Medicinal Plant Research 2025, Vol.15, No.2 http://hortherbpublisher.com/index.php/mpr © 2025 HortHerb Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Medicinal Plant Research 2025, Vol.15, No.2 http://hortherbpublisher.com/index.php/mpr © 2025 HortHerb Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Publisher HortHerb Publisher Editedby Editorial Team of Medicinal Plant Research Email: edit@mpr.hortherbpublisher.com Website: http://hortherbpublisher.com/index.php/mpr Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Medicinal Plant Research (ISSN 1927-6508) is an open access, peer reviewed journal published online by HortHerb Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all aspects of medicinal plant research, including plant growth and development, plant biology, plant nutrition, medicinal properties, phytochemical constituents, fitoterapia, pharmacognosy, essential oils, ethno- pharmacology agronomic management, and phytomedicine, as well as chemistry, pharmacology and use of medicinal plants and their derivatives. HortHerb Publisher is an international Open Access publisher specializing in horticulture, herbal sciences, and tea-related research registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. All the articles published in Medicinal Plant Research 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. HortHerb Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Medicinal Plant Research (online), 2025, Vol. 15, No.2 ISSN 1927-6508 http://hortherbpublisher.com/index.php/mpr © 2025 HortHerb Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Research Insight into Enhancing Triterpenoid Content through Genetic Modification in Ganoderma lucidum Baofu Huang, Guangman Xu Medicinal Plant Research, 2025, Vol. 15, No. 2, 51-61 Application of Heat Treatment and Tissue Culture Combined Detoxification Technology in the Healthy Seedling Propagation of Lindera aggregata Xiazhen Huang, Yufen Wang Medicinal Plant Research, 2025, Vol. 15, No. 2, 62-70 Research on Off-season Efficient Cultivation Technology of Leonurus japonicus Based on Environmental Control Yali Deng, Meifang Li Medicinal Plant Research, 2025, Vol. 15, No. 2, 71-79 Effects of Different Transplanting Substrates on the Survival Rate and Growth Quality of Anoectochilus roxburghii Tissue Culture Seedlings Chunyu Li, Jiayao Zhou Medicinal Plant Research, 2025, Vol. 15, No. 2, 80-87 Research on the Extraction of Flavonoids from Hangbaiju (Chrysanthemum morifolium) and the Development of Functional Foods Jianli Lu, Chuchu Liu Medicinal Plant Research, 2025, Vol. 15, No. 2, 88-98
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 51 Research Insight Open Access Research Insight into Enhancing Triterpenoid Content through Genetic Modification inGanoderma lucidum Baofu Huang, Guangman Xu Traditional Chinese Medicine Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, Zhejiang, China Corresponding author: guangman.xu@cuixi.org Medicinal Plant Research, 2025, Vol.15, No.2 doi: 10.5376/mpr.2025.15.0006 Received: 15 Dec., 2024 Accepted: 20 Jan., 2025 Published: 10 Mar., 2025 Copyright © 2025 Huang and Xu, 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 B.F., and Xu G.M., 2025, Research insight into enhancing triterpenoid content through genetic modification in Ganoderma lucidum, Medicinal Plant Research, 15(2): 51-61 (doi: 10.5376/mpr.2025.15.0006) Abstract Ganoderma lucidumhas been widely studied and applied due to its high medicinal value. Its triterpenoid compounds are one of the main active ingredients. But, it is not easy to stably increase the triterpenoid content using traditional cultivation methods, which is a common bottleneck in the industrialization process. In order to solve this problem, many studies have begun to focus on gene-level regulation. For example, using new gene editing technologies such as CRISPR/Cas9, the key genes responsible for triterpenoid synthesis in G. lucidumcan be accurately regulated. At the same time, transcription factors such as GlbHLH5 have also been shown to promote triterpenoid synthesis, indicating that the content can be increased from multiple links. Overall, this type of genetic improvement method has indeed opened up a new situation, which is expected to increase triterpenoid production and provide a more stable source of raw materials for the development of functional foods and medicinal products of G. lucidum. Keywords Ganoderma lucidum; Triterpenoids; Gene editing; Transcription factors; Functional foods; Medicinal value 1 Introduction In traditional Chinese medicine, Ganoderma lucidumis a highly valuable medicinal mushroom. People value it not only for its tonic effects, but also for the various active ingredients it contains. The most noteworthy of these are triterpenoids, such as ganoderic acid, ganoderic acid, and ganoderic acid, which have been shown to have multiple pharmacological activities such as anti-inflammatory, antiviral, and anti-tumor (Ahmad et al., 2021a; b; Sheikha, 2022). It is precisely because of the presence of these triterpenes that G. lucidumhas changed from a traditional medicinal material to a hot target in modern nutritional products and drug development, and has received continuous attention from the scientific research community. Wang et al. (2015) mentioned that triterpenoids can inhibit the activity of some key enzymes, such as neuraminidase and HIV protease, which may be one of the reasons why they have antiviral ability. In terms of immunomodulation and anti-cancer, this type of compound also shows considerable potential. However, the content of triterpenoids in G. lucidumhas always been an important factor limiting the efficacy of the