eChapters

Introduction

This chapter gives first hand information on PCR for beginners. PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments; usually up to 10 kb (kb stands for kilo base pairs). Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a human cell contains about three billion base pairs (Kary Mullis, 1983).

History of PCR

A 1971 Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F) environments) paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

Developed the PCR in 1984 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

Polymeric nanoparticles are nanoparticles which are prepared from polymers. The drug is dissolved, entrapped, encapsulated or attached to nanoparticles and depending upon the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained. Nanocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a polymer membrane, while nanospheres are matrix systems in which the drug is physically and uniformly dispersed.

In recent years, biodegradable polymeric nanoparticles have attracted considerable attention as potential drug delivery devices in view of their applications in drug targeting to particular organs/tissues, as carriers of DNA in gene therapy, and in their ability to deliver proteins, peptides and genes through oral route of administration.

In spite of development of various synthetic and semi synthetic polymers, natural polymers still enjoy their popularity in drug delivery, some of them are: gums (Eg. Acacia, Guar, etc.,) chitosan, gelatin, sodium alginate, and albumin. A range of materials have been employed to delivery of bioactive agents. A polymer used in controlled drug delivery formulation, must be chemically inert, non-toxic and free of leachable impurities. It must also have an appropriate physical structure, with minimal undesired aging, and be readily processable. Some of the polymeric materials are: cellulosics., poly(2-hydroxyethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid).

Microsatellites are highly polymorphic repetitive DNA sequences that are randomly distributed throughout eukaryotic genomes displaying high levels of variation and consequently, are ideal for deciphering genetic variability. They are having high mutation rates (1-4 per generation). Among the microsatellite, (CA) n repeats occurring about one in every 30 kb of mammalian genome constitute useful genetic markers, as their polymorphism is sufficiently stable and their mutation rate are low enough. Out of innumerable microsatellite, ZuBeCa3 microsatellite shows huge variability in the canine genome (Switonski and others 1998). This microsatellite is located in chromosome 9q21-q22 and vastly studied in different dog breeds depicting its heterogeneous distribution over the chromosome (Switonski and others 1998).

Any organism with other than two basic sets of chromosomes, that is, monoploid, triploid, tetraploid, and various aneuploids is termed as polyploid. There are several economically important crops which are natural polyploids emphasizing the economic importance of polyploids in agriculture. These are:

Triploid-Banana

Autotetraploid-Alfalfa, potato, coffee, peanut or groundnut Autohexaploid- Sweet potato

Amphidiploid/allopolyploid-Wheat, cotton, oats, Brassica species (B. carinata- Ethiopian mustard, B. juncea-Indian mustard, B. napus--rapeseed), sugarcane

Man-made commercially successful allopolyploid- Triticale

The newly synthesized autopolyploids, having few or more undesirable features, e. g; meiotic instability, low seed setting and late maturity etc, are usually not suitable for immediate commercial use, are known as raw polyploids. This is specifically more applicable for those sexually propogated polyploids which are developedfor seed. In early generations due to polyploid chromosome constitution and polysomic inheritance they predominanantly do not breed true. Further more, due to meiotic instability chromosome number in their progeny is not always constant or equal to as in synthesized autotetraploid which may vary from few more to few less than the standard autotetraploid. The raw polyploids usually reverts back to diploid parental constitution due chromosome elimination (Randolph and Fischer, 1939). The mechanism responsible for reverting back to diploid status is similar to that of which give rise haploids from diploids. Presently cases are known where either spontaneously or due to specific treatments the chromosome number was reduced to half in somatic tissues. The phenomenon causing such chromosomal reduction is termed as somatic reduction or reduced mitosis Earlier reports suggested it probably to be caused due to spindle organizer abnormalities or some other unknown mechanism/s. The treatment of seed or seedling by colchicine, chloramphenicol and paraflurophenylalanine are reported to have induced chromosome reduction and production of haploids.

