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Agriculture continues to be the occupation and way of life for more than half of the Indian population even today. Sustainable prosperity of this class-the farmers, the landless agricultural labourers holds the key for improving the overall human resource development scenario in the country. Indian agriculture had been on traditional lines till the first waves of Green Revolution gave a sudden boost to the production and productivity of major cereals in the assured irrigated areas. Quick dissemination of technological information from the agriculture research system to the farmers in the field and reporting of farmers feed back to the research system is one of the critical inputs in transfer of agricultural technology. The information and communication support during last 50 years has mainly been conventional. The extension personnel of the departments of agriculture disseminated the technological message to the farmers through personal approach. This approach has not been able to reach and satisfy majority of the farmers who are spread across the whole country with varied interests. This gap remains a challenge for the Extension System even today.

To reach over 110 million farmers, spread over 500 districts and over 6000 blocks is an uphill task. The diversity of agro-ecological situations adds to this challenge further. The success of Green revolution was mainly achieved due to concerted homogenous extension strategy. Farmer’s needs are much more diversified and the knowledge required to address them is beyond the capacity of the grass root level extension functionaries.

There are basically two aspects of extension: the education function which aims to help farmers gain knowledge, acquire skills and change attitudes for their own benefit; and the communication function which transfers information about suitable technologies to farmers and growers and offers them alternative solutions to improve their situations. The aim is to transform the information into knowledge timely and accurately to create an environment for farmers to use it. IT will be indispensable in transferring information between research and extension organizations.

After preparing the desired DNA molecule using E.coli, the plasmid can be transferred into Agrobacterium by this method. Efficiency is low (~10³ transformants per mg of DNA) but the method is reliable and very rapid. This method also eliminates much of the plasmid rearrangement that occurs during triparental mating.

Procedure

Grow an Agribacterium strain containing an appropriate helper Ti plasmid in 5 ml of LB medium 16 hours at 28ºC.

Add 2 ml of the culture to 50 ml LB medium in a 250 ml flask and shake (~250 RPM) at 28ºC until an OD₆₀₀ of 0.5 to 1.0 is obtained.

Chill the culture on ice. Centrifuge the cell suspension at 1000 g for 30 minutes at 4ºC.

Discard the supernatant. Resuspend the cells in 1 ml of 20 mM CaCl₂ (ice cold). Dispense 100 mL aliquots into pre-chilled 1.5 ml tubes.

Add about 1 µg of the desired plasmid DNA (pure or crude) to the cells in each tube.

This protocol is designed for potato stem internodes on ZIG medium, but might work for leaf pieces as well.

Plant Material

Cut stem internode explants from 5-6-week-old in vitro plants (or leaf explants from 3-4-week-old in vitro plants) grown on 15 ml PROP in 25 x 150 mm test tubes.

Day 1

Streak out desired Agrobacterium strain on YEP + antibiotic to obtain a single colony. Grow at 28 C for 2 days. Alternatively, skip this step and use frozen stock for next step.

Day 3

Use a single Agrobacterium colony to inoculate 20 ml YEP + antibiotic in 250-ml flask. Grow overnight at 28 C with vigorous shaking (~200 rpm). Some Agrobacterium strains require an additional day to reach sufficient density.

Principle

Micropropagation is especially useful for include propagation of high value plants that grow slowly (bulbs, ferns, orchids, palms, etc), new cultivars having limited stock and high market demand (new cultivars produced via conventional breeding or genetic engineering), cultivars that are difficult to root by conventional techniques, and endangered plant species. In addition, virus elimination is possible via culture initiation from meristem explants. Also, micropropagation provides a source of sterile tissues that can be used for other tissue culture protocols, including genetic transformation.

Micropropagation is possible using multiple types of cultures; however, shoot cultures are typically employed, in which case five stages to the micropropagation process are typically recognized as outlined below. Nodal explants of rose are used for culture initiation. Three media, differing in hormone content, is employed and the explants should be evaluated for differences in axillary shoot growth and proliferation.

