INTRODUCTION
The term “Environment” is defined as our surroundings which includes the abiotic component (the non living) and biotic component (the living) around us. The abiotic environment includes water, air and soil while the biotic environment consists of all living organisms – plants, animals and microorganisms. Environmental pollution broadly refers to the presence of undesirable substances in the environment which are harmful to man and other organisms. In the past decade or two, there has been a significant increase in the levels of environmental pollution mostly due to direct or indirect human activities. The major sources of environmental pollution are –Industries, Agricultural sources (mainly rural area), anthropogenic sources (man related activities mainly in urban areas), biogenic sources etc. The pollutants are chemical, biological and physical in nature. The Chemical pollutants include- gaseous pollutants (hazardous gases like sulfur dioxide, nitrogen oxide), toxic metals, pesticides, herbicides toxins and carcinogens
Etc. The physical pollutants are- heat, sound, radiation, and radioactive substances. The pathogenic organisms and some poisonous and dangerous biological products are the biological pollutants.
Controlling the environmental pollution and the conservation of environment and biodiversity and controlling environmental pollution are the major focus areas of all the countries around the world. In this context the importance and impact of biotechnological approaches and the implications of biotechnology has to be thoroughly evaluated. There have been serious concerns regarding the use of biotechnology products and the impact assessment of these products due to their interaction with the environmental factors.
A lobby of the environmentalists have expressed alarm on the release of genetically engineered organisms in the atmosphere and have stressed on thorough investigation and proper risk assessment of theses organisms before releasing them in to the environment. The effect of the effluents from biotechnological companies is also a cause of concern for everyone. The need of the hour is to have a proper debate on the safety of the use of the biotechnological products.
The efforts are not only on to use biotechnology to protect the environment from pollutionbut also to use it to conserve the natural resources. As we all know that microorganisms are known natural scavengers so the microbial preparations (both natural as well as genetically engineered) can be used to clean up the environmental hazards.
Development of alternate cleaner technologies using biotechnology
Biotechnology is being used to provide alternative cleaner technologies which will help to further reduce the hazardous environmental implications of the traditional technologies. E.g. some Fermentation technologies have some serious environmental implications. Various biotechnological processes have been devised in which all nutrients introduced for fermentation are retained in the final product, which ensures high conversion efficiency and low environmental impact.
In paper industry, the pulp bleaching technologies are being replaced by more environmentally friendly technologies involving biotechnology. The pulp processing helps to remove the lignin without damaging valuable cellulosic fibres but the available techniques suffer from the disadvantages of high costs, high energy use and corrosion. A lignin degrading and modifying enzyme (LDM) was isolated from Phanerochaete chrysosporum and was used, which on one hand, helped to reduce the energy costs and corrosion and on the other hand increased the life of the system. This approach helped in reducing the environmental hazards associated with bleach plant effluents.
In Plastic industry, the conventional technologies use oil based raw materials to extract ethylene and propylene which are converted to alkene oxides and then polymerized to form plastics such as polypropylene and polyethylene. There is always the risk of these raw materials escaping into the atmosphere thereby causing pollution. Using biotechnology, more safer raw materials like sugars (glucose) are being used which are enzymatically or through the direct use of microbes converted into alkene oxides.e.g. Methylococcus capsulatus has been used for converting alkene into alkene oxides.
INTEGRATION OF BIOLOGICAL STEPS IN PULPING PROCESS LEADING TO LIGNIN DEGRADATION
Bioremediation
Bioremediation is defined as ‘the process of using microorganisms to remove the environmental pollutants where microbes serve as scavengers. The removal of organic wastes by microbes leads to environmental cleanup. The other names/terms used for bioremediation are biotreatment, bioreclamation, and biorestoration. The term “Xenobiotics” (xenos means foreign) refers to the unnatural, foreign and synthetic chemicals such as pesticides, herbicides, refrigerants, solvents and other organic compounds. The microbial degradation of xenobiotics also helps in reducing the environmental pollution.
Pseudomonas which is a soil microorganism effectively degrades xenobiotics. Different strains of Pseudomonas that are capable of detoxifying more than 100 organic compounds (e.g. phenols, biphenyls, organophosphates, naphthalene etc.) have been identified. Some other microbial strains are also known to have the capacity to degrade xenobiotics such as Mycobacterium, Alcaligenes, Norcardia etc.
Factors affecting biodegradation
The factors that affect the biodegradation are: the chemical nature of xenobiotics, the concentration and supply of nutrients and O2, temperature, pH, redox potential and the capability of the individual microorganism. The chemical nature of xenobiotics is very important because it was found out that the presence of halogens e.g. in aromatic compounds inhibits biodegradation. The water soluble compounds are more easily degradable whereas the presence of cyclic ring structure and the length chains or branches decrease the efficiency of biodegradation. The aliphatic compounds are more easily degraded than the aromatic ones.
Biostimulation
It is a process by which the microbial activity can be enhanced by increased supply of nutrients or by addition of certain stimulating agents like electron acceptors, surfactants etc.
Bioaugmentation
It is possible to increase biodegradation through manipulation of genes i.e. using genetically engineered microorganisms and by using a range of microorganisms in biodegradation reaction.
Depending on the method followed to clean up the environment, the bioremediation is carried out in two ways:
A) In situ bioremediation - In situ bioremediation involves a direct approach for the microbial degradation of xenobiotics at the site of pollution which could be soil, water etc. The adequate amount of essential nutrients is supplied at the site which promotes the microbial growth at the site itself. The in situ bioremediation is generally used for clean up of oil spillages, beaches etc. There are two types of in situ bioremediation-
1) Intrinsic bioremediation- The microorganisms which are used for biodegradation are tested for the natural capability to bring about biodegradation. So the inherent metabolic ability of the microorganisms to degrade certain pollutants is the intrinsic bioremediation. The ability of surface bacteria to degrade a given mixture of pollutants in ground water is dependent on the type and concentration of compounds, electron acceptor and the duration of bacteria exposed to contamination. Therefore, the ability of indigenous bacteria degrading contaminants can be determined I laboratory by using the techniques of plate count and microcosm studies. The conditions of site that favour intrinsic bioremediation are ground water flow throughout the year carbonate minerals to buffer acidity produced during biodegradation, supply of electron acceptors and nutrients for microbial growth and absence of toxic compounds.
2) Engineered in situ bioremediation- When the bioremediation process is engineered to increase the metabolic degradation efficiency (of pollutants) it is called engineered in situ bioremediation. This is done by supplying sufficient amount of nutrients and oxygen supply, adding electron acceptors and maintaining optimal temperature and pH. This is done to overcome the slow and limited bioremediation capability of microorganisms.
Advantages of in situ bioremediation
a) The method ensures minimal exposure to public or site personnels.
b) There is limited or minimal disruption to the site of bioremediation.
c) Due to these factors it is cost effective.
d) The simultaneous treatment of contaminated soil and water is possible.
Disadvantages of in situ bioremediation
a) The sites are directly exposed to environmental factors like temperature, oxygen supply etc.
b) The seasonal variation of microbial activity exists.
c) Problematic application of treatment additives like nutrients, surfactants, oxygen etc.
d) It is a very tedious and time consuming process.
B) Ex-situ bioremediation - In this the waste and the toxic material is collected from the polluted sites and the selected range of microorganisms carry out the bioremediation at designed place. This process is an improved method over the in situ bioremediation method. On the basis of phases of contaminated materials under treatment ex-situ bioremediation is classified into two : a) Solid phase system and (b) Slurry phase systems.
A) Solid phase treatment- This system includes land treatment and soil piles comprising of organic wastes like leaves, animal manures, agricultural wastes, domestic and industrial wastes, sewage sludge, and municipal solid wastes. The traditional clean-up practice involves the informal processing of the organic materials and production of composts which may be used as soil amendment. Composting is a self heating, substrate-dense, managed microbial system which is used to treat large amount of contaminated solid material. Composting can be done in open system i.e. land treatment and/or in closed treatment system. The hazardous compounds reported to disappear through composting includes aliphatic and aromatic hydrocarbons and certain halogenated compounds. The possible routes leading to the disappearance of hazardous compounds include volatilization, assimilation, adsorption, polymerization and leaching.
B) Slurry phase treatment- This is a triphasic treatment system involving three major components- water, suspended particulate matter and air. Here water serves as suspending medium where nutrients, trace elements, pH adjustment chemicals and desorbed contaminants are dissolved. Suspended particulate matter includes a biologically inert substratum consisting of contaminants and biomass attached to soil matrix or free in suspending medium. The contaminated solid materials, microorganisms and water formulated into slurry are brought within a bioreactor i.e. fermenter. Biologically there are three types of slurry-phase bioreactors: aerated lagoons, low shear airlift reactor, and fluidized-bed soil reactor. The first two types are in use of full scale bioremediation, while the third one is in developmental stage.
Advantages of ex-situ bioremediation
a) As the time required is short, it is a more efficient process.
b) It can be controlled in a much better way.
c) The process can be improved by enrichment with desired and more efficient microorganisms.
Disadvantages of ex-situ bioremediation
a) The sites of pollution remain highly disturbed.
b) Once the process is complete, the degraded waste disposal becomes a major problem.
c) It is a costly process.
Several types of reactions occur during the bioremediation/microbial degradation
a) Aerobic bioremediation- When the biodegradation requires oxygen O2 for the oxidation of organic compounds, it is called aerobic bioremediation. Enzymes like monooxygenases and dioxygenases are involved and act on aliphatic and aromatic compounds.
b) Anaerobic bioremediation-This does not require oxygen O2. the degradation process is slow but more cost effective since continuous supply of oxygen is not required.
c) Sequential bioremediation- Some of the xenobiotic degradation requires both aerobic as well as anaerobic processes which very effectively reduces the toxicity e.g. tetrachloromethane and tetrachloroethane undergo sequential degradation.
Use of genetic engineering and genetic manipulations for more efficient bioremediation
In recent years, efforts have been made to create genetically engineered microorganisms (GEMs) to enhance bioremediation. This is done to overcome some of the limitations and problems in bioremediation. These problems are:
a) Sometimes the growth of microorganisms gets inhibited or reduced by the xenobiotics.
b) No single naturally occurring microorganisms has the capability of degrading all the xenobiotics present in the environmental pollution.
c) The microbial degradation is a very slow process.
d) Sometimes certain xenobiotics get adsorbed on to the particulate matter of soil and thus become unavailable for microbial degradation.
As the majority of genes responsible for the synthesis of enzymes with biodegradation capability are located on the plasmids, the genetic manipulations of plasmids can lead to the creation of new strains of bacteria with different degradative pathways. In 1970s, Chakrabarty and his team of co-workers reported the development of a new strain of bacterium Pseudomonas by manipulations of plasmid transfer which they named as “superbug”. This superbug had the capability of degrading a number of hydrocarbons of petroleum simultaneouslysuch as camphor, octane, xylene, naphthalene etc. In 1980, United States granted the patent to this superbug making it the first genetically engineered microorganism to be patented.
In certain cases, the process of plasmid transfer was used. E.g. The bacterium containing CAM (camphor degrading ) plasmid was conjugated with another bacterium with OCT (octane degrading) plasmid. Due to non-compatibility, these plasmids cannot coexist in the same bacterium. However, due to the presence of homologous regions of DNA, recombination occurs between these two plasmids which results in a single CAM-OCT plasmid giving the bacterium the capacity to degrade both camphor as well as octane.
A new strain of Pseudomonas sp. (strain ATCC 1915) has been developed for the degradation of vanillate (which is a waste product from paper industry) and sodium dodecyl sulfate (SDS, a compound used in detergents).
