Appl Microbiol Biotechnol (2012)93:941-963 D0110.1007/s00253-011-3780-7 MINI-REVIEW Applied modern biotechnology for cultivation of Ganoderma and development of their products Xuan-Wei Zhou.Kai-Qi Su.Yong-Ming Zhang Received:21 October 2011/Revised:18 November 2011 /Accepted:21 November 2011/Published online:15 December 2011 C Springer-Verlag 2011 Abstract A white-rot basidiomycete Ganoderma spp.has (or Ganodermaceae)of Aphyllophorales.is one of the most long been used as a medicinal mushroom in Asia,and it has popular medicinal mushrooms in China,Japan,Korea,and an array of pharmacological properties for immunomodula- other Asian countries.It has been under modern biochemi- tory activity.There have been many reports about the bio- cal and pharmacological research during the last 30 years active components and their pharmacological properties.In (Gao et al.2006).However,in the earliest Chinese litera- order to analyze the current status of Ganoderma products, tures,this medicinal mushroom is not called "Lingzhi", the detailed process of cultivation of Ganoderma spp.and instead of "Rui Cao”(means auspicious herbs)or“Zhi” development of their products are restated in this review After the Han dynasty,the name of Lingzhi started to appear article.These include the breeding,cultivating,extracting in the ancient Chinese literatures.It should be noted that bioactive component,and processing Ganoderma products, Lingzhi mentioned in ancient Chinese literatures is different etc.This article will expand people's common knowledge from the Ganoderma described in fungal classification to- on Ganoderma,and provide a beneficial reference for re- day.Besides the Ganoderma and its relatives,Lingzhi de- search and industrial production. scribed in ancient Chinese literatures also included some fungi belonging to Polyporales and Agaricales,etc.,espe- Keywords Ganoderma spp.biotechnology.Breeding cially in Taoism history books(Zhou and Lin 1999). and cultivation.Development and utilizations. In the opinion of modern traditional Chinese medicine Ganoderma-based products.Quality control (TCM),Lingzhi presents three characters for prevention or treatment of diseases.Firstly,the usage of Lingzhi is without any toxicity and apparent absence of side effects;secondly, it has no pertinence on a special organ;and the last one is its Introduction improvement effects on normalization of the organ function. With the development of biotechnology,many researchers Ganoderma lucidum (Fr.)Karst (named as Lingzhi in China). have intensively studied the bioactive components of a species of basidiomycetes which belongs to Polyporaceae Lingzhi and many Lingzhi-based products.Modern phar- X.-W.Zhou·Y.-M.Zhang(☒ macological and clinical trials have demonstrated that College of Life and Environment Sciences, Lingzhi showed a significant effect on the prevention and Shanghai Normal University, treatment of various diseases.For example,the anti-cancer Shanghai 200234,People's Republic of China effects of Lingzhi were associated with triterpenes,polysac- e-mail:zhym@shnu.edu.cn charides,and fungal immunomodulatory proteins (FIPs)by X.-W.Zhou(☒)·K.-Q.Su the mechanisms of DNA polymerase inhibition,post- Plant Biotechnology Research Center,Shanghai Key Laboratory translation modification inhibition of the Ras oncoprotein, of Agrobiotechnology,School of Agriculture and Biology, or cytokine production stimulation(Sliva 2006;Ding et al. Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, 2009a,b;Ogbe et al.2011).Nowadays,there is an increas- Shanghai 200240,People's Republic of China ing public interest on the secondary metabolites of Lingzhi e-mail:xuanweizhou@sjtu.edu.cn for exploring new drugs or leading compounds.Therefore,a ②Springer
MINI-REVIEW Applied modern biotechnology for cultivation of Ganoderma and development of their products Xuan-Wei Zhou & Kai-Qi Su & Yong-Ming Zhang Received: 21 October 2011 /Revised: 18 November 2011 /Accepted: 21 November 2011 / Published online: 15 December 2011 # Springer-Verlag 2011 Abstract A white-rot basidiomycete Ganoderma spp. has long been used as a medicinal mushroom in Asia, and it has an array of pharmacological properties for immunomodulatory activity. There have been many reports about the bioactive components and their pharmacological properties. In order to analyze the current status of Ganoderma products, the detailed process of cultivation of Ganoderma spp. and development of their products are restated in this review article. These include the breeding, cultivating, extracting bioactive component, and processing Ganoderma products, etc. This article will expand people’s common knowledge on Ganoderma, and provide a beneficial reference for research and industrial production. Keywords Ganoderma spp. biotechnology . Breeding and cultivation . Development and utilizations. Ganoderma-based products. Quality control Introduction Ganoderma lucidum (Fr.) Karst (named as Lingzhi in China), a species of basidiomycetes which belongs to Polyporaceae (or Ganodermaceae) of Aphyllophorales, is one of the most popular medicinal mushrooms in China, Japan, Korea, and other Asian countries. It has been under modern biochemical and pharmacological research during the last 30 years (Gao et al. 2006). However, in the earliest Chinese literatures, this medicinal mushroom is not called “Lingzhi”, instead of “Rui Cao” (means auspicious herbs) or “Zhi”. After the Han dynasty, the name of Lingzhi started to appear in the ancient Chinese literatures. It should be noted that Lingzhi mentioned in ancient Chinese literatures is different from the Ganoderma described in fungal classification today. Besides the Ganoderma and its relatives, Lingzhi described in ancient