World Water Day 2014

World Water Day 2014: Holistic actions needed

SKT Nasar‏

The UN World Water Day 2014 with the theme ‘Water and Energy’ has been celebrated on 22 March 2014 as a part of International Decade for Action ‘Water for life 2005-2015’. Nations have been talking for long but many of the countries sulk when it comes to fair action. Only piecemeal programmes for patchwork are not enough. The challenges are extraordinary that require holistic actions now.

Cyclic existence of everything is an established principle valid for all aspects from God Particle to Multiverse. These cycles overlap and are intricately interlaced.  Damage or snapping off of the natural chains of cycles causes severe distortions, even disasters. Until functional cycles are invented de novo, the only option left to humanity is to repair the damages to re-establish the snapped chains. So is it with liquid water on madre terra.

Life on earth cannot exist without water. The universal right to life indeed includes the right to liquid water. It is a massive task to provide physically, chemically and biologically clean water to quench the thirst of over seven billion peoples as of now; and demands are growing exponentially. Industries and urbanised landscapes require billions of gallons of clean water per day to ultimately gush out contaminated effluents. Intensive and extensive agriculture, in addition, require enormous amounts of clean water for irrigation. Erratic rainfalls or over-irrigation causes flash flooding that drains out top soil contaminating already silted water bodies. Agrochemicals of modern agriculture are washed out causing environmental pollution. Mammoth drawls of ground water for irrigation and other purposes disseminate polluting compounds such as arsenic otherwise sequestered in deep aquifers. These waters, thrown centrifugally off course their natural cycles, are not reinstated to natural states. This phenomenon sets off water-related disasters.

Distribution of water for all needs from source to destination involves high energy costs. Cleaning of water for consumption by humans, agriculture, livestock, and industry involves additional costs in terms of energy. Unfortunately, the stakeholders, both producers and consumers, of these processes are apathetic to recycling the abused water.

Water is used to produce hydroelectric energy. Large dams for harnessing hydro-power have shown potential for catastrophe of varied intensity. The hydro-energy is used for providing water to all and also for decontamination of water. Fossil, nuclear and renewable energy is consumed in addition to hydroelectricity to meet escalating demands. Nuclear energy has shown the capacity for tragedies. Each nation state should, by intention, laws, programmes and strict execution, make it compulsory for each stakeholder to conserve water, reduce its consumption, and to restore used water to its natural state at well-researched low energy costs. All industries, including bottled water and cold-drink industries should be made to comply.

The principle of ‘Polluter Must Pay’ needs to be modified to ‘Polluters Must Restore and Compensate Damage’. Moist agriculture rather than flooding- or wet-agriculture should become the order of the day. Polluting industry and agriculture must be discouraged. A palpable shift to using renewable forms of energy which is costlier in the present-day economy needs to be made compulsory.

Water and energy have become inseparable. While World Water Day is more or less of an annual ritual, the real challenge is to make each day, 24x7, the water-&-energy day a habit for every inhabitant of planet earth. Global zero-tolerance common-goal policy and action programmes should be: ‘Sustain water; Slash energy’.

S.K.T. Nasar

Former Director (Research)

Bidhan Chandra Krishi Viswavidyalaya

(= BC Agricultural University)

West Bengal, INDIA

E-mail: sktnasar (at) hotmail.com

Crop Plant Agriculture vis-ã-vis Environment

Crop Plant Agriculture vis-ã-vis Environment

S.K.T. Nasar

The is a magazine article to create awareness about environment; hence, details of technical aspects are avoided.

Published in Jeebaner Paribesh - Environment of Life; 2nd Year, 9th Issue, 15 April 2014, pp 15-19

Agriculture Sustains Life and Livelihood of Billions

Many perceive Agriculture as too muddy for respectability which, in fact, it is not. Agriculture includes all operations from sowing seeds to harvesting and making agricultural products available for use as food, feed, aesthetics and industrial raw materials. Agriculture encompasses almost all spheres of human life.

The enormity of agriculture as a sub-system of biology, society, economy and environment emanates from data that more than seven billion people globally, 1.27 billion in India, are eating food and using agricultural products. Demand for contaminant-free food-cum-nutrition is rising as never before. Requirements for agricultural products for services and as industrial raw materials are growing exponentially.

Agricultural production is prone to environmental conditions. Spells of drought or flash floods cause devastations that make the landless farmers to the members of the United Nations Organisation to sit up in alarm. Earth quakes, Tsunamis and typhoons spell disasters not only for local communities but trigger international ripples in prices and in transborder businesses.

Agriculture contributes to making or marring the environment. Agriculture and environment maintain a two-way traffic.

Sunlight Energy and Agriculture

The fundamental tenet of capturing and utilising sunlight energy through agriculture has not changed over millennia. Nor is it likely to alter in the foreseeable future. Agriculture in India as elsewhere has modified from subsistence- to surplus- to commercial agriculture giving way to internationalisation of farming systems. This sunlight energy captured by all green cells including those of crop plants passes through food webs on to other organisms that lack the mechanism of capturing sunlight energy. Solar energy ultimately reaches the human species from different sources from crop products to edible animals - their meat, milk and egg.

Agro-ecosystems like natural ecosystems recycle all substances created by life processes and from dead organisms.  The process is bio-geo-chemical recycling. Breakdown of the recycling processes has far reaching consequences for the environment.

Glocal (i.e. global plus local) Concerns

No wonder then that Agriculture is glocal business and trade at global to local levels. Policy concerning self-sufficiency in agriculture and its sustainability besides equitable accessibility to food, and profit-making to satisfy human needs and, of course, greed are bothering all stake holders. Agricultural operations transcend national, environmental, geographical and generational boundaries.

Location specificity and seasonality of agricultural commodities are determined by the environment. Sustainable agriculture needs to adjust to climate change and unprecedented environmental fluctuations. That is a formidable challenge.

Massive intensive and extensive agricultural operations to assure the escalating needs of exponentially growing human population indeed modify the environment in diverse ways.

Agriculture and environment are inseparable components affecting each other.

Facets of agriculture

Agriculture has been defined as ‘the systematic and controlled use of living organisms and the environment’. It includes cultivation of terrestrial and aquatic plants, animals and fish. Major operational schemes of agriculture are related to where, when and how of cultivation methods, choice of the right varieties and essential practices to be followed. Inputs like seeds, implements, irrigation water, energy in the form of fuel and electricity, fertilisers and devices for pest protection have to be made available to grower-farmers.  These are non-farm operations. Non-farm agricultural activities have a direct bearing on the environment and vice versa.

