eChapters

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:

Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation.

Introduction

The isolation of mutants is often critical to the study of cellular process, especially when such studies are performed at the molecular level. The phenotype of a particular mutant is attributable to the loss of a particular cellular function; the loss of that function is actually attributable to a mutation in a gene necessary for that function. In this experiment we will isolate auxotrophic mutant which can grow only if provided with specific amino acid. For example arg^- mutant have lost the catalytic function of an enzyme involved in arginine biosynthesis due to mutation in the structural gene that encodes the enzyme.

Mutagenic Agent

Mutation is an alteration in the coding sequence of a gene. Mutation occur spontaneously at a frequency of about 10^-8 per bacterial cell per generation. The rate of mutation can be increased with mutagenic agents. Mutants are strains with any structural alteration of their DNA that gives rise to a mutant phenotype. Mutagens are chemical agents (e.g. nitorsoguanidine, 5 bromouracil), physical (e.g.UV) and biological agents (e.g. trnasposons) that cause mutation to occur.

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.

Introduction

Mutation refers to any heritable change in the base sequence of a DNA molecule. A mutagen is a physical of chemical agent that causes mutations to occur (or increase the frequency of their occurrence). Mutagenesis is the process of producing a mutation. If the mutation occurs in nature without the addition of a mutagen, it is referred as a spontaneous mutation, and the resulting mutants are spontaneous mutants. If it caused by a mutagen, the process is induced mutagenesis.

Ultraviolet light is a fairly potent mutagen. The main DNA lesions are two chemically different types of covalent dimers: cyclobutane-pyrimidine dimers and pyrimidine dimers. The pyrimidine dimers only account for 20% of the total dimers but are correspondingly more mutagenic. In E. coli the number of mutants induced by ultraviolet (UV) light can be reduced by exposure to visible light (photoreactivation), which implicates cyclobutane pyrimidine dimers since dimers can not be photoreactivated.

Aromatic amino acid of proteins, and purine as well as and pyrimidine bases of nucleic acid absorb ultra violet radiation. Pyridine bases, particularly thymine of DNA is the main target compound involved in bactericidal action of ultra violet radiation. The energy absorbed by thymine causes it to under go a photochemical reaction with adjacent ( adjoining) thymine molecule in same DNA strand. DNA replication is blocked because the formation of thymine: thymine (T:T) dimmers. As the dosage increase, cytosine-thymine (C:T) to (C:C) cytosine-cytosine dimmers can be found to add the lethality of ultra violate radiation.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

RNA is transcriptional product of DNA. The total RNA includes three classes, rRNA, tRNA and mRNA. The ribonucleic acids exists as ribonucleo protein particles in intact cells. The ribonucleo protein complex is dissociated by SDS in to RNA and protein deproutinised by phenol and the free RNA left in the aqueous solution is precipitated in cold by adding alcohol. 

Introduction

This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors.

Principle

The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

What is DNA

DNA is a simple molecule

DNA is negatively charged

DNA has lots of ring structures; ring structures are generally quite hydrophobic.

DNA is a very robust molecule.

DNA work has lots of toys.

Although it can be argued that due to its overall trendiness, they have been a lot of recent developments in making it more fun to work with. As a molecule, it is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get rid of.

Plasmid is a double stranded, self replicating, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. The sizes of plasmids range from 1-1000 kbps. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation.

There are various methods available for isolation of high molecular weight genomic DNA from plants. The basic steps involved in all these methods are removal of cell wall, nuclear membrane, proteins, lipids, carbohydrates, cell debris, RNA without affecting the integrity of DNA. The two major factors that affect DNA integrity are action of endogenous nucleases and impact of mechanical shearing.

ABSTRACT

This paper discusses IPR concepts in the light of Indian culture and issues emanating from enforcement of those concepts in developing countries such as India.

INTRODUCTION

The adage “Knowledge is wealth” suits the present era well than any other period. India is known for its innovative thoughts since ages. However, it never monopolized the knowledge it possessed. In India, the knowledge of a person is thought to be utilized for the benefit of the society as an obligation of the knowledge holder. Does this mindset of Universal humanity (Vasudevo Kudumbagam) and common good (Sarva Sugino Bavandu) would benefit us in the materialistic society? This paper is an attempt to answer this query.

Genetically engineered crops refer to alterations in the genetic makeup of the crop by introgression new traits such as herbicide tolerance, virus resistance, drought, flood and frost resistance, delay in maturation time of the crop and increased crop yield. They can be made resistant to pests and diseases which can significantly reduce the consumption of insecticide. Biodiversity is the feedstock for biotechnology industries. The issues relating to the Genetically Modified (GM) Foods have generated intense public debate in many parts of the world. Even though the issues under debate include the costs and benefits of the GM crops and the inherent safety concerns, the outcome of the debate differs from country to country, depending on its geographical location, strength and resilience of the farm sector, attitudes of people towards food, and so on (Anonymous, 2009). In India also, this debate has engaged the attention not only of the Government but also of the farming community and the civil society.