drug. Once the content can be further increased, it will not only be more effective in treating diseases such as viral infections or cancer, but will also have more advantages in anti-inflammatory applications. Sheikha (2022) also pointed out that the improvement of this type of compound also means that the value of functional foods and nutritional products will increase accordingly, thereby driving the medicinal and commercial development of G. lucidumto a further level. The potential of triterpenoids should not be underestimated, but it is not easy to stably increase their content in G. lucidum. The problem lies in multiple levels. On the one hand, the synthesis process of triterpenes is complex, which is regulated by genes and easily disturbed by the external environment. Traditional cultivation methods often make it difficult to control these variables, resulting in large fluctuations in yield and composition (Hennicke et al., 2016). On the other hand, the classification of G. lucidumvarieties is not clear enough. Some strains with high triterpenoid production may be mixed in populations with "similar names", which brings troubles to strain screening and standardization. The key to solving these problems is to figure out the genetic pathway of triterpenoid synthesis and establish a stable and reliable strain identification and cultivation system.
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 52 In recent years, the development of genetic modification methods has brought new possibilities for increasing the content of triterpenoid compounds in G. lucidum. Technologies such as CRISPR/Cas9 and RNA interference (RNAi) have been used to regulate key genes in the triterpene synthesis process, with the aim of allowing G. lucidumto produce more triterpenes (Mirmazloum et al., 2021). In addition, researchers have also screened strains that are already high-yielding and suitable for commercial cultivation through molecular phylogenetic analysis and metabolite detection (Hennicke et al., 2016). Although these methods have high technical barriers, they show great application potential in increasing triterpene production and stabilizing the quality of G. lucidumproducts (Hennicke et al., 2016; Mirmazloum et al., 2021). This study looked at whether genetic improvement could help raise the amount of triterpenoids in G. lucidum. It focused on how key genes control the making of these compounds. The goal is to give a scientific basis for breeding G. lucidumstrains that have stable and high levels of triterpenoids. The study also aims to solve some problems with traditional growing methods, help develop better G. lucidum-based treatments and health foods, and increase its medical and market value. 2 Triterpenoid Compounds inGanoderma lucidum 2.1 Types of triterpenoids inG. lucidum The most representative triterpenoid component in G. lucidum is ganoderic acid. It is an oxygenated lanostane-type triterpene. There are many types, and more than 150 types have been discovered so far, each with its own structural characteristics. In a word, their basic structure is a steroid skeleton, and the difference is that these skeletons have oxidative modifications such as hydroxyl groups added to certain carbon atoms (Cai et al., 2020; Chen et al., 2023). For example, the CYP512U6 P450 family gene in G. lucidumcan add a hydroxyl group to the C-23 position of the triterpene molecule to generate derivatives such as ganoderic acid A and ganoderic acid Jc (Yang et al., 2018). There is also a triterpenoid compound called gingodermic acid, which is also common in G. lucidum. The biggest difference between this type of compound and ganoderic acid is that their degree of oxidation and modification are different. Ganoderic acid is often modified in a more "complex" way, while gingodermic acid may have other changes in the way of hydroxylation (Ye et al., 2018; Ahmad et al., 2021b). It is precisely because of this structural diversity that these triterpenoid compounds have rich biological activities, such as anti-cancer, antiviral, and immunomodulatory effects. 2.2 Pharmacological properties of triterpenoids The triterpenoids in G. lucidum are quite outstanding in anti-inflammatory and antiviral properties. These compounds can interfere with a variety of enzymes and pathways involved in inflammatory responses or viral replication (Wu et al., 2019). For example, studies have found that they can inhibit neuraminidase, HIV protease, and even the NS2B-NS3 protease of dengue virus (Ahmad et al., 2021b). Behind these mechanisms, in fact, is to interrupt the key links of viral replication, so that the virus cannot proliferate normally. For example, it has a clear inhibitory effect on viruses such as HSV. Triterpenoids also have a "boosting" effect on the immune system. They can activate immune cells and improve the body's ability to respond to infection and inflammation. In the field of anti-cancer, the potential of triterpenoids is also gaining more and more attention. Studies have shown that they can induce apoptosis of tumor cells, inhibit metastasis, and affect some key signal transduction pathways in cells, such as those that regulate cell division and growth (Ding et al., 2023). Not only that, these ingredients can also enhance the function of antigen-presenting cells (Ahmad et al., 2021b). It is worth mentioning that triterpenoids are not only "medicines", but also a promising "anti-aging" nutrient. Some studies have mentioned that their long-term use can help slow down physiological decline and improve brain function, and may even bring certain benefits to neurodegenerative diseases such as Alzheimer's disease (Zeng et al., 2021).
