eChapters

Abstract

Nitrogen is the most essential nutrient for all plants and is also the major limiting factor in plant productivity. High yielding varieties of all crops are responsive to nitrogen but its ever-increasing use has shown detrimental impact on the environment along with extremely low use efficiency. Nitrogen use efficiency (NUE) in crop plants depends on various internal and external factors, which are dealt in detail. There has been a significant interest in genetic engineering of the crops to improve NUE. The use of biotechnological interventions by manipulating genes of the nitrogen utilization pathway to improve NUE has not been very successful. But transgenics/mutants with modified capacities for nitrate uptake, assimilation and remobilization have enhanced our understanding of the genetic control of NUE. In both cellular, and at whole plant level the mechanisms involved in N remobilization from the senescing organs towards the grain have recently gained importance but their understanding is still preliminary. Recent evidences have shown that grain filling during later stages of crop growth is supported by N recycling.

Although thousands of plant species exist on the earth, only those that were found to be useful for humans as food, feed, fiber, timber and other utility materials have been selected for domestication. When these plant species were affected by various biotic and abiotic causes, the necessity arose to determine the ways and means of protecting the valuable plants species and to understand the host plant defense mechanisms and pathogenic potential of the microbial pathogens causing enormous losses to the cultivators. All plant species have built-in resistance to most microbial plant pathogens existing in the environment. Susceptibility of plants to some of them is an exception, since they are endowed with genes which effectively nullify the possible adverse conditions created by the microorganism. Likewise, microbial pathogens possess genes for survival and pathogenicity to survive in the absence of susceptible plant species and to successfully establish infection in suitable plant species. The microbial pathogens have evolved highly specialized tactics to overcome the defense responses and to rapidly multiply in plants for their survival and perpetuation in a geographic location / ecosystem

Introduction
• Arrival of the nerve stimulus at the motor end plate result in reversal of the polarization of the sarcolemmal surfaces and the muscle gets activated
• This causes momentarily loss of potassium and sodium ions impermeability by the sarcolemma and Ca++ ions combined with calsequestrin in the sarcotubular system gets dissociated bringing Ca++ ion equilibrium with the sarcoplasm
• The junctional foot protein (JFP) and the L-type calcium channel (LTC) are the two big protein complexes responsible for the calcium release mechanism (Fig. 10.1)
• This leads to increase in level of Ca++ ion from nearly 0.10 mM to 10 mM
• Thus, troponin C, the calcium-binding site of the troponin complex becomes saturated bringing a structural modification resulting in interaction of actin with the MgATP2- on the H-meromyosin heads of the myosin molecule as the troponin I, the inhibitory protein, no more inhibits this interaction
• The interaction of actin with the MgATP2- intensely activate contractile ATP-ase near the bonding leading to break down of MgATP2- to MgADP– at a greater rate and liberating energy for inward (towards the sarcomere center) pulling of the actin filament
• This leads to breaking of bond between actin and myosin and thus, the myofibrillar part involved in above mentioned activity gets contracted

• General considerations

 • Polyacrylamide gel electrophoresis (PAGE)

 • SDS-polyacrylamide gel electrophoresis (SDS PAGE)

• Nucleic acid hybridization

 • Radio labelled probes

 • Biotin labelled probes

 • DNA finger printing

 • Polymerase chain reaction (PCR)

 • Reverse transcriptase polymerase chain reaction (RT PCR)

The shift from vegetative to reproductive phase in plants is the result of multiple gene cascades in response to photoperiodic and temperature dependent pathways, gibberellic acid pathway, vernalization pathway and autonomous pathway. The environmental cues are initially detected in the leaves and the mRNA of the flowering signal integrator FT gene, moving to the shoot apical meristem, acts as a long-distance signal for floral meristem identity. At SAM, FT-FD protein complex, based on signals from above said pathways in association with the signals from age-dependent and miRNA pathways, leads to the activation of master regulator gene of flowering AP1. The activation of genes involved in ABCDE model of floral organ differentiation, based on the directions of various cadastral genes lead to the development of floral organs. Thus, the sequential action of floral meristem identity genes, floral organ developmental genes, floral meristem size and organ number genes, floral organ pattern genes and cadastral genes leads to the flower development. This chapter sums up the gene interactions happening in leaf and SAM, leading to the development of a complete flower.