Ixora is an evergreen flowering shrub valued for its attractive clusters of brightly coloured flowers and its suitability for loose flower production, religious offerings, garlands and landscaping. The crop performs well under tropical and subtropical conditions and is appreciated for its extended flowering period. This chapter presents the botanical characteristics, origin, distribution, genetics and important varieties of Ixora. It discusses suitable climate and soil conditions, propagation, land preparation, planting, nutrient management, irrigation and cultural practices. Special attention is given to pruning and canopy management for improving flowering. The chapter also covers important insect pests and diseases, harvesting, postharvest handling and yield, providing practical guidance for commercial Ixora production.

Jasmine is one of India’s most important fragrant loose flower crops, valued for garlands, Veni, Gajra, religious ceremonies, festivals and perfume production. Flowers and buds are also important raw materials for jasmine oil, cosmetics, soaps and other fragrance products. The chapter describes jasmine’s origin, distribution, genetics and commercially cultivated species, particularly Jasminum sambac, J. auriculatum and J. grandiflorum. It discusses climate and soil requirements, propagation, planting, nutrient management, irrigation, pruning and other cultural practices. Major pests and diseases and their management are included. The chapter also explains harvesting, postharvest handling, marketing and the value-addition potential of jasmine flowers and essential oil.

The exile of Rama and his journey to the forests (vanvasa) for 14 years was started from Ayodhya (Uttar Pradesh, India) and ended to Lanka (Sri Lanka), through different forests, territorial regions, lands, hills, lakes of old India. The whole journey to those places was candidly portrayed in Ramayana with vivid descriptions, from narrations of the night sky, the position of different stars to descriptions of the flora, fauna and landscape. In this chapter, a retrospection of the places, forests, Rama passed through during his entire journey has been documented. The present status of those forests and places has been considered to evaluate a comparison over the thousands of years and the changes taken place besides significance they hold till date (Anonymous, 2024; Iyer, 2014).

Journey to the Forest and Route Map

Ayodhya, the capital city, kingdom of Kosala, was situated on the banks of holy river Saryu and was the birthplace of Rama. To fulfill the wish of his father, king Dasharatha, Rama left Ayodhya for 14 years of exile to the forest, along with Sita and Lakshmana. Their journey started from Ayodhya and passed through different places / forests.

I. INTRODUCTION & OVERVIEW

Intellectual property rights may be defined as the legal relationships that arise out of an individual’s creative gifts and the communication to society of his or her intellectual creations. Intellectual property is challenging as well as rapidly widening horizon of law. The purpose of law is to encourage and reward creativity and intellectual effort. The prominence of this branch of law in India is partly due to India’s economic structure and its geographical position in the world. Many international companies operate in India either directly or through licensees or agents. Numerous goods, services, and technological developments have overseas origins. Foreign companies often control their interests through contract but they invariably do so by means of copyright law, obtaining of patents or by registration of trade-marks and designs. It follows that in particular circumstances intellectual property remedies are most convenient method of maintaining control, or at least a useful adjunct to other remedies based on contract. Another important factor is the upsurge in “piracy” that is occurring not only in India but also in almost every other part of the world. Piracy may originate outside India, or, it may also originate in lndia. While devising legal apparatus for the welfare and protection of creative people, we must not lose sight of the public interest in the greatest measure of access to information and culture. Intellectual property rights are significant wealth- creating assets and their protection and commercialization are of increasing economic significance. In past the value of such rights was often overlooked and it is only in recent years that the economic potential of intellectual property has started receiving greater recognition. The dawn of the third millennium which has led to the emergence of knowledge capitalism with emphasis on intangible assets such as knowledge, competencies and intellectual property is a defining movement in the history of mankind. It is a defining moment in the history of mankind because the historical forces set in motion by the digital revolution has spawned a new civilization characterized by path breaking inventions and technological innovations and the opening up of the electronic frontier. It is an age of digital renaissance which combines the marvels of the Age of Discovery and the excellence of medieval Renaissance rolled into one. As a result of the information technology revolution there is a transition from industrial society to information society. Convergence of computer networks and communication technologies facilitated by the digital revolution has given birth to Cyberspace which in turn has ushered in knowledge economy. Hence, the intellectual property law faces challenges. In this background the concept, nature and development of intellectual property rights are proposed to be examined. The paper will be divided into V more parts. Part II of the paper will outline the meaning concept of intellectual property and range of subject matter protected by law, part III will examine the jurisprudential justifications of intellectual property protection, part-IV will explore the evolution and development of intellectual property rights under international law, part-V will demonstrate the evolution and development of intellectual property rights under Indian law. And part-VI will conclude the subject.