eChapters

Food is the basic requirement for maintaining the life process. Plants supply the bulk of foods required for human and animal. With limited cultivable area and ever increasing pressure of human population, the demand for food is constantly rising which can not be alone met by conventional plant breeding technologies. Novel techniques like plant genetic engineering are gaining momentum for production of superior plants or yield enhancement of a species.  It has been observed that the most commercially successful application of recombinant DNA technology is in genetic engineering of plants, which led development of multinational seed companies deriving monetary benefit from genetically modified crop and food products. From production of herbicide resistant or insect pest tolerant plants to alter the inherent quality composition of human food, plant genetic engineering has established firmly the commercial viability of recombinant DNA technology.

Introduction
Plant genetic engineering has revolutionized modern agriculture, enabling the development of crops that not only meet the demands of a growing global population but also address the challenges posed by climate change, pests, and diseases. As traditional breeding methods often face limitations in terms of time, precision, and scope, genetic engineering offers a powerful alternative. By directly manipulating plant genomes, scientists can introduce specific traits with greater accuracy and speed, leading to significant improvements in crop quality and productivity. This chapter explores the protocols and methodologies employed in plant genetic engineering aimed at enhancing the quality of crops, providing insights into the techniques that have transformed agricultural practices.
The importance of plant genetic engineering cannot be overstated. With the global population projected to reach nearly 10 billion by 2050, the agricultural sector must find ways to increase food production while ensuring sustainability and environmental conservation (Tilman et al., 2011). Conventional breeding methods, which rely on the natural variation present in plant populations, can be slow and imprecise. In contrast, genetic engineering allows for the targeted introduction of desirable traits, such as disease resistance, improved nutritional content, and enhanced stress tolerance (Gurian-Sherman, 2009). These advancements are crucial for maintaining food security in the face of growing environmental challenges.

The National Bureau of Plant Genetic Resources, commonly known as NBPGR was established by the Indian Council of Agricultural Research (ICAR) in 1976 with its main campus at New Delhi. Being the nodal organization in India it has been given the national mandate to plan, conduct, promote and coordinate all activities concerning plant exploration and collection and also for safe conservation and distribution of both indigenous and introduced genetic variability in crop plants and their wild relatives. The Bureau is also vested with the authority to issue Import Permit and Phytosanitary Certificate and conduct quarantine checks on all seed materials and plant propagules (including transgenic material) introduced from abroad or exported for research purposes.

Plants are vital part of the world’s biological diversity and essential resource for human well being. Besides meeting our basic food and fibre requirement, many thousands of wild plants have immense economical and cultural importance and potential for providing food, medicine, fuel, clothing and shelter for vast number of people at the global  level. Traditional Chinese medicines use around 5000 plant species and about 7000 different plants are used in traditional medicines in India. Plants play an important role in maintaining basic ecosystem functions essential for survival of animal life. Thus, it is of vital importance to collect and conserve the plant genetic resources on a global basis. It is all the more important as several cases of genetic vulnerability of plants that are grown for food and other uses have been recorded (National Research Council, 1972, Granett et al., 1991). Genetic diversity in the germplasm of crop plants is an effective defense against genetic vulnerability. The genetic diversity is strategically used as modern plant breeding intervention to help increase stability and sustainability of food production using modern cultivars.

