Raman intensities for ID, IG, I2D, ID/IG and I2D / IG of Figure 10.
\r\n\tIn sum, the book presents a reflective analysis of the pedagogical hubs for a changing world, considering the most fundamental areas of the current contingencies in education.
",isbn:"978-1-83968-793-8",printIsbn:"978-1-83968-792-1",pdfIsbn:"978-1-83968-794-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"b01f9136149277b7e4cbc1e52bce78ec",bookSignature:"Dr. María Jose Hernandez-Serrano",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10229.jpg",keywords:"Teacher Digital Competences, Flipped Learning, Online Resources Design, Neuroscientific Literacy (Myths), Emotions and Learning, Multisensory Stimulation, Citizen Skills, Violence Prevention, Moral Development, Universal Design for Learning, Sensitizing on Diversity, Supportive Strategies",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 14th 2020",dateEndSecondStepPublish:"October 12th 2020",dateEndThirdStepPublish:"December 11th 2020",dateEndFourthStepPublish:"March 1st 2021",dateEndFifthStepPublish:"April 30th 2021",remainingDaysToSecondStep:"3 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dr. Phil. Maria Jose Hernandez Serrano is a tenured lecturer in the Department of Theory and History of Education at the University of Salamanca, where she currently teaches on Teacher Education. She graduated in Social Education (2000) and Psycho-Pedagogy (2003) at the University of Salamanca. Then, she obtained her European Ph.D. in Education and Training in Virtual Environments by research with the University of Manchester, UK (2009).",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"187893",title:"Dr.",name:"María Jose",middleName:null,surname:"Hernandez-Serrano",slug:"maria-jose-hernandez-serrano",fullName:"María Jose Hernandez-Serrano",profilePictureURL:"https://mts.intechopen.com/storage/users/187893/images/system/187893.jpg",biography:"DPhil Maria Jose Hernandez Serrano is a tenured Lecturer in the Department of Theory and History of Education at the University of Salamanca (Spain), where she currently teaches on Teacher Education. She graduated in Social Education (2000) and Psycho-Pedagogy (2003) at the University of Salamanca. Then, she obtained her European Ph.D. on Education and Training in Virtual Environments by research with the University of Manchester, UK (2009). She obtained a Visiting Scholar Postdoctoral Grant (of the British Academy, UK) at the Oxford Internet Institute of the University of Oxford (2011) and was granted with a postdoctoral research (in 2021) at London Birbeck University.\n \nShe is author of more than 20 research papers, and more than 35 book chapters (H Index 10). She is interested in the study of the educational process and the analysis of cognitive and affective processes in the context of neuroeducation and neurotechnologies, along with the study of social contingencies affecting the educational institutions and requiring new skills for educators.\n\nHer publications are mainly of the educational process mediated by technologies and digital competences. Currently, her new research interests are: the transdisciplinary application of the brain-based research to the educational context and virtual environments, and the neuropedagogical implications of the technologies on the development of the brain in younger students. Also, she is interested in the promotion of creative and critical uses of digital technologies, the emerging uses of social media and transmedia, and the informal learning through technologies.\n\nShe is a member of several research Networks and Scientific Committees in international journals on Educational Technologies and Educommunication, and collaborates as a reviewer in several prestigious journals (see public profile in Publons).\n\nUntil March 2010 she was in charge of the Adult University of Salamanca, by coordinating teaching activities of more than a thousand adult students. She currently is, since 2014, the Secretary of the Department of Theory and History of Education. Since 2015 she collaborates with the Council Educational Program by training teachers and families in the translation of advances from educational neuroscience.",institutionString:"University of Salamanca",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Salamanca",institutionURL:null,country:{name:"Spain"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"23",title:"Social Sciences",slug:"social-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"301331",firstName:"Mia",lastName:"Vulovic",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/301331/images/8498_n.jpg",email:"mia.v@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"6942",title:"Global Social Work",subtitle:"Cutting Edge Issues and Critical Reflections",isOpenForSubmission:!1,hash:"222c8a66edfc7a4a6537af7565bcb3de",slug:"global-social-work-cutting-edge-issues-and-critical-reflections",bookSignature:"Bala Raju Nikku",coverURL:"https://cdn.intechopen.com/books/images_new/6942.jpg",editedByType:"Edited by",editors:[{id:"263576",title:"Dr.",name:"Bala",surname:"Nikku",slug:"bala-nikku",fullName:"Bala Nikku"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"45025",title:"Laser Based Fabrication of Graphene",doi:"10.5772/55821",slug:"laser-based-fabrication-of-graphene",body:'Graphene, two dimensional (2D) carbon-carbon atoms arranged in a hexagonally honeycomb lattice structure, is the latest carbon (C) allotrope to be discovered. It has gained tremendous scientific research interests due to some unique properties, such as quantum hall effect and nearly ballistic electronic transportation in ambient.[1-4] These properties have stimulated intense activity among physicists, chemists and material scientists. Much research has focused on developing routs for the controllable growth of high quality graphene. Historically, graphene can be produced via micromechanical cleavage and SiC decomposition methods.[2, 5, 6] In order to meet even higher requirements, such as good crystallinity, less impurities and large area coverage, the present most successful fabrication technique focuses on chemical vapor deposition (CVD) on copper [5 - 8] Some other graphene growth methods include growth of graphene from solid carbon source, graphene synthesis by ion beam implantation, and graphene formation by decomposition of C60, have also been reported in the recent years.[9, 10] Apart from these, pulsed laser deposition (PLD), which is one of the unique physical vapor deposition (PVD) methods, represents a completely new fabrication way in this field.
For many reasons, PLD is a versatile material fabrication technique. There are many laser parameters regarding sample preparation can be tuned in the PLD system, which have great influence on the sample qualities. First, for laser itself, the laser fluence, wavelength, repetition rate, and pulse duration can be altered. The second aspect includes target to substrate distance, background gas and pressure, and substrate temperature can be varied depending on certain requirements. Since with this technique the energy source, laser, is located outside the vacuum system, it is possible to adopt ultrahigh vacuum (UHV) as well as ambient conditions. It allows growing many kinds of materials; for example, oxides, nitrides, carbides, semiconductors, metals, superconductors, superlattice structures, and even fullerenes and polymers. The pulsed nature of PLD process even allows preparing complex polymer-metal compounds and multylayers. For example, one of the promising realizations of the PLD system is to introduce oxygen to the chamber for oxides based fabrication. It is inevitable for achieving a sufficient amount of oxygen during film growth.
