Recent advances in plant biotechnology: Applications in Agriculture. Ashwani Kumar Professor of Botany, Department of Botany and P G School of Biotechnology University of Rajasthan Jaipur 302004. firstname.lastname@example.org Tel 0141 2711654 ( Off) 0141 2654100 ( Res) Mob (0) 9414057484 Abstract: Biotechnology is an area of production and research in which biological systems and biological principles are employed to solve technological problems. In this sense it becomes all inclusive. And during the last decade the advancements in biology have led to the development newer areas like , cellular engineering, biochips and biomaterial science, stem cells, nanobiotechnology etc. Biotechnology is a vast subject and covers Gene and genome analysis: analysis of genes and gene networks showing the potential for industrial application; gene expression studies; biotech plant breeding, e.g. marker assisted breeding. Transgenic technologies: Production and analysis of transgenic crops; gene insertion studies; gene silencing; factors affecting gene expression; post-translational analysis; molecular farming; field trial analysis; commercialisation of modified crops; safety and regulatory affairs Functional genomics: bioinformatics; gene function studies for applied uses Comparative genomics: applications to crop species; use of current crop databases Physiological studies: pathways relevant to an application; secondary metabolites; manipulations of physiology for stress resistance – abiotic and biotic stress resistance including salinity and drought stress. Development of salt resistance plant using plant biotechnology. Host pathogen interaction and role of plant biotechnology for developing resistant corps Developmental studies: developmental mechanisms leading to a further understanding of an industrial use of plants. Plant tissue culture and its role in plant biotechnology. Introduction: Scientists have been improving plants by changing their genetic makeup since the late 1800s. Typically, this has been accomplished through crossbreeding and hybridization, in which two related plants are cross-fertilized and the resulting offspring have characteristics of both parent plants. In the breeding process, however, many undesirable traits often can appear in addition to the desirable ones. Some of those undesirable traits can be eliminated through additional breeding, which is time consuming. Breeders can then further select and reproduce the offspring that have the desired traits. Many of the foods that are already common in our diet are obtained from plant varieties that were developed using conventional genetic techniques of breeding and selection. Today, by inserting one or more genes into a plant, scientists are able to produce a plant with new, advantageous characteristics. The new gene splicing techniques are being used to achieve many of the same goals and improvements that plant breeders historically have sought through conventional methods. They give scientists the ability to isolate genes and introduce new traits into foods without simultaneously introducing undesirable traits. This is an important improvement over traditional breeding. Because of the increased precision offered by the bioengineered methods, the risk of introducing detrimental traits is actually likely to be reduced. Gene and genome analysis: Detailed studies have been conducted on plant genome and physical and genetic maps are available for several plants. As an example of studies on genome the rice genome is discussed here. Rice has a much smaller genome (430 Mbp per haploid genome) than many other crops that belong to the Poaceae family. Due to the genome colinearity, high similarity in gene order and gene content, among the Poaceae family, the importance of rice genetics has been emphasized, and comparative analyses among rice, wheat, and maize have been intensively studied. As a result, rice becomes the model crop for the molecular genetic approach. This crop is available for many applications, including the construction of a high dense map, expressed sequence tag (EST) and full genomic sequence database, bacterial and yeast artificial chromosome (BAC and YAC) libraries, quantitative traits loci (QTL) mapping for yield and morphology, functional genomics by knockout mutagenesis using T-DNA insertion, map-based cloning, and genetically modified rice using transformation techniques. ( see review Cho et al. 2007 cited in Kumar and Sopory 2007). State-of-the-art Genome Profiling (GP) : The traditional approach for species identification is exclusively based on phenotypic traits such as morphological, anatomical, chemical properties and others, which are often affected by environmental factors and thus are difficult to analyze and unreliable. Interspecies homogeneity, intraspecies variability and the existence of undescribed species often lead to phenotypic misidentification. Moreover, species, which