eChapters

Introduction

Ligation of DNA fragment is one prime important in genetic engineering because without ligation we could not transfer the interest gene.DNA cloning requires the DNA sequence of interest to be inserted in a vector DNA molecule. For this, both the vector as well as insert DNA is prepare by digestion with compatible restriction enzymes, so that the ends produced during digestion is complementary in both. When setting up ligation, it is important to consider the permutations that can occur and bias the relative concentration of DNA accordingly. Usually a 5- to 10-fold excess of insert over the vector DNA is the norm. This ensures that enough ligated product will be produced in the right orientation.

Introduction

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

Introduction

The AFLP amplified fragment polymorphism also known as selective restriction fragment amplification (SRFA) technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously. The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an isolate or the degree of similarity among isolates. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry. produces highly complex DNA profiles by arbitrary amplification of restriction fragments ligated to double-stranded adaptors with hemi-specific primers harboring adaptor-complementary 5' termini.The technique has been widely used in the construction of genetic maps containing high densities of DNA marker loci.

The AFLP protocol amplifies restriction fragments obtained by endonuclease digestion of target DNA using “universal” AFLP primers complementary to the restriction site and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain selective nucleotides at the 3' termini that extend into the amplified restriction fragments. These arbitrary terminal sequences result in the amplification of only a small subset of possible restriction fragments. The number of amplified fragments can therefore be “tailored” by extending the number of arbitrary nucleotides added to the primer termini. Alternatively, the use of endonuclease combinations that vary in their restriction frequency can also be used to tune the number of amplicons. Generally, the abundant restriction fragments produced from complex genomes require of AFLP primers with longer selective regions. Conversely, analysis of small genomes require of only few arbitrary nucleotides added at the primer 3' termini. The resulting AFLP fingerprints are usually a rich source of DNA polymorphisms that can be used in mapping and general fingerprinting endeavors.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

Introduction

This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors.

Principle

The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

DNA can be easily isolated and purified after size selection on an agarose gel. The fragment of interest is simply cut out of the gel with a razor blade and purified by a number of different methods.The easiest is to use a method that involves first dissolving the agarose slice in a solution at 50°C, then binding the DNA from the melted agarose to a silica-gel membrane.

Principle

In this method following gel electrophoresis using low gelling/ melting temperature agarose, the band of interest is removed from the gel. The agarose slice is then melted and subjected to phenol extraction.

Introduction

The primary tools used by the molecular biologists in manipulating DNA are restriction enzymes and other DNA/RNA modifying enzymes. Over the years, the number and uses of these enzymes have increased as new molecules have been discovered. Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA. They are present in bacteria presumably to destroy DNA from foreign sources. By cleaving the foreign DNA at specific sites. The host bacterial DNA os protected from cleavage because specific recognition sites are modified, usually by methylation form methylase at one of the bases in the site, making the site no longer a substrate for RE cleavage. Host bacteria used to propagate cloned DNA in the laboratory arte usually mutant in the host restriction genes, thus their intracellular enzyme activities will not destroy the foreign recombinant sequences. The endonuclaase with its accompanying methylase is called a restriction modification (R-M) system.

At least four different kinds of R-M systems exist characterized on the basis of the subunit composition, cofactor requirements and type of DNA cleavage. Most characterized enzymes belong to type II class, together with type IIS class. They comprise the commercially available restriction enzymes used for DNA analysis and manipulation. Type I and Type III enzymes are relatively uncommon and a few additional enzymes fit none of the classes.

Among more than 3000 different enzymes isolated from bacterial strains many share common specificities. Restriction enzymes that recognize identical sequences have been called as isoschizomers.

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.

Introduction

Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. SDS-PAGE is the most widely used method for qualitatively analyzing any protein mixtures. The method is based on the separation of proteins according to their size and then locating by binding them to a dye. It is particularly useful for monitoring protein purity and determines their relative mass. The SDS-PAGE enables the students to understand the theory behind the techniques in checking the purity of the protein and identifying the protein in a test sample.

Separation of Proteins

Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e.: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

Electrophoresis is a method of separating out large molecule ( DNA or protein). An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins.

What is Gel Electrophoresis?

A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Principle

In this method the estimation of pentose sugar is carried out. Acid hydrolysis of RNA releases ribose (a pentose sugar) which in the presence of strong acid undergoes dehydration to yield furfural. Orcinol, in the presence of ferric chloride as a catalyst, reacts with furfural producing a green colored compound which can be quantitatively estimated at A_{665 nm}. DNA gives a limited positive reaction with Orcinol test.