Until now, there have fewer studies on PLD based graphene growth. Indeed, such method used in graphene fabrication originates from the growth of carbon thin film by the PLD system. If a single carbon layer with aromatic ring structure in plane can be obtained, it gives a result of a single layer graphene. For the PLD method, the amorphous C layer can be easily deposited at room temperature. However, in order to fabricate C layer or graphene with a desirable crystalline quality by PLD, some additional conditions are required. For example, the substrate temperatures, vacuum level inside the PLD chamber, appropriate laser operation conditions (i.e. laser fluence and repetition rate) and the choice of catalytic metals. Wang, et. al. have demonstrated that few layer graphene that is bi-layer to multi-layer graphene can be fabricated on catalytic nickel (Ni) thin film by PLD system.[11] The number of graphene layers is found depending on the laser ablation time, and the crystallinity of the graphene depends on the substrate temperature during laser ablation. During the graphene formation, it involves several steps, such as C atoms adsorption, precipitation, segregation and recrystallization. All these processes happen due to the interaction between C atoms and metals. The formation of the graphene on the metal surfaces was first observed during the preparations of platinum (Pt) and ruthenium (Ru) single crystal surfaces. [12, 13]
In fact, the studies of the interaction of the C and the metals has a long history, however, the graphene fabricated by PLD method is realized in the recent years. By comparing with the conventional CVD method which usually involves high processing temperature (>1000 oC) and chemical reactive hydrocarbon gas flow, PLD can reach the same goal at relatively low temperatures and the C target is always in a solid form. Koh, et. al. have demonstrated that few layer graphene can be fabricated at 750 oC on a Ni plate (Figure 1).[14] Apart from the temperature issue, they also showed the cooling rate and laser energy are crucial in fabricating such graphene layers. Afterwards, Wang, et. al. did more systematic studies on the formation of the graphene on the Ni thin film. The significant contribution from their work is the number of graphene layer can be controlled by altering the number of the laser pulses (Figure 2). [11]
Apart from the demonstration of this PLD fabrication technique in this chapter, the characterizations and quantitative studies of graphene mainly rely on fast and non-destructive micro-Raman spectroscopy.[6, 15-17] In the past four decades, it has witnessed that Raman spectroscopy plays an important role in characterizing pyrolytic graphite, glassy carbon, graphitic foams, carbon fibers, nanographite ribbons, carbon nanotubes and fullerences. Owing to the presence of sp2 bond graphene, Raman spectroscopy gives plenty of inspired information about crystallite size, the introduction of chemical impurities, the magnitude of the mass density, the optical energy gap, the elastic constants, the doping, defects, the crystal disorder, the strain, and the number of the graphene layers. With this respect, the discussion about PLD fabricated graphene here will provide a taste of the power of Raman spectroscopy.
Crossing sectional TEM showing (a) the graphene layers above Ni. (b) Ni/nickel silicide/Si layered structure. Inset c is an overview optical image showing rather uniform coverage [ref. 13. figure 2].
TEM characterization of PLD-grown graphene films. (a) Low magnification TEM image showing PLD-grown graphene on Ni thin film. High resolution TEM images for (b) bi-layer graphene, (c) tri-layer graphene and (d) multi-layer graphene.
Metal induced crystallization method, also known as metal mediated or metal catalyzed crystallization method, is a crystalline growth technique to fabricate mono- or poly-crystalline materials via the interdiffusion, precipitation, segregation and recrystallization of two materials upon thermal annealing. One of them performs as the catalyst; it is usually a metal, such as aluminum (Al), gold (Au), platinum (Pt), nickel (Ni) and cobalt (Co); [11, 12, 18, 19] while the other is always chosen as a semiconductor material, such as germanium (Ge), and silicon (Si). This technique has been widely explored in the past, in particular for the polycrystalline Si thin film based solar cell investigation.[20] It has been proposed and experimentally demonstrated in fabricating high crystalline quality and large area polycrystalline Si thin film on various kinds of substrates at low temperature. For both CVD and PVD method based graphene fabrications, metal induced crystallization method acts as a dominating role during the graphitization process at elevated temperature. One way to realize this method in graphene growth is amorphous C layer can be easily changed to crystalline graphene layer based on thermal annealing process. Such process can be understood from figure 3.[21] This process utilizes solid phase sources of C. In this approach, the C is introduced in the amorphous phase with the Ni thin film forming bi-layer stack. Upon high temperature annealing, the C atoms from the a-C layer would dissolve into the Ni layer and be expelled from solution after cooling below the solid solubility limit. By comparing with previous studies about metal-induced Si crystallization, similar mechanism is involved. The driving force for crystallization is thermodynamic stability of the crystalline C and Si phases relative to the amorphous phase.
The process schematics for the metal-catalyzed crystallization of amorphous C to graphene by thermal annealing [ref. 19 figure 1].
PLD is a thin film fabrication technique using high energy pulsed laser beam to bombard one or more targets at a certain vacuum pressure.[22, 23] The laser shooting areas of the targets experience the transition from the solid to the vapor phase, and subsequently been coated onto a substrate. This growth technique was first used by Smith and Turner in 1965 for the preparation of semiconductors and dielectric thin films.[24] It was further established by Dijkkamp and coworkers on high temperature superconductors in 1987.[25] Afterwards, this technique has been extensively optimized, investigated, and used in oxides, nitrides, carbides, metallic thin films and even organic polymers.[26]
PLD is a form of physical vapor deposition (PVD). The system design is somewhat different from other PVD systems such as, thermal evaporation, electron beam evaporation, molecular beam epitaxial growth and magnetron sputter systems, because an external laser source is required. The useful range of laser wavelengths for thin films growth by PLD lies between 200 nm and 400 nm because most materials exhibit strong absorption in this spectral region. Therefore, most photon energies come from laser source can be absorbed by materials. Within this range, there are few commercially available laser sources capable of easily delivering the high energy densities (1 J/cm2), in relatively large areas (10 mm2 or larger), which are required for PLD works. A homogenous uniform laser output is also important for high quality thin films fabrications. Most PLD systems work these days use excimer lasers, in which the lasing medium is a mixture of some reactive gases such as krypton (Kr), fluorine (F) and neon (Ne). It is also known that neodymium (Nd): YAG laser can also be used for graphite fabrication on Si substrate, but no one has demonstrated the fabrication of graphene using this laser source so far.
An experimental setup for a typical PLD system is shown in figure 4. It consists of two major components, one is the external laser source and another is the stainless steal vacuum chamber. The vacuum chamber can be placed direct facing the output laser pulse or be set at certain angles. For the latter case, a reflecting mirror is necessary. Inside the PLD chamber, a target and a substrate holder are aligned on the same line but are separated by a distance of 3 cm to 5 cm. Such distance range has been well experimentally confirmed for efficient laser ablation. When the incident focusing laser beam bombards the rotating target, the rise of the localized temperature causes vaporization of the material. It is a feature of plasma plume with high energetic species, for example, ions, electrons, atoms, molecules, clusters, particulates and molten globules. For an ideal PLD based thin films fabrication, people hope that the clusters, particulates and molten globules can be avoided. The film growths depend on several parameters, such as laser fluence, laser repetition rate, substrate temperature and vacuum level. By adjusting the number of laser pulses on the targets, different layers with controllable thicknesses can reach.
PLD system setup.