23.1 Introduction

An organism or individual having more than two basic sets of chromosomes is called polyploid and the condition is known as polyploidy. Polyploidy is an important tool to increase genetic diversity by altering the chromosome number (2x) and consequently the number of genes within a single cell. Polyploidy has been one of the important mechanisms in the evolution of many cultivated crop plants.

23.2 Types of Polyploidy

23.2.1 Autopolyploidy

The condition that arises due to multiplication of the same chromosomes of a single species is known as autopolyploidy (also called ‘simple polyploidy’ or ‘single species polyploidy’). Autopolyploids have generally larger leaves, stem and other vegetative parts and flower, fruit and seeds than their normal diploids.

Polyploidy, the condition in which a normal diploid cell or organism acquires one or more additional sets of chromosomes. In other words, the polyploid cell or organism has three or more times the haploid chromosome number. Polyploidy arises as the result of total nondisjunction of chromosomes during mitosis or meiosis. The somatic chromosome number of any species, whether diploid or polyploidy is designated as 2n. The somatic cells of plants are diploid that they posses two complete set of chromosomes, one received from seed parent and another from pollen parent. The gametic chromosome number is said to be haploid and denoted by n. Haploid is defined as cell with gametic chromosome number. While monoploid has the basic chromosome number and represented by x.

Classification of Polyploidy

Polyploids are classified in to two groups, euploidy and aneuploidy. Euploidy have chromosome number which is an exact multiple of basic number. Euploidy includes monoploids, diploids and polyploids. In aneuplod individual with other than exact multiple of basic chromosome number or change in chromosome number which posses one or few chromosomes of the genome.

Refers to induced chromosome manipulation

Aneuploidy-One or few chromosomes extra or missing from 2n.

Autoploidy-Genomes identical with each other.

Polyploid is any organism with other than two basic sets of chromosomes, that is, monoploid, triploid, tetraploid, and various aneuploids. Species that have their chromosomes as multiples of a basic number (x) are polyploids. Many of the species which have been developed most recently are polyploids. Autoploidy is not known to have significantly impacted the evolution of species. Autoploids of commercial importance include banana, a triploid which is seedless (diploid bananas have hard seeds not desirable for consumption). Other important autoploids are tetraploid crops, namely, alfalfa, peanut, potato and coffee. Spontaneous autoploids are important in horticultural crops where gigas feature has produced superior varieties of flowering ornamentals of narcissus, tulip, hyacinth, gladiolus, and dahlia among others. Triticale is the most common example of man-made cereal through allopolyploidy. A number of economically important crops are natural alloploids. These include wheat, oat, tobacco, cotton, sugarcane, strawberry and blueberry, wild mustard, black mustard, rutabaga/rape, etc.

A. Define/Explain the following

1. Heteroploidy: Any change in chromosome number from the diploid state.

2. Euploidy: Numerical change in the entire genome.

Polysaccharides comprise several monosaccharide units (more than ten) linked by glycosidic bonds; therefore, they are high molecular weight carbohydrates. They are also called polyanhydrides of simple sugars. The most common monosaccharide unit of polysaccharides is D-glucose. The polysaccharides may differ in their monosaccharide units, molecular weight, chain nature, glycosidic bond types (alpha, beta), and linkages (1-2, 1-3, 1-4, or 1-6). They are tasteless, colorless, amorphous powders that are a little water-soluble. Some may form colloidal solutions. The polysaccharides provide structural stability (structural polysaccharides) to cells, prevent cells and tissues from drying out due to their water-binding properties, and reserve food for the future. Reserved polymeric carbohydrates are osmotically less active and can be stored in large quantities within the cell.
The right and left end of polysaccharides, respectively, are called the reducing and non-reducing ends. Based on the functional aspects, they are grouped in two ways.
• The first way depends on its digestibility—1. Nutrient polysaccharides are (digestible) starch, dextrin, and glycogen, and 2. Structural polysaccharides are (indigestible), chitin and cellulose.
• The second way, depending upon the constituent monosaccharide units, they are 1. Homoglycans (same units), and 2. Heteroglycans (different units).