are phenotypically far less prominent, cannot be always identified in this way. To overcome these problems, genotypebased (nucleic acid-based) techniques have been employed as an alternative or complementary approach and have continuously been developed including RFLP ,AFLP, RAPD, 16S rRNA or 16S-23S internal transcribed spacer (ITS) sequence analysis and others. These methods provide a possible way to identify species directly based on their genomic sequences but none of them have been shown to identify species in general, mainly because of the insufficiency in the amount of information which they can provide. In this stream, the whole genome sequencing is surely the most definitive solution for species identification though simply too redundant for such purposes and impossible in practice to analyze all the constituents of a heavily dense population. On the other hand, the information obtained from the comparison of a single gene is often not sufficient to place a species at the appropriate position on the phylogenetic tree. In order to deal with above issues, previously Nishigaki and co-workers have described a realistic solution conforming to the notion of the amount of information sufficient for species identification and demonstrated this by inventing a novel method called Genome Profiling (GP), which is a temperature-gradient gel electrophoresis (TGGE) analysis of random-PCR products. Next, the complexity of the generated data, genome profiles, can be simplified by extracting feature points in GP, i.e., species identification dots (spiddos) which can be used for further processing of measuring the similarity of two species by calculating Pattern Similarity Score (PaSS). Further, the technical advances by constructing internet-based GP databases (named On-web GP), and developing a highly reproducible and miniaturized system (micro-TGGE) have moved this technology towards being a universal, general and global tool for species identification( see review Biyani 2007 cited in Kumar and Sopory 2007).. Recombinant DNA technology: Gene targeting (GT) Gene targeting (GT) is a key technology for the rational, accurate and safe exploitation of plantsthrough genetic manipulation. Moreover, it offers the potential to completely knockout the expression of target genes or to make specific changes to gene function, objectives that cannot be achieved by conventional transgenesis. The ability to target DNA integration would permit the locus-specific integration of a transgene into a predetermined site of the host genome, avoiding the accidental inactivation of an endogenous gene localized at the insertion site or the unexpected expression profiles of the transgene itself, the so-called position effect. Systematic isolation and sequencing of genomic DNA flanking the insertion sites (known as FSTs or Flanking Sequence Tag offers the opportunity to rapidly characterize plants altered in a candidate gene sequence. This approach is notably most useful in fully sequenced genomes such as in Arabidopsis thaliana. With 125 Mbp and 26,422 genes, the Arabidopsis genome shows very limited synteny with the 420–466 Mbp and 60,000 predicted genes of the rice genome. The recombination machinery has been well conserved throughout evolution, as an essential component of cell survival. In nature, homologous recombination is a DNA maintenance pathway that protects chromosomes against damage affecting both DNA strands, such as double strand breaks (DSBs) or interstrand crosslinks. DSB repair (DSBR) has been one of the most investigated homologous repair pathways see SHRIVASTAVA1, SHARMA2 AND KUMAR 2007 cited in Kumar and Sopory 2007). Recently plastid genome transformation technique has gained prominence due to its better integration and less chances of random spread. The genome propagated by higher plant plastids, the plastome, is typically a double stranded DNA molecule of 130 to 160 kb. Over one hundred copies of this genome can be present in a single plastid. It is ideally represented as a circular monomer containing 2 inverted repeats, even though reality is more complex since linear and circular multimers have been frequently detected The complete sequence of this highly polyploidy genome is available for about 20 different species of angiosperms http://megasun.bch.umontreal.ca/ ogmp/projects/other/cp_list.html). The first successful transformation of tobacco was performed using as marker a mutant plastid DNA fragment covering the 16SrRNA gene derived from a line resistant to spectinomycin and streptomycin. Major improvements in the selection process were soon obtained with the dominant aadA marker gene, inactivating spectinomycin or streptomycin. When fused to GFP, this marker can be used to track the selection process. Genes encoding resistance to kanamycin, nptII and more recently aphA-6 are also possible options, and could be more appropriate for some species (Kumar et al., 2004a see review Dubald. 