eChapters

Principle

Cloning experiments would be very difficult if we do not screen those very few cells that took up the recombinant plasmid DNA, as the transformation efficiency is very low as stated before (usually less than 1 in 10⁵ cells). Hence it is necessary that there should be a selection mechanism (i.e. conditions where cells grow if they express a gene, but die if they do not) to sort out which are transformed cells and which are not make the job easy.

A commonly used selection mechanism is antibiotic resistance. If the E. coli strain used is not resistant to the antibiotic, but the plasmid DNA contains a gene coding for resistance, then only the cells that have taken up the plasmid will be able to grow. Most commonly used plasmids contain a gene for resistance to ampicillin; only cells that have taken up the plasmid will be able to grow and form colonies.

Note: Uptake of plasmid DNA is rare; hence each group of cells visible on a plate represents the offspring of a single cell that will contain a single molecule of plasmid DNA.

Zerovalent (nZVI) Ionic Compounds

Although Ag⁺ contains a filled d¹⁰ shell, the structures of Ag compounds can in many instances be understood only if one assumes the existence of weak attractive Ag⁺ - Ag⁺ interactions. Similar attractive forces have been shown to be responsible for the formation of gold cluster compounds. In Ag⁺ solid state compounds the unoccupied 5s and 5p orbitals give rise to empty bands which may be partially occupied thus resulting in subvalent Ag compounds; examples are Ag₃O and Ag₂F. The compound Ag₅SiO₄ prepared from Ag and Si0₂ at 350 °C under a high pressure of oxygen, contains [Ag₆]⁴⁺ clusters (average oxidation state 2/3) and should be formulated at [Ag₆]⁴⁺ (Ag⁺)₄ (SiO₄ ^{4 -})₂.

Ag and Au forms numerous cluster compounds in which the metals are zero valent or subvalent. Examples are Ag₆ Fe₃ (CO)₁₂ {HC(PPh₂)₃} and paramagnetic cluster ions [Ag₁₃ Fe₈ (CO)₃ ²]^4-.

Polymer Clay Nanocomposites

Polymer clay nanocomposites has been used as to affect permeability of the maternal to gases and vapours. Majority of papers on nanocomposites have been focused on the use of smectitic type clays as nanoparticles. They are a group of swelling type of clay minerals including montmorilonite, nontronite, saponite, sauconite and hectorite.

A number of polymer-layered silicates (PLS) preparation methods has been reported in literature. The three most common methods to synthesize PLS nanocomposite are intercalation of suitable monomer and subsequent in situ polymerization, intercalation of polymer from solution and polymer melt intercalation.

In general, layered silicates have layer thickness on the order of 1 nm and a very high aspect ratio (e.g. 10–1000). A few weight percent of layered silicates that are properly dispersed throughout the polymer matrix thus create much higher surface area for polymer/filler interaction as compared to conventional composites. Depending on the strength of interfacial interactions between the polymer matrix and layered silicate (modified or not), three different types of PLS nanocomposites are thermodynamically achievable (Fig. 1 and 2 ) as outlined by Sinha Ray and Okamoto (2003). They are as follows:

Biological synthesis of Zinc Oxide (ZnO) nanoparticles

Aim

To synthesize nano ZnO particles using fungal isolates.

Introduction

The ZnO nanoparticle are effective anti-bacterial and antiodur agent. Biological synthesis of ZnO nanoparticle is a new approach for environmentally benign protocol in context to green nanotechnology, (Raliya and Tarafdar, 2013).

Aim

To synthesize phosphorus (P) nanoparticles from tricalcium phosphate using fungal mycelium.

Introduction

Green nanotechnology includes a range of processes that reduce or eliminate toxic substances to restore environment. Microorganisms like fungi and bacteria having naturally bestowed property of reducing and oxidizing nanoparticles, are used for synthesis.

Aim

To synthesize gold (Au) nanoparticle using fungal mycelium.

Introduction

Biosynthesis of nanoparticles is a kind of bottom up approach where the main reaction occurring is reduction/oxidation. The fungal enzymes are usually responsible for reduction of metal compounds into their respective nanoparticles.

Aim

Phytosynthesis of silver (Ag) nanoparticles using Cassia roxburghii aqueous leaf extract.

Introduction

The Ag nanoparticles constantly exhibit adverse effects on microbes such as inhibition and inactivation (Nasrollahi et al., 2011). The Ag nanoparticles synthesized by using various biological sources such as herbs, plants and biological organisms display excellent properties in various fields i.e., non-linear optics and intercalation materials for electrical batteries as optical receptors, catalysts in chemical reactions biolabelling and as antibacterials (Sanghi and Verma, 2009).

