Plant Cell Tissue And Organ Culture Biotechnology Biology Essay

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Without claiming to deal with all aspects of this topic, this editorial article aims to provide a brief overview of the status quo of the contribution of plant cell tissue and organ biotechnology in the field of the valorization and conservation of medicinal and aromatic plants and their secondary metabolites. Several textbooks and review articles have been described this topic more comprehensively than is possible in this editorial [1, 2].

Medicinal and aromatic plants (MAPs) share the common feature of having a high level of active principles or chemical substances with highly specific chemical, biochemical or organoleptic properties, which enables us to use them for therapeutic purposes (medicinal plants), aromatic purposes (aromatic plants or essences), and dietetic or culinary purposes (condimental plants). The importance of their use depends not only in the richness of their active principle content, but also on the rarity of their occurrence in the nature and the extraction difficulty of their secondary compounds.

MAPs are increasing worldwide and continue to attract growing interest for farmers, traders, economists, teachers, professionals, health officials and various industries. The MAPs are natural biological resources that have a great potential to synthesize a huge variety of important secondary compounds, also referred to as natural product, far more than animal and even microorganisms. Are defined as secondary compounds ²metabolites² all substances specific (characteristic of a species) and which are not synthesized by metabolic pathways of primary metabolism they are common to large groups of plants. These natural compounds are used as pharmaceuticals, agrochemicals, and cosmeceuticals. Recently, MAPs are finding a new, expanding market as herbal components of health foods and preventative medicines, especially under the terms functional foods, nutraceuticals and natural health products [3]. The supply of the source plants however, is often limited due to disease, changes in climate, and changes in the economic development in the growing regions.

It is well known that MAPs have been traditionally harvested in the wild state, in only few regions, and they are not used equally worldwide. Till date, these phytoresources have been exploited nearly without any major limitation which may easily result in the exhaustion of plant genetic resources, biotope destruction and loss of wild populations. Thus, also threatening valuable incomes for rural households, especially in developing countries; consequently, the sustainable use of natural resources has become an unavoidable necessity from both environment protection and socio-economic points of view. Currently, between 4,000 and 10,000 MAP are on the endangered species list and this number is expected to increase [4]. These problems could be overcome through MAP selection and cultivation under agricultural conditions which could respond to increasing demands in terms of plant security, socio-economic development; biodiversity conservation and sustainable use of genetic phytoresources as basic inputs for the future.

Sometimes MAP species selected from the wild population may be suitable for the cultivation and there is no immediate necessity for any improvement programs in it. Selection in wild population is the most common method of MAP breeding. Furthermore breeding research provides the prerequisites of efficiently breeding, for increasing the levels of desired compounds, decreasing the undesired compounds, more resistance, and most homogeneity/uniformity, of new varieties. Inheritable traits of MAPs can be adapted by breeding to the special demands of the costumers and of the stakeholders in the supply chain. MAP breeding can help to create different strains of a species that have more suitable characteristics, both in terms of chemical and agronomically profiles and these strains can increase yield and reduce cost. Evidence of the increasing importance MAP breeding has been demonstrated by the growing number registered varieties in Germany during the past decade from only 10 to 66 in 1990 and 2004 respectively[5].

Cultivated material, which could be easily certified 'organic', is more appropriate for large scale uses such as the production of bioactive compounds by pharmaceutical, cosmeceutical and aromatherapy entities, which require standardized products of guaranteed or known content and quality. A fully traceable MAP production protocol is essential for a quality product. Properties of the plant should be identified and measured against the target profile of the ultimate product before plantation in the field. The production chain starts with the selection of the seeds which must meet quality parameters, such as those related to the availability of seeds from specified origins, breeds and cultivars, seed levels of infection (fungi), foreign material, moisture content, germination rate, and purity and viability.

In fact, cultivation decidedly guarantees a steady source of feedstock. Wholesalers and specialized companies could agree on volumes and prices over time with the grower and allows controlled post-harvest handling and, therefore, quality controls can be assured, and product standards can be adjusted to regulations and consumer preferences [6-8]. Nonetheless, many problems persist including genetics (origin, variation), morphogenesis (leaf position and age, harvest, flowering), environment (temperature, day length and light intensity), and finally agricultural practices (nutrition, irrigation, propagation, harvesting and extraction). In order to overcome some of these various problems, the agrobiotechnological approach by utilizing plant cell tissue and organ culture (PCTOC) technology has been expected to be an efficient and useful tool for the breeding (selection) of high-quality MAPs [9-11] and for the preservation of endangered species as well [10, 11].while providing an alternative source of controlled production of phytochemical products [11, 12]