2007 cited in Kumar and Sopory 2007). There are three different fates for the external DNA to get integrated into the native genome. They are homologous recombination, illegitimate recombination or nonhomologous end joining, and single-strand annealing. . Single-strand annealing (SSA), a third path of repair, requires the presence of repeated sequences on both sides of a break. After exonuclease degradation of the 5’ ends, repair occurs by annealing of the two complementary sequences, a process leading to the loss of the genetic information contained between these repeats. With respect to the species preferential DSB repair pathway, HR but also IR mediates transgene integration. This second aspect explains the inefficiency of GT in higher plants, which use HR as a minor pathway of repair. Thus, despite the fact that transgene integration processes are still unclear in plants transgenic DNA would be preferentially integrated by end joining whether or not sharing homology within the host genome. GM crops: The U.S. Food and Drug Administration (FDA) has found no evidence to indicate that either ordinary plant deoxyribonucleic acid (DNA) or the DNA inserted into plants using bioengineering presents food safety problems. Nor are the small amounts of the newly expressed proteins likely to change dramatically the safety profile of the plant. If safety concerns should arise, however, they would most likely fall into one of three broad categories: allergens, toxins, or anti-nutrients. FDA has extensive experience in evaluating the safety of such substances in food. It is important to note that the kinds of food safety testing typically conducted by developers of a bioengineered food crop to ensure that their foods meet all applicable requirements of the Food, Drug and Cosmetics Act (FD&C Act) address these potential concerns. In the event that something unexpected does occur, this testing provides a way to detect such changes at the developmental stage and defer marketing until any concern is resolved. As aforementioned, some of the food safety concerns that could arise include: Allergens: Foods normally contain many thousands of different proteins. While the majority of proteins do not cause allergic reactions, virtually all known human allergens are proteins. Since genetic engineering can introduce a new protein into a food plant, it is possible that this technique could introduce a previously unknown allergen into the food supply or could introduce a known allergen into a “new” food. Toxins: It is possible that a new protein, as introduced into a crop as a result of the genetic modification, could cause toxicity. Anti-nutrients: It is possible that the introduction of anti-nutrients, such as molecules like phytic acid, could reduce essential dietary minerals such as phosphorus. The use of genetic engineering techniques could also result in unintended alterations in the amounts of substances normally found in a food, such as a reduction of Vitamin C or an increase in the concentration of a naturally occurring toxicant in the plant food. LEGAL AND REGULATORY ISSUES: One important component in ensuring food safety is the U.S. regulatory structure. The FDA regulates bioengineered plant food in conjunction with the United States Department of Agriculture (USDA) and the Environmental Protection Agency (EPA). FDA has authority under the FD&C Act to ensure the safety of all domestic and imported foods for man or animals in the United States market. The exceptions to this are meat, poultry and certain egg products, which are regulated by USDA. The safety of animal drug residues in meat and poultry, however, is regulated by FDA. Pesticides, including those bioengineered into a food crop, are regulated primarily by EPA. USDA’s Animal and Plant Health Inspection Service (APHIS) oversees the agricultural and environmental safety of planting and field testing bioengineered plants. Bioengineered foods and food ingredients must adhere to the same standards of safety under the FD&C Act that apply to their conventionally bred counterparts. This means that these products must be as safe as the traditional foods in the market. FDA has the power to remove a food from the market, or sanction those marketing the food if the food poses a risk to public health. It is important to note that the FD&C Act places a legal duty on developers to ensure that the foods they market to consumers are safe and comply with all legal requirements. Area under the commercialization of genetically modified (GM), often called biotech crops continued to grow for the ninth consecutive year at a sustained double-digit growth rate of 20% in 2004 (James, 2004). The estimated global area of approved GM crops for 2005 was 90.0 million hectares with $4.70 billion global market value—based on the sale price of GM seed plus any technology fees that apply. The