Chinese literatures also included some fungi belonging to Polyporales and Agaricales, etc., especially in Taoism history books (Zhou and Lin 1999). In the opinion of modern traditional Chinese medicine (TCM), Lingzhi presents three characters for prevention or treatment of diseases. Firstly, the usage of Lingzhi is without any toxicity and apparent absence of side effects; secondly, it has no pertinence on a special organ; and the last one is its improvement effects on normalization of the organ function. With the development of biotechnology, many researchers have intensively studied the bioactive components of Lingzhi and many Lingzhi-based products. Modern pharmacological and clinical trials have demonstrated that Lingzhi showed a significant effect on the prevention and treatment of various diseases. For example, the anti-cancer effects of Lingzhi were associated with triterpenes, polysaccharides, and fungal immunomodulatory proteins (FIPs) by the mechanisms of DNA polymerase inhibition, posttranslation modification inhibition of the Ras oncoprotein, or cytokine production stimulation (Sliva 2006; Ding et al. 2009a, b; Ogbe et al. 2011). Nowadays, there is an increasing public interest on the secondary metabolites of Lingzhi for exploring new drugs or leading compounds. Therefore, a X.-W. Zhou : Y.-M. Zhang (*) College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, People’s Republic of China e-mail: zhym@shnu.edu.cn X.-W. Zhou (*) : K.-Q. Su Plant Biotechnology Research Center, Shanghai Key Laboratory of Agrobiotechnology, School of Agriculture and Biology, Fudan–SJTU–Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China e-mail: xuanweizhou@sjtu.edu.cn Appl Microbiol Biotechnol (2012) 93:941–963 DOI 10.1007/s00253-011-3780-7
942 Appl Microbiol Biotechnol (2012)93:941-963 number of bioactive constituents have been isolated from quality has an effect not only on the yield of Lingzhi-based Lingzhi,including small molecule compounds,polysacchar- product but also its quality.Therefore,selection of a good ides,proteins,enzyme,polysaccharide-protein complexes, Lingzhi strain is a very important task.At any time,high etc.(Zhong and Xiao 2009;Xu et al.2010a,b;Ferreira et al. production and good quality are always the principal goals 2010;Xu et al.2011).Because of the unique pharmacolog- for agriculturally important crops,without the exception of ical function and apparent absence of side effects,it has medicinal mushrooms.There are a lot of breeding methods. attained a reputation in the East and some African countries such as mass selection,programmed mutation,cross-breeding as the ultimate herbal substance.Now Lingzhi has been and transgenic breeding,etc.Up to now,with the development added not only to the Chinese Pharmacopoeia(Zhou et al. of modern biotechnology,the protoplast fusion techniques 2007a)but also to the American Herbal Pharmacopoeia and widely applied on mushroom breeding have made greater Therapeutic Compendium (Sanodiya et al.2009). progress.Transgenic engineering techniques applied on the In an overview of previous literatures,there was a larger medicinal mushroom breeding are a technological innovation collection of papers on Lingzhi's bioactive components and on the molecular level.However,the selection and transgenic their pharmacological properties,and a number of reviews breeding are more objective and promising,and have made a had appeared on these aspects (Luo and Lin 2002;Shiao lot of progress from theory to practice in the last 20 years.The 2003:Yuen and Gohel 2005:Zhou et al.2007a,b:Sanodiya breeding strategy of Lingzhi is summarized in Fig.1. et al.2009;Olaku and White 2011;Xu et al.2011).The reason why Lingzhi draws so much attention is because it is Artificial selection Artificial selection,also known as selec- a potential pharmacological macrofungi (Sanodiya et al. tive breeding,is a primitive breeding method,which uses 2009)and it plays an important role in disease prevention artificial means to choose superior strains from nature,for and treatment in folk medicine;meanwhile,modern phar- biologically obtaining the new species and reproducing it macological tests have also demonstrated some actions and selectively.The basic methods of artificial selection were properties of Lingzhi,including immunomodulating,induc- the tissue separation and spore separation methods for obtain- ing cytokine production,anti-allergic,anti-radiation,anti- ing the pure strain,followed by optimization of this strain. tumor,anti-inflammatory,anti-parasitic,anti-oxidant, and then the required strain is obtained (Chen and Su 2008). benefiting on the cardiovascular system,respiratory system, In fact,during the procedure of mushroom production,tissue endocrine and metabolic systems,etc.(Wasser 2002;Gao et separation method is often employed in getting the strain al.2004;Hong et al.2004;Zhou et al.2007a;Mahajna et al. because Lingzhi spore is hard to germinate (Lin and Zhou 2009).However,Lingzhi products processed by bioactive 1999a).Artificial selection is more commonly used in the components depend on the upstream cultivation,such as the breeding of other edible mushrooms than in Lingzhi fruiting bodies,mycelia,and culture broths;meanwhile, Lingzhi-based products also rely on the downstream pro- Mutation breeding Mutation breeding is a new and more cess.In summary,the qualitative and quantitative differ- effective method compared to artificial selection method, ences in the chemical composition of Lingzhi products are which changes genes of the strains,and achieves genic dependent on the strain,origin,extracting process,and recombination.A general procedure of mutation breeding cultivation conditions (Mizuno 1995:Zhou and