eChapters

History of PCR

A 1971 Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F) environments) paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

Developed the PCR in 1984 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

Polymeric nanoparticles are nanoparticles which are prepared from polymers. The drug is dissolved, entrapped, encapsulated or attached to nanoparticles and depending upon the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained. Nanocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a polymer membrane, while nanospheres are matrix systems in which the drug is physically and uniformly dispersed.

In recent years, biodegradable polymeric nanoparticles have attracted considerable attention as potential drug delivery devices in view of their applications in drug targeting to particular organs/tissues, as carriers of DNA in gene therapy, and in their ability to deliver proteins, peptides and genes through oral route of administration.

In spite of development of various synthetic and semi synthetic polymers, natural polymers still enjoy their popularity in drug delivery, some of them are: gums (Eg. Acacia, Guar, etc.,) chitosan, gelatin, sodium alginate, and albumin. A range of materials have been employed to delivery of bioactive agents. A polymer used in controlled drug delivery formulation, must be chemically inert, non-toxic and free of leachable impurities. It must also have an appropriate physical structure, with minimal undesired aging, and be readily processable. Some of the polymeric materials are: cellulosics., poly(2-hydroxyethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid).

Any organism with other than two basic sets of chromosomes, that is, monoploid, triploid, tetraploid, and various aneuploids is termed as polyploid. There are several economically important crops which are natural polyploids emphasizing the economic importance of polyploids in agriculture. These are:

Triploid-Banana

Autotetraploid-Alfalfa, potato, coffee, peanut or groundnut Autohexaploid- Sweet potato

Amphidiploid/allopolyploid-Wheat, cotton, oats, Brassica species (B. carinata- Ethiopian mustard, B. juncea-Indian mustard, B. napus--rapeseed), sugarcane

Man-made commercially successful allopolyploid- Triticale

The newly synthesized autopolyploids, having few or more undesirable features, e. g; meiotic instability, low seed setting and late maturity etc, are usually not suitable for immediate commercial use, are known as raw polyploids. This is specifically more applicable for those sexually propogated polyploids which are developedfor seed. In early generations due to polyploid chromosome constitution and polysomic inheritance they predominanantly do not breed true. Further more, due to meiotic instability chromosome number in their progeny is not always constant or equal to as in synthesized autotetraploid which may vary from few more to few less than the standard autotetraploid. The raw polyploids usually reverts back to diploid parental constitution due chromosome elimination (Randolph and Fischer, 1939). The mechanism responsible for reverting back to diploid status is similar to that of which give rise haploids from diploids. Presently cases are known where either spontaneously or due to specific treatments the chromosome number was reduced to half in somatic tissues. The phenomenon causing such chromosomal reduction is termed as somatic reduction or reduced mitosis Earlier reports suggested it probably to be caused due to spindle organizer abnormalities or some other unknown mechanism/s. The treatment of seed or seedling by colchicine, chloramphenicol and paraflurophenylalanine are reported to have induced chromosome reduction and production of haploids.

Polyploidy, the condition in which a normal diploid cell or organism acquires one or more additional sets of chromosomes. In other words, the polyploid cell or organism has three or more times the haploid chromosome number. Polyploidy arises as the result of total nondisjunction of chromosomes during mitosis or meiosis. The somatic chromosome number of any species, whether diploid or polyploidy is designated as 2n. The somatic cells of plants are diploid that they posses two complete set of chromosomes, one received from seed parent and another from pollen parent. The gametic chromosome number is said to be haploid and denoted by n. Haploid is defined as cell with gametic chromosome number. While monoploid has the basic chromosome number and represented by x.

Classification of Polyploidy

Polyploids are classified in to two groups, euploidy and aneuploidy. Euploidy have chromosome number which is an exact multiple of basic number. Euploidy includes monoploids, diploids and polyploids. In aneuplod individual with other than exact multiple of basic chromosome number or change in chromosome number which posses one or few chromosomes of the genome.

