eChapters

1. Introduction

Microbial diversity is becoming increasingly essential with the requirement to preserve the integrity, function and long-term sustainability of natural and managed ecosystems. However, our knowledge of the diversity of microbes remains obscure due to lack of appropriate approaches to evaluate the contribution of different components of the soil microbes to ecosystem. This lack of approaches together with the complex and heterogeneous matrix that soil microbes are embedded in, poses great challenges to understand the dynamics of microbial communities in soils (Nautiyal et al. 2010; Chauhan et al. 2013).

In classical terms, diversity is described as the total number of species present, i.e. species richness or species abundance, and the distribution of individuals among those species i.e. species evenness or species equitability. However, the incorporation of these two components into diversity indices has led to much controversy among ecologists (Atlas 1984). The Shannon-Weaver index (Shannon and Weaver 1949) is probably the most widely used index for measuring diversity of a microbial community. But this index, as many others that have been proposed, has its own limitations. A major difficulty is that both richness and evenness play a role in determining the value of the index. Therefore, any diversity index is a single value and thus cannot indicate the total make-up of a community. It is recommended that evenness, richness and diversity values should be calculated in order to obtain an objective assessment of community structure (Atlas 1984). From molecular point of view, diversity often refers to the number of different sequence type present in a habitat e.g. a soil (Ogram 2000). Measuring this type of diversity does not give information about the frequency of individual species, but it can give valuable information about changes in the community structure and species richness in response to changes in the physico-chemical properties of the soil and soil management practices (Nautiyal et al. 2010).

All separation procedures depend mainly on some physical characteristics of the compounds. It is relatively easy to separate compounds that have significantly different physical characteristics by simple techniques such as solvent extraction. If the various compounds are similar to each other in their molecular size, any slight difference in any one of their physical properties is exploited to achieve separation. 

Seed is a basic input to raise the crop plants. Healthy seeds produces the healthy crop. However, the seed carries different pathogens either externally or sometimes internally in the seed to affect the seed emergence and healthy seedling growth. Therefore, the seed treatment with fungicides/bactericides, based on the infection of the pathogen, is highly essential to counter the attack and infection of these pathogens to the germinating seedlings.

Objectives

1. To know the methods of essential oil extraction of spices and condiments.
2. To know the various chemicals present in the essential oil of different spices and condiments.

Essential oil: Essential oils are the liquids that are isolated from plants when introduced to solvents. They are liquefied versions of the plants.

Extraction methods of essential oil

1. Steam Distillation
2. Solvent Extraction
   1. CO₂ Extraction
   2. Maceration
   3. Enfleurage
3. Water Distillation
4. Water and Steam Distillation

Extraction is a process whereby the desired constituents of a plant are removed using a solvent. The primary ways for extraction of organic molecules of interest to biologists and medical investigators involve breaking open the cells. Cell rupturing is carried out in a variety of ways. The method used depends on the type of tissue used. For plant cells grown in cell suspension culture or for plant callus tissue, a French press or a sonicator can be used to break open the cell.

Objective

1. To know the different types of fertilizer application methods applied in spices and condiments.

Methods of fertilizer application: The different methods of fertilizer application are as follows:

a) Broadcasting

1. It refers to spreading fertilizers uniformly all over the field.
2. Suitable for crops with dense stand, the plant roots permeate the whole volume of the soil, large doses of fertilizers are applied and insoluble phosphatic fertilizers such as rock phosphate are used.

Broadcasting of fertilizers is of two types

1. Broadcasting at sowing or planting (Basal application): The main objectives of broadcasting the fertilizers at sowing time are to uniformly distribute the fertilizer over the entire field and to mix it with soil.

After isolation and purification of the compound, it has to be identified. It is necessary to find out the class of the compound first and then to find out the specific compound within the class. The class of compound is usually determined from the colour tests, solubility, Rf properties and UV spectral characteristics of the compound. Further identification within the class depends on measuring physical properties and spectral measurements and then comparing these data with those in the literature.

