1 Introduction

Lindera aggregata, which is a Chinese herbal medicine, is highly medicinally active and finds widespread applications in medicine and healthcare (Salleh, 2020). It is a key component of traditional medicines such as the Suoquan pill that is used in the management of chronic kidney disease (CKD) (Cai et al., 2020). The plant's bioactive phytoconstituents, namely the isoquinoline alkaloids, have extensive applications in the food and drug industries due to their drug potential (Peng et al., 2020). Lindera aggregata has also been used traditionally to treat gastrointestinal diseases since centuries, which exhibits the drug's extensive medicinal applications (Lai et al., 2021).

Nevertheless, seedling propagation of Lindera aggregata is highly limited by the incidence of systemic infections, such as viral pathogens, that cannot be eliminated through regular cultivation techniques (Wang et al., 2012). Not only do these infections lower productivity but also hinder industrial manufacturing of quality Lindera aggregata products. Heat treatment and tissue culture are among the new methods that hold the solution to decontamination of infected plants and producing healthy seedlings. Heat treatment suppresses viral activity by exposing plant tissues to elevated temperatures, and tissue culture re-establishes pathogen-free plants from meristematic tissues under aseptic conditions. Combination of the two technologies has been highly promising in detoxifying crops and supporting sustainable seedling propagation (Linck et al., 2019).

Detoxification technology when combined with tissue culture methods is a solution for addressing the pathogenic infection challenges of Lindera aggregata cultivation. Tissue culture technology has been successfully implemented in other species such as Lindera glauca, for the aim of increasing propagation levels and ensuring quality seedlings production. It can be eliminated with the combined heat treatment and tissue culture technology to make their resistance and general health in Lindera aggregata seedlings stronger. It can be done to retain their medicinal properties and attain sustainable production.

This study will explore the application of combined heat treatment and tissue culture detoxification technology for producing healthy Lindera aggregata seedlings. The study is intended to evaluate the efficacy of these procedures in pathogen elimination, seedling quality improvement, and upholding the medicinal material stability of the plant. The objective of this study is to develop an understanding of how to maximize cultivation practice on Lindera aggregata, thereby making it accessible in traditional medicine, as well as modern medicine.

2 Overview of Lindera aggregata Diseases and Current Detoxification Techniques

2.1 Common diseases of Lindera aggregata

Lindera aggregata is susceptible to a variety of viral diseases that significantly affect its growth, yield, and active compound content such as essential oils, flavonoids, and alkaloids. Viral infection symptoms include stunted growth, chlorosis, and leaf and stem deformation, which ultimately reduce the value of the plant from a pharmacological and economic perspective. Potyviruses and tobamoviruses are prevalent viral pathogens infecting Lindera aggregata and thriving under nursery and cultivation conditions.

The most significant modes of viral transmission are external infection and seedling transmission. Infected seedlings represent the largest source of viral spread under cultivation. External infection modes include mechanical transmission of viruses through contaminated tools and machinery and vector transmission through vector insects such as whiteflies and aphids. These infection modes make it difficult to maintain virus-free plants, especially in intensive planting systems (Rubio et al., 2020).

Lindera aggregata is highly susceptible to viral diseases, which affect the growth, yield, and production of pharmacologically active compounds such as sesquiterpenoids and alkaloids. Viral disease in the crop is better defined by symptoms such as chlorosis, growth retardation, and biomass reduction, which lowers the medicinal as well as economic value of the crop. The viral diseases are transmitted by two primary modes: seedling transmission, where the virus-infected mother plants infect the progeny, and external infection via arthopod and whitefly vectors. Mechanical transmission occurs due to the utilization of unsterilized equipment that leads to increased disease transmission, especially under intensive propagation systems (Huang et al., 2023).

2.2 Limitations of traditional detoxification techniques

Lindera aggregata is a bitter-tasting traditional Chinese medicinal herb, which is warm in nature and predominantly associated with the lung, spleen, kidney, and bladder meridians (Chen and Yu, 2024). Its richness in chemical constituents provides the rationale for its vast applications in medicine and public health. The distribution ratio of its major chemical constituents consists of 127 sesquiterpenoids, 37 alkaloids, 32 flavonoids, and 35 others (Figure 1). Traditional detoxification processes, such as chemical treatment, are seriously disadvantageous in Lindera aggregata detoxification. Chemical compounds applied to suppress viral infection would typically create adverse environmental impacts, such as soil contamination and residues of chemicals on the plant. Moreover, the process can alter the physiological state of the plant, reducing biosynthesis of active compounds associated with Lindera aggregata's medicinal activity (Lv et al., 2023).

