eChapters

Introduction to Molecular Markers in Vegetable Breeding
As global demand for food continues to rise, the agricultural sector faces mounting challenges from climate change and resource limitations. These pressures necessitate an urgent transformation in farming practices, making molecular markers vital for enhancing crop improvement strategies (Tester and Langridge, 2010). Traditional breeding techniques, which often rely on phenotypic selection and protracted breeding cycles, find it increasingly difficult to produce resilient, high-yield cultivars (Bernardo, 2016). Molecular markers, including Single Nucleotide Polymorphisms (SNPs), Simple Sequence Repeats (SSRs), and Amplified Fragment Length Polymorphisms (AFLPs), provide direct access to plant genomes, allowing for the precise identification and manipulation of desirable traits regardless of environmental variability (Varshney et al., 2005). This approach is essential for expediting breeding programs and enhancing genetic gains, ultimately leading to crops better suited for diverse and evolving climates (Jannink et al., 2010).Additionally, the integration of genome-wide association studies (GWAS) and genomic selection (GS) harnesses high-density marker data to deepen our understanding of complex trait genetics, fostering sustainable agricultural practices and securing future food supplies (Hickey et al., 2019).

Abstract

While, life on Earth is dependent on microbes for many essential services, microbes are intimate partners in virtually every life process of plants by way of mobilization/acquisition of nutrients, out-competition and antagonism of pathogens, plant stress alleviation, plant growth promotion, co-metabolism, degradation of toxins, etc. Exploitation of such interactions between microbes and plants and optimization of plant microbiome could allow farmers to apply less chemical fertilizer, and pesticides besides improving overall plant health which paves the way for sustainable development. What is less appreciated, and less well understood, is the mechanism of microbes exerting such benefits to the plants. A greater understanding of how plants and soil microbes live together, communicate, build innate plant immunity, triggers hormone synthesis and benefit each other can therefore provide new strategies to improve plant productivity, while helping to protect the environment and maintain global biodiversity.

Nanotechnology has emerged as a revolutionary force in the food industry, providing innovative solutions to improve food quality, safety, and usefulness. Researchers have devised novel ways to numerous issues in food production and preservation by harnessing nanoparticles' unique traits, such as their high surface area-to-volume ratio and customizable physicochemical characteristics. Nanotechnology-enabled food additives provide a wide range of benefits, including improved texture, taste, color, and nutritional value, as well as increased shelf life and microbiological safety. Furthermore, nanoparticles are effective transporters for bioactive chemicals, allowing for targeted and regulated release within the food matrix.

Different basic as well as advanced laboratory equipment need to be used in a laboratory while conducting experiments. Many of them like pH meter, laminar air-flow hood, viscometer, thermometer, magnetic stirrer, microwave ovens, incubators, sand-bath, water-bath, oil-bath, etc. are the basic and simple equipment which require to be used routinely while conducting experiments in a laboratory. However, basic knowledge of their operation, handling procedure, when to be used, how to be used, and more importantly what not to be done with it, etc. are essential for keeping them in serviceable condition as well as for the accuracy/precision in their operation. In this chapter, we will discuss about some of the routinely used laboratory equipment along, while some other sophisticated equipment will be discussed in other chapters.

pH Meter

A pH meter measures exactly the H+ concentration of a solution; thus indicates acidity or alkalinity of a solution with greater precision. Actually, it measures the difference in electrical potential between a pH electrode and a reference electrode, and display the results converted into the corresponding pH value. Electrode is the key part (rod-like structure, usually made of glass) of a pH meter, having a bulb at the bottom containing a sensor. The pH meters range from simple and inexpensive pen-like devices (portable/pocket pH meter) to complex and expensive laboratory instruments with computer interface (Figure 5.1).

Frequent calibration of a pH meter using the solutions of known pH (standard solution of pH 4.01, 7.00, 10.01, Figure 5.2), generally before each use, ensures the accuracy of the instrument. For very precise work the pH meter should be calibrated before each measurement. For normal use calibration should be performed at the beginning of each day. This is necessary because the glass electrode does not give a reproducible measurement over a longer period of time.