Biotechnological method to reduce atmospheric carbon dioxide (CO2)
Carbon dioxide is the gas that is the main cause of green house effect and rise in the atmospheric temperature. During the past 100-150 years, the level of CO2 has increased about 25% with an increase in the atmospheric temperature by about 0.5% which is a clear indication that CO2 is closely linked with global warming. There is a steady increase in the CO2 content due to continuous addition of CO2 from various sources particularly from industrial processes. It is very clear that the reduction in atmospheric CO2 concentration assumes significance. Biotechnological methods have been used to reduce the atmospheric CO2 content at two levels:
a) Photosynthesis- Plants utilize CO2 during the photosynthesis which reduces the CO2 content in the atmosphere. The equation for photosynthesis is:
sunlight
6CO2 + 6H2O---------->C 6 H12 O6 + 6O2
Chlorophyll
The fast growing plants utilize the CO2 more efficiently for photosynthesis. The techniques of micropropagation and synthetic seeds should be used to increase the propagation of such fast growing plants.
Further, the CO2 utilization can be increased by enhancing the rate of photosynthesis. The enzyme ribulose biphosphate carboxylase (RUBP-case) is closely linked with CO2 fixation. The attempts are being made to genetically manipulate this enzyme so that the photosynthetic efficiency is increased.
Some microalgae like Chlorella pyrenodiosa, Spirulina maxima are known to be more efficient than higher plants in utilizing atmospheric CO2 for photosynthesis and generate more O2 than the amount of CO2 consumed.
The growing of these microalgae near the industries and power plants (where the CO2 emission in to atmosphere is very high) will help in the reduction of polluting effects of CO2. Using genetic engineering, attempts are going on to develop new strains of these microalgae that can tolerate high concentrations of CO2. A limited success has already been reported in the mutants of Anacystis nidulans and Oocystis sp.
b) Biological Calcification- Certain deep sea organisms like corals, green and red algae store CO2 through a process of biological calcification. As the CaCO3 gets precipitated, more and more atmospheric CO2 can be utilized for its formation. The process of calcification is as follows:
H2O + CO2---------->H2CO3
H 2CO3 + Ca 2+----------------> CaCO3 + CO2 + H2O
Treatment of sewage using microorganisms
The sewage is defined as the waste water resulting from the various human activities, agriculture and industries and mainly contains organic and inorganic compounds, toxic substances, heavy metals and pathogenic organisms. The sewage is treated to get rid of these undesirable substances by subjecting the organic matter to biodegradation by microorganisms. The biodegradation involves the degradation of organic matter to smaller molecules (CO2, NH3, PO4 etc.) and requires constant supply of oxygen. The process of supplying oxygen is expensive, tedious, and requires a lot of expertise and manpower. These problems are overcome by growing microalgae in the ponds and tanks where sewage treatment is carried out. The algae release the O2 while carrying out the photosynthesis which ensures a continuous supply of oxygen for biodegradation.
The algae are also capable of adsorbing certain heavy toxic metals due to the negative charges on the algal cell surface which can take up the positively charged metals. The algal treatment of sewage also supports fish growth as algae is a good source of food for fishes. The algae used for sewage treatment are Chlorella, Euglene, Chlamydomnas, Scenedesmus, Ulothrix, Thribonima etc.
Bio-technology and Agriculture
INTRODUCTION
Genetic engineers can now transfer useful genes into farm stock, to improve their growth rates, milk or meat production. In genetic engineering there are several different techniques which can be used to improve the yields from crop plants and increase plant variation. Using micropropagation it is easy to obtain a large numbers of genetically identical plants.
TRANSGENIC FARM ANIMALS
A transgenic animal is an individual in which a gene (or genes) from another individual has been artificially inserted with the new genetic information forming a permanent part of the genome and being passed on to their offspring according to the Mendelian laws of inheritance. So far, transgenic cattle, sheep, pigs, rabbits, and chicken have been produced.
After fertilization a mammalian embryo passes through many stages of development such as formation of pronuclei, morula formation, blastocyst formation, implantation etc. Using these early development stages of mammalian embryos, genetic engineers have used different methods to introduce foreign genes into the genome.
TABLE A LIST OF TRANSGENIC ANIMALS PRODUCED WITH THEIR PROMOTER, ENHANCER AND STRUCTURAL TRANSGENES
ANIMAL GENES TANSFERRED (PROMOTER OR ENHANCER/STRUCTURAL TRANSGENES)
Goat A variant of tPA gene (LAtPA)
Sheep mMT/hGH, mMT/TK, mMT/bGH, mMT/hGRF, oBLG/hFIX, oBLG/alpha1AT, oMT/oGH
Rabbit mMT/hGH, hMT/hGH, rbEu/rb,c-myc
Pig mMT/hGH, mMT/bGH, hMT/pGH, MLV/rGH, MLV/rGH, bPRL/bGH
Fish hGH, mMT/hGh, mMT/bGal, cd-crystallin SV/hygro, AFP
Cow BPV, lactoferrin
Chicken ALV, REV
Mouse mMT/rGH, mMT/bGH, mMT/oGH, mMT/hGh, mMT/hGRF, mMT/hFIX
Methods to induce foreign genes into the genome
a) Micro-injection in to a pronucleus
A fertilized egg is held in a pipette by suction and several copies of the foreign genes are injected into one of the pronuclei via a micropipette. Although this has been the most widely used and most successful method, 30% of the treated embryos degenerate and die with in a few hours.
b) Using retroviruses as vectors to carry foreign DNA into morulas.
A morula is placed on a culture of fibroblasts infected with retrovirus after removing the
zona pellucida layer. The virus cannot penetrate this layer therefore it is essential to get rid of it. The retrovirus, genetically engineered to carry foreign DNA, infect the cells of the morula as they are shed from the fibroblast. This technique often creates ‘mosaic offspring’, where some cells contain the foreign gene while others do not.
One of the limitations of this technique is that the modified virus cannot leave the transgenic species and under certain conditions it could mutate regaining its ability to cause disease in the tissues where it occurs.
c) Retroviral infection of the stem cells, which are then injected into the cavity of another blastocyst.
In this method, stem cells, from the inner cell mass of an embryo, are infected with genetically-engineered retroviruses, then injected into the central cavity of a different blastocyst. The injected cells colonise the new embryo and participate in the formation of all the tissues, including the ovaries and testes from which cells are formed. This technique has been used to produce transgenic mice and hamsters.
The first transgenic animals produced were mice, into which a gene cloning for rat growth hormone was inserted by microinjection. Two techniques were used to introduce the new gene into the mouse genome:
a) As many as 10000 copies of the rat gene were injected into a pronucleus of a fertilized mouse egg, using a microneedle.
b) After treatment with calcium chloride, to make them more permeable, the rat DNA was applied to the outside of a 2-8- celled mouse embryo in culture.
The treated eggs, or embryos were then transplanted into foster mothers. Embryos with the inserted genes were identified by DNA hybridization with small samples of DNA from white blood cells or skin cells. Transformed adult mice produced the growth hormone mainly in their livers rather than pituitary gland where it is normally synthesized.
This technique can be used successfully to transfer other genes such as those affecting the properties of hair, hides, cold tolerance, disease-resistance and milk production which might help to produce strains of farm animals with economic advantage over existing breeds and stocks. In this regard, research is going on to use techniques which can improve the quality of the farm animals. In this regard, following two techniques are of great importance.
TECHNIQUES TO IMPROVE THE QUALITY OF FARM ANIMALS
a) Embryo manipulation
In this method, the fertilized egg is bisected at the two celled stage and each half is transplanted into different regions of the uterus. Using this method the reproductive rate is doubled as we know that female sheep and cattle produce, on an average, one offspring per pregnancy. Hence the farmers can easily increase their farmstock.
This technique can also be used to conserve rare breeds. After fertilizing their eggs in the laboratory , the young embryos or rare animals are dissected into anything from 2-8 cells. Each cell, is then transplanted into a surrogate mother of a common breed where it grows to produce a new individual of the rare type.
Another variation of this technique allows surrogate mothers to carry embryos of a different species e.g. Horses giving birth to zebras from zebra embryos implanted at the blastocyst stage. The outer layer of zebra embryo is exchanged with the trophectoderm of the horse embryo in order to make it acceptable to the surrogate mother. This exchange of the layer does not affect the development of the inner cell mass which is the true embryo-forming region.
b) Embryo cloning
Using this technique it is possible to produce many genetically identical copies of an animal. This method has been used to clone mouse however, research is going on to standardize this method to obtain clones of cattle with desirable traits e.g. cows with high milk production etc.
Following steps are used in this method:
a) an egg from the donor is grown to blastocyst stage under laboratory conditions.
b) The egg is dissected to remove the inner cell mass.
c) The mass cells are separated into individual cells.
d) Injection of a nucleus from each of these cells into a one-celled embryo containing two pronuclei.
e) Removal of pronuclei followed by culturing of embryos in the laboratory until the blastocyst stage.
f) Transplantation of these embryos into surrogate mothers.
CHIMERAS
Early research into the production of transgenic animals revealed a simple method for producing hybrids, called Chimeras, between closely related species. Sheep-goat chimeras were produced by mixing four celled sheep embryos with eight-celled goat embryos. After removing the zona pellucida from each egg, the eggs were pressed together and incubated at 370C. The cells reorganized and formed hybrid blastocysts. These hybrid blastocysts were then transferred to sheep foster mothers, where they continued their growth and development until the sheep gave birth. Each hybrid offspring contained both sheep and goat cells in all it’s tissues which resulted in the coats of these animal having a patchwork kind of pattern with irregular patches of sheep and goat fur.
TRANSGENIC PLANTS
Transgenic plants are genetically engineered varieties containing one or more artificially inserted genes. The aim of producing transgenic plants is to
a) improve crop yields
b) increase variety
c) give cultivated plants more protection against their pests, parasites and harsh weather conditions.
Most of the techniques used to produce transgenic plants only demonstrate that genes could be transferred in to plants. Transgenic plants resistant to herbicides, insects, viruses and a variety of other stresses have already been produced. Besides this, transgenic plants suitable for food processing have also been produced such as bruise resistance, delayed ripening in tomato. Initially, the techniques used only helped in the production of transgenic plants in dicotyledons but no it has been possible to produce transgenic plants in monocotyledons like wheat, maize, rice and oats.
The safety of these transgenic plants for human consumption should be thoroughly evaluated before they are commercially used. The safety of growing and using these transgenic plants is of concern for the public at large.
Table A list of higher plants where transgenic plants have been produced using different methods
Nicotiana tabacum (tobacco) Picea glauca (white spruce)
N. plumbaginifolia (wild tobacco) Avena sativa (oats)
Petunia hybrida (petunia) Zea mays (corn)
Lycopersicon esculentum (tomato) Triticum aestivum (wheat)
Solanum tuberosum (potato) Oryza sativa (rice)
Solanum melongena (eggplant) Secale cereale (rye)
Arabidopsis thaliana Dactylis glomerata (orchard grass)
Lactuca sativa (lettuce) Asparagus sp. (asparagus)
Apium graveolens (celery) Vitis vinifera (grape)
Helianthus annuus (sunflower) Carica papaya (papaya)
Linum usitatissimum (flax) Actinidia sp. (Kiwi)
Brassica napus (oilseed rape; canola) Fragaria sp. (strawberry)
Brassica oleracea (cauliflower) Ipomoea purpurea (morning glory)
Brassica rapa (syn. B. campestris) Ipomoea batatas (sweet potato)
Gossypium hirsutum (cotton) Digitalis purpurea (foxglove)
Beta vulgaris (sugarbeet) Glycorrhiza glabra (licorice)
Glycine max (soybean) Armoracia sp. (horse radish)
Pisum sativum (pea) Daucus carota (carrot)
Chrysanthemum sp. (chrysanthemum) Cichorium intybus (chicory)
Rosa sp. (rose) Cucumis melo (muskmelon)
Populus sp. (poplar) Cucumis sativus (cucumber)
Malus sylvestris (apple) Lotus corniculatum (lotus)
Pyrus communis (pear) Medicago sativa (alfalfa)
Azadirachta indica (neem)
METHODS TO TRANSFER GENES IN PLANTS
Use of Ti plasmids of Agrobacterium for gene transfer
The transgenic plants were created using the bacterium Agrobacterium tumefaciens which causes crown gall disease and carries tumour-inducing (Ti) plasmids. Genetic engineers have exploited the discovery that any piece of foreign DNA, inserted between the left and right borders of the plasmid’s T- DNA region is transferred to one of the plant’s chromosomes, where it also becomes integrated. This natural system used for transferring DNA into plants was further improved by deleting the genes that made cells to produce more hormones, followed by adding gene for antibiotic resistance, and then attaching “sticky ends” for the insertion of foreign DNA.