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 53 2.3 Importance of triterpenoid content enhancement The efficacy of G. lucidumoften depends on the amount of triterpenoids in it. The higher the content, the more obvious the therapeutic effect. This is not only a laboratory speculation, but also supported by experimental evidence. For example, Ye et al. (2018) found that spraying salicylic acid (SA) during the formation of G. lucidum fruiting bodies can increase the triterpenoid content by 23.32%. This improvement directly leads to the enhancement of pharmacological effects. But the significance is not just "curing diseases". For the market, increasing the triterpenoid content is equivalent to increasing the added value of the product. G. lucidumproducts are not only pharmaceutical raw materials, but also enter the field of functional foods and nutritional supplements. For these products, triterpenoids are like a "selling point", the higher the better. They not only provide basic nutrition, but also enhance immunity, anti-inflammatory, and even help prevent certain chronic diseases (Ahmad et al., 2021b). 3 Genetic Basis of Triterpenoid Biosynthesis 3.1 Key genes involved in triterpenoid pathway In recent years, the identification of key genes in the triterpenoid biosynthesis pathway of G. lucidumhas become a research focus. For example, the cytochrome P450 gene CYP512U6 is considered to be an important gene in the biosynthesis of ganoderic acid (oxidized triterpenoids). Studies have found that the CYP512U6 gene is involved in the synthesis of ganoderic acid, generating new ganoderic acid compounds through hydroxylation, increasing the diversity of triterpenoid compounds produced by G. lucidum(Yang et al., 2018). Due to the difficulty of genetic manipulation of G. lucidumand its slow growth, researchers have achieved heterologous synthesis of ganoderic acid by expressing the cytochrome P450 gene cyp5150l8 in G. lucidumin Saccharomyces cerevisiae to produce anti-tumor ganoderic acid. This provides a new approach for the industrial production of ganoderic acid (Wang et al., 2018). Xu et al. (2022a) first identified the transcription factor GlbHLH5, which can positively regulate the biosynthesis of triterpenes in G. lucidum. In particular, under the induction of methyl jasmonate, GlbHLH5 significantly responded and promoted the expression of key enzyme genes (such as HMGR, SQS and LS), thereby increasing the production of triterpenoids (Figure 1). In addition, genes such as hmgr, hmgs, mvd, fps, sqs and ls are involved in the early stages of triterpenoid synthesis, especially in the mevalonate pathway, which is crucial for the synthesis of triterpenoid precursors (Ye et al., 2018). Figure 1 Schematic of the predicted regulatory effect of GlbHLH5 in Ganoderma triterpenoids biosynthesis. The solid box represents the main pathway (MVA pathway) of Ganoderma triterpenoids. AACT, acetyl-CoA acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase, SQS, squalene synthase; SE, squalene monooxygenase; LS, lanosterol synthase (Adopted from Xu et al., 2022a)
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 54 3.2 Regulatory mechanisms of triterpenoid biosynthesis Transcription factors play an important role in the regulation of triterpenoid biosynthesis in G. lucidum. For example, the transcription factor GlbHLH5 was found to be a key regulatory factor that responds to the induction of methyl jasmonate (MeJA) and plays an important role in the synthesis of triterpenoids (Xu et al., 2022a). Its role in regulating the expression of key enzyme genes was verified by gene overexpression and silencing experiments, thereby increasing the accumulation of