Potential of plant cell tissue and organ culture biotechnology

There is a widespread interest in the application of PCTOC techniques and biotechnological approaches to the production of MAPs and isolation of secondary metabolites. PCTOC systems have been successfully developed for many MAP species especially endangered species [9, 10]. PCTOC systems may offer certain advantages over traditional agricultural methods of propagation, including: the rapid production and propagation of high quality, disease-free and uniform planting material. The MAP propagation can be done under a controlled environment, anywhere, independent of climatic changes and soil conditions, on a year-round basis. For MAPs that have long generation time, low levels of seed production, or seeds that otherwise have very low chances of germinating and growing, rapid multiplication is possible. The easy selection of desirable traits directly from the in vitro culture, thus reducing the amount of space required for field trials. The time shortness required in the MAP selection processes is possible, no need to wait for the lifecycle of seed development. It allows for the elimination of plant diseases through careful stock selection and sterile techniques. It enables plants preservation by pollen and cell collections form which plants may be propagated (like a seed bank). It enables cold storage of large numbers of viable plants in a small space. It allows for the international exchange of sterilized plant materials (eliminating the need for quarantine.).

In addition, use of PCTOC systems offers a tremendous tool for dissecting the physiological, biochemical and molecular regulation of plant development and stress response phenomena. Furthermore, they are extensively utilized for clonal propagation, as a gateway for genetic engineering of a vast majority of valuable economic plants and as an economical and large scale production platform for various pharmaceutics, drugs, flavors, colors, enzymes and medicinal compounds under controlled conditions [9-14].

PCTOC biotechnology is a corpus of techniques designed for the growth and multiplication of plant cells tissues and organs using nutrient solutions under aseptic and in a defined physical and chemical environments in vitro. Provided that an appropriate phytohormone regime is chosen, explants such as plant leaves, stems, roots, meristems etc cultivated in vitro can undergo coordinated division and development resulting in the formation of various and complex structures such as cotyledons, shoot tips, hypocotyls, anthers, internodes, leaf disks, roots, stem and thin cell layers and ultimately complete plants. PCTOC is based on the principle of totipotency, which states that every cell within the plant has the potential to regenerate into a complete plant [11, 13, 15, 16].

Briefly, PCTOC is now a proven technology for the rapid clonal propagation [15] preservation of endangered species and valuable germaplasms, regeneration and multiplication of genetically modified fertile clones [10, 11, 15, 16] enzymes production [14] and economically valuable chemicals [11, 16-18]. The empirical approach that has been extensively used in numerous studies on PCTOC has shown that success is largely dependent on three factors: explant choice, medium composition, especially phytohormone or synthetic growth combinations and control of the physical environment. The composition of a medium, preferable to a certain plant species, is nearly the main task for the establishment of successful plant PCTOC techniques. This biotechnology has been developed to such a level that any MAP species can be in vitro regenerated through one of the following methods: embryo culture, anther culture, callogenesis, somatic or asexual embryogenesis, and organogenesis. The choice of either method depends on the species, the success rate of the method for producing plants at a realistic cost, and local production conditions.

Many plant species containing high-value compounds are difficult to cultivate and the chemical synthesis of plant-derived compounds is often not economically feasible because of their highly complex structures. The PCTOC of MAPs can provide an alternative way of consistent medicinal chemical isolation from plants materials [17-18].

After approximately four decades of the study and numerous attempts by several laboratories around the world, it has been demonstrated that PCTOC systems will produce many unusual secondary metabolites. The major advantages of a PCTOC system over the conventional cultivation of whole plants are:

Useful compounds can be produced by PCTOC under controlled conditions independent of climatic changes or soil conditions;

Organ, tissue and cell culture would be free of microbes and insects;

The cells of any plants could easily be multiplied to yield their specific metabolites,

Organ, tissue and cell can be selected for high production of many compounds;

Organ, tissue and cell can be stored for long periods;

Plant cells can be grown in bioreactors containing thousands of liters of medium;

Automated control of tissue and cell growth and rational regulation of metabolite processes would reduce labor costs and improve productivity;

Cultured plant cells often produce different quantities with different profiles of secondary metabolites and this production can be more reliable, simpler, and more predictable;

Product harvest can be rapid and efficient, as compared to extraction from complex whole plants;

Secondary metabolites produced in vitro can directly parallel compounds in the whole plant;

Interfering compounds that occur in the field-grown plant can be avoided in tissue and cell cultures;

Organ, tissue and cell cultures can yield a source of defined standard phytochemical in large volumes;

Tissue and cell cultures can be radio labeled, such that the accumulated secondary products, when provided as feed to laboratory animals, can be traced metabolically.