In a typical experiment for fabricating graphene layer via PLD, people can select different substrates based on certain requirements. For convenient purpose, we use 300nm SiO2 coated Si as a typical example to illustrate this fabrication method (figure 5(a)). Furthermore, it is also a good candidate for making bottom gate graphene based field effect transistor (FET) due to the present of sufficient thick SiO2 layer. Prior to the deposition, the substrate can be cleaned by the conventional chemical means. The fabrications for those metal thin films and C were carried out inside a stainless steal PLD chamber. The SiO2/Si substrate was firmly attached onto a substrate holder, which is 4 cm in a distance to a PLD target holder. The laser beam can be guided to the target via a focusing lens. More details about the experimental condition can be found from reference 10. A schematic diagram which shows the graphene formation on a thin metal thin film is given in figure 5. The metal thin film is fabricated at room temperature (figure 5(b)). Without breaking the high vacuum, the substrate temperatures were rapidly increased to 650 oC and 600 oC. After the deposition of C (figure 5(c)), the samples were naturally cooled down to room temperature in an ultra-high vacuum. For some PLD systems, the cooling rate can also be controlled, for example by the flow of the liquid nitrogen or pass through the inert gas into the chamber. During this cooling process, the C atom will segregate from the C containing C-metal solid solution and form a continued layer on the topmost surface (figure 5(d)). This is due to the reduction of the solubility of C-metal solution. The metal which locates below graphene layer after fabrication is not a pure thin metal film anymore, because there are still sufficient amount of C atoms which participate the interdiffusion process remain and form C-metal eutectic alloy.
Schematic diagram shows metal-induced graphene formation process by PLD technique. (a) Preparation of SiO2/Si substrate. (b) Ni thin film deposition at room temperature. (c) C deposition at elevated temperature. (d) Graphene formation at the topmost surface.
Figure 6 shows the Raman spectra for the carbon layers fabricated at 5 difference temperatures, 300 oC, 400 oC, 500 oC, 600 oC and 650 oC respectively on Ni thin film. In figure 6(a), there is no distinct peak can be observed for crystalline C phase. This indicates 300 oC is insufficiently to obtain crystalline carbon layer on Ni thin film. As the deposition temperature for carbon increases, such as shown in figure 6(b) and (c), two remarkable bands tend to emerge at 1350 cm-1 and 1550 cm-1 respectively. Both of them represent disorder-induced (D) and graphitic (G) bands. For the one locates at 1350 cm-1, its occurrence is due to the breathing mode of sp2 atoms or A1g symmetry in hexagonal graphitic rings. From both figure 6(b) and (c), a broad bandwidth of such D mode indicates the nonorganized C.[16] Thus, the large full width at half maximum (FWHM) of the D band yields the highly disordered characteristic of the C layer. This mode is forbidden in perfect graphite and become active in the presence of disorder. Furthermore, the D mode is dispersive and varies with photon excitation energy. Therefore, its intensity is strictly related with the presence of sixfold aromatic ring. As the atomic mechanism concerns, the rise of G band is ascribed to the in-plane stretching of the C-C bond in graphitic materials.[17] By comparing with the one of D mode, this one does not require the presence of sixfold ring so that it occurs at all pairs of sp2 C sites. In the Raman spectra, the G band is due to the doubly degenerate zone center E2g mode. The Raman spectroscopy is very sensitive to this strain effect in sp2 C based materials. When the interaction between one graphene layer and substrate or another graphene layer is encountered, the induced strain is due to the modification of the C-C bond lengths and angles. As we can see from figure 6(d) and (e), further increase in the deposition temperature of C leads a clear distinguish of D and G bands. The relative intensity ratio of ID to IG has been significantly reduced at the deposition temperature of 650 oC for C. It results in the reduction of the crystalline defects. In addition, there is another band gradually become noticeable at 2700 cm-1. It is the second order of zone-boundary phonons, but it has nothing to do with the G band. Because the Raman shift at this point is approximately doubled when comparing with the one of the D band, it is conventionally denoted as 2D or G´ band. Owing to the G´ band is the second order process associated with a phonon close to the K point in graphene, there has a strong dependence on any perturbation to the electronic and phonon structure of graphene. Therefore, G´ band plays an important role for differentiating single, bi- and few- layer graphene upon Raman spectroscopy.[6, 15] The results of the temperature dependence for C deposition suggests that 650 oC is an optimum temperature to fabricate the crystalline C layer on the Ni thin films by the PLD method in this experiment. Apart from Ni, it is also possible to fabricate graphene layer on a Co thin film. Figure 7 shows the temperature dependent graphene fabrication on the Co thin film. By comparing with figure 6, similar results were obtained for these two 3d transition metals.
Temperature dependent Ni-induced few layer graphene formation. (a) at 300 °C. (b) at 400 °C. (c) at 500 °C. (d) at 600 °C and (e) at 650 °C.
Temperature dependent Co-induced few layer graphene formations. (a) at 300 °C. (b) at 350 °C. (c) at 400 °C. (d) at 450 °C and (e) at 500 °C. (f) at 550 oC. (g) at 600 oC. (h) at 650 oC.
Figure 8(a) shows the photographic image during graphene layer transferring process. The PLD made graphene transfer method is very similar to the one used in CVD method. A very thin protective poly[methyl methacrylate] (PMMA) layer was initially coated on the top of the graphene/Ni or Co/SiO2/Si sample by spin coater. The catalytic Ni or Co can be etched away by chemical wet-etching, for instance using an aqueous HCL solution. In figure 8(a), After the Ni thin film was completely dissolved in FeCl3 solution; a 1 × 1 cm2 few layer graphene coated with the PMMA was detached from the Ni thin film and forms a free-standing layer in the HCl solution. The diluted HCl solution can be further utilized for removing the residual Ni flakes. Afterward, the few-layer graphene can be transferred onto another clean SiO2/Si substrate and the top as-coated PMMA layer can be dissolve quickly by putting the sample directly into acetone. The corresponding photographic picture of such graphene layer on the SiO2/Si substrate after successful transfer is shown in figure 8(b). Moreover, the graphene sample preparation method for the transmission electron microscopy (TEM) characterization is slightly different. In this case, after the sample is closely attached to the TEM copper grid, the PMMA can be dissolved by exposing to the acetone vapor for approximately 4 to 5 hours. This transfer process allows maintaining the continuity of the graphene. Figure 8(c) displays the feature of the few-layer graphene and the blue dotted circle denotes the presence of the graphene wrinkles. The formation is primarily due to lattice mismatch between Ni and C and the wrinkles have high possibility to be found at the grain boundaries of as-prepared Ni thin films. Similar with CVD technique, the morphology of fabricated graphene tend to replicate the one of substrate materials. At the edge of the sample which is shown in figure 8(d), the graphene layer by layer feature can be clearly observed. In figure 8(e), the hexagonally symmetric selected area electron diffraction (SADP) for this few-layer graphene was captured indicating the monocrystalline structure of our fabricated few-layer graphene. Two red dotted circles highlight the presence of (0002) lattice plane in the d space. This diffracted spots only appear when few layer or bulk graphite is encountered.
Graphene transfer process after PLD fabrication. (a) Detach of graphene layer from the substrate. (b) Graphene is transferred onto a new piece of SiO2/Si substrate. (c) Low magnification TEM image shows graphene wrinkles. (d) High resolution TEM image shows few layer graphene edges. (e) The corresponding selected area electron diffraction pattern of few layer graphene.
