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Science, Technology and Medicine open access publisher.Publish, read and share novel research. Instead of using expensive artificially created water ionizers, use ? of fresh-squeezed lemon in two cups of lukewarm water.
Take this drink in the morning in order to clean your digestive system, balance the acids in the system and boost your metabolism.
Leafy greens, vegetables, wheat grass, seeds, nuts and certain fruits can be used for creation of tasty and highly effective organic green juices and green smoothies. Their ingredients like minerals, vitamins, phytonutrients, oxygen and chlorophyll, alkalize the body and even prevent occurrence of damaged, unhealthy cells, bacteria and other potentially dangerous microorganisms.
On the other hand, consuming processed food stimulates the appearance of these harmful organisms. Engage in physical activity, control your stress, sleep better and get rid of your nasty habits.
Although many people consider stress to be a myth when it comes to developing diseases, the truth is that emotional stress triggers release of acid-forming hormones like adrenaline and cortisol, which disturb the normal functioning of the system.
Some claim that certain type of food is more acidic while others say that food is less acidic. Throw Away Your Glasses By Using Only THIS One Ingredient to Increase Your Vision Up To 97%!
Micropropagation of Orchids (a) callus culture (b) shoot regeneration (c) rooted plantlets17. Tissue culture of Potato (a) nodal segment (b) regenerated shoots and roots (c) tissue culturedpotato20.
Tissue culture of Jatrophacurcas (a) callus of Jatropha (b) shoot regeneration (c) root induction.21. Lahore, Pakistan[3] Plant tissue Culture Lab, Qarshi Herb Research Center, Qarshi Industries (Pvt.) Ltd.
What most people don’t know is that lemons have acidic properties in their natural form, but when they are mixed with water they have alkaline properties. It is very important to take care of our diet, but it is also very important to pay attention to the physical activity we have during the day. There are many ways to avoid this situation – meditation, pilates, yoga, exercising in a gym or at home, breathing exercises, developing stress management strategy etc.
In addition, stress, drugs and cigarettes increase the level of acids in the body and lead to inflammation. Scientists usually burn samples of food to ashes when they want to check the acidic or alkaline foods properties. IntroductionTissue culture is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions [1] often to produce the clones of plants. The pH in your system is affected by the unhealthy modern lifestyle which many people practice. Since only a small number of people have access to scientific labs I have added some sources that can help you make the right choice. It is good to point out that people can find different sources arguing about certain foods and their alkalizing or acidic properties.
The controlled conditions provide the culture an environment conducive for their growth and multiplication.
These conditions include proper supply of nutrients, pH medium, adequate temperature and proper gaseous and liquid environment. Apart from their use as a tool of research, plant tissue culture techniques have in recent years, become of major industrial importance in the area of plant propagation, disease elimination, plant improvement and production of secondary metabolites.Small pieces of tissue (named explants) can be used to produce hundreds and thousands of plants in a continuous process.
A single explant can be multiplied into several thousand plants in relatively short time period and space under controlled conditions, irrespective of the season and weather on a year round basis [2].
Endangered, threatened and rare species have successfully been grown and conserved by micropropagation because of high coefficient of multiplication and small demands on number of initial plants and space. In addition, plant tissue culture is considered to be the most efficient technology for crop improvement by the production of somaclonal and gametoclonal variants. The micropropagation technology has a vast potential to produce plants of superior quality, isolation of useful variants in well-adapted high yielding genotypes with better disease resistance and stress tolerance capacities [3].
Certain type of callus cultures give rise to clones that have inheritable characteristics different from those of parent plants due to the possibility of occurrence of somaclonal variability [4], which leads to the development of commercially important improved varieties. Commercial production of plants through micropropagation techniques has several advantages over the traditional methods of propagation through seed, cutting, grafting and air-layering etc. It is rapid propagation processes that can lead to the production of plants virus free [5]. Coryodalisyanhusuo, an important medicinal plant was propagated by somatic embryogenesis from tuber-derived callus to produce disease free tubers [6].
Meristem tip culture of banana plants devoid from banana bunchy top virus (BBTV) and brome mosaic virus (BMV) were produced [7].
Increase in yield up to 150% of virus-free potatoes was obtained in controlled conditions [8]. The main objective of writing this chapter is to describe the tissue culture techniques, various developments, present and future trends and its application in various fields.2. History of plant tissue cultureThe science of plant tissue culture takes its roots from the discovery of cell followed by propounding of cell theory.