Materials and Reagents

1. Spectrophotometer
2. Boiling water bath
3. 5% chloroform (HClO₄)
4. Standard RNA solution : Dissolve yeast RNA (500ìg/ml) in 5% HClO₄ . make different dilutions to obtain solutions containing 100 – 500ìg RNA/ml with 5% HClO₄ .
5. Orcinol reagent : Dissolve 100mg of ferric chloride (FeCl₃.6H₂O) in 100 ml of concentration HCl and then add 3.5 ml of 6% solution of Orcinol prepared in alcohol.

To Estimate the DNA Using Diphenylamine

Introduction

DNA estimation is helpful to know about the total amount of DNA of prokaryotic as well as eukaryotic system. One method used for DNA estimation is Diphenylamine assay, is that of Burton who modified the original procedure of Dische. This procedure is based on treatment of nucleic acid with hot perchloric acid to hydrolyze DNA nucleotides. Diphenylamine reacts specifically with deoxyribose in DNA to form a blue-colored complex. Since diphenylamine does not react with ribose, one can assay for DNA in the presence of contaminating RNA.

Principle

The DPA method is based on the colorimetry measurement of products formed by the reaction of ù- hydroxylevulinaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 2.5 N perchloric acid.

When DNA is treated with diphenylamine under acidic condition (PCA) a blue complex is formed with a sharp absorption maximum at 595 nm (600 nm). In acid solution, the straight chain form of the deoxypentose is converted to highly reactive ω – hydroxylevulinaldehyde that reacts with diphenylamine to give blue complex. The reaction yields two mixture of product which together have absorbance maximum at 595 nm (600 nm). Interferences include proteins, lipids, saccharides and RNA. In DNA the only deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present.

Introduction

DNA denaturation, also called DNA melting, is the process by which double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands during the breaking of hydrogen bonding between the bases. Both terms are used to refer to the process as it occurs when a mixture is heated, although “denaturation” can also refer to the separation of DNA strands induced by chemicals like urea. For multiple copies of DNA molecules, the melting temperature (T_m) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the “random-coil” states. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.

Applications of DNA Denaturation

The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenosine / thymine base-pairing, the amount of cytosine and guanine in a genome (called the “GC content”) can be estimated by measuring the temperature at which the genomic DNA melts. Elevated temperatures are associated with high GC content. GC ratios within a genome are found to be markedly variable. These variations in GC ratio within a genome of higher organisms results in a mosaic like formation with islet regions called isochores. This results in the variations in staining intensity in the chromosomes. The isochores include in them essential protein coding genes, termed housekeeping genes and thus determination of ratio of these specific regions contributes in mapping these essential genesbacterial systematics has recommended use of GC ratios in higher level hierarchical classification. For example, the Actinobacteria are characterised as “high GC-content bacteria”. In Streptomyces coelicolor A3(2), GC content is 72%. The GC-content of Yeast (Saccharomyces cerevisiae) is 38%, and that of another common model organism Thale Cress is 36%. Because of the nature of the genetic code, it is virtually impossible for an organism to have a genome with a GC-content approaching either 0% or 100%. A species with an extremely low GC-content is Plasmodium falciparum (GC% = ~20%), and it is usually common to refer to such examples as being AT-rich instead of GC-poor.

Procedure

Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube.

Heat the tube in a boiling water bath for 1 hour.

Cool the tube, open it and place the contents into a centrifuge tube. Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA.

Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography.

Using a micropipette, spot 20 µl of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than one large drop). Allow the spots to completely dry before proceeding.

Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20 cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40° C for about 2 hours.

Locate the spots of nucleotides by their fluorescence under an ultra-violet light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid and finally, uridylic acid.

After cafefully marking the spots, cut them out with cutters and place the paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add 5.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

DNA Migration in Agarose and Polyacrylamide Gels

Recommended Gel Percentages for Separation of Linear DNA

Mole (mol)

1 mole of any substance contains 6.02 x 10²³ units. The number of moles in a given weight of substance can be calculated using following equation.

1. pH Meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

2. UV Spectrophotometer

Perhaps there is no other technique except UV-visible spectroscopy, which has been so extensively employed and has such a profound impact on progress in biochemical investigations. UV-visible spectroscopy has undoubtedly become an indispensable tool and is routinely used for identification and quantitative estimation of most of the biomolecules. Besides other applications, most of the enzyme assays and the screening of biomolecules recovered in different fractions by separation processes such as column chromatography, density gradient centrifugation etc, rely heavily on spectrophotometer or colorimetric technique.

Search for genetic material—nucleic acid or protein/DNA or RNA?