The high resolution TEM crossing sectional view of bi- and tri- layer graphene are displayed in figure 9(a) and (b) respectively. During the deposition of C at 650 oC, the interdiffusion between C and Ni leads to the formation of the solid solution. Such interdiffusion proceeds until C reaches its saturation status at this temperature. Once the C saturation status reaches, further deposition of C causes a formation of amorphous C layer. Therefore, a well control of the thickness ratio between C and Ni is crucially important in achieving a high quality, bi-, tri- and few layer graphene. When C saturation status reaches at 650 oC, the natural reduction of the substrate temperature to room temperature in high vacuum causes the decrease in the Ni solubility. For this reason, C atoms segregate from the solid solution and form a crystalline and continue graphene layer on the most top of the sample. Figure 9(c) shows the AFM image of the bi-layer graphene. After scratching the sample for depth profile measurement, some graphene parts fold and the relative contrast can be seen from this AFM image. A location across the surface of this piece of graphene was randomly chosen and the corresponding depth profile is shown in figure 9(d). The lateral height which is around 2.5 nm indicates the presence of bi-layer graphene.
High resolution TEM images show (a) crossing sectional view of tri-layer graphene, (b) crossing sectional view of bi-layer graphene. (c) The AFM image for few layer graphene. (d) The lateral height measurement for few layer graphene.
Further studies in fabricating graphene by the PLD include the thickness ratio dependence between Ni and C. In this scheme, we found both the number of graphene layers and the D band can be greatly affected. From the Raman spectra in figure 10, C with 3 different thicknesses or laser ablation time were deposited onto 250 nm Ni thin films at 650 oC. The D band is the greatest among those three when the laser ablation time is 120 s (figure 10(a). Moreover, the intensity ratio of I2D/IG is 1.24 (Data is summarized in table 1). Thus, both of them imply the poor crystallinity of C layers and the layers. By decreasing the amount of C, such as the resultant Raman spectrum shown in figure 10(b) which corresponds to the laser ablation time of 90 s, the intensity ratio of I2D/IG is 0.47 which indicates the existence of bi- or tri-layer graphene. Nevertheless, the intensity of the D band is over half of the intensity of the G band. However, when the laser ablation time for C is further decreased to 60 s (figure 10(c)), we observe a significant reduction of the D band Raman intensity and the intensity ratio of I2D/IG becomes even larger. Similar experiment was done for the graphene fabricated on 250 nm Co thin film. The Raman spectra are shown in figure11 for the different laser ablation times of C, 240 s, 180 s, 120 s, 60 s, and 30 s respectively. The Raman intensities which correspond to each band are summarized in table 2. In contrast to table 1, the graphene which is fabricated on the Co thin film shows similar trend as the laser ablation time for C tends to decrease.
\n\t\t\t\tC deposition time (s) / thickness (nm)\n\t\t\t | \n\t\t\t\n\t\t\t\tD-band position\n\t\t\t | \n\t\t\t\n\t\t\t\tD-band intensity\n\t\t\t | \n\t\t\t\n\t\t\t\tG-band position\n\t\t\t | \n\t\t\t\n\t\t\t\tG-band intensity\n\t\t\t | \n\t\t\t\n\t\t\t\t2D-band position\n\t\t\t | \n\t\t\t\n\t\t\t\t2D-band intensity\n\t\t\t | \n\t\t\t\n\t\t\t\tID/IG\n\t\t\t\t\n\t\t\t | \n\t\t\t\n\t\t\t\tI2D/IG\n\t\t\t\t\n\t\t\t | \n\t\t
120s | \n\t\t\t1359.30 | \n\t\t\t1011.76 | \n\t\t\t1596.04 | \n\t\t\t814.71 | \n\t\t\t2704.16 | \n\t\t\t130.39 | \n\t\t\t1.24 | \n\t\t\t0.16 | \n\t\t
90s | \n\t\t\t1359.30 | \n\t\t\t178.43 | \n\t\t\t1588.48 | \n\t\t\t261.52 | \n\t\t\t2719.27 | \n\t\t\t122.79 | \n\t\t\t0.68 | \n\t\t\t0.47 | \n\t\t
60s | \n\t\t\t1346.70 | \n\t\t\t154.17 | \n\t\t\t1580.93 | \n\t\t\t327.43 | \n\t\t\t2688.21 | \n\t\t\t183.99 | \n\t\t\t0.47 | \n\t\t\t0.56 | \n\t\t
Raman intensities for ID, IG, I2D, ID/IG and I2D / IG of Figure 10.
\n\t\t\t\tC deposition time\n\t\t\t | \n\t\t\t\n\t\t\t\tD-band position\n\t\t\t | \n\t\t\t\n\t\t\t\tD-band intensity\n\t\t\t | \n\t\t\t\n\t\t\t\tG-band position\n\t\t\t | \n\t\t\t\n\t\t\t\tG-band intensity\n\t\t\t | \n\t\t\t\n\t\t\t\t2D-band position\n\t\t\t | \n\t\t\t2D-band intensity | \n\t\t\t\n\t\t\t\tI2D/IG\n\t\t\t\t\n\t\t\t | \n\t\t
240s | \n\t\t\t1355.19 | \n\t\t\t358.81 | \n\t\t\t1591.07 | \n\t\t\t403.40 | \n\t\t\t2724.95 | \n\t\t\t97.86 | \n\t\t\t0.24 | \n\t\t
180s | \n\t\t\t1355.19 | \n\t\t\t202.91 | \n\t\t\t1581.97 | \n\t\t\t277.00 | \n\t\t\t2724.95 | \n\t\t\t76.17 | \n\t\t\t0.27 | \n\t\t
120s | \n\t\t\t1364.30 | \n\t\t\t85.57 | \n\t\t\t1581.97 | \n\t\t\t141.12 | \n\t\t\t2724.95 | \n\t\t\t58.97 | \n\t\t\t0.41 | \n\t\t
60s | \n\t\t\t1364.30 | \n\t\t\t65.95 | \n\t\t\t1581.97 | \n\t\t\t113.52 | \n\t\t\t2716.76 | \n\t\t\t55.83 | \n\t\t\t0.49 | \n\t\t
30s | \n\t\t\t1364.30 | \n\t\t\t43.17 | \n\t\t\t1581.97 | \n\t\t\t84.08 | \n\t\t\t2707.65 | \n\t\t\t52.29 | \n\t\t\t0.62 | \n\t\t
Raman intensities for ID, IG, I2D, ID/IG and I2D / IG of Figure 11.
