eChapters

Nanotechnology offers unique opportunities for the development of novel materials and applications. It is set to offer a platform to revolutionize agriculture sector from production, protection, processing and storage (Kuzma and Verhage, 2006). Bulk of nanomaterials produced is used in other sectors; only ten per cent is of use in food, beverages and packaging sector. Studies have projected the opportunities that nanotechnologies might bring to developing countries in the fields of water, energy, food and agriculture and health (Salamanca- Buentello et al., 2005) The key contributions due to application of nanotechnology in agriculture are delivery systems that aid in slow release and efficient delivery of agroinputs.

In recent years application of nanotechnology in various fields including agriculture has received increased attention. Nanotechnology offers an important role in improving the existing crop management techniques especially in plant disease management by controlled and targeted release of agrochemicals to enhance their efficiency. The nano-based delivery systems are beneficial because of improvement in efficacy due to their higher surface area, higher solubility, induction of systemic activity, higher mobility and lower toxicity (Sasson et al., 2007). The pharmacokinetic parameters of nano particles may be altered according to size, shape and surface functionalization. They can also be used to alter the kinetic profiles of drug release leading to more sustained release of drug there by reducing the frequent application (Sharon et al., 2010). The successful use of metal nanoparticles in medical streams as antimicrobial agents has also led to their applications in controlling phyto pathogens (Nair et al., 2010).

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.

Variation in Chromosome Structure

Natural variation exists in chromosome structure

Changes in chromosome structure include deletions, duplications, inversions, and translocations

The loss of genetic material in a deletion tends to be detrimental to an organism

Duplications tend to be less harmful than deletions

Duplications provide additional material for gene evolution, sometimes leading to the formation of gene families

Copy number variation is relatively common among members of the same species

Comparative genomic hybridization is used to detect chromosome deletions and duplications

Inversions often occur without phenotypic consequences

Inversion heterozygotes may produce abnormal chromosomes due to crossing over

Translocations involve exchanges between different chromosomes

Individuals with reciprocal translocations may produce abnormal gametes due to the segregation of chromosomes

A successfully developed new cultivar should have stable performance and broad adaptation over a range of environments, seasons and locations, in addition to high yield potential. Evaluating performance stability and range of adaptations is becoming increasingly important in breeding programs. Since stability and adaptability are important selection criteria in breeding programs, in depth research on stability is needed for a better estimate of crop performance and adaptability (Lin and Binns, 1988).

Having realized the importance of genotype × environment, several biometrical models have been developed to analyse the stability of genotypes. Such models have been discussed, critically reviewed comprehensively by Knight (1970), Freeman (1973), Hill (1975), Brain (1986) and Gautam et al. (1986).

However, the various methods proposed for the statistical analysis of genotype × environment are based on one of following approaches (Jain, 1982). In the first the variance interactions are estimated by equating the observed mean squares in the analysis of variance to their expectations on the random model. The second approach called phenotypic approach by Hill (1975) used the technique of partitioning the genotype _× environment component of variability into its linear and non-linear portions of assessing the stability of genotypes over a range of environments. This approach which is very widely adopted in plant breeding, came to be known as “Joint regression analysis”.

Many workers contributed for the development of this approach, notably Finlay and Wilkinson (1963), Eberhart and Russell (1966), Freeman and Perkins (1971) and Shukla (1972). The third approach called genotypic approach which also suggested by Hill (1975) is based on fitting of general models in which it specify, the contribution of genetic, environmental and genotype × environment to the generation means and variance. The contributions for he development of this approach were from Bucio (1966), Bucio and Hill (1966), Bucio et al. (1969), Perkins and Jinks (1968), Breese (1969), Hill and Perkins (1969) and Perkins (1970). Linear regression and joint regression models were more commonly used due to their simplicity and reliability in biological experiments. In general, these approaches fall conveniently into two parts.

Ramya (2012) studied stability of 12 hybrids in three environments. The researcher selected the 12 hybrids based on the standard heterosis. The hybrids are: P₂ × P₅, P₁ × P₅, P₃ × P₅, P₂ × P₇, P₂ × P₃ and P₁ × P₂. Among the twelve hybrids, the cross combinations viz., P₂ × P₅, P₂ × P₇ and P₂ × P₃ showed non-significant reciprocal effects for most of the traits including fruit yield per plant. The direct hybrid P₁ × P₂ showed less than 40 per cent standard heterosis for fruit yield per plant. It also exhibited low standard heterosis for number of fruits per plant. Eight hybrids viz., P₂ × P₅, P₅ × P₁, P₃ × P₅, P₂ × P₇, P₁ × P₅, P₂ × P₃, P₂ × P₁, P₅ × P₃, based on standard heterosis (>40 per cent) and their parents viz., P₁, P₂, P₃, P₅, P₇ were selected and grown in three different locations for finding their stable performance. Several authors have suggested different types of models to identify the stable genotypes. The model most commonly preferred by breeders is Eberhart and Russell (1966) model which was used in the present investigation. Various stability parameters were worked out on the available data and the results were discussed below.