global value of the GM crop market is projected a more than $5.0 billion for 2005 (James, 2004). Commercialization of genetically modified (GM) crops continued to grow for the ninth consecutive year. It reflects the substantial improvements in productivity, the environment, economics, health and social benefits realized by farmers, consumers and society. At the same time the growing controversy over GM food products increased interest in food labelling and identity preservation (IP) of GM crops. Hence, an IP system must be designed to provide assurances that the desired traits are present (or absent) in a product from the seed source, through all steps of production and delivery, to the end user. There are numerous regulatory issues related to GM crops. These include the testing and acceptance of new GM crops for commercial introduction, both domestically and internationally. Nearly every country has different approaches and many have their own regulatory framework, with an intent to prevent cross-contamination of the conventional food and feed industries. IP tracking software is also available in market to ease the burdens associated with precise record-keeping requirements. The economics of IP has been calculated by various scientists depending on different applied IP systems. Niche-marketing opportunities will grow, because of the availability of GM crops and finally, IP of agricultural commodities from GM crops can provide greater choice and value desired by both agricultural producers and consumers (DOSHI AND FRANÇOIS EUDES, 2007 cited in Kumar and Sopory 2007) Based on annual percentage growth in area, of the eight leading GM crop countries, India had the highest percentage year-on-year growth in 2004 with an increase of 400% in Bt cotton area over 2003, followed by Uruguay (200%), Australia (100%), Brazil (66%), China (32%), south Africa (25%), Canada (23%), Argentina (17%) and USA at 11%. India increased its area of approved Bt cotton, introduced only two years ago, from approximately 38,038 hectares in 2002/ 03 to 560,000 hectares in 2004/05 seasons with Bt coverage of 11.65% and approximately 300,000 small farmers benefited from Bt cotton( see review DOSHI AND EUDES, 2007 cited in Kumar and Sopory 2007). . REGULATORY ISSUES RELATED TO GM CROPS: There are numerous regulatory issues related to GM crops. These include the testing and acceptance of new GM crops for commercial introduction and the introduction of food products containing ingredients from GM crops, both domestically and internationally. Nearly every country has different approaches and many have their own regulatory framework. Regulation is a very dynamic issue with changes being reviewed and proposed in many countries on an ongoing basis. Numerous regulatory actions are consequently being proposed as governments react to consumer concerns and pressures. Several countries have or have proposed to create new agencies to specifically cover GM crops. Approaches range from cautious acceptance to attempts to ban (growing and even imports), at least for the foreseeable future, all crops and products with GM traits. Each is approaching the testing, introduction, and acceptance of GM crops in its own manner and on its own time schedule. Table 6 summarizes the current status of some of the regulations related to introduction, approval, and commercial acceptance of GM crops. Table 6. Status of regulations over GM products. Abiotic and biotic resistance: Coat Protein Mediated Resistance: CP is an important structural protein as it not only protects the viral nucleic acid from degradation, but also plays an important role in virus infection. Its functions includes acquisition and transmission of virus by vectors, cell to cell and long distance spread of the virus in host plants, and for some viruses, it regulates one or more steps of virus replication.Coat Protein (CP) mediated resistance has been demonstrated for 17 groups of viruses, and so far this strategy has shown best promise. CP transgenes have been shown to be effective in preventing or reducing infection and diseases caused by homologous and closely related viruses (Gonsalves, et al., 1998). Coat protein-mediated protection has been reported for Tobacco mosaic virus, TMV,Tomato mosaic virus, ToMV, (Sa), Cucumber mosaic virus, CMV, Alfalfa mosaic virus, AlMV, (Loesch-Fries et al., 1987; Tumer et al., 1997), Potato virus X, PVX, Potato virus Y, PVY, Potato leaf roll virus, PLRV, Papaya ringspot virus (PRSV) and a number of other viruses. CP-mediated resistance in Cantaloupe, Papaya, Potato, Squash and Tomato has been tested under the field conditions with fair degree of protection against most of the viruses (Table 4). ( see review Verma and Parveen 2007 cited in Kumar and Sopory 2007) Anti-HIV Agents Among Desert Plants Around 40 million people are affected due to the Human Immuno-deficiency Virus globally. During the past decades, a