• Griffith’s Transformation Experiment
• Avery’s Transformation Experiment
• Hershey-Chase Bacteriophage Experiment
• Tobacco Mosaic Virus (TMV) Experiment

Timeline of Events

• 1890 Weismann - substance in the cell nuclei controls development.
• 1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.
• 1928 Griffith’s Transformation Experiment
• 1944 Avery’s Transformation Experiment
• 1953 Hershey-Chase Bacteriophage Experiment
• 1953 Watson & Crick propose double-helix model of DNA
• 1956 Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material

BC

1750 The Sumerians brew beer.

500 The Chinese use moldy soybean curds as an antibiotic to treat boils.

250 The Greeks practice crop rotation to maximize soil fertility.

100 Powdered chrysanthemum is used in China as an insecticide.

AD: Before the 20^th Century

1590 The microscope is invented by Janssen.

1663 Cells are first described by Robert Hooke.

1675 Leeuwenhoek discovers protozoa and bacteria.

1745 Maupertuis proposes an adaptationist account of organic design

1797 Jenner inoculates a child with a viral vaccine to protect him from smallpox.

1802 The word “biology” first appears.

The Laboratory Worker is Surrounded by Many Dangers such as from..........

Handling of infectious materials.

Handling of broken glassware.

Accidental spill of corrosive reagents.

Swallowing of corrosive reagents such as concentrated Sulphuric acid,

Hydrochloric acid, sodium hydroxide, trichloroacetic acid etc.

Swallowing of infectious specimen.

Inhalation of poisonous fumes.

Infection in the conduct of necropsies.

Potential hazards in the form of inflammable chemicals and gas leakages.

Due to increase in population, rapid urbanization, industrialization and shrinking land holdings, there is tremendous pressure on availability of land resources for agriculture, which results in decreased arable land area year after year. The huge gap between present production and future requirements necessitates intensive vegetable production and availability at affordable prices. Vegetable production can be enhanced either by increasing productivity, use of improved cultivars and ago-techniques, bringing additional land under vegetable crops or by promotion of protected cultivation. By adopting protected cultivation, year round availability of quality produces both for domestic use and export can be assured.

Several biotic and abiotic stresses are the major factors responsible for low productivity and poor quality in large number of vegetable crops under open field cultivation more specifically during rainy and post rainy season. The increasing demand of off-season and high quality vegetables in various markets of the big cities had call the attention of the vegetable growers for diversification from traditional ways of vegetable cultivation to modern methods of vegetables cultivation in an agri-business entrepreneurial models. Under the new era of FDI (Foreign Direct Investment) in retail these kinds of models are having high potential for betterment of the farmers opting for quality and offseason vegetable cultivation through protected cultivation. Protected cultivation structures are useful for combating both biotic and abiotic stresses that limits the productivity and quality of vegetable crops. This requires careful planning, attention and details about timing of production and moreover, harvest time to coincide with high market prices, choice of varieties adopted to the off season environments, and able to produce economical yields of high quality produce etc.

Introduction

Organic farming per se is not a new concept rather is an age old practice of cultivation crops without synthetics. However, in modern usage it is a type of farming without synthetics but at the same time taking advantage of all the modern agriculture technologies like soil testing, nutrient management, ecofriendly pest management, etc. developing and developed world have different incentives for taking up organic farming. Growing health consciousness among the people of developed world is the prime motive behind interest in organic farming. However, conversion of “by default” organic agriculture into “certified organic” is perspective in the developing world so that farmers get price premium. Whatever the case may be, the area under organic farming as well as demand for organic food/product is increasing both in developing as well as in the developed world.

Organic production is a systems approach. Although many aspects of vegetable production remain the same in both “organic” and “non-organic” or “conventional” systems, there are differences. The major components of production which differ between these two systems include transplant production, soil fertility management, and insect, disease, and weed management. “Organic” usually refers to a crop management system that promotes biodiversity, biological cycles, and soil biological activity. Organic production concentrates on natural processes and how to manage them. Other materials and products are additions to, not replacements for, management. An organic system takes time to develop. It may be achieved in stages, for example by starting with organic soil amendments and other soil improving procedures. Then, try biological control of pests, some companion plants, etc. Eventually, the entire system will be changed. It will take years to convert to a healthy organic system.

A successful vegetable production industry is very dependent on a sustainable supply of seeds to the growers. At the present time, the seed industry plays an important role in both production and distribution of high quality vegetable seeds.