triterpenes. Xu et al. (2022b) conducted a transcriptome analysis of transcription factors in G. lucidumunder MeJA induction and found a total of 103 differentially expressed transcription factors, including members of 22 families such as C2H2, HTH, MADS and HMG. The study further showed that the expression of these transcription factors is closely related to the content of triterpenoids in G. lucidum. In addition, Meng et al. (2021) studied the function of the MADS-box transcription factor GlMADS1 in G. lucidumand found that after the gene was silenced by RNA interference technology, the content of ganoderic acid and flavonoids in G. lucidum increased, indicating that GlMADS1 may act as a negative regulatory factor to inhibit the accumulation of these metabolites. Epigenetic changes also help control how triterpenoids are made. The activity of key genes in this process can be influenced by signals from the environment or other molecules (Bai et al., 2018). Research has shown that sodium acetate can increase the amount of ganoderic acid, a type of triterpenoid, by turning on important genes like hmgs, fps, and sqs that help make it (Meng et al., 2019). Sodium acetate also affects how sodium and calcium ions move in cells by acting through the calmodulin and calcineurin pathways. This helps boost ganoderic acid production evenmore. 3.3 Challenges in identifying genetic targets Finding genes that can directly improve the efficiency of triterpenoid synthesis is easy to say but difficult to do. The problem is that the entire biosynthesis process is not completed by a few genes alone, but a very complex network system involving many genes and regulatory levels, and there are cross-influences between each other (Aminfar et al., 2019; Paramasivan et al., 2022). Take the relationship between carbon metabolism and triterpenoid synthesis as an example. Under conditions of limited nitrogen supply, the integration between the two involves multiple metabolic pathways. This complex "collaboration model" makes it difficult for us to see at a glance which gene is really worthy of priority transformation (Lian et al., 2020). In addition to the complex mechanism itself, higher fungi such as G. lucidum are also not easy to manipulate genetically. They have a slow growth cycle and a genetic system that is not easy to manipulate. It takes a long time to do a mutation experiment. In order to circumvent this problem, researchers have come up with a way to put some target genes into a system such as Saccharomyces cerevisiae for heterologous expression (Wang et al., 2018). Although this method speeds up the progress of the experiment, it also has shortcomings - the yeast environment is different from the metabolic background of G. lucidumitself, and it is often impossible to truly restore the regulation of genes in the original background. This still brings certain obstacles to understanding the true function of genes. 4 Current Approaches to Genetic Modification inGanoderma lucidum 4.1 Traditional methods and their limitations The breeding of G. lucidum actually started a long time ago, but the initial methods were more traditional. Excellent varieties such as "Hunong 5" were selected through conventional hybrid breeding. This variety has higher content of polysaccharides and triterpenoids, and has stronger medicinal value (Chao et al., 2018). In addition to breeding, some treatment methods have also been tried, such as spraying salicylic acid (SA) during the fruiting stage of G. lucidum. The results showed that this treatment not only significantly increased the content of triterpenoids, but also activated the expression of some related synthetic genes (Ye et al., 2018).
