eChapters

Abstract

Various cropping systems and ecologies illustrate the positive use of chemicals both as fertilizers and pesticides and organic/biological sources within the frame work of Integrated Plant Nutrition and Plant Protection System (IPPS). While considering the organic/biological sources, green manures seem to be the best alternative that may largely suffice to the organic matter requirements of the soil under crop cultivation. Green manuring can be defined as a practice of ploughing or turning into the soil undecomposed green plant tissues for the purpose of improving physical structure as well as fertility of the soil. “Green manure” refers to fresh plant matter, which is added to the soil largely for supplying the nutrients contained in its biomass. The agricultural practices consisting of growing and ploughing under a green crop or addition of a green organic matter in the form of leaves and other succulent parts from outside to improve soil productivity is known as green manuring. In this review, advantages of green manuring, decomposition of green manure,   process of decomposition, factors affecting decomposition and effects of green manuring have been discussed in detail.

Abstract

Production technology has been worked out for crops and several inputs have been recommended which are to be applied in proper quantity at most suitable time to get the maximum return. Nutrition in the form of fertilizers is a must. However, injudicious use of agro-chemicals has resulted in diseases called ‘iatrogenic plant diseases’. Under the circumstances it is important to know the relationship between soil nutrients and plant pathogens which are causing the diseases of economic importance. Author has given adequate emphasis to micronutrients as at a very minor quantity they reduce the disease to an acceptable level. He has also discussed in detail about all the major and minor nutrients required by the plants and how their deficiency or over doses affect the plant pathogens and in turn the diseases.

Keywords:  Soil nutrients, plant pathogens, diseases, relationship

Abstract
 
Much attention has been paid to mycorrhza as a tool for improving the growth and health of plants.  The use of mycorrhiza in agricultureal, horticultural and forestry has been described. The significance of AM fungi in sustainable agriculture has been a subject of growing interest for the past several decades. In sustainable agriculture it is imperative to maximize benefits with low input costs. The fact remains that stable and lower human population is an integral component of sustainable agriculture and more so in an Indian context. Therefore, it is imperative to collect further information on the different aspects of AM fungal symbiosis as discussed in this article so as to utilize this plant microbe symbiotic system for the increased production and productivity in a sustainable manner. This may become possible when the integrated approach is made to study AM fungi right from the isolation of AM fungal spores to the high quality inoculum production and its application in the field. It is also imperative to stimulate new mutualism between mycorrhizal scientists and ecologists. It this regard, recent advancement made in the molecular techniques should be encouraged in order to obtain more authentic results. Adequate field testing of mycorrhizal inoculation and commercial exploitation of the potential benefits of mycorrhiza still rests on the development of suitable technology for mycorrhizal inoculum preparation.

Abstract

Higher plants can be exploited for the discovery of new bioactive products that could serve as lead compounds in pesticide development because of their novel modes-of-action. Different secondary metabolites of higher plants have been reported to exhibit efficacy as fungitoxicants as well as mycotoxin inhibitors. Pest controls using the botanicals are safer to the user and the environment because they break down into harmless compounds within hours or days in the presence of sunlight. The article presents different plant products found efficacious as antimicrobial and inhibitory to mycotoxin secretions by fungi. Their prospect as safe alternatives to synthetic antimicrobials in plant protection has been discussed.

Abstract

Eight species including 1 variety, 2 macro and 6 micro-lichens from Garhwal Himalaya, c 1500-2700 m, have been found to glow and emit fluorescent light, when exposed to long wave-length (254 nm) ultra-violet radiataions. Taxonomy and chemistry of all these species have been critically evaluated. The recorded taxa belong to families Physciaceae (4 species); Pertusariaceae, Rhizocarpaceae (1 species each) and Lichen imperfecti (2 species). The reported taxa are: Buellia hemispherica Singh and Awasthi, B. sub-glaziouana Singh and Awasthi; Pertusaria pertusa (Weig.) Tuck., Pyxine berteriana (Fee) Imsh. var. himalayensis Awasthi; P. cocoes (Swartz) Nyl., Rhizocarpon geographicum (L.) DC.; Lepraria taxonomic species I and II.