For the graphene fabricated by the PLD method, the D and 2D bands of bi-layer graphene show some unique characteristics by comparing with few-layer graphene and bulk graphite. As we can see from figure 12(a), the D band of bi-layer graphene possesses a non-symmetric band at 1346.70 cm-1 while the few-layer graphene and bulk graphite give symmetric D bands at 1359.30 cm-1. In addition, the Raman spectrum of the asymmetric 2D band of the bi-layer graphene shows red shift relative the ones of few-layer graphene and bulk graphite. Such 2D mode of the bi-layer graphene composes of 4 components, 2D1B, 2D1A, 2D2A, and 2D2B; in which, 2D1A and 2D2A have higher intensities than the other two, as shown in figure 12(b). These four components of bi-layer graphene are attributed to the evolution of the electronic band structure. Raman scattering is a fourth order process involving four virtual transitions: [1] a laser induced excitation of an electron-hole pair; [2] electron-phonon scattering with an exchanged momentum; [3] electron phonon scattering with an exchanged momentum; and [4] electron-hole recombination. Based on both TEM and Raman spectroscopic studies, the graphene which is fabricated by the PLD method can be achieved. Nevertheless, there are still some aspects have to be encountered. As we have discussed previously, the number and the quality of graphene layers are decisive by the ratio between C layer and catalytic metals, substrate temperature and laser operation conditions. In order to obtained desirable property of graphene, careful understandings of the graphene and metal interaction, interdiffusion and interface property are crucial.
Raman spectroscopic study of different laser ablation time for C deposited onto 250 nm Ni thin film at 650 oC. The corresponding quantities of Raman intensities for each spectrum are summarized in table 1. (a) 120 s laser ablation time. (b) 90 s laser ablation time. (c) 60 s laser ablation time.
Raman spectroscopic study of different laser ablation time for C deposited onto 250 nm Co thin film at 650 oC. The corresponding quantities of Raman intensities for each spectrum are summarized in table 2. (a) 240 s laser ablation time. (b) 180 s laser ablation time. (c) 120 s laser ablation time. (d) 60 s laser ablation time. (e) 30 s laser ablation time.
The D and 2D bands for graphene fabricated on Ni thin film by the PLD method.
By the PLD method, other nanostrcutural materials, for instance crystalline Si nanodots, can also be fabricated together with single or multi-layer graphene. Because it is possible to place multi-target within a PLD system, crystalline Si nanodots can then be fabricated via Ni-induced crystallization method as well.[27] Figure 13 (a) and (b) show the SEM images of the crystalline Si nanodots fabricated on top of the graphene layer via the PLD method. Owing to the fact that Si usually required much higher processing temperature (>1000 oC) to be completely crystallized, it has experimentally demonstrated that metal induced crystallization method can reach the same goal with relatively low temperature (< 500 oC). Such kind of Si nanodots structure combined with graphene is very attractive and interesting for studying semiconductor-graphene interface property and eventually reaching the purpose of modern nanoelectronic device design. With regard of such nanostruture, an additional template is necessary in order to define the wide distribution of the Si nanodots. Therefore, a so-called ultra-thin anodic porous alumina (UAPA) template was used in this case. However, the photolithographic and electron beam lithographic techniques can also be applied to graphene. Apparently, it inevitably proves the functionality of PLD based graphene fabrication method in today’s graphene research field.
a) Top-view FESEM image of the Ni-induced crystalline silicon nanodots on graphene with remaining UAPA template. (b) The well-ordered nanodots arrays on graphene after lift-off.
In this chapter, PLD method has been reviewed for graphene fabrication in this chapter. The foundation of this technique is metal-induced crystallization mechanism. Some catalytic metals need to participate during the crystallization process of C. The advantage of using PLD is to prompt C atoms diffuse into metal thin film at a certain high temperature because PLD can generate high energetic species. After the sample is cooled down, the C atoms segregate from the carbon-metal solid solution and subsequently forming a continuous C thin layer on the top of a carbon containing metal film. The number graphene layer can be controlled by the PLD method, for example by tuning the number of laser pulses, the thickness ratio of C to metal thin film. Besides, we also see the Raman spectroscopy and TEM are both important in characterizing the as-prepared graphene made by the PLD technique. It is expected that PLD based graphene fabrication technique is very promising in current graphene related nanotechnology research.
The plant kingdom has provided a wide variety of natural products with diverse chemical structures and a vast array of biological activities, many of which found applications in health sciences. Over 80% of the approximately 30,000 known natural products are of plant origin. In 1985, 3500 new chemical structures were identified out of which 2600 were derived from higher plants and 121 clinically useful drugs were derived from plants [1]. Plants will continue to provide novel products as well as chemical models for new drugs in the near future [2].
Many of the plant species that produce medicinal herbs have been scientifically evaluated for their possible medical applications. The economic importance of phytopharmaceuticals in plants has led to their inevitable collection from their natural habitats and thus creating environmental and geopolitical instabilities posing a threat to their survival. The reckless collection of plants has put several of them under the categories of endangered or at the verge of extinction. This has prompted industries and scientists to find the alternative technologies for the production of phytopharmaceuticals so that the natural habitat of plants can be preserved.
Plant cell cultures have served as potential renewable resources for the production of valuable medicinal compounds, flavors, fragrances, pigments, dyes, cosmetics and fine chemicals. All these compounds belong to a group collectively known as secondary metabolites. The commercial importance of secondary metabolites and the possibilities of their production by means of cell culture technologies have gained great interest in the recent years. The current review is a survey and analysis of current status of various plant cell culture technologies used for the production of medicinally important metabolites. The future prospects of cell culture technologies in light of successful case studies have been reviewed and possible improvements are suggested.
The capacity of plant cell, tissue and organ cultures to produce and accumulate many of the valuable chemical compounds has been recognized almost since the inception of in vitro technology. The strong and growing demand in today’s market place for natural, renewable products has refocused attention on in vitro cell cultures as potential factories for phytochemical production. The advantage of producing plant metabolites in vitro has been in understanding the biology of their biosynthetic activity which ultimately can be enhanced by regulating physical, chemical, nutritional and genetic parameters. Medicinal compounds localized in morphologically specialized tissues or organs of native plants have been produced in culture systems not only by inducing specific organized cultures but also by undifferentiated callus/cell cultures.
The advances in plant cell culture technologies has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavonoids, and amino acids. The production of plant metabolites through cell cultures offer several advantages such as it makes possible to select genotypes with higher production of secondary metabolites, which can be generated on a continuous year round basis under controlled environment. Plant cell cultures eliminate potential political boundaries or geographic barriers which are otherwise to the production of a crop, such as the restriction of natural rubber production to the tropics or anthocyanin pigment production to climates with high light intensity. Many cost effective parameters have been tried for their economic production at large scale or by possible use of plant cell cultures for biotransformation of natural compounds [3].
Callus/cell suspension cultures have been the prime focus of various studies aimed at the production of phytochemicals of not only medicinal value but also of other industrially important metabolites. Callus is a proliferating mass of undifferentiated cells, which can be established from different explants of a plant species under in vitro conditions on suitable nutrient media. Once the callus is derived from high metabolite producing explants, their suspension cultures can be established by transferring those calli into liquid media under continuous agitation. Zenk [4] successfully established cell lines of different plants capable of producing high yields of secondary compounds in cell suspension cultures. The production of solasodine from calli of Solanum elaeagnifolium and cephaeline and emetine from callus cultures of Cephaelis ipecacuanha were successfully achieved [5]. Some of the notable cell culture methods which have been employed for large scale production of metabolites are production of taxol from cell suspension cultures of Taxus mairei [6]; production of paclitaxel and its related taxanes from different Taxus species; production of berberine through cell suspension culture of Coptis japonica; production of vincristine and vinblastine from Catharanthus roseus [7, 8], and production of taxoids from cell suspension cultures of Taxus cuspidate [9] (Table 1).