Introduction to Molecular Biology Techniques Molecular biology is the domain of biology concerned with DNA replication, RNA synthesis (transcription), translation, and the role of proteins in cellular function, while molecular biology techniques are the keystones of modern biology, facilitating scientists to decode these molecular foundations of life. These molecular techniques aim to analyze and understand the structure, function, and interactions of biomolecules such as DNA, RNA, and proteins. These techniques facilitate an understanding of complex life processes such as gene expression, genetic variations, and cellular mechanisms. Researchers have used these molecular tools to unravel complex biological processes from genetic inheritance to cellular signaling pathways. The knowledge gained has been applied to solve challenges in various domains of biological science such as medicine, agriculture, and environmental science. During 1960’s methods to identify, isolate and manipulate molecular components were developed by molecular biologists. The field of molecular biology emerged in mid 20th century, driven by groundbreaking discoveries such as the deduction of the double-helix of DNA

Introduction

Biochemical techniques are fundamental tools that allow scientists to investigate the molecular intricacies of biological systems, including DNA, RNA, and proteins. These techniques are essential for understanding cellular processes, genetic functions, and disease mechanisms. By employing biochemical methods, researchers can dissect complex biological systems at the molecular, cellular, and genomic levels, paving the way for advances in basic biology and therapeutic interventions.

Introduction

Nucleic acids, primarily DNA and RNA, are essential for the storage and expression of genetic information. Under physiological conditions, the amino and oxo tautomers of purines and pyrimidines predominate. Adenine a., guanine (G), cytosine c., thymine (T), and uracil (U) are the main building blocks of nucleic acids. Other building blocks include 5-methylcytosine, 5-hydroxymethylcytosine, pseudouridine, and N-methylated heterocycles. A -glycosidic bond connects D-ribose or 2-deoxy-D-ribose to the N-1 of a pyrimidine or the N-9 of a purine, with the syn conformers being the most common.

Nucleotides are the monomeric units of nucleic acids that contain a phosphate group attached to the sugar’s 3’ or 5’ hydroxyl. Extra phosphate groups form nucleoside diphosphates and triphosphates. These have high group transfer potential and help make covalent bonds. Cyclic phosphodiesters cAMP and cGMP act as second messengers. 3′ 5′ phosphodiester bonds link nucleotides to form polynucleotides, which have a directional 5′ and 3′ end. Four bases—A, G, C, and T—compose DNA, and hydrogen bonds (A to T, G to C) hold them together to form a double helix. Human DNA consists of approximately 3 billion base pairs organized into 23 chromosomes, encoding around 25,000 protein-coding genes and various non-coding RNAs (ncRNAs).

Introduction

Improvement of wheat grain quality depends on the knowledge of quality requirements of different end-use products and the genetic components controlling different quality traits. In previous chapters we have dealt in-depth with quality requirements of different end-use products and their genetic components. Depending upon the goal of quality improvement, different strategies are adopted to improve wheat quality. It depends upon the number of genes controlling the traits as well as the availability of the simple methods for phenotyping the traits especially during early segregating generations. In addition stability analysis of the traits across the environments is essential before we go for breeding. Constraints in time, cost, and sample availability usually necessitate the use of predictive tests during early generations, followed by milling and baking tests on advanced lines that have met a number of agronomic, disease and predictive quality criteria. Knowledge of germplasm lines having specific quality traits is needed to initiate breeding for the improvement of wheat grain quality for different end-use products. The quality requirements of different major end-use products are provided in the following table (Table 1).

Introduction

Food supply is a major concern in the near future as the human population touches 9 billion in 2050 and therefore, more food has to be produced. This has to be achieved in the face of climate change amounting to constant pressure of biotic and abiotic stresses. However, Swaminathan (2007) opined that science and technology can play a very important role in stimulating and sustaining an evergreen revolution leading to long-term increases in productivity without associated ecological harm. Rice is considered as global grain since it is consumed by more than half of the world population. In India rice supplies calorie requirement to more than 70 per cent of its population. This is evident from the fact that rice is cultivated in all the 30 states and 5 union territories of the Indian subcontinent of diverse ecologies ranging from below sea level to mountainous regions. Though India tops in rice acreage (43.61 mha) its productivity of 3.19 t/ha (rough rice) is far below world average of 4.12 t/ha and that of Asia’s average of 4.19 t/ha (Shobharani et al., 2010). This low productivity is mainly due to unfavourable ecologies of the varied ecosystems which, rice encounters during critical stages of its growth. Salinity and submergence are major abiotic stresses, next to drought, limiting rice production in South East Asia including India.

In India nearly 8 mha are salt affected of which 2 mha are coastal saline and 3.4 mha are sodic (Singh, 1994). Rice is predominant crop in these areas especially in vast stretches of eastern and western coasts of India. The presence of salts, in excess to plant growth, in the soil solution makes the soil detrimental to crop growth. The source of the excess salt may be from soil or irrigation water. The content and composition of salts (ions) in tandem with types of soil and weather conditions prevailing during the crop period decides the severity of salt stress. Similarly, f lash floods or short-term submergence regularly affect rice growing areas in South and Southeast Asia. Even more favorable irrigated areas experience flooding problems during the monsoon season. It is also prevalent in rainfed lowlands where most of the present day rice varieties do not survive complete submergence for more than a week. In India 30% of the rice growing area is prone to such menace resulting in severe yield losses (Neeraja et al., 2007).