Abstract

C. albicans is a diploid, dimorphic yeast producing three morphologic forms: yeast cells, pseudohyphae and true hyphae. It is an opportunistic pathogen causing various types of candidiasis which are on the increase around the globe. It is a polymorphic yeast with its genome organized in eight diploid chromosome with its genome size ranging between 14.3 and 16 Mb having 5,733 to 6, 318 genes. It has two mating-type like (MTL) alleles that control the mating behavior. In its genetic makeup it resembles with Saccharomyces cerevisiae hence classified in the Saccharomycetales. The chromosome number from tetraploid to diploid is brought about by the parasexual mechanism. Keeping in view the increase of incidence of candidiasis due to C. albicans around the globe, it is important to identify the causative organisms to the species level correctly. Morphological, biochemical, germ tube test and growth on different media are often used for identifying isolated yeasts which sometimes takes days to week to develop in culture hence, rapid identification of Candida species in clinical laboratory is becoming increasingly important. Molecular techniques utilizing amplification of target DNA or PCR amplification of conserved regions of the genome and sequencing of the resulting PCR product and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA, provides quick and precise methods for diagnostics or, identification of Candida species.

Abstract

Agroforestry is an intensive land use management system that integrates trees, shrubs, crops, etc. on a landscape level to achieve optimum benefits. Agroforestry is not only concerned with beneficial effects of one component on another, but it also involves the manipulations of negative effects to minimize their influence on the productivity of the overall system. In an agroforestry, trees and crops compete inevitably for light, nutrients, moisture and other resources. The interactions between tree and crop roots can also have an effect on soil organisms, which play an essential role in the functioning and productivity of agroforesty systems. Arbuscular mycorrhizal fungi (AMF) are one of the most important components of the soil biota in monoculture and agroforestry systems. AM fungi can rehabilitate degraded lands subjected to agroforestry systems and common mycorrhizal network may further enhance the benefits of agroforestry through vertical niche expansion of AMF. However, management practices of agroforestry affect mycorrhization, AMF community composition and diversity of AM fungi. This review focuses on AMF diversity, which plays a significant role in agroforestry systems.

Abstract

A field study of seven arid districts of Rajasthan was undertaken to evaluate the occurrence of selected tree species and arbuscular mycorrhizal fungal (AMF) associations with them. Five genera were identified in the rhizosphere of these selected tree species. A high diversity of AMF was observed which varied between different host tree species. Among the five genera, Glomus occurred most frequently, with four species, Acaulospora and Gigaspora were found with three species, while Entrophospora and Scutellospora was detected with two species. Gigaspora margarita, Gigaspora rosea, Glomus deserticola, Glomus constrictum, Glomus fasciculatum, Glomus mosseae and Scutellospora calospora were the most dominant species. The AMF spore density was not clearly affected by the host tree suggesting that biotic factors may be relatively less important than abiotic/edaphic factors for establishing population pattern. The spore density of AMF had a strong positive correlation with soil pH and organic carbon content and a negative correlation with Olsen’s P content of the soil. The association with AMF of these tree species native to the harsh environmental conditions of the Indian Thar Desert may play a significant role in the re-establishment and conservation of these multipurpose desert tree species.

Abstract

The ability of life to dwell in extreme environments on our mother planet earth right from a thirsty desert environment to highly water saturated oceans, gave the scientists a new area to work in during the 1990’s. The discovery of microbial life which had the ability to survive in conditions which were highly inhospitable for the normal life gave rise to a revolution in the field of science and technology. Extremophiles are one of the most primitive forms of life on earth since they have accustomed themselves to the present day harsh conditions compared to the normal environment. The last few years have seen a massive increase in their discovery, since the areas which once were thought barren by the scientists are now found inhabited by the microbial life. The extremophiles endure conditions right from extreme temperatures, pressure, salt concentration to radiations, metal tolerance, pH etc. During the past few years the industrial applications associated with the extremophiles and their products have led to their intensive exploration. Keeping in view the present and the future scenario, extensive efforts are still required in order to exploit the extreme environments for extremophiles and their products.