Figure 1 Proportion of chemical compounds in L. aggregata (Adopted from Lv et al., 2023)

Single detoxification processes, such as heat treatment or tissue culture, also achieve limited success. Heat treatment effectively prevents some viruses but causes thermal stress in plant tissue, which will affect viability. Tissue culture, which allows for rapid growth of pathogen-free plants, does not offer protection against systemic infection or viruses at latent periods. This inefficacy has a tendency to render detoxification incomplete and necessitates the establishment of integrated approaches (Zhao et al., 2022).

2.3 Synergistic advantages of heat treatment and tissue culture

Tissue culture combined with heat treatment synergistically detoxifies Lindera aggregata seedlings. Heat treatment involves the exposure of plant tissues to a controlled high temperature range (generally 35°C-40°C) for a period of time that inhibits or inactivates heat-sensitive viral nucleic acids and proteins. In the process, the content of viruses in infected tissues is reduced without utilizing chemical agents, making it an eco-friendly method (Gan et al., 2009).

Tissue culture facilitates heat treatment with the cultivation of virus-free plants on sterile, controlled conditions. Meristematic tissues, in which there is minimal possibility of virus infection, are excised and cultured on nutrient media to generate healthy seedlings. The synthesis leads to virus elimination as well as pathogen-free plant production at a higher rate. The combined approach has been found to have high detoxification efficiency, increased seedling vigor, and increased scalability for commercial propagation (Huang et al., 2023).

3 Application of Heat Treatment in Lindera aggregata Detoxification

3.1 Principles of heat treatment

Heat treatment is a well-documented virus suppression and elimination method in plants. The primary mechanism is inoculating plant tissue with high temperature, disrupting viral proteins and nucleic acid and hindering replication. Experiments have confirmed that in Lindera aggregata detoxification, viruses exhibit differential temperature and treatment time sensitivity. For instance, thermotherapy at 37 °C-40 °C has been effective in reducing the activity of well-known plant viruses like Tobacco mosaic virus and Potato virus Y (Wang et al., 2018).

3.2 Optimization of heat treatment conditions

Optimizing the conditions of heat treatment is the key to achieving high detoxification efficiency and less tissue damage in Lindera aggregata. Temperature gradient tests confirmed that a temperature gradient from 35°C to 45°C is the optimal for suppressing most viral infections in plants. Longer exposure for extended durations at higher temperatures, for instance, 45°C for 15-30 minutes, is able to enhance detoxification more effectively but may cause reduced survival rates of sensitive plant tissues (Torres et al., 2000).

The duration of heat treatment of Lindera aggregata is also crucial. One must find a balance between the time for inactivation of the virus and avoid thermal stress. For example, in the thermotherapy of garlic, optimum detoxification was at 37°C for 35 days (Lizárraga et al., 2017).

3.3 Effects of heat treatment on lindera aggregata growth

Although heat treatment has been proven to reduce viral loads greatly, it may have some effect on Lindera aggregata development and vigor. Excessive exposure to high temperature may lead to reduced survival rates of seedlings and delayed growth. Studies in other plant species showed that the intensity and duration of heat treatment can be calibrated precisely to reduce these side effects. For instance, water treatments under heat and follow-up tissue culture have been used for vigour restoration and viability maintenance in treated plants (Langens-Gerrits et al., 2004).

In Lindera aggregata, combining mild heat treatment with employing fast propagation techniques, such as tissue culture, enables proper recovery of the plant. The two-approach method minimizes the adverse impacts of heat stress while effectively eliminating viruses, making way for disease-free healthy seedlings with an enhanced capability for growth.