Introduction

Over decades, improvement of domestic animal species has been done by selecting superior animals, mainly based on individual performance and their subsequent breeding to get progenies with higher production potential. Within the 20th century, understanding of inheritance of economic traits, factors influencing genetic potential and their interactions provided better criteria for selection and this has resulted into faster genetic gains. Several selection criteria which were composite ones based on the individuals as well as its relative performances were being used.

Introduction
Aquaculture has emerged as the fastest-growing sector in global agriculture. The total fish production worldwide includes both wild catches and aquaculture outputs. In recent years, fish production has consistently risen, reaching around 130.9 million tons, which accounts for 52% of the fish available for human consumption (FAO, 2024). Molecular breeding in fisheries and aquaculture utilizes cutting-edge genetic technologies to enhance favourable traits in aquatic species. This approach encompasses the application of molecular markers, genome-wide association studies (GWAS), and gene editing techniques to improve attributes such as growth rates, disease resistance, and resilience to environmental stressors. For example, marker-assisted selection (MAS) facilitates the identification and selection of superior broodstock by leveraging genetic markers associated with advantageous traits.

Introduction

Genetic programs in livestock have been extensively used to improve their productivity over the last 50 years. The traditional approach of breeding, based on quantitative genetics principle, has been very successful (Schaeffer, 2006), without knowledge of genes increasing or decreasing in herds. Effective progeny testing program and use of genetically proven bulls have made drastic genetic improvement in livestock populations. Further advances in computation for genetic merit have further added to improvement process. Livestock producers presently are focusing on overall development of livestock with improved health, production, reproduction and longevity and therefore, the traits are becoming the part of selection program (Decker et al., 2009). Advancement in molecular biology along with high-throughput genotyping has led to discovery of a number of single nucleotide polymorphisms (SNPs) in livestock (Van Tassell et al., 2008). SNP arrays covering the entire genome may explain most of the genetic variation in important traits of livestock (Meuwissen et al., 2001). These SNPs may act as molecular markers and be used as a substitute for phenotypic selection. This process, in a broad sense is known as molecular or marker-assisted breeding and describes selection strategies including marker-assisted selection (MAS) and genome wide selection (GWS) or genomic selection (GS).

Spectacular productivity gains have been achieved in wheat and rice in south and south-east Asia and also several other developing countries in Latin America and central Asia due to introduction of dwarfing genes in these two crops purely through conventional plant breeding approaches and these developments were christened as “Green Revolution” (Reynolds and Borlaug, 2006). The wheat breeding progrmme was being spearheaded by Norman Borlaug at CIMMYT (International Maize and Wheat Improvement Centre, Mexico) supported by Sanjay Rajaram and the rice breeding programme was under leadership of Gurdev Singh Khush at IRRI (International Rice Research Institute, Manila, Philippines) coinciding with these exciting developments during mid-sixties to mid-seventies. In recognition of these quantum jumps in yield of wheat and rice, Borlaug was awarded Nobel Peace Prize for Peace in 1970. Later on World Food Prize was instituted by joint efforts of Borlaug and his colleagues to honour Agricultural Scientists and others on pattern of Nobel Prize who had made significant contributions to sustainable food production and food and nutritional security globally. M. S. Swaminathan happened to be Director General, Indian Council of Agricultural Research when Green Revolution was taking roots in India and because of his enormous contributions to provide effective leadership to ensure that Green Revolution happened in India, M. S. Swaminathan is credited as “Father of Green Revolution in India” and he was the first recipient of World Food Prize in 1987 for spearheading the introduction of high-yielding wheat and rice varieties in India. G. S. Khush along with Henry Beachell received 1996 World Food Prize for achievements in enlarging and improving the global supply of rice during a time of exponential population growth and Sanjay Rajram was the 1914 World Food Prize recipient for his scientific research that led to huge increase in world wheat production.