Abstract

Today more than 70% of energy is generated by coal-based thermal power plants. Indian coal contains about 40% ash, these power plants generate enormous amounts of fly ash, which is dumped in the nearby areas. This creates not only economy constraints but also imposes serious environmental problems, which are the potential hazards to the ecosystem. However, recent attempts in India and abroad are being made for biological association of fly ash e.g. in soil improvement, in wasteland management and also as a source of essential plant nutrients for nutrient deficient soil in terrestrial eco-system and agro-ecosystem. Fly ash has been found to have manifold advantages in agriculture especially in modification of soil texture and bulk density, in optimization of soil pH, in improvement of water holding capacity of soil, in the improvement of yield as a micronutrient supplement to soil and in creation of conducive condition for better plant growth. In this article utilization of fly ash along with VAM fungi and other microbes especially nitrogen fixers and phosphate solubilizers in removing the constraints of salt affected soils has been discussed.

Abstract

Extensive occurrence of alkaline soils has been reported from the Indo-Gangetic plains of Northern India, which is commonly known as ‘usar’ or ‘reh’. High alkalinity and high exchangeable sodium percentage (ESP) of such soil render it inhospitable for normal crop production. Research efforts over the past few decades have resulted in evolving a number of practices for the reclamation and management of such soils. However, application of biological inputs in combination with some moderately salt tolerant plant species seems to be a better option. AM fungi is well known to enhance the ability of plants to cope with environmental stresses generally prevalent in the degraded ecosystems and stress conditions by providing a number of nutritional and non-nutritional benefits. Since isolates of AM fungi differ in their tolerance to adverse physical and chemical conditions in soil and are better adapted to their native stressed microhabitat, selection of appropriate native AMF isolate is necessary for any large scale reclamation program. Taxonomic diversity of AM fungi in the alkaline soils of Upper Gangetic plains of district Allahabad and adjoining areas has been investigated and it was found that such soils have a detrimental effect on the AM spore population, distribution and diversity. Maximum spore population occurred in winter and minimum in summer. Glomus followed by Acaulospora was the most dominant genus. Acaulospora scrobiculata and Glomus fasciculatum were the most frequent species followed by Glomus mosseae and Glomus aggregatum. Glomus fasciculatum was the most abundant species followed by Glomus mosseae and Acaulospora longula.

Abstract

Microorganisms that harbour in the root region are the most befitting candidates for use as biocontrol agents. The rhizosphere provides the frontline defense for root against attack by pathogens. Pathogens face a complicated phenomena of antagonism from roots. They also compete with each other for site of colonization on the root surface and plant nutrients. The ideal biocontrol agent introduces and /or promotes the antagonists only, whenever required and are most effective in minimizing the wasteful application of inoculum to non-targets. Many genera belonging to bacteria, fungi, actinomycetes and viruses are used as biocontrol agents to combat several important plant diseases. The present review focuses on the use of bacterial antagonists ,particularly rhizobacteria, as biocontrol agents of fungal diseases of plants , their mechanism of action, molecular and genetic basis of antagonistic property and possible genetic manipulations to bring about desirable changes. 

Abstract

The traditional forage production systems and grazing lands are under extreme pressure due to the population growth of both human and livestock, decreasing area and inputs. In the central India, especially the Bundelkhand region, the soil is highly weathered, degraded and have low inherent nutrient, which was further worsened due to use of chemical fertilizers and pesticides, for enhancing crop production to meet the escalating demand of the population.