4 Application of Tissue Culture Technology in Healthy Seedling Propagation

4.1 Principles and processes of tissue culture

Tissue culture is an aseptic method where cells, tissues, or organs are propagated in regulated environmental conditions (Grout, 2017). For Lindera aggregata, meristem culture and shoot tip culture are the two prominent detoxification methods. These methods are based on the fact that meristematic tissues will have lesser opportunities of virus harbouring, and virus-free plants will be produced. The tissue culture process entails explant harvesting and sterilization, culture medium preparation, and differentiation induction and proliferation. Microelements, PGRs such as cytokinins (e.g., 6-benzylaminopurine [6-BA]), and auxins (e.g., naphthaleneacetic acid [NAA]) are all significant constituents to be incorporated in inducing shoot development and initiation (Espinosa-Leal et al., 2018).

4.2 Optimization of culture media

Optimization of the culture medium composition is to be made for enhanced efficiency in Lindera aggregata tissue culture. The concentration of sucrose as a source of energy and agar as structural support for the plantlet are the most significant. Concentrations of these have been found to play a vital role in inducing growth, differentiation, and proliferation of the tissues in culture. Besides, some PGR concentrations, such as 6-BA for shoot and NAA for rooting, also enhance the efficiency of regeneration and quality of detoxified seedlings (Hussain et al., 2012).

4.3 Detoxification efficiency of tissue culture

Tissue culture improves detoxification effectiveness significantly by regeneration of virus-free plants from meristematic tissues. The efficiency of removal of viruses by detection through reverse transcription PCR (RT-PCR) in pre- and post-tissue culture has established high levels of removal of viruses from treated crops. Tissue culture also facilitates the promotion of quick development in seedlings to allow for the production of healthy plants on a regular basis. In Lindera aggregata, higher rates of proliferation and survival of detoxified seedlings have been established through maximized tissue culture protocols (Thorpe, 2012).

5 Application of Combined Heat Treatment and Tissue Culture Technology

5.1 Implementation Process of Combined Technology

The combined use of heat treatment and tissue culture represents a promising detoxification process for Lindera aggregata. Heat-treated shoot tips or stem segments are utilized as beginning materials, where virus is inhibited or killed by heat without extreme tissue damage in the plant. The treated tissue is then transferred into a sterile tissue culture medium where virus-free seedling multiplication and differentiation can occur (Pasternak and Steinmacher, 2024). Through the use of both methods' advantage, the process guarantees healthy pathogen destruction and healthy regeneration (Linck et al., 2019).

5.2 Advantages of combined technology

Double heat treatment combined with tissue culture enhances significantly the effectiveness of detoxification in Lindera aggregata. Double treatment provides higher detoxification percentages than separate treatments by affecting more than a single viral activity stage. Heat treatment suppresses active viral replication, and tissue culture grows plants from virus-free meristematic tissues. The process also allows for the mass production of seedlings with retained resistance characteristics and vigor and is therefore scalable for commercial purposes (Torres et al., 2000).

5.3 Optimization of technical parameters

Control of the conditions of combined heat treatment and tissue culture is crucial to allow the maximum efficiency and least damage to the tissue in Lindera aggregata. Synergy of heat treatment temperature and time is also required; for example, heating at 37 °C-40 °C for 2-3 weeks has been effective in breaking down viral activity without lowering tissue viability. Subsequently, precise regulation of culture conditions, such as media composition and light patterns, subsequently enhances regeneration success. The process also readily supports diverse virus strains, and hence is a useful tool in plant detoxification programs (Linck et al., 2019).

6 Effectiveness Evaluation of Combined Detoxification Technology

6.1 Detoxification rate and health assessment

The effectiveness of virus elimination in Lindera aggregata through the combined application of heat treatment and tissue culture techniques is primarily evaluated by detecting viral loads before and after treatment. Techniques such as reverse transcription-polymerase chain reaction (RT-PCR) are widely used for precise quantification of viral content. Studies have shown that after the application of combined virus elimination methods, viral loads are significantly reduced, and the health status of plant tissues is improved. In addition, growth parameters such as plant height, leaf area, and root length are commonly measured to comprehensively assess the health level of virus-free seedlings (Linck et al., 2019).