Introduction

To accomplish experimentations in laboratories, sometimes we need microscope to test/validate our ideas/findings. A microscope is needed to examine objects which are very small which cannot be visualised by naked eyes. However, microscopic study requires some expertise in operating the microscope, particularly the sophisticated microscopes, as well as to prepare (peeling, sections, cutting, fixation, staining, dehydration, mounting, etc.) the sample, so that it can be appropriately examined for the anticipated details.

According to the scientific requirements different types of microscopes are used in the laboratories for research purpose. In this chapter, our aim is to acquaint you with the basics of using and handling of microscopes, particularly a compound microscope that is commonly used by the graduate and post graduate students for basic research.

Microscopy is often designated as the scientific investigation that is employed to review and analyse minute structures and objects using a microscope. The term “microscope” has been derived from ancient Greek words “micros” meaning “small” and “skopeîn” meaning “to look at or see,”.

Microscope

A microscope is a laboratory instrument used to examine objects (biological specimens) which are too small to be seen by the naked eye. The microscope must accomplish three tasks:

• Magnification- to provide a magnified image of the specimen.

• Resolution- to separate the details in the image.

• Contrast- to render the details clearly visible to the eyes.

Although we will tnroduce to you the different types of microscopes used for different speciments, our main focus is to discuss about compound microscope, which is commonly used in the laboratories. Compound microscope was developed around the beginning of 17th century.

A variety of glassware like burettes, pipettes, measuring cylinders, flasks, separating funnel etc. are used for experimental purposes in a laboratory. All of these need to be used and handled correctly, otherwise the chances of experimental error and damage to the glassware increase significantly. Being made of glass, they need special care while washing and using in experiments because even a minor mistake in handling of glassware may result in breakage of the apparatus. With this precautionary note, let us discuss about some of the specific glassware used in the laboratories.

Burette

Burette is a long graduated glass tube with a stopcock at one end for delivering a known/desired volume of a liquid, especially in titration experiments. It contains a stopcock at the lower end and a tapered capillary tube as the stopcock’s outlet. The flow rate of liquid from the tub stopcock’s outlet can be controlled with the help of stopcock valve. Burette is used for measuring the volume of a solution/chemical added in a quantitative (titrimetric) estimation experiment.

Procedure of using a Burette

The burette reading must be noted before and after delivering the chemical/ solution from the burette. The difference in these two readings is the volume of the chemical/solution released out of the burette. The solution should be released slowly/drop-by-drop to avoid any experimental error/mistake. If the solution/chemical is allowed to pour out too fast, the wall of the burette will not drain properly and some liquid may remain sticking to the surface of the walls. Moreover, excessive amount of chemical may be released/dropped during the experiment, particularly during the titration experiment wherein we need to know the exact amount of acid/base needed to get neutralization point. Thus, any minor mistake may lead to get a faulty reading. The measuring capacity of a burette usually used in a laboratory is 50 mL.

Since mouth pipetting of liquid reagents in a laboratory is not advisable, different tools are needed for safe, easier, and precise dispensing of liquid reagents. This chapter is devoted to make you aware of the tools like vacupette and micropipette which can be used for the handling of liquid reagents in a safe and precise way.

Vacupette

Glass pipettes have been traditionally used by sucking liquid solvent/solution directly using mouth. For safety reasons, mouth pipetting is no longer permitted in a laboratory. Hence, to fill a pipette vacupette is used by placing it on the top of glass or plastic pipette. It acts as a device to create vacuum for filling reagent in the pipette as well as for releasing the reagent with mechanical regulation on the flow out of the liquid. Vacupette can be of different types.

1. Rubber Bulb Vacupette

Rubber bulb vacupette is often used to transfer (pump up and discharge) liquid using a pipette when a larger quantity of chemicals is to be transferred. Small/tiny rubber bulb is attached to glass/plastic pipette for drawing little amount of reagents. Small vacupette is used to precisely discharge smaller volume/ drops.

Handling of Vacupette

• Insert the top of a pipette in the bottom of the rubber bulb vacupette (Figure 6.1).

• Remove air from the pipette by squeezing valve “A” at the top of the vacupette while simultaneously squeezing the rubber bulb. The amount of air released depends on the size of the rubber bulb.

Genomics-driven approaches to Genebank management

• Pre-breeding can facilitate the characterization and utilization of PGRs.