On-Farm Plant Crop Production in Relation to Environment

A quick look at the production technology and package of practice for plant crops highlights the two-way relationship between agriculture and environment.

Vital agricultural operations of plant crop are land preparation, sowing of desired seeds, providing irrigation water, adding fertilisers, taking pest control measures, and finally harvesting products. Timeliness of each operation is crucial. 

Manoeuvres are different for the production of human food and livestock feed, plantation crop, flower, fibre, wood-fuel, biofuel, medicines, and industrial raw material. Molecular farming for specific molecules is in high demand. Agriculture uses, in addition to plant species, organisms such as microbes, animals, fish, insects with similar goals of production and services. Agricultural procedures may combine the benefits of different organisms for specific production targets. These operations depend on local climatic conditions and on the environment at large.

Photosynthesis is fundamental to all life and the essence of agriculture

Agricultural production is dependent entirely on photosynthesis, the natural phenomenon in which living green cells capture energy from sunlight and convert it to chemical energy. Leaves of crop plants appear green due to the presence chlorophyll contained in special compartments within cells, the chloroplasts. Crop plant agriculture is the only managed system that brings in energy from extraterrestrial source, the Sun.

During photosynthesis, sunlight energy, the photon, is first captured by the chloroplast/chlorophyll which then, through a series of complex biochemical processes, combines carbon dioxide (CO₂) and water (H₂O) to produce carbohydrate such as sugars and starch which are ultimately converted into protein, fat etc. However, all organisms must respire to sustain life. The biochemical process of respiration breaks down photosynthetic products but then, in balance, the accumulation of biomass i.e. biological mass is the outcome. Photosynthetic products during the prehistoric past formed fossil fuel that are presently contributing to the production of food, feed and other products that humans utilise.

The photosynthetic CO₂ comes from the atmosphere while H₂O, nutrients and minerals are mined from the soil by plant roots.

Agriculture contributes greatly to reducing CO₂ load of the atmosphere and to raising O₂ levels thus assisting the maintenance of environmental equilibrium.

Ecosystem and agro-ecosystem

Different kinds of organisms co-exist in nature. Living organisms form the biological or biotic factor of an ecological system called ecosystem. Air, water, soil, weather, and non-living matter together make the non-biological or abiotic component of an ecosystem. Agro-ecosystem is a managed ecosystem as opposed to unmanaged natural ecosystem such as a forest ecosystem.

Human societies began shifting from hunting edible animals or fish and gathering edible forest products such as cereals, fruits and fuel wood to domesticating animals and sowing seeds of plants for food and services more than twelve thousand years ago. This process gradually gave rise to organised agriculture and by about eleven thousand or more years ago societies were established as food producers.

Natural forest lands had to be cleared irreversibly for organised agriculture, human settlements, plantations, pastures for livestock, mining etc. It was from this time on that naturally occurring landscape continued to be converted into managed farm lands. This phenomenon impacted glocal environment. Yet, agricultural fields continue to be significantly influenced by the environment at local levels depending upon climate and topography. While climate determines the energy from sunlight and CO₂, the supply of water is determined by local geography, rainfall pattern and soil type. 

Ploughing of Farmland

Agricultural practices include tillage by ploughing to primarily loosen up the soil for aeration and smothering of weeds to achieve good germination of crop seeds, easy penetration of roots and desired plant growth. For the cultivation of wet or lowland rice (Oryza sativa), the ploughed land is puddled to retain water in the crop field. On the other hand, water must not be retained in the field for upland or dry cultivation of wheat (Triticum vulgare) for example.

Soil operations are confined mostly to the uppermost layer. The top soil is the most fertile soil rich in micro-organisms, nematodes and mites, earthworms, organic matter and crop nutrients. The top soil shows high biological activity.

The soil contains large quantities of essential nutrients for crop plants.  Living soil organisms convert crop nutrients to forms in which these are utilised by the plants. Soil moisture supplies water to the crop.

Ploughed soil is removed from cultivated fields with the outflow of excess water or is blown away from dry and dusty crop land with high velocity wind. Erosion moves soil into water bodies adding enormous amount of sediments. Sedimentation over decades causes choking of dams and irrigation canals. Soil erosion depletes fertility in farmland. Soil organisms including seeds of noxious weed are spread out to large areas. Ecosystem equilibrium is thus disturbed every cropping season. Emission of Green House Gas (GHG) is another international concern to which wetlands and wet agriculture is a contributor.

Quenching Crop Thirst – A Necessary Evil?

Crop plants need water at different stages of growth. Irrigation is resorted to where rain is scanty or when rain water is unavailable as required.

Irrigation water is sourced from open water bodies, for instance rivers, streams, lakes and ponds, or from groundwater aquifers by tube wells. Excessive withdrawal of ground water for irrigation besides for municipal and industrial supplies causes subsidence of the groundwater table.

Intensification of agriculture has caused depletion of groundwater leaving the soil dry with crusts of unwanted salts. Desertification or salinisation of once fertile arable land is not very uncommon in some areas. Desertification, salinisation, alkalisation and acidification of soils impact the environment in several ways.  

Many aquifers contain iron, arsenic and selenium. Groundwater contaminants are spread by irrigation and absorbed by farmland, water bodies and crops. Agricultural products are contaminated to unacceptable levels. The case of arsenic contamination is well known.  Chemical contaminants reach humans and livestock via the ecosystem food web. Health hazards and diminished saleability of commodities are rampant in vast geographical areas.

Periurban farm fields are largely sewage-irrigated with urban effluents overloaded with chromium (Cr), cadmium (Cd), arsenic (As), lead (Pb), copper (Cu), zinc (Zn), and antibiotic-resistant bacteria and fungi.

These toxic chemicals and disease-causing biological entities infused into agricultural products adversely affect the well being of consumers.

Dams and labyrinthine canals have been constructed to provide irrigation to crop plants at huge public costs. The total area of agricultural land in India is about 8.32 million hectares out of which about 2.67904 million hectares are under irrigation. Dams are evidenced to create environmental and social problems. Silting of canals and choking of dams lead not only to huge costs for restoration but cause floods and disasters on large scales.

Channelling of river water for agriculture and industries is already creating inter-state and inter-nation conflicts. Water war is likely to follow Oil war at international levels.