6.2 Seedling quality and genetic stability

Compound detoxification technology not only improves the quality of Lindera aggregata seedlings but also ensures genetic stability. Experiments on medicinal components such as detoxified seedlings' essential oils and polysaccharides have revealed equivalent active ingredient levels, which confirm that detoxification does not have any negative impact on their pharmacological activity. Genetic stability analysis, which is typically done under the direction of molecular markers like SSR and SNP, ensures that tissue culture does not generate any significant genetic variation in the seedlings produced (Torres et al., 2000).

6.3 Field performance and adaptability

Field trials are of utmost importance in assessing the performance and viability of detoxified seedlings. Healthy seedlings produced using the combined detoxification technology display improved growth performance and tolerance to environmental stresses. This work shows lowered disease incidence rates and improved yields in the detoxified plants compared to the untreated controls. These findings demonstrate the potential for this integrated technology in enhancing the vigor and productivity of Lindera aggregata under different conditions of cultivation.

7 Promotion and Industrial Application of the Technology

7.1 Large-scale propagation of healthy seedlings

The heat treatment and tissue culture technology has established a repeatable protocol for propagation of Lindera aggregata healthy seedlings. The protocol follows sterilization protocol, optimized heat treatment time and temperature for every step, and plant growth regulators in the culture media preparation. Additionally, callus culture and suspension cell culture provide platforms for controlled synthesis of secondary metabolites, and gene editing technologies such as CRISPR-Cas9 enable the efficient synthesis of target compounds in addition to metabolic pathway enhancement. The yields of compounds can be significantly enhanced by exogenous elicitors and environmental stresses (Figure 2). Future opportunities plan to develop efficient and low-cost cultivation techniques, together with state-of-the-art synthetic biology, to meet industrial demands and allow sustainable manufacture of green chemicals and pharmaceuticals from Lindera aggregata. The process ensures homogeneity of seeds and fulfills small-scale local and large-scale industrial cultivation production needs (Hasnain et al., 2022).

Figure 2 Recent methods used for industrial production of bioactive compounds via plant tissue culture (Adopted from Hasnain et al., 2022)

In tissue culture process efficiencies, for example, usage of automated bioreactors in propagation and enhanced sterilization practices has drastically reduced labor and material expenses. In addition, effective resource allocation management (such as culture medium components and energy use) enhances cost-effectiveness even more, making the technology economically attractive to growers as well as industry stakeholders (Gulzar et al., 2020).

7.2 Extension of the industrial chain

Healthy seedlings acquired through the integrated detoxification technology are used to produce high-quality planting bases for Lindera aggregata. The planting bases are intended to improve the medicinal quality of plants through an assurance of even growth, reduced rates of disease, and enhanced levels of bioactive compounds. Such planting bases provide high-grade and standard raw materials for pharmaceutical and nutraceutical companies (Loyola-Vargas and Ochoa-Alejo, 2018).

Employment of healthy seedlings leads to products of higher medicinal quality that meet high local and foreign markets' standards. Higher product uniformity and quality translate to greater market worth, enhancing higher competitiveness and profitability of Lindera aggregata products in global business (Hasnain et al., 2022).

7.3 Analysis of ecological and economic benefits

By eliminating viral diseases through integrated detoxification technology, chemical pesticide application is significantly reduced. This green approach reduces soil and water pollution, lowers input costs, and conserves biodiversity in agricultural ecosystems (Gulzar et al., 2020).

Detoxified seedlings are healthier and more productive and disease-resistant, an immediate increase in farmer revenue through reduced loss of production. In addition, the quality of Lindera aggregata products increases, which augments market value, ensuring economic sustainability in the long term for cultivation and processing investors (Loyola-Vargas and Ochoa-Alejo, 2018).

8 Challenges and Future Directions

8.1 Limitations in technical applications

The heat treatment quality among different virus strains is also quite inconsistent since they vary in their heat resistance. Heterogeneity makes it difficult to standardize detoxification processes, particularly in infected plants with a mixed population of viruses (Torres et al., 2000). Strain-specific thermal responses necessitate specially tailored treatments, and this makes operations more complex.

Tissue culture techniques have their development to industrial scales marred by issues like risk of contamination, consumption of resources, and poor scope for automation. Despite the advent of bioreactors, human interventions in activities such as explant preparation and transplanting remain the main bottlenecks (Hasnain et al., 2022).