Various insect and mite pests infest fruit and plantation crops in different stages of crop growth directly or indirectly causing significant losses, hence need their management. The major pests of different fruit and plantation crops, their identification characters, life history, nature of damage and symptoms alongwith its management practices are described crop wise:

Temperate fruits

Pome fruits and stone and nut fruits are mainly cultivated in the temperate regions, which usually requires chilling requirements. Among stone and nut fruits cherry, apricot, peach and plum are the main crops whereas among the nut fruits, walnut, almond are major crops which are a source of handsome income to the orchardists of those areas. The insect and mite pests of pome, stone and nut fruits are described hereunder:

Pomegranate  (Punica granatum L.)  also  known as anar, dalim  is   an ancient  fruit and cultivated in semi-arid to arid regions of the country. However, pomegranate plants are found throughout the country from temperate to tropical climate. It has enjoyed a reputation for its healthy dietetic and medicinal properties. Pomegranate has versatile adaptability, drought tolerance, high value fruits, steady and high yields of the crop (Vishalnath et al., 2007). The pomegranate fruit has therapeutic value (Julie Jurenka, 2008); good keeping quality, high export potential and is mainly grown for table purpose, though several value added products are also available.

Pomegranate (Punica granatum L.) belongs to the family Punicaceae and is one of the favaourite table fruits of tropical and subtropical regions. The fresh fruit is of delicate quality while its processed products are highly appreciative. It is now extensively grown in Arid regions. Pomegranate is in fact native to Iran and is extensively cultivated in Mediterranean countries. It is distributed in Spain, Morocco, Egypt, Afghanistan, Arabian countries, Pakistan, Myanmar, China, Japan, Chile, Argetina, Mexico, USA, Bulgaria, Italy, India and Iraq.

Cultivars: Important pomegranate varieties cultivated in India are Bhagwa, Ganesh, Alandi or Vadki, Dholka, Kandhari, Kabul, Muskati Red, Paper Shelled, Spanish Ruby, Ganesh (GB I), G 137, P 23, P 26, Mridula, Aarakta, Jyoti, Ruby, IIHR Selection, Yercaud 1 and Co 1.

Uses

The pomegranate is commercially grown for its sweet acidic taste arils. The fresh fruit has exquisite quality, while its processed products such as bottled juice, syrups and jelly are highly appreciated. Pomegranate is a delicious fruit consumed for its medicinal value. Its juice is sweet with pleasant aroma and is refreshing. The fruit is widely used for Ayurvedic and Unani medicines. Some of the commercial products of pomegranates are juice, squash, syrup, jelly. Anardana is obtained by drying the seed along with pulp and sun. The rind is used as a valuable tanning material. The flowers yield a red dye.

Pomegranate is emerging as one of the important fruit crops owing to its hardiness and ability to withstand adverse soil and climatic conditions. India contributes 40-45 percent of total 10 lakh tons production in the world. Currently the export of pomegranate is around 10,000 tons, which is just 5 percent of the total export. With the increase in population, the domestic demand of fruits has also increased substantially. As on date, out of total area of 1, 25,000 ha. under pomegranate, major area under the crop is found in Maharashtra. The state has 85,000 ha. (68 % area) under the crop, of which only 51,000 ha is under production, followed by Karnataka 12,727 ha. Gujarat 3,787 ha, up to 2003 –2004 (Anon, 2005). Earlier orchards were raised mainly with variety Ganesh, which was the leading variety. However, the variety is fast being replaced with the new cultivars like Bhagawa and Arakta, which have attractive skin and aril colour and are suitable for export.

The pomegranate is an ancient and favorite table fruit of tropical countries. It is commercially grown for its sweet-acidic fruits, which provide a cool refreshing juice, and is valued for its medicinal properties. The fruit rind, juice, leaf and roots are used in the preparation of various ayurvedic medicines. Its popularity is also due to ornamental nature of the plant especially due to bearing bright red flowers throughout the year. The fruit juice is refreshing and contains both glucose and fructose. The juice and seed contain large quantities of tannin and agolic acid, which is essential in curing several diseases. The fruit can be successfully grown even under purely rain fed conditions. In the recent years pomegranate cultivation has become an economically viable proposition.