19.1 Introduction

In self-pollinated crops, the methods of selection aim at improving individual plants. On the other hand, in cross-pollinated crops they aim at improving a population of plants. A population is a large group of interbreeding individuals. Hence, selection is called population improvement in cross-pollinated crops. Selection in self-pollinated crops results in isolation of pure breeding lines which is the end product. But in cross-pollinated crops, it results in an improved population which is used as the commercial cultivar. Improved inbred lines (also called inbreds) for producing hybrids can be obtained from such populations. Again, like many rounds of selection being practised in different inbreeding generations in self-pollinated crops we are also practising many rounds of selection in cross-pollinated crops. But, unlike self-pollinated crops selection in cross-pollinated crops is followed by intermating to develop a new population before starting another round of selection. One cycle of selection consists of selection of individual/family and intermating of the selected units. Such cycle is repeated till we get the improved population. This method of practising the cycles of selection and recombination in a population is called recurrent selection or population improvement. Frey (1983) prefers the use of recurrent selection for closed populations where no new germplasm is added to the population during selection. Population improvement was used for a group of breeding techniques where all aspects are similar to recurrent selection but with the provision of adding new sources of germplasm whenever feasible. It is only a matter of coincidence that past history of application of recurrent selection has been limited to closed populations, but its basic aim has always been the accumulation of favourable genes, without any restriction on the migration of new genes during the course of selection. Apart from a slight nuance, population improvement and recurrent selection are two titles of the same principle and procedure of manipulating the genetic potential of populations. Recurrent selection can also be applied in self-pollinated crops.

17.1 Introduction

Self-pollinated vegetable, spice and ornamental crops may be heterogeneous. It is exemplified by landrace cultivars. They may have originated from single plant selections but have been grown over a long period of time. Landrace cultivars are a mixture of genotypes, perhaps because of breeders’ lack of appreciation of selection techniques to produce a pure line, spontaneous mutations, chance hybridization, or a combination of factors. Natural selection in landraces has resulted in a cultivar that is well adapted to the local environment. Depending on amount of human selection, differences among genotypes in a landrace cultivar may be small and inconspicuous. On the other hand, a lack of human intervention may mean that differences among plants in a landrace cultivar will be conspicuous. After years of self-pollination, individual plants are homozygous. Selection of such individuals would result in a true-to-type reproduction of their phenotype within the limitations imposed by the environment.

18.1 Introduction

The genetic variability present in populations of self-pollinated vegetable, spice and ornamental crops is rapidly exhausted by pureline selection. For further improvement, genetic variability must be created. This is generally achieved by hybridization. After the cross has been made, F₁ is grown and seeds set on it are harvested. This makes the seed source for raising F₂ population, and so on. The segregating populations from crosses are handled according to a suitable method. The three basic methods available for this purpose are: (1) pedigree method, (2) bulk method, and (3) backcross method. There are also certain modifications of these methods.

18.2 Pedigree Method

Pedigree method is developed from the pureline system and widely used method of plant breeding for self-pollinated crops. A main difference between pedigree selection and mass selection or pureline selection is that hybridization is used to create variability for the base population in case of the former, whereas in mass and pureline selections variability is naturally present in the base populations. The pedigree method was first described by H.H. Lowe (1927). This method predominated the crop improvement programme throughout the world until 1980s. Pedigree method is common in horticultural crops, especially where readily identifiable qualitative traits are involved. Vegetable crops in which pedigree system is used include tomato, cucumber, pepper, pea, lettuce, snap and field beans, all self-pollinated crops. In general, selection is done on F₂ plants. Yield is important in many horticultural plants, but quality is equally or even more important. Quality may be associated with identifiable traits, making the pedigree system ideal for self-pollinated vegetable crops.

Methylene-cyclo-propyl-glycine (MCPG), is a cyclopropyl derivative, grouped into the amino acids that have been isolated from the higher plants (Fowden, 1960). MCPG was identified to be closely related to two other cyclopropyl derivatives (Fig. 1), 1-aminocyclopropane – 1 – carboxylic acid from Vacciniumvitis-idaea and perry pears (Vahatalo and Viertanen, 1957; Burroughs, 1957) and â- methylenecyclopropyl alanine (MCPA or Hypoglycin A) from Blighia sapida (Hassall and Rayle, 1955; Anderson et al., 1958; Ellington, Hassall and Plimmer, 1958).