8.2 Directions for technology optimization

Developing heat treatment protocols that require less energy inputs, such as lower exposure times or cycling heating, for instance, will enhance sustainability. Meanwhile, the optimization of culture media composition to regenerate tissues at a higher rate and with better seedling quality will reduce cost and increase scalability (Gulzar et al., 2020).

The integration of molecular breeding technologies, such as CRISPR/Cas9 gene editing, with in-built detoxification technology offers promising avenues towards the production of resistant germplasm. The integration can enhance the efficiency of detoxification mechanisms and create virus-resistant Lindera aggregata cultivars (Loyola-Vargas and Ochoa-Alejo, 2018).

8.3 Future research prospects

Characterizing the genetic mechanism of resistance to disease through genome sequencing and transcriptomics will result in identifying the major resistance genes. The information can be utilized to guide molecular marker development for resistance breeding and enhanced detoxification efficiency (Linck et al., 2019; Fang, 2024).

Installation of intelligent systems for real-time monitoring and predictive analytics in tissue culture laboratories can optimize growth conditions and reduce human intervention. Emerging automation technologies, such as AI-based bioreactors, have the ability to automate mass seedling propagation (Hasnain et al., 2022).

Acknowledgments

The authors thank the colleagues and research partners for their support and assistance in literature compilation, data analysis, and other aspects of this study.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Cai H., Wang J., Luo Y., Wang F., He G., Zhou G., and Peng X., 2020, Lindera aggregata intervents adenine-induced chronic kidney disease by mediating metabolism and TGF-β/Smad signaling pathway, Biomedicine & Pharmacotherapy, 134: 111098.

https://doi.org/10.1016/j.biopha.2020.111098

Chen M.Y., and Yu S.Y., 2024, The classification study and categorization of medicinal properties in traditional Chinese medicine, International Journal of Clinical Case Reports, 14(1): 14-22.

https://doi.org/10.5376/ijccr.2024.14.0002

Espinosa-Leal C., Puente-Garza C., and García-Lara S., 2018, In vitro plant tissue culture: Means for production of biological active compounds, Planta, 248: 1-18.

https://doi.org/10.1007/s00425-018-2910-1

Fang J., 2024, Breeding 4.0: The breeding revolution of genetic information integration and editing, Molecular Plant Breeding, 15(1): 15-26.

https://doi.org/10.5376/mpb.2024.15.0003

Gan L.S., Zheng Y.L., Mo J.X., Liu X., Li X.H., and Zhou C.X., 2009, Sesquiterpene lactones from the root tubers of Lindera aggregata, Journal of Natural Products, 72(8): 1497-1501.

https://doi.org/10.1021/np900354q

Grout B., 2017, General principles of tissue culture, in Comprehensive Biotechnology (2nd ed.), 4: 437-443.

https://doi.org/10.1016/B978-0-12-394807-6.00143-X

Gulzar B., Mujib A., Malik M., Mamgain J., Syeed R., and Zafar N., 2020, Plant tissue culture: Agriculture and industrial applications, in Transgenic Technology Based Value Addition in Plant Biotechnology, 9-28.

https://doi.org/10.1016/B978-0-12-818632-9.00002-2

Hasnain A., Naqvi S., Ayesha S., Khalid F., Ellahi M., Iqbal S., Hassan M., Abbas A., Adamski R., Markowska D., Baazeem A., Mustafa G., Moustafa M., Hasan M., and Abdelhamid M., 2022, Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries: Current scenario and future approaches, Frontiers in Plant Science, 13: 1009395.

https://doi.org/10.3389/fpls.2022.1009395

Huang Q., Liu K., Qin L., and Zhu B., 2023, Lindera aggregata (Sims) Kosterm: A systematic review of its traditional applications, phytochemical and pharmacological properties, and quality control, Medicinal Plant Biology, 2(1): 1-11.

https://doi.org/10.48130/mpb-2023-0011

Hussain A., Qarshi I., Nazir H., and Ullah I., 2012, Plant tissue culture: Current status and opportunities, Recent Advances in Plant In Vitro Culture, InTechOpen, 1(1): 1-28.