Chemical Synthesis Methods

Several methods can be used for the production of these nanoparticles, namely: (i) top-down methods (Li et al., 2006) such as vacuum sputtering (Kuhn et al., 2002) or the decomposition of iron penta carbonyl (Fe(CO)5) in organic solvents (Karlsson et al., 2005); and (ii) bottom-up methods that promote the ‘growth’ of the nanostructures via chemical synthesis, for example through the reaction of iron(II) or iron(III) salts with sodium borohydride (Wang and Zhang, 1997). However, these methods present several limitations and problems; the top-down methods are generally expensive and require specific and costly equipment while the drawbacks of the bottom-up approaches are related to safety issues due to the toxicity of sodium borohydride, the production of flammable hydrogen gas during the process (Li et al., 2006), and the tendency to form large agglomerates, very fast and at a high extent, and therefore have a reduced reactivity and degradation efficiency.

The Madrid System for the International Registration of Marks is governed by two treaties. The first one is he Madrid Agreement, which was concluded in 1891 and revised at Brussels (1900), Washington (1911), The Hague (1925), London (1934), Nice (1957) and Stockholm (1967), and amended in 1979. The other one is the Protocol related to this agreement which was concluded in 1989. The aims of this protocol are to make the Madrid system more flexible and more compatible with the domestic legislation of certain countries or intergovernmental organizations that had not been able to accede to the Agreement. 

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

Characterization Tools: Microscopy

It is the most significant international convention in the field of protecting copyright. The Berne Convention deals with the protection of works and the rights of their authors. It is based on three basic principles and contains a series of provisions determining the minimum protection to be granted, as well as special provisions available to developing countries that want to make use of them. The Berne Convention, signed on 9 September, 1886, was revised at Paris in 1896 and at Berlin in 1908, completed at Berne (Switzerland) in 1914, revised at Rome in 1928, at Brussels in 1948, at Stockholm in 1967 and at Paris in 1971, and was amended in 1979.

Principle

Orchid seeds are produced in large numbers but are exceedingly small, hence are also called “dust seeds” may be as small as 0.01 by 0.05 mm in dimension and weigh as little as 10-30 ug. Seedling development is generally a slow process in orchids, with the seedlings passing through an initial stage, termed the protocorm stage, prior to the production of distinct vegetative organs (leaves, roots, etc). The protocorms of terrestrial orchids are subterranean and entirely heterotrophic, with all of the nutritional needs of the orchid being supplied by the mycorrhizae (the symbiotic fungus). The length of the protocorm stage is variable, however, in many cases, months, or even years, elapse before leaves and roots are produced.

The combination of a small seed size and fungal symbiont requirement largely precludes the propagation via conventional seed propagation protocols. Accordingly, most of the early research on the development of orchid seed germination protocols focused on in vitro germination with the seeds and fungal symbiont being sown on the same petri plate. Successful protocols were developed for several species; however, the process was complicated by the diversity of fungal symbionts required (across orchid taxa) and the difficulty in isolating and culturing these fungi.

In the 1920’s the process of orchid seed propagation was revolutionized by Lewis Knudson with the demonstration that nutrient media that could be used as a substitute for fungal infection. The isolation and culture of multiple fungal strains was no longer required, and improvements in germination frequency, germination speed, and seedling vigor were generally observed. To date, successful asymbiotic germination techniques, termed ‘flasking’, have been developed for many epiphytic orchids, with several species being saved from extinction via the application of these techniques.

Principle

Micropropagation is especially useful for include propagation of high value plants that grow slowly (bulbs, ferns, orchids, palms, etc), new cultivars having limited stock and high market demand (new cultivars produced via conventional breeding or genetic engineering), cultivars that are difficult to root by conventional techniques, and endangered plant species. In addition, virus elimination is possible via culture initiation from meristem explants. Also, micropropagation provides a source of sterile tissues that can be used for other tissue culture protocols, including genetic transformation.

Micropropagation is possible using multiple types of cultures; however, shoot cultures are typically employed, in which case five stages to the micropropagation process are typically recognized as outlined below. Nodal explants of rose are used for culture initiation. Three media, differing in hormone content, is employed and the explants should be evaluated for differences in axillary shoot growth and proliferation.