Plant genetic resources (PGR) are the genetic material of plants which are of value for present and future generations of humankind. Often used as a synonym to plant germplasm, it can be defined as a seed, a plant or plant part including cell cultures, genes and DNA sequences that are held in a repository or collected from wild as the case may be and that are useful in crop breeding, research or conservation because of genetic attributes. The term is used to describe a collection of genetic resources for an organism or genetic material which forms the physical basis of inherited qualities. In brief, germplasm is the sum total of the hereditary materials in a species. Germplasm is the foundation and lifeblood of plant breeding without which plant breeding activities cannot be conducted. Germplasm is the genetic material that can be used to perpetuate a species or a population. The base germplasm can be improved for better performance of the crop. In the initial stage of a breeding programme, breeder evaluates the available germplasm lines and makes a selection from them for further test and final release to the farmers. Later on, the germplasm serves as a source of the parental lines which are used to initiate another phase of the breeding programme. Besides, using the base germplasm, breeders generate new variability using a variety of breeding methods such as crossing parental lines and developing new lines, induced mutations and more recently gene transfer. This new variability is again subjected to appropriate selection methods leading to identification and further evaluation of new and promising genotypes for release as cultivars. Generally, germplasm includes following types:

Allard (1960) defines germplasm as “the sum total of the hereditary  materials in  species” The word germplasm in context of plants is now referred to as plant genetic resources i.e. PGR.PGR is a component of the world’s biological diversity. The term biological diversity is a vast and often undervalued resource. Biological diversity includes every form of life, from the smallest microbe to the largest beast. Biodiversity is the variety and variability of all plants, animals and microorganisms and the ecological complexes of which they are part. The earth’s biodiversity – its ecosystems, species and genes – are the product of over 3000 million years of evolution. Throughout this time, small changes have accumulated in populations, resulting in a multitude of living forms closely adapted to the physical conditions they face and to each other. They supply all our food, much of our raw materials and energy and many of our medicines. Intact eco-systems also play a central role in the functioning of biosphere. Plants are of crucial importance in stabilizing climate, protecting watersheds on soils and maintaining the chemical balance of the earth. When key species are lost, vital ecological services are disrupted (Zedan, 1995).

Introduction

The objec tive of genomic research in any species is to sequence the whole genome and to decipher functions of all the different coding and non-coding seq,uences. The technology for large-scale DNA sequencing has enabled the scientists to undertake genome sequencing project in a realistic time scale. Since the time of first ‘large’ genome sequencing in bacteriophage λ in 1983, the projects on different groups have been completed. Eg. Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Pan troglodytes, Homo sapiens.

There are certain species which have been chosen for genome projects as model organisms. A model organism is a non-human species that is used to understand biological phenomena with a thought of gaining insight into the working of other organisms. They are chosen on the basis of their small genome in size, diploid genetics with a few chromosomes, rapid life cycle, easily transformed, well positioned in plant phylogeny, small stature for plant growth in a small space, large number of seeds produced, convenient for discovery of gene-trait discovery at low cost and speed and well investigated. They include,

• Monocots – Oryza sativa

• Dicots – Arabidopsis thaliana

• Temperate grasses – Brachypodium

Abstract

Plant Bioinformatics is a new and rapidly evolving field driven by the advances in ‘Omics’ technologies. This chapter is intended to help both beginners and experienced researchers to develop and apply bioinformatics tools to specific areas of plant genomics research. This chapter will also help plant biologists to access and implement comparative genomics studies of the plant genes of interest. Also discussed are the databases which house the data on plant genes and genomes. The chapter ends with a review of basic Perl Programming for sequence motif analysis beneficial for plant biologists.

Introduction

Ever since the discovery of DNA structure, the field of plant genomics has experienced a dramatic change in the ways scientific problems are approached. With the recent advances in High Throughput analysis clubbed with methods like Physical mapping of genes, RFLP, RAPD, large scale EST sequencing and mRNA protein profiling, there is an enormous amount of biological data available for various analysis. As the amount of data grows tremendously, there is a demand for developing tools and methods to effectively access these assembled data for analysis, modeling, visualization and prediction. In this chapter, we emphasize on some of the key concepts in bioinformatics, tools and databases relevant to plant genomics.