large number of anti-viral screening experiments on medicinal plant extracts have been reported and have led to the selection of several extracts active towards herpes viruses. A promising result of a naturally occurring antiherpetic agent was given by n- docosanol (a natural 22 carbon saturated fatty alcohol) which is undergoing phase III clinical trials in patients. Clinical testing of the topical formulation, or systemic administration of drug suspensions has demonstrated a good therapeutic index, since high doses of n- docosanol do not elicit appreciable toxicity. The findings show that natural products are still potential sources in the search for new antiherpatic agents (Hattori et al., 1995). Various plant extracts used in Ayurvedic medicine for inhibitory effects on HIV virus have been studied (Hattori, personal communication). A large number of such plants occur in semi-arid and arid climate of Rajasthan. Acquired immunodeficiency syndrome (AIDS) , the great pandemic of the second half of the 20th Century, is still a threatening disease world wide. Many research approaches are currently aimed at developing novel agents to arrest the replication of HIV through various targets. These may include the inhibition of reverse transcriptase (RT), protease (PR), membrane fusion and integrase. HIV PR enzyme has been demonstrated to play an essential role in viral replication ( see review Kumar 2007 cited in Kumar and Sopory 2007) A range of HIV PR inhibitors have been designed and applied in clinical trials such as Sanqunavir, Ritonavir and Indinavir. However, the development of drug resistance by virus, irrespective of the target, remains as an overwhelming problem in AIDS chemotherapy. Thus there is great need to search for and develop new and different anti-HIV candidates from plants and natural products are of considerable importance. In search for anti-HIV active agents from natural products, many attempts at screening traditional medicines have been made. Biotic and abiotic stress: Environmental abiotic stress conditions, and especially drought and salinity, are currently the major factors which reduce crop yields world-wide leading to the fact that more than 800 million people are chronically undernourished.. The United Nations Environment Program estimates that approximately 20% of agricultural land and 50% of cropland in the world is saltstressed This salinity, in particular, is an increasing problem and nearly half of the area under irrigation, is at risk to be lost due o building up of salinity. Therefore genetic improvement of salt tolerance has become an urgent need for the future of agriculture in arid and semiarid regions.. One way of solving this problem would be breeding tolerant varieties of crop plants that can be grown on saline soils, but these breeding programs are time consuming and remained elusive . Hence, many metabolic changes are known to occur in plants subjected to salt stress, physiological parameters such as ionic relations have been suggested to be used as tolerance indicators since they can be related to salt tolerance mechanisms. Biofuel production: India has over 180 million of wasteland out of which 90 million ha is uncultivable. The degraded and denuded lands arise due to soil erosions as well as secondary salinizations. However Calotropis procera is a potential plant for bioenergy and biofuel production in semi arid regions of the country because it is able to grow on such lands. The plant has a growth potential of 2 dry tones to 40 dry tones per ha depending on the agro climatic conditions of it’s growth. The plant has high level of regeneration potential and could be harvested up to 4 times a year. The plant yields valuable hydrocarbons which could be converted into diesel substitutes. The bio-diesel derived from Calotropis procera is free from NOx gases, S02 and Suspended Particulate Matter (SPM) and has high cetane value. Due to it’s enormous potential for growth under adverse climatic conditions Calotropis procera is suggested as potential plant for bio-diesel production under semi-arid and arid conditions ( Anita and Kumar,2005, Anita et al. 2005). Jatropha curcas also provides non-edible oil which could be converted into methyl ester with a gain of glycerine ( Anita and Kumar 2007) . The JME is used as mix in the ratio of 05, to 20 percent blend to fossil fuel diesel and in Germany the use of Rape seed methyl ester is increasing. Soil salinity: Soil salinity affects plant production in many parts of the world, particularly on irrigated land. NaCl is the predominant salt in most saline environments. Many crop species are sensitive to high concentrations of salt with negative impacts on agricultural production. Maize (Zea mays L.) is considered a moderately salt-sensitive plant.. Salt resistance of plants is a complex phenomenon that involves biochemical and physiological processes as well as