eChapters

Deep learning emerged as a serious contender in the field from a 10-year explosive computational development. Deep learning is therefore a special form of machine learning, the algorithms of which are based on the structure and function of the human brain.

Machine learning VS Deep learning

Deep learning is arguably the most efficient form of machine learning. This is so important because they know the best way to solve the problem and how to fix everything. The following is a description of deep learning and machine Learning:

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. 

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.

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: 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.

Introduction

Flowering plants (Angiosperms) are identified by their morphological characteristics like flower, fruit and seeds that help their taxonomic characterization into different species, genera, families and orders. The floral characters which are used for taxonomic characterization of plants include the type of inflorescences, perianth structure, floral symmetry, arrangement of floral leaves, type of androecium, stamens, gynoecium, carpel, ovule, etc. The type of fruit and seed also play important roles in taxonomic classification. In this chapter we will briefly discuss about various morphological features of flowering plants (in botanical terms) that are useful in taxonomy.

Nomenclature of Plant

Nomenclature of plant is the formal and scientific naming of plants. It is related to but distinct from taxonomy for grouping and classifying plants and provide botanical name.

The scientific/botanical nomenclature started with Species Plantarum by Carolus Linnaeus in 1753. Botanical nomenclature is governed by the International Code of Nomenclature for algae, fungi, and plants (ICN), which replaces the International Code of Botanical Nomenclature (ICBN).

ICBN and ICN

International Code of Botanical Nomenclature, (ICBN) was adopted by the VIIth International Botanical Congress held in Stockholm, July1950. While the International Code of Nomenclature for algae, fungi, and plants (ICN) was adopted by the XVIIIth International Botanical Congress held in Melbourne, Australia in July 2011.

12.1 Monitoring Strategies: Pre- and Post-Release
12.1.1 Introduction
Monitoring of genetically modified organisms (GMOs) or transgenic crops is a critical component of biosafety governance. It involves the systematic collection of scientific data to assess the presence, behavior, and potential impacts of transgenic organisms in controlled and natural environments. Monitoring strategies are divided into two principal stages:
1. Pre-release monitoring, which takes place during confined field trials, and
2. Post-release monitoring, conducted after commercial or environmental release.
These strategies ensure that transgenic technologies are introduced responsibly, maintaining human safety, ecosystem integrity, and regulatory compliance.
12.1.2 Objectives of Monitoring
• To evaluate the biosafety and efficacy of GMOs in different environments.
• To detect unexpected adverse effects on human health or the environment.
• To monitor gene flow, resistance development, or weediness.
• To ensure compliance with biosafety regulations, stewardship programs, and licensing agreements.
• To maintain public trust and provide transparency in GM deployment.

Introduction

Spoilage in agricultural products due to mould growth can occur at various stages of production and storage and is significant in terms of trade economics, food safety and public health (Shephard, 2008). It has been reported that 5–10 % of agricultural products in the world are spoiled by mould contamination to the extent that they cannot be consumed by humans or animals. Mould growth decreases the quality of food and also creates a potential risk for human health because of the production of toxic secondary metabolites known as “mycotoxins”. Foods contaminated by mycotoxins, when consumed by humans and animals causes a disease called “mycotoxicosis”, resulting in death (Wagacha and Muthomi, 2008).

Introduction

The most common method of bacterial transformation utilizes high levels of calcium chloride to facilitate the entry of plasmid DNA vectors into E.coli in conjunction with structural alteration of the bacterial cell wall. In general, there are three basic steps in the introduction of plasmid DNA unto cells.

1. Preparation of “competent” cells to enable cells to accept DNA.
2. Transformation of these competent cells, and
3. Selection of transformants.

Most E.coli strains exhibit transformation efficiency (measured as transformants per microgram plasmid DNA) in the range of 10⁵ to 10⁸.

The factors influencing this efficiency depends upon the condition that render cells competent.

Introduction

In order to use a fine quality of DNA for various molecular biological work like sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (random primer labeling), etc., quality and quantity of DNA is extremely important to have the defined template concentration. Failure to produce results from the above techniques can sometimes be attributed to an incorrect estimate of the DNA template used.

Example: Isolation and Characterization of Protease Inhibitor Gene and Promoter from Black gram (Vigna mungo L.) and Genetic Transformation and Molecular Analysis of Transgenic Tobacco

Introduction

Crops diseases are caused by bacteria, virus, fungi and phytoplasmas. The detection of pathogenic bacteria and viruses in plants, planting material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The molecular based techniques, have evolved significantly during recent past that  allows the rapid and reliable detection of pathogens. The detection and diagnosis of plant pathogens is essential to take control measures and subsequently for eradication of the disease that causes.  Molecular detection is largely based on PCR or RT-PCR amplification following purification of nucleic acids from the samples, with the extraction of the target DNA.

Initially, exploitation of heteosis was confined to corn and a few other open- pollinated crops but presently heterosis is being exploited practically in all species cutting across mode of reproduction. More and more evidences are emerging that heterotic expression in a cross is a function of genetic backgrounds and genes. Heterotic grouping would remain a key strategy in management of diverse genetic backgrounds and genes for full exploitation of heterosis. The commonly used methods in developing hybrids in self- pollinated crops are listed and explained as follows:

Manual emasculation and Pollination Method

In this method, female parent flower buds are emasculated manually and the emasculated buds are pollinated by the pollen grain collected from protected flowers of the male parent. This method is effective and successful where the crossed fruits produce plenty of seeds and the seed requirement for planting the commercial hybrid seed is much less. Consequently, this method is being used in a routine manner on a commercial scale in vegetable crops mostly by private sector seed companies in India and south-east Asian countries. The crops covered under this method of hybrid seed production are as follows:

Tomato, hot-pepper, sweet pepper, eggplant, okra, bottle gourd, bitter gourd, ridge gourd, smooth gourd, cucumber, pumpkin, water melon and muskmelon. Cucurbits are included here as although they are cross-pollinated but behave like self-pollinated crops in terms of almost no inbreeding depression.