Protecting crop plants from pests

Crop plants are vulnerable to pest attacks. Destructive pests may include species and strains of virus, bacterium, fungus, nematode, mite and insect either singly or in combination.  Rodents and birds also destroy standing crops. Large quantities of pesticides are employed to ward off or kill pests to save pre- and post-harvest losses in production.

Weeds are another group of common agricultural pests. Herbicides and weedicides are applied to control infestation of weed plants that compete with crop plants for water, nutrient and also harbour other pests.

All pesticides are toxic to life forms. These chemicals not only kill unwanted target pests but also non-target crop-friendly organisms such as pollinators (honey bees, butterflies), root zone-dwelling bacteria and fungi.

Many pesticides are retained in agricultural products consumed by humans and livestock. There is a build up of pesticides in the agricultural soil carried to water bodies through run off movements. Over time, pesticides also leach into aquifers. Finally, pesticides are consumed by people. We pay to eat poison!

Feeding the Food Producing Crop Plants!

Fertilisation is the timely supply of nutrition to hungry crop plants to produce food. Some chemical elements vital to crop survival and growth are the main ingredients in fertilisers. Atmospheric air supplies hydrogen (H), oxygen (O), and carbon (C) as non-mineral nutrients. Mineral nutrients comprising macronutrients and micronutrients come from the soil and are absorbed through roots. Among these, nitrogen (N), phosphorus (P), and potassium (K) are primary nutrients, whereas calcium (Ca), magnesium (Mg), silicon (Si) and sulphur (S) are secondary macronutrients. In addition, boron (B), copper (Cu), iron (Fe), chlorine (Cl), manganese (Mn), molybdenum (Mo) and zinc (Zn) are micronutrients needed only in small quantities.

For crops under nature farming or organic farming, the nutrition is made available by the soil through recycling of organic matter or by converting soil nutrients into forms suited for uptake by plants.

Modern intensive and extensive farming entails the use of factory manufactured fertilisers. If not applied appropriately, excess N and P can have negative environmental consequences. Surplus N from synthetic fertilisers as highly soluble nitrate can lead to groundwater nitrate contamination. Nitrate with P contaminates areas causing eutrophication i.e. over dominance of a single species turning the area ultimately into dead zones.

The major inputs of heavy metals such as lead (Pb), cadmium (Cd), arsenic (As), selenium (Se) and mercury (Hg) into agricultural systems also derive from fertilisers, organic wastes and industrial effluents. These accumulate in downstream reservoirs contaminating water creating a toxic web.

Genetically Modified Organisms (GMOs) in Agriculture and Environment

GMOs in Indian agriculture are fiercely debated. Scientists and public are arguing. The Parliament is considering the issue. The National Biodiversity Authority (NBA) and Karnataka Biodiversity Board (KBB) have ordered prosecution under Biodiversity Act 2002 of senior representatives of the University of Agricultural Sciences, Dharwar, M/s Mahyco/Monsanto and M/s Sathguru.  The High Court of Karnataka dismissed on 11th October 2013 petitions that sought quashing of criminal prosecution. Higher courts are weighing options. The hullabaloo is all about Bt brinjal.

The Genetic Engineering Appraisal (formerly Approval) Committee (GEAC), created under the Environment Protection Act 1986 in the Union Ministry of Environment and Forests, is eager to order release of Bt brinjal for large scale cultivation. Curiously, Bangladesh has allowed commercial cultivation of Bt brinjal giving a talking point to proponents of Bt brinjal in India.

Bt brinjal is a brinjal (Solanum melongena; baigan, begun) variety created by inserting a gene which is an engineered stretch of DNA segments from bacteria and virus. The crystal protein gene, Cry1Ac, is extracted from a soil bacterium Bacillus thuringiensis and inserted into the genome, the complete genetic makeup of cultivated brinjal varieties. The process is known as transgenesis. The transgenic brinjal plants show resistance against specific insect pests, namely Brinjal Fruit and Shoot Borer, Leucinodes orbonalis, and Fruit Borer, Helicoverpa armigera. 

Bt cotton, the first non-edible transgenic crop, is being cultivated in India for some years with the claim that Bt reduces heavy pesticide use. However, long term results need to be scrutinised.

Insect resistant crop varieties have been created through conventional plant breeding techniques for over a century. In most cases the resistance breaks down chiefly due to the fact that the species of insect pests alter their genetic makeup over time. Preliminary data indicate that insects mutate to breakdown resistance of GM crops.

GM crops such as herbicide resistant Canola, a Canadian oilseed crop, result in genetic contamination of native plant species through unintended natural hybridisation. Feral communities of canola have been reported recently. Feral species are the species cropped in agricultural fields but escape to establish in natural ecosystems.

Non-target insects such as pollinators and natural enemies of other pests are also killed by Bt-toxin produced by GM crops.  The Bt gene composed of DNA is also known to move to other native organisms. These processes are likely to lead to genetic contaminations of cropped and wild plant species with far reaching consequences.

Future is Tricky but not Bleak, Provided........

The future of agriculture will come with unprecedented challenges. Sustainability with higher production in consonance with the environment can be achieved, provided we act diligently no later than now. Agriculture must conserve ecological balance and retain natural resources sustainable and provide biologically and chemically clean food-cum-nutrition, feed and other products. Technology supported by science is available to minimise contamination in, and by agriculture.

Climate change has emerged as a formidable challenge. Weaponising the weather is likely to aggravate problems for future agriculture. Water wars along with energy wars have begun to be felt across the globe.  National policy to combat such challenges must be in place before it is too late.

Agriculture is a multi-level multi-factorial system that requires new approaches for data gathering and statistical analyses and for multi-level multipronged actions. The debates on chemical agriculture versus organic and restorative agriculture must end and well thought out actions must begin. Development of new generation pest control mechanisms including biotechnology approaches already in use should be strengthened, augmented and advanced. Application of nanotechnology to agriculture is immediately called for to meet the requirements of future agriculture.

Advancements in new biology are required to be used in agriculture. Recent knowledge on panspermia, extracellular DNA, 4-stranded DNA, genomics, proteomics and biotechnology must be applied on a much bigger scale to face challenges ahead. Public funded and freely accessible GM crops that pass bias-free and scientific risk assessments are needed now as also for future agriculture. Nanotechnology applied to agriculture has begun to show promising results. New methods of agriculture should sustain environment and natural resources.