morphological and developmental changes.. In addition to general osmotic stress, high concentrations of Na+ are toxic to maize and molecular mechanisms for salt resistance have not been fully identified or characterized (Zoerb et al 2004). The analysis of the plant’s proteome is an important amendment to the analysis of the genome, because gene expression is altered under salinity stress. The proteome, in contrast to the genome, is not static but rather dependent on a number of responses influenced by internal and external factors. The plant adaptation to environmental stress, such as soil salinity, is expected to have a strong influence on proteins. One approach to study the molecular mechanisms of plant responses to salinity is to use 2D polyacrylamide gel electrophoresis. Furthermore, the identification of differentially regulated proteins can lead to the identification of proteins and their corresponding genes which are involved in the physiology of salt resistance. The high resolution achieved by 2D gels and computer-assisted analysis of the differentially regulated proteins were used to examine those proteins whose synthesis was modulated by salt treatment and to quantify these changes. As far as we know, our investigations are the first to characterize the differentially expressed proteins from roots and shoots of maize after treatment with low and high salt stress. Plant material was an efficiently Na+-excluding maize inbred line developed in our laboratory. According to Munns the growth response to salt stress consists of two phases, first, a water-deficit that results from the relatively high solute concentrations in the root medium and, second, ion-specific stress resulting from altered K+/Na+ ratios or Na+ and Cl− concentrations that are toxic to plants. The aim of this study was to elucidate biochemical and physiological reactions of glycophytes to salt stress in the first phase of salinity. While all major crops, as well as most wild species, are glycophytes, i.e. sensitive to relatively low salt concentrations, there are plants naturally adapted to conditions of high salinity in the soil. These plants, known as halophytes, include a large taxonomic variety and occupy diverse habitats, from extreme dry to temporarily waterlogged sites or salt marshes, and can tolerate NaCl concentrations similar, or even higher than that of sea water, ca. 500 mM (Figure 1). It is - without doubt - necessary to develop sustainable biological production systems which can tolerate higher water salinity because freshwater resources will not come up with increasing demands of agricultural practice in near future. The sustainable use of halophytic plants is a promising approach to valorize strongly salinised zones unsuitable for conventional agriculture and mediocre waters. The development of cash crop halophytes and the breeding of salt resistant crop varieties will require a clear understanding of the complex mechanisms of salt stress tolerance, which we are still lacking despite intensive research during the last decade (see review KOYROAND HUCHZERMEYER 2007 cited in Kumar and Sopory 2007). Recombinant proteins: It has been estimated that 1 g of recombinant antibody could be produced in leaves of a plant crop for only about US$100 while the current prices for monoclonal antibodies range from US$2000 to US$5000 per gram. Indeed the cost of producing 1 kg of recombinant protein from most major field crops is estimated to be 10 to 50 times lower than the cost of producing the same amount by E. coli fermentation. Whole plants also have an advantage when tissues such as a fruit, tuber, etc. can be used to express the protein of interest (James and Lee 2001), and an area of undisputed advantage occurs when the oral delivery of pharmaceuticals, as well as feed and food enzymes, is possible. However, there are also some evident obstacles that arise when a whole plant is used for large-scale protein production( see review SODERQUIST and LEE 2007). Cell-free systems have proved to have high utility at the genomic, transcriptomic and proteomic levels and to form a vital component of many aspects of recombinant gene expression, and of both structural and functional proteomics.. Compared with DNA microarrays, protein bio-chips provide more challenges and have yet to be perfected due to the complexity and inherent difficulties with protein immobilization. Novel cell free translation system is unique discovery: A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA–protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA–protein fusion is ligated to the 3′ ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA–protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of ‘RNA chip-to-protein chip’ using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution (Biyani et al. 2006). Plant tissue culture: Another area of biotechnology is micropropagation of plants. The aim