There is need to find better ways and solutions to mitigate future agricultural challenges toward increasing productivity across the crops and equip them with the desirable genes for resistance to biotic and abiotic stresses and to mitigate the impact of climate change and also to make them nutritionally superior. Here comes “Plant Genomics”—a newly evolved discipline of plant sciences—targeting to decode, characterize, and study the genetic composition, structures, organizations, functions, and interactions/networks of all plant genes in a genome-wide scale. Being evolved from plant molecular genetics, biology, and biotechnology, plant genomics represents the key sub- divisions of structural, functional, comparative, evolutionary, physiological, and genetical genomics. Its development and advances, however, are tightly interconnected with plant science sub-disciplines, such as proteomics, metabolomics, epigenomics, phenomics, metagenomics, transgenomics, breeding-assisted genomics, bioinformatics and system biology as well as modern instrumentation and robotics sciences.

Plant genomics is a recent convergence of many sciences. Simply stated “Genomics” is the new science that deals with the discovery and noting of all the sequences in the entire genome of a particular organism. The genome can be defined as the complete set of genes inside a cell. Genomics, is, therefore, the study of the genetic make-up of organisms. Determining the genomic sequence, however, is only the beginning of genomics. Once this is done, the genomic sequence is used to study the function of the numerous genes (functional genomics), to compare the genes in one organism with those of another (comparative genomics), or to generate the 3-D structure of one or more proteins from each protein family, thus offering clues to their function (structural genomics). In crop agriculture, the main purpose of the application of genomics is to gain a better understanding of the whole genome of plants. Agronomically important genes may be identified and targeted to produce more nutritious and safe food while at the same time preserving the environment. Genomics is an entry point for looking at the other ‘omics’ sciences. The information in the genes of an organism, its genotype, is largely responsible for the final physical makeup of the organism, referred to as the “phenotype”. However, the environment also has some influence on the phenotype. DNA in the genome is only one aspect of the complex mechanism that keeps an organism running – so decoding the DNA is one step towards understanding the process. However, by itself, it does not specify everything that happens within the organism. The basic flow of genetic information in a cell is as follows. The DNA is transcribed or copied into a form known as “RNA”. The complete set of RNA (also known as its transcriptome) is subject to some editing (cutting and pasting) to become messenger-RNA, which carries information to the ribosome, the protein factory of the cell, which then translates the message into protein.

The methods of plant germplasm preservation or conservation depend  upon type of germplasm i.e. seed or non-seed clonal materials. 

Storage of Seeds

According to the storage tolerance of seeds to low temperature and dry environments seeds are further divided into storable (orthodox) and non-storable (recalcitrant) types. The orthodox seeds are those seeds which are fully desiccation tolerant. These basically include small seeded grain crops and vegetables. They can be stored for a long time under low temperature and low humidity environment and are separately stored in long, medium and short term storage conditions in accordance with the purpose of storage.

The diversity collection and making it available for future use is one of the most important activities related to management to plant germplasm resources. The collection of plant germplasm from their natural habitat is broadly known as plant germplasm exploration. The main reasons for collecting germplasm of given gene pool in a given geographical location are as follows (Engels et al., 1995)

Marshall and Brown (1975) have considered the subject of optimal sampling strategies for use in the genetic conservation of crop plants. The theme of this paper has been relatively narrow. These authors have defined a strategy for populations under imminent threat of extinction applicable to particularly the traditional landraces of some major crops such as wheat, rice and barley on account of danger of replacement of the landraces on a very broad scale due to the spread of modern cultivars. According to Brown and Marshall (1995) the emphasis in collecting of crop genetic resources has  changed radically over the years. Instead of crisis-driven broad scale crop specific programmes, the collection of plant genetic resources now involves 3 important elements as given below :

Abstract

The greatest challenge facing us in the coming years is to produce adequate amounts of basic necessities of food, feed, fibre, fuel and raw materials for the rapidly shrinking per capita agricultural land. One alternative is to make a sustainable use of existing marginal lands. In many regions of the tropics and sub-tropics, the need to use marginal low-input soils with usually inadequate nutrient(s) supply permits only very low or unreliable yields to be achieved. It is frequently not possible  to supply required amounts of mineral fertilizers or chemical pesticides because of the low purchasing power of the marginal farmers. In particular, it is not possible to compensate for the deficient nutrient supply and this constitutes a yield limiting factor. The role of soil microorganisms in sustainable productivity has been well construed. In recent years, root inhabiting (rhizozosphere) bacteria (rhizobacteria) is gaining importance.