The method involves the following steps:
a) Insertion of the desired foreign gene into a Ti plasmid.
b) Then the naked plant cells or protoplasts are placed into a Petri dish and covered by a nutrient solution.
c) Addition of Agrobacterium tumefaciens containing genetically engineered plasmids.
d) Incubation of all the contents for several days at 25-30oC.
e) Plating of cells on nutrient agar with an appropriate antibiotic.
f) Only the plant cells that have taken up the gene for antibiotic-resistance with it’s foreign DNA will grow on this medium.
g) Harvesting the living cells after some growth period.
h) Each cell is then grown to produce a complete plant by cultivation on nutrient media by using different plant hormones.
Agrobacterium infection method has been extensively used to transfer foreign DNA into a number of dicotlyledonous species with the exception of soybean (Glycine max). However, this technique of gene transfer was not successful in monocotyledons for reasons yet unknown most probable being the lack of wound response of monocotyledonous cells.
Protoplast fusion
Protoplast fusion is an additional technique for inducing variation in plant crops. By fusing protoplasts from different strains of species, it is possible to transfer genes from one strain to another. The protoplasts are prepared by immersing sterilized plant material in a solution of the enzyme cellulose, either from fungi or the alimentary canal of snails. One can use ethylene glycol or apply electric field to fuse the protoplasts of different species or closely related species. The technique of protoplast fusion was used in transferring genes for resistance to late blight fungus from one variety of potato to others.
However there are limitations in using this technique especially for transferring genes for salt tolerance and disease-resistance from wild rice into cultivated varieties. Research is still going on to make this technique a reliable tool to use for inducing variations in plants.
Strategies used for Protoplast Fusion
Use of Polyethylene glycol (PEG) for DNA uptake
Direct DNA uptake by protoplasts can be stimulated by chemicals like polyethylene glycol (PEG). PEG is also used to stimulate the uptake of liposomes and to improve the efficiency of electroporation. PEG at high concentration (15-25%) precipitates ionic macromolecules such as DNA and stimulate their uptake by endocytosis without any gross damage to protoplasts.
This method has been successfully used in Petunia, Nicotiana, rice, maize etc. However there are problems related to plant regeneration from protoplasts subjected to this treatment for gene transfer.
Use of chemicals like Colchicine to introduce genetic variation in plants
The flowering plants are diploid (2n) and sexual reproduction involves fertilization of a haploid egg or ovum (n) by a haploid male gamete (n), formed in a germinating pollen grain. Haploid plants (n) can be formed by growing pollen grains or anthers on solid or liquid cultures with appropriate nutrients and growth hormones. By using Colchicine, a mutagen which causes chromosomes numbers to double during cell division, polyploidy plants are obtained with entirely new homozygous features and traits. The advantage of this technique is that it takes less than half of the time taken by conventional cross-breeding to produce pure-breeding lines.
Use of Liposomes for gene transfer
Liposomes are small lipid bags, in which large number of plasmids are enclosed. They can be infused with protoplasts using devices like PEG, and therefore have been used for gene transfer especially in plant species like tobacco, petunia, carrot etc. The method involves the following steps:
1. Adhesion of the liposomes to the protoplast surface.
2. Fusion of liposomes at the site of adhesion
3. Release of plasmids inside the cell.
The advantages of using this method are:
1. protection of DNA/RNA from nuclease digestion
2. low cell toxicity
3. stability and storage of nucleic acids due to encapsulation in liposomes
4. applicability to a wide variety and range of cells
Agroinfection
Cereals are important as they are the major food crop for us. Under natural circumstances, Agrobacterium tumefaciens does not attack cereals therefore it cannot be used to modify the genome of these plants. However, it was observed that if the DNA of wheat dwarf virus is inserted into a Ti plasmid, the bacteria carrying Ti plasmids will attack wounded wheat plants. Similarly, bacteria carrying Ti plasmids with DNA from maize streak virus will attack wounded maize plants. This technique is called agroinfection which was first used in 1987. In this mature cereal plants are infected with plasmid-carrying bacteria. Transformed cells develop symptoms of the viral disease, and do not need to be identified by selection. The infection spreads from cell to cell until all the cells of the cereal plant have been transformed. Efforts are being made to use this technique to introduce foreign DNA into cereals.
Electroporation
This is a technique which relies on direct uptake of DNA for gene transfer with out the use of bacterial vector. In this technique of electroporation, after mixing the protoplasts with foreign DNA, an electric shock is given. This electric pulse causes pores in the cell membranes to open up which increases the amount of exogenous DNA that enters the cell. Once the current is switched off the pores reseal. A small amount of the foreign DNA becomes incorporated into the chromosomes which causes some of the protoplasts to undergo transformation. This method has been successful in maize and rice protoplasts. Instead of electric pulse, ethylene glycol can also be used to make the membrane more permeable to exogenous DNA.
Macro- and micro-injection of foreign DNA
Results obtained from the direct microinjection of foreign DNA into young embryos have been mixed with limited success. The method of macroinjection involves the use of a syringe to inject foreign DNA into the space around the young inflorescence. Success for this approach has been claimed in the transfer of a gene for resistance to the antibiotic kanamycin.
Bombardment of intact plant cells with DNA coated spheres of tungsten or gold.
This method involves the bombardment of intact plants with very small (1-4um in diameter) DNA-coated spheres of tungsten or gold. These micro-projectiles are shot from a macro-projectile, resembling a bullet with an open tip, which is itself held by a stopping plate. The propulsive force gives the micro-projectiles sufficient acceleration. This force comes from either a shotgun ‘explosion’ or an electrical discharge. This method has been successful in transforming soyabeans.
All these methods are being used to give crop plants better protection against pests and parasites and also making them resistant to harsh environmental conditions.
TRANSGENIC PLANTS IN DICOTYLEDONS
Transgenic plants in dicotyledons have been produced for crop improvement, to become herbicide, insect and viral resistant.
Herbicides normally affect processes like photosynthesis or biosynthesis of amino acids. One can develop resistant plants either by making target molecules insensitive to herbicide or by overproducing the target protein. Another approach is introduction of a pathway that will detoxify the herbicide. All these methods have been tried. To induce insect resistance the toxin gene (bt2) from bacteria B. thuringiensis was isolated. The Bt toxins produced by this bacteria has been used as a biological insecticide since long time. The bt2 gene was used for Agrobacterium Ti plasmid mediated transformation of tobacco, cotton, and tomato plants. The transgenic plants were resistant to the Manducta sexa which is a pest of tobacco.
Induction of resistance to storage pests was clearly demonstrated in cowpea, Vigna unguiculata. In this plant a trypsin inhibitor (CpTI) is responsible for its resistance to attack by the major storage pests and insects. The gene CpTI was transferred to induce insect resistance by using binary vectors. The vector was mobilized into Agrobacterium, which was used to infect tobacco leaf discs. This lead to the production of transgenic tobacco plants expressing high level of CpTi thereby making the plant resistant against a variety of insects.
TRANSGENIC PLANTS IN MONOCOTYLEDONS
As mentioned earlier the production of transgenic plants in monocotyledons was not so successful due to two reasons:
a) the Ti plasmids could not be used to transform monocots because monocots are not ordinarily infected by Agrobacterium , which is generally used to transform dicots. And
b) the regeneration of plants from protoplasts or single cells which is generally used for transformation was not possible.
These limitations have been solved by using alternative and new methods of DNA uptake and regeneration protocols for crops like rice and maize. In rice (both in japonica and indica varieties, there have been successful production of transgenic plants. In Maize, a reporter gene for neomycin phosphotransferase (NPT II) associated with 35S promoter region of the cauliflower mosaic virus (CMV) was used for production of transgenic plants.
USES OF TRANSGENIC PLANTS
Transgenic plants for Molecular Farming
Many plant products useful for humans such as sugars, fatty acids, starches, celluloses, rubber, and wax are obtained by using the traditional methods. The efforts are going on to use genetic engineering to increase their production. In this regard, the transgenic plants can play an important role as ‘factories’ for manufacturing specialty chemicals and pharmaceuticals. Some of the examples are: increase in the level of mannitol in transgenic tobacco plants following the transfer of the gene for mannitol dehydrgenase from E.Coli to tobacco. Similarly Chimeric genes having CaMV promoter and encoding human serum albumin (HSA) were transferred and transgenic potato and tobacco plants were obtained. The secretion of protein was achieved by using either the human preprosequence or the signal sequence from extra cellular PR-S protein from tobacco. HSA was secreted in transgenic leaf tissue.
Transgenic plants to study regulated gene expression
Transgenic plants have been used to study the expression of genes in different environmental conditions or at different stages of development which can lead to induction or suppression of gene expression. Using transgenic plants it was possible to recognize the regulatory sequences involved in differential expression of gene activity. The regulatory sequences of a number of structural genes was studied using this method. E.g. In order to study heat shock genes which start transcription under thermal stress and reduces the expression of many other vital genes, a gene construct, carrying NPT II reporter gene was fused with upstream region of heat shock gene hsp70 from Drosophila. This was then introduced into Tobacco. The expression of NPT II due to heat shock was comparable to that of endogenous plant heat shock genes. Similarly when hsp 70 gene of maize, with 1.1 kilobases of upstream sequence was introduced in petunia, it exhibited heat inducibility. Similar studies were carried out using different systems such as the gene for small subunit of ribulose bisphosphate carboxylase (rbcS) was transferred from pea plant to petunia and tobacco plants and the soybean rbcS gene was transferred to petunia. The gene expressed itself in transgenic plants. The gene for chlorophyll a/b binding protein (Cab gene) was transferred from pea plant and wheat to tobacco.
Transgenic plants suitable for food processing
Transgenic plants suitable for food processing have also been developed. Tomatoes showing ‘delayed ripening’ were developed either by using antisense RNA against enzymes involved in ethylene production (e.g. ACC synthase) or by using gene for ACC deaminase, which degrades 1 aminocyclopropane-1 carboxylic acid (ACC) which is an immediate precursor to ethylene. This not only increases the shelf life of tomato but also the tomatoes can stay longer on the plant which gives more time for accumulation of sugars and acids for improving flavour. Therefore they are described as ‘Flavr Savr. Another example is the development of bruise resistant tomatoes which express antisense RNA against polygalacturonase (PG), which attacks pectin in the cell walls of ripening fruit and softens the skin.
Tomatoes with elevated sucrose and reduced starch could also be produced using sucrose phosphate synthase gene.
Some pathogens for which resistance has been transferred in some crop plants
Pathogens Disease Resistance gene Source of gene Transgenic Crop
Pseudomones
syringae Wild fire Acetyl transferase gene - Tobacco
Alternaria
longipes Brownspot Chitinase gene Serratia
Marcescens (soil bacterium) Tobacco
Rhizoctonia
solani - Chitinase gene Bean Tobacco
Phytophthora
infestans Late blight Osmotin gene Potato Potato
BIOETHICS IN PLANT GENETIC ENGINEERING
The GM crops are fast becoming a part of agriculture throughout the world because of their capacity for increased crop productivity and their use in health-care and industry. However, there are conflicting schools of thought regarding the safety issues related to the use of GM crops and foods. The major concerns about GM crops and GM foods are:
- Are GM foods fit for human and animal consumption?
- What will be the effect of GM crops on biodiversity and environment?
- the risk of transgenes escaping through pollen to related plant species (gene pollution) which may lead to the development of highly resistant super weeds.
-The GM crops may change the fundamental vegetable nature of plants as the genes from animals (e.g. fish or mouse) are being introduced into crop plants.
- The transfer of antibiotic resistance marker genes present in transgenic crops into microbes which can induce the problem of antibiotic resistance in human and animal pathogens.
