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The technique of preserving fruit by the use of low temperature dates from the eighteenth century with the first use of ice house in 1750. Later in 1800s commercial cold storages have come up for the storage of perishables like meat, dairy and fruit products.  The main change took place in the cold store in recent years has been the introduction of multi temperature chambers with humidity control for specific products. Cold preservation can be either refrigeration or freezing. Household refrigerators usually run at 4-7^ºC. Chilling or cold storage usually uses a slightly lower temperature on the basis of the food to be refrigerated. In freezing a temperature of -18^ºC or below is used. Chilling will preserve perishable foods for days or weeks and freezing will preserve foods for months or even years.

Drying is the oldest known method of preserving food. Drying or dehydration is removal of majority of water contained in fruit or vegetable. Although preservation is the primary reason for dehydration, it also lowers cost of packaging, storing and transportation by reducing both weight and volume of final product. In addition, dried foods add variety to our diets. In the process of drying, sufficient moisture is removed to protect product  from  spoilage. Processing should be done in such a way that food value, natural flavour and characteristic cooking quality of fresh material are retained after drying.

Processing of fruits  and vegetables into various products which could be preserved for a long time and add to value of product using proper cost effective technology will be a viable tool for improving   economic status of  farmer as well as our country. Modern methods of fruit and vegetable preservation are  broadly classified as follows:

Storage is one of the most important aspects of post harvest handling of fruits and vegetables. The main objective of storage is to extend storage life of fruits and vegetables and increase their period of availability. A substantial quantity of fruits go waste in India due to lack of proper storage facilities. Storage of fruits and vegetables is practiced for various reasons. Proper post harvest handling of fresh horticultural produce helps in extending  period of availability of wide range of  fruits and vegetables round the year, prevention of seasonal market gluts, orderly marketing, better return to grower and  preservation of  nutritional qualities. The   long  term  storage  of  fruits  and  vegetables is expensive and requires high level of technical  knowledge of crop. However, an orderly marketing reduces storage period considerably. 

Various systems were developed to take produce from field to consumer and selection of a particular method depends on several factors, including perishability and value of crop.  One method is to harvest the crop and take it to a packhouse for various unit operations like grading, cleaning, post-grading treatments, and packing for transport and marketing. When deciding upon where to locate a packinghouse, access to field and market point, adequate space for vehicles to enter and leave  packinghouse and ease of access to labor will all be considered.
 
In the simplest packinghouse, produce is delivered in picking containers, immediately after harvest, directly to packers.  Packers then sort, grade, size and pack the produce directly into appropriate transport containers. In this case, each worker must be aware of produce defects, grade and size requirements, and packing methods.

Maturity at harvest is the most important determinant of storage life and final fruit quality. Maturity indices are determined for many fruit, vegetable and floral crops. Harvesting crops at proper maturity allows handlers to begin their work with the best possible quality produce. Produce harvested too early may lack flavour and may not ripen properly, while produce harvested too late may be fibrous or over ripe.
 
Almost all fruits reach their best eating quality when allowed to ripen on the tree or plant (exceptions e.g., pears, avocados, and bananas). In general, fruits become sweeter, more colourful, and softer as they mature. A few fruits are usually picked mature but unripe so that they withstand post harvest handling system when shipped long distances.  Most of  currently used maturity evaluation is based on a compromise between those methods that would ensure the best eating quality and those that provide flexibility in transportation and marketing.  Optimum harvesting stage depends upon type of fruit or vegetable and their final use. Such a division is usually based on their classification according to physiology, environmental conditions, ripening bahaviour and nutritional aspect. Some of the classifications, important for their harvesting and storage are briefed in  subsequent sections.

Structure

Structural unit of edible portion of most fruits and vegetables is the parenchyma cells and makes up bulk of cells. A parenchyma cell is rather thin walled and may be polygonal or cubical in shape.  Walls of parenchyma cells in young plants are composed almost entirely of fibrils of cellulose. In parenchyma cells of plants actively metabolising protoplast portion represents only about 5% of total cell volume. This protoplast is rather film like and is pressed against cell wall by large water filled central vacuole.  Protoplast has inner and outer semi permeable membrane layers between which is confined cytoplasm and its nucleus. 

Fruits and vegetables are very important food commodities. India has become the largest producer of fruits and the second largest producer of vegetables. Though the production of fruits and vegetables make us believe in our strength for self-sufficiency, a significant qualitative and quantitative loss occur in the produce from harvest till consumption.  For self sufficiency and also for processing, export and to meet additional requirements, a lot of emphasis need to be given to reduce post harvest losses, besides increasing  production and productivity of horticultural produces. Processing plays an important role in  conservation and effective utilisation of these perishable commodities. However, only less than 2% of total production of fruits and vegetables are processed.  The new industrial policy has placed this sector in the list of high priority areas.

Tissue culture consists of growing plants cells as relatively on organized masses of cells on an agar medium (callus culture) or as a suspension of free cells and small cell masses in a liquid medium (suspension culture). In other words, In plant cell culture, plant tissues and organs are developed in vitro on artificial media, under aseptic and controlled environment. The procedure depends mainly on the concept of totipotentiality of plant cells (Haberlandt, 1902) which refers to the ability of a single cell to express the full genome by cell division.

1. Microscope

The microscope is a precious instrument. There are many small objects or details of objects which cannot be seen by the unaided human eye.
 
A microscope (from Greek: mikrós, means “small” and skopeîn, means “to look”) is an instrument used to see objects that are too small for the naked eye.  In another word microscope is an optical instrument that uses a lens or a combination of lenses to produce magnified images of small objects, especially of objects too small to be seen by the unaided eye.