https://doi.org/10.5772/50568

Lai H., Yang Z., Lou Z., Li F., Xie F., Pan W., Xu C., Zhang L., Zhang S., Zhang L., and Huang M., 2021, Root extract of Lindera aggregata (Sims) Kosterm. modulates the Th17/Treg balance to attenuate DSS-induced colitis in mice by IL-6/STAT3 signaling pathway, Frontiers in Pharmacology, 12: 615506. https://doi.org/10.3389/fphar.2021.615506Langens-Gerrits M., Albers M., and De Klerk G., 2004, Hot-water treatment before tissue culture reduces initial contamination in Lilium and Acer, Plant Cell, Tissue and Organ Culture, 52: 75-77.

https://doi.org/10.1023/A:1005997202225

Linck H., Lankes C., Krüger E., and Reineke A., 2019, Elimination of phytoplasmas in Rubus mother plants by tissue culture coupled with heat therapy, Plant Disease, 103(6): 1252-1255.

https://doi.org/10.1094/PDIS-08-18-1372-RE

Lizárraga A., Ascasíbar J., and González M., 2017, Fast and effective thermotherapy treatment for in vitro virus eradication in apple and pear trees, American Journal of Plant Sciences, 8: 2474-2482.

https://doi.org/10.4236/AJPS.2017.810168

Loyola-Vargas V., and Ochoa-Alejo N., 2018, An introduction to plant tissue culture: Advances and perspectives, Methods in Molecular Biology, 1815: 3-13.

https://doi.org/10.1007/978-1-4939-8594-4_1

Lv Y., Zou Y., Zhang X., Liu B., Peng X., and Chu C., 2023, A review on the chemical constituents and pharmacological efficacies of Lindera aggregata (Sims) Kosterm., Frontiers in Nutrition, 9: 1071276.

https://doi.org/10.3389/fnut.2022.1071276

Pasternak T., and Steinmacher D., 2024, Plant growth regulation in cell and tissue culture in vitro, Plants, 13(2): 327.

https://doi.org/10.3390/plants13020327

Peng X., Luo Y., Wang J., Ji T., Yuan L., and Kai G., 2020, Integrated analysis of the transcriptome, metabolome and analgesic effect provide insight into potential applications of different parts of Lindera aggregata, Food Research International, 138(Part B): 109799.

https://doi.org/10.1016/j.foodres.2020.109799

Rubio L., Galipienso L., and Ferriol I., 2020, Detection of plant viruses and disease management: Relevance of genetic diversity and evolution, Frontiers in Plant Science, 11: 1092.

https://doi.org/10.3389/fpls.2020.01092

Salleh W., 2020, Lindera aggregata (Sims) Kosterm: Review on phytochemistry and biological activities, Latin American and Caribbean Bulletin of Medicinal and Aromatic Plants, 19(6): 527-541.

https://doi.org/10.37360/blacpma.20.19.6.37

Thorpe T., 2007, History of plant tissue culture, Molecular Biotechnology, 37(2): 169-180.

https://doi.org/10.1007/S12033-007-0031-3

Torres A., Fajardo T., Dusi A., Resende R., and Buso J., 2000, Shoot tip culture and thermotherapy for recovering virus-free plants of garlic, Horticultura Brasileira, 18(3): 192-195.

https://doi.org/10.1590/S0102-05362000000300010

Wang M., Cui Z., Li J., Hao X., Zhao L., and Wang Q., 2018, In vitro thermotherapy-based methods for plant virus eradication, Plant Methods, 14: 79.

https://doi.org/10.1186/s13007-018-0355-y

Wang Q., Jin Z., and Peng L., 2012, Effects of nitrogen deposition on leaf physiological and ecological characteristics of Lindera aggregata seedlings, Ying Yong Sheng Tai Xue Bao (Journal of Applied Ecology), 23(10): 2766-2772.

Zhao X., Tan M., He Y., Pan Y., Fan X., Ye L., Peng X., Wang J., Li F., Zhang S., Zhang L., and Fu L., 2022, The ethanolic extract of Lindera aggregata modulates gut microbiota dysbiosis and alleviates ethanol-induced acute liver inflammation and oxidative stress via SIRT1/Nrf2/NF-κB pathway, Oxidative Medicine and Cellular Longevity, 2022(1): 6256450.

https://doi.org/10.1155/2022/6256450