• Allele mining involves searching useful alleles of genes from a wide germplasm collection.

• Transformation of germplasm collections into bio-digital resource centres like

• DNA Barcoding,

• DNA fingerprinting.

Pre-breeding

• Pre-breeding is the step before practical breeding.

• Aim - To introduce new desirable traits/genes into an adapted genetic background. This will broaden the genetic base in a breeding material in pace with environmental changes.”

• The main link between the germplasm conservation and its use in plant breeding

• A vital contribution of pre-breeding is increasing the total genetic diversity in crops and finding specific genes and traits.

• Main link between conservation of PGR in gene bank collections and utilisation of these resources in agriculture and horticulture.

• Essential for linking genetic diversity arising from wild relatives and other unimproved materials for utilization.

• These activities are a collaboration between germplasm curator and the plant breeder who need to work germplasm collections and how new traits from these collections can bred into new varieties.

11.1 Introduction

Nanotechnology is the manipulation or self-assembly of individual atoms, molecules, or molecular clusters into structures to create materials and devices with new or vastly different properties. Nanotechnology can work from the top down (which means reducing the size of the smallest structures to the nanoscale e.g. photonics applications in nanoelectronics and nanoengineering) or the bottom up (which involves manipulating individual atoms and molecules into nanostructures and more closely resembles chemistry or biology). The definition of nanotechnology is based on the prefix “nano” which is from the Greek word meaning “dwarf”. In more technical terms, the word “nano” means 10-9, or one billionth of something. It is a very promising new branch of small-scale technology named for the unit of measure at which it operates: the nanometer. Nanotechnology enables us to create functional materials, devices, and systems by controlling matter at the atomic and molecular scales, and to exploit novel properties and phenomena. Consider that most chemical and biological sensors, as well as many physical sensors, depend on interactions occurring at these levels and you’ll get an idea of the effect nanotechnology will have on the sensor world.

VAM fungi are of worldwide distribution. They are present in the soil in form of chlamydospores, zygospores and azygospores and have been recovered from the soils of a variety of habitats e.g. agricultural fields, nutrient deficient soils, sand dunes and deserts, industrial wastes, sodic soils, polluted sites, sewage, eroded sites and others like forests, open woodlands, scrub, savana, heaths, grasslands, anthracite and bituminous coal wastes. There are several reports of VAM fungi from diverse habitats from India.

VA-mycorrhizal associations occur widely throughout the plant kingdom. They have  been reported to be present in Bryophytes, Pteridophytes, Gymnosperms and Angiosperms in nearly all the geographical regions of the world. Most of the agricultural and horticultural crops belonging to diverse families have been shown to form VA-mycorrhizae. Many species of plants of economically important families have been reported to be mycorrhizal. Likewise, many of the plantation crops like coffee, tea, citrus, timber trees, many of the fruit trees and medicinal plants have been shown to form VA-mycorrhiza.

Favourable effect of VAM association in plants has been attributed to an increased nutrient uptake by mycorrhizal roots. The transfer of ion from the soil to the roots by mass flow or by diffusion depends on the mobility of that particular ion in the soil. Ions such as NO₃^- ,SO ₄^— and Ca⁺⁺ may move rapidly through the soil to the roots by mass flow, and uptake would be limited by the absorbing capacity of the roots. On the other hand, ions such as H₂PO₄^-, NH₄⁺, Zn⁺⁺ and Cu⁺⁺ are poorly mobile and move to the roots by diffusion, the rate limiting steps is likely to be the movement to the root surface. Under such conditions, it is expected that uptake would be increased if ions could be moved rapidly through extramatrical hyphae of the VAM fungi rather than diffusing slowly through the soil. It is now well established that VA mycorrhizae enhance the uptake of phosphate by the roots but it is likely that they can also assist in the uptake of other ions. The following account provides an information on the findings of Indian mycorrhizasts on the effects of VA mycorrhizal association on nutrient uptake in the plants and its physiological implications.