Among the ten traits studied, days to first flowering, fruit weight and fruit girth showed non-significant genotype × environment interaction. Hence, the remaining seven traits were taken for further analysis. ANOVA across environment revealed highly significant mean sum of square for genotypes, indicating the presence of wide genotypic variability among the genotypes involved in the study for all the characters studied. High significant differences were observed for environments for the traits viz., number of branches per plant, fruit length and fruit yield per plant, indicating the divergence among the growing environments and differential response of genotypes to the various environments. The main factors contributing to the divergence of the environments might be temperature differences during crop season, intensity and duration of sun shine hours. Therefore, the genotypes must be tested over an extensive range of environments for proper assessment of stability of genotypes (Salesh Kumar Jindal et al.,2008).

This is a procedure for transforming bacterial cells with circular DNA plasmids containing an origin of autonomous DNA replication and a selective marker (antibiotic resistance). Logarithmically growing cells are made permeable to DNA by incubation in CaCl2 and the DNA is taken up from the surrounding buffer.

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

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

The polymerase chain reaction (PCR) is a very powerful new method used for in vitro to amplify a deoxynibonuclic specific region that lies between two regions of known DNA sequence single molecule to million folds in short time. This is achieved by an enzyme taq. DNA polymerase which is thermostable using this enzyme can synthesize microgram quantities of DNA of specified length and sequence from pictogram quantities of complex template in a few hours time.

Objectives

• Differentiate between fixatives and stains.
• Describe methods of staining.
• List the commonly used fixatives and stains and their composition.
• Describe the uses of above mentioned solutions and stains.

Introduction

Every time a part of an organism is removed, the action of bacteria will soon disintegrate, dry or putrefy it. They must be kept as close to normal as possible or in other words preserved and fixed in order to study these. Chemicals are required in many practical of Biology. Some of these chemicals preserve and specifically colour certain cell parts while others are used as solutions. You will learn about certain fixatives (preservatives) and stains in this chapter.

Objectives

• Detection of linkage using Chi-square test for goodness of fit.

Introduction

The tendency of two or more genes to stay together during inheritance is known as linkage and it is the consequence of the concerned genes being located in the same chromosome. Linked genes do not show independent segregation, as a result the ratios obtained in F₂ and test cross generation are significantly different from the expected ratios of 9:3:3:1 and 1:1:1:1, respectively.

Detection of linkage χ² using test

In genetic experiments, when the material under study is segregating simultaneously for two characters, it may be of interest to know whether the two characters are inherited independently or they are linked. The χ² test can be used for this purpose. χ² test is used in detecting linkage in back crosses and inter crosses.

First the χ² is computed based on the theoretical segregating ratios. If χ² is not significant it may be concluded that the loci segregate independently. If χ² is significant we may suspect linkage between the loci. The χ² is then partitioned into components assignable to different factors.

Objectives

• Study of gene mapping.

Introduction

All the genes that are linked together form a linkage group. Genes of a linkage group may be presented on a single straight line in the same order in which they are normally present on the concerned chromosome. The number of different linkage groups in a species is, as a rule, equals to its gametic chromosome number (n). The distance between two neighbouring genes is proportional to the frequency of recombination (%) between them depicted on a line drawing. Such a line drawing depicting the linked genes the recombination frequencies between them is known as Linkage map, Genetic map or Chromosome map. Linkage maps provide information on the genes that are linked together and the frequencies of recombination that may be expected between them. The frequency of recombination between two linked genes cannot exceed 50% which is the frequency in case of independent segregation.

Objectives

• Better understand the phenomenon of probability.

Introduction

The concept of probability is basis to many principles of classical genetics (segregation, independent assortment etc.) The principals of population genetics are based on the considerations of laws of probability. Probability is the likelihood of occurrence of an event.

Trial

A trial refers to an opportunity provided for an event to occur, e.g. tossing of a coin once constitute one trial as it provides an opportunity for head or tail to occur. A new borne can be either a boy or a girl.