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 55 But the issue is that these traditional methods are often guided by natural mutations or external stimuli, and the operation process is quite slow. If you want to select a good strain with stable performance, you may have to cultivate several generations, and each generation must be carefully selected, which takes a lot of time and manpower. Moreover, sometimes the effect is not stable, and it is difficult to accurately control the improvement of the target trait (Ye et al., 2018; Zhou et al., 2021). 4.2 CRISPR/Cas9-based gene editing inGanoderma lucidum Compared with traditional methods, the emergence of CRISPR/Cas9 has undoubtedly accelerated the genetic modification of G. lucidum. This technology, in the final analysis, can precisely destroy or modify specific DNA sites. For example, the cytochrome P450 monooxygenase (CYP450) gene is closely related to the synthesis of ganoderic acid. Once it is knocked out, the content of ganoderic acid immediately drops a lot, which shows that this editing system can indeed work (Wang et al., 2020). Although CRISPR is very effective, it is not without its challenges in higher fungi such as G. lucidum. The biggest problem is that its homologous recombination efficiency is too low. This results in a low success rate when you want to insert or replace a gene at a specified location. To solve this problem, Tu et al. (2021) tried to suppress the non-homologous end joining (NHEJ) repair mechanism and found that doing so could improve the efficiency of gene insertion and replacement. Tan et al. (2023) further simplified the CRISPR system into a ribonucleoprotein (RNP) form and delivered it directly into G. lucidumcells. This method does not require any carriers. The pure "protein + RNA" combination achieved 100% editing efficiency on the selective culture medium right away. More importantly, they successfully targeted multiple genes related to triterpenoid synthesis. This achievement means that if G. lucidumstrains with high triterpenoid production are to be cultivated, this type of RNP system is likely to become a key technical support. 4.3 RNA interference and other gene silencing techniques RNA interference (RNAi) is not really a "new technology", but it still has unique value in the study of G. lucidum. It is a biological process that inhibits gene expression by neutralizing target mRNA molecules. dsRNA is cut into small interfering RNA (siRNA) by Dicer enzyme, and then these siRNAs carry RNA-induced silencing complex (RISC) to accurately identify and cut off the target mRNA, thereby effectively silencing the gene. RNAi technology has been used to silence certain key genes in G. lucidum, thereby enhancing the synthesis of triterpenoid compounds. For instance, Wang et al. (2018) conducted an interesting experiment: instead of directly enhancing the genes for triterpenoid synthesis, they chose to silence some metabolic pathway genes that "grab resources". The results showed that this could significantly increase the production of ganoderic acid, proving that this is an "indirect acceleration" strategy. Lu et al. (2020) also selected the LAG1 gene, which is closely related to lipid synthesis. After being interfered with by RNAi, lipid metabolism was weakened, and resources were freed up for triterpenoid synthesis, resulting in an increase in the accumulation of ganoderic acid. RNAi has also been used to silence P450 enzyme genes, such as CYP5150L8, which plays an important role in the multi-step biotransformation of ganoderic acid. Wang et al. (2020) confirmed this by silencing this gene. After silencing this gene, the production of ganoderic acid was greatly reduced, which basically confirms that CYP5150L8 is the key to the synthesis of ganoderic acid. Now there are many studies focusing on the upstream regulatory level, such as the role of signal molecules. Ye et al. (2018) found that signal substances such as calcium ions and salicylic acid can actually indirectly increase the level of triterpene synthesis by regulating the expression of a series of metabolic genes. This shows that the factors affecting triterpene synthesis are far more than just the genes in the synthesis pathway, and the entire signal transduction network is also playing a role behind the scenes.
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 56 5 Genetic Modification and Optimization Strategies of G. lucidum 5.1 Promoter selection and optimization for higher expression In the genetic improvement of G. lucidum, the selection of promoters is a key step. Whether the promoter can drive the stable expression of the target gene directly affects the synthesis efficiency of triterpenoid compounds. For example, studies have found that the endogenous u6 promoter works well in the CRISPR-Cas9 system and is very helpful in improving editing efficiency (Wang et al., 2019). If more stable and efficient promoters like this can be found, it will be helpful to promote the manipulation of G. lucidumfunctional genes. Some promoters can also respond to induction signals, such as methyl jasmonate (MeJA). Such promoters can enhance the expression of genes related to triterpene synthesis under specific induction conditions (Xu et al., 2022b). In addition, there are also studies on engineering existing promoters, such as introducing self-cleaving ribozymes HDV or performing site-directed mutagenesis to improve their activity and specificity. With the support of transcriptome data, promoters with obvious expression under different environmental conditions can also be screened, providing a basis for subsequent optimization (Xu et al., 2022b). 