Mycorrhiza research is relatively recent. Although, it was in 1885 that A.B. Frank made reference for the first time to the term “Mycorrhiza”, it took him nine more years to establish the symbiotic relationship between the fungus and its host plant. It took a much longer time to convince the rest of the scientific community of the validity of Frank’s theory. Researches carried out between 1917 to 1950 confirmed that “Mycorrhizal colonization is a beneficial symbiosis between tree and fungus”. It is an unique partnership in nature, where the higher plants share their photosynthates with their fungal partners and provide them shelter, the fungal partners in return provide protection to the root system and assist it in performing its functions especially anchorage and absorption of nutrients / water from soil more efficiently.

The ectomycorrhizal fungi have been in use as a biological tool in improving the performance of forest trees since long. Better performance of forest trees is ensured by making them mycorrhizal, employing efficient strains of ectomycorrhizal fungi.

Extensive studies have been carried out to explore the possibility of using mycorrhizal model system in inducing suppressions of potential soil-borne pathogens. Its relevance becomes all the more important since most of the soil-borne diseases, by and large, are not easily controlled by conventional methods.

Diseases due to Fungal Pathogens

Most of the studies conducted on the interaction of VAM fungi and fungal pathogens suggest that mycorrhizae decrease the severity of disease, while a few studies suggest that there is either no effect or there is an increase in the severity of disease. It has been pointed out that in view of the fact that VAM fungi deter the severity of disease they may be used as biocontrol agents. Various mechanisms by which VAM could suppress the fungal pathogens have been suggested on the basis of experimental evidences.

During the last thirty years, there has been a growing appreciation of the impor tance of VAM fungi in improving the plant growth and yield. They have been extensively studied also in India. Considering the potential importance of these organisms in the recovery of disturbed sites and their possible use in land reclamation, researches regarding their relationship to ecosystem recovery have now been initiated also in India.

Large areas of land in the country called wastelands are degraded and lying unutilized due to various constraints. Part of it called culturable, has the potential for the development of vegetation cover. On the other hand, part of it called unculturable, has no potential for the development of vegetation cover. In addition to these natural wastelands, there are large tracts of wastelands created due to human activities such as mining, deforestation etc. Such wastelands are also culturable and a vegetation cover can be developed on them after removing the constraints with some efforts.

Since a method to cultivate VAM fungi in pure culture is not available as yet, clean and high quality VAM inoculum production is not possible. However, VAM technology is gradually getting a place in agriculture as well as reclamation and afforestation programmes. The inoculum currently being used includes infested fields soils, sievings from such soils, soil and roots of greenhouse plants, or pot culture inoculum derived from surface disinfected spores of a single VAM fungus on a host plant grown in a sterilized growth medium. Attempts are being made to manipulate VAM fungi, rooting media, hosts and fertilizers in order to secure superior quality of inoculum by these methods on a large scale. In addition, other techniques like tissue culture, hydroponic system etc. are also being attempted to cultivate VAM fungi in pure culture. The Indian mycorrhizasts have also attempted to devise and develop improved methods for production and storage of commercial VAM inoculum. The following description gives an account of Indian work related to production and storage of consistently high quality disease free VAM inoculum.

Of the various microorganisms present in and around roots, VAM fungi occupy unique ecological position as they are partly inside and partly outside the host. Their internal phase encounters competition and antagonism from other beneficial symbiotic microbes while their external phase faces competition and antagonism from beneficial non-symbiotic microbes present around the roots. Since they have an ensured source of nutrients from the host, they have comparatively better conditions for their growth and may exert greater effect on plants with other symbiotic and non-symbiotic microbes in and around their roots. It has been pointed out that investigations are needed on the interactions between VAM fungi and these beneficial microbes, and their effect on growth and productivity in plants. A bulk of information has been collected related to this aspect; however, authentic generalization has not been possible as yet. Following description gives an account of the work done by Indian mycorrhizasts on the interactions between VAM fungi and other beneficial symbiotic/non-symbiotic microbes in and around the roots.