Plant species | Active ingredient | Reference |
---|---|---|
Agave amaniensis | Saponins | Andrijany et al. (1999) |
Allium sativum L. | Alliin | Malpathak and David (1986) |
Coptis japonica | Berberine | Suzuki et al. (1988) and Morimoto et al. (1988) |
Duboisia leichhardtii | Tropane alkaloids | Yamada and Endo (1984) |
Gentiana sp. | Secoiridoid glucosides | Srrzypezak et al. [10] |
Panax ginseng | Saponins and sapogenins | Furuya et al. [11] |
Papaver bracteatum | Thebaine | Day et al. (1986) |
Rauvolfia serpentine × Rhazya stricta hybrid plant | 3-Oxo-rhazinilam | Gerasimenko et al. (2001) |
Scutellaria columnae | Phenolics | Stojakowska and Kisiel (1999) |
Tecoma sambucifolium | Phenylpropanoid glycosides | Pletsch et al. (1993) |
Taxus mairei | Taxol | Wu et al. [6] |
Taxus spp. | Terpenes, sterols, flavonoids | Lish et al. (2002) |
Catharanthus roseus | Catharanthine | Zhao et al. [7, 8] |
Panax notoginseng | Gensenoside | Zhong and Zhong (1995) |
Papaver somniferum | Morphine and codeine | Shia and Doran (1991) |
Podophyllum hexandrum royle | Podophyllotoxin | Chattopadhay et al. [12] |
Salvia fruticosa | Rosmarinic acid | Karam et al. [13] |
Picrorhiza kurroa | Picroside-1 | Sood et al. (2010, 2011) and [14] |
Taxus cuspidate | Taxoids | Ketchum et al. [9] |
Bioactive secondary metabolites produced through shoot/callus cultures/suspension cultures.
Off late, the cultivation of hairy roots has been seen as a sustainable strategy for the production of medicinally important metabolites of plants not only due to the reason that harvesting roots has been destructive for the plants in nature but also due to the ease of growing hairy roots in mass cultures in the absence of external hormones, absence of geotropism and high branching, etc. Furthermore, hairy roots produce secondary metabolites for larger periods of time, unlike natural roots which are not only in limited supply but are available at specific times in a year. For these reasons, switching from culturing natural plant-organs to hairy roots is considered as an attractive alternative for the production of many valuable natural secondary metabolites [15].
For establishing hairy root cultures, the plants are infected by Agrobacterium rhizogenes which induces hairy roots by the transfer of T-DNA from Ri plasmid into the plant genome. This ability of A. rhizogenes has led to studies on it as a source of root-derived pharmaceuticals [16]. Important metabolites produced through hairy roots are serpentine production from Catharanthus roseus, ajmalicine from Rauvolfia micrantha [17] and ginkgolides from hairy roots of Gingko biloba [18]. Large scale production of ginsenoside from Panax ginseng hairy roots has been achieved by optimizing organic nutrients in bioreactor for enhancing their production. Recent developments have indicated that hairy root culture technology has moved from small laboratory scale to a large scale industrial production. For example, the German Co. RooTec has been carrying out production of camptothecin and podophyllotoxin through hairy root cultures. In a cross-species co-culture system, hairy roots of Linum flavum have been found to increase the production of podophyllotoxin by 240% in the cell suspensions of Podophyllum hexandrum. It has been reported that secondary metabolites accumulating in aerial plant have also been accumulated in the hairy roots such as artemisinin which was thought to accumulate only in the aerial parts of Artemisia annua also accumulated in the hairy roots. Higher production of forskolin in transformed roots of Coleus forskolli was achieved by using various concentrations of auxins and auxin conjugates, cytokinins and GA3 [19]. The enhanced production of picroside-1 has been reported through hairy root cultures of P. kurroa [20].
The lower yield of phytochemicals in plant cell cultures prompted researchers to look into various other means of enhancing their production. The recognition that certain specific secondary metabolites such as phytoalexins are produced by plants in response to microorganisms has led to the concept of using such stimulators (known as elicitors) for in vitro cultures. The substances used as elicitors can be of biotic or abiotic origin [21]. The plants also elicit the same response when challenged by compounds of pathogenic origin [22]. The elicitation of cell suspension cultures or hairy root cultures with biotic or abiotic elicitors has been found to enhance the rate of production as well as the yields of plant secondary metabolites [23].
The biotic elicitors are substances of biological origin, which include fungal homogenate, chitosan, microorganisms (Pseudomonas aeruginosa, Bacillus cereus), glycoprotein or intracellular proteins whose function are coupled to receptors and act by activating or inactivating a number of enzymes or ion channels [24]. Abiotic elicitors include physical and chemical stresses such as UV radiations, temperature, antibiotics, salts of heavy metals, etc. [22].
Various fungal elicitors including cell wall fragments, polysaccharides, glycoproteins and oligosaccharides have been used for the production of secondary metabolites in many plant spp. and their cell cultures. The cell extracts and filtrates of four species of fungi were used for the production of taxol from elicited cell cultures of Taxus sp. [25]. The cell wall fractions of Aspergillus niger have been used as an elicitor in cell suspension cultures of Taxus chinensis thereby resulting in more than two fold increase in taxol yield and about six fold increase in total secretion.
Jasmonic acid (JA) and its methyl esters, methyl jasmonate (MJ) have been reported as key signaling compounds in the process of elicitation leading to the accumulation of various secondary metabolites. Lu et al. (2001) reported 28 fold higher saponin production in the elicited cultures of Panax ginseng by using yeast extract and methyl jasmonate as elicitors. Production of many valuable secondary metabolites using various elicitors have been reported successfully in various other plant species [26, 27, 28, 29]. Enhanced production of podophyllotoxin in suspension cultures of Linum album was reported by using biotic (yeast extract) and abiotic (Ag+, Pb2+ and Cd2+) elicitors.
Methyl jasmonate, vanadyl sulphate and chitosan were used for enhancing the production of ginsenoside from hairy root cultures of P. ginseng [23].
Pitta-Alvarez and Giulietti et al. (2000) used jasmonic acid and aluminium chloride as elicitors for enhancing the production of scopolamine and hyoscyamine in hairy root cultures of Brugmansia candida. Bacterial elicitors like Bacillus cereus, Staphylococcus aureus, etc. have been used for enhancing scopolamine production from the adventitious hairy roots of Scopolia parviflora (Table 2).