Principle

Orchid seeds are produced in large numbers but are exceedingly small, hence are also called “dust seeds” may be as small as 0.01 by 0.05 mm in dimension and weigh as little as 10-30 ug. Seedling development is generally a slow process in orchids, with the seedlings passing through an initial stage, termed the protocorm stage, prior to the production of distinct vegetative organs (leaves, roots, etc). The protocorms of terrestrial orchids are subterranean and entirely heterotrophic, with all of the nutritional needs of the orchid being supplied by the mycorrhizae (the symbiotic fungus). The length of the protocorm stage is variable, however, in many cases, months, or even years, elapse before leaves and roots are produced.

The combination of a small seed size and fungal symbiont requirement largely precludes the propagation via conventional seed propagation protocols. Accordingly, most of the early research on the development of orchid seed germination protocols focused on in vitro germination with the seeds and fungal symbiont being sown on the same petri plate. Successful protocols were developed for several species; however, the process was complicated by the diversity of fungal symbionts required (across orchid taxa) and the difficulty in isolating and culturing these fungi.

In the 1920’s the process of orchid seed propagation was revolutionized by Lewis Knudson with the demonstration that nutrient media that could be used as a substitute for fungal infection. The isolation and culture of multiple fungal strains was no longer required, and improvements in germination frequency, germination speed, and seedling vigor were generally observed. To date, successful asymbiotic germination techniques, termed ‘flasking’, have been developed for many epiphytic orchids, with several species being saved from extinction via the application of these techniques.

Media Preparation

Before mixing medium, determine the quantity of each medium and the amount of plant growth regulator needed for each experiment. Make slightly more medium than is required. A media check off list is helpful in making medium to keep track of ingredients.

Remove media stock solutions and plant growth regulator stock solutions to be used from the refrigerator. Make up fresh stock solutions if adequate quantities are not available or if solutions have precipitated.

Plant growth regulators stock solutions are prepared biweekly and stored at 4º C in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and NAA) are dissolved in 100% ethanol prior the addition of water.

Fill the volumetric flasks upto ½ to ¾ with deionized water. Add proper a of stock solution and plant growth regulators and mark each item on the check off list. Add 30 g/l sucrose, stir until dissolved. Make up the volume of the flask with deionized water and later por into a larger beaker.

Principle

Western blot analysis (also called protein immunoblot) is a useful tool for the detection of proteins in biological samples. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose), where they are probed (detected) using antibodies specific to the target protein.  It is a technique for analyzing and identifying protein antigens: the proteins are separated by electrophoresis in polyacrylamide gel, then transferred (“blotted”) onto a nitrocellulose membrane or treated paper, where they bind in the same pattern as they formed in the gel. The antigen is overlaid first with antibody, then with anti-immunoglobulin or protein A labeled with radioisotope, fluorescent dye, or enzyme. 

The method originated from the laboratory of George Stark at Stanford. The name “Western blot” was given to the technique by W. Neal Burnette and is a play on the name “Southern blot”, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting and the detection of posttranslational modification of protein is termed “Eastern blotting”.

Cell Lysis Procedure

Pellet the cells in a microfuge tube: add 1.5 ml culture, centrifuge for 15 seconds at maximum speed, discard the supernatant, and then repeat the procedure. This will result in the cells from 3 ml of culture in a single tube.

Resuspend the cells in 1 ml Lysis buffer.

Take 300 µl aliquot for Bradford protein assay.

Add 50 µl lysozyme, and mix gently.

After 10-20 minutes at room temperature, microfuge for 10 minutes to pellet cell debris.

Use supernatant for LDH assay.

Principle

The SDS polyacrylamide gel electrophoresis (PAGE) performs:

Separation of proteins according to their molecular weight

Ascertain whether a given protein is actually present in a sample,

Assessment of purity of the preparation

Estimation of the approximate quantity of the protein, and

Measurement of the size of the protein.

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

The principle of silver nitrate staining of proteins is based on the reduction of silver ions to its metallic form. (Silver ions in alkaline conditions bind with proteins through å-amino group of lysine and sulphur groups of cysteine and methionine residues). When the complexed silver ions are reduced in the presence of formaldehyde, they become metallic silver, which is seen on the gel as bands. Staining of proteins with silver nitrate after SDS-PAGE is quicker and ~ 10 times more sensitive than Coomassie brilliant blue staining and that is the reason it is widely used in research laboratories these days.