In 1838, Schleiden and Schwann proposed that cell is the basic structural unit of all living organisms. They visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plant. Based on this premise, in 1902, a German physiologist, Gottlieb Haberlandt for the first time attempted to culture isolated single palisade cells from leaves in knop’s salt solution enriched with sucrose.The cells remained alive for up to one month, increased in size, accumulated starch but failed to divide.
Though he was unsuccessful but laid down the foundation of tissue culture technology for which he is regarded as the father of plant tissue culture. Basics of plant cell and tissue culture In plant cell culture, plant tissues and organs are grown in vitro on artificial media, under aseptic and controlled environment. The technique depends mainly on the concept of totipotentiality of plant cells [9] which refers to the ability of a single cell to express the full genome by cell division. Along with the totipotent potential of plant cell, the capacity of cells to alter their metabolism, growth and development is also equally important and crucial to regenerate the entire plant [1]. Plant tissue culture medium contains all the nutrients required for the normal growth and development of plants. It is mainly composed of macronutrients, micronutrients, vitamins, other organic components, plant growth regulators, carbon source and some gelling agents in case of solid medium [10].
Murashige and Skoog medium (MS medium) is most extensively used for the vegetative propagation of many plant species in vitro. The pH of the media is also important that affects both the growth of plants and activity of plant growth regulators.
The composition of the medium, particularly the plant hormones and the nitrogen source has profound effects on the response of the initial explant.Plant growth regulators (PGR’s) play an essential role in determining the development pathway of plant cells and tissues in culture medium.
The type and the concentration of hormones used depend mainly on the species of the plant, the tissue or organ cultured and the objective of the experiment [11]. Auxins and cytokinins are most widely used plant growth regulators in plant tissue culture and their amount determined the type of culture established or regenerated. The high concentration of auxins generally favors root formation, whereas the high concentration of cytokinins promotes shoot regeneration.
A balance of both auxin and cytokinin leads to the development of mass of undifferentiated cells known as callus.
Cytokinins generally promote cell division and induce shoot formation and axillary shoot proliferation.
High cytokinin to auxin ratio promotes shoot proliferation while high auxin to cytokinins ratio results in root formation [13]. Tissue culture in agricultureAs an emerging technology, the plant tissue culture has a great impact on both agriculture and industry, through providing plants needed to meet the ever increasing world demand. It has made significant contributions to the advancement of agricultural sciences in recent times and today they constitute an indispensable tool in modern agriculture [5].


Tissue culture allows the production and propagation of genetically homogeneous, disease-free plant material [37]. Cell and tissue in vitro culture is a useful tool for the induction of somaclonal variation [38]. Genetic variability induced by tissue culture could be used as a source of variability to obtain new stable genotypes. Interventions of biotechnological approaches for in vitro regeneration, mass micropropagation techniques and gene transfer studies in tree species have been encouraging.
Genetic engineering can make possible a number of improved crop varieties with high yield potential and resistance against pests. Germplasm conservationIn vitro cell and organ culture offers an alternative source for the conservation of endangered genotypes [40].
Germplasm conservation worldwide is increasingly becoming an essential activity due to the high rate of disappearance of plant species and the increased need for safeguarding the floristic patrimony of the countries [41]. Tissue culture protocols can be used for preservation of vegetative tissues when the targets for conservation are clones instead of seeds, to keep the genetic background of a crop and to avoid the loss of the conserved patrimony due to natural disasters, whether biotic or abiotic stress [42].
The plant species which do not produce seeds (sterile plants) or which have ‘recalcitrant’ seeds that cannot be stored for long period of time can successfully be preserved via in vitro techniques for the maintenance of gene banks. Cryopreservation plays a vital role in the long-term in vitro conservation of essential biological material and genetic resources. It involves the storage of in vitro cells or tissues in liquid nitrogen that results incryo-injury on the exposure of tissues tophysical andchemical stresses. Successful cryopreservation is often ascertained by cell and tissue survival and the ability to re-grow or regenerate into complete plants or form new colonies [43]. It is desirable to assess the genetic integrity of recovered germplasm to determine whether it is ‘true-to-type’ following cryopreservation [44].