Situation specific agricultural mechanisation based on zero/renewable energy technology is another essential for a successful agricultural future. GMOs are a gift to agriculture but it has to be applied with utmost caution with special reference to monopolisation of technology, environmental costs and ecosystem disturbances besides genetic health of both target and non-target organisms. Rural and urban water harvesting systems need to be enlarged.

Agriculture cannot afford to go in conflict with the environment. Only environment friendly agriculture will save humanity.

Extraction of Genomic DNA from Lantana camara Leaf: Simple Protocol for Freshers in Molecular Biology and Biotechnology

This research note has been accepted for publication by Harmony Newsletter published online from Patna, Bihar, INDIA

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Extraction of Genomic DNA from Lantana camara Leaf: Simple Protocol for Freshers in Molecular Biology and Biotechnology

Sourav Datta¹, Suman Roy², Sourav Kumar Das³, Palash Mukherjee⁴ and SKT Nasar*

Department of Biotechnology, Bengal College of Engineering and Technology

(Affiliated to West Bengal University of Technology), Durgapur-713212, West Bengal

Abstract

We present here an ultrasimple, effective and inexpensive procedure to extract genomic DNA from plants and other organisms with special focus on a common weed Lantana camara L. This report also presents a brief discussion on some selected downstream applications of genomic DNA extracted by this protocol. 

Introduction

Genomic DNA comprising nuclear, mitochondrial and chloroplast DNA is basic to molecular biology and biotechnology and application of genetic technologies. Extraction of DNA from organisms is the first step for the application of molecular biology. Shirazu et al. (2009), Nasar and Nasar (2009, 2010) and others demonstrated that extraction of DNA from different organisms is easy, inexpensive and fun.

Lantana camara (L.) is a common invasive weed plant showing wide diversity. Lantana biodiversity has been used to produce commercially available ornamental varieties for hedges and gardens. We have selected Lantana camara for the present work for two main reasons: Durgapur shows wide variability and DNA extraction is rendered difficult due to high content of pentacyclic terpenoids and volatile oil in leaves.

We show here that gDNA extraction from Lantana leaf is effectively as easy as for other plant species. We also show that its applicability in selected downstream procedures is feasible.

Materials and Methods

Planting selected materials in pots

Selected disease-free plants of Lantana camera L. were maintained live in earthen pots at the departmental garden. Healthy fresh leaves were collected for DNA extraction experiments.

Extraction of genomic DNA

The procedure standardised by Nasar & Nasar (2010) and Nasar, Mukherjee & Trivedi (2010, unpublished; personal comm.) has been used here. This protocol requires household items such as refrigerator, mortar-pestle, table salt, dish washing liquid soap and alcohol.

The method of gDNA extraction from leaf of Lantana camera L. constituted the following steps:

(i) All items – leaves, glassware, and chemicals – were pre-cooled in the freezing chamber of a refrigerator and all steps were carried out on a bed of ice cubes or in ice box; (ii) Only   healthy and young leaves were collected and washed with clean water (Fig 1 A); (iii) leaves were cut into small pieces and surface dirt of the material removed by washing  repeatedly with distilled or drinkable water (Fig 1 B); (iv) A tablespoonful of clean water, a pinch each of ethylenediaminetetraacetic acid (EDTA) and Tata® salt (NaCl) were added to the material and it was pulverised with mortar-pestle to make a paste of tissue; (v) The paste was filtered through fibre-free cloth and the filtrate was collected in a test tube or small glass container; the residue was discarded; (vi) 2-4 drops of Vim® liquid dish washing soap were carefully added to the suspension of pulverised tissue; very gentle stirring with a pre-cooled glass rod avoided lathering; (vii) Finally, pre-cooled ethanol was poured slowly along the wall of test tube/container without disturbing the tissue suspension; (viii) The test tube was kept in the refrigerator at 14-15^O C for 20-30 minutes and progress was watched intermittently; (ix) Bubbles began rising through alcohol layer raising DNA threads that aggregated as a white cloud floating in the ethanol layer (Fig 1 C & D); (x) DNA, at his stage, was carefully sucked out with a long-nozzle glass dropper and was then expelled from the dropper in pre-cooled 70% ethanol in a glass container for storing in a refrigerator (Fig 1 E).

Fig. 1. Steps of gDNA extraction from leaves of Lantana camara L. from preparing the material to DNA isolation to storing

Sufficient amounts of DNA were collected in about 30 minutes as can be seen in Fig. 1 D and E.

Confirmation of the extracted substance being DNA

Several protocols exist for confirmation of the extracted substance being DNA. We used ethidium bromide [EtBr] in a simple experiment. The extracted DNA stored in 70% ethanol was poured on a clean watch glass (#1) and kept in a refrigerator for four-five days till the alcohol evaporated. Air-dried extracted DNA clung to glass surface at the centre. A drop of EtBr was placed over the substance. Another drop of EtBr was placed on a clean watch glass (#2) to act as experimental control. Watch glasses #1 and #2 were exposed to UV light in a transilluminator. The fluorescence patterns were photographed [Fig. 2 (a)]. A teaspoonful of water each was added to another set of #1 and #2 and similarly fluoresced [Fig. 2 (b)].

Fig. 2. Ethidium bromide (EtBr) fluorescence and EtBr-stained air-dried DNA and DNA dissolved in water

It was observed that EtBr alone fluoresced with lesser intensity than in conjunction with the extracted substance [Fig. 2(a) #2 and (b) #2]. EtBr is known to intercalate with dsDNA and produce intense fluorescence. On the other hand, ssDNA, RNA and stretches of dsRNA fluoresce with much lower intensity.  This mini-experiment confirmed that the extracted substance is indeed DNA.

Confirmation of gDNA

Leaf cell contains nuclear, chloroplast and mitochondrial DNA. It was important to verify if the extracted DNA was a mixture of nuclear and organellar DNA since downstream applications would depend upon this information.  To this end, the extracted DNA was subjected to agarose gel electrophoresis in the laboratory.

Agarose gel electrophoresis for analysis of gDNA

The basic information utilised for this section was based on Ogden and Adams (1987) and Brody and Kern (2004). The extracted DNA was placed in pre-cooled water in eppedorf tube and rinsed by centrifugation several times before use. Washing with water would remove salt, ethanol and lighter segments of sheared DNA, 

The extracted DNA samples were electrophoresed in 1% agarose gel with EtBr at 75 v and, 60 mA for 1 hr.  The EtBr-DNA fluorescent bands under transilluminator were captured in digital pictures.