Polyploid is any organism with other than two basic sets of chromosomes, that is, monoploid, triploid, tetraploid, and various aneuploids. Species that have their chromosomes as multiples of a basic number (x) are polyploids. Many of the species which have been developed most recently are polyploids. Autoploidy is not known to have significantly impacted the evolution of species. Autoploids of commercial importance include banana, a triploid which is seedless (diploid bananas have hard seeds not desirable for consumption). Other important autoploids are tetraploid crops, namely, alfalfa, peanut, potato and coffee. Spontaneous autoploids are important in horticultural crops where gigas feature has produced superior varieties of flowering ornamentals of narcissus, tulip, hyacinth, gladiolus, and dahlia among others. Triticale is the most common example of man-made cereal through allopolyploidy. A number of economically important crops are natural alloploids. These include wheat, oat, tobacco, cotton, sugarcane, strawberry and blueberry, wild mustard, black mustard, rutabaga/rape, etc.

A. Define/Explain the following

1. Heteroploidy: Any change in chromosome number from the diploid state.

2. Euploidy: Numerical change in the entire genome.

Genes in Populations

• A population is a group of interbreeding individuals who share a gene pool
• Some genes are monomorphic, and others are polymorphic
• Population genetics is concerned with allele and genotype frequencies

The Hardy-Weinberg Equilibrium

The Hardy-Weinberg equation can be used to calculate genotype frequencies based on allele frequencies

Nonrandom mating may occur in natural and human populations

This protocol is designed for potato stem internodes on ZIG medium, but might work for leaf pieces as well.

Plant Material

Cut stem internode explants from 5-6-week-old in vitro plants (or leaf explants from 3-4-week-old in vitro plants) grown on 15 ml PROP in 25 x 150 mm test tubes.

Day 1

Streak out desired Agrobacterium strain on YEP + antibiotic to obtain a single colony. Grow at 28 C for 2 days. Alternatively, skip this step and use frozen stock for next step.

Day 3

Use a single Agrobacterium colony to inoculate 20 ml YEP + antibiotic in 250-ml flask. Grow overnight at 28 C with vigorous shaking (~200 rpm). Some Agrobacterium strains require an additional day to reach sufficient density.

I. INTRODUCTORY OUTLINE

The post-Vienna legal phase is remarkably known for revolutionary changes across the global wherein the third generation of human rights became the hallmark for the comity of nations and a liberational instrument to come out of nightmarish experiences of poverty and hunger.1 On the other hand the post-GATTised legal ordering is known for much publicized and debatable conclusion of Trade Related Aspect of Intellectual Property Rights (TRIPs) and creation of World Trade Organisation (WTO).2 This new jurisprudential polemics soon evoked fierce scrutiny from the stand point of human right and poverty among third world countries.3 At home, it is dubbed as ‘bleeding operation with the dubious promise of benign global competition’, ‘de facto nullity of constitutional goal of social welfare’ and ‘contra- constitutional coup.4 Since India has become a member of WTO it indulged into massive rehabilitation and fundamental mutation of IPR regime. Despite the fact that it has a stark distinction of being a grain- surplus and food and livelihood insecure country. An inventory of over forty five million tonnes of wheat and rice in granaries remains ready for consumption. On the other hand over two hundred fifty million people generally go to bed partially hungry every day. There is a near consensus on the need to banish hunger but the realities on the ground remain that even at the risk of raising hopes the goal of food security may not be realized for generations.5 At home under the cover of food security the government is keeping millions of tonnes of food out of reach of the poor people.6 In fact India’s record breaking 200 plus million tonnes harvest is the result of steep fall in purchasing power of poor. The myth of surplus is based on sending hundreds of millions of human being hungry to their beds.7 Taken to its logical end we have abundance of food, surplus of hunger and scarcity of vision under the garb of food security.8