Foodgrains are a fine example of diversity of nature with many varieties and varied features. They are storable food commodities and lend themselves to numerous processing manipulations. Nutritionally foodgrains are rich source of almost all known nutrients and several phyto-chemicals. The increasing incidence of metabolic diseases are attributed to unbalanced energy rich diets lacking in fiber and protective bioactive compounds such as micronutrients and phyto-chemicals (Prakash, 2013). Cereals along with pulse combinations constitute an important part of human diet in many parts of the world because of easy availability, low cost, long shelf life and nutritional balance. During recent past, India has made significant progress in improving the food security of its masses. The green revolution of 60’s helped the country in achieving the food security by improving the availability and access components (Kaul et al., 2016). However, surplus quantity of food produced is not uniformly distributed and the quality component is not addressed properly which has a direct impact on nutrition. In spite of surplus production of foodgrains still malnutrition persists. However, it can suitably be overcome by combining cereals with good quality pulses and other value addition techniques which will go long way in solving the problem of malnutrition. Even though country is producing large quantity of coarse grains particularly maize but its utilization for direct consumption is very low which is mainly due to poor quality of protein. Although maize contains higher proportion of protein (10-12 %), its quality is poor as compared to that of protein in rice due to presence of high concentration of alcohol soluble protein fraction ‘prolamine’ also known as ‘Zein’ in the endosperm. Zein is very low in some essential amino acids, mainly lysine and tryptophan which contribute to more than 50% of the total protein. On the other hand, very high amount of leucine and imbalanced proportion of isoleucine contribute to poor quality of protein in normal maize. To overcome such deficiencies, scientists developed Quality Protein Maize (QPM) where the quality of protein is not only higher than the common maize, but also significantly higher than that of other cereal grains. In the recent past QPM has got special distinction among the cereals due to presence of high amount of essential amino acids like lysine and tryptophan. QPM can be utilized for diversified purposes in food and nutritional security as infant food, health food, convenience food, specialty food and emergency ration. In India maize is consumed mainly in the form of boiled corn or roasted as pop corn. In order to increase the proportion of maize for direct consumption, several traditional food-processing and preparation methods can be used at household level to enhance the bioavailability of micronutrients in maize based diets. These methods include alkali cooking, thermal processing, mechanical processing, soaking, germination/ malting and fermentation. In countries like Central America and Africa, it is converted into food products by grinding, alkali processing, boiling, cooking and fermentation (Srilakshmi, 2008).

5.1 Introduction
One of the main methods for cost avoidance and prevention is value engineering/ value analysis. “Society of American Value Engineers (SAVE)” states that value Analysis is the systematic use of known procedures which determine the function of a product or service, define a monetary value for the function, and provide the essential function reliably at the lowest overall cost. If these are applied to a new product, it is called value engineering. By analysing the function of any product, material part, component, system, or service, it is possible to find extraneous expenditures connected with them and effectively remove them without compromising the product’s quality, functional dependability, or ability to provide services. The principles and steps of value analysis/ value engineering will supplement the effort of design thinking.
5.2 Symptoms for Introducing Value Analysis (VA)
If one or more of the following symptoms exist, starting a VA programme is expected to yield very positive effects.
i. Sales of the company’s products are declining.
ii. The prices of the products of the company are greater than those of the products of its rivals.

Knowledge on genetic variability of the available population is very essential for any crop improvement programme, as it will positively enhance the efficiency of selection. The variability in quantitative characters increase considerably by treating the biological materials with different mutagenic agents. It is possible to identify mutants for different characteristics which can be useful in further programme. Variability is measured by different statistics viz., phenotypic coefficient of variation (PCV) and genotypic co-efficient of variation (GCV). GCV would be more useful for the assessment of inherent variability as it exhibits the heritable portion only.

Minisatellite is a section of DNA that consists of a short series of bases 10– 60bp. These occur at more than 1000 locations in the human genome. Some minisatellites contain a central (or “core”) sequence of letters “GGGCAGGANG” (where N can be any base) or more generally a strand bias with purines (Adenosine (A) and Guanine (G)) on one strand and pyrimidines (Cytosine (C) and Thymine (T)) on the other. It has been proposed that this sequence per se encourages chromosomes to swap DNA. In alternative models, it is the presence of a neighbouring cis-acting meiotic double-strand break hotspot which is the primary cause of minisatellite repeat copy number variations. Somatic changes are suggested to result from replication difficulties (which might include replication slippage, among other phenomena). When such events occur, mistakes are made, this causes minisatellites at over 1000 locations in a persons genome to have slightly different numbers of repeats, thereby making them unique. The most highly mutable minisatellite locus described so far is CEB1 (D2S90) described by Vergnaud.