The fidelity of recovered plants can be assessed at phenotypic, histological, cytological, biochemical and molecular levels, although, there are advantages and limitations of the various approaches used to assess genetic stability [45]. Cryobionomics is a new approach to study genetic stability in the cryopreserved plant materials [46].
The embryonic tissues can be cryopreserved for future use or for germplasm conservation [47].6. Embryo cultureEmbryo culture is a type of plant tissue culture that is used to grow embryos from seeds and ovules in a nutrient medium.
In embryo culture, the plant develops directly from the embryo or indirectly through the formation of callus and then subsequent formation of shoots and roots. The technique has been developed to break seed dormancy, test the vitality of seeds, production of rare species and haploid plants [59, 119]. It is an effective technique that is employed to shorten the breeding cycle of plants by growing excised embryos and results in the reduction of long dormancy period of seeds.
Intra-varietal hybrids of an economically important energy plant “Jatropha” have been produced successfully with the specific objective of mass multiplication [62]. Somatic embryogenesis and plant regeneration has been carried out in embryo cultures of Jucara Palm for rapid cloning and improvement of selected individuals [60]. In addition, conservation of endangered species can also be attained by practicing embryo culture technique.
Recently a successful protocol has been developed for the in vitro propagation of Khayagrandifoliola by excising embryos from mature seeds [61]. This technique has an important application in forestry by offering a mean of propagation of elite individuals where the selection and improvement of natural population is difficult. Genetic transformationGenetic transformation is the most recent aspect of plant cell and tissue culture that provides the mean of transfer of genes with desirable trait into host plants and recovery of transgenic plants [63]. The technique has a great potential of genetic improvement of various crop plants by integrating in plant biotechnology and breeding programmes.
It has a promising role for the introduction of agronomically important traits such as increased yield, better quality and enhanced resistance to pests and diseases [64].Genetic transformation in plants can be achieved by either vector-mediated (indirect gene transfer) or vectorless (direct gene transfer) method [65].
Among vector dependant gene transfer methods, Agrobacterium-mediated genetic transformation is most widely used for the expression of foreign genes in plant cells. Successful introduction of agronomic traits in plants was achieved by using root explants for the genetic transformation [66].
Virus-based vectors offers an alternative way of stable and rapid transient protein expression in plant cells thus providing an efficient mean of recombinant protein production on large scale [67].Recently successful transgenic plants of Jatropha were obtained by direct DNA delivery to mature seed-derived shoot apices via particle bombardment method [68]. This technology has an important impact on the reduction of toxic substances in seeds [69] thus overcoming the obstacle of seed utilization in various industrial sector. Regeneration of disease or viral resistant plants is now achieved by employing genetic transformation technique. Researchers succeeded in developing transgenic plants of potato resistant to potato virus Y (PVY) which is a major threat to potato crop worldwide [70]. In addition, marker free transgenic plants of Petunia hybrida were produced using multi-auto-transformation (MAT) vector system. The plants exhibited high level of resistance to Botrytis cinerea,causal agent of gray mold [71].8.
Protoplast fusionSomatic hybridization is an important tool of plant breeding and crop improvement by the production of interspecific and intergeneric hybrids. The technique involves the fusion of protoplasts of two different genomes followed by the selection of desired somatic hybrid cells and regeneration of hybrid plants [48]. Protoplast fusion provides an efficient mean of gene transfer with desired trait from one species to another and has an increasing impact on crop improvement [3]. Somatic hybrids were produced by fusion of protoplasts from rice and ditch reed using electrofusion treatment for salt tolerance [49].In vitro fusion of protoplast opens a way of developing unique hybrid plants by overcoming the barriers of sexual incompatibility. The technique has been applicable in horticultural industry to create new hybrids with increased fruit yield and better resistance to diseases. Successful viable hybrid plants were obtained when protoplasts from citrus were fused with other related citrinae species [50].