Wide hybridization (interspecific/intergeneric) is basically attemped to introduce new genetic variability from wild or distantly related species into cultivated ones or to synthesize new plant type/s like Triticale and Raphanobrassica. Majority of the commercial crop plants are usually deficient for one or the other desirable traits which are usually available in their wild relatives. However, crossability between commercial varieties and their wild relatives is very limited which is naturally imposed by certain genetically controlled reproductive barriers. These reproductive barrier/s usually operate either at pre or post fertilization stage/s in plant species (Stebbins1958). Since a species is constituted of individuals who share a common gene pool, reproduce among themselves only / exchange genes and share a common habitat. Hence at natural level gene flow remains strictly confined only among members of a particular species otherwise among the members of unrelated species a genetic barrier exists to create reproductive isolation. Therefore, wide crossing basically requires the overcoming of such barriers whch are imposed by temporal and spatial isolation of parents. The reproductive isolation consists of premating and post mating isolation mechanisms like habitat isolation or geographical isolation, sexual isolation and mechanical barrier depending upon incompatibility or mismatching of ganitalia (animals). Sexual isolation in animal kingdom is usually enforced by type of sexual displays and mating calls which are attractivce only to particular members of a species , however other potential intermating species also do exist in the same habitat or locality at that particular period of time.

Introduction

Crop improvement lies at the heart of ensuring food security, agricultural sustainability, and the resilience of our farming systems. As we confront a world characterized by climate variability, emerging pests, and a growing global population, the need for innovative approaches to crop enhancement becomes paramount. This is where the concept of pre-breeding steps in: a fundamental phase that acts as a bridge between the untamed genetic diversity of wild plants and the refined traits of cultivated crops. Pre-breeding represents the exploration, identification, and incorporation of valuable genetic traits from the vast array of plant species that often remain on the fringes of agricultural attention. These “wild relatives” and underutilized varieties possess traits that have evolved over time to withstand diverse environmental stresses, combat diseases, and adapt to changing conditions. Pre-breeding aims to tap into these reservoirs of genetic potential, merging them with the qualities we value in our cultivated crops. At its core, pre-breeding is a scientific journey of discovery and innovation. It involves the strategic selection and hybridization of plant materials, guided by a vision of enhancing crop performance across multiple fronts. The goal is to create crop varieties that are not only productive and high yielding, but also equipped to thrive in the face of evolving challenges. This involves a delicate dance between tradition and cutting-edge techniques as well as collaboration between breeders, geneticists, and researchers (Qureshi et al. 2014).

In this Chapter the precision farming/ Agriculture is discussed along with its steps of implementation. It also includes how artificial Intelligence plays role to perform precision farming/ agriculture in various way along with its scope, limitation and challenges.

Introduction

The primary source of livelihood is agriculture in India, where two thirds of India’s population, services and the private sector account for the remainder. Agricultural land accounts for about 43 percent of the geographical area of India. In earlier days, India was largely dependent in terms of food imports, but over the years, the country’s production of grain and seeds was self-sufficient, and this effort led to the formation of the Green Revolution. In fact, there was strong effort to achieve food self-sufficiency. This trend has continued up to now and continuous improvements have occurred. Precision agriculture or precision farming means the right thing to do, in the right way, on the right place and at the right time. Precision agriculture is expected to suit the agro-climate activities to improve application precision. Farming land has declined a bit in the last 40 years but the number of farmers has increased. According to the 2010–11 Agriculture Census, a total of 138.35 million operating holdings were estimated (single farmers) and 159,59 million hectares were covered by operations.

Precision agriculture is a management technique that collects, processes and analyzes temporary, spatial and individual data and integrates this with other information in order to support management decisions based on an estimated variability to enhance efficiency of use of agricultural production resources, productivity, quality, profitability and sustainability. Precision Agriculture manages every input of crop production (fertilizer, specifically found calestone, herbicide, seeds, insecticide, etc. Reduce waste, boost income and maintain efficiency Place. Precision Agriculture adapts soil and crop management carefully to suit the different field conditions.

Predictive modelling of plant growth can help farmers and researchers improve crop management, resource utilization and production planning. This chapter examines machine learning approaches for predicting important plant growth characteristics. It introduces plant growth fundamentals and the environmental and management factors that influence development. The chapter covers the machine learning workflow, including data acquisition, preprocessing, feature engineering, model development and evaluation. Supervised learning, unsupervised learning and deep learning approaches are considered for predicting plant height, biomass, canopy development, leaf area and yield-related characteristics. Data from IoT devices, drones and satellite imagery can strengthen prediction models. The chapter also discusses precision agriculture applications, limitations and emerging technologies such as explainable AI, digital twins and edge AI.