Abstract

Photosynthetic blooms of toxin producing marine algae are often referred to as “red tides”. Proliferations of freshwater toxin-producing cyanobacteria are simply called “cyanobacterial blooms” or “toxic algal blooms.” Biotoxins of cyanobacteria are water-soluble and heat stable and they are released upon aging or lysis of the cells. The primary types of cyanobacterial biotoxins involves three types of toxins: hepatotoxins (microcystin; cylindrospermopsin and nodularin), which are taken up by the liver and cause weakness and anorexia (loss of appetite); neurotoxins (usually anatoxin and saxitoxin), which effect the nervous system and dermatotoxins (aplysiatoxin and lyngbyatoxin, LPS), which cause skin and mucous irritations upon contact. Unlike freshwater toxic algal blooms, marine algal toxins become a problem primarily because they may concentrate in shellfish and fish that are subsequently eaten by humans, causing syndromes known as paralytic shellfish poisoning/PSP (paralysis due to saxitoxin and gonyaulatoxins), diarrhetic shellfish poisoning/DSP (diarrhea due to okadaic acid) , amnesic shellfish poisoning /ASP(memory disturbances due to domoic acid), neurotoxic shellfish poisoning/NSP (paresthesia due to brevetoxin; also bronchial spasms). Coral reef fishes are the vectors of another algal poisoning, the ciguatera fish poisoning/CFP (paresthesia, warm-cold sense inversion etc., due to ciguatoxins and maitotoxin).

Abstract

The word “mycorrhiza” literally means “fungus root” to describe the mutualistic association between roots of higher plants and certain fungi. Arbuscular mycorrhizae (AM) are the most common and widely occurring of all the mycorrhizal associations and have great economic significance. They belong to the phylum Glomeromycota. Mycorrhizal associations are potential factors in determining the diversity in ecosystems. They can probably modify the structure and functioning of a plant community in a complex and unpredictable way. Currently there is considerable resistance against the use of chemical pesticides and fertilizers, because of their hazardous influence on the environment, and on soil, plant, animal, and human health. Hence, use of biofertilizers and biocontrol agents are recommended in practical agriculture/horticulture/forestry.

Abstract

Ectomycorrhizal symbionts are important natural resources to revive degraded and deforested areas. Artificial inoculum of these mycobionts improves the growth of tree seedlings in nutrient poor soils due to the formation of extensive network of fungal mycelium responsible for the transfer of water and nutrients to the plants. Seedlings of tree species like, Pinus insularis, Quercus griffithii and Schima wallichii with ectomycorrhizae formed by Suillus bovinus, Boletus edulis and Scleroderma citrinum were studied in field condition. The study was conducted at two different stages of degraded sites. Results of the study indicated that seedlings with ectomycorrhizae were able to generate numerous lateral roots of greater length and thereby utilize water and nutrients more effectively than non- mycorrhizal ones The degree of mycorrhizal development varied in different host species with different fungal species. There was a strong relation between the colonization by mycorrhiza and the survival of seedlings. The study has suggested that mycorrhiza has a potential in reclamation of soil and reforestation of degraded land. S. bovinus., B. edulis and S. citrinum successfully colonized and promoted the growth of P. insulates Q. griffithii and S. wallichii respectively in reforestation programmes of degraded lands of Nagaland.

Fruits and vegetables are very important food commodities. India has become the largest producer of fruits and the second largest producer of vegetables. Though the production of fruits and vegetables make us believe in our strength for self-sufficiency, a significant qualitative and quantitative loss occur in the produce from harvest till consumption.  For self sufficiency and also for processing, export and to meet additional requirements, a lot of emphasis need to be given to reduce post harvest losses, besides increasing  production and productivity of horticultural produces. Processing plays an important role in  conservation and effective utilisation of these perishable commodities. However, only less than 2% of total production of fruits and vegetables are processed.  The new industrial policy has placed this sector in the list of high priority areas.

Structure

Structural unit of edible portion of most fruits and vegetables is the parenchyma cells and makes up bulk of cells. A parenchyma cell is rather thin walled and may be polygonal or cubical in shape.  Walls of parenchyma cells in young plants are composed almost entirely of fibrils of cellulose. In parenchyma cells of plants actively metabolising protoplast portion represents only about 5% of total cell volume. This protoplast is rather film like and is pressed against cell wall by large water filled central vacuole.  Protoplast has inner and outer semi permeable membrane layers between which is confined cytoplasm and its nucleus. 