Successful seed production of a crop depends on a thorough knowledge of the reproductive process of a particular crop. The techniques of seed production must take into account several features of reproduction: whether it is sexual, asexual, or a combination of the both, the nature of floral structures, the amount of pollen transfer, the degree and means of self-incompatibility and the effect of inbreeding on vigour. For most purposes the important consideration to the seed producers is the extent of cross-pollination. It is, therefore, necessary that before engaging himself in seed production, a producer must acquaint himself with the following details of reproduction in the particular crop:

Methods of Reproduction

The method of reproduction in vegetable crop plants may be broadly grouped in to two categories, asexual/vegetative and sexual:

Improved Varieties of Tuber Vegetables

A. POTATO

Improved Varieties of Asparagus

(1) Perfection

Source : Developed at IARI, New Delhi

Maturity : Early

Spears : Large, green, succulent, heavy cropper and uniform in maturity

Production Technology of Asparagus

Climate

Asparagus is temperate season vegetable and bears excellent crop between a temperature range of 15-24ºC for most of the growing season. The ideal day temperature is 25-30^oC and night temperature 15-20^oC. It can tolerate frost up to some extent but continuous freezing is harmful for its growth. Spear initiation occurs at soil temperature of more than 10ºC. Spear elongation is faster at high air temperature. Root and shoot development is reduced below 13ºC and above 29ºC. Due to warm weather, spear tips open prematurely and quality of spears is reduced. Elevation of more than 1000m is good for high yield.

Improved Varieties of Leafy Vegetables

A. AMARANTHUS

Improved Varieties of Root Crops

A. RADISH

A. Asiatic/Tropical Varieties

Improved Varieties of Bulb Crops

A. ONION

Improved Varieties of Peas and Beans

A. VEGETABLE PEA

A. Early Varieties

Improved Varieties of Cucurbits

A. CUCUMBER, MELONS, PUMPKIN AND SQUASHES

Cucumber (Khira)

Improved Varieties of Cole Crops

A. CAULIFLOWER

Improved Varieties of OKRA (Bhindi)

Vegetable research in India is of very recent origin. Research on vegetable crops was started by ICAR with the sanctioning of a nucleus ‘Plant Introduction Scheme’ at IARI, New Delhi in 1947-48. Simultaneously, the ICAR also started such ad-hoc schemes in different states like Punjab, Uttar Pradesh, West Bengal, Maharashtra, Himachal Pradesh, Jammu & Kashmir and Tamil Nadu.

Prior to World War II, entire seeds of temperate vegetables were imported from European countries. When, during World War II, supply of vegetable seeds from Europe suffered a serious setback, the government of India felt the need for production of these seeds within the country. As a result of this, the government of India established a Vegetable Breeding Station at Katrain in Kullu Valley, Himachal Pradesh in 1949. This station was transferred to Indian Agriculture Research Institute in 1955 to undertake research on improvement of temperate vegetable crops, standardization of their seed production technology and to produce seeds of identified improved varieties of temperate vegetables. This research station is carrying out intensive research on all aspects of temperate vegetables including improvement, production technology and their seed production.

Further, a division of Horticulture was created at the Indian Agriculture Research Institute, New Delhi in 1956-57. However, vegetable research was organized on sound footing with the establishment of the Indian Institute of Horticulture Research (IIHR) at Bangalore with a full fledged Division of Vegetable Crops in 1968. The vegetable research in India received a further boost when a separate division of Vegetables Crops and Floriculture was started at IARI, New Delhi in 1970. The floriculture work was separated from the division of Vegetable Crop and Floriculture, IARI, New Delhi in 1982 and the division was then named as Division of Vegetable Crops, which was assigned the research work exclusively on different vegetable crops. In the Sixth Five-year Plan, IIHR, Bangalore started two Central Horticulture Experiment Stations, one each at Ranchi, (Bihar) and Godhra (Gujarat) to intensify research on horticultural crops in important tribal areas of the country where research on vegetable crops was also started to meet the specific requirements of these regions. Establishment of Agricultural Universities and All India Coordinated Vegetables Improvement Project gave further fillip to vegetable research in India.

Significant achievements have been made in the food availability throughout the world over the last few decades. The estimated number of people in developing regions suffering from chronic malnutrition has declined (from 946 million people in 1969-71 to 870 million people in 2010-12), as the proportion of malnourished people from 36 percent to 20 percent, even though the world population has increased. Global food supplies (if distributed according to individual requirements) are sufficient to fulfill the energy needs. An estimated 20 percent of the people in developing world are chronically under nourished, consuming too little food to meet even the minimal energy needs. Approximately 192 million children under the age of five years suffer from acute or chronic protein energy malnutrition. According to WHO, 6.9 million children under the age of five died in 2011, from direct or indirect result of hunger and malnutrition. Moreover, more than 2 billion people, mostly women and children are deficient in one or more micronutrients, babies continue to be born mentally retarded as a result of iodine deficiency, children go blind from vitamin-A deficiency and enormous number of women and children are adversely affected by iron deficiency.