Plant species | Secondary metabolites | Elicitor | Reference |
---|---|---|---|
Catharanthus roseus | Ajmalicine, vincristine, vinblastine |
| DiCosmo et al. (1987), Menke et al. (1999), Zhao et al. [7, 8] and Namdeo et al. (2004) |
Picrorhiza kurroa | Picroside-1 | Seaweed extract | [14] |
Datura stramonium | Alkaloids (tropane) | Phytophthora megasperma | Kurosaki et al. (2001) and Dorenburg et al. (1994) |
Azadirachta indica | Azadirachtin | Jasmonic acid, salicylic acid | Satdive et al. [30] and Funk et al. [31] |
Papaver somniferum | Codeine, morphine | Fungal spores | Heinsterin et al. (1985) |
Dioscorea deltoidea | Diosgenin | Rhizopus arrhizus | Rokem et al. (1984) |
Hyoscyamus niger, H. muticus | Hyoscyamine, scopolamine | Fungal elicitor, MeJA | Singh (1995) |
Rauwolfia canescens | Raucaffrincine | Yeast elicitor, MeJA | Gundlach et al. (1992) and Parchmann et al. (1997) |
Panax ginseng | Saponin | Oligogalacturonic acid low energy ultra sound | Threfal and Whitehead (1988) and Hu et al. (2003a,b) |
Hyoscyamus muticus | Sesquiterpenes | Rhizoctonia solani | Singh (1995) |
Lithospermum erythrorhizon | Shikonin | Endogenous source | Fukui et al. [32] |
Taxus chinensis | Taxol | Fungal elicitation | Wang et al. (2001) |
Taxus brevifolia, T. cuspidate | Taxol, Baccatin III | Fungal elicitor | Yukimuni et al. [33], Hefner et al. (1998) and Luo et al. (2001) |
Elicitors used for the production of secondary metabolites by cell cultures of medicinal plants.
Knowledge about biosynthetic pathways for secondary metabolite production open avenues for the targeted production of medicinal compounds as reported by Varun et al. [34] where he proposed the biosynthetic pathways for the production of picroside-1 and picroside-2 of Picrorhiza kurroa an endangered herb of North-Western Himalayas, having hepatoprotective iridoid compounds. Varun et al. [35, 36] optimized preparative RP-HPLC method for the isolation and purification of picrosides in Picrorhiza kurroa.
For maximizing the production and accumulation of secondary metabolites through plant cell cultures, specific physical conditions such as type and composition of nutrient media, type and source of explant for initiating cell cultures, incubation temperatures and intensity of light, etc. are of paramount importance.
The tissue culture media are the basic support system for the growth and development of plant cell cultured in vitro. The activities of basic primary metabolism are largely influenced by the basal media considered to be common to most of the plant species. However, the differentiation or dedifferentiation of plant tissue cultures is influenced by the combinations of growth hormones mainly auxins and cytokinins (Table 3). The manipulation of media components have been reported to influence the biosynthesis and accumulation of secondary metabolites in plant cell cultures. Different strategies have been employed for improving secondary metabolite production in suspension cultures. The influence of media constituents and nutrient stress affect the production of diosgenin from callus cultures of Dioscorea deltoidea. The production of gentipicroside and swertiamarin was enhanced on MS medium supplemented with kinetin, NAA and 3% sucrose in suspension cultures of Gentiana davidii [44].
Plant species | Metabolites | Reference |
---|---|---|
Camptotheca acuminate | Camptothecin | Lorence et al. [37] |
Gingko biloba | Ginkgolides | Ayadi et al. [18] |
Gmelina arborea | Verbascoside | Dhakulkar et al. [38] |
Linum flavum | Coniferin | Lin et al. [39] |
Papaver somniferum | Morphine, sanguinarine, codeine | Le Flem et al. [40] |
Panax ginseng | Ginsenoside | Palazon et al. [23] |
Pueraria phaseoloides | Puerarin | Shi and Kintizos et al. [41] |
Rauvolfia micrantha | Ajmalicine, ajmaline | Sudha et al. [17] |
Saussurea medusa | Jaceosidin | Zhao et al. [42] |
Solidago altissima | Polyacetylene (cis-dehydromatricaria ester) | Inoguchi et al. [43] |
Pharmaceutical metabolites produced by hairy root cultures.
The productivity of picroside-1 was increased by optimizing the concentration of nutrients in growth medium and levels of phytohormones in the shoot cultures of Picrorhiza kurroa [45, 46]. Elevated sucrose levels from 3 to 6% were favourable in some cultures whereas addition of fructose promoted paclitaxel production in Taxus cell cultures [6]. Supplementation of MS medium with seaweed extract also contributed in enhancement of picroside accumulation in shoot cultures of Picrorhiza species [14].
The type and source of explant has been of major importance in not only establishing successful tissue cultures in any plant species but also of significant importance in producing phytochemicals in vitro. The prime importance of choosing a right explant for the production of phytochemicals lies in the fact that the biosynthesis and accumulation of metabolites is very specific to tissues and organs along with their developmental stages. The tissue and developmental specific accumulation of phytochemicals thus makes it important that appropriate explant be selected for starting plant cell cultures for the production of phytochemicals.
Production of diosgenin has been carried out from cell suspension cultures of different explants of Dioscorea doryophora like stem-node, microtuber and intact tuber, etc., along with varying concentrations of sucrose in MS liquid media supplemented with 2 mg/L 2,4-D (0.3–3.5%). Increase in diosgenin production was obtained from tuber derived cell suspensions as compared to intact tuber explant.
Different cell lines were established on B5 medium supplemented with NAA by using stem- and needle-derived callus of Taxus mairei and taxol yield of upto 200 mg/L was obtained in precursor feeded cell suspensions [47].
Plants tissue cultures are largely influenced by the quality and duration of light treatments. There are various case studies in the literature wherein manipulation of light parameters or the temperature regimes has resulted in the alteration in the production of secondary metabolites. Zhang et al. (2005) gave heat shocks of 35–50°C for 30–60 min in the suspension cultures of Taxus yunnanensis for enhancing the production of paclitaxel. Production was increased to six fold by pretreatment with abscisic acid. The production of swertiamarin and gentipicroside was enhanced in cell suspension cultures of Gentiana davidii by incubating at 25°C and light intensity of 2.33 Lux [44]. Increase in the concentration of glycyrrhizin was found in the root tissue of Glycyrrhiza uralensis grown under red light or under low and high intensity of UV-B radiations [48].
Exogenous supply of a biosynthetic precursor to culture medium also increases the yield of the desired products. The concept is based on the idea that any compound which is an intermediate, or is in the beginning of a secondary metabolite biosynthetic route, proves to be a good candidate for increasing the final yield of secondary metabolite. Varun et al. [49] has carried out exogenous feeding of immediate biosynthetic precursor, i.e., cinnamic acid and catalpol in the shoot cultures of Picrorhiza kurroa hence stimulated 4.2 fold production of picroside-1. The production of monoterpene alkaloids was increased in cell suspension cultures of Catharanthus roseus fed with precursor mevalonic acid, secologanin [50]. Callus cultures of Dioscorea balcanica fed with cholesterol, norflurazon as precursors were used for the production of diosgenin, phytosteroids [51]. Hallard et al. [52] used secologanin and tryptamine in cell suspension cultures of Nicotiana tabaccum for the production of strictosidine. Phenolics compounds were elicited from micropropagated plants of Calligonum polygonoides by Owis et al. [53].