5.2 Gene stacking To put it simply, gene stacking is to integrate multiple target genes at one time to form a "combined force" to make the entire triterpene synthesis pathway more efficient. Now tools like CRISPR-Cas9 can already achieve this kind of precise editing of multiple genes. For example, researchers used CRISPR-Cas9 to knock out P450 genes such as cyp5150l8 and cyp505d13, and found that the types and proportions of triterpene compounds changed significantly (Wang et al., 2020). This shows that it is indeed effective to regulate triterpene synthesis by multiple genes at the same time. The advantage of this method is that it can "link" multiple links in one step, making the entire metabolism flow more smoothly. But it is not without difficulties. It is common for multiple genes to "hinder" each other in the body, and if you are not careful, it may also interfere with other metabolic pathways. But in general, gene stacking is still the most worthwhile way to increase the production of triterpenes in Ganoderma lucidum. 5.3 Use of transcription factors to boost triterpenoid production The triterpenoid synthesis pathway is coordinated by a series of genes, and many of these genes are regulated by transcription factors. Transcription factors such as GlbHLH5 have been found to positively regulate the synthesis of triterpenes. Studies have shown that when GlbHLH5 expression is enhanced in G. lucidum, it will drive the gene expression of multiple key enzymes in the synthesis pathway, thereby allowing more triterpenoid compounds to accumulate (Xu et al., 2022a). In addition, transcription factors that respond to methyl jasmonate (MeJA) are also receiving more and more attention. As regulatory points under exogenous induction, such factors themselves can also become targets for overexpression to increase triterpene production (Xu et al., 2022b). Except for the direct regulation of a single transcription factor, signaling pathways such as HO-1/CO may also indirectly promote triterpenoid synthesis by affecting the expression of related genes in the mevalonic acid (MVA) pathway (Cui et al., 2021). After all, the MVA pathway is the main route for triterpenoid synthesis, and regulating it is equivalent to regulating the basic flow of triterpenoids. At the same time, transcriptome technology can also help us screen out transcription factors whose expression levels change significantly under specific induction or stress conditions, which may become a breakthrough for subsequent gene editing (Xu et al., 2022b). 6 Case Studies 6.1 CRISPR/Cas9 for increasing triterpenoid yield The CRISPR/Cas9 system, as a third-generation gene editing tool, has been widely applied in gene editing research in fungi, plants, and animals due to its efficiency and precision. However, the genetic diversity of G. lucidum and codon preference variations among different strains limit the application of plasmid-dependent CRISPR systems (Tu et al., 2021).
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 57 In order to overcome these limitations, a research team specifically selected the widely cultivated monokaryotic strain L1 of "Shanghai Agricultural No. 1" G. lucidum, established and optimized a CRISPR/Cas9 system based on RNP (i.e., ribonucleoprotein) (Tan et al., 2023). In this system, the researchers used ura3 as the target gene and achieved a considerable editing efficiency: more than 35 mutants were successfully obtained for every 107 protoplasts. This shows that the system is stable in inducing DNA double-strand breaks and can complete gene editing through repair mechanisms such as NHEJ and MMEJ. In addition, the study further edited key genes related to triterpenoid metabolism, such as cyp512a3 and cyp5359n1, providing new tools for regulating triterpenoid synthesis. In another experiment, the researchers tried to add introns to the promoter region of the Cas9 gene, which improved the overall editing efficiency of the system and ultimately achieved a 36.7% ura3 gene deletion rate in G. lucidum(Figure 2) (Liu et al., 2020). These studies provide a technical basis for subsequent functional gene verification and directed breeding, and also make the genome manipulation of G. lucidummore feasible. Figure 2 An effective platform for disruption and deletion of target genes inG. lucidum(Adopted from Liu et al., 2020) Image caption: The figure illustrates the overall process and results of gene knockout and fragment deletion in G. lucidumusing the CRISPR/Cas9 system. It describes the design and steps of two different Cas9 vectors (opCas9 and intron-opCas9) for gene editing, including protoplast transformation, sgRNA targeting design, gene disruption, and fragment deletion processes. The results indicate the superiority of the Cas9 system with an intron, significantly enhancing the efficiency of gene disruption and fragment deletion, validating the effectiveness of the CRISPR/Cas9 system for precise gene editing inG. lucidum(Adopted from Liu et al., 2020) 6.2 Overexpression of key enzymes in triterpenoid pathways In order to increase the content of triterpenoids in G. lucidum, researchers have tried to overexpress key enzymes in the triterpene biosynthesis pathway. In a study, scientists identified for the first time the transcription factor GlbHLH5 as an important regulator of triterpene synthesis and regarded it as a potential metabolic engineering target (Xu et al., 2022a). The experiment found that methyl jasmonic acid (MeJA) treatment not only significantly promoted the accumulation of triterpenoids, but also upregulated the expression level of GlbHLH5, thereby further enhancing its regulatory effect. By constructing transgenic G. lucidum strains that overexpressed and silenced GlbHLH5, the researchers found that the triterpene production of the former increased significantly, while the latter showed a phenomenon of blocked synthesis (Figure 3), verifying its key role.