The overall growth in food availability resulting from the green revolution over the last two decades has eliminated the threat of famine for India’s entire population. Country’s production of food grains has reached over 257.07 million tonnes. However, malnutrition is still a serious problem in this region. Evidences suggest that micronutrient deficiency is far more prevalent in the area than protein energy malnutrition. The International Food Policy Research Institute has reported a rising trend in micronutrient deficiencies, which is becoming more serious than malnutrition from lack of major nutrients. Thus, the emphasis is now shifting from supply of basic food (calories & protein) towards supply of balanced diet (calories, protein & micronutrients which includes vitamins and minerals).

Vegetables occupy an important place in diversification of agriculture and have played a pivotal role in the food and nutritional security of every growing population of our country. Vegetables are becoming important as cash crops for urban and export markets. Looking at the national scenario, vegetables have tremendous strength in terms of natural and genetic resources. A large number of wild taxa and indigenous germplasm are available in the country and many of them have not being fully capitalized. Though efforts have been made to tap the variability present among the vegetable crops in the country and many varieties have been released, it needs further attention so that the problems of biotic and abiotic stresses can also be minimized. The vast diversity of land, soil and agro climatic conditions prevalent in India also offers a unique competitiveness to grow wide range of vegetables. During the last four decades, India has made commendable progress in vegetable production (from 23.45 million tonnes in 1961-65 to 166.47 million tonnes in 2015-16), securing the position of second largest production of vegetables in world next only to China. The total world vegetable production is 1159.88 million tonnes from an area of 59.16 million hectares. In this, India contributes about 15.88 % with the production equal to 166.47 million tonnes from an area of 9.29 million hectares (NHB, 2015-16).

1. Potato

Scientific name: Solanum tuberosum L.

Family: Solanaceae

Origin: South America

Chromosome No.: 2n = 4x = 48

Introduction

Potato is one of the four major food crops of the world and is also a very popular vegetable crop. It is extensively cultivated in China, Russian Federation, Ukarine, Poland, Ireland, Great Britain, Germany, Netherlands, France, Spain, South America, India and USA. Potato was brought from Peru to Spain by the Spaniards in 1565 when it was widely grown in Spain and Italy. They were probably brought to England about 1586 by Sir Francis Drake. Potato was introduced in India in early 17^th century probably by the Portuguese. Now it is one of the principal cash crops of India.

Importance and use

• Potato is the fourth major food crops of the world after rice, wheat and maize.
• Being a major source of carbohydrate it is often used as a substitute for cereals.
• It is one of the major vegetable crops of the world and is grown in almost every country.
• In India, it is considered as a staple vegetable and used either alone or mixed with other vegetables.
• Potato is used as vegetable in various forms such as, boiled, mashed, baked, fried and cooked in sambar and different curries of vegetable, fish and meat.
• Potato is processed into different commercially used dehydrated, canned and fried products like, shredded potato, sliced potato, chips, flakes, French fries, finger chips, granules, disc, cubes, flour, etc.
• It is used for several industrial purposes such as production of starch, alcohol, dextrin, glucose, etc.
• Potato starch is a large grained starch and contains 25 % amylase and 73 % amylopectin and high phosphate content.

1. Seedling management in the nursery

Seedlings of many seed propagated vegetable crops like, tomato, brinjal, chillies, sweet pepper, cauliflower, cabbage, knolkhol, broccoli, Brussels sprout, kale, endive, chicory, celery, lettuce, parsley, onion, etc. are first raised in the nursery beds before transplanted in the main field although, many of these crops like, tomato, sweet pepper, onion, cabbage, cauliflower, broccoli, etc. are also direct seeded using precision seed drills in the USA, Japan, the Netherlands, Italy and other European countries. Many seed propagated vegetable crops viz., okra, peas, beans, radish, carrot, beet, turnip and all the cucurbits (e.g. bitter gourd, bottle gourd, pumpkin, cucumber, watermelon, muskmelon, snake gourd, sponge gourd, ridge gourd, etc.) are conventionally grown through direct seeding in the main field.

Transplant groups

1. Plants easy to transplant: They form new roots rapidly, put forth shoots rather slowly and absorb water readily e.g. beet, broccoli, Brussels sprouts, cabbage, cauliflower, Swiss chard, lettuce and tomato.

2. Plants transplanted with moderate ease: This group replace roots and grow shoots a bit slower than the previous group and do not absorb water very efficiently e.g. celery, brinjal, onion, pepper, leek and cauliflower.

Introduction

The book has been framed as a text book cum vegetable production manual for both under graduate and post graduate students of Agriculture/ Horticulture as well as commercial vegetable production professionals. This book has been oriented on different management practices of vegetable culture in open field condition viz., Classification of vegetable crops, Management of seedlings in the nursery, Water managent, Plant nutrient management, Pollination management, Integrated pest management, Biological management of diseases, Weed management and Production technology of different vegetable crops.

