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2. Methods to increase the yields of secondary metabolites in plant cell culture

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2. Methods to increase the yields of secondary metabolites in plant cell culture
  2. Methods to increase the yields of secondary metabolites in plant cellculture The plant cell culture is a way of producing secondary metabolites such aspharmaceuticals, agrochemicals, flavors, colorants, fragrances and food additives.Plant cells are totipotent, and therefore are able to produce all compounds found inthe whole plant (Ramachandra Rao and Ravishankar, 2002). Cell culture has someadvantages over conventional agricultural production: • Independence of geographical variables and the envir  onment. • Provide defined production, quality and uniform yield.   • Production of novel compounds not found in the srcinal plant.   • Manufacturing of the product is more quickly and efficiently.   • Production of compounds with specific stereochemical require ments.In plant cell culture there are several strategies to increase production of secondary metabolites and are listed below: • Obtaining fast growing cell lines and production of the metabolite of interest. • Immobilization of cells to enhance the production of metabolites. • Use of elicitors to increase productivity in a short time. • Membrane Permeation to facilitate diffusion of the metabolite. • Adsorption inhibitory metabolites by feedback. • Scaling of cell culture bioreactors. • Genetic engineering for production of specific metabolites.The following describes some of these strategies. 2.1 Plant cell culture (Somaclonal variation). Somaclonal variation is generated by genetic modification in vitro culture, whichmanifest as inheritable mutations regenerated plantlets (Larkin and Scowcroft,1982). The term somaclonal variation is used universally for all variants of CTV(Bajaj, 1990), and is described as one of the major problems in the regeneration of plants grown in vitro. Plant cell growth in vitro is an asexual process that involves  only mitotic cell division and theoretically should not cause any mutation (Larkin,1998). However, these mutational effects have utility in improving the crop throughthe creation of novel plant varieties with characteristics that increase their commercial value as disease resistance, improved biomass yield and increasedproduction of secondary metabolites (Karp, 1994). 2.1.1 Causes of somaclonal variation. Unlike the mutations in vivo , in vitro mutations occur more often and more easilydetected by the variants found in a limited space and are observable in a shortperiod of time. Mutations may be due to exposure of plant material to the chemicalcomponents of the culture medium, as is the habituation a new balance hormoneauxin / cytokinin, as also the way in which the plant material is rearranged to form de novo organ. May also be due to natural variation already present in somaticcells of the plant material before CTV caused by alterations in cell cycle pathways,transposons and chromosomal aberrations (Vasil, 1990). Somaclonal variationmay be present in the nuclear DNA, mitochondrial and chloroplast. 2.1.2 Methods for detecting somaclonal variations. Somaclonal variation can be a major problem in operating large scalemicropropagation, whereby early detection and elimination of variants is essentialto reduce losses to the producers. Conversely, detection of variations also used tofind lines useful agronomic characteristics or producing compounds of commercialinterest (Karp, 1995). Several types of techniques for detecting somaclonalvariations that falls into molecular, biochemical and morphological characteristics.Detection techniques rely on recognizing morphological changes in plant anatomysuch as the height, leaf area pigmentation abnormalities, including (Israeli et al.,1995). Biochemical detection to identify variations in early stages of the plant andreduce economic losses. These techniques seek to detect various plant responsesto physiological factors such as hormones, light, carbon dioxide assimilation andalteration of pigment synthesis (Shahijram et al  ., 2003; Peyvandi et al  ., 2009).Molecular detection techniques are used to determine the genetic fidelity of   micropropagated species. Some of these techniques are the karyotypes andmolecular markers, RFLP, RAPD, AFLP and SSR (MW. Bairu et al., 2011). 2.2. Genetic transformation of plant cells. Genetic transformation involves the introduction and integration of DNA into thenuclear genome, mitochondrial and chloroplast. Genetic transformation methodsare classified into two types: direct when DNA is introduced into the plant cells or tissues by imbibition, microinjection, electroporation or biolistic and indirect whenusing the soil bacterium  Agrobacterium tumefaciens to transfer foreign DNA to theplant material (S. Naqvi et al  ., 2010; Barampuram and Zhang, 2011). Strategies onsecondary metabolite production using genetic transformation are: • Increased expression of precursors of secondary metabolites. • Increased gene expression limited by the metabolic pathway of interest. • Creation of a new metabolic pathway from an existing route. • Inhibit competitive routes or catalytic steps of the metabolite of interest onthe route using antisense DNA or interference RNA. • Handling regulatory genes function as transcriptional activators or repressors. • Selection of mutants on producers of secondary metabolites. • Reorientation of secondary metabolite production to organs or tissues of interest by using specific promoters. 