Micro propagation in also called as in vitro propagation. In vitro is a Latin word, meaning ‘in glass’. It refers to procedures done in controlled artificial environment, outside a living organism. These procedures naturally occur in the living system. Opposite to in vitro is in vivo which is also a Latin word, meaning ‘in living’. Biological processes that occur within the living system.

Micro propagation refers to the multiplication of plants, in aseptic conditions in containers under controlled nutritional and environmental condition. Micropropagation is done in artificial growth medium from various plant parts like meristem tip, embryos, anthers, axillary buds, leaf section, shootlips etc. It is a method by which a true to type and disease free plant can be regenerated from a miniature piece of plant throughout the year. Some popularly grown crops from tissue culture are as follows

Ornamental plants - orchids, lilium, gerbera, cordyline, begonia, ficus, etc.

In the area of pharmaceutical discovery, screening methods continue to play a major role in drug discovery. The screening process is simply testing molecular entities (compounds/natural products) for activity to advance those that exhibit the desired activity. This process has evolved over time in both the testing methods and in the degree of testing. Originally, the screening was done entirely by dosing animals and studying their responses. Then came test tube experiments and now, testing is conducted either with cell lines or with purified proteins in miniaturized test tubes called microplate wells. Screening by microplate has allowed the degree of testing to increase from a very small selected set of compounds to libraries, which contain thousands of test substances. Technological improvements in many areas of science and information technology have led to the evolution of screening methods from time consuming, animal dosing studies to high throughput screening (HTS) methods that have the potential to generate very large datasets of information in a very short time. Today, developments in bioinformatics have opened up the possibility of virtual (in silico) screens for targets where there is sufficient knowledge of the target or of its ligand. High throughput screening has become a pivotal part of drug discovery for identifying potential lead molecules for specific disease targets. The success of screening is measured by several factors including the throughput and costs of the assays, but ultimately by how many of the HTS hits progress forward through the drug discovery pipeline. Miniaturization of assays by conversion from 96- to 384- to 1536- wells to well-less platforms, such as microarrays and the utilization of automated equipments are methodologies implemented to increase the throughput and reduce the cost of the screen.

ABSTRACT

Wood apple is a hardy tree grown in arid and semiarid regions of the country. It is generally propagated through seeds. The traditional methods like budding and inarch grafting require 9 to 12 months for the root stock to come buddable size and thus 1½ to 2 years to become ready for transplanting. The present study was initiated to prepare a grafts within eight months by adopting the micrografting technique. The seeds were sown in the month of April in polybags filled with FYM, sand and soil in equal proportions with a pinch of blitox. The micrografting was done on 1½, 2, 2½ and 3 month old seedling root stock having a diameter of 0.13 to 0.15 cm, 0.17 to 0.19 cm, 0.20 to 0.22 cm and 0.23 to 0.26 cm, respectively. The seedling root stocks were decapitated with the surgical blade and 1 to 2 cm deep split was made. The scion micro bud (1 to 2 cm length and 1 to 2 mm width) of previous season’s growth was inserted in the split. The micropipette cap (0-200 μl) was inserted on the grafted portion. After sprouting of the micro bud, the micro pipette cap was removed. The study revealed that, micro bud grafting success was significantly influenced by the root stock age. Early bud take was observed when the micrografting was done on 2½ month (10.73 days) and 3-month (11.00 days) old root stock. The percent bud take on 3-month old root stock was significantly higher (99.33 per cent) than the rest of root stocks. Micrografting done on 3-month old seedling root stocks in June-July is suggested for quick multiplication of woodapple.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008).

Experimental Procedure

Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).

ABSTRACT

Dissected shoot tips from healthy suckers of banana var. Meitei Hei (local variety) were inoculated on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators viz., NAA and BAP. After 15 days of incubation 50% of the shoot tips turned green in the medium with 5.0 mgl^-1 BAP. A maximum of 85.7% of the explants produced phenolic exudates in MS + 5.0 mgl^-1 BAP. After one month the explants were subcultured in fresh medium with the respective concentrations and combinations of plant growth regulators. After 75 days of incubation an average of 2.20 microshoots were induced in 78.57% of the explants in MS + 5.0 mgl^-1 BAP. The regeneration of shoots, leaves and roots was more proportionate in MS + 0.1 mgl^-l NAA + 0.5 mgl^-1 BAP. Under the treatment the microshoots had an average height of 1.19 cm with 4.13 leaves and 2.40 roots. The multiple microshoots were separated and subcultured on MS + 0.1 mgl^-l NAA + 0.5 mgl^-1 BAP for proper development. After 120 days the completely regenerated plantlets were hardened under green house environment.