Classification of vegetable crops

Vegetables are defined as herbaceous plant origin material with generally low calorific value providing micronutrients, vitamins, antioxidants, fibre and induce alkaline reaction inside human body. Vegetable crops are not only botanically different but also their cultural and climatic adaptation shows wide variability. It is, therefore, necessary to classify the vegetable crops for their easy recognition. Vegetable crops can conveniently be classified “Botanically” for the purpose of vegetable breeding and according to “Method of culture” from the point of crop husbandry.

Botanical classification

The crops used for vegetable purpose in the world belong to 1200 species under 78 families and of them, more than 860 species under 59 families belong to dicotyledoneae and about 340 species under 19 families belong to monocotyledoneae. In the tropical and subtropical parts of the world, about 90 species of vegetable crops are cultivated, but hardly 20 of them are commercially important.

In the present botanical classification, over 140 different vegetable crops under 6 monocotyledoneae and 20 dicotyledoneae families have been included.

Maize (Zea mays L.) has tropical origin and is traditionally grown in monsoon. The crop is grown in diverse geographical and climate conditions around the world and in India maize is grown in kharif (rainy season), rabi (winter season) and spring seasons where more than 70 per cent of the production happen in kharif alone. Of late, the area under rabi maize has increased because of higher productivity and ever increasing demand.

Insect pests of maize

Compared to most cereals, maize faces fewer biotic and abiotic constraints in production. Among biotic stresses, insect pests are major constraints in the production and productivity of maize crop. The earlier literature cites over 160 insect and mite species which attack maize crop (Fletcher, 1914, 1917; Ayyar, 1940; Bhutani, 1961; Pant and Kalode, 1964) but later Mathur (1987) observed over 250 species of pests associated with maize in field and storage conditions. Dick and Guthrie (1988) identified 87 species that directly or indirectly exert severe stress on corn culture in tropical and temperate regions throughout the world. Excluding stored grain insects, Luckman (1978) lists 34 pests or pest groups for which chemical controls are recommended on corn in the United States. There have been more than 130 insect pests reported to cause damage to maize in India but only about a dozen cause economic losses (Sarup et al., 1987).

Shrinking land resources, vulnerable market of cash crops along with climatic aberrations have adversely affected the Indian agriculture and as a result, small farmers are increasingly alienating from this profession. To sustain their livelihood, farmers are increasingly adopting the alternative sources and dairy farming is one of the best-suited businesses, which can rightly fit at this stage. It is fast emerging as a potential business in rural India, particularly in the peri-urban belt. Among the major inputs required to set up a dairy unit, round the years availability of good quality fodder is the foremost requirement. Green forages are rich and cheapest source of carbohydrates, protein, vitamins and minerals for dairy animals. The importance of forages in our country is well recognized since feeding forages alone accounts for over 70% of the cost of milk production. Hence by providing sufficient quantities of green fodder instead of costly concentrates and feeds to the milch animals, the economics of dairy unit can easily be maintained. Milch animals need around 40 kg of green fodder daily for better milk production and for maintaining good health of the animal. There is a large number of fodder crops grown in our country, the majority being dominated by pearl millet, sorghum, maize and oats in the non-leguminous and berseem and lucerne in the leguminous category. Pearl millet and sorghum are extensively grown as cereals in the southern and dryer regions of the country and the stover remained is used as animal fodder. However, the nutritional quality of the stover is very poor due to higher content of indigestible lignin and low levels of cellulose, whereas, the cultivated green fodders are mostly confined to the North-Western plain zones of Punjab, Haryana and Western Uttar Pradesh. The availability of round the years quality green fodder, therefore, seems to be a tough task. At present, the country faces a net deficit of 61.1% green fodder, 21.9% dry crop residues and 64% feeds (IGFRI Vision, 2030).