Fig. 3. DNA bands of Lantana camara (L.) after agarose gel electrophoresis. Heavy band likely nuclear DNA, Light band likely chloroplast band and Lightest band likely mitochondrial DNA

The electrophoresis yielded three distinct bands. A comparison of the results (Fig. 3) with other results showed that Lantana leaf DNA bands were similar to those of DNA extracted from different plant species. The heaviest band represents nuclear DNA (nDNA), the light band is chloroplast DNA (ctDNA) and the lightest band comprises mitochondrial DNA (mtDNA).

The result shows that extraction of DNA from Lantana camara (L.) leaf is easy, quick and inexpensive. It also confirms that the extracted ‘white cloud’ in ethanol is indeed DNA as further confirmed by the electrophoresis result.

Discussion

Experiments conducted at Department of Biotechnology, Bengal College of Engineering and Technology, Durgapur, confirmed that the ultrasimple procedure of gDNA  extraction from Aloe vera leaf (Mala 2010), Oryza sativa leaf (Ganguly 2010; Tewari 2011), giant tiger prawn (Penaeus monodon Fab.) muscle tissue (Nasar and Trivedi, pers. comm.),  goat liver and Lactobacillus (Nasar and Mukherjee, pers. comm.) are uniformly effective.

Four basic steps are essential for all DNA extraction protocols: (a) breaking cell membranes by grinding to expose cellular contents and DNA, (b) getting rid of intracellular boundaries by disrupting lipo-protein membranes by a detergent containing sodium dodecyl sulphate, (c) removing proteins by protease such as papaya juice and (d) precipitating DNA with alcohol, preferably ethanol. The experiment must be conducted in cool conditions to decrease endonuclease activity. Procedures must be carried out gently to avoid shearing of DNA.

An inexpensive gDNA extraction protocol (Shirazu et al. 2009; Nasar & Nasar 2010) that consumes less time without compromising with quality has been considered handy for this work. This report has shown that the ultrasimple protocol of gDNA extraction is inexpensive, quick and effective as compared with standard procedures such as the hexadecyltrimethylammonium bromide (CTAB) method (Ausubel et al. 1994, Tewari 2011) are elaborate, costly and time consuming. This report corroborates our other experiments. The total time duration for the experiment from harvesting plant tissues to harvesting gDNA has been found to be typically less than an hour.

Extraction of DNA from plant tissue varies with experimental materials. Required modifications adopted for DNA extraction from Lantana camara without changing the fundamentals of different steps yields sufficient amounts of gDNA amenable to downstream analysis.

Addition of table salt in lieu of lab-grade NaCl is important. The negative charge of one of the oxygen atoms linked to the phosphorous provides high polarity to DNA that dissolves in water at neutral pH.  The negative charge of phosphodiester group of DNA is neutralised on addition of salt. DNA then becomes much less soluble in water. Iodised Tata® table salt contains enough NaCl for the purpose of the protocol under report. Table salt worked well in all our experiments.

Ethylenediaminetetraacetic acid (EDTA) provided better results when used in our experiments. EDTA is a chelating agent for metal ions such as Ca²⁺, Mg²⁺ and Fe³⁺. Nucleases need divalent cations such as Mg²⁺ which when depleted deactivates the enzymes.

Experiments reported here used Vim® dishwashing liquid soap containing sodium dodecyl/lauryl sulphate (SDS/SLS). SDS in the soap is a strong anionic detergent that solubilises and breaks lipo-protein membranes and nuclear envelope. In addition to breaking down membranes, SDS emulsifies lipid bi-layer structure of cell and nuclear membranes. SDS also helps the release of chromosomal DNA from histones and other DNA binding proteins by denaturing them.

After careful examination of DNA precipitation in isopropyl alcohol, denatured alcohol or ethanol in pilot experiments, cold ethanol was found to yield the best results.  Addition of alcohol and salt causes DNA to precipitate while other soluble cell components remain in solution in the aqueous phase. Alcohol also removes alcohol-soluble-salt (see Kurabo PI-80X 2010; CTAB, CIMMYT 2005).

Experiments in our laboratory revealed three bands of DNA extracted from leaf of Rice (Oryza sativa L. cv. MTU 7029 ‘Swarna’; Ganguly 2010 and Tewari 2011), Aloe vera L.  (Mala 2010) and Lantana camara L. (present report) as can be seen in the collage presented at Fig. 4.

Ganguly (2010), in another experiment on the DNA of fresh and senesced leaf and fresh root of rice (Oryza sativa L. cv. MTU 7029) successfully showed that the three bands as seen in Fig. 4 represent nuclear DNA (nDNA; heavy band), chloroplast DNA (ctDNA; light band) and mitochondrial DNA (mtDNA; lightest band). The parallel illustrated in Fig. 4 confirms that the ultrasimple simple protocol is undeniably effective for gDNA extraction from Lantana leaf.

The gDNA extracted by the ultrasimple procedure is fit for downstream application. Tewari (2011) subjected gDNA extracted by standard (CTAB) and ultrasimple protocols to digestion with two restriction enzymes i.e. Hind 111 and EcoR1 for RFLP studies on rice (Oryza sativa L. cv. MTU 7029). He observed that gDNA extracted by these procedures yielded the same results.  Similarly, our PCR studies on gDNA extracted by standard and ultrasimple protocols from giant tiger prawn (Penaeus monodon Fab.) muscle tissue (Nasar and Trivedi, pers. comm.) showed equally acceptable result.

Fig. 4 (A, B, C & D) Collage of leaf DNA from (A & B) Oryza sativa L. (C) Lantana camara L. and (D) Aloe vera L. after agarose gel electrophoresis showing three bands each. (A & B) DNA bands of Oryza sativa L. cv. MTU 7029 leaf; (A) DNA extracted by the ultrasimple protocol as in the present report; (B) DNA extracted by standard (CTAB) protocol; (C) DNA bands of Lantana camara L.; Present work, DNA extracted by ultrasimple protocol under this report; (D) DNA bands of Aloe vera L.; DNA extracted by the ultrasimple protocol as in the present report;

(1% Agarose gel, 20µl EtBr; electrophoresis running time 3 hr 30 minutes at 50 volts)

Although gDNA extracted by ultrasimple protocol has an OD of 1.4-1.5 at 260nm/280nm UV absorbance (Tewari 2011), our conclusion that the ultrasimple protocol for extraction of gDNA good for downstream applications is inescapable. This is corroborated by Chum et al. (2012) who showed that DNA extracted directly from tissues can be used for PCR analysis.