Introduction

Plants are considered as immobile organisms with poor sensitivity and limited ability to respond. Within the plant kingdom, Mimosa pudica (also called the sensitive plant), Drosera (sundews), Dionea muscipula (flytraps) and tendrils of climbing plants which showed rapid and purposeful movements were considered as exceptions. These sensitive plants attracted attention of outstanding pioneer researchers like Pfeffer (1845-1920), Burdon-Sanderson (1828-1905), Darwin (1809-1882), Haberlandt (1854-1945) and Bose (1858-1937). Stahlberg, 2006 reported the presence of various mechanoreceptors in such species which could trigger action potentials (APs) which activated these sudden movements.

The first extracellular recording of a plant action potential (AP) development was initiated by Charles Darwin; later, the animal physiologist Burdon-Sanderson in 1873 performed similar studies on leaves of the Venus flytrap (Dionea muscipula Ellis). Haberlandt (1884) demonstrated that phloem strands were the actual paths for excitation by destroying the external, non-woody part of vascular bundles. This notion was confirmed in Mimosa and other plant species by a number of recent studies. Houwink (1935) carried out experiments with two species of Vitis (grape) and obtained electrical fluctuations which was similar and comparable with the action potentials he obtained with Mimosa. In 1907, Bose, used a D’arsonval galvanometer to demonstrate propagating electrical effects in different genera of plants such as Ficus, Artocarpus, Cucurbita, Corchorus, “fern” etc. APs have their largest amplitude in phloem sieve cells and cells adjacent to this area (Sibaoka, 1969). Other studies found that AP-like signals are propagated with equal rate and amplitude throughout the cells of vascular bundles.

3.1. What is the International Plant Protection Convention (IPPC)

The International Plant Protection Convention (IPPC) is a 1951 multilateral treaty deposited with the Food and Agriculture Organization of the United Nations (FAO) that aims to secure coordinated, effective action to prevent and to control the introduction and spread of pests of plants and plant products. The Convention extends beyond the protection of cultivated plants to the protection of natural flora and plant products. It also takes into consideration both direct and indirect damage by pests including weeds.

The IPPC as an intergovernmental treaty signed by over 180 countries, aiming to protecting the world’s plant resources from the spread and introduction of pests, and promoting safe trade. The Convention introduced International Standards for Phytosanitary Measures (ISPMs) as its main tool to achieve its goals, making it the sole global standard setting organization for plant health.

The IPPC is one of the “Three Sisters” recognized by the World Trade Organization’s (WTO) Sanitary and Phytosanitary Measures (SPS) Agreement, along with the Codex Alimentarius Commission for food safety standards and the World Organization for Animal Health (OIE) for animal health standards.

1.1. Plant Quarantine

The literary meaning of the word quarantine is” a state, period or place of isolation in which the people or animals or plants or acquacultures that have arrived from elsewhere or been exposed to infectious or contagious disease are placed.

Plant quarantine is a technique of keeping the imported or introduced plants or planting material in isolation for detecting the exotic pests, diseases and disease pathogens and weeds which may be harmful to the importing country for its crop plants, agriculture or the ecosystem. These plants/planting material are kept in isolation for a specified period which differ from plant species to plant species depending on the type of pathogen/pests for which the material has to be quarantine. This is based on the Rules and regulations promulgated by governments to regulate the introduction of plants, planting materials, plant products, soil, living organisms, etc. with a view to prevent inadvertent introduction of exotic pests, weeds and pathogens harmful to the agriculture or the environment of a country/region, and if introduced, to prevent their establishment and further spread. Plant quarantine is thus designed as a safeguard against harmful pest /pathogens exotic to a country or a region.