1) Bacterial Blight
Diagnostic Symptoms
• Bacterial blight of pomegranate was first reported in India from Delhi in 1952.
• First water-soaked lesions develop on fruits within 2–3 days and appear as spots.
• Initially, spots are black and round and surrounded by bacterial ooze.
• Under favorable conditions, spots enlarge to become raised, dark brown lesions with indefinite margins that cause the fruit to crack.
• Disease may cause up to 90% yield reduction (Fig.1).
Etiology
Domain : Bacteria
Phylum : Proteobacteria
Class : Gammaproteobacteria
Order : Xanthomonadales

Introduction

Pomegranate is one of the most remunerative fruit crops that grow well under arid and semi-arid agro eco-system with severe scarcity of water. The popularity of pomegranate has grown tremendously due to its health beneûts, high returns on investment, less water requirement, therapeutic and functional properties. Growing health consciousness among the consumers and acquisition of super fruit status by pomegranate has made marketing of pomegranate fruits and derived value added products quite easy. As the result of all these benefits, India is witnessing a pomegranate revolution with more than 80 % expansion in area and 250 % increase in production during last one decade. However, there are many challenges to be overcome so as to double the income of pomegranate growers, these challenges include market glut due to unprecedented increase in production coupled with limited export resulting into suboptimal price of the harvested produce; intensification of diseases and insects and pest due to huge unplanned expansion; dominance of only one variety; very limited value addition; hampered export due to suboptimal quality, unwanted sprays, issue of pesticide residues and lack of sufficient number pesticide label claims in pomegranate; inadequate supply chain management. So, in order to overcome these challenges and to increase the profitability of pomegranate stakeholders, we need to focus on scientifically sound cultivation practices to reduce the cost of cultivation, breeding for improved varieties, improved packaging, transport, storage, marketing, supply chain management, facilitating and encouraging export, practicing and promoting climate resilient horticulture, formation of farmers’ cooperatives and farmers producers organization (FPOs), processing and value addition with the aim of total utilization and use of information and communication technologies. This article deals with opportunities and strategies in pomegranate for doubling farmers’ income through nursery management; improved horticultural practices; integrated disease, insets pests and nutrient management; use of information and communication technologies; creation of FPOs; processing and value addition.

Pomegranate commonly known as ‘anar’ is an ancient favourite table fruit of tropical and sub-tropical regions of the world.  The pomegranate tree is native from Iran to the Himalayas in northern India and has been cultivated since ancient times throughout the Mediterranean region of Asia, Africa and Europe. The word pomegranate comes from the Latin “Pomum granatum” which means “apple of many seeds.” The Romans called it the Punic apple. Ancient Romans not only enjoyed the succulent flesh of this fruit but due to the high amount of tannic acid in the skins, they also used the skins in the process of tanning leather. Perhaps due to the fruit’s princely blossom crown, it has gained distinction as a royal fruit. “Pomum granatum” or seeded apple was the name which was known in the middle ages. The French named their hand-tossed explosive a grenade after the seed-scattering properties of the pomegranate fruit.

Genus : Punica 
Species : granatum L.
Family : Punicaceae
Chromosome No. : 2n = 16,18

Pomegranate has originated in Afghanistan and Iran the abode of prophet Mohammad and for very long has been associated with grape and fig and is still held in higher esteem. Acknowledging the multi usefulness of pomegranate Prophet Mohammad said ‘Eat the pomegranate sententiously for it progress the system of envy and hatred’. The crop was very much in demand and liked by the inhabitants prior to the days of Mohammad in the Orient, as pomegranate has something special in the form of refreshing which the people of hot arid region like and adore enormously. The ancient Semitic name rimmon was adopted by Arabs as rumman. The Potruguese roma or roman was formed from rumman. The present botanical name Punica granatum has been derived from Roman name malum punicum (apple of Carthage) and granatum.