Electrophoresis is a method used to separate charged particles from one another based on differences in their migration speed. In the course of electrophoresis, two electrodes (typically made of an inert metal, e.g. platinum) are immersed in two separate buffer chambers. Gel electrophoresis is a technique used to separate molecules based on physical characteristics such as size, shape, or isoelectric point. A gel is composed of agarose or polyacrylamide. Agarose is used for separating DNA fragments in size range of 100bp to 20kb. Polyacrylamide is preferred for smaller DNA fragments. Separation is achieved by moving the negatively charged nucleic acid molecules through agarose matrix with an electric field. Rate of migration of DNA molecules is inversely proportional to their molecular weight. Smaller molecules move faster than larger ones. But conformation of the DNA molecule is also an important factor. Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds such as: supercoiled plasmid DNA will move fastest followed by linearised DNA, while open circular DNA will move at the slowest pace. To visualise DNA, gels are stained with ethidium bromide which intercalates in DNA and fluorescse under UV.

Blotting is the procedure through which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. We can identify inherited disease, infectious agent present in the sample by using blotting technique. It is also used to mapping restriction sites in single copy gene.

Experiment - 1
 
Aim: Molecular analysis of putative transformed plants by Polymerase Chain Reaction

The expression of transgenes are not identified at that time but it can be achieved through DNA analysis of that plant. The presence of transgene are generally identified of PCR-based detection followed by gel electrophoresis and comparison with standard samples. The main achievement of PCR-based detection is that it is very sensitive. The Polymerase Chain Reaction (PCR) provides a rapid and sensitive method to amplify a specific gene sequence. 

Experiment - 1 
Aim: Mobilization of recombinant Ti plasmid (with gene of interest) from  
(E. coli) to an Agrobacterium tumefaciens strain. 

Principle: Due to ease of cloning and harvesting plasmids, E. coli is most popular for maintenance of plant transformation vectors. Consequently, the plant transformation vector with cloned gene of interest is mobilized to recipient Agrobacterium strain harbouring vir plasmid. Plasmid are closed, circular DNA molecules that are able for autonomous replication with a host cell. 

Experiment- 1

Aim: Direct DNA delivery to Plant by Particle Bombardment
 
In direct gene transfer method, the foreign gene of interest is delivered into the host plant cell without the help of vector. Klein and Coworkers (1987) evolved a procedure through which the delivery of DNA into cells of intact plant organs or cultured cells is done by a process called Projectile Bombardment. The microprojection (Small and high density particles) are accelerated to high velocity by particle gun apperatus. These particles with very high kinetic energy penetrate the cells and membranes and transmit foreign desired DNA inside of the bombardment cells.

Experiment - 1

Aim: Isolation of plant genomic DNA through modified CTAB method

Principle: Genomic DNA is extracted from plant material requires cell lysis, inactivation of cellular nucleases and separation of the desired genomic DNA from cellular debris. Ideal lysis method is rigorous enough to disrupt the complex starting material (plant tissue), yet gentle enough to preserve the target nucleic acid. The cetyltrimethylammonium bromide (CTAB) procedure is sufficient for the extraction and purification of DNA from plants and plant derived food stuff.  This is particularly suitable for the elimination of polysaccharides and polyphenolic compounds otherwise affecting the DNA purity and hence quality.

Isolation of Protoplasts

Protoplast is the living material of the cell however an isolated protoplast is the cell from which the  cell wall is removed. In plant breeding programme many desirable combination of characters could not be transmitted through the conventional method of genetic manipulation. Higher plants that could lead to the genetic process involving fusion between the subsequent developments of a product to a hybrid plant is known as somatic hybridization. Plant protoplasts can be isolated from cells by two methods:

A. Somatic Embryogenesis
 
In plant tissue culture, the developmental pathway of various well organised, small embryoids resembling the zygotic embryos form the embryogenic potential somatic cell of callus tissue or cells of suspension cultures is known as somatic embryogenesis. The capability of somatic cell of a culture to produce embryoids is known as embryogenic potential.

Embryoid: Embryoid is a small, well organised structure comparable to the sexual embryo, which is produced in tissue culture of dividing embryogenic potential somatic cells.

The ultimate success of commercial propagation depends on the capacity to transfer plants out of culture on a huge range, at low cost and with high survival rates. The plants multiplied in vitro are exposed to a unique set of growth conditions (high levels of inorganic and organic nutrients, growth regulators, sucrose as carbon source, high humidity, low light, poor gaseous exchange) which may support rapid growth and multiplication but also induce structural and physiological abnormalities in the plants, rendering them unfit for survival under in vivo conditions. Poor control of water loss and heterotrophic mode of nutrition are main cause of deficient growth of in vitro plants.  Hence, gradual acclimatization is necessary for these plants to survive transition from culture to the greenhouse or field. During acclimatization the in vitro formed leaves do not recover but the plant develops normal leaves and functional roots. Once roots are well formed the plants are ready to be transferred into soil.The procedure of acclimatization of regenerated plantlets are: 

A. Anther and Pollen Culture (Production of Haploid Plants)

Haploid plants possess a single set of chromosomes (gametophytic no of chromosomes i.e. n) in the sporophyte in contrast to diploids which contain two sets of chromosomes (2n). The existence of haploid plants was reported by Bergner (1921) in Datura stramonium. Guha and Maheswari (1964) reported the direct development of haploid embryos and plantlets from microspores of Datura inoxia by the cultures of excised anthers. After this, haploid plants have been produced via anther culture in more than 170 species. The anther culture technique is useful in haploid production.