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 58 Except for the regulatory factors, direct overexpression of enzyme genes in metabolic pathways has also achieved positive results. A research team introduced the cytochrome P450 gene cyp5150l8 into Saccharomyces cerevisiae for expression and successfully synthesized a ganoderic acid derivative with anti-tumor activity, 3-hydroxylanostere-8,24-diene-26-acid (HLDOA), with a yield of 14.5 mg/L (Wang et al., 2018). These results show that by overexpressing key enzymes or regulatory factors, not only can the triterpenoid production in G. lucidumbe increased, but it also has the potential to achieve industrial production in heterologous host systems. Figure 3 (A) The phenotypes of the GlbHLH5 overexpression and control lines of Ganoderma lucidumand the coloration differences between extracts of different lines. (B) The quantitative analysis of triterpenoids and the key genes transcriptions of the biosynthetic pathway in the GlbHLH5 overexpression and the control lines. "**" means the difference is extremely significant (P < 0.01) (Adopted from Xu et al., 2022a) Image caption: The figure shows the effect of GlbHLH5overexpression on the biosynthesis of triterpenoids and the expression of key genes in Ganoderma lucidum. (A) illustrates the phenotypic differences between GlbHLH5 overexpression lines and the wild-type, with the overexpression lines exhibiting a significant increase in triterpenoid content. (B) Quantitative analysis reveals that triterpenoid content in the GlbHLH5 overexpression lines increased by 21%~45%, along with a significant upregulation in the expression levels of key genes in the triterpenoid biosynthetic pathway, including HMGR, SQS, and LS. The results indicate that GlbHLH5 overexpression can significantly promote the accumulation of triterpenoids, confirming its positive regulatory role in triterpenoid biosynthesis (Adopted from Xu et al., 2022a) 7 Potential Applications and Commercial Impacts 7.1 Pharmaceutical applications of triterpenoid-enrichedG. lucidum The medicinal value of G. lucidumhas long been widely recognized, and its triterpenoids and polysaccharides are regarded as its core pharmacological substances. In particular, triterpenoids have shown unique and extensive
Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 59 pharmacological activities in multiple disease areas. These activities include antioxidant, anti-inflammatory, anti-tumor, liver protection and hypoglycemic effects, and have the potential to intervene in a variety of chronic and metabolic diseases (Ahmad et al., 2021b; Zeng et al., 2021; Swallah et al., 2023). Studies have shown that triterpenoids can effectively inhibit the activity of key enzymes such as neuraminidase and HIV protease, thereby interfering with the viral replication process and showing good antiviral effects (Ahmad et al., 2021b). Triterpenes also show important value in the intervention of neurodegenerative diseases. For example, in animal models of Alzheimer's disease, long-term use of triterpenes can significantly slow down neurological degeneration and improve cognitive performance, showing its potential in neuroprotective therapy (Zeng et al., 2021). With the realization of technologies such as genetic modification to increase the content of triterpenes, the medicinal effects of this type of compound will be further enhanced, making G. lucidum an important candidate source for the development of new natural drugs. 7.2 Market demand and future prospects In recent years, the global medicinal mushroom market, including G. lucidum, has continued to show a growth trend. This development is mainly due to the public's increased awareness of natural health products, especially the recognition of G. lucidum's efficacy in enhancing immunity, anti-oxidation and anti-inflammatory. At the same time, the rising demand for functional foods by consumers has also provided a good market foundation for the promotion of G. lucidum products. Increasing the content of triterpenoids through genetic modification technology will not only help improve the efficacy of the product, but may also accelerate the penetration of G. lucidumproducts in the health care products and food industries and enhance their competitiveness (Ekiz et al., 2023; Swallah et al., 2023). At the same time, the antiviral properties of triterpenoids have also opened up new possibilities for their use in disease prevention and treatment. For example, studies have shown that these compounds may fight new viral infections including COVID-19, further expanding the scope of their market applications (Ahmad et al., 2021b; Ekiz et al., 2023). As scientific research progresses, triterpenoid-rich G. lucidumproducts will not only be limited to traditional fields in the future, but will also have broader application prospects in precision nutrition, chronic disease management, and even auxiliary treatments, and the market size and innovation potential are expected to continue to be released. Acknowledgments We sincerely thank Ms. Li from the Horticultural and Herbal Research Center for her valuable support in data analysis and material collection, which contributed to the successful completion of this study. Additionally, we would like to extend my heartfelt gratitude to the two anonymous peer reviewers for their thorough evaluation of the manuscript. 