Maturity at harvest is the most important determinant of storage life and final fruit quality. Maturity indices are determined for many fruit, vegetable and floral crops. Harvesting crops at proper maturity allows handlers to begin their work with the best possible quality produce. Produce harvested too early may lack flavour and may not ripen properly, while produce harvested too late may be fibrous or over ripe.
 
Almost all fruits reach their best eating quality when allowed to ripen on the tree or plant (exceptions e.g., pears, avocados, and bananas). In general, fruits become sweeter, more colourful, and softer as they mature. A few fruits are usually picked mature but unripe so that they withstand post harvest handling system when shipped long distances.  Most of  currently used maturity evaluation is based on a compromise between those methods that would ensure the best eating quality and those that provide flexibility in transportation and marketing.  Optimum harvesting stage depends upon type of fruit or vegetable and their final use. Such a division is usually based on their classification according to physiology, environmental conditions, ripening bahaviour and nutritional aspect. Some of the classifications, important for their harvesting and storage are briefed in  subsequent sections.

Various systems were developed to take produce from field to consumer and selection of a particular method depends on several factors, including perishability and value of crop.  One method is to harvest the crop and take it to a packhouse for various unit operations like grading, cleaning, post-grading treatments, and packing for transport and marketing. When deciding upon where to locate a packinghouse, access to field and market point, adequate space for vehicles to enter and leave  packinghouse and ease of access to labor will all be considered.
 
In the simplest packinghouse, produce is delivered in picking containers, immediately after harvest, directly to packers.  Packers then sort, grade, size and pack the produce directly into appropriate transport containers. In this case, each worker must be aware of produce defects, grade and size requirements, and packing methods.

Storage is one of the most important aspects of post harvest handling of fruits and vegetables. The main objective of storage is to extend storage life of fruits and vegetables and increase their period of availability. A substantial quantity of fruits go waste in India due to lack of proper storage facilities. Storage of fruits and vegetables is practiced for various reasons. Proper post harvest handling of fresh horticultural produce helps in extending  period of availability of wide range of  fruits and vegetables round the year, prevention of seasonal market gluts, orderly marketing, better return to grower and  preservation of  nutritional qualities. The   long  term  storage  of  fruits  and  vegetables is expensive and requires high level of technical  knowledge of crop. However, an orderly marketing reduces storage period considerably. 

Processing of fruits  and vegetables into various products which could be preserved for a long time and add to value of product using proper cost effective technology will be a viable tool for improving   economic status of  farmer as well as our country. Modern methods of fruit and vegetable preservation are  broadly classified as follows:

Drying is the oldest known method of preserving food. Drying or dehydration is removal of majority of water contained in fruit or vegetable. Although preservation is the primary reason for dehydration, it also lowers cost of packaging, storing and transportation by reducing both weight and volume of final product. In addition, dried foods add variety to our diets. In the process of drying, sufficient moisture is removed to protect product  from  spoilage. Processing should be done in such a way that food value, natural flavour and characteristic cooking quality of fresh material are retained after drying.

The technique of preserving fruit by the use of low temperature dates from the eighteenth century with the first use of ice house in 1750. Later in 1800s commercial cold storages have come up for the storage of perishables like meat, dairy and fruit products.  The main change took place in the cold store in recent years has been the introduction of multi temperature chambers with humidity control for specific products. Cold preservation can be either refrigeration or freezing. Household refrigerators usually run at 4-7^ºC. Chilling or cold storage usually uses a slightly lower temperature on the basis of the food to be refrigerated. In freezing a temperature of -18^ºC or below is used. Chilling will preserve perishable foods for days or weeks and freezing will preserve foods for months or even years.

Fruit beverages

Fruit beverages are highly refreshing, thirst quenching, appetizing, easily digestible and nutritionally far superior to synthetic drinks. The fruit beverages includes natural and sweetened juices, squash, syrup, cordial, nectar, Ready to Serve (RTS) beverages, fruit juice concentrate and citrus juice barley water. Fruits most commonly used to prepare beverages are oranges, lime, pineapple, grapes, cashew fruit, mango, jack fruit, apple etc. To prepare fruit beverages select fresh, fully ripe, tart, juicy and seasonal fruits.