of this technique is a fast production of a great number of genetically identical plants from a highly valuable mother plant or e. g. monosexual male and female plants. These plants can be either directly sold on the market for planting, used for breeding purposes, for genetechnology or the technique is used as a method for basic science studies. Using petiole explants from transgenic plants containing the auxin responsive MAS promoter linked to the GUS reporter gene (Fig. 15, 16) the distribution of auxin within the cultured petiole could be followed during the induction phase of somatic embryogenesis (Neumann 2000 and Neumann 2006). Interestingly, the cells forming the glandular canal contain high concentrations of auxins as shown by using transgenic plants containing the auxin sensitive MAS-promoter coupled to the GUS-gene ( Fig see below ), whatever the significance. Rhizogenic centers develop near vascular bundles prior to those embryogenic centers. Fig. 2 Plasmid pPCV812 with the MAS promoter and the GUS reporter gene, hyg=Hygromycin resistance, Ap/Cb=Ampicillin/Carbenicillin resistance (courtesy of Dr. Z. Koncz, Max-Planck- Institut Cologne, Germany, for providing the plasmid) Genetic factors play a central role to induce somatic embryos, i.e. to provide the competence of the species for the process. Here, great variation can be found even within a genera such as Daucus. Eight of twelve Daucus species cultured in identical conditions produced somatic embryos (D. halophilus, D. capillifolius, D. commutatus, D. azoricus, D. gadacei, D. maritimus, D. maximus, D. carota ), whereas four species (D. montevidensis, D. pussillus, D. muricatus, D. glochidiatus) were not competent to do so. Under identical culture conditions, only 8 out of 12 species and subspecies of the genus Daucus proved capable of somatic embryogenesis. Random amplified polymorphic DNA analysis indicated a polymorphism between the genomes of individual species that were capable of embryogenesis and those that were not. Two specific bands (1.1 kbp, 0.68 kbp) were detected only inthe genomes of individuals with the capacity for embryogenesis. These were cloned and sequenced, and the homology of the nucleotide sequences of the various species was detected: this ranged from 74% to 92% for the larger sequence and from 92% to 97% for the smaller one. These DNA sequences would appear to be useful as a marker of the capacity for somatic embryogenesis in the genus Daucus (Imani et al.2001) The sequences obtained in this study have been registered with the European Bioinformatics Institute (EMBL). The access numbers for the sequences are: AJ278039 DCA78039; AJ278040 DCA78040; AJ278041 CA78041; AJ278042 DCA78042; AJ278176 DCA278176; AJ278177 DCA278177; AJ278178 DCA278178; AJ278179 DCA278179. No open reading frames were detected. We performed later additional studies with other Daucus species (D. capillifolius; D. carota ssp. Azoricus and gadecaei) as shown in Table 1 to determine the use-fulness of these RAPD products as markers for identifying the ability of Daucus species to generate somatic embryos (Fig. 3b). There was a 100% correlation between the embryogenic potential of the species (Table 1) and the occurrence of the 1.1-kbp and 0.68-kbp band (Imani et al.2001). Micropropagation Technique in Enhancing the Productivity of Crops have been taken up at large scale at TERI ( see review Saxena, 2007 see Kumar and Shekhawat 2007) Some of the activities undertaken at MTP include: • Large-scale production of superior quality planting material of various economically important plant species using tissue culture technology • Mass multiplication of those species which are difficult to regenerate by conventional methods of propagation or where conventional methods of propagation are inadequate to meet the demand of planting material • Development of new micropropagation protocols and refining of others so as to make them suitable for large-scale production of plants • Helping the entrepreneurs/industry through technology transfer, mother cultures and training of staff • Assisting clients in setting-up their own tissue culture labs • Creating awareness Till date over 15 million plants of forest species, cash crops, medicinal and aromatic plants, and ornamentals (foliage and flowering) have been dispatched to various state forest and horticulture departments, private entrepreneurs, nurseries, farmers etc. for field demonstrations and routine plantations. In addition, MTP is in possession of micropropagation protocols for over 90 economically important plant species. Field demonstration plots of tissue cultured plants have been laid at different locations to evaluate and compare their growth performances with conventional plants. Besides transfer of technologies to industry for commercialization, MTP has been instrumental in capacity building and creating awareness about tissue culture technology