Furthermore, new molecules which have not been found previously in plants or have even been synthesized chemically have been produced by cell cultures. Thus this technology constitutes a genuinely new means of achieving production of novel metabolites. In this context, these techniques include the following sequential stages or developments: selection among wild plants of a high-producing genotype, in vitro culture or callogenesis, which involves the selection and stabilization of producing calli with a view to identifying a high-producing line or strain; maximizing growth of callus or cell suspension, culture conditions and isolation of the best producing line; industrial scaling-up, mass cultivation in bioreactors; downstream processing, i.e. extraction and purification of the compounds [1, 12, 17, 18]. Cell suspension cultures are the most used plant cell culture type in the research and production of secondary metabolites. Unfortunately, in many cases low quantities of the desired compounds are accumulated by undifferentiated cell cultures and often the biosynthesis of a certain compound is not produced in cell culture and remains organ or tissue specific [19, 20].

In many cases the biosynthesis of a certain compound is organ or tissue specific. In such cases the organ cultures, i.e. shoot or root cultures, may provide a better alternative than undifferentiated cell cultures. For example, cardenolide biosynthesis was achieved in cultures regenerating shoots or somatic embryos more than undifferentiated cells of Digitalis sp [21, 22]. Fritillaria unibracteata can be rapidly propagated, directly from small cuttings of the bulb by the technique of organ culture. The growth rate of cultured bulb was about 30-50 times higher than that under natural wild growth conditions. The content of alkaloid and beneficial microelements in the cultured bulbs was higher than found in the wild bulb [23].

Otherwise, the research is going for the application of plant transformation and genetic modification using Agrobacterium rhizogenes, in order to boost production of those secondary metabolites, which are naturally synthesized in the roots of the mother plant. Once produced, hairy roots can be excised from the explant and placed on a medium containing a suitable antibiotic to free them from the bacteria. It is characterized by high growth rate, genetic stability and growth in hormone free media. Transformed hairy roots mimic the biochemical machinery present and active in the normal roots, and in many instances transformed hairy roots display higherproduct yields. The greatest advantage of hairy roots is that their cultures often exhibit approximately the same or greater biosynthetic capacity for secondary metabolite production as compared to their mother plants [24]. They can be a promising source for the continuous and standardized production of secondary metabolites under controlled conditions without losing genetic or biosynthetic stability [25].

In addition, whatever in vitro systems used for secondary metabolites production or harvest and depending on the usage in the in vitro studies, different approaches and strategies are being used to obtain higher yields of secondary metabolites in cultures through biotic and/or abiotic elicitors (methyl jasmonate, salicylic acid, chitosan, autoclaved pathogen phytoalexins, heavy metal ions, osmotic stress, ultraviolet irradiation, or gamma irradiations, high salinity,…), gene technology, media manipulation, adsorption of the metabolites to overcome feedback inhibition, permeation of metabolites to facilitate downstream processing, phytohormone combinations, precursor feeding, plant cell immobilization, biotransformation, and bioconversion, many of them are completely interactive. The basic idea behind the usage of elicitors focuses on the stress that they induce upon administration in the cell cultures, which concomitantly affects the yield and quality parameters of the secondary products accumulated [11, 12, 17, 18].

Attempts have been made to manipulate pharmaceutically important medicinal plants for their secondary metabolic pathways by using transgenic technique. The development of transgenic plants is the result of integrated application of rDNA technology, gene transfer and PCTOC techniques. These technologies have enabled the production of transgenic plants in more than 150 species, which include MAPs and most major valuable economic crops. Examples of direct DNA transfer methodology to engineer medicinal plants and cultures have been reviewed [26, 27]. Direct genetic transformation, a technology that is progressing fast in other areas of plant improvement, can only be exploited for secondary metabolite formation, if we have information about the gene(s) controlling the synthesis of the desired product or genetic markers associated with it. Knowledge of the genetics of biosynthetic pathways and their regulation is thus of crucial importance to bypass the low yield of various secondary metabolites in plant cells. Since secondary products however are often biosynthesized in multi-step enzymatic reactions in specifically differentiated cells, manipulations of such pathways to alter metabolic production is complex, complicated and unpredictable.

Scale-up of in vitro mass production of secondary metabolites

Large-scale PCTOC is found to be an attractive alternative approach to traditional methods of plantation as it offers a controlled supply of phytochemical compounds independent of plant availability.

