC3, which are crucial for mediating resistance to Bt toxins in pests like the diamondback moth and cotton bollworm. These genetic modifications have resulted in significantly increased resistance levels, providing insights into the molecular mechanisms underlying Bt toxin resistance. Additionally, this study highlights the potential of CRISPR-mediated gene deletions in Bt strains to enhance their pesticidal protein profiles, thereby broadening their spectrum of activity against multiple insect pests. The findings underscore the importance of CRISPR technology in developing next-generation biopesticides with improved insecticidal properties and reduced likelihood of resistance development. Keywords CRISPR/Cas9; Bacillus thuringiensis; Insecticidal proteins; ABC transporter genes; Pest resistance 1 Introduction Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacterium widely recognized for its insecticidal properties. It produces crystal (Cry) proteins during sporulation, which are toxic to a variety of insect pests upon ingestion. These Cry proteins have been effectively utilized in agriculture for pest control, reducing the reliance on chemical pesticides and thereby mitigating environmental pollution and health risks (Nair et al., 2020; Arsov et al., 2023; Sauka et al., 2023). Bt's specificity towards target pests, such as moths, beetles, and mosquitoes, makes it a valuable tool in integrated pest management programs (Nair et al., 2020; Arsov et al., 2023). Despite the success of Bt-based insecticides, several limitations hinder their long-term efficacy. One significant challenge is the rapid evolution of resistance in target insect populations. For instance, resistance to Bt Cry1 toxins has been observed in various lepidopteran pests, which compromises the effectiveness of Bt crops and biopesticides (Guo et al., 2019). Additionally, the narrow spectrum of activity of certain Bt strains limits their utility against a broader range of pests (Reyaz et al., 2019; Sauka et al., 2023). Environmental factors, such as soil enzymatic activities, can also influence the persistence and efficacy of Bt proteins, necessitating careful evaluation of their environmental impact (Li et al., 2019; Li et al., 2022). Genetic modification offers a promising approach to overcome the limitations of traditional Bt insecticides. Traditional methods of genetic modification in Bt have been labor-intensive and time-consuming (Liu and Zhang, 2024). By employing techniques such as CRISPR/Cas9, researchers can enhance the insecticidal properties of Bt strains, broaden their spectrum of activity, and mitigate resistance development in target pests (Guo et al., 2019). For example, the construction of chimeric Bt proteins with novel domain combinations has shown enhanced activity against multiple soybean pests, demonstrating the potential of genetic engineering in developing more effective biopesticides (Chen et al., 2021). Additionally, the identification and characterization of novel Cry proteins, such as Cry78Ba1, provide new avenues for specific and safe pest control (Cao et al., 2020). This study provides a comprehensive overview of the advancements in CRISPR-based gene editing of Bt for improved insecticidal properties, including summarizing the current state of Bt as a biopesticide and its role in pest control, identifying the limitations of traditional Bt insecticides, exploring the potential of genetic
Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 217 modification techniques, particularly CRISPR/Cas9, in enhancing Bt's insecticidal properties, and evaluating the environmental and ecological implications of genetically modified Bt strains. By addressing these objectives, this study aims to highlight the potential of CRISPR-based gene editing in developing next-generation Bt biopesticides with improved efficacy and sustainability. 2 CRISPR-Cas9 Technology: A Tool for Genetic Enhancement 2.1 Introduction to CRISPR-Cas9 gene editing CRISPR-Cas9, derived from a bacterial immune defense mechanism, has revolutionized genetic engineering by providing a precise, efficient, and versatile tool for genome editing. Initially recognized for its role in bacterial immunity against viruses, CRISPR-Cas9 has been adapted for use in a wide range of organisms, including plants and animals, to facilitate targeted genetic modifications (Demirci et al., 2018; Li et al., 2021). This technology employs a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it introduces double-strand breaks (DSBs). These breaks are then repaired by the cell's natural repair mechanisms, leading to targeted mutations or insertions (Bao et al., 2019). 2.2 Mechanism of CRISPR-Cas9 in gene editing The CRISPR-Cas9 system operates through a simple yet highly effective mechanism. The Cas9 protein, guided by a single-guide RNA (sgRNA), binds to a complementary DNA sequence and introduces a DSB at the target site. The cell's repair machinery then attempts to fix the break, typically through non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ often results in small insertions or deletions (indels) that can disrupt gene function, while HDR can be used to introduce specific genetic changes using a donor template (Bao et al., 2019). This precise targeting capability allows for the modification of specific genes, enabling the study of gene function and the development of organisms with desirable traits (Eş et al., 2019; Erdoğan et al., 2023). 2.3 Advantages of CRISPR in agricultural biotechnology CRISPR-Cas9 offers several advantages over traditional breeding and earlier genome editing technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). These advantages include higher efficiency, ease of design, lower cost, and the ability to target multiple genes simultaneously (multiplexing) (Demirci et al., 2018; Eş et al., 2019). In agriculture, CRISPR-Cas9 has been used to enhance crop yield, quality, and resistance to diseases and environmental stresses. For instance, it has been employed to develop crops with improved resistance to pests and pathogens, increased tolerance to abiotic stresses like drought and salinity, and enhanced nutritional profiles (Bisht et al., 2019; Chen et al., 2019; Zhu et al., 2020a). The technology's ability to create transgene-free plants also addresses regulatory and public acceptance issues associated with genetically modified organisms (GMOs) (Erdoğan et al., 2023). 2.4 Specific considerations for applying CRISPR toBt When applying CRISPR-Cas9 to Bacillus thuringiensis (Bt), several specific considerations must be taken into account. Bt is widely known for its insecticidal properties, which are primarily due to the production of crystal (Cry) proteins that target specific insect pests. Enhancing Bt's insecticidal properties through CRISPR-Cas9 involves precise modifications to the genes encoding these Cry proteins to increase their efficacy or broaden their spectrum of activity (Figure 1) (Komal et al., 2023). Additionally, CRISPR can be used to engineer Bt strains with improved stability and environmental persistence, ensuring sustained pest control (Bisht et al., 2019; Komal et al., 2023). However, challenges such as off-target effects, delivery methods, and regulatory hurdles must be carefully managed to ensure the successful application of CRISPR technology in Bt (Rao and Wang, 2021; Erdoğan et al., 2023). Komal et al. (2023) highlights the use of genome-editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 to develop insect-resistant crops, offering a novel approach to pest management. By targeting specific genes in either plants or insects, these technologies can increase plant resistance and reduce insecticide resistance in pests. For plants, gene editing can enhance their natural defense mechanisms, such as increasing salicylic acid levels or altering volatile compounds to repel pests. In insects, modifying genes responsible for detoxifying insecticides can make them more susceptible, reducing pest populations. These strategies present a promising path for sustainable agriculture by minimizing the reliance on chemical pesticides and improving crop productivity.
Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 218 Figure 1 Depicting emerging tools of genome editing for resistance to insect pests. Genome editing of either plant or insect can make the insect susceptible plant to a resistant plant. The potential tools can be ZFNs, TALENs, or CRISPR/Cas9 (Adopted from Komal et al., 2023) 3 Genetic Modifications inBt for Improved Insecticidal Properties 3.1 Target genes inBt for enhanced toxin production Genetic modifications in Bacillus thuringiensis (Bt) have primarily focused on enhancing the production and efficacy of its insecticidal toxins. Key target genes include those encoding ATP-binding cassette (ABC) transporters, which play a crucial role in the susceptibility of insects to Bt toxins. For instance, mutations in the ABCC2 and ABCC3 genes have been linked to high levels of resistance to Cry1Ac toxin in the diamondback moth,
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