through seminars/ workshops/training programmes, exhibitions, etc. (Dhawan and Saxena 2004; Saxena and Dhawan, 2004). Secondary metabolites: Since the establishment of plant tissue culture techniques in 1960’s, significant contributions have been made to the development of biochemical studies on secondary metabolism such as structural elucidation, biosynthesis, enzymology, metabolic regulation system, intracellular distribution of metabolites and relevant enzymes, metabolite transportation, molecular biology and many others . However, one of the greatest difficulties and challenges in the application of plant tissue culture to metabolism research has been that unorganized callus tissues have often failed to accumulate metabolites usually detected in the mother plant. In some cases, metabolic potential was recovered through the development of a production medium, change in culture conditions or selecting cell strains of high productivity (Fujita and Tabata, 1987). It is commonly observed that recalcitrant callus tissues begin to synthesize secondary metabolites after organ - such as shoots and roots - differentiation .Although somatic embryogenesis occurs in cultured cells of numerous plant species, it has rarely been applied to secondary metabolite production. Recently secondary metabolite production by somatic embryo cultures and especially by those of Corydalis species has been reviewed by HIRAOKA and Bhatt, 2007) . References: Anita Kumari and Ashwani Kumar (2005) SOME POTENTIAL BIOFUEL PLANTS FOR SEMI-ARID AND ARID REGIONS AND IMPROVING THEIR GROWTH AND PRODUCTIVITY In : CARRASCO J.E., L. SJUNNESSON, P. HELM, A. GRASSI (eds) BIOMASS FOR ENERGY, INDUSTRY AND CLIMATE PROTECTION. pp 279-281. Anita Kumari, Ashwani Kumar, V.R. Kumar (2005) PRODUCTIVTY OF CALOTROPIS PROCERA IN SEMI-ARID REGIONS OF RAJASTHAN AND ITS USE AS RENEWABLE SOURCE OF ENERGY In : CARRASCO J.E., L. SJUNNESSON, P. HELM, A. GRASSI (eds) BIOMASS FOR ENERGY, INDUSTRY AND CLIMATE PROTECTION. pp 276-278 Dhawan V and Saxena S (2005) Production of superior quality disease-free planting material. In: Chadha KL, Ahloowalia Imani, J., (1999): In situ- Nachweis der Auxinverteilung in kultivierten Petiolenexplantaten von transgenen Pflanzen während der Induktion der somatischen Embryogenese bei Daucus carota L. Diss. Justus Liebig Universität, Gießen, Germany J. Imani • L. Tran Thi • G. Langen,B. Arnholdt-Schmitt • S. Roy • C. Lein • A. Kumar K.-H. Neumann (2001) Somatic embryogenesis and DNA organization of genomes from selected Daucus species. Plant Cell Rep 20:537–541 Prasad, BS KV and Singh SK (Eds.) Crop Improvement and Production Technology of Horticultural Crops Proceedings of First Indian Horticulture Congress - 2004. pp 174-184. Kumar A. (2004) Calotropis Procera: a Potential Plant for Hydrocarbons from Semi-Arid and Arid Regions In : Van Swaaij, Fjallstrom, Helm and Grassi (eds):. Biomass for energy, industry, and climate protection. Proceedings of the Second World Conference ETA-Florence, Rome Italy WIP-Munich , Germany pp 173. Kumar, A. and Sudhir Sopory ( eds) ( 2007) Recent advances in plant biotechnology. IK International New Delhi Kumar A and N S Shekhawat ( eds) (2007) Plant tissue culture, Molecular markers and their role in agriculture production. IK International. New Delhi Neumann, K.-H. (1995): Pflanzliche Zell- und Gewebekulturen. Ulmer Verlag, Stuttgart,304 pages Neumann KH (2000) Some studies on somatic embryogenesis, a tool in plant biotechnology. http://bibd.uni-giessen.de/ghtm/ 2000/uni/p000004.htm Neumann, K.-H. (2006): Some studies on somatic embryogenesis: a tool in plant biotechnology. In: Kumar and Roy (eds) Plant biotechnology and its applications in tissue culture. I.K. International, New Delhi . pp 1-14. Shekhawat V.P.S. and A. Kumar 2006 Somaclonal variants for salt tolerance and in vitro propagation of peanut. In: (Eds.) A. Kumar, S. Roy and S.K. Sopory. Plant Biotechnology & its Application in Tissue Culture. I.K. International New Delhi, Mumbai, Bangalore. pp. 177-196 Shekhawat, V.P.S., Kumar, A., and K.H. Neumann. 2005. Bio-reclamation of secondary salinized soils using halophytes. Biosaline Agriculture & Salinity tolerance in Plants. (Eds.) M.Ozturk, Y. Waisal, M.A. Khan and G. Gork, Birkhäuser Verlag , Switzerland. pp 145-152. Shekhawat, V.P.S., Kumar, A., and K.H. Neumann. 2006. Effect of NaCl salinity on growth and ion accumulation in some chenopodiaceous halophytes. Communication in Soil Science and Plant analysis 13-14 (37), 1933 – 1946 Manish Biyani, Yuzuru Husimi, and Naoto Nemoto (2006) Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA Nucleic Acids Res. 2006 November; 34:140-. Saxena, S and Dhawan V (2004) Changing Scenarios in Indian Horticulture In : PS Srivastava, A Narula and S Srivastava (Eds.) Plant Biotechnology and Molecular Markers. Anamaya Publishers, New Delhi. pp. 261-277.
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