- The GM crops may cause changes in the evolutionary pattern.
There is a need for public debate on these aspects of using GM foods and crops. The researchers and scientists are accumulating a large number of authentic and reproducible evidence about the safety of these products by doing field trials. The transgenic crops e.g. cotton, tomato, corn and soybean are already being used commercially after the risk assessment for environmental safety. However one cannot deny the importance of the assessment of the risks associated with the use of transgenic plants for animals and humans before they are released in to the environment. According to some people the use of GM crops and plant genetic engineering will be a very effective tool to sole the problems of poverty and hunger.
Bio-Technology and Animals
INTRODUCTION
An important aspect of any biotechnological processes is the culture of animal cells in artificial media. These animal cells in culture are used in recombinant DNA technology, genetic manipulations and in a variety of industrial processes. Animal cells e.g. egg cells are used for multiplication of superior livestock using a variety of techniques like cloning of superior embryonic cells, transformation of cultured cells leading to the production of transgenic animals. The animal cells are also used in vitro fertilization and transfer of embryos to surrogate mothers. Hence the establishment and maintenance of a proper animal culture is the first step towards using them as tools for biotechnology.
HISTORY OF ANIMAL CELL CULTURE
It was Jolly, who (1903) showed for the first time that the cells can survive and divide in vitro. Ross Harrison, (1907) was able to show the development of nerve fibres from frog embryo tissue, cultured in a blood clot. Later, Alexis Carriel (1912) used tissue and embryo extracts as cultural media to keep the fragments of chick embryo heart alive.
For about 50 years, mainly tissue explants rather than cells were used for culture techniques, although later after 1950s, mainly dispersed cells in culture were utilized. In 1966, Alec Issacs discovered Interferon by infecting cells in tissue culture with viruses. He took filtrates from virus infected cells and grew fresh cells in the filtered medium. When the virus was reintroduced in the medium, the cells did not get infected. He proposed that cells infected with the virus secreted a molecule which coated onto uninfected cells and interfered with the viral entry. This molecule was called Interferon.
For animals, if the explant maintains its structure and function in culture it is called as an ‘organotypic culture’. If the cells in culture reassociate to create a three dimensional structure irrespective of the tissue from which it was derived, it is described as a ‘histotypic culture’
ANIMAL CELL CULTURE
Salient Features of Animal cell culture
a) Animal cells can grow in simple glass or plastic containers in nutritive media but they grow only to limited generations.
b) Animal cells exhibit contact inhibition.
c) There is a difference in the in vitro and in vivo growth pattern of cells.
d) The maintenance of growth of cells under laboratory conditions in suitable culture medium is known as PRIMARY CELL CULTURE.
e) Cells are dissociated form tissues by mechanical means and by enzymatic digestion using proteolytic enzymes.
f) Cells can grow as adherent cells (anchorage dependent) or as suspension cultures (anchorage independent).
g) The primary culture is subcultured in fresh media to establish SECONDARY CULTURES.
h) The various types of cell lines are categorized into two types as Finite cell line and Continuous cell line.
i) Finite cell lines are those cell lines which have a limited life span and grow through a limited number of cell generations.
j) Cell lines transformed under in vitro conditions give rise to continuous cell lines.
k) The physical environment includes the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses.
l) Nutrient media is the mixture of inorganic salts and other nutrients capable of sustaining cell survival in vitro.
m) Serum is essential for animal cell culture and contains growth factors which promote cell proliferation. It is obtained as exuded liquid from blood undergoing coagulation and filtered using Millipore filters.
n) Cryo preservation is storing of cells at very low temperature (-180oC to -196oC) using liquid nitrogen.
o) DMSO is a cryopreservative molecule which prevents damage to cells.
p) In order to maintain the aseptic conditions in a cell culture, a LAF hood is used.
q) Based on the nature of cells and organism the tissue culture hoods are grouped into three types: Class I, Class II, and Class III.
r) CO2 incubators are used and designed to mimic the environmental conditions of the living cells.
s) An inverted microscope is used for visualizing cell cultures in situ.
t) For most animal cell cultures low speed centrifuges are needed.
Animal cells can grow in simple glass and plastic containers in a nutritive medium. As pointed earlier there are certain features typical of animal cell cultures.
A) Mortality- Animal cells, depending on the tissue they have been isolated from can grow to only limited generations in spite of growing them in the best nutritive media.
B) Contact Inhibition- The animal cells in culture divide and fill the surface of the container they are growing in and then stop growing. This phenomenon is similar to what happens in the normal body where the body grows to a certain size after which it stops. This is due to Contact Inhibition. In cultures when the cells come in close contact with each other, they stop growing.
C) In vivo and in vitro environment difference- The cells in culture behave differently from the cells in vivo environment. There is an absence of cell-cell interaction, cell matrix interaction, lack of three dimensional architecture and alteration in hormonal and nutritional environment. Due to these differences, the cells in culture adhere to the glass or plastic container in a different manner, the shape of the cells change and the way cells proliferate or grow also changes.
In fact these parameters help us to distinguish the cancer cells in culture from the normal cells because the cancer cells in culture, change shape (more rounded), loose contact inhibition, pile on each other due to overgrowth and uncontrolled growth.
REQUIREMENTS FOR ANIMAL CELL CULTURE
Among the essential requirements for animal cell culture are special incubators to maintain the levels of oxygen, carbon dioxide, temperature, humidity as present in the animal’s body. The synthetic media with vitamins, amino acids and fetal calf serum. Following parameters are essential for successful animal cell culture:
a) Temperature- In most of the mammalian cell cultures, the temperature is maintained at 370C in the incubators as the body temperature of Homo sapiens is 370C.
b) Culture media- The culture media is prepared in such a way that it provides-
1) The optimum conditions of factors like pH, osmotic pressure, etc.
2) It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions. Glucose is added as energy source-it’s concentration varying depending on the requirement. Phenol Red is added as a pH indicator of the medium.
3) Natural Media - The Natural Media used to promote cell growth fall in three categories.
i) Coagulant, such as plasma clots.
ii) Biological fluids such as serum. Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors.
iii) Tissue extracts for example Embryo extracts- Other biological fluids used as natural media include amniotic fluids, ascetic and pleural fluids, aqueous humour (from eye), serum ultra filtrate insect haemolymph etc.
4) pH- Most media maintain the pH between 7 and 7.4. The optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular enzymes and binding of hormones and growth factors to cell surface receptors in the cell cultures. The regulation of pH is done using a variety of buffering systems. Most media use a bicarbonate-CO2 system as its major component.
5) Osmolality- A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in the growth media determine the osmolality of the medium. All commercial media are formulated in such a way that their final osmolality is around 300 mOsm.
CELL BASED THERAPY
The animal cell culture techniques are used in replacing the damaged and dead cells with normal and healthy cells using the stem cell technology. This therapy is called Cell-Based therapy which involves the use of stem cell technology involving the replacement of damaged and dead cells with normal and healthy cells. This is used to treat blood cancer, and other neuro-degenerative diseases etc.
Equipments Required for Animal Cell Culture
Laminar Flow Cabinets
Depending on the nature of the cells and organisms being handled, tissue culture hoods can be grouped as follows :
a) Class I hoods are found with in specially designed sterile work areas and give good protection to the operator and, to a lesser degree, the cell culture. There is an open front from which the air is drawn over the cell culture and goes out through the top of the hood.
b) Class II hoods offer protection to both operator and the cell culture and is the most common type found in a tissue culture laboratory. The cell culture is protected in a stream of sterile air and the operator is protected from contamination by the inflow of air into the base of the work area.
c) Class III hoods contains a full physical barrier which screens the worker, and is mainly used for working with highly pathogenic organisms.
The Incubators
Generally CO2 incubators are used in animal cell cultures. This is
a) to maintain the sterility of the chamber for which filtered High Efficiency Particulate Air (HEPA) is used.
b) to maintain constant temperature the incubators is made airtight using a silicon gasket on the inner door.
c) to keep an atmosphere with a fixed level of CO2 and high relative humidity which prevents the dessication of the medium and maintains the osmolality?
Inverted Microscope
This type of microscope is used for visualizing cell cultures in situ.
Centrifuges
Only low speed centrifuges are used generally at 20oC to avoid disruption of the separated bands of cells.
APPLICATIONS OF ANIMAL CELL CULTURE
The animal cell cultures are used for a diverse range of research and development. These areas are:
a) production of antiviral vaccines, which requires the standardization of cell lines for the multiplication and assay of viruses.
b) Cancer research, which requires the study of uncontrolled cell division in cultures.
c) Cell fusion techniques.
d) Genetic manipulation, which is easy to carry out in cells or organ cultures.
e) Production of monoclonal antibodies requires cell lines in culture.
f) Production of pharmaceutical drugs using cell lines.
g) Chromosome analysis of cells derived from womb.
h) Study of the effects of toxins and pollutants using cell lines.
i) Use of artificial skin.
j) Study the function of the nerve cells.
Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated.
Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.
FIG SHOWING THE PRODUCTION OF T-PA
rDNA technology has been used to produce Factor VIII which causes blood clotting. This factor VIII is absent in the people suffering from genetic disorder called Haemophilia A.
Amgen Inc. holds US patent for preparation of, eErythropoietin, by recombinant method using Chinese Hamster Ovary cell lines. Erythropoietin (EPO) is a hormone-like substance released by the kidney under hypoxic or anoxic conditions caused by anaemia. r-HUEPO- recombinant human erythro- protein has been effectively used to treat anemia associated with AIDS, renal failure etc.
The production of Monoclonal Antibodies using hybridoma technology
Hybridoma are obtained by using an antibody producing lymphocytes cell and a single myeloma cell. Monoclonal antibodies bind very specifically to an epitope (specific domains) on an antigen and by using them it is possible to detect the presence of specific antigens. The Monoclonal antibodies are used for the treatment of patients with malignant leukaemia cells, B cell lymphomas and allograft rejection after transplantation. OKT3 is a monoclonal antibody which has been licensed for clinical use for the treatment of acute renal allograft rejection. OKT3 removes antigen bearing cells from circulation thereby helps in accepting the graft.
FIG SHOWING THE STEPS INVOLVED IN THE PRODUCTION OF MONOCLONAL ANTIBODIES
When Monoclonal antibodies are used as enzymes using the technique of enzyme engineering, then they are called abzymes.
Using animal cell cultures, it is also possible to produce Polyclonal Antibodies. Polyclonal antisera are derived from many cells therefore contains heterogeneous antibodies that are specific for several epitopes or an antigen.
SCALE-UP OF ANIMAL CELL CULTURE
Modifying a laboratory procedure, so that it can be used on an industrial scale is called scaling up. Laboratory procedures are normally scaled up via intermediate models of increasing size. The larger the plant, the greater the running costs, as skilled people are required to monitor and maintain the machinery.
Mainly Roller Bottles with Micro Carrier Beads are used in Scale-up of animal cell culture process.
Roller Bottles
The Roller bottles provide total curved surface area of the micro carrier beads for growth. The continuous rotation of the bottles in the CO2 incubators helps to provide medium to the entire cell monolayer in culture.
DIAGRAM SHOWING THE ROLLER BOTTLE CELL CULTURE
Micro Carrier Beads
Micro Carrier beads, increase the number of adherent cells per flask. The beads are either dextran or glass-based and come in a range of densities and sizes. The cells grow at a very high density which rapidly exhausts the medium and therefore the medium has to be replaced for the optimum cell growth.
Spinner cultures
Spinner cultures are used for scaling up the production of suspension cells. The flat surface glass flask is fitted with a Teflon paddle that continuously turns and agitates the medium. This stirring of the medium improves gas exchange in the cells in culture.
TYPES OF CELL CULTURES
Primary cell culture
The maintenance of growth of cells dissociated from the parental tissue (such as kidney, liver) using the mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers is called Primary Cell Culture.