Fermentation was known to mankind from time immemorial. But, development of biochemical principles of  fermentation  was originated by  Lavoisier in France in 1789 by way of analysing chemical composition of  sugar and its fermentation products such as ethanol, carbon dioxide and  acetic acid. Many products are produced by fermentation process. In fermentation, controlled action of selected microorganisms is used to alter  texture of foods, preserve foods by production of acids or alcohol or to produce fine flavours and aromas which increase value of raw materials.

Minimally processed fruits and vegetables are products that have the attributes of convenience and fresh like quality. Their forms vary widely, depending on nature of unprocessed commodity and how it is normally consumed. In many cases, minimally processed product is raw and cells of tissues are alive, but these characteristics are  not regarded as essential. It is important that products have fresh-like character, regardless of   state of cells.  Minimally processed products for both retail and food service applications have increasingly appeared in market place recently.  In coming years, it is commonly perceived that this industry will have unprecedented growth.

Packaging is an integral part of fruit and vegetable processing. It has  major influence on storage life and on marketability of fresh as well as processed products. Packaging fulfills several functions including containment, facilitating transportation, protection of fruit/vegetable from further damage, protection of environment from contents of package (for example, if the contents are dirty), marketing, product advertising, and stock control.

Transportation and distribution of fruits and vegetables are important stages   of post-harvest loss. In India,  transportation of perishable commodities is in the most precarious stage. For local market, the produce is brought either by bullock cart or tractor trollies. The long distance transportation is mainly by rails and trucks, which is very costly. The basic reason for preference to road transportation is that it takes short transit period.

Quality is an increasingly important factor in the production and marketing of biological products. Consumers are becoming more discerning as their affluence increases, and would be suppliers of products must meet these demands if they are to maintain or increase market share. The term ‘quality’ is one of the most defined terms in use in food industry today. Quality may be defined as ‘The totality of features and characteristics of a product that bear on its ability to satisfy a given need’. The first part, ‘The totality of features and characteristics of a product…..’ concerns objective factors related to the product.  The second part, ‘…… to satisfy a given need’, concerns subjective factors related to user or consumer of goods.

Practical have a significant role in the study of any theoretical subject. Whatever a student learns in theory, he verifies it by actual doing with his own hands.  Thus, these reduce any doubts and misbelieve from his mind. These verifications by actually doing the things are performed in the laboratories.  A laboratory is a place with the basic and essential equipments and adequate facilities for students and teachers to perform and observe experiments.  Plants will continue to provide novel products as well as chemical models for new drugs in the coming centuries.  The arrival of chemical analysis and the characterization of molecular structures have helped in precisely identifying these plants and correlating them with their activity under controlled experimentation.

Year    Contributor    Description
1756    Duharmel du Monceau H.L.    Discovered callus formation in decorticated elm tree. 
1839    Schwann, T.H.    Proposed the view that each living cell of a multicellular organism would be capable of developing independently if provided with proper external conditions.
1853    Trecul, A.    Performed experiment on callus formation by decorticated trees such as Robinia, Pawlonia and Ulmus.

A tissue culture laboratory can be placed in any geographical area. The internal controlled environmental conditions permit isolation with a minimum of external influence. To decide where to build the tissue culture laboratory, the following factors must be taken into consideration:

Common rules for fine laboratory techniques call for:

    ·    A laboratory should have a record and a complete up to-date record of all the equipments and their operating manual.
    ·    A laboratory should have a record and detailed up to-date record of all the chemicals including the name of manufacturer with their     grade and arranged alphabetically in specific area
    ·    Strong acid and bases in safe area.
    ·    Chloroform, alcohol which are volatile or toxic must be stored in fume hood.