DNA fragment to be used as a probe should be purified by gel electrophoresis. The band can be located after staining with ethidium bromide and electroeluted.

The in vitro radioactive labeling of DNA fragment is accomplished by the incorporation of a radioactive nucleotide during strand synthesis with Klenow enzyme (large subunit of DNA polymerase-I).

Enterobacteria phage ? is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli (E.coli). It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

Introduction

For agricultural applications as well as for research work, often we need to prepare solutions of different agrochemicals of varying strength, as per the requirements. Agrochemicals are used to help crop plants grow, produce, and protect them from biotic/abiotic stresses. Many of the agrochemicals (insecticides, herbicides, fungicides, rodenticides, molluscicides, nematicides, etc.) are used to protect the crop from insect pests and diseases. However, some of them (fertilizers and soil conditioners) are used to augment crop yield. While in the previous chapter (Chapter 8) we discussed about preparation of laboratory solutions, in this chapter we will briefly discuss about the preparation of different agrochemicals.

Agrochemical/agrichemical is a generalized term used for the chemicals applied in agriculture. In general, it also includes the feed-supplements used in f isheries and animal husbandry.

Agrochemicals

Fertilizers

Fertilizers are used to augment growth of crop plants and to mitigate nutrient deficiency in the soil. Fertilisers can be organic (containing natural substances, prepared through natural processes) and inorganic fertilisers (synthetic fertilizers, manufactured artificially using chemical processes using natural deposits, by chemical alterations).

Soil Conditioners

Soil conditioners include manure, livestock and leaf compost, peat that help making the soil in good condition. They are laid on the top soil and then mixed properly. They not only increase nutrient contents in the soil but also improve aeration and water holding capacity.

Introduction

Different types of culture media are used for culturing microbes and plant tissues. Preparing as well as sterilizing them properly following appropriate method with safety for the desired purpose are important parts of research work. In this chapter, we will discuss briefly about different types of media, their composition, preparation and sterilization in laboratory for microbial or plant tissue culture.

Culture Medium

Ingredients mixed to prepare solid, liquid or semi-solid substance to support the growth of microorganism or plant tissue in vitro (outside the body) is called culture medium. Frequently, it becomes important to grow microorganism or plant/animal tissue in vitro for any or all of the following purposes:

• To study characteristic features or properties of microorganisms.

• To identify the causal organism(s) of infection in a clinical sample, so that appropriate remedy can be determined.

• To culture/multiply microorganism or plant/animal tissues in vitro for research or commercial purposes.

• To prepare/produce biological molecules, vaccines, antigens, proteins, metabolites, etc. in vitro for research or industrial purposes.

Objectives

• Better understand the process and different stages of mitosis.
• Prepare your own specimens of onion root in which you can visualize all of the stages of mitosis.

Principle

The genetic information of plants, animals and other eukaryotic organisms resides in several (or many) individual DNA molecules, or chromosomes. For example, each human cell possesses 46 chromosomes, while each cell of an onion possesses 8 chromosomes. All cells must replicate their DNA when dividing.

DNA replication in eukaryotes is followed by the process called mitosis which assures that each daughter cell receives one copy of each of the replicated chromosomes. During the process of mitosis, the chromosomes pass through several stages known as prophase, metaphase, anaphase and telophase. The actual division of the cytoplasm is called cytokinesis and occurs during telophase. During each of the preceding stages, particular events occur that contribute to the orderly distribution of the replicated chromosomes prior to cytokinesis.

An onion root tip is a rapidly growing part of the onion and thus many cells will be in different stages of mitosis. The onion root tips can be prepared and squashed in a way that allows them to be flattened on a microscopic slide, so that the chromosomes of individual cells can be observed easily. The super coiled chromosomes during different stages of mitosis present in the onion root tip cells can be visualized by treating with DNA specific stains, like Feulgen stain and Acetocarmine stain.