Preface Biotechnology is an applied branch of biology with relation to technology. It plays an advance role in plant and animal breeding. Biotechnology has broad practical applications in agriculture, industry, and medicines. Now, Biotechnology has emerged as a separate discipline at undergraduate and postgraduate at all universities at worldwide level. There are various books of foreign and Indian origin on descriptive aspect of Biotechnology. However, it was felt deficiency in practical based literatures of “Principle of Biotechnology” since its introduction into the course curricula as a major subject of Plant Breeding and Genetics in agricultural institutions. The prime objective of writing the book ‘Practical Plant Biotechnology and Genetics’ is to fill up this gap and help students. I have tried to arrange the topics and the contents in such a manner that students get simple reading with due continuity and thus understanding. Therefore, this book is expected to appeal undergraduate and postgraduate students and to the field practitioners engaged in agricultural research. I humbly thank authors, editors, and publishers of the books, journals, etc which have been consulted during the prepration of the work. I am also grateful to my husband Dr. Sanjeev Kumar for jubilant inspiration and keen co-operation and cute children Saumya and Adyan for bearing with me during the preparation of this book.

Practical have a significant role in the study of any theoretical subject. Whatever a student learns in theory, he verifies it by actual doing with his own hands.  Thus, these reduce any doubts and misbelieve from his mind. These verifications by actually doing the things are performed in the laboratories.  A laboratory is a place with the basic and essential equipments and adequate facilities for students and teachers to perform and observe experiments.  Plants will continue to provide novel products as well as chemical models for new drugs in the coming centuries.  The arrival of chemical analysis and the characterization of molecular structures have helped in precisely identifying these plants and correlating them with their activity under controlled experimentation.

Year    Contributor    Description 1756    Duharmel du Monceau H.L.    Discovered callus formation in decorticated elm tree.  1839    Schwann, T.H.    Proposed the view that each living cell of a multicellular organism would be capable of developing independently if provided with proper external conditions. 1853    Trecul, A.    Performed experiment on callus formation by decorticated trees such as Robinia, Pawlonia and Ulmus.

A tissue culture laboratory can be placed in any geographical area. The internal controlled environmental conditions permit isolation with a minimum of external influence. To decide where to build the tissue culture laboratory, the following factors must be taken into consideration:

Common rules for fine laboratory techniques call for:     ·    A laboratory should have a record and a complete up to-date record of all the equipments and their operating manual.     ·    A laboratory should have a record and detailed up to-date record of all the chemicals including the name of manufacturer with their     grade and arranged alphabetically in specific area     ·    Strong acid and bases in safe area.     ·    Chloroform, alcohol which are volatile or toxic must be stored in fume hood.

One of the most essential factors governing the growth and morphogenesis of plant tissues in culture is the composition of the culture medium. The basic nutrient requirements of cultured plant cells are very similar to those of whole plants. The basic nutritional requirements of cultured plant cells as well as plants are very similar. However, the nutritional composition varies according to the cells, tissues, organs and protoplasts and also with respect to particular plant species.  The appropriate composition of the medium largely determines the success of the culture. A wide variety of salt mixtures  have been reported in various media. A nutrient medium is defined by its mineral salt composition, carbon source, vitamins, plant growth regulators (PGR) and other organic supplements. When referring to a particular medium, the intention is to identify only the salt composition unless otherwise specified. Any number and concentration of amino acids, vitamins, growth regulators and organic supplements can be added in an infinite variety of compositions to a given salt composition in order to achieve the desired product.

Sterilization Techniques Sterilization: All the materials, e.g., vessels, instruments, medium, explants, etc., used in culture work must be freed from microbes. This can be achieved by one of the following approaches:

Explant The tissue obtained from plant to be cultured is known as Explant. It may include the parts of shoots, leaves, stems, roots, flowers and callus. Maintenance of Aseptic Environment All culture vessels, media and instruments used in handling tissues as well as the explants must be sterilized. The importance is to keep the air surface and floor free of dust. All operations are carried out in laminar air-flow, a sterile cabinet.In general, the methods of elimination of these sources of infection can be grouped under different categories of sterilization procedures: 

Modes of Culture  Callus is a mass of undifferentiated plant tissues formed from plant cells or tissue cuttings when grown in culture. Tissues and cells of plant cultured in a liquid medium aerated by agitation grow as suspension of single cells and cell clumps. For growth, the cells require to divide, whereas, the cells breaking up from explant are mature, nondividing. Hence, the differentiated tissue undergoes modifications to become meristematic. This phenomenon of a mature cell reverting back to meristematic state to form undifferentiated callus tissue is called dedifferentiation.

Micropropagation /Clonal propagation Micropropagation or clonal propagation is the growing of plants from meristematic tissue or somatic cells of superior plants on nutrient suitable media under controlled aseptic physical conditions. Sexually propagated plants (through generation of seeds) demonstrate a high amount of heterogeneity since their seed progenies are not true to type whereas asexual reproduction (by multiplication of vegetative parts) gives rise to genetically identical copies of parent plant. Thus, it permits perpetuation of the parental characters of the cultivars among the plants resulting from micropropagation. Micropropagation proves useful for propagation of: 

Experiment - 1   Aim: Micropropagation of Tobacco plant by leaf disc culture Principle: The totipotency of the plant cells and tissues form the basis for in vitro cloning i.e., generation or multiplication of genetically identical plants in in vitro culture. The ability to propagate new plants from a cells or tissues of parent plant has many interesting possibilities.

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.

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. 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.

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:

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.

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: 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: 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. 

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.

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.

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.

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.

Glossary A ABA -Abscisic acid, a phytohormone.  Adenine - Aminopurine; exhibits cytokinin activity in bud initiation.  Adventitious - Adventitious roots can originate from leaf or stem tissue  initiation of a  structure out of its usual place, i.e., arising sporadically. Agar - A polysaccharide powder derived from algae used to gel a medium / Natural gelling agent.  Agarose Gel Electrophoresis - Electrophoresis carried out on agarose gel to separate DNA fragments. Agrobacterium Tumefaciens - A rod shaped bacterium that causes crown gall disease by inserting it’s DNA into plant cells. Amino Acids - The building blocks or monomeric units of protein. Annealing - The pairing of complimentary single strands of DNA to form a double helix. Anticodon - A set of three nucleotides in tRNA molecule that are complementary to a set of three nucleotides (codon) in mRNA. Aseptic - Sterile; free from contamination by microorganisms. Autoradiography - The process of detection of radioactively labeled molecules by exposure of an X-ray sensitive film.