ABSTRACT

Black pepper (Piper nigrum L.), known as the ‘King of Spices’ is an important export- oriented spice crop, valued for its medicinal and condimental properties. A perfect protocol for the rapid in vitro multiplication of this plant is available. However, systemic bacterial contamination is the single major constraint in the large scale in vitro production of plantlets. Based on morphological and biochemical characters, this bacterium was identified as Beijerinckia indica It grows profusely on nitrogen-free Jensen’s agar and solubilizes CaCO in the medium by acid production. It is resistant to a range of antibiotics and produces indole acetic acid. The bacterium is slow growing and has pH optima of 5.5 and temperature optima of 27oC. It has both endophytic as well as epiphytic association with the plant, the colonization being more in the stem and petiole. A single plasmid of 4.0 kb size was isolated from the bacterium and characterized. Competent cells of E. coli DH5a, transformed with this plasmid survived on Jensen’s nitrogen free agar, indicating its ability to fix atmospheric nitrogen. We stipulate that the nif genes are located on the plasmid of the bacterium. Since endophytic bacteria are known to stimulate plant growth through antagonistic activity against pathogens, production of growth promoting substances and inducing systemic resistance in several other agricultural crop species, the symbiotic association between Bejerinckia indica and black pepper could be further exploited through integrated nutrient management for improved growth and yield of this spice crop.

ABSTRACT

An effort was made to characterize twelve ber genotypes using RAPD markers. Of the twenty random decamer primers tested, 17 generated polymorphic bands, while rest of the primers showed monomorphic bands. The primers that amplified all the 12 genotypes were OPC-20, OPD-1, OPD-3 and OPD-4. The size of amplified DNA fragments varied from 0.56-4.26 kilobase pair. In total, 109 bands were obtained, of which 85 bands were polymorphic, while 24 bands were monomorphic. For the cultivars tested between 2-9 bands were obtained for each primer with an average of 5.45 bands per primer. The highest number of bands (9) were generated by OPA-4 and OPD-3. With un-weighted group method using arithmetic mean (UPGMA) cluster analysis, the twelve ber genotypes fell into four major clusters. The pair wise dissimilarities of ber genotypes showed that genotypes G-9 and G-8 were close to each other. Maximum dissimilarity was observed between the genotypes G-8 and G-12.

Molecular characterization involves the use of techniques that are available to analyze variation in plants and animals at the DNA level. Differences in gene sequences can be directly observed and described with a precision previously impossible to achieve. Many of the techniques that have been developed have already been used in evolutionary and taxonomic studies. They have immense value in studies of accessions identity and for the detection of novel useful variation in the plant germplasm. Molecular markers provide genetic information of direct value in key areas of conservation both ex situ and in situ. For ex situ conservation the key issues are:

ABSTRACT

The study was conducted to fingerprint and to analyze genetic diversity among 20 genotypes. of Psidium guajava and two species P. friedrichsthalianum and P. cattleianum using random amplified polymorphic DNA. Genomic DNA was isolated using modified CTAB method and polymerase chain reaction was carried out using standard protocols. In all 41 primers were used, 39 of which amplified 376 alleles with 347 polymorphic alleles. All the primers discriminated between two and more genotypes. Primers produced 2-16 alleles. The size of amplified DNA alleles ranged from 0.12-2.2 kilobase pairs. Polymorphic index content value was found to be maximum for primer OPB-12 (0.916). In the dendrogram all the 22 genotypes were grouped into two major clusters. Cluster 1 comprised of Spear Acid, Apple Colour and P. friedrichsthalianum, while Cluster 2 constituted rest of the 19 genotypes. Thus, RAPD markers detected a high level of polymorphism and can be used for breeding of improved guava varieties.

ABSTRACT

Piper colubrinum is a wild relative of the cultivated species of black pepper, Piper nigrum. The enzyme hydroxy methyl glutaryl Co A reductase which catalyses mevalonate synthesis, is the key enzyme in isoprenoid biosynthesis.This is the rate limiting step in the pathway which leads to the formation of sesquiterpene phytoalecins, a group of plant defense metabolites. Piper colubrinum is known to be tolerant to the dreaded disease called foot rot, incited by Phytophthora capsici. The hmgr gene is reported to confer resistance to fungal pathogens. Hence we made an attempt to isolate gene encoding this enzyme. cDNA was synthesised by reverse transcription from total RNA of P.colubrinum using oligo dt primers. Specific degenerate primers designed based on the conserved boxes among various plant species were used for amplifying hmgr gene from the cDNA. The 700 bp amplicon was cloned in pGEMT vector and sequenced with T7 primer. Multiple sequence alignment using clustal W 1.8 revealed homology of the cDNA fragment with hmgr genes in several other plant species. Maximum similarity was observed among P. colubrinum, Solanum xanthocarpum and Nicotiana tabacum. The cDNA library of P. colubrinum constructed in lTriplEx2 when screened with radiolabelled hmgr amplicon, yielded positive signals. Attempts are being made to isolate full length gene from these clones, which could be later used for imparting biotic stress tolerance to cultivated crop species.