The primary cell culture could be of two types depending upon the kind of cells in culture.
k) Anchorage Dependent /Adherent cells- Cells shown to require attachment for growth are set to be Anchorage Dependent cells. The Adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. They grow adhering to the cell culture.
l) Suspension Culture/Anchorage Independent cells- Cells which do not require attachment for growth or do not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes.
Secondary cell cultures
When a primary culture is sub-cultures, it becomes known as secondary culture or cell line.
Subculturing- Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the growth media, washing the plate, disassociating the adhered cells, usually enzymatically. Such cultures may be called secondary cultures.
Cell Line
A Cell Line or Cell Strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. Cell lines are categorized into two types:
a) Finite cell Lines - The cell lines which have a limited life span and go through a limited number of cell generations (usually 20-80 population doublings) are known as Finite cell lines. These cell lines exhibit the property of contact inhibition, density limitation and anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours.
b) Continuous Cell Lines - Cell lines transformed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines. The cell lines show the property of ploidy (aneupliody or heteroploidy), absence of contact inhibition and anchorage dependence. They grow in monolayer or suspension form. The growth rate is rapid and doubling time is 12-24 hours.
The cell lines are known by:
a) A code e.g. NHB for Normal Human Brain.
b) A cell line number- This is applicable when several cell lines are derived from the same cell culture source e.g. NHB1, NHB2.
c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two doublings.
FIG SHOWING THE SALIENT FEATURES OF CELL CULTURE WITH EVOLUTION OF
A CELL LINE
CHARACHTERIZATION OF CELL LINES
The cell lines are characterized by their a) growth rate and b) karyotyping.
a) Growth Rate- A growth curve of a particular cell line is established taking into consideration the population doubling time, a lag time, and a saturation density of a particular cell line. A growth curve consist of:
1) Lag Phase: The time the cell population takes to recover from such sub culture, attach to the culture vessel and spread.
2) Log Phase: In this phase the cell number begins to increase exponentially.
3) Plateau Phase: During this phase, the growth rate slows or stops due to exhaustion of growth medium or confluency.
b) Karyotyping- Karyotyping is important as it determines the species of origin and determine the extent of gross chromosomal changes in the line. The cell lines with abnormal karyotype are also used if they continue to perform normal function. Karyotype is affected by the growth conditions used, the way in which the cells are subcultured and whether or not the cells are frozen.
TABLE-SOME ANIMAL CELL LINES AND THE PRODUCTS OBTAINED FROM THEM
Cell line Product
Human tumour Angiogenic factor
Human leucocytes Interferon
Mouse fibroblasts Interferon
Human Kidney Urokinase
Transformed human kidney cell line, TCL-598 Single chain urokinase-type plasminogen activator (scu-PA)
Human kidney cell (293) Human protein (HPC)
Dog kidney Canine distemper vaccine
Cow kidney Foot and Mouth disease (FMD) vaccine
Chick embryo fluid Vaccines for influenza, measles and mumps
Duck embryo fluid Vaccines for rabies and rubella
Chinese hamster ovary (CHO) cells 1. Tissue-type plasminogen activator (t-PA)
2. B-and gamma interferons
3. Factor VIII
STEM CELL TECHNOLOGY
Stem cells retain the capacity to self renew as well as to produce progeny with a restricted mitotic potential and restricted range of distinct types of differentiated cell they give rise to. The formation of blood cells also called haematopoiesis is the classical example of concept of stem cells. Indirect assay methods were developed to identify the haematopoietic stem cells. The process of haematopoeis is occurs in the spleen and bone marrow in mouse. In human beings about 100,000 haematopoietic stem cells produce one billion RBC, one billion platelets, one million T-cells, one million B cells per kg body weight per day.
Several methods have been developed to study haematopoiesis and stem cells:
a) Repopulation assay- Edmens Snell’s group created mice which were genetically identical by mating of sibling mice after 21 generations. Two groups of mice were lethally X- irradiated to destroy their blood cell forming capacity. One of this group was injected with marrow cells from the femur bone of a normal and healthy albino mice. It was observed that this group survived whereas the mice in the other group died. The spleen of mice which survived had the colonies of the bone marrow cells just like bacterial colonies on a Petri plate. This came to be known as colony forming units of spleen (CFU-S) and the technique is known as repopulation assay.
b) The in vitro clonal assay- In this assay, the stem cells proliferate to form colonies of differentiated cells on semi-solid media. This assay helps in identifying growth factors required for the formation of blood cells from the primitive stem cells. One of the first commercialized biotechnology product – erythropoietin was assayed by this procedure.
c) Long term marrow culture- In this method, the marrow cells from femur bone were grown under in vitro conditions on plastic surfaces. These techniques were helpful in bone marrow transplantation and treatment of blood cancer by releasing immature blood cells into the blood stream.
d) Embryonic stem cell culture- Embryonic stem cells are cell lines derived from the inner cell mass of fertilized mouse embryo without the use of immortalizing or transforming agents. The Inner cell mass (ICM) are the cells that are maintained in tissue culture in the presence of irradiated fibroblast cells. These cells are often used in creating chimeric mice. In 1998, J.A. Thomson developed the method to multiply the human embryonic stem cells. Human ICM can also be now derived either by IVF or from germ cell precursors and cultured on a Petri plate. The differentiation of these cells into lineage restricted (neuronal and glial) cells can be accomplished by altering the media in which the cells grow.
e) The ICM cells could be used to create chimeric mice. In chimeric mice it was possible to take ES cells from a black mouse and implant it into the embryo of an albino mouse (white). The progeny so developed had skin colour of black and white ( a chimera).
BIOETHICS IN ANIMAL GENETIC ENGINEERING
There are some serious issues related to genetic modification of animals using animal genetic engineering techniques. One is not sure of the consequences of these genetic modifications and the further interaction with the environment. Proper clinical trials are also necessary before one can use it for commercial purposes. In the recent past people have raised objections on some of the methods used e.g. the transfer of a human genes into food animals, use of organisms containing human genes as animal feed. Some religious groups have expressed their concern about the transfer of genes from animals whose flesh is forbidden for use as food into the animals that they normally eat. Transfer of animal genes into food plants that may be objectionable to the vegetarians.
Besides this, there are several other aspects of this issue have to be sorted out.
a) What will be the consequences, if a modified animal will breed with other domestic or wild animals thereby transferring the introduced genes to these populations?
b) What are the health risks to human on consumption of genetically modified animals and their products?
c) With the production of disease resistant animals, what will be the effect on ecology?
d) There is also wide spread concern about the risks of human recipients getting infected with animal viral diseases after a xenotransplantation., which might infect the population at large.
e) There are also concerns about the risk that drug resistance gene markers used in genetic engineering procedures might inadvertently be transferred and expressed.
The need of the hour is to formulate clear guidelines which should be followed while using genetic engineering techniques in bio-medical research. e.g. products from transgenic organisms should be clearly marked to give choice to people who follow dietary restrictions due to religious beliefs. In fact all the ethical and moral issues raised by some aspects of biotechnology should be addressed by open discussion and dialogue.
INTRODUCTION
An important aspect of all biotechnology processes is the culture of either the plant cells or animal cells or microorganisms. The cells in culture can be used for recombinant DNA technology, genetic manipulations etc.
Plant cell culture is based on the unique property of the cell-totipotency. CELL-TOTIPOTENCY is the ability of the plant cell to regenerate into whole plant. This property of the plant cells has been exploited to regenerate plant cells under the laboratory conditions using artificial nutrient mediums. With the advances made in genetic engineering, it became possible to introduce foreign genes into cell and tissue culture systems. This led to the development of GENETICALLY MODIFIED (GM) OR TRANSGENIC CROPS which had improved traits and characteristics.
History of cell culture
In the early 19th century, Schleiden and Schwann proposed the concept of the 'cell theory'. In 1902, Gottlieb Haberlandt, the german botanist and regarded as the father of plant tissue culture, first attempted to cultivate the mechanically isolated plant leaf cells on a simple nutrient medium. He did not succeed in achieving the growth and differentiation of the cultured cells, however, he predicted the concept of growth hormones, the use of embryo sac fluids, the cultivation of artificial embryos from somatic cells, etc.
During the period 1902 - 1930, attempts were made to culture the isolated plant organs such as roots and shoot apices (organ culture). Hanning (1904) isolated embryos of some crucifers and successfully grew on mineral salts and sugar solutions. Simon (1908) successfully regenerated a bulky callus, buds, roots from a poplar tree on the surface of medium containing IAA which proliferated cell division. Gautheret, White and Nobecourt (1934-1940) largely contributed to the developments made in plant tissue culture. White (1939) cultured tobacco tumour tissue from the hybrid Nicotiana glauca, and N. Langsdorffii.
The period of 1940 - 1970s saw the development of suitable nutrient media to culture plant tissues, embryos, anthers, pollen, cells and protoplasts, and the regeneration of complete plants (in vitro morphogenesis) from cultured tissues and cells. In 1941, van Overbek and co-workers used coconut milk (embryo sac fluid) for embryo development and callus formation in Datura. Steward and Reinert (1959) first discovered somatic embryo production in vitro. Maheswari and Guha (1964) developed the anther culture for the production of haplid plants. Skoog and Miller (1957) advanced the hypothesis of organogenesis in cultured callus by varying the ratio of auxin and cytokinin in the growth medium. Muir (1953) developed a successful technique for the culture of single isolated cells wich is commonly known as paper-raft nurse technique (placing a single cell on filter paper kept on an actively growing nurse tissue). In 1952, the Pfizer Inc., New York (U.S.A) got the US patent and started producing industrially the secondary metabolites of plants. The first commercial production of a natural product shikonin by cell suspension culture was obtained.
In 1980s using Genetic engineering, for the first time, it was possible to introduce foreign genes into cell and tissue culture systems to develop plants with improved characteristics (transgenic crops) which may contribute to the path towards the second green revolution.
PLANT CELL AND TISSUE CULTURE TECHNIQUES
The whole plants can be regenerated virtually from any plant part (referred to as explant) or cells.
Plant tissue culture techniques involve the following steps:
a) Preparation and selection of suitable nutrient media
b) Selection of explants such as shoot tip.
c) Surface sterilization of the explants by disinfectants e.g. sodium or calcium hypochorite solution 0.3- 0.6% ) followed by washing the explants with sterile distilled water.
d) Inoculation or Transfer of the explants onto the suitable nutrient medium (sterilized by autoclaving) in culture vessels under sterile conditions (using laminar flow hood).
e) Incubation or Growing the cultures in the growth chamber or plant tissue culture room at optimum physical conditions of light (16 hours of photoperiod), diurnal illumination, temperature (25+/- 20C and relative humidity (50%-60%).
f) Regeneration of plants from cultured plant tissues.
g) Hardening: it is the gradual exposure of plantlets for acclimatization to environmental conditions
h) Transfer of plants to the field conditions following the acclimatization/ hardening of the regenerated plants.
APPLICATIONS OF CELL AND TISSUE CULTURE
Micropropagation /Clonal Propagation
Clonal propagation refers to the process of asexual reproduction by multiplication
of genetically identical copies of individual plants. The vegetative propagation of plants is labour-intensive, low in productivity and seasonal. The tissue culture methods of plant propagation, known as 'micropropagation' utilizes the culture of apical shoots, axillary buds and meristems on suitable nutrient medium.The regeneration of plantlets in cultured tissue was described by Murashige in 1974. Fossard (1987) gave a detailed account of stages of micropropagation.
The micropropagation is rapid and has been adopted for commercialization of important plants such as banana, apple, pears, strawberry, cardamom, many ornamentals (e.g. Orchids) and other plants.The micropropagation techniques are preferred over the conventional asexual propagation methods because of the following reasons: (a) In the micropropagation method, only a small amount of tissue is required to regenerate millions of clonal plants in a year., (b) micropropagation is also used as a method to develop resistance in many species., (c) in vitro stock can be quickly proliferated as it is season independent,. (d) long term storage of valuable germplasm possible.