The environment in witch plants cells tissues and organ grow usually changes when cultures are scaled-up from shake flasks to bioreactors, and this may result in reduced productivity. The bioreactors are the key step towards commercial production of secondary metabolites by plant biotechnology. However, this technology is still being developed and despite the advantages, there is a variety of problems to be overcome before it can be adopted for the production of useful plant secondary metabolites. With the ultimate aim of implementing an industrial-scale process, the behavior of cell and tissue culture in bioreactor has received much attention. Engineers are currently developing improved and appropriate bioreactors for the improvement of production systems by adopting techniques of growth cells and metabolite production.

It is noteworthy to point out that PCTOC have proven successful for the commercial production of several important plant constituents. Scale up of the process from laboratory scale, through pilot to industrial scales was successful, adopting either continuous or semicontinuous cultures and using stirred bioreactors with a capacity of up to 75000 liters, with full automation of medium preparation and sterilization, transfers, aeration, stirring, growth monitoring and harvesting [28]. This has been successfully achieved for the production of immunostimulant polysaccharides by Echinacea purpurea cultures [28] and several other plant constituents by commercial or semi-commercial large scale procedures [29, 30].

It should be mentioned that literature reports provide only an incomplete picture of the actual commercial progress, since current industrial investigations fall well under the realm of proprietary and patentable research [30]. Briefly, in 1983, the anti-inflammatory drug shikonin was produced by plant cell cultures of Lithospermum erythrohizon on an industrial scale for the first time by Mitsui Petrochemical Industries Ltd. [31]. Screening for clones derived from individual high-producing cells, and the use of two state cultures-one for growth and another for production has resulted in the commercial production of the antihypertensive drug, ajmalicine by Catharanthus roseus cultures [31, 32]. Other examples include the production of rosmarinic acid from cell suspension cultures of Coleus blumei [33, 34], digoxin by cell cultures from Digitalis sp. [35] and ginsenosides from Panax ginseng cultures [36]. The successful industrial production of paclitaxel, an anti-cancer drug originally extracted from the bark of 50-year-old Pacific yew trees, Taxus brevifolia, by plant cell cultures [37] will trigger research into other plant-based chemotherapeutics such as podophytoxin and comptothecin etc

Concluding remarks and Outlooks

Even with the advances in microbial and chemical productions process, plants remain an indispensable source for a number of chemical substances that are difficult to synthesize chemically owing to their complicated structures. They represent a vast source of untapped chemicals, with a wide variety of proteins and secondary products that have already been isolated from plant tissues.

Nowadays, most of MAPs are still harvested from the wild in a no sustainable way. Such practice can lead to over-exploitation of endangered and vulnerable species as well as to biotope destruction. Therefore, cultivated material is more suitable for large scale uses and breeding (selection) of MAPs is undoubtedly linked to the future success of the exploitation.

Cultivation of medicinal plants is not only a means for meeting current and future demands for large volume production of MAPs and their secondary products, but also a means of relieving harvest pressure on wild populations. Otherwise, due to the structural complexity of natural products, total synthesis does not present an economical option for industrial production for many of these metabolites. Thus, PCTOC is a power biotechnological tool that provides a renewable, easily scalable source of natural product. PCTOC has been established for a wide variety of species, a particularly useful strategy for secondary metabolites production from endangered or rare plants.

Despite the enormous progress achieved in secondary metabolites production into PCTOC systems, several important challenges remain. These include the low productivity, only a small fraction of plant natural products can be expressed at industrially useful levels in culture, recalcitrance of some plant genotype or cell line systems to express their biosynthetic potential, knowledge of the biosynthetic pathways of desired compounds in plants as well as of cultures is often still rudimentary, and strategies are consequently needed to develop information based on a cellular and molecular level. Most notable are cases where some of the new compounds are formed in cells cultures that are not present in whole plants into the same species. This raises a very simple question how is biosynthetic pathways for the production of various secondary compounds in plants? How many of these compounds produced are genetically controlled and how many of them are being attributed to environmental factors?

There has been remarkable progress in understanding of the biochemistry of MAPs due to the development of new analytical tools such as metabolomic, proteomics, and genomics. These newly designed techniques would not only provides data on phenotypic variation, caused by in vitro environmental factors, but also yield data on the genes involved in the biosynthesis metabolic pathways. As well, these new tools could facilitate discovery of new compounds in MAPs, and more interestingly, may identify compounds of medicinal efficacy in yet unexploited in untapped plant species. Progress in this way will open new avenues for high volume production of pharmaceuticals, neutraceuticals, and other beneficial substances.

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