Plant quarantine is a most important activity in the introduction of plants and planting material as various insects, including mites and nematodes and diseases caused by fungi, bacteria, viruses, viroids, phytoplasmas,rickettsialike organisms are known to attack various crops of economic importance to not only reduce the quantity but also spoil the quality of the produce to a considerable extent and if these pests and pathogens are introduced with the planting material in its non ecosystem areas may prove fatal to the agriculture or ecosystem

Plant quarantine is a government endeavor enforced through legislative measures to regulate the introduction of planting materials, plant products, soil, living organisms, etc., in order to prevent inadvertent introduction of insect-pests, pathogens and weeds harmful for the agriculture of a country/state/region, and if introduced, prevent their establishment and further spread. Various methods of pest disease control are: exclusion, eradication, protection, therapy, resistance, and biological control. Exclusion or ‘keeping out’ is fundamental to the concept of plant quarantine while eradication methods are employed to eliminate a newly established pest/pathogen. Plant quarantine may, therefore, be defined as ‘Rules and regulations promulgated by governments to regulate the introduction of plants, planting materials, plant products, soil living organisms, etc. with a view to prevent inadvertent introduction of exotic pests, weeds and pathogens harmful to the agriculture or the environment of a country/region, and if introduced, to prevent their establishment and further spread’. Plant quarantine is designed as a safeguard against harmful pests/pathogens exotic to a country or a region (Ram Nath, 1991, Khetrapal et al., 2004).

Summary

Plant cell culture systems produce myriads of valuable compounds which cannot be synthesized by microbial cells or chemical synthesis. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The burgeoning demand for secondary metabolites in recent years as therapeutic agent has resulted in a great interest, in secondary metabolism, and particularly in the possibility to alter the production of bioactive compounds using cell culture technology. Various strategies such as cell line selection, use of precursors and elicitors, mass culture in bioreactors and others have proved effective in enhancing the production of these metabolites so as to meet the market demand but space for improvement still exists. In order to stretch the boundary, recent advances, new directions and opportunities in plant cell based processes are being examined in this section.

Summary

Plant tissue culture is the technique of growing plant cells, tissues or organs in isolated from the mother plant on artificial media. It has opened up unprecedented opportunities in many areas of basic and applied biological research. This includes techniques and methods used to research into many botanical disciplines and has several practical objectives of plant tissue culture and micropropagation for sustainable development. Indeed, the landmark publication by Gottlieb Haberlandt who is arguably referred to as the “Father of Tissue Culture”, is often cited as the origin and emergence of plant tissue culture and its subsequent applications. Success of biotechnological approaches is dependent on regeneration of intact plants following genetic modification, generally by micropropagation. Potential uses of plant tissue culture in biotechnology to further our understanding of plant physiology, how plants function, resolution and expression of use full characters form in vitro to in vivo condition. Agro bacterium-mediated genetic transformation along with other modes of transformation such as microprojectile bombardment has been accomplished mainly through plant tissue culture.

Media Preparation

Before mixing medium, determine the quantity of each medium and the amount of plant growth regulator needed for each experiment. Make slightly more medium than is required. A media check off list is helpful in making medium to keep track of ingredients.

Remove media stock solutions and plant growth regulator stock solutions to be used from the refrigerator. Make up fresh stock solutions if adequate quantities are not available or if solutions have precipitated.

Plant growth regulators stock solutions are prepared biweekly and stored at 4º C in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and NAA) are dissolved in 100% ethanol prior the addition of water.

Fill the volumetric flasks upto ½ to ¾ with deionized water. Add proper a of stock solution and plant growth regulators and mark each item on the check off list. Add 30 g/l sucrose, stir until dissolved. Make up the volume of the flask with deionized water and later por into a larger beaker.