Gene transformation
To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the necessary controlling sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two kinds of gene transfer methods in plants are:
Vector-mediated or indirect gene transfer
Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.
Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.
Vectorless or direct gene transfer
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are:
Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.
Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are:
Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.
Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues.
Transformation - This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The transformation frequency (the fraction of cell population that can be transferred) is very good in this method. E.g. the uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-50C) followed by heat shock treatment at 37-450C for about 90 sec. The transformation efficiency refers to the number of transformants per microgram of added DNA. The CaCl2 breaks the cell wall at certain regions and binds the DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a method for gene transfer. In conjuction, two live bacteria come together and the single stranded DNA is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria.
The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues.
Transformation - This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The transformation frequency (the fraction of cell population that can be transferred) is very good in this method. E.g. the uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-50C) followed by heat shock treatment at 37-450C for about 90 sec. The transformation efficiency refers to the number of transformants per microgram of added DNA. The CaCl2 breaks the cell wall at certain regions and binds the DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a method for gene transfer. In conjuction, two live bacteria come together and the single stranded DNA is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria.
Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells.
Selection of transformed cells from untransformed cells
The selection of transformed plant cells from untransformed cells is an important step in the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is introduced into the plant along with the transgene followed by the selection of an appropriate selection medium (containing the antibiotic). The segregation and stability of the transgene integration and expression in the subsequent generations can be studied by genetic and molecular analyses (Northern, Southern, Western blot, PCR).
During the last decades, a tremendous progress has been made in the development of transgenic plants using the various techniques of genetic engineering. The plants, in which a functional foreign gene has been incorporated by any biotechnological methods that generally are not present in the plant, are called transgenic plants. As per estimates recorded in 2002, transgenic crops are cultivated world-wide on about 148 million acres (587 million hectares) land by about 5.5 million farmers. Transgenic plants have many beneficial traits like insect resistance, herbicide tolerance, delayed fruit ripening, improved oil quality, weed control etc.
Some of the commercially grown transgenic plants in developed countries are: “Roundup Ready” soybean, ‘Freedom II squash’, ‘High- lauric’ rapeseed (canola), ‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing insect resistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA. Some of the traits introduced in these transgenic plants are as follows:
Some of the commercially grown transgenic plants in developed countries are: “Roundup Ready” soybean, ‘Freedom II squash’, ‘High- lauric’ rapeseed (canola), ‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing insect resistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA. Some of the traits introduced in these transgenic plants are as follows:
Stress tolerance
Biotechnology strategies are being developed to overcome problems caused due to biotic stresses (viral, bacterial infections, pests and weeds) and abiotic stresses (physical actors such as temperature, humidity, salinity etc).
Abiotic stress tolerance
Biotechnology strategies are being developed to overcome problems caused due to biotic stresses (viral, bacterial infections, pests and weeds) and abiotic stresses (physical actors such as temperature, humidity, salinity etc).
Abiotic stress tolerance
The plants show their abiotic stress response reactions by the production of stress related osmolytes like sugars (e.g. trehalose and fructans), sugar alcohols (e.g. mannitol), amino acids (e.g. proline, glycine, betaine) and certain proteins (e.g. antifreeze proteins). Transgenic plants have been produced which over express the genes for one or more of the above mentioned compounds. Such plants show increased tolerance to environmental stresses. Resistance to abiotic stresses includes stress induced by herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone and intense light. These environmental stresses result in the destruction, deterioration of crop plants which leads to low crop productivity. Several strategies have been used and developed to build ressitance in the plants against these stresses.
Herbicide tolerance
Weeds are unwanted plants which decrease the crop yields and