2.2.1. Methods for detecting genetically transformed cells. a) Marker genes.Once transformed cells have been obtained, they have to select cell lines thatexpress the integrated DNA. There are a number of marker genes that haveproven effective in transforming plant selection, such as neomycinphosphotransferase ( NPT  II) that confers kanamycin resistance and is most oftenused as a selectable marker gene for dicots. There are also other systems basedon glyphosate resistance or spectinomycin (Goldschmidt and Day, 2011).  b) Reporter genes.Reporter genes are used to demonstrate the transient or stable transformation of plant material. Reporter genes encode enzymes whose substrate is not normallyfound in plants, such as the uid   A gene coding for β -glucuronidase enzyme whichproduces a blue color to degrade the substrate X-gluc in the transformant tissue(RA. Jefferson, et al  ., 1987). Green fluorescent protein (GFP) from the jellyfish  Aequorea victoria has also been used as a marker gene and allows visualization of cells and transformed plastids by light excitation, and without supplying the plantsubstrate (W. Chiu et al  ., 1996). Can also perform other tests, such as Southernblot analysis to determine how many copies of the gene of interest have beeninserted in the plant genome or isolating the gene of interest by PCR (RG. Birch,1997). 2.2.2 Transfer of multiple genes through improving vector. The transformation with multiple genes simultaneously allows researchers to studyand manipulate an entire metabolic pathway to produce secondary metabolites andproteins of interest. However, there are several barriers to transformation withmultiple genes simultaneously since the first plant transformation methods weredeveloped to introduce only one or two genes. Due to this, as more genes areintroduced lower the probability that all of them are integrated and express (S.Naqvi et al  ., 2010).To solve this problem, the transfer of multiple genes may be achieved usingconventional methods such as crossing transgenic lines homozygous or sequentialprocessing of the same transgenic line; however these methods are very time andlabor intensive in addition to the laboratory transgenes may progeny segregateindependently. This problem has been partially resolved with the co-transformationand design vectors that enable the insertion of multiple genes in a singletransformation event. The transfer of multiple genes may be mediated by A. tumefaciens usingbinary vectors, wherein a vector has the T-DNA region with the gene of   interest and a vector containing a vir  region. Ti binary vectors have an srcinof replication for E. coli which gives the multiplication in vitro and another source for A. tumefaciens. This method is effective when the size of DNA tobe transferred is less than 50 kb (Valderrama et al., 2005). With conventionaltransfer vectors allows the introduction of multiple genes in a single DNA segment,and was efficient to introduce segments of 50 to 80 kb. Also be used artificial chromosomes  A. tumef  aciens (BIBAC) as a transformationvector. The system is similar to that of the bacterial artificial chromosomes or BACs. This vector was developed based on the knowledge generated from BACs,but it also contains an srcin of replication for  E. coli  and one in A. tumefaciens   besides allowing the introduction of multiple genes of interest and selection. This system is useful for transferring DNA segments between 150-200 kb(Hamilton et al  ., 1997; YG. Liu et al  1999).There are also methods of direct transfer of high capacity using protectivestructures such as calcium alginate particles for encapsulating the plasmidDNA (Sone et al  ., 2002). This method enables to introduce DNA fragments up to200 kb; however does not support and biolistic transformation efficiency is very lowmaking it very applicable in large-scale experiments (N. Wada et al  ., 2009).Furthermore, the use of biolistic and A. tumefaciens  has allowed the geneticmanipulation of different plants of commercial interest. However, these methodshave several limitations when inserting multiple genes, as the insertion occurs inan uncoordinated way, disrupting the integrity of the host genome.These limitations stimulated the development of plant mini-chromosomes whichenables gene transfer large complex genes and multiple genes, along withregulatory elements for expression safe, controlled and persistent, avoidingrearrangements are often associated with insertion events. Must contain artificialchromosomes telomeres, centromeres, a replication srcin and an insertionsite for genes of interest and regulatory (Yu et al  ., 2007). This technology
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