Supplementation of media with amino acids has been found to enhance the production of indole alkaloids tropane alkaloid in cell suspension cultures [54, 55]. Addition of phenylalanine to cell suspension culture of Salvia officinalis enhanced the production of rosmarinic acid. The production of taxol from Taxus cultures was also increased by using the same precursor [56]. Nicotinic acid was used as a precursor in the hairy root cultures of Nicotiana rustica for the production of nicotine. Hakkinen et al. [57] used hyoscyamine as a foreign substrate for enhancing the production of scopolamine in the hairy roots of N. tabaccum and found that 85% of the converted scopolamine was released into the medium.
The biosynthesis and accumulation of secondary metabolites or phytochemicals of medicinal value is influenced by the genotype of the target plant species [58]. There are examples wherein genotypic variations have been reported for phytochemical content. However, there has been a technical problem in most of these studies because genotype collections are made from different locations, which vary in altitude, climatic conditions, etc. thus resulting in variation in accumulation of phytochemicals. It would be highly desirable and practically viable if the influence of genotypic variation on phytochemical content is investigated by collecting genotypes of a particular plant species and then growing under uniform environmental conditions. The variation for metabolite content can be done on those genotypic collections.
True metabolic engineering of plant secondary metabolite pathways has been hampered due to the lack of thorough knowledge of biosynthetic pathways and their regulatory mechanisms leading to the formation of desired compounds. Methods like labeled precursor feeding, induced expression of regulatory genes and block competitive pathways and metabolism by antisense genes have been used for enhancing the production of desired metabolites. Yun et al. [59] cloned the hyoscyamine 6-beta hydroxylase gene (h6h) of Hyoscyamus niger and introduced into Atropa belladonna and collected scopolamine from engineered plant. In a later study, Hashimoto et al. (1993) reported fivefold higher concentration of scopolamine from A. belladonna hairy roots expressing the same gene than the wild-type hairy roots. Increased alkaloid production by overexpression of genes encoding key enzymes of tropane alkaloid biosynthesis pathway was reported by Palazon et al. [23] and Moyano et al. [60] in Duboisia hybrid, Datura metel and Hyoscyamus muticis hairy roots, respectively. Similarly, Zang et al. (2004) produced 411 mg/L scopolamine in cultivated hairy roots from the simultaneous over expression of pmt and h6h genes in H. niger. Elevated nicotine alkaloid production was achieved in Nicotiana tabaccum hairy roots carrying h6h gene [57]. Neha et al. [61] reported 2.6 fold increase in picroside-1 production by modulating four integrated secondary metabolic pathways, i.e., methyl erythritol phosphate, mevalonate, iridoid and phenylpropanoid pathway using seaweed extract. Moreover Sharma et al. [62] defined many strategies through metabolic engineering for stimulating the production of bio-active compounds from medicinal plants.
The production of phytopharmaceuticals in cell cultures coupled with their low yield from natural sources and supply concerns of plant species has renewed interest in up scaling cell culture technology for large scale production. Bioreactors are the key step towards their commercial exploitation because it provides defined parameters for up scaling the production of phytochemicals or secondary metabolites from plant cell cultures.
Bioreactor is a large culture vessel fitted with microprocessor control unit for the control of pH, temperature, light, dissolved oxygen, gas flow rate, agitation speed, nutrient factors, cell density for optimal growth or production, handling of cultures, nutrient uptake and product harvestation, etc. The success of Mitsui Petrochemical Industry Co. Ltd. in Japan in producing shikonin on a commercial scale from Lithospermum erythrorhizon and that of Nitto Denko Corp. Japan in mass production of Panax ginseng or ginseng cells have demonstrated the practical feasibility of using cell cultures in the large scale production of secondary metabolites of pharmaceutical importance. Commercial companies like Phyton and Samyang Genex are successfully producing paclitaxel and its related taxanes on large scale [63].
Heble and Chadha (1985) reported the successful cultivation of Catharanthus roseus cells in 7–20 L capacity of airlift bioreactor for the production of ajmalicine and serpentine by judicious use of air lift and low agitation. Significant amounts of sanguinarine were produced in cell suspension cultures of Papaver somniferum using bioreactors [64]. Ginseng root tissue cultures in 20 ton bioreactor produced 500 mg/L of saponin per day [65]. Hahn et al. [66] have produced gensinoside from adventitious root culture of Panax ginseng through large scale bioreactor system. Chattopadhyay et al. [12] produced podophyllotoxin through cell cultures of Podophyllum hexandrum in a bioreactor.
Different types of culture systems have been successfully used such as airlift bioreactors were used for scaling up hairy root production of Astragalus membranaceus [67] and Solanum chrysotricum [68] and mist bioreactor for hairy root of Tagetes patula [69]. Flow diagram of a process for the production of picrosides from Picrorhiza kurroa is given below wherein callus cultures/suspension cultures have been established from different explants and accumulation of picrosides is being investigated by HPLC [46, 70] (Figure 1).
Pictorial representation for picroside-1 production through plant tissue culture.
The research on in vitro production of phytochemicals has been carried for the past 20 years, however, there are very few case studies where technologies have been upscaled successfully. There has been several shortcomings some of which are mentioned below:
Lack of understanding about the physical environmental and genetic factors controlling the production of pharmaceuticals
Low yields of pharmaceuticals in tissue cultures
Lack of information on cost effectiveness in the production of pharmaceuticals through cell cultures
Poor amenability of most of the plant species producing pharmaceuticals to in vitro conditions
Use of high sugar concentration (3–8%) or addition of elicitors or precursors increases the production cost considerably
Infections due to contamination limit the progress of cell cultures
Lack of knowledge of various molecular events that occur in secondary metabolite biosynthesis
Cell sedimentation and death due to mass transfer of cells in large vessels limits the supply of oxygen and nutrients
Plant cells are extremely sensitive to shear forces
Plant cells have very low doubling time (16–24 h) therefore produce less biomass and relatively produce small amount of secondary metabolites
For aeration of cells stirring is needed which sometimes cause damage to cells and lower the yield of products
In spite of bottlenecks in the large scale production of phytopharmaceuticals many technological advancements and refinements have been made in the recent years right from the selection of high yielding cell lines to manipulation of basic chemical, physical and biological parameters. The identification of right explant of proper developmental stage, standardization of optimum nutrient medium resulting in maximum accumulation of pharmaceuticals, optimization of low-cost production technology are some of the areas which warrant immediate attention. Knowledge of the biosynthetic pathways of desired compounds in plants as well as in cell cultures is still rudimentary, therefore emphasis need to be made generate information based on a cellular and molecular level. Major breakthrough in the metabolomics and its integration with genomics and transcriptomics technologies will help in discovering potential genes of biosynthetic pathways so that closer understanding of the links between different levels in biological systems can lead to better understanding of the molecular biology of secondary metabolite production in plants.
The author is thankful to Prof. R.S. Chauhan, PhD scholars of Plant Biotechnology and the administration of the JUIT for providing infrastructural support in carrying out R&D on phytopharmaceuticals.
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