The procedure is as follows:

Denaturing polyacrylamide/urea gels in TBE buffer

Prepare 20ml of a 5% polyacrylamide gel containing 7M urea by adding:

47.5% acrylamide:

2.5% bis-acrylamide solution 2ml

10M urea 14ml

10X TBE buffer 2ml

10% freshly prepared ammonium persulfate 0.2ml

deionized water 1.8ml

Principle

Monomeric acrylamide (which is neurotoxic) is polymerised in the presence of free radicals to form polyacrylamide. The free radicles are provided by ammonium persulphate and stabilised by TEMED (N’N’N’N’-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of crosslinking. The chain length is proportional to the acrylamide concentration: usually between 3.5 and 20%. Cross-linking bisacrylamide is usually added at a ratio of 2g BIS 38g acrylamide.

Principle

RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode. RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form. The following protocol is based on Formaldehyde Agarose Gel Method.

Principle

To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.

Time required

Restriction digest - minimum of 2 hours

Electrophoresis - approx. 4 hours

Elution - 2-5 hours depending on fragment size

DNA Purification -2 hours

here are many methods for extracting DNA from gel apart from using commercial kits, some methods however yield cleaner DNA than others.

1. Freeze squeeze

Excise band and place pieces of it in a home-made spin-column (a piece of filter placed in a PCR 0.5ml eppendorf, pierced the bottom of the PCR tube with a needle). Place the PCR tube at -20 °C for 15-20 mins or freeze in liquid N2. Then place the PCR tube into the 1.5 ml eppendorf and spin at top speed in a microfuge for 15 mins, collect DNA solution which can be EtOH ppt or use directly.

2. Powdered silica

Melt excised bands at 70°C cool and add equal volume of TE-buffered phenol and mix. Spin for 5 min, remove aqueous phase, repeat and EtOh ppt. the DNA.

3. Electroelution

Agarose gel electrophoresis is the easiest and commonest way of separating, capable of resolving mixtures of DNA fragment that cannot be separated conveniently by other procedures. The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band. The DNA is visualised in the gel by addition of ethidium bromide. This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light.

Agarose, which is extracted from seaweed, is a linear polymer whose basic structure is shown in figure.

Principle

The principle of Electrophoresis involves the motion of a particle in a fluid medium, where the gravitational attraction (on a flowing liquid) is the sedimenting force, and the frictional force is the opposing force and is proportional to the velocity of the particle. These forces balance each other acting in opposite directions and hence balance each other. This will lead to the uniform motion of the particle in the mobile liquid medium (i.e. the particle moves at constant velocity). In case the external field is an electric field instead of a gravitational field, a macromolecule will respond to the external field in two ways. In case the molecule is charged, it will migrate in an electric field to the electrode of opposite charge. Under this principle the technique of electrophoresis functions.

All electrophoresis operations are governed by the principle:

Principle

Cloning experiments would be very difficult if we do not screen those very few cells that took up the recombinant plasmid DNA, as the transformation efficiency is very low as stated before (usually less than 1 in 10⁵ cells). Hence it is necessary that there should be a selection mechanism (i.e. conditions where cells grow if they express a gene, but die if they do not) to sort out which are transformed cells and which are not make the job easy.

A commonly used selection mechanism is antibiotic resistance. If the E. coli strain used is not resistant to the antibiotic, but the plasmid DNA contains a gene coding for resistance, then only the cells that have taken up the plasmid will be able to grow. Most commonly used plasmids contain a gene for resistance to ampicillin; only cells that have taken up the plasmid will be able to grow and form colonies.

Note: Uptake of plasmid DNA is rare; hence each group of cells visible on a plate represents the offspring of a single cell that will contain a single molecule of plasmid DNA.

Transformation

The uptake of DNA by competent cells is called transformation. Transformation involves:

Mix a small volume of DNA (usually 1-3 µl) with 100 µl of competent cells.

The mixture is incubated on ice for 30 minutes to allow the cells to take up the DNA.

The cells are then heat-shocked at 37 to 42°C for 1 minute, and cold shocked on ice for 2 minutes.

The heat shock/cold shock reverses the effect of the competent cell process, by healing the damage inflicted by the competent cell solution.

Add 0.5 ml of LB culture mediumz

Incubate the cells for 10 - 30 minutes at 37°C to aid the healing process.

This cell/medium mixture is then spread onto bacterial culture plates containing agar, LB, and a selection mechanism.