The discovery of mutagenic effects of X-rays on the fruit fly (Drosophila) by H. Muller in 1927 and by L. J. Stadler in barley and maize in 1928 paved the way for researchers to experiment with its effects on various organisms. In 1928, H. Stubbe showed the use of mutagenesis in producing mutants in crop plants like tomato, soybean and others. The first commercial mutant was produced in tobacco in 1934. Reports by B. Sigurbjornsson and A. Micke mentioned 77 cultivars that were developed through mutagenesis prior to 1995. In 1995, this number was 484. This number has since been significantly increased covering a large number of crops, namely, corn, wheat, pea, chrysanthemum, poinsettia, dahlia, citrus, apple, peach and the modified traits include plant maturity, winter hardiness, lodging resistance, protein and lysine content and numerous ornamental mutants. By the end of 20th century, the number of mutant varieties rose to 2252 covering a range of crops like cereals, oilseeds, pulses, vegetables, fruits, fibres, and ornamentals. Majority of these mutants were released as direct mutants and the rest as a result of cross breeding with the mutants. Most of the mutant varieties have come as a result of using physical mutagens (X-rays, gamma rays, thermal and fast neutrons) with gamma rays alone accounting for more than 60% of the mutant varieties. By 2017, nearly 3250 mutant varieties belonging to 175 plant species have been developed and released where China, Japan, India, Russia, the Netherlands, Germany and USA are the leading countries. India with 345 commercialized mutants stands third after China (810 mutants) and Japan (481 mutants). Thus mutation breeding is a significant contributor to the development and release of commercial cultivars in several crops and for several traits.

The concept of recurrent selection grew out of need to increase the number of superior genotypes that could be obtained from breeding stock of corn. This could have been possible by increasing the frequency of superior genes in the gene pool and by increasing the chances for genetic recombination to occur. Both can be achieved by recurrent selection which is defined as “a method of breeding designed to concentrate favourable genes scattered among a number of individuals by selecting in each generation among the progeny produced by mating inter se of the selected individuals (or their selfed progeny) of the previous generation.” The original idea behind recurrent selection was proposed first by Hayes and Gerber in 1919 and independently by East and Jones in 1920. Jenkins (1940) was the first to describe the method in detail and Hull (1945) proposed the name ‘recurrent selection’. Each cycle of recurrent selection requires (i) evaluation and selfing of selections and (ii) crossing of the progenies of superior selfed plants in all combinations and bulking of equal amount of seed from each cross. The selection of plants before selfing is possible for maturity, plant height, ear/head characters, kernel characteristics and resistance to diseases and insects. In general, these are the characters which have high heritability. It is clear that the selection of plants before selfing is more for morphological traits and these evaluations do not include yield. Yield evaluations are done on the top cross progenies (crossing of selected plants to a tester). Such top crosses will be grown in the second year of the cycle during which time the selfed seed is held over until the evaluations for yield are over. Or the selfed seed may be grown out as S₁ lines which in turn are selfed. After yield evaluation is over, the selfed seed of superior plants is grown out for crossing. Progenies of selfed plants are crossed in all possible combinations. This is done manually or by growing progenies together in an isolated block. In isolation planting, random mating should be ensured. After manual crossing or random mating in isolation, equal amounts of seed of each are bulked and this bulk seed is used to start the second cycle of selection, selfing and crossing.

When conditions in the plant growing environment are less than the optimum, the plants experience stress which is broadly termed as abiotic stress. Major abiotic stresses impacting crop yield significantly are as follows (Grover et al., 2004; Acquaah, 2007):

Drought: This is the environmental condition caused by lack of adequate rainfall. This is also termed as water stress or desiccation stress. Effects of drought on crop plants are complex and variable and are greatly accentuated by a number of interacting factors. The onset of drought in general has been observed to reduce germination, emergence, hypocotyl length, water uptake and the mobilization of dry matter reserves even at the early growth stages. Drought stress causes a marked reduction in leaf area. Soil moisture availability at the seed germination, anthesis and at post-anthesis growth stages determines as to what extent production potential of crops would be expected in drought-prone situations.

The yield losses in field crops may be attributed to inadequate water availability at the critical growth stages. Crops therefore differ in their susceptibility of growth stages to water stress. Stress exposed plants immediately lower down relative water content (RWC) of their leaves, leaf water potential, osmotic potential. Turgor pressure may also be decreased unless plants have finally adapted to drought by opening of stomata again and improving the water potential. It has been reported that low leaf expansion rate at the early stage and accelerated leaf senescence accompanied by mortality of older leaves at later stage decreases total leaf green area.

When there is indication of linkage from F or testcross data through significant deviation from 9: 3: 3: 1 and 1: 1: 1: 1 ratio, it is implied that independent assortment is not taking place and this is taken as an indication of linkage. The inverse of linkage strength is the recombination value and is often symbolized by ‘p’ whose maximum value is 50% because out of four strands of a tetrad, two are always parental types and two are recombinant types when all the cells show crossing over.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.