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 Ahmad R., Riaz M., Khan A., Aljamea A., Algheryafi M., Sewaket D., and Alqathama A., 2021a, Ganoderma lucidum (Reishi) an edible mushroom; a comprehensive and critical review of its nutritional, cosmeceutical, mycochemical, pharmacological, clinical, and toxicological properties, Phytotherapy Research, 35(11): 6030-6062. https://doi.org/10.1002/ptr.7215 Ahmad F., Ahmad F., Khan M., Alsayegh A., Wahab S., Alam M., and Ahmed F., 2021b, Ganoderma lucidum: A potential source to surmount viral infections through β-glucans immunomodulatory and triterpenoids antiviral properties, International Journal of Biological Macromolecules, 183: 2180-2191. https://doi.org/10.1016/j.ijbiomac.2021.06.122 Aminfar Z., Rabiei B., Tohidfar M., and Mirjalili M., 2019, Identification of key genes involved in the biosynthesis of triterpenic acids in the mint family, Scientific Reports, 9(1): 14826. https://doi.org/10.1038/s41598-019-52090-z Bai J., Mu R., Dou M., Yan D., Liu B., Wei Q., Wan J., Tang Y., and Hu Y., 2018, Epigenetic modification in histone deacetylase deletion strain of Calcarisporium arbuscula leads to diverse diterpenoids, Acta Pharmaceutica Sinica B, 8(4): 687-697. https://doi.org/10.1016/j.apsb.2017.12.012
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Medicinal Plant Research 2025, Vol.15, No.2, 62-70 http://hortherbpublisher.com/index.php/mpr 62 Research Insight Open Access Application of Heat Treatment and Tissue Culture Combined Detoxification Technology in the Healthy Seedling Propagation of Lindera aggregata Xiazhen Huang1 , Yufen Wang 2 1 Tropical Medicinal Plant Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China 2 Traditional Chinese Medicine Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, Zhejiang, China Corresponding author: xiazhen.huang@hitar.org Medicinal Plant Research, 2025, Vol.15, No.2 doi: 10.5376/mpr.2025.15.0007 Received: 02 Jan., 2025 Accepted: 08 Feb., 2025 Published: 15 Mar., 2025 Copyright © 2025 Huang and Wang, 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 X.Z., and Wang Y.F., 2025, Application of heat treatment and tissue culture combined detoxification technology in the healthy seedling propagation of Lindera aggregata, Medicinal Plant Research, 15(2): 62-70 (doi: 10.5376/mpr.2025.15.0007) Abstract This study discusses the application of integrated heat treatment and tissue culture technology for detoxifying Lindera aggregata seedlings, determining its efficiency in detoxification and healthy seedling development, and its usability and value for further implication. The study has revealed that tissue culture technology readily increases the rate of propagation of Lindera aggregata seedlings using shoot tip culture technology with optimal media conditions promoting shoot proliferation and differentiation. Integrated technology enhances substantially detoxification efficacy along with genetic stability and resistance characteristics of Lindera aggregata seedlings. Thermal treatment suppresses viral replication at elevated temperatures, whereas tissue culture utilizes sterile environment and shoot tip isolation to remove viruses. Combined, these treatments attain detoxification efficacy of over 90%. Detoxified seedlings show better growth characteristics and increased quantities of medicinal constituents, including essential oils and polysaccharides, than control seedlings. The detoxified seedlings also have enhanced growth vigor and stress resistance as well as remarkable active ingredient content promotion. The integration of heat treatment and tissue culture technology in the cultivation of healthy Lindera aggregata seedlings not only increases detoxification efficiency and seedling multiplication but also reduces the risk of disease transmission, rationalizes cultivation management, and guarantees the quality of Lindera aggregata products. This research provides theoretical foundations and technical backstopping for Lindera aggregata germplasm resource preservation and utilization in industry. Keywords Lindera aggregata; Detoxification technology; Heat treatment; Tissue culture; Healthy seedlings 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
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