ABSTRACT

The present experiment was conducted to standardize the composition of nutrient media for callus regeneration. Shoot tip, internodal segment, leaf and hypocotyls of seedling grown under controlled condition were used as explants for callus induction. The study revealed that explants collected from younger seedlings showed greater proliferation of callus as compared to those collected from older one. The basal MS medium as well as WP medium fortified with different concentrations of auxins and cytokinins were tried. Initial better results of profuse callus growth with green colour and compact texture having regenerative capacity were obtained by using leaf explants. In all the treatments, the callus initiation occurred at the cut ends and then spread over the surface of the explant as a result of injury. The level of auxins presumable rises and quiescent cells were stimulated to divide rapidly producing callus. The leaf segment explant induced the callus after 9.50 days in medium WPM + NAA 2.5 mg/l with maximum callus growth (976.75 mg) with green, friable callus which was difficult to subculture. White and friable callus occurred within 12.00 days in MS + 2, 4 - D 5 mg/l. The green and compact callus having vigorous growth was observed in MS + NAA 2.5 mg/l within 12.75 days when subcultured on different regeneration media proved recalcitrant for shoot regeneration. On the regeneration medium the callus turned brown while some callus proliferates to produce white growth around it and dried later on.

ABSTRACT

Myriophyllum aquaticum (Velloso) Verdcourt, commonly known as ‘parrot feather milfoil’ is the aquatic plant belonging to the family Haloragaceae. The plant gets the name ‘parrot feather’ from its feather-like leaves arranged around the stem in whorls of 4 to 6 giving it the value of a beautiful aquarium plant. It is a commercially valuable imported aquatic plant used in aquarium plant trade. These plants are exclusively females and as such no seeds are produced. Moreover, current macropropagation techniques are insufficient to produce sufficient propagules for the domestic and export market. Tissue culture techniques offer great opportunities in this regard and attempts were made for micropropagation of this valuable species. A viable protocol has been outlined for the in vitro mass multiplication of Myriophyllum aquaticum. Shoot regeneration was induced from nodal segments and leaves in half strength Murashige and Skoog (1962) medium supplemented with 1.0 mg l^-1 benzyl adenine. Incorporation of 0.2 mg l^-1 kinetin along with benzyl adenine in the culture medium resulted in a marked increase in the frequency (1:20) of multiple shoot formation. The in vitro raised plantlets were successfully rooted, hardened and planted out. The protocol is ready for commercialization.

ABSTRACT

Cultivated turmeric consists of fertile short duration types with a chromosome number of 2n=84. In vivo crossing was done between two short duration types VK 70 and VK76 to develop a high yielding variety with high curcumin and curing percentage. Seed set and seed development was obtained by in vivo stigmatic pollination. Hybrid seed germination was obtained under in vitro in dark on moist filter paper and the seedlings were micropropagated to establish a population of six plantlets /seedling with in a period of 10 months. The multiple shoots were obtained in the medium of half strength MS + 3 per cent sucrose+ BA 2.5 mg l^-1+ NAA 0.5 mgl^-1. Coupling conventional breeding with tissue culture reduces the breeding time.

The term micropropagtion is also known as tissue culture. Micropropagation refers to aseptic culture of various plant parts in artificial growth medium to produce new plant. This means is recently applied in propagation of horticultural crops e.g. Orchid, banana, strawberry, papaya, grape and many other plants.

Requirements for tissue culture: It includes basic facilities including equipment, asepsis, culture medium and culture environment.

• Basic facilities including equipment: Provision of working space, large sinks, washing machine to wash glassware, oven, dryers, dust free storage, space for glass house and chemicals, autoclave for sterilizing media, solutions, water culture and instruments, supply of distilled or deionized water and various essential instruments and appliances.