The steps in micropropagation method are: a) Initiation of culture - from an explant like shoot tip on a suitable nutrient medium, b) multiple shoots formation from the cultured explant, c) rooting of in vitro developed shoots and, d) transplantation - transplantation to the field following acclimatization.
The factors that affect micropropagation are: (a) genotype and the physiological status of the plant e.g. plants with vigorous germination are more suitable for micropropagation., (b) the culture medium and the culture environment like light, temperature etc. For example an illumination of 16 hours a day and 8 hours night is satisfactory for shoot proliferation and a temperature of 250C is optimal for the growth.
The benefits of micropropagation this method are:
a) rapid multiplication of superior clones can be carried out through out the year, irrespective of seasonal variations.
b) multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatus), cassava (Manihot esculenta)
c) multiplication of sexually derived sterile hybrids
d) It is a cost effective process as it requires minimum growing space.
Somaclonal variation
The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variation and the plants derived from such cells are called as ‘somaclones’. It has been observed that the long-term callus and cell suspension culture and plants regenerated from such cultures are often associated with chromosomal variations. It is this property of cultured cells that finds potential application in the crop improvement and in the production of mutants and variants (e.g. disease resistance in potato).
Larkin and Scowcroft (1981) working at the division of Plant Industry, C.S.I.R.O., Australia gave the term 'somaclones' for plant variants obtained from tissue cultures of somatic tissues. Similarly, if the tissue from which the variants have been obtained is having gametophytic origin such as pollen or egg cell, it is known as 'gametoclonal' variation.They explained that it may be due to: (a) reflection of heterogeneity between the cells and explant tissue, (b) a simple representation of spontaneous mutation rate, and (c) activation by culture environment of transposition of genetic materials.
Shepard et al. (1980) also contributed by screening about 100 somaclones produced from leaf protoplasts of Russet Burbank. They found that there was a significant amount of stable variation in compactness of growth habit, maturity, date, tuber uniformity, tuber skin colour and photoperiodic requirements.
Somaclonal Variations has been used in plant breeding programmes where the genetic variations with desired or improved characters are introduced into the plants and new varieties are created that can exhibit disease resistance, improved quality and yield in plants like cereals, legumes, oil seeds tuber crops etc. Somaclonal variation is applicable for seed
APPLICATIONS OF SOMACLONAL VARIATIONS
a) Methodology of introducing somaclonal variations is simpler and easier as compared to recombinant DNA technology.
b) Development and production of plants with disease resistance e.g. rice, wheat, apple, tomato etc.
c) Develop biochemical mutants with abiotic stress resistance e.g. aluminium tolerance in carrot, salt tolerance in tobacco and maize.
d) Development of somaclonal variants with herbicide resistance e.g. tobacco resistant to sulfonylurea
e) Development of seeds with improved quality e.g. a new variety of Lathyrus sativa seeds (Lathyrus Bio L 212) with low content of neurotoxin.
f) Bio-13 – A somaclonal variant of Citronella java (with 37% more oil and 39% more citronellon), a medicinal plant has been released as Bio-13 for commercial cultivation by Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow, India.
g) Supertomatoes- Heinz Co. and DNA plant Technology Laboratories (USA) developed Supertomatoes with high solid component by screening somaclones which helped in reducing the shipping and processing costs.
Production of virus free plants
The viral diseases in plants transfer easily and lower the quality and yield of the plants. It is very difficult to treat and cure the virus infected plants therefore te plant breeders are always interested in developing and growing virus free plants.
In some crops like ornamental plants, it has become possible to produce virus free plants through tissue culture at the commercial level. This is done by regenerating plants from cultured tissues derived from a) virus free plants, b) meristems which are generally free of infection - In the elimination of the virus, the size of the meristem used in cultures play a very critical role because most of the viruses exist by establishing a gradient in plant tissues. The regeneration of virus-free plants through cultures is inversely proportional to the size of the meristem used., c) meristems treated with heat shock (34-360C) to inactivate the virus, d) callus, which is usually virus free like meristems.e) chemical treatment of the media- attempts have been made to eradicate the viruses from infected plants by treating the culture medium with chemicals e.g. addition of cytokinins suppressed the multiplication of certain viruses.
Among the culture techniques, meristem-tip culture is the most reliable method for virus and other pathogen elimination.
Viruses have been eliminated from a number of economically important plant species which has resulted in a significant increase in the yield and production e.g. potato virus X from potato, mosaic virus from cassava etc. These virus free plants are not disease resistant so there is a need to maintain stock plants to multiply virus free plants whenever required.
Production of synthetic seeds
In synthetic seeds, the somatic embryos are encapsulated in a suitable matrix (e.g. sodium alginate), along with substances like mycorrhizae, insecticides, fungicides and herbicides. These artificial seeds can be utilized for the rapid and mass propagation of desired plant species as well as hybrid varieties. The major benefits of synthetic seeds are:
a) They can be stored up to a year with out loss of viability
b) Easy to handle and useful as units of delivery
c) Can be directly sown in the soil like natural seeds and do not need acclimatization in green house.
Mutant selection
An important use of cell cultures is in mutant selection in relation to crop improvement. The frequency of mutations can be increased several fold through mutagenic treatments and millions of cells can be screened. A large number of reports are available where mutants have been selected at cellular level. The cells are often selected directly by adding the toxic substance against which resistance is sought in the mutant cells. Using this method, cell lines resistant to amino acid analogues, antibiotics, herbicides, fungal toxins etc have actually been isolated.
Production of secondary metabolites
The most important chemicals produced using cell culture are secondary metabolites, which are defined as’ those cell constituents which are not essential for survival’. These secondary metabolites include alkaloids, glycosides (steroids and phenolics), terpenoids, latex, tannins etc. It has been observed that as the cells undergo morphological differentiation and maturation during plant growth, some of the cells specialize to produce secondary metabolites. The in vitro production of secondary metabolites is much higher from differentiated tissues when compared to non-differentiated tissues.
The cell cultures contribute in several ways to the production of natural products. These are: (a) a new route of synthesis to establish products e.g. codeine, quinine, pyrethroids, (b) a route of synthesis to a novel product from plants difficult to grow or establish e.g. thebain from Papaver bracteatum, (c) a source of novel chemicals in their own right e.g. rutacultin from culture of Ruta, (d) as biotransformation systems either on their own or as part of a larger chemical process e.g. digoxin synthesis.
The advantages of in vitro production of secondary metabolites
a) The cell cultures and cell growth are easily controlled in order to facilitate improved product formation.
b) The recovery of the product is easy.
c) As the cell culture systems are independent of environmental factors, seasonal variations, pest and microbial diseases, geographical location constraints, it is easy to increase the production of the required metabolite.
d) Mutant cell lines can be developed for the production of novel and commercially useful compounds.
e) Compounds are produced under controlled conditions as per the market demands.
f) The production time is less and cost effective due to minimal labour involved.
APPLICATIONS OF SECONDARY METABOLITES
Many of these secondary products especially various alkaloids are of immense use in medicine. The yield of these chemicals in cell culture, is though generally lower than in whole plants, it is substantially increased by manipulating physiological and biochemical conditions.
Shikonine is a dye produced by the cells Lithospermum erythrorhizon on a commercial scale. Besides this there are a number of secondary metabolite products that are being widely used for various purposes. Vincristine is used as anticancer agent, digoxin controls cardiovascular disorders, pyrithrins is an insecticide etc. The production of specialty chemicals by plants has become a multibillion industry.
Please refer to the table for some secondary metabolites and their uses.
TABLE SHOWING PLANT SPECIES AND SECONDARY METABOLITES OBTAINED FROM THEM USING TISSUE CULTURE TECHNIQUES
Product Plant source Uses
Artemisin Artemisia spp. Antimalarial
Azadirachtin Azadirachta indica Insecticidal
Berberine Coptis japonica Antibacterial, anti inflammatory
Capsaicin Capsicum annum Cures Rheumatic pain
Codeine Papaver spp. Analgesic
Camptothecin Campatotheca accuminata Anticancer
Cephalotaxine Cephalotaxus harringtonia Antitumour
Digoxin Digitalis lanata Cardiac tonic
Pyrethrin Chrysanthemum cinerariaefolium Insecticide (for grain storage)
Morphine Papaver somniferum Analgesic, sedative
Quinine Cinchona officinalis Antimalarial
Taxol Taxus spp. Anticarcinogenic
Vincristine Cathranthus roseus Anticarcinogenic
Scopolamine Datura stramonium Antihypertensive
Production of Somatic hybrids and cybrids
The Somatic cell hybridization/ parasexual hybridization or Protoplast fusion offers an alternative method for obtaining distant hybrids with desirable traits significantly between species or genera, which can not be made to cross by conventional method of sexual hybridization.
SOMATIC HYBRIDIZATION
Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant. The process involves: a) fusion of protoplasts, (b) Selection of hybrid cells, (c) identification of hybrid plants.
During the last two decades, a variety of treatments have been used to bring about the fusion of plant protoplasts. Protoplast fusion can be achieved by spontaneous, mechanical, or induced fusion methods.. These treatments include the use of fusogens like NaNO3, high pH with high Ca2++ ion concentration, use of polyethylene glycol (PEG), and electrofusion. These inducing agents used in protoplast fusion are called ‘fusogen’.
PEG treatment is the most widely used method for protoplast fusion as it has certain advantages over others. These are : (a) it results in a reproducible high-frequency of heterokaryon formation., (b) The PEG fusion is non specific and therefore can be used for a wide range of plants., (c) It has low toxicity to the cell and (d) The formation of binucleate heterokaryons is low.
MECHANISM OF FUSION
The fusion of protoplasts takes place in three phases- agglutination, plasma membrane fusion and formation of heterokaryons. When the two protoplasts come in close contact with each other, they adhere to each other. This agglutination can be induced by PEG, high pH and high Ca2+. The protoplast membranes get fused at localized sites at the point of adhesion. This leads to the formation of cytoplasmic bridges between protoplasts. High pH and high Ca2+ ions neutralize the surface charges on the protoplasts which allows closer contact and membrane fusion between agglutinated protoplasts. The fused protoplasts become round as a result of cytoplasmic bridges which leads to the formation of spherical homokaryon or heterokaryon.
SELECTION OF HYBRID CELLS
The methods used for the selection of hybrid cells are biochemical, visual and cytometric methods using fluorescent dyes. The biochemical methods for selection of hybrid cells are based on the use of biochemical compounds in the medium. The drug sensitivity method is useful for the selection hybrids of two plants species, if one of them is sensitive to a drug. Another method, auxotrophic mutant selection method involves the auxotrophs which are mutants that cannot grow on a minimal medium. Therefore specific compounds are added in the medium. The selection of auxotropic mutants is possible only if the hybrid cells can grow on a minimal medium. The visual method involves the identification of heterokaryons under the light microscope. In some of the somatic hybridizations, the chloroplast deficient protoplast of one plant species is fused with the green protoplast of another plant species. The heterokaryons obtained are bigger and green in colour while the parental protoplasts are either small or colourless. The cytometric method uses flow cytometry and flourescent-activated cell sorting techniques for the analysis of plant protoplasts.
APPLICATIONS OF SOMATIC HYBRIDIZATION
a) Creation of hybrids with disease resistance - Many disease resistance genes (e.g. tobacco mosaic virus, potato virus X, club rot disease) could be successfully transferred from one species to another. E.g resistance has been introduced in tomato against diseases such as TMV, spotted wilt virus and insect pests.
b) Environmental tolerance - using somatic hybridization the genes conferring tolerance for cold, frost and salt were introduced in e.g. in tomato.
c) Cytoplasmic male sterility - using cybridization method, it was possible to transfer cytoplasmic male sterility.
d) Quality characters - somatic hybrids with selective characteristics have been developed e.g. the production of high nicotine content.
CHROMOSOME NUMBER IN SOMATIC HYBRIDS
The chromosome number in the somatic hybrids is generally more than the total number of both of the parental protoplasts. If the chromosome number in the hybrid is the sum of the chromosomes of the two parental protoplasts, the hybrid is said to be symmetric hybrid. Asymmetric hybrids have abnormal or wide variations in the chromosome number than the exact total of two species.