One of the most essential factors governing the growth and morphogenesis of plant tissues in culture is the composition of the culture medium. The basic nutrient requirements of cultured plant cells are very similar to those of whole plants. The basic nutritional requirements of cultured plant cells as well as plants are very similar. However, the nutritional composition varies according to the cells, tissues, organs and protoplasts and also with respect to particular plant species.  The appropriate composition of the medium largely determines the success of the culture. A wide variety of salt mixtures  have been reported in various media. A nutrient medium is defined by its mineral salt composition, carbon source, vitamins, plant growth regulators (PGR) and other organic supplements. When referring to a particular medium, the intention is to identify only the salt composition unless otherwise specified. Any number and concentration of amino acids, vitamins, growth regulators and organic supplements can be added in an infinite variety of compositions to a given salt composition in order to achieve the desired product.

Sterilization Techniques

Sterilization: All the materials, e.g., vessels, instruments, medium, explants, etc., used in culture work must be freed from microbes. This can be achieved by one of the following approaches:

Explant

The tissue obtained from plant to be cultured is known as Explant. It may include the parts of shoots, leaves, stems, roots, flowers and callus.

Maintenance of Aseptic Environment

All culture vessels, media and instruments used in handling tissues as well as the explants must be sterilized. The importance is to keep the air surface and floor free of dust. All operations are carried out in laminar air-flow, a sterile cabinet.In general, the methods of elimination of these sources of infection can be grouped under different categories of sterilization procedures: 

Modes of Culture 

Callus is a mass of undifferentiated plant tissues formed from plant cells or tissue cuttings when grown in culture. Tissues and cells of plant cultured in a liquid medium aerated by agitation grow as suspension of single cells and cell clumps. For growth, the cells require to divide, whereas, the cells breaking up from explant are mature, nondividing. Hence, the differentiated tissue undergoes modifications to become meristematic. This phenomenon of a mature cell reverting back to meristematic state to form undifferentiated callus tissue is called dedifferentiation.

Micropropagation /Clonal propagation

Micropropagation or clonal propagation is the growing of plants from meristematic tissue or somatic cells of superior plants on nutrient suitable media under controlled aseptic physical conditions. Sexually propagated plants (through generation of seeds) demonstrate a high amount of heterogeneity since their seed progenies are not true to type whereas asexual reproduction (by multiplication of vegetative parts) gives rise to genetically identical copies of parent plant. Thus, it permits perpetuation of the parental characters of the cultivars among the plants resulting from micropropagation. Micropropagation proves useful for propagation of: 

Experiment - 1
 
Aim: Micropropagation of Tobacco plant by leaf disc culture

Principle: The totipotency of the plant cells and tissues form the basis for in vitro cloning i.e., generation or multiplication of genetically identical plants in in vitro culture. The ability to propagate new plants from a cells or tissues of parent plant has many interesting possibilities.

A. Anther and Pollen Culture (Production of Haploid Plants)

Haploid plants possess a single set of chromosomes (gametophytic no of chromosomes i.e. n) in the sporophyte in contrast to diploids which contain two sets of chromosomes (2n). The existence of haploid plants was reported by Bergner (1921) in Datura stramonium. Guha and Maheswari (1964) reported the direct development of haploid embryos and plantlets from microspores of Datura inoxia by the cultures of excised anthers. After this, haploid plants have been produced via anther culture in more than 170 species. The anther culture technique is useful in haploid production.

The ultimate success of commercial propagation depends on the capacity to transfer plants out of culture on a huge range, at low cost and with high survival rates. The plants multiplied in vitro are exposed to a unique set of growth conditions (high levels of inorganic and organic nutrients, growth regulators, sucrose as carbon source, high humidity, low light, poor gaseous exchange) which may support rapid growth and multiplication but also induce structural and physiological abnormalities in the plants, rendering them unfit for survival under in vivo conditions. Poor control of water loss and heterotrophic mode of nutrition are main cause of deficient growth of in vitro plants.  Hence, gradual acclimatization is necessary for these plants to survive transition from culture to the greenhouse or field. During acclimatization the in vitro formed leaves do not recover but the plant develops normal leaves and functional roots. Once roots are well formed the plants are ready to be transferred into soil.The procedure of acclimatization of regenerated plantlets are: 

A. Somatic Embryogenesis
 
In plant tissue culture, the developmental pathway of various well organised, small embryoids resembling the zygotic embryos form the embryogenic potential somatic cell of callus tissue or cells of suspension cultures is known as somatic embryogenesis. The capability of somatic cell of a culture to produce embryoids is known as embryogenic potential.