A major criticism of teaching in molecular biology and biotechnology is that practicals are not conducted appropriately (Lakhotia 2008). Such inadequacies are due to the prohibitive costs of infrastructure coupled with fund crunch. Experiments in selected areas with innovative and cost cutting measures (Nasar 2009) can lessen the burden. The present work has been undertaken to address this issue.

The present work shows that, with a good understanding of fundamental principles of protocols, innovative experiments in molecular biology and biotechnology can be cost-effective and quick. Students can, for example, extract DNA at residences and bring samples to laboratory for further analysis.

Acknowledgement

Authors are indebted to Bengal College of Engineering and Biotechnology, Durgapur for providing facilities and to Mr. Prasenjit Tewari, Mrs. Kanchan Mala and Mr. Joydev Ganguly, all former students of SKTN, for allowing us to use data from their dissertations.

References

1.     Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. 1994 Current Protocols in Molecular Biology. New York City, NY: John Wiley & Sons Inc.

2.     Brody, JR and Kern SE (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13. Review.

3.     Chum, P. Y., Haimes, J. D., André, C. P., Kuusisto, P. K., Kelley, M. L. 2012 Genotyping of Plant and Animal Samples without Prior DNA Purification. J. Vis. Exp. (67), e3844, DOI: 10.3791/3844.

4.     Ganguly, Joydev 2010 Genomic DNA of Developing and Senescing Paddy (Oryza sativa L.) Plants: Evaluation with Innovative Agarose Gel Cast and Inexpensive Extraction Protocol; B. Tech. (Biotechnology) Dissertation, (Biotechnology Department, Bengal college of Engineering and Technology, Durgapur), West Bengal University of Technology, Kolkata.

5.     Lakhotia S. C. 2008 Are biotechnology degree courses relevant? Curr. Sci, 94 (10), 1244-1245.

6.     Mala, Kanchan 2010 Genomic DNA of Aloe vera L.: Pot Grown vis-à-vis Tissue Cultured Plants; M. Tech. (Biotechnology) Dissertation, (Biotechnology Department, Bengal college of Engineering and Technology, Durgapur), West Bengal University of Technology, Kolkata.

7.     Nasar S. K. T. 2009 Degree courses in biotechnology; Curr. Sci, 96 (3), 323.

8.     Nasar S.K.T. and Nasar S. Farzaan D. 2009 Genomic DNA Extraction is About the Playing; The Science Creative Quarterly. Available at http://www.scq.ubc.ca/genomic-dna-extraction-is-about-the-playing/; Accessed on 10 November 2012.

9.     Nasar S.K.T. and Nasar S. Farzaan D. 2010 Genomic DNA Extraction is Play http://futureagricultureindia.blogspot.com/2010/04/genomic-dna-extraction-is-play.html; Accessed on 30 April 2010.

10.  Ogden, R.C., and Adams, D.A., (1987) Electrophoresis in agarose and acrylamide gels. Meth. Enzymol. 152, 61-87.

11.  Shirazu Yas, Lee Donna and Abd-Elmessih Esther 2009 The MacGyver Project: genomic DNA extraction and gel electrophoresis experiments using everyday materials. The Science Creative Quarterly. Available at http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/; Accessed on 10 November 2012.

12.  Tewari, Prasenjit 2011 Genomic DNA of Rice Cultivar: Ultrasimple Extraction and RFLP; M. Tech. (Biotechnology) Dissertation, (Biotechnology Department, Bengal college of Engineering and Technology, Durgapur), West Bengal University of Technology, Kolkata.

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^{1, 2 & 3}Former B. Tech. (Biotechnology) students; ⁴Lab In-charge; ¹sourav.aka.peter@gmail.com, ²suman.biotech1411@gmail.com, ³srvhell@gmail.com, ⁴palash_muk@rediffmail.com,

*skt.nasar@gmail.com; Corresponding author, Former Honorary Professor (Biotechnology)

======================

Nasar comments briefly on Bt Brinjal

COMMENT on

1. Group hails PM’s take on Bt Brinjal

SATURDAY, 25 FEBRUARY 2012 00:32 PNS | NEW DELHI

The Pioneer

http://www.dailypioneer.com/nation/45400-group-hails-pms-take-on-bt-brinjal.html

and

2. Manmohan blaming NGOs for Bt brinjal moratorium criticised

THE HINDU

HYDERABAD, February 28, 2012

http://www.thehindu.com/news/national/article2943368.ece

Hon'ble Prime Minister is right in supporting the "use of genetic engineering to increase the productivity of our agriculture”. No one denies "the need for science-based decisions on agri-biotechnology" either. 

Regrettably, however, the case of Bt brinjal satisfies neither "science-based " nor need-based decision for its commercial release. A biopiracy case under BD Act is pending decision by a SBB and NBA on court orders. Let Bt lobbyists kindly inform us about the locus/loci of 'their' cry gene/genes on brinjal chromosomes. Does their 'panacea-of-all FSB destruction' gene jump within and out of brinjal genome? Why, despite being a Bt crop, it should require insecticide sprays? What is the fate of the surviving pest infesting a Bt crop? And, so on. 

Why do the 'owners' of Bt brinjal shy away from labeling 'their' products? Are they prepared to pay compensation for contamination of other than their 'own' genome or metagenome? 

NO to Bt brinjal of Monsanto-Mahyco for now!

S.K.T. Nasar

Posted on: Mar 15, 2012 

IPR in Agriculture with Special Reference to India

This is a comment by SKT  Nasar on the opinion by Howard D. Grimes et alia (2011) on ‘Food Security Needs Sound IP’ published in The ScienceDaily On-line

So long as agriculture continues to be practised on land and partly in water, it shall remain a sensitive issue for socio-politico-economy of world’s peoples. There is as yet no factory-based mass scale industrial system of agricultural production that draws raw inputs from freely available natural resources and specific advantage of agro-ecosystems.

Intellectual Property Rights (IPR) regime originated initially for trade-related industrial production systems which agriculture is considered to be not. That is why global application of IPR to agriculture took long and circuitous paths through decades. IPR in agriculture and other biology-based technologies faces controversies till date.

The opinion by Howard D. Grimes et alia (2011) on ‘Food Security Needs Sound IP’ skips ground realities in India and similarly situated countries.  The primary contention of Howard D. Grimes et alia is that protection of IPR needs improvement to help promote transfer of sustainable agricultural technologies remodelled into inexpensive tools accessible to ‘world’s poorest populations’ and that such technologies are ‘most likely to be developed by the industrialised countries’. Their contention is unacceptable. 