Foodgrains are a fine example of diversity of nature with many varieties and varied features. They are storable food commodities and lend themselves to numerous processing manipulations. Nutritionally foodgrains are rich source of almost all known nutrients and several phyto-chemicals. The increasing incidence of metabolic diseases are attributed to unbalanced energy rich diets lacking in fiber and protective bioactive compounds such as micronutrients and phyto-chemicals (Prakash, 2013). Cereals along with pulse combinations constitute an important part of human diet in many parts of the world because of easy availability, low cost, long shelf life and nutritional balance. During recent past, India has made significant progress in improving the food security of its masses. The green revolution of 60’s helped the country in achieving the food security by improving the availability and access components (Kaul et al., 2016). However, surplus quantity of food produced is not uniformly distributed and the quality component is not addressed properly which has a direct impact on nutrition. In spite of surplus production of foodgrains still malnutrition persists. However, it can suitably be overcome by combining cereals with good quality pulses and other value addition techniques which will go long way in solving the problem of malnutrition. Even though country is producing large quantity of coarse grains particularly maize but its utilization for direct consumption is very low which is mainly due to poor quality of protein. Although maize contains higher proportion of protein (10-12 %), its quality is poor as compared to that of protein in rice due to presence of high concentration of alcohol soluble protein fraction ‘prolamine’ also known as ‘Zein’ in the endosperm. Zein is very low in some essential amino acids, mainly lysine and tryptophan which contribute to more than 50% of the total protein. On the other hand, very high amount of leucine and imbalanced proportion of isoleucine contribute to poor quality of protein in normal maize. To overcome such deficiencies, scientists developed Quality Protein Maize (QPM) where the quality of protein is not only higher than the common maize, but also significantly higher than that of other cereal grains. In the recent past QPM has got special distinction among the cereals due to presence of high amount of essential amino acids like lysine and tryptophan. QPM can be utilized for diversified purposes in food and nutritional security as infant food, health food, convenience food, specialty food and emergency ration. In India maize is consumed mainly in the form of boiled corn or roasted as pop corn. In order to increase the proportion of maize for direct consumption, several traditional food-processing and preparation methods can be used at household level to enhance the bioavailability of micronutrients in maize based diets. These methods include alkali cooking, thermal processing, mechanical processing, soaking, germination/ malting and fermentation. In countries like Central America and Africa, it is converted into food products by grinding, alkali processing, boiling, cooking and fermentation (Srilakshmi, 2008).

Maize or corn (Zea mays L.), which was first domesticated in Mexico, is the most widely grown cereal crop in the world. Maize is consumed as feed and food, and also has myriads industrial applications, including the production of bioethanol and starch. Due to its highest genetic potential, production and productivity among the cereal crops, maize is called as “Queen of the Cereals”. Noble Laureate Dr. Norman E. Borlaug, the Father of Green revolution stated 15 years ago that “The past two decades saw the revolution in rice and wheat, the next few decades will be known as maize era”. In India, maize is the third largest grain crop and its production and demand is increasing rapidly. In India, it is primarily a non-food cereal crop, with only 10% being consumed as direct human food, while the rest being used for feed, industrial and export purposes. The effective deployment of various frontier technologies viz. genetic engineering, doubled haploids, molecular markers, phenomics etc.has greatly helped in raising productivity of maize crop worldwide in the last decade.The present chapter provides an overview on role of frontier technologies in maize crop improvement.

Seed in agriculture recognized as one of the most vital and crucial input in agriculture ever since the human kind has started tilling the land to produce the food for his existences. Seed technologist defined seed as matured fertilized ovule consisting of an intact embryo, endosperm and or cotyledon with protective covering (seed coat). A quality hybrid maize seed production system ensures design of the production technology which enables to maintain the healthy embryo, endosperm and /or cotyledon with protective covering. In fact the search of quality seed had acted as a major force to evolve the science of plant breeding. Ever since the farmers understood the importance of seed, they started trading the quality seed within community and now between geographies. Self-pollinated crops possess the natural mechanisms to maintain the intactness of genetic constitution of a seed during multiplication as there is very minimal chance of natural crossings. On the contrary, cross pollinated crops like maize requires special techniques in seed multiplication to maintain the genetic constitution of a F₁ hybrid. Hybrid Maize seed production techniques are well defined and are used by public and private sectors to produce the required hybrid seed in India. In this chapter, I will be highlighting the process, potential agro-climatic zones, hybrid maize seed production capacity and its future perspective in India. In addition, it also reviews the current system and possible strategic move in order to support the maize seed production system and steps required to meet future demand.