The potential of somatic hybridization in important crop plants is best illustrated by the production of intergeneric hybrid plants among the members of Brassicaceae [51]. To resolvethe problem of loss of chromosomes and decreased regeneration capacity, successful protocol has been established for the production of somatic hybrid plants by using two types of wheat protoplast as recipient and protoplast of Haynaldiavillosa as a fusion donor. Haploid productionThe tissue culture techniques enable to produce homozygous plants in relatively short time period through the protoplast, anther and microspore cultures instead of conventional breeding [53]. Haploids are sterile plants having single set of chromosomes which are converted into homozygous diploids by spontaneous or induced chromosome doubling. The doubling of chromosomes restores the fertility of plants resulting in production of double haploids with potential to become pure breeding new cultivars [54]. The term androgenesis refers to the production of haploid plants from young pollen cells without undergoing fertilization. The haploidy technology has now become an integral part of plant breeding programs by speeding up the production of inbred lines [56] and overcoming the constraints of seed dormancy and embryo non-viability [57]. The technique has a remarkable use in genetic transformation by the production of haploid plants with induced resistance to various biotic and abiotic stresses. Introduction of genes with desired trait at haploid state followed by chromosome doubling led to the production of double haploids inbred wheat and drought tolerant plantswere attained successfully [58].10. Current and future status of plant tissue cultureThe past decades of plant cell biotechnology has evolved as a new era in the field of biotechnology, focusing on the production of a large number of secondary plant products.
During the second half of the last century the development of genetic engineering and molecular biology techniques allowed the appearance of improved and new agricultural products which have occupied an increasing demand in the productive systems of several countries worldwide [31, 32, 33, 34]. Nevertheless, these would have been impossible without the development of tissue culture techniques, which provided the tools for the introduction of genetic information into plant cells [35]. Nowadays, one of the most promising methods of producing proteins and other medicinal substances, such as antibodies and vaccines, is the use of transgenic plants [36].
Transgenic plants represent an economical alternative to fermentation-based production systems.
Plant-made vaccines or antibodies (plantibodies) are especially striking, as plants are free of human diseases, thus reducing screening costs for viruses and bacterial toxins. MicropropagationMicropropagation starts with the selection of plant tissues (explant) from a healthy, vigorous mother plant [15].Any part of the plant (leaf, apical meristem, bud and root) can be used as explant.


Stage I: Initiation stageIn this stage an explant is surface sterilized and transferred into nutrient medium.
The surface sterilization of explant in chemical solutions is an important step to remove contaminants with minimal damage to plant cells [17]. The most commonly used disinfectants are sodium hypochlorite [18, 19], calcium hypochlorite [20], ethanol [21] and mercuric chloride (HgCl2) [17]. Stage II: Multiplication stageThe aim of this phase is to increase the number of propagules [22]. Stage III: Rooting stageThe rooting stage may occur simultaneously in the same culture media used for multiplication of the explants. Hardening is done gradually from high to low humidity and from low light intensity to high light intensity. Somatic embryogenesis and organogenesisSomatic embryogenesis: is an in vitro method of plant regeneration widely used as an important biotechnological tool for sustained clonal propagation [23].
These somatic embryos can develop into whole plants without undergoing the process of sexual fertilization as done by zygotic embryos. There are various factors that affect the induction and development of somatic embryos in cultured cells.
A highly efficient protocol has been reported for somatic embryogenesis on grapevine [25] that showed higher plant regeneration sufficiently when the tissues were cultured in liquid medium. Plant growth regulators play an important role in the regeneration and proliferation of somatic embryos. Highest efficiency of embryonic callus was induced by culturing nodal stem segments of rose hybrids on medium supplemented with various PGR’s alone or in combination [26].
This embryonic callus showed high germination rate of somatic embryos when grown on abscisic acid (ABA) alone.
Somatic embryogenesis is not only a process of regenerating the plants for mass propagation but also regarded as a valuable tool for genetic manipulation.
The process can also be used to develop the plants that are resistant to various kinds of stresses [27] and to introduce the genes by genetic transformation [28]. A successful protocol has been developed for regeneration of cotton cultivars with resistance to Fusarium and Verticillium wilts [29]. Plant regeneration via organogenesis involves the callus production and differentiation of adventitious meristems into organs by altering the concentration of plant growth hormones in nutrient medium. Tissue culture in pharmaceuticalsPlant cell and tissue cultures hold great promise for controlled production of myriad of useful secondary metabolites [72].
Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures for the production of valuable therapeutic secondary metabolites [73]. In the search for alternatives to production of medicinal compounds from plants, biotechnological approaches, specifically plant tissue cultures, are found to have potential as a supplement to traditional agriculture in the industrial production of bioactive plant metabolites [74].
Exploration of the biosynthetic capabilities of various cell cultures has been carried out by a group of plant scientists and microbiologists in several countries during the last decade [75]. Cell suspension culture: Cell suspension culture systems are used now days for large scale culturing of plant cells from which secondary metabolites could be extracted.