In 1972, Carlson and his associates produced the first inter-specific somatic hybrid between Nicotiana glauca and N. langsdorffii. In 1978, Melchers and his co-workers developed the first inter-genetic somatic hybrids between Solanum tuberosum (potato) and Lycopersicon esculentum (tomato). The hybrids are known as ‘Pomatoes or Topatoes’.
LIMITATIONS OF SOMATIC HYBRIDIZATION
a) Somatic hybridization does not always produce plants that give fertile and visible seeds.
b) There is genetic instability associated with protoplast culture.
c) There are limitations in the selection methods of hybrids, as many of them are not efficient.
d) Somatic hybridization between two diploids results in the formation of an amphidiploid which is not favourable therefore haploid protoplasts are recommended in somatic hybridization.
e) It is not certain that a specific character will get expressed in somatic hybridization.
f) Regenerated plants obtained from somatic hybridization are often variable due to somaclonal variations, chromosomal elimination, organelle segregation etc.
g) Protoplast fusion between different species/genus is easy, but the production of viable somatic hybrids is not always possible.
CYBRIDS
The cytoplasmic hybrids where the nucleus is derived from only one parent and the cytoplasm is derived from both the parents are referred to as cybrids. The process of formation of cybrids is called cybridization. During the process of cybridization and heterokaryon formation, the nuclei are stimulated to segregate so that one protoplast contributes to the cytoplasm while the other contributes nucleus alone. The irradiation with gamma rays and X-rays and use of metabolic inhibitors makes the protoplasts inactive and non-dividing. Some of the genetic traits in certain plants are cytoplasmically controlled. This includes certain types of male sterility, resistance to certain antibiotics and herbicides. Therefore cybrids are important for the transfer of cytoplasmic male sterility (CMS), antibiotic and herbicide resistance in agriculturally useful plants. Cybrids of Brassica raphanus that contain nucleus of B. napus, chloroplasts of atrazinc resistant B. capestris and male sterility from Raphanus sativas have been developed.
IN VITRO PLANT GERMPLASM CONSERVATION
Germplasm refers to the sum total of all the genes present in a crop and its related species.
The conservation of germplasm involves the preservation of the genetic diversity of a particular plant or genetic stock for it’s use at any time in future. It is important to conserve the endangered plants or else some of the valuable genetic traits present in the existing and primitive plants will be lost. A global organization- International Board of Plant Genetic Resources (IBPGR) has been established for germplasm conservation and provides necessary support for collection, conservation and utilization of plant geneic resources through out the world. The germplasm is preserved by the following two ways:
(a) In-situ conservation- The germplasm is conserved in natural environment by establishing biosphere reserves such as national parks, sanctuaries. This is used in the preservation of land plants in a near natural habitat along with several wild types.
(b) Ex-situ conservation- This method is used for the preservation of germplasm obtained from cultivated and wild plant materials. The genetic material in the form of seeds or in vitro cultures are preserved and stored as gene banks for long term use.
In vivo gene banks have been made to preserve the genetic resources by conventional methods e.g. seeds, vegetative propagules, etc. In vitro gene banks have been made to preserve the genetic resources by non - conventional methods such as cell and tissue culture methods. This will ensure the availability of valuable germplasm to breeder to develop new and improved varieties.
The methods involved in the in vitro conservation of germplasm are:
(a) Cryopreservation- In cryopreservation (Greek-krayos-frost), the cells are preserved in the frozen state. The germplasm is stored at a very low temperature using solid carbon dioxide (at -790C), using low temperature deep freezers (at -800C), using vapour nitrogen (at- 1500C) and liquid nitrogen (at-1960C). The cells stay in completely inactive state and thus can be conserved for long periods. Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses. Certain compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide etc are added during the cryopreservation. These are called cryoprotectants and prevent the damage caused to cells (by freezing or thawing) by reducing the freezing point and super cooling point of water.
(b) Cold Storage- Cold storage is a slow growth germplasm conservation method and conserves the germplasm at a low and non-freezing temperature (1-90C). The growth of the plant material is slowed down in cold storage in contrast to complete stoppage in cryopreservation and thus prevents cryogenic injuries. Long term cold storage is simple, cost effective and yields germplasm with good survival rate. Virus free strawberry plants could be preserved at 100C for about 6 years. Several grape plants have been stored for over 15 years by using a cold storage at temperature around 90C and transferring them in the fresh medium every year.
(c) Low pressure and low oxygen storage- In low- pressure storage, the atmospheric pressure surrounding the plant material is reduced and in the low oxygen storage, the oxygen concentration is reduced. The lowered partial pressure reduces the in vitro growth of plants. In the low-oxygen storage, the oxygen concentration is reduced and the partial pressure of oxygen below 50 mmHg reduces plant tissue growth. Due to the reduced availability of O2, and reduced production of CO2, the photosynthetic activity is reduced which inhibits the plant tissue growth and dimension. This method has also helped in increasing the shelf life of many fruits, vegetables and flowers.
The germplasm conservation through the conventional methods has several limitations such as short-lived seeds, seed dormancy, seed-borne diseases, and high inputs of cost and labour. The techniques of cryo-preservation (freezing cells and tissues at -1960c) and using cold storages help us to overcome these problems.
TRANSGENIC PLANTS AS BIOREACTORS (MOLECULAR FARMING)
Plants can be used as cheap chemical factories that require only water, minerals, sun light and carbon dioxide to produce thousands of sophisticated chemical molecules with different structures. By transferring the right genes, plants can serve as bioreactors to modified or new compounds such as amino acids, proteins, vitamins, plastics, pharmaceuticals (peptides and proteins), drugs, enzymes for food industry and so on. The transgenic plants as bioreactors have some advantages such as the cost of production is low, there is an unlimited supply, safe and environmental friendly and there is no scare of spread of animal borne diseases.
Tobacco is the most preferred plant as a transgenic bioreactor because it can be easily transformed and engineered. Tobacco is an excellent biomass producer with about 40 tons of fresh leaf production as against e.g. rice with 4 tons. The seed production is very high (approx. one million seeds per plant) and it can be harvested several times in a year.
Some of the uses of transgenic plants are:
Improvement of Nutrient quality
Transgenic crops with improved nutritional quality have already been produced by introducing genes involved in the metabolism of vitamins, minerals and amino acids.
A transgenic Arabidopsis thaliana that can produce ten-fold higher vitamin E (alpha-tocopherol) than the native plant has been developed. The biochemical machinery to produce a compound close in structure to alpha-tocopherol is present in A. thaliana. A gene that can finally produce alpha-tocopherol is also present, but is not expressed. This dormant gene was activated by inserting a regulatory gene from a bacterium which resulted in an efficient production of vitamin E.
Glycinin is a lysine-rich protein of soybean and the gene encoding glycinin has been introduced into rice and successfully expressed. The transgenic rice plants produced glycinin with high contents of lysine.
Using genetic engineering Prof Potrykus and Dr. Peter Beyer have developed rice which is enriched in pro-vitamin A by introducing three genes involved in the biosynthetic pathway for carotenoid, the precursor for vitamin A. The aim was to help millions of people who suffer from night blindness due to Vitamin A deficiency, especially whose staple diet is rice. The presence of beta-carotene in the rice gives a characteristic yellow/orange colour, hence this pro-vitamin A enriched rice is named as Golden Rice.
The genetic engineering is also being used to improve the taste of food e.g. a protein ‘monellin’ isolated from an African plant (Dioscorephyllum cumminsii) is about 100,000 sweeter than sucrose on molar basis. Monellin gene has been introduced into tomato and lettuce plants to improve their taste.
Improvement of seed protein quality
The nutritional quality of cereals and legumes has been improved by using biotechnological methods. Two genetic engineering approaches have been used to improve the seed protein quality. In the first case, a transgene (e.g. gene for protein containing sulphur rich amino acids) was introduced into pea plant (which is deficient in methionine and cysteine, but rich in lysine) under the control of seed-specific promoter. In the second approach, the endogenous genes are modified so as to increase the essential amino acids like lysine in the seed proteins of cereals.
These transgenic routes have helped to improve the essential amino acids contents in the seed storage proteins of a number of crop plants. E.g. overproduction of lysine by de-regulation. The four essential amino acids namely lysine, methionine, threonine, and isoleucine are produced from a non-essential amino acid aspartic acid. The formation of lysine is regulated by feed back inhibition of the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DHDPS). The lysine feedback- insensitive genes encoding the enzymes AK and DHDPS have been respectively isolated from E. Coli and Cornynebacterium. After doing appropriate genetic manipulations, these genes were introduced into soybean and canola plants. The transgenic plants so produced had high quantities of lysine.
Diagnostic and therapeutic proteins
Experiments are going on to use transgenic plants in diagnostics for detecting human diseases and therapeutics for curing human and animal diseases. Several metabolites and compounds are already being produced in transgenic plants e.g. the monoclonal antibodies, blood plasma proteins, peptide hormones, cytokinins etc. The use of plants for commercial production of antibodies, referred to as plantbodies, is a novel approach in biotechnology. The first successful production of a functional antibody, namely a mouse immunoglobulin IgGI in plants, was reported in 1989. This was achieved by developing two transgenic tobacco plants-one synthesizing heavy chain gamma- chain and other light kappa- chain, and crossing them to generate progeny that can produce an assembled functional antibody. In 1992, C.J. Amtzen and co-workers expressed hepatitis B surface antigen in tobacco to produce immunologically active ingredients via genetic engineering of plants.
Several other therapeutic proteins have also been produced like haemoglobin and erythropoietin in tobacco plants, lactoferrin in potato, trypsin inhivitor in maize etc. The first proteins/enzymes that were produced in transgenic plants (maize) are avidin and beta-glucuronidase and are used in diagnostic kits.
Edible vaccines
Crop plants offer cost-effective bioreactors to express antigens which can be used as edible vaccines. The approach is to isolate genes encoding antigenic proteins from the pathogens and then expressing them in plants. Such transgenic plants or their tissues producing antigens can be eaten for vaccination/immunization (edible vaccines). The expression of such antigenic proteins in crops like banana and tomato are useful for immunization of humans since banana and tomato fruits can be eaten raw.
Transgenic plants (tomato, potato) have been developed for expressing antigens derived from animal viruses e.g. rabies virus, herpes virus. In 1990, the first report of the production of edible vaccine (a surface protein from Streptococcus) in tobacco at 0.02% of total leaf protein level was published in the form of a patent application under the International Patent Cooperation Treaty (Mason and Arntzen,1995).The first clinical trials in humans, using a plant derived vaccine were conducted in 1997 and were met with limited success. This involved the ingestion of transgenic potatoes with a toxin of E. coli causing diarrhea.
The process of making of edible vaccines involves the incorporation of a plasmid carrying the antigen gene and an antibiotic resistance gene, into the bacterial cells e.g. Agrobacterium tumefaciens. The small pieces of potato leaves are exposed to an antibiotic which can kill the cells that lack the new genes. The surviving cells with altered genes multiply and form a callus. This callus is allowed to grow and subsequently transferred to soil to form a complete plant. In about a few weeks, the plants bear potatoes with antigen vaccines.
The bacteria E.coli, V. cholerae cause acute watery diarrhea by colonizing the small intestine and by producing toxins. Chloera toxin (CT) is very similar to E.Coli toxin. The CT has two subunits, A and B. Attempt was made to produce edible vaccine by expressing heat labile enterotoxin (CT-B) in tobacco and potato.
Another strategy adopted to produce a plant-based vaccine, is to infect the plants with recombinant virus carrying the desired antigen that is fused to viral coat protein. The infected plants are reported to produce the desired fusion protein in large amounts in a short duration. The technique involves either placing the gene downstream a subgenomic promoter, or fusing the gene with capsid protein that coats the virus.