Maize is a miracle crop and is popularly known as “Queen of Cereals” due to its very high yield potential. It’s a leading crop worldwide in terms of area, production and productivity and is suitable to diverse agro-climates. Its extent of adaptability is unmatched by any other crop. Due to this adaptability to diverse climates, maize had been grown by many generations in different parts of the world. Earlier, the primary utilisation of maize was focused towards human consumption and even today it is a staple food of many underprivileged societies especially in African countries and certain tribal pockets in India. It is playing an important role as a cheap source of carbohydrate in daily meals due to high productivity and wider adaptability. With the passage of time the productivity increased due to conscious selection towards high yield by farmers and researchers. This enhanced production and productivity opened new doors of utilisation of this wonderful crop. Maize found its place as a feed crop for economically important anima ls like chicken and swine. This shift towards feed industry got encouragement by change in the food habits of people. Looking at the recent consumption pattern of maize as a food crop displays declining trend. As per a NSSO survey (2014) maize consumption in rural areas in India has seen a decline of around 35% from 3.7 kg/year in 2004-05 to 2.4 kg per year in 2009-10, and further declined to 1.56 kg during the year 2011-12. Continued efforts towards yield enhancement and decline in human consumption made its way to utilise surplus maize in economically viable industrial products. Maize is a crop where every part of its plant can be utilised to produce food and non-food products. Oil, starch, glucose, dextrose and sorbitol are few important products produced by processing of maize grains. Apart from these, maize has been very widely used for production of ethanol which is used as a fuel in many developed countries. Direct utilisation of maize grains in poultry and other feed industries is on the rise with increasing demand of such foods. Maize oil is of high quality and is being widely used in developed countries. Maize is a major source of industrial starch which finds its way to diverse utilization such as food and beverage, pharmaceutical, textile, paper and other industries. Processing of maize is done by two ways- Dry Milling and Wet Milling. Wet milling produces starch, gluten and husk and Dry Milling produces corn meal, germ, grits and animal feed. Apart from these processed products, maize is also being developed to be utilised as a biopharmaceutical crop. Maize crop is being bioengineered to extract products of therapeutic and medicinal importance. Such a diverse utilisation of maize has generated a necessity to improve morpho-physiological traits which are important with respect to its industrial utilisation. In the present chapter, we will discuss various aspects of maize improvement for industrially important traits which will include oil, starch, methionine, biofuel and biopharmaceuticals.

The ever mounting population in 21^st century is a great challenge for the researchers to provide ample food and that has to be achieved within the context of existing challenges such as deteriorated arable land along with increasing adverse affects of changing climate. There is demand to increase food production to the tune of 70% by 2050 (Wani and Sah, 2014). Although major crops have achieved good progress, vulneRability to climatic variability emerged as a serious concern in crop improvement which could be attributed to higher sowing densities that in turn lead to more competition for water and nutrients. Abiotic stresses can be considered as major yield restraining factors among different crops. The projected estimations for changes in abiotic stresses such as drought, water logging and heat reveals far serious concerns for crop productivity (Qin et al., 2011; Bailey-Serres et al., 2012). Therefore, the concern of global food security in the era of climate change has compelled plant scientists in different crops to shift to breeding for climate resilient genotypes. Maize, being a hardy crop has an inherent potential to perform well under sub-optimal environments with minimum crop management (Kole et al., 2015). Abiotic stresses such as drought, heat and water logging affects maize yield adversely.

Although, conventional breeding approaches have exploited its fullest potential for production of elite maize hybrids but still little success attained for breeding climate resilient genotypes or varieties owing to the fact that stress tolerance is multi-genic or quantitative in nature. Therefore, the best option is to utilize the molecular breeding along with the conventional approaches for development of climate smart varieties.

Maize occupies an important position in the world economy, and serves as an important source of food and feed (Gupta et al., 2015a). Together with rice and wheat, it provides at least 30% of the food calories to more than 4.5 billion people in 94 developing countries (Shiferaw et al., 2011). In India, maize is an important cereal too, and is grown on an area of 9.2 million hectares with production of 24.2 million tonnes (www.indiastat.com). The demand for cereals will continue to increase as a consequence of the expanding human population. The world will have around 7.7 billion people by 2020, and it will reach up to 9.3 billion by 2050, and the demand for maize between now and 2050 will double in the developing world (Rosegrant et al., 2009). By 2025, India too would require to double the production (50 million tonnes) of maize grain to meet the domestic demand (Yadav et al., 2016).

Malnutrition caused by inadequate consumption of balanced diet has emerged as one of the alarming problems especially in the under-developed and developing world. Mostly the resource-poor suffer from ‘hidden hunger’, a term often used to describe malnutrition caused due to micronutrient deficiencies in staple food diet (Bouis and Welch, 2010).

An estimated two billion people suffer from micronutrient deficiency, while 795 million people are undernourished (IFPRI, 2016a). Of the 667 million children under the age of five worldwide, 159 million are stunted, while 50 million do not weigh enough for their height (IFPRI, 2016b). An estimated 45% of deaths of children under age five are linked to malnutrition (Black et al., 2013). Malnutrition contributes to global burden of disease, and loss in annual gross domestic product (GDP) in Asia and Africa to extent of 11%. South Asia is home to more than 35% of the world’s poor, and 21.9% of the population of India lives in poverty, and thus vulnerable to various health problems (IFPRI, 2016a). 15.2% of the people in India are undernourished, and more than one third of the world’s underweight malnourished children live in India, posing severe socio-economic loss to the country.