Bulk population breeding developed by the Swede H. Nilsson Ehle in 1908 and later on supported and practiced by H. V. Harlan in barley breeding in 1940s is a breeding strategy in crop improvement in which natural selection is allowed to play its full role where the F2 generation is planted in a plot large enough to accommodate several hundreds of plants. Planting density and cultural practices are usually the normal ones followed for commercial crop planting. At maturity, the plot is harvested in bulk and the seeds are used to plant a similar plot in the next season. This process is repeated till at least F5 where the plants are space planted to allow individual plant evaluation for effective selection. During period of bulk propagation natural selection plays a key role in shifting gene frequencies in the bulk population. This role is of crucial importance as bulk handling is continued over a number of generations. Bulk population handling can be aided by artificial selection at any time during the period of bulk propagation. Aggressive and highly competitive but undesirable plants may be physically rogued out of the population to avoid increasing the frequency of undesirable genes. For example, determinate bush types of beans do not compete well against indeterminate, vine types. And if the breeder is interested only in determinate types, artificial selection against the indeterminate types is certainly called for. There is also good reason to apply artificial selection against seed colour, pubescence, awns or other characters that are undesirable in the variety to be developed but may be more or less neutral in survival. Bulk population handling often provides opportunities to make use of artificial aids to selection. For example, bulk harvesting when only part of plants are mature is an effective and inexpensive way to select for earliness. If particular sizes of seeds are required, screens or mechanical devices provide a rapid way of eliminating inferior types. Naturally occurring or artificially induced disease or insect infestation, of course, provide opportunities to rogue susceptible individuals from the bulk plot. In F6, the plants selected in F5 are progeny rowed and superior progenies are selected from where again few plants can be selected and seed bulked within a selected row. Depending upon seed requirement for yield trial in the following years, even the seed from selected entire row can be bulked. The follow up yield evaluation trials are as given in case of pedigree selection. This method is operationally best suited for seed crops such as small grains, beans and soybean and is almost totally unsuited for fruit crops and for most vegetable crops.

Centre of origin are also known as centre of diversity. N. I. Vavilov in 1926 has proposed that crop plants evolved from wild species in the areas showing diversity and termed them as primary centers of origin. Primary centers, crops moved to other areas with the movements of man. But in some areas, certain crop species show considerable diversity of forms although they did not originate there. Such areas are known as secondary centers of origin of these species. Genetic diversity is essential for crop improvement. Vavilov has suggested eight main centers of origin of crop plants.

1. Chinese Centre

This centre is considered to be one of the earliest and largest independent center of origin of cultivated plants. This centre includes mountain regions of central and western China. The endemic species listed from this centre include, soya bean, radish, turnip, pear, peach, plum, colocasia, buckwheat, opium poppy, brinjal, apricots, oranges, china tea etc.

2. Himalayan Centre

This centre is also known as the Indian centre of origin. This centre includes regions of Assam, Burma, Indo-china and Malayan Archipelago. The crops originated from this centre are rice, red gram, chick pea, pea, mung, brinjal, cucumber, sugar cane, black pepper, moth bean, rice bean, cotton, turmeric, indigo, millets etc.

Germplasm is the sum total of all the genes and their alleles present in a crop and its related species. Germplasm is also termed as genetic resources. It may be release as a variety, subjected to selection for developing a variety, and may be used as a parent in hybridization programme. They may be obtained from gene banks, center of diversity, gene sanctuary, farmer's field and seed companies.

Types of Germplasm

1. Land races

These are primitive varieties/cultivars. They are storehouse of genetic variability. Land races were first collected and studies by N.I.Vavilov in rice. They are the source of development of varieties in many valuable crops.

2. Obsolete varieties

The improved varieties of recent past are called as obsolete varieties. These varieties were breed by systematic breeding methods, were once commercially cultivated, but are no more grown. For example, wheat varieties K65, Ph591, many NP series etc.

Pollination can be defined as the pre-fertilization event or process where pollen grains from anther are transferred to the stigma of a flower. Pollination is the process that helps to unite the male and female gametes and thus helps in fertilization. It can be broadly classified into two, cross-pollination and self-pollination.

1. Self-Pollination

Self-pollination is the process by transferring the pollen grains directly from anther into the stigma of the same flower. It is called as self-pollination. This form of pollination is common in hermaphrodite or dioecious plants which have both male and female sexual organs on the same flower. In self-pollinating flowers, the anthers and stigma are of similar lengths to facilitate the transfer of pollen. Self – pollination can be further divided into two types:

The mating or crossing between two genetically dissimilar plants or lines is known as hybridization. In other words, hybridization is an artificial controlled pollination in which pollen grains of desired male parent is transferred on the stigma of the desired female parent.

Objectives of Hybridization

To create genetic variability, which is pre-requisite for crop improvement programme.

To transfer one or more characters into single plant.

To know the inheritance of the characters.

To estimate the general combining ability of the parents and specific combining ability of the crosses.

To exploit and utilize the hybrid vigour.

To produce hybrid/synthetic varieties.

Reproduction that does not involve the union of male and female gametes is known as asexual reproduction. Reproduction in which sexual organs or related structures take part but fertilization does not occur, or crops which are propagated asexually or by vegetative means are known as asexually propagated crops. Most of the fruit plants are propagated asexually which consist of large number of clones. Example: sugarcane, potato, sweet potato, banana, mango, citrus, pears, peaches, litchi, crops that propagates by asexual means. The procedure of selection used for asexually propagated crops is known as clonal selection, since the selected plants are used to produce new clones or superior clones can be isolated from local variety, introduced variety or inter crossed population.