Embryoid: Embryoid is a small, well organised structure comparable to the sexual embryo, which is produced in tissue culture of dividing embryogenic potential somatic cells.

Isolation of Protoplasts

Protoplast is the living material of the cell however an isolated protoplast is the cell from which the  cell wall is removed. In plant breeding programme many desirable combination of characters could not be transmitted through the conventional method of genetic manipulation. Higher plants that could lead to the genetic process involving fusion between the subsequent developments of a product to a hybrid plant is known as somatic hybridization. Plant protoplasts can be isolated from cells by two methods:

Experiment - 1

Aim: Isolation of plant genomic DNA through modified CTAB method

Principle: Genomic DNA is extracted from plant material requires cell lysis, inactivation of cellular nucleases and separation of the desired genomic DNA from cellular debris. Ideal lysis method is rigorous enough to disrupt the complex starting material (plant tissue), yet gentle enough to preserve the target nucleic acid. The cetyltrimethylammonium bromide (CTAB) procedure is sufficient for the extraction and purification of DNA from plants and plant derived food stuff.  This is particularly suitable for the elimination of polysaccharides and polyphenolic compounds otherwise affecting the DNA purity and hence quality.

Experiment- 1

Aim: Direct DNA delivery to Plant by Particle Bombardment
 
In direct gene transfer method, the foreign gene of interest is delivered into the host plant cell without the help of vector. Klein and Coworkers (1987) evolved a procedure through which the delivery of DNA into cells of intact plant organs or cultured cells is done by a process called Projectile Bombardment. The microprojection (Small and high density particles) are accelerated to high velocity by particle gun apperatus. These particles with very high kinetic energy penetrate the cells and membranes and transmit foreign desired DNA inside of the bombardment cells.

Experiment - 1 
Aim: Mobilization of recombinant Ti plasmid (with gene of interest) from  
(E. coli) to an Agrobacterium tumefaciens strain. 

Principle: Due to ease of cloning and harvesting plasmids, E. coli is most popular for maintenance of plant transformation vectors. Consequently, the plant transformation vector with cloned gene of interest is mobilized to recipient Agrobacterium strain harbouring vir plasmid. Plasmid are closed, circular DNA molecules that are able for autonomous replication with a host cell. 

Experiment - 1
 
Aim: Molecular analysis of putative transformed plants by Polymerase Chain Reaction

The expression of transgenes are not identified at that time but it can be achieved through DNA analysis of that plant. The presence of transgene are generally identified of PCR-based detection followed by gel electrophoresis and comparison with standard samples. The main achievement of PCR-based detection is that it is very sensitive. The Polymerase Chain Reaction (PCR) provides a rapid and sensitive method to amplify a specific gene sequence. 

Blotting is the procedure through which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. We can identify inherited disease, infectious agent present in the sample by using blotting technique. It is also used to mapping restriction sites in single copy gene.

Electrophoresis is a method used to separate charged particles from one another based on differences in their migration speed. In the course of electrophoresis, two electrodes (typically made of an inert metal, e.g. platinum) are immersed in two separate buffer chambers. Gel electrophoresis is a technique used to separate molecules based on physical characteristics such as size, shape, or isoelectric point. A gel is composed of agarose or polyacrylamide. Agarose is used for separating DNA fragments in size range of 100bp to 20kb. Polyacrylamide is preferred for smaller DNA fragments. Separation is achieved by moving the negatively charged nucleic acid molecules through agarose matrix with an electric field. Rate of migration of DNA molecules is inversely proportional to their molecular weight. Smaller molecules move faster than larger ones. But conformation of the DNA molecule is also an important factor. Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds such as: supercoiled plasmid DNA will move fastest followed by linearised DNA, while open circular DNA will move at the slowest pace. To visualise DNA, gels are stained with ethidium bromide which intercalates in DNA and fluorescse under UV.