A suspension culture is developed by transferring the relatively friable portion of the callus into liquid medium and is maintained under suitable conditions of aeration, agitation, light, temperature and other physical parameters [76]. Cell cultures cannot only yield defined standard phytochemicals in large volumes but also eliminate the presence of interfering compounds that occur in the field-grown plants [77]. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products [78].
The major advantage of the cell cultures include synthesis of bioactive secondary metabolites, running in controlled environment, independently from climate and soil conditions [79].A number of different types of bioreactors have been used for mass cultivation of plant cells.
The first commercial application of large scale cultivation of plant cells was carried out in stirred tank reactors of 200 liter and 750 liter capacities to produce shikonin by cell culture of Lithospermumerythrorhizon [80]. Cell of Catharanthusroseus, Dioscoreadeltoidea, Digitalis lanata, Panaxnotoginseng, Taxuswallichiana and Podophyllumhexandrum have been cultured in various bioreactors for the production of secondary plant products.A number of medicinally important alkaloids, anticancer drugs, recombinant proteins and food additives are produced in various cultures of plant cell and tissues. Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavanoids and amino acids [72, 81]. Some of these are now available commercially in the market for example shikonin and paclitaxel (Taxol).
Until now 20 different recombinant proteins have been produced in plant cell culture, including antibodies, enzymes, edible vaccines, growth factors and cytokines [73].
Advances in scale-up approaches and immobilization techniques contribute to a considerable increase in the number of applications of plant cell cultures for the production of compounds with a high added value. Hairy root culturesThe hairy root system based on inoculation with Agrobacterium rhizogenes has become popular in the last two decades as a method of producing secondary metabolites synthesized in plant roots [99]. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. High stability [101] and productivity features allow the exploitation of hairy roots as valuable biotechnological tool for the production of plant secondary metabolites [102]. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants [103]. Hairy root technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. Optimizing the composition of nutrients for hairy root cultures is critical to gain a high production of secondary metabolites [100]. Tissue culture facilities at QarshiindustriesPlant tissue culture Lab was established in 2004 with the objectives to raise endangered medicinal plant species and the plants difficult to raise through traditional methods for conservation and mass propagation. The protocols developed for The Moth Orchid, Tobacco, Honey Plant, Potato and Physic nut are presented as case studies.16.
Micropropagation of Phalaenopsis “The Moth Orchids”Orchids are usually grown for the beauty, exoticism and fragrance of their flowers. In addition, the desired characteristics of seedlings and uniformity are not attained.In vitro propagation studies of phalaenopsis “the moth orchids” had the objective to develop a protocol for plant regeneration from callus. Thus in vitro culture techniques are adopted for quick propagation of commercially important orchid species. The callus of phalaenopsis previously obtained from the mature orchid plant was used as explant source. For shoot regeneration and elongation, the callus was transferred to MS medium supplemented with BAP and GA3 at different concentrations. Further research work will focus on different potting medium compositions best suited for acclimatization of regenerated plants.As a high value crop, the mass production of orchids will provide a good opportunity of marketing locally as a good source of income.
Figure 3.Micropropagation of Orchids (a) callus culture (b) shoot regeneration (c) rooted plantlets17. Tissue culture of Tobacco (NicotianatabacumL.) Tobacco is an important crop of Pakistan which covers a large area under cultivation. Fresh leaves of the plants are processed to obtain an agricultural product that is commercially available in dried, cured and natural forms. PGH-01, PGH-02, PGH-04 and PGH-09 was carried out with the special objective of commercialization of tissue cultured plants to the farmers and industry. Callus induction and proliferation was carried out on MS medium supplemented with different concentrations of 2,4-D.
Case study 3In vitro propagation of Honey Plant (Stevia rebaudianaBertoni) The in vitro clonal propagation of Stevia rebaudiana was conducted by inoculatingseeds on MS medium [10] and placing under photoperiod of 16 hrs light and 8hrs dark in growth room. The seedlings with four nodes have beendivided into 0.5 cm pieces of nodal segments and used as explants. The rooted plantlets were acclimatized successfully and transferred to greenhouse under low light intensity. This protocol for in vitro clonal propagation of Stevia rebaudiana has been optimized for thelocal environment, as a consequence it will be helpful to establish and cultivate Steviarebaudiana for commercial scale production in various environmental conditions in Pakistan.



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