Attention is drawn here to just two among many international instruments: World Trade Organisation (WTO) and Convention on Biological Diversity (CBD).

India became a member of the WTO on 1 January 1995. It also brought to reality in an updated form the failed attempt in 1948 to create an International Trade Organization (ITO). WTO is concerned with all trade-related aspects among and between countries. Member countries are required to frame laws for equity in transborder trade. One major aspect of WTO is Trade Related Aspects of Intellectual Property Rights (TRIPS). In conformity with WTO, TRIPS and Agreement on Agriculture (AoA), a number of acts have been promulgated in India. Several of existing laws have been amended or extended in scope and application. A few of such laws are Indian Patents Act, Protection of Plant Varieties and Farmers’ Rights Act, Geographical Indication Act, Seed Act etc.  

The CBD is another international legally binding treaty with three principal goals: conservation of biological diversity (biodiversity), sustainable use of its components and fair and equitable sharing of benefits arising from genetic resources. India is a party to the United Nations CBD signed at the Earth Summit, Rio de Janeiro on 5 June 1992. The BD Act 2002 is in existence in India as Law. Rules (BD Rules 2009) under this law have been implemented.

Global IPR regime initiated by WTO-TRIPS-AoA should be considered together with CBD in the Indian context. Government of India, on the recommendations of the National Biodiversity Authority, framed rules in 2009 for ground level implementation of the provisions of BD Act. An important feature is elaboration of Traditional Knowledge (TK).  These rules are called the ‘Rules for Protection, Conservation and Effective Management of Traditional Knowledge Relating to Biological Diversity’. TK has been defined under these rules as:

“the collective knowledge of a traditional community including of a group of families, on a particular subject or a skill and passed down from generation to generation, either orally or in written form, relating to properties, uses and characteristics of plant and animal genetic resources; agricultural and healthcare practices, food preservation and processing techniques and devices developed from traditional materials; cultural expressions, products and practices such as weaving patterns, colors, dyes, pottery, painting, poetry, folklore, dance and music; and all other products or processes discovered through a community process including by a member of the community individually but for the common use of the community”. Despite the enormous volume of TK-based products, their production systems have not been categorised as industry simply because these belong to the unorganised sector and benefits thereof are public good freely accessible to all. Unknown or unrecorded individuals and communities are TK inventors which is why IPR system disregarded TK as patentable. This simple fact illustrates that IPR is basically meant to channel profit to inventors and their mentors but not to all as the common property. Normal patent laws tend to provide time-bound ownership to individual inventors and for inventions demonstrating novelty and capability of industrial application. Agrodiversity-suited agricultural practices, location-specific biodiversity and community-based TK, on the other hand, have collective ownership held in perpetuity from generation to generation, are incremental, informal and occur over time. India and the international community, following long and tortuous debates, finally accepted to recognise and protect TK by suitable laws. Since common laws were inadequate, a sui generis (i.e. unique unto itself) IPR law was considered indispensable to protect TK. The question was how to make appropriate laws for TK protection. India provides for collective rights of communities over TK, location-specific or community-specific innovative products, forest management and biodiversity through separate laws.  These rights must be used for the common good of all. Other uses for commercial, scientific and research purposes may be allowed only with prior informed consent (PIC) to be obtained from the traditional community. Misappropriation of TK (in any form of appropriation, monopolisation, including claims of private ownership and/or intellectual property rights etc.) which deprive the concerned traditional community from using, conserving and protecting TK is not permissible. TK and biological resources must be utilised for equitable access and benefit sharing (ABS). Under BD Act, local BD Management Committees (BMC) of the panchayat level is responsible for creating and updating local-level BD Registers. India has already put on the web the Traditional Knowledge Digital Library (TKDL). Such digital database enables Indian citizens and Patent Offices around the world to search and examine any TK to prevent grant of erroneous patents and to deter biopiracy. It is conceded here that, regrettably, Indian laws driven by commitments to WTO and CBD are not implemented as these ought to have been. The reasons for this lacuna are lack of awareness by scientists and other stakeholders in agriculture and absence of the required infrastructure. 

Indian agriculture is a gigantic producing-consuming market. It is faced with unprecedented challenges. The global developments in cutting edge technologies in conjunction with open market economy encourage the national potentials to gain a comparative advantage. With WTO and CBD related commitments to an altering World Order, Indian agriculture calls for commensurate action to national benefit. Agriculture has grown larger than mere cropping of organisms. India needs manpower to steer the agriculture sector through international disputes, trade intrigues and local-level litigation. People’s Biodiversity Registers and documentation of innovations of all kinds and at all levels should become indispensable through mass awareness and action. 

An improved protection of IPRs recommended by Howard D. Grimes et alia shall fail to provide the public good that ‘the world’s poorest populations’ actually need. IPRs in all its forms, shades and extensions, including Community and Farmers’ Rights, must be concurrently considered en bloc to create common public good. Only then shall IPRs benefit world’s agriculture.

SKT Nasar

Kolkata, India

sktnasar@hotmail.com

Protection of Traditional knowledge in India

By

Sunil Prasad and SKT Nasar 

Department of Biotechnology

Bengal College of Engineering and Technology, Durgapur

A write up for the IPR Workshop Proceedings.

IPR Workshop was held at Bengal College of Engineering and Technology, Durgapur

and

organised by WBCS&T and Department of Biotechnology on 30 March 2011

Introduction 

Human communities have generated, refined and relayed knowledge, technology, inventions and skills from generation to generation. The age-old production of wine, vinegar, organic manures, biofertilisers, traditional medicines, drugs, cosmetics, food additives, agricultural implements and distillation apparatuses for perfume making are acknowledged instances. Farmers have selected, bred, used and maintained varieties and types of crop plants, domesticated animals and fish since the beginning of organised human settlements. Selection of microbes for fermentation for homestead cheese production, yeast for pre-cooking fermentation and bacterial strains for curd making are common examples. Vast populations depend on traditional medicines, including ayurveda and unani remedies, for healthcare needs. 

Such traditional knowledge (TK) is an important component of cultural identity, food-nutrition security, medical need and livelihood of millions of people in India. Legal protection against exploitation of TK is minimal under common laws. The need for separate laws for protecting TK became essential.