Advantages of edible vaccines
The edible vaccines produced in transgenic plants will sole the storage problems, will ensure easy delivery system by feeding and will have low cost as compared to the recombinant vaccines produced by bacterial fermentation. Vaccinating people against dreadful diseases like cholera and hepatitis B, by feeding them banana, tomato, and vaccinating animals against important diseases will be an interesting development.
Biodegradable plastics
Polythenes and plastics are one of the major environmental hazards. Efforts are on to explore the possibility of using transgenic plants for biodegradable plastics. Transgenic plants can be used as factories to produce biodegradable plastics like polyhydroxy butyrate or PHB. Genetically engineered Arabidopisis plants can produce PHB globules exclusively in their chloroplasts without effecting plant growth and development. The large-scale production of PHB can easily be achieved in plants like Populus, where PHB can be extracted from leaves.
Molecular Breeding
The term molecular breeding is frequently used to represent the breeding methods that are coupled with genetic engineering techniques. Up till now, conventional breeding methods have been used to meet the food demands of the growing world population and the challenges of poverty and improved crop production and yields. However in the years to come, the development in the agriculture yields and techniques is going to be due to the use of molecular breeding programme.
Linkage analysis which deals with the studies to correlate the link between the molecular marker and a desired trait is an important aspect of molecular breeding programme. In the past, linkage analysis was carried out by use of isoenzymes and the associated polymorphisms. Now a days, molecular markers are being used.
Molecular breeding involves breeding using molecular (nucleic acid) markers. A molecular marker is a DNA sequence in the genome which can be located and identified therefore molecular markers can be used to identify particular locations in the genome.
Due to mutations, insertions, deletions, etc. the base composition at a particular location may be different in different plants. These differences, termed polymorphisms, allow DNA markers to be mapped in a genetic linkage group.
Generally, there are three types of markers used in screening/selection:
a) Morphological marker based on visible character (phenotypic expression) e.g. flower color, seed color, height, leaf shapes, etc. Morphological markers could be dominant or recessive. There are certain constraints in using these markers as the morphological markers are easily influenced by environmental factors and thus may not represent the desired genetic variation. Some of the visible markers have not much role to play in the plant breeding programme.
b) Biochemical marker: The proteins produced by gene expression are also used as markers in plant breeding programmes. The most commonly used are isozymes, the different molecular forms of the same enzyme. Each individual variety has its own isozyme variability (profiles) which can be detected by electrophoresis on starch gel.
c) Molecular marker based on DNA polymorphism detected by DNA probes or amplified products of PCR, e.g.Restriction fragment length polymorphism (RFLP), Randomly Amplified polymorphic DNA (RAPD), variable Number Tandom Repeats (VNTR), Microsatellites, etc. Plant breeders always prefer to detect the gene as molecular marker, although it is not always possible. Molecular markers provide a true representation of the genetic make up at the DNA level. They are consistent and free from environmental factors, and can be detected much before the development of plants occur. The advantage with a molecular marker is that a plant breeder can select a suitable marker for the desired trait which can be detected well in advance. A large number of markers can be generated as per the needs. The molecular markers to be used in plant breeding programme should have the following characteristics: (a) the marker should be closely linked with the desired trait, (b) the marker screening methods should be effective, efficient, reproducible and easy to carry out, (C) the entire analysis should be cost effective.
Molecular makers are of two types: (a) based on nucleic acid (DNA) hybridization- This involves the cloning of the DNA piece followed by the hybridization with the genomic DNA, which is later detected.
The Restriction fragment length polymorphism (RFLP) was the very first technology employed for the detection of polymorphism, based on the DNA sequence differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombinations of genomic DNA. The procedure involves the isolation of genomic DNA and it’s digestion by restriction enzymes. The fragments are separated by electrophoresis and finally hybridized by incubating with cloned and labeled probes.
(b) Molecular markers based on PCR amplification.
Polymerase chain reaction (PCR) is a novel technique for the amplification of selected regions of DNA. The most important advantage is that even a minute quantity of DNA can be amplified and the PCR- based molecular markers require only a small quantity of DNA to start with. Random amplified polymorphic DNA (RAPD) markers use PCR amplification where the DNA is isolated from the genome and is denatured. The template molecules are annealed with primers and amplified by PCR. The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bends on gel electrophoresis.
Amplified fragment length polymorphism (AFLP) is a novel technique involving a combination of RFLP and RAPD. AFLP is based on the principle of generation of DNA fragments using restriction enzymes and oligonucleotide adaptors (or linkers), and their amplification by PCR.
Microsatellites
Microsatellites are the tandemly repeated multiple copies of mono-, di-, tri-, and tetra nucleotide motifs. In some instances, there are unique flanking sequences present in the repeat sequences. Primers are designed for such flanking sequences to detect the sequence tagged microsatellites (STMS) which is done by PCR.
Commercial use of transgenic plants
The main goal of producing transgenic plants is to increase the productivity. In 1995-96, transgenic potato and cotton plants were used commercially for the first time in USA. By the year 1998-99, five other major transgenic crops cotton, maize, canola, soybean, and potato were introduced to the farmers. These accounted for about 75% of the total area planted by crops in USA. There are still a lot of concerns regarding the harmful environmental and hazardous health effects of transgenic plants. The major areas of public concern are- the development of resistance genes in insects, generation of a super weeds by mutation etc. Certain other legal and regulatory hurdles pertaining to commercial use of transgenic plants, needs to be addressed.
Bioethics in Plant genetic Engineering
There are issues and concerns regarding the use of transgenic crops and their effects on the health and the environment in general. The major concerns about GM crops and GM foods are:
a) Effect of GM crops on biodiversity and environment- As the GM crops are created artificially, there is no natural process of evolution in their development. Hence, there is a question of this affecting the biodiversity and overall effect on the environment.
b) The risk of transfer of transgene from GM crops to pathogenic microbes- Antibiotic marker genes are used to identify and select the modified cells. If GM food containing antibiotic resistance marker gene is consumed by animals and humans, there is a risk that the transgene will transfer from GM food to microflora of human and animals. This may lead to the gut microbes to become resistant to antibiotics.
c) The transfer of genes from animals into Gm crops for molecular farming may change the fundamental vegetable nature of plants.
d) The GM crops may bring about changes in evolutionary patterns. The plants adapt to the changing environment in the natural way by changing their genes and developing better races with superior traits which ultimately leads to the development of evolved races and varieties. What will be the evolutionary pattern of the GM crops? There are concerns about the effect of transgene flow from GM crops to other non-GM plants and the alteration of these non-GM crops.
e) There is a risk of transferring allergens (usually glycoproteins) from GM food to human and animals.
f) There is a risk of “gene pollution” i.e. transfer of transgene of GM crop through pollen grains to related plant species and development of super weeds.
g) There are also some religious issues related to the consumption of transgenic plants with animal genes introduced into them, especially, for some strict vegetarian people and some ethnic groups with certain food preferences and restrictions.
h) There is a need to study thoroughly as to how the genetically engineered plants will affect the ecological balance, once they are released in the environment.
Bio-Technology and Society
INTRODUCTION
The field of biotechnology has had a lot of beneficial contribution in the area of healthcare, agriculture, food production, manufacture of industrial enzymes, and appropriate environmental management. However, the advancement in this field has also lead to some concerns and controversies raised by a number of groups, NGOs etc. ELSI is the short form to represent the ethical, legal, and social implications of biotechnology. ELSI broadly covers the relationship between BIOTECHNOLOGY and SOCIETY with particular reference to ethical and legal aspects.
Concerns about the Genetically modified organisms (GMOs)
There are concerns regarding the biosafety, ethics and issues related to the release of GMOs in the environment. Many contries and NGOs have opposed the release of the GMOs due to these reasons. In order to address theses issues, the UNIDO/WHO/FAO/UNEP has built up an Informal Working Group on Biosafety. In 1991, this group prepared the “ Voluntary Code of Conduct for the release of Organisms into the Environment”. The ICGEB organizes annual workshops on biosafety and on risk assessment for the release of GMOs. It collaborates with the management of UNIDO’s BINAS (Biosafety Information Network and Advisory Service), whose aim is to monitor the global development in regulatory issues in biotechnology. An on-line bibliographic data-base on biosafety and risk assessment has also been created by ICGEB to evaluate the environmental release of GMOs.
Besides this, the ICGEB also assists its member states in developing the national biosafety framework.
The main areas of consideration for safety aspects in biotechnology are the following:
a) How to dispose off spent microbial biomass and purify the effluents from biotechnological processes?
b) The toxicity of the allergy associated with microbial production.
c) How to deal with the increase in the number of antibiotic resistant pathogenic microorganisms?
d) How to evaluate the pathogenicity of the genetically engineered microorganisms to infect humans, plants and animals?
e) How to prevent contamination, infection or mutation of the processed strains?
f) The evaluation of the interaction of the genetically engineered microbes with the elements of natural environment.
In the past, time and again, there has been public outrage against the use of genetically modified or transgenic plants and other organisms. In 1999, a British medical journal published the adverse effect of genetically modified (GM) potato (which was produced by Rowett Research Insitute). This potato was found to contain snow drop lectin which affected the small intestine of the rats and stunted the growth and damaged their immune system. This led to worldwide public concern about this issue and created a lot of controversy about the safety of GM foods.
The transgenic Bt-plants such as cotton, corn, soybean, and potato were approved for cultivation in USA. However, some countries did not allow Bt-plants in their fields e.g. Br-rice was not allowed in Philippines, Bt-cotton in France. Many Governments are also suspicious of the use of GMOs due to various reason-risks, societal beliefs, and economic concerns.
Biological Warfare?
Most of the countries of the world are signatories to the Biological Weapons Conventions of 1972. As a signatory, it is a voluntary pledge by a nation “never to produce microbial or other biological agents or toxins, whatever may be their method of production, for use in wars. However, many people have expressed their concerns about the possible use of genetic manipulations for military purposes in the near future.
Intellectual Property
With the fast pace development in the field of biotechnology, the issues related to legal characterization and the treatment of trade related biotechnological processes and products are of immense importance. These are popularly known as Intellectual Property. Intellectual Property includes Patents, trade secrets, copyrights, and trademarks. Intellectual Property Rights (IPR) is a collective term applied to a number of different types of legal rights granted by each country.
The rights to protect this property prohibits others from making, copying, using or selling the proprietary subject matter.
In biotechnology, the intellectual property covers the processes and products which result from the development of genetic engineering techniques through the use of restriction enzymes to create recombinant DNA.
Another example of intellectual property is the development of crop varieties which are protected through “plant breeder’s rights or PBRs. The PBRs ensures that the plant breeder who developed a particular variety gets the exclusive rights for marketing the variety.
Agriculture for the first time was included in the trade related intellectual property rights (TRIPS) and TRIPS is a major concern for developing countries. The following two major steps were taken in consideration of PBRs:
(a) The Food and Agriculture Organisation (FAO) has an International treaty on plant genetic resources for food and agriculture. This treaty consists of a particular classes which refers to operation of farmer’s rights.
(b) The ‘Plant Varietal Protection and Farmer’s Rights Act 2001 agrees for the right of farmers, breeders, and researchers. The protection is provided by making compulsory licensing of rights, and inhibiting the import of plant varieties consisting of ‘genetic use of restriction technology’ (GURT) e.g. terminator technology of Monsanto.
Following conditions should be fulfilled to grant protection to the new varieties:
a) the new variety must always be new i.e. it should not have ever been exploited commercially.
b) It should be biologically distinct and possess different characters.
c) The new variety of the plant must have uniform characters.
d) The distinguishing character of new variety must be stable for generations.
e) The new variety should have taxonomic validity i.e. systematic position, generic and species names etc.
Recently Utility patents for both plant and animal genetic materials, have been allowed in some countries. This forbids the use of patented material for further breeding. The farmers are allowed to use and save the seeds for cultivation only after paying a fee to the patent holder.
Some concerns have been voiced regarding the implications of IPR on the genetic diversity and the conservation of genetic resources. IPRs will directly or indirectly affect the food security and distribution around the globe, biological diversity and ecological balance, employment avenues in the poor and developing countries, and the use of new and effective agricultural practices.
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