Lin 1999: involves the following steps:selection of original strain- Zhou et al.2008a:McKenna et al.2002).In this review preparation of spore (or protoplast)suspension liquid article,the cultivation methods and conditions of Lingzhi viable count and mutagenizing-spreading plate for culti- have been overviewed.Subsequently,the current status of vation-picking up strain and inoculation-initial screen- products process would be addressed.Finally,problems and ing→slope culture→re-screening→selection of superior prospects were analyzed and viewed.This review is benefi- strain.The protoplast is usually chosen for mutation breeding cial to researchers and producers (Li et al.2001),and the increased bioactive components,such as polysaccharides (Gao et al.2008),triterpenoids (Li et al. 2001),and organic germanium (Dong et al.2009),are Cultivation of Ganoderma species regarded as the breeding objectives.Mutation breeding could not only increase strain mutation rate through simple opera- Wild Lingzhi is difficult to collect and to control its quality.In tion but also provide genetic markers for further cross- 1970,a Chinese technician used"spore separation cultivation breeding and cell fusion breeding.However,it also has some method"to successfully cultivate Lingzhi.From then on. disadvantages.For example,the mutation generation is ran- artificial cultivation of Lingzhi has been available in China. dom and the work on selecting the mutant is complicated,etc Breeding of Lingzhi The good quality of Lingzhi strain is Cross-breeding Cross-breeding technology is the most the precondition or key for Lingzhi production.The strain widely used and effective breeding method in breeding the ②Springer
number of bioactive constituents have been isolated from Lingzhi, including small molecule compounds, polysaccharides, proteins, enzyme, polysaccharide–protein complexes, etc. (Zhong and Xiao 2009; Xu et al. 2010a, b; Ferreira et al. 2010; Xu et al. 2011). Because of the unique pharmacological function and apparent absence of side effects, it has attained a reputation in the East and some African countries as the ultimate herbal substance. Now Lingzhi has been added not only to the Chinese Pharmacopoeia (Zhou et al. 2007a) but also to the American Herbal Pharmacopoeia and Therapeutic Compendium (Sanodiya et al. 2009). In an overview of previous literatures, there was a larger collection of papers on Lingzhi’s bioactive components and their pharmacological properties, and a number of reviews had appeared on these aspects (Luo and Lin 2002; Shiao 2003; Yuen and Gohel 2005; Zhou et al. 2007a, b; Sanodiya et al. 2009; Olaku and White 2011; Xu et al. 2011). The reason why Lingzhi draws so much attention is because it is a potential pharmacological macrofungi (Sanodiya et al. 2009) and it plays an important role in disease prevention and treatment in folk medicine; meanwhile, modern pharmacological tests have also demonstrated some actions and properties of Lingzhi, including immunomodulating, inducing cytokine production, anti-allergic, anti-radiation, antitumor, anti-inflammatory, anti-parasitic, anti-oxidant, benefiting on the cardiovascular system, respiratory system, endocrine and metabolic systems, etc. (Wasser 2002; Gao et al. 2004; Hong et al. 2004; Zhou et al. 2007a; Mahajna et al. 2009). However, Lingzhi products processed by bioactive components depend on the upstream cultivation, such as the fruiting bodies, mycelia, and culture broths; meanwhile, Lingzhi-based products also rely on the downstream process. In summary, the qualitative and quantitative differences in the chemical composition of Lingzhi products are dependent on the strain, origin, extracting process, and cultivation conditions (Mizuno 1995; Zhou and Lin 1999; Zhou et al. 2008a; McKenna et al. 2002). In this review article, the cultivation methods and conditions of Lingzhi have been overviewed. Subsequently, the current status of products process would be addressed. Finally, problems and prospects were analyzed and viewed. This review is beneficial to researchers and producers. Cultivation of Ganoderma species Wild Lingzhi is difficult to collect and to control its quality. In 1970, a Chinese technician used “spore separation cultivation method” to successfully cultivate Lingzhi. From then on, artificial cultivation of Lingzhi has been available in China. Breeding of Lingzhi The good quality of Lingzhi strain is the precondition or key for Lingzhi production. The strain quality has an effect not only on the yield of Lingzhi-based product but also its quality. Therefore, selection of a good Lingzhi strain is a very important task. At any time, high production and good quality are always the principal goals for agriculturally important crops, without the exception of medicinal mushrooms. There are a lot of breeding methods, such as mass selection, programmed mutation, cross-breeding and transgenic breeding, etc. Up to now, with the development of modern biotechnology, the protoplast fusion techniques widely applied on mushroom breeding have made greater progress. Transgenic engineering techniques applied on the medicinal mushroom breeding are a technological innovation on the molecular level. However, the selection and transgenic breeding are more objective and promising, and have made a lot of progress from theory to practice in the last 20 years. The breeding strategy of Lingzhi is summarized in Fig. 1. Artificial selection Artificial selection, also known as selective breeding, is a primitive breeding method, which uses artificial means to choose superior strains from nature, for biologically obtaining the new species and reproducing it selectively. The basic methods of artificial selection were