TK versus industry

Despite the enormous volume of TK-based products, their production systems have not been categorised as industry simply because these belong to the unorganised sector. Unknown individuals or communities are the inventors of unrecorded TK and invention which is why Intellectual Property Rights (IPR) system disregarded TK as being patentable. Normal patent laws tend to provide time-bound ownership to individual inventors and for inventions demonstrating novelty and capability of industrial application. TK, on the other hand, has collective ownership, is held in perpetuity from generation to generation and is incremental, informal and occurs over time. 

India and the international community, following long and tortuous debates, finally accepted to recognise and protect TK by suitable laws. Since common laws were inadequate, a sui generis (i.e. unique unto itself) IPR law was considered indispensable to protect TK. The question was how to make appropriate laws for TK protection.

Country laws and international instruments

Legislatures enact laws for the welfare of the people. Laws in India are made within the Constitutional framework by the legislature – Parliament and State Assemblies. Such laws are known as Acts divided into sections.  Every Act authorises the government to frame rules consistent with connected laws. Acts and rules are announced through Gazette Notifications to be in effect. Designated executives issue orders.

Many a time, legislations are prompted by international commitments made by India in different fora notwithstanding anything contrary to Constitutional provisions. International fora are also referred to as international instruments. 

WTO and CBD

Attention is drawn here to just two among many such international instruments: World Trade Organisation (WTO) and Convention on Biological Diversity (CBD).

India became a member of the WTO on 1 January 1995. It also brought to reality — in an updated form — the failed attempt in 1948 to create an International Trade Organization (ITO). 

WTO is concerned with all trade related aspects among and between countries. It requires member countries to frame laws for equity in transborder trade. One major aspect of WTO is Trade Related Aspects of Intellectual Property Rights (TRIPS).

The Convention on Biological Diversity (CBD) is another international legally binding treaty with three main goals: conservation of biological diversity (or biodiversity); sustainable use of its components; and fair and equitable sharing of benefits arising from genetic resources. 

Biological resources as a global asset are vital to economic and social development. India is a party to the United Nations Convention on Biological Diversity signed at the Earth Summit, Rio de Janeiro on 5 June 1992 when the Convention was opened for signature at the United Nations Conference on Environment and Development. 

Indian laws based on international commitments

India amended its Patent Act 1970 and enacted the Protection of Plant Varieties and Farmers’ Rights Act 2001 in conformity with TRIPS. The Geographical Indications of Goods (Registration & Protection) Act, 1999 came into force in 2003 with the Geographical Indications (Registrations & Protections) rules were notified earlier in 2002.          

The Biological Diversity (BD) Act 2002 was made to provide for conservation of biological diversity, sustainable use of its components and fair and equitable sharing of the benefits arising out of the use of biological resources, knowledge and for matters connected therewith or incidental thereto.

Traditional Knowledge (TK) 

Biological Diversity Agreement of CBD specifically provides that every member country “-- will respect, preserve and maintain, the innovations and practices of indigenous and local communities that entail traditional life styles which are pertinent for the preservation and sustainable use of biological diversity and will promote its broadest application, with the approval and participation of those who posses said knowledge, innovations and practices, and will encourage that benefits derived from the use of that knowledge, innovations and practices be shared fairly.”

Government of India, on the recommendations of the National Biodiversity Authority, framed rules in 2009 for ground level implementation of the provisions of BD Act. These rules are called the ‘Rules for Protection, Conservation and Effective Management of Traditional Knowledge Relating to Biological Diversity’.

Traditional Knowledge (TK) has been defined under these rules as:

“the collective knowledge of a traditional community including of a group of families, on a particular subject or a skill and passed down from generation to generation, either orally or in written form, relating to properties, uses and characteristics of plant and animal genetic resources; agricultural and healthcare practices, food preservation and processing techniques and devices developed from traditional materials; cultural expressions, products and practices such as weaving patterns, colors, dyes, pottery, painting, poetry, folklore, dance and music; and all other products or processes discovered through a community process including by a member of the community individually but for the common use of the community”. 

Recognition of collective rights

India provides for collective rights of communities over TK, location-specific or community-specific innovative products, forest management and biodiversity through separate laws. 

These rights must be used for the common good of all. Other uses for commercial, scientific and research purposes may be allowed only with prior informed consent (PIC) to be obtained from the traditional community.  

Misappropriation of TK (in any form of appropriation, monopolisation, including claims of private ownership and/or intellectual property rights etc.) which deprive the concerned traditional community from using, conserving and protecting TK is not permissible. TK and biological resources must be utilised for equitable access and benefit sharing (ABS).

TK and BD Registers

Communities and their government are required to maintain registers showing details of TK and biological diversity. 

Under BD Act, local BD Management Committees (BMC) of the panchayet level is responsible for creating and updating local-level BD Registers.

India has already put on the web the Traditional Knowledge Digital Library (TKDL). This is an easily navigable computerised database of documented TK. Such digital database enables Indian citizens and Patent Offices around the world to search and examine any TK to prevent grant of erroneous patents and to deter biopiracy.

Government owned portals related to GI Act, Seed Act, PPV&FR Act, BD Act et cetera are of great help to those wishing to be updated on TK protection and related matters. The Innovation Foundation of India (IFI) web sites are also handy tools for understanding TK.

TK and Biotechnology

Biotechnology is divided into two major categories – the first generation non-rDNA biotechnology and the second generation rDNA biotechnology. As mentioned above, the first generation biotechnology is based fundamentally on TK. The two generations of biotechnologies are interdependent. For example, ‘gene hunt’ begins with biodiversity at the levels of extracellular DNA and that of higher organisms. Biotechnology has expanded in scope and has entered the era of nanobiotechnology.

Detailed knowledge about and the protection of TK blocks exploitation by unscrupulous commercial organisations within India and at the international scale. Biopiracy has taken different forms such as unauthorized use of just one gene or a cell or an organism, traditional techniques for utilisation of biodiversity, the granting of wrong patents, deliberate exploitative patents, granting patents without prior informed consent (PIC) etc. 

TK registers must, therefore, include molecular information on systems of biodiversity obtained through biotechnology tools and techniques.

Bioterrorism has gained current importance. This menace can be thwarted only by combining biotechnology and TK to enable quick and mass action by communities at the ground level.

Epilogue

Indian science and technology should take advantage of TK to launch new areas of research for the good of humanity. 

Technology institutions should include courses in TK and involve with the people to protect TK as a national heritage and treasure.