Invertebrate Development

The early stages of embryonic development determine the pattern of structures in the adult organism

The study of Drosophila mutants with disrupted development patterns has identified genes that control development

The generation of a body pattern depends on the positional information that each cell receives during development

The gene products of maternal effect genes are deposited asymmetrically into the oocyte and establish the anteroposterior and dorsoventral axes at a very early stage of development

Gap, pair-rule, and segment-polarity genes act sequentially to divide the Drosophila embryo into segments

The expression of homeotic genes controls the phenotypic characteristics of segments

The developmental fate of each cell in the nematode Caenorhabditis elegans is known

Heterochronic mutations disrupt the timing of developmental changes in C. elegans

Diagnostic biomarkers are measurable biological molecules that provide information about physiological or pathological conditions. This chapter examines the use of metabolomics for discovering, evaluating, and validating biomarkers for disease diagnosis, prognosis, therapeutic monitoring, and personalized medicine. It discusses different types of biomarkers, metabolomic biomarkers, biological samples, analytical platforms, biomarker-discovery workflows, and statistical analysis. Applications in cancer, diabetes, cardiovascular, liver, kidney, neurological, infectious, and metabolic diseases are considered. The chapter also explains the role of metabolomics in drug discovery, toxicology, nutrition, and clinical research. Challenges involving biological variability, analytical reproducibility, data complexity, and clinical validation are addressed alongside future opportunities involving AI and multi-omics.

12.1. Introduction

The protocols provides guidance on the structure and content of the International Plant Protection Convention (IPPC) diagnostic protocols for regulated pests. The protocols describe procedures and methods for the official diagnosis of regulated pests that are relevant for international trade. They provide at least the minimum requirements for reliable diagnosis of regulated pests.

This standard sets the framework for the content of diagnostic protocols, their purpose and use, their publication and their development. Diagnostic protocols for specific regulated pests are included as annexes to this standard. Information relevant for diagnosis is provided in the diagnostic protocol on the specified regulated pest, its taxonomic position, and the methods to detect and identify it. Diagnostic protocols contain the minimum requirements for reliable diagnosis of the specified regulated pests and provide flexibility to ensure that methods are appropriate for use in the full range of circumstances.

The methods included in diagnostic protocols are selected on the basis of their sensitivity, specificity and reproducibility, and information related to these factors is provided for each of these methods. Detailed information and guidance for the detection of pests is provided on, for example, signs and/or symptoms associated with the pest, illustrations (where appropriate), developmental stages of the pest, and methods for detecting the pest in a commodity, as well as methods for extracting, recovering and collecting the pests from plants.

Introduction
Plant breeding, according to Sansern et al. (2010), is the process of genetically altering plants to meet human demands. Prehistoric humans in various parts of the world domesticated only a small number of the hundreds of thousands of species that were then available. They did this by genetically altering these species’ components, which entailed both conscious and unconscious selection as well as differential reproduction of variants (Allard, 1999). A relatively small number of plant species have evolved over many years of trial and error to become the backbone of agriculture and, consequently, the world’s food supply (Mackay, 2001). This method of domestication includes the identification of specific valuable wild species along with a system of selection that resulted in alterations in the quality and quantity of cultivated crops.The modifications brought about by domestication involved altered organ size and shape, loss of many survival traits, such as bitter or toxic substances, the disintegration of seeds in grains protective structures, such as spines and thorns, seed dormancy, and change in life span increased for crops grown for roots or tubers and decreased for crops grown for seed or fruit. The domestication process followed mostly by mass selection (selection by bulking preferred kinds) is a potent method for facilitating quick changes while preserving genetic variability in the population (Okporie and Obi 2002).
Populations can exist in constantly changing surroundings due to genetic variation. More variation increases the likelihood that certain individuals in a group will have allele variants that are appropriate for the environment. Those individuals have a higher chance of survival to have offspring which carry that allele. Because of their success, the population will endure for additional generations. Selecting the best parents to utilize in artificial crosses is one of the key concerns for plant breeders since it encourages the expression of the largest genetic variability and the production of superior recombinant genotypes. The objective of plant breeding is to simultaneously improve all the desired traits. This task is complicated by correlations between various traits, which may result from pleiotropic genes, physical chromosomal links between genes, or population genetic structure. Choosing one feature will alter associated qualities, sometimes in a positive or in a negative way. As a result, selection may result in unexpected modifications, provided they fall within the typical range of variations seen in the crop and are therefore thought to be useful to consumers or the environment. Breeding practices in the 20th century improved a variety of genetic techniques, including methods for identifying and preserving more genetic diversity, creating variable populations for selection, and testing how to differentiate between the effects of genes and the environment. Naturally self-pollinating plants like soybeans or tomatoes, naturally cross-pollinated plants like maize, and plants whose vegetative propagation (typically cross-pollinated) allowed the direct fixing of improved types, all required different precise steps in the production process (Ubi, 2013).

Reagents

Nuclease-Free Water: 14µl

10X Restriction Buffer: 2µl

Acetylated BSA (1mg/ml): 2µl

DNA (~1µg): 1µl

Restriction Enzyme (10u): 1µl

Final Volume: 20µl