the tissue separation and spore separation methods for obtaining the pure strain, followed by optimization of this strain, and then the required strain is obtained (Chen and Su 2008). In fact, during the procedure of mushroom production, tissue separation method is often employed in getting the strain because Lingzhi spore is hard to germinate (Lin and Zhou 1999a). Artificial selection is more commonly used in the breeding of other edible mushrooms than in Lingzhi. Mutation breeding Mutation breeding is a new and more effective method compared to artificial selection method, which changes genes of the strains, and achieves genic recombination. A general procedure of mutation breeding involves the following steps: selection of original strain→ preparation of spore (or protoplast) suspension liquid→ viable count and mutagenizing→spreading plate for cultivation→picking up strain and inoculation→initial screening→slope culture→re-screening→selection of superior strain. The protoplast is usually chosen for mutation breeding (Li et al. 2001), and the increased bioactive components, such as polysaccharides (Gao et al. 2008), triterpenoids (Li et al. 2001), and organic germanium (Dong et al. 2009), are regarded as the breeding objectives. Mutation breeding could not only increase strain mutation rate through simple operation but also provide genetic markers for further crossbreeding and cell fusion breeding. However, it also has some disadvantages. For example, the mutation generation is random and the work on selecting the mutant is complicated, etc. Cross-breeding Cross-breeding technology is the most widely used and effective breeding method in breeding the 942 Appl Microbiol Biotechnol (2012) 93:941–963
Appl Microbiol Biotechnol (2012)93:941-963 943 Fig.1 The breeding strategy of Ganoderma species Set up the breeding Define the regions and types that objectives of Lingzhi Lingzhi being origin from Identification,purification and construction of gene pools of Lingzhi Conventionality Radiating and Cell engineering Gene engineering breeding mutant breeding breeding breeding Evaluation of the agronomic traits,physiological and biochemical characteristic,genetic rule of generation ★ Generation area Comparison of Identification Selection of species area area breeding area Multi-site test,demonstration base,popularization and transformation new species of edible or medicinal mushroom.The principle protoplast-regenerating and culturing the fusion-detecting of cross-breeding is to achieve genetic recombination and selecting fusion.In the 1970s,this method has been through haploid mating,and then strains from generation widely applied in basidiomycetes breeding (Ferenczy et al. with parent's good traits are selected.After the 1980s,cross- 1974).At the beginning of the 1980s,it has been used in breeding technology was widely used in the breeding edible mushroom breeding in some Asian countries.Initially, researches of edible fungi in China and other Asian most of the researches focused on isolation and preparation of countries (Zhao and Chang 1993;Chiu et al.2005).The Lingzhi protoplast(Choi et al.1987;Li and Li 1999;Chen et Lingzhi spores are difficult to germinate under artificial al.2007a,b),and then it was gradually applied in breeding the conditions,so the monokaryotic strains cannot be obtained, new strains by fusing intragenus protoplast(Park et al.1988) which is necessary for the breeding of the Lingzhi.As a and intergeneric protoplast (Yoo et al.2002).Some research- result,the hybridization process of Lingzhi is restricted. ers also successfully obtained the new variety of Lingzhi by Therefore,the protoplast monokaryogenesis method to ob- protoplast monokaryogenesis method (Wu et al.2009a). tain the new strain are used in practice (Wu et al.2009a). There have been some reports about artificial cross-breeding Genetic engineering breeding Genetic engineering is a tech- where most of them were selected using protoplast as mate- nological innovation in the area of molecular biology.Using rials(Chiu et al.2005). this technology,a DNA sequence from one species can be isolated and then transferred to another.Meanwhile,the Cell fusion breeding Cell fusion breeding is an important good characters of donor strain can be expressed in host part of modem biotechnology and is also a significant leap strain,which will become a high-production and good- on genetic breeding.In the fusion of cell protoplasts,the quality strain.This novel breeding method provides a new different genotypes of protoplasts from various organisms breeding solution for edible or medicinal mushroom,espe- are fused with each other,which is induced by the fusion cially for those limited by conventional breeding means. agent after breaking the cell wall.In that case,the fusions The general procedure of genetic engineering breeding is could make the cell genomes from different genus mix effec- presented as follows:selecting donor strains-separating tively,which produces a whole set of genetic exchange and gene-genes reconstruction in vitro-transfer the gene into restructures to generate a new individual(Tan et al.2005).A the recipient cell-reproduction and expression of recom- common method of cell fusion engineering breeding involves binant DNA-selection of new individual.Transformation the following procedure:selecting parent strain-ensuring is the key to Lingzhi genetic engineering breeding.Up to genetic markers of parent strain-isolating protoplast from now,six kinds of transformation methods have been applied parent strain-regenerating and culturing protoplast-fusing for filamentous fungi,which includes protoplast-mediated ②Springer
new species of edible or medicinal mushroom. The principle of cross-breeding is to achieve genetic recombination through haploid mating, and then strains from generation with parent’s good traits are selected. After the 1980s, crossbreeding technology was widely used in the breeding researches of edible fungi in China and other Asian countries (Zhao and Chang 1993; Chiu et al. 2005). The Lingzhi spores are difficult to germinate under artificial conditions, so the monokaryotic strains cannot be obtained, which is necessary for the breeding of the Lingzhi. As a result, the hybridization process of Lingzhi is restricted. Therefore, the protoplast monokaryogenesis method to obtain the new strain are used in practice (Wu et al. 2009a). There have been some reports about artificial cross-breeding where most of them were selected using protoplast as materials (Chiu et al. 2005). Cell fusion breeding Cell fusion breeding is an important part of modern biotechnology and is also a significant leap on genetic breeding. In the fusion of cell protoplasts, the different genotypes of protoplasts from various organisms are fused with each other, which is induced by the fusion agent after breaking the cell wall. In that case, the fusions could make the cell genomes from different genus mix effectively, which produces a whole set of genetic exchange and restructures to generate a new individual (Tan et al. 2005). A common method of cell fusion engineering breeding involves the following procedure: selecting parent strain→ensuring genetic markers of parent strain→isolating protoplast from parent strain→regenerating and culturing protoplast→fusing protoplast→regenerating and culturing the fusion→detecting and selecting fusion. In the 1970s, this method has been widely applied in basidiomycetes breeding (Ferenczy et al. 1974). At the beginning of the 1980s, it has been used in edible mushroom breeding in some Asian countries. Initially, most of the researches focused on isolation and preparation of Lingzhi protoplast (Choi et al. 1987; Li and Li 1999; Chen et al. 2007a, b), and then it was gradually applied in breeding the new strains by fusing intragenus protoplast (Park et al. 1988) and intergeneric protoplast (Yoo et al. 2002). Some researchers also successfully obtained the new variety of Lingzhi by protoplast monokaryogenesis method (Wu et al. 2009a). Genetic engineering breeding Genetic engineering is a technological innovation in the area of molecular biology. Using this technology, a DNA sequence from one species can be isolated and then transferred to another. Meanwhile, the good characters of donor strain can be expressed in host strain, which will become a high-production and goodquality strain. This novel breeding method provides a new breeding solution for edible or medicinal mushroom, especially for those limited by conventional breeding means. The general procedure of genetic engineering breeding is presented as follows: selecting donor strains→separating gene→genes reconstruction in vitro→transfer the gene into the recipient cell→reproduction and expression of recombinant DNA→selection of new individual. Transformation is the key to Lingzhi genetic engineering breeding. Up to now, six kinds of transformation methods have been applied for filamentous fungi, which includes protoplast-mediated Set up the breeding objectives of Lingzhi Define the regions and types that Lingzhi being origin from Identification, purification and construction of gene pools of Lingzhi Conventionality breeding Radiating and mutant breeding Cell engineering breeding Gene engineering breeding Evaluation of the agronomic traits, physiological and biochemical characteristic, genetic rule of generation Generation area Comparison of species area Identification area Selection of breeding area Multi-site test, demonstration base, popularization and transformation Fig. 1 The breeding strategy of Ganoderma species Appl Microbiol Biotechnol (2012) 93:941–963 943
944 Appl Microbiol Biotechnol (2012)93:941-963 transformation (PMT),agrobacterium-mediated transforma- cultivation.In the developed countries and developing tion,electroporation,biolistic transformation,restriction countries,the effects of mushroom breeding rely on the enzyme-mediated integration (REMD),and lithium acetate, amount of cultivation test for verification.In any case,from etc.(Zhou et al.2010).Most of these methods have suc- the viewpoint of the study level of mushroom genetics,the cessfully worked in breeding of Lingzhi (Park et al.1991: method of DNA special analysis cannot be applied directly Sun et al.2001a:Kim et al.2004a). to explain and decide on the detection of strain quality and For example,Li et al.(2004)constructed fungal expres- other problems relative to the strain quality (Zhang et al. sion plasmid pAN7-1(6.7 kb),which carried the promoters 2005a,b).Today,the quality standards of Lingzhi strains of hph(hygromycin phosphate dehydrogenase)gene from have been addressed in some provinces in China where the Escherichia coli and the gpd(glyceraldehyde-p-dehydroge- Lingzhi could be produced on a large scale (Fujian Bureau nase)gene from Aspergillus nidulans.The plasmid could of Quality and Technical Supervision 2002;Anhui Bureau express hygromycin B resistance in fungal culture and of Quality and Technical Supervision 2004).The local achieve the transformation of Lingzhi protoplasts with standards were used for the evaluation of Lingzhi strains. 60%polyethylene glycol (PEG)4000.So it provided a In different opinions,TCM fingerprint generates from foundation for transforming Lingzhi protoplast by the different names,such as chemical fingerprints and biologi- PMT method (Li et al.2004).Kim et al.(2004a,b)reported cal fingerprint,spectral fingerprint and chromatographic the studies on genetic transformation and mutant isolation fingerprint,etc.From the view of modern biotechnology based on REMI technology in G.lucidum.They constructed analysis,it includes the DNA fingerprint,protein finger- a plasmid pJS205-1(6.5 kb)carrying the resistance gene of print,and chemical fingerprints.At present,there are many geneticin (geneticin,an aminoglycoside antibiotic)and studies about chemical fingerprints of Lingzhi.However, phosphinothricin.After using restriction enzyme EcoRV, chromatography methods were popular and were studied Notl,and Xhol,plasmid pJS205-1 was transformed into in depth during the past decade (Xing et al.2004;Huang the Lingzhi protoplasts.Then a series of mutants were et al.2004a,b;Zhang et al.2009;Chen et al.2010a,b; obtained and its preliminary identification on biochemical Dejaegher and Heyden 2010).With the development of characteristics was observed (Kim et al.2004a).For exam- molecular biology technology,DNA fingerprint technology ple,Li and Chen(2002)used a Ti plasmid vector containing was firstly introduced to fungi to research genetic diversity an exogenous gene to transform the exogenous gene into the related to the genus,species,strain,etc.(Anderson et al fungus protoplasts and made it stably replicated and expressed 1987;Hwang and Kim 1995).Subsequently,it was intro- (Li and Chen 2002).Zhang et al.(2011)over-expressed rice duced to variety identification and authentication in Lingzhi OsUgp2 gene in Ganoderma sinensis and increased the con- (Hseu et al.1996;Shi et al.2008;Wu et al.2009b;Zheng et tent of intracellular and extracellular polysaccharide(EPS)in al.2009).Some scholars tried to use the function gene such G.sinensis (Zhang et al.2011).In all,gene engineering as FIPs to identify Lingzhi (Zhou et al.2008b).However, techniques could contribute to the new vigor for using sec- there are many different opinions about how to build an ondary engineering to breed Lingzhi. effective fingerprint(Gottlieb et al.2000;Zhou et al.2005). The total strategies are shown in Fig.2(Zhou et al.2006). Setting up the fingerprint of good quality species It is cru- DNA fingerprint has two main purposes:identification of cial for both growers and researchers to understand the the TCM authenticity and analysis of the TCM quality.The features and qualities of the best strains.The choice of a DNA molecular marker technology,which DNA fingerprint proper strain can determine success or failure.Therefore,the depends on,can be divided into three groups:(1)restriction analysis and evaluation of the strain qualities have always fragment length polymorphism(RFLP)and other technolo- been the focus of the producer and researcher of medicinal gies,in which the core is based on the electrophoretic mushroom.Up to now.in China.the evaluated content of techniques and molecular hybridization techniques(Tanksley medicinal mushroom(including Lingzhi)includes six major et al.1989;Deragon and Landry 1992);(2)DNA fingerprints indices of determinations and assays,which are the appear- and DNA sequencing technology such as random amplified ance of mycelia or strains,microorganism examination, polymorphic DNA (RAPD)(Bardakci 2001),simple se- microbe testing,determination of the growing speed,the quence repeats (SSR),arbitrarily primed polymerase chain esterase isozyme analysis,and the cultivated character.The reaction(AP-PCR),etc.,which depend on the electrophoretic abovementioned cultivated character includes the shape of techniques and PCR techniques;and(3)amplified fragment fruit bodies,mycelia growing speed,vegetative stage (and length polymorphism (AFLP)(Vos et al.1995),sequence reproductive stages),yield,etc. characterized amplified regions(SCAR).direct amplification The existing knowledge and technique for identification of length polymorphism(DALP),RFLP-PCR,RAPD-PCR, of strain qualities are based on the cultivation test.determi- and the latest gene chip diagnostic technique,which combine nation,and assay of agronomic traits in the course of the two abovementioned techniques.Of course,all kinds of ②Springer
transformation (PMT), agrobacterium-mediated transformation, electroporation, biolistic transformation, restriction enzyme-mediated integration (REMI), and lithium acetate, etc. (Zhou et al. 2010). Most of these methods have successfully worked in breeding of Lingzhi (Park et al. 1991; Sun et al. 2001a; Kim et al. 2004a). For example, Li et al. (2004) constructed fungal expression plasmid pAN7-1 (6.7 kb), which carried the promoters of hph (hygromycin phosphate dehydrogenase) gene from Escherichia coli and the gpd (glyceraldehyde-p-dehydrogenase) gene from Aspergillus nidulans. The plasmid could express hygromycin B resistance in fungal culture and achieve the transformation of Lingzhi protoplasts with 60% polyethylene glycol (PEG) 4000. So it provided a foundation for transforming Lingzhi protoplast by the PMT method (Li et al. 2004). Kim et al. (2004a, b) reported the studies on genetic transformation and mutant isolation based on REMI technology in G. lucidum. They constructed a plasmid pJS205-1(6.5 kb) carrying the resistance gene of geneticin (geneticin, an aminoglycoside antibiotic) and phosphinothricin. After using restriction enzyme EcoRV, NotI, and XhoI, plasmid pJS205-1 was transformed into the Lingzhi protoplasts. Then a series of mutants were obtained and its preliminary identification on biochemical characteristics was observed (Kim et al. 2004a). For example, Li and Chen (2002) used a Ti plasmid vector containing an exogenous gene to transform the exogenous gene into the fungus protoplasts and made it stably replicated and expressed (Li and Chen 2002). Zhang et al. (2011) over-expressed rice OsUgp2 gene in Ganoderma sinensis and increased the content of intracellular and extracellular polysaccharide (EPS) in G. sinensis (Zhang et al. 2011). In all, gene engineering techniques could contribute to the new vigor for using secondary engineering to breed Lingzhi. Setting up the fingerprint of good quality species It is crucial for both growers and researchers to understand the features and qualities of the best strains. The choice of a proper strain can determine success or failure. Therefore, the analysis and evaluation of the strain qualities have always been the focus of the producer and researcher of medicinal mushroom. Up to now, in China, the evaluated content of medicinal mushroom (including Lingzhi) includes six major indices of determinations and assays, which are the appearance of mycelia or strains, microorganism examination, microbe testing, determination of the growing speed, the esterase isozyme analysis, and the cultivated character. The abovementioned cultivated character includes the shape of fruit bodies, mycelia growing speed, vegetative stage (and reproductive stages), yield, etc. The existing knowledge and technique for identification of strain qualities are based on the cultivation test, determination, and assay of agronomic traits in the course of cultivation. In the developed countries and developing countries, the effects of mushroom breeding rely on the amount of cultivation test for verification. In any case, from the viewpoint of the study level of mushroom genetics, the method of DNA special analysis cannot be applied directly to explain and decide on the detection of strain quality and other problems relative to the strain quality (Zhang et al. 2005a, b). Today, the quality standards of Lingzhi strains have been addressed in some provinces in China where the Lingzhi could be produced on a large scale (Fujian Bureau of Quality and Technical Supervision 2002; Anhui Bureau of Quality and Technical Supervision 2004). The local standards were used for the evaluation of Lingzhi strains. In different opinions, TCM fingerprint generates from different names, such as chemical fingerprints and biological fingerprint, spectral fingerprint and chromatographic fingerprint, etc. From the view of modern biotechnology analysis, it includes the DNA fingerprint, protein fingerprint, and chemical fingerprints. At present, there are many studies about chemical fingerprints of Lingzhi. However, chromatography methods were popular and were studied in depth during the past decade (Xing et al. 2004; Huang et al. 2004a, b; Zhang et al. 2009; Chen et al. 2010a, b; Dejaegher and Heyden 2010). With the development of molecular biology technology, DNA fingerprint technology was firstly introduced to fungi to research genetic diversity related to the genus, species, strain, etc. (Anderson et al. 1987; Hwang and Kim 1995). Subsequently, it was introduced to variety identification and authentication in Lingzhi (Hseu et al. 1996; Shi et al. 2008; Wu et al. 2009b; Zheng et al. 2009). Some scholars tried to use the function gene such as FIPs to identify Lingzhi (Zhou et al. 2008b). However, there are many different opinions about how to build an effective fingerprint (Gottlieb et al. 2000; Zhou et al. 2005). The total strategies are shown in Fig. 2 (Zhou et al. 2006). DNA fingerprint has two main purposes: identification of the TCM authenticity and analysis of the TCM quality. The DNA molecular marker technology, which DNA fingerprint depends on, can be divided into three groups: (1) restriction fragment length polymorphism (RFLP) and other technologies, in which the core is based on the electrophoretic techniques and molecular hybridization techniques (Tanksley et al. 1989; Deragon and Landry 1992); (2) DNA fingerprints and DNA sequencing technology such as random amplified polymorphic DNA (RAPD) (Bardakci 2001), simple sequence repeats (SSR), arbitrarily primed polymerase chain reaction (AP-PCR), etc., which depend on the electrophoretic techniques and PCR techniques; and (3) amplified fragment length polymorphism (AFLP) (Vos et al. 1995), sequence characterized amplified regions (SCAR), direct amplification of length polymorphism (DALP), RFLP-PCR, RAPD-PCR, and the latest gene chip diagnostic technique, which combine the two abovementioned techniques. Of course, all kinds of 944 Appl Microbiol Biotechnol (2012) 93:941–963
Appl Microbiol Biotechnol(2012)93:941-963 945 Fig.2 Formation strategy of DNA fingerprint of Ganoderma Component identification Ganoderma Activity (revised from Zhou et al.2006) Pesticide residue detection lucidum analysis DNA extraction The choice of Sample selection target gene Expasion of DNA amplification target gene Fingerprint DNA sequencing Restriction endonuclease enzyme digestion Specific-primer PCR Fingerprint molecular marker methods have its advantages and disadvan- Artificial cultivation of Lingzhi fruit bodies It is necessary to tages (Jones et al.1997).Any kind of marker technologies meet the requirement of Lingzhi fruit body growth,which cannot overall analyze all the medicinal materials.The selec- includes the nutrients components and the environmental con- tion of methods must lie on the actual need,different features, ditions.Meanwhile,we must also master the culture methods. and advantages of the molecular marking techniques.How- The nutrition conditions of Lingzhi growth mainly include ever,the fingerprint techniques are better or advanced,but not carbon resources and nitrogen resources,together with inor- exact.When selecting the specific technologies,the popularity ganic salts and growing factors.The Lingzhi growth generally of the technology and the cost of samples analysis also should uses organic carbon resources such as sugar,starch,cellulose. be taken into consideration(Wu et al.2009b;Zhou et al.2006; hemicelluloses,and lignin.The glucose and sucrose are usu- Blundeoll 2006;Urbanelli et al.2007). ally used in culture of mycelia,the sawdust of broad-leaved tree (Jiang 2001;Xia et al.2003;Liao and Xiao 2006),and the agricultural by-products,such as cotton seed husk,straw,and corn cob,in cultivation of fruit bodies (Wei et al.2005;Yan Cultivation procedures 2000).Some small molecular weight compounds,such as amino acid,urea,nitrogen,etc.,can be utilized by Lingzhi The methods of Lingzhi cultivation can be divided into two mycelia.During the process of Lingzhi cultivation,yeast major types,including liquid-state cultivation (LSC)and powder and peptone are usually added in the media of cultur- solid-state cultivation (SSC).The SSC also can be divided ing mycelia,while wheat bran,corn powder,coarse powders into two methods based on the used raw materials.which are of rice bran,ammonium sulfate,urea,etc.are used in the called as the log (or basswood)cultivation and substituted media of culturing fruit bodies.When the rate of carbon to cultivation.At the beginning of the artificial cultivation of nitrogen is 15-45:1 in the substrate,the Lingzhi mycelia may this valuable medicinal mushroom,only four species was grow well.The appropriate rate of carbon to nitrogen on the used for trials,which included G.lucidum (Leyss.Ex.Fr.) substrate for cultivation of fruit bodies is 30-40:1 (Lin and Karst,G.lucidum (Leyss.Ex.Fr)Karst vat.,G.japonicum Zhou 1999b;Han et al.2003;Wu et al.2008). (Fr.)Lloyd,and G.capense (Lloyd)Teng (Zhou and Lin Besides carbon and nitrogen resources,Lingzhi growth 1999).Through more than 40 years of development,the also needs inorganic salts and other inorganic elements,in- cultivation techniques have achieved significant progress. cluding kalium,natrium,calcium,magnesium,phosphorus, Currently,the various methods are widely used for commer- sulfur,zinc,etc.Among them,phosphorus,kalium,and mag- cial production (Hou and Liao 2009;Zhou et al.2010). nesium are the three main nutrition elements.Their ②Springer
molecular marker methods have its advantages and disadvantages (Jones et al. 1997). Any kind of marker technologies cannot overall analyze all the medicinal materials. The selection of methods must lie on the actual need, different features, and advantages of the molecular marking techniques. However, the fingerprint techniques are better or advanced, but not exact. When selecting the specific technologies, the popularity of the technology and the cost of samples analysis also should be taken into consideration (Wu et al. 2009b; Zhou et al. 2006; Blundeoll 2006; Urbanelli et al. 2007). Cultivation procedures The methods of Lingzhi cultivation can be divided into two major types, including liquid-state cultivation (LSC) and solid-state cultivation (SSC). The SSC also can be divided into two methods based on the used raw materials, which are called as the log (or basswood) cultivation and substituted cultivation. At the beginning of the artificial cultivation of this valuable medicinal mushroom, only four species was used for trials, which included G. lucidum (Leyss. Ex. Fr.) Karst, G. lucidum (Leyss. Ex. Fr) Karst vat., G. japonicum (Fr.) Lloyd, and G. capense (Lloyd) Teng (Zhou and Lin 1999). Through more than 40 years of development, the cultivation techniques have achieved significant progress. Currently, the various methods are widely used for commercial production (Hou and Liao 2009; Zhou et al. 2010). Artificial cultivation of Lingzhi fruit bodies It is necessary to meet the requirement of Lingzhi fruit body growth, which includes the nutrients components and the environmental conditions. Meanwhile, we must also master the culture methods. The nutrition conditions of Lingzhi growth mainly include carbon resources and nitrogen resources, together with inorganic salts and growing factors. The Lingzhi growth generally uses organic carbon resources such as sugar, starch, cellulose, hemicelluloses, and lignin. The glucose and sucrose are usually used in culture of mycelia, the sawdust of broad-leaved tree (Jiang 2001; Xia et al. 2003; Liao and Xiao 2006), and the agricultural by-products, such as cotton seed husk, straw, and corn cob, in cultivation of fruit bodies (Wei et al. 2005; Yan 2000). Some small molecular weight compounds, such as amino acid, urea, nitrogen, etc., can be utilized by Lingzhi mycelia. During the process of Lingzhi cultivation, yeast powder and peptone are usually added in the media of culturing mycelia, while wheat bran, corn powder, coarse powders of rice bran, ammonium sulfate, urea, etc. are used in the media of culturing fruit bodies. When the rate of carbon to nitrogen is 15–45:1 in the substrate, the Lingzhi mycelia may grow well. The appropriate rate of carbon to nitrogen on the substrate for cultivation of fruit bodies is 30–40:1 (Lin and Zhou 1999b; Han et al. 2003; Wu et al. 2008). Besides carbon and nitrogen resources, Lingzhi growth also needs inorganic salts and other inorganic elements, including kalium, natrium, calcium, magnesium, phosphorus, sulfur, zinc, etc. Among them, phosphorus, kalium, and magnesium are the three main nutrition elements. Their Component identification Pesticide residue detection Ganoderma lucidum Activity analysis Sample selection DNA extraction The choice of target gene DNA amplification Expasion of target gene Fingerprint DNA sequencing Fingerprint Specific-primer PCR Restriction endonuclease enzyme digestion Fig. 2 Formation strategy of DNA fingerprint of Ganoderma (revised from Zhou et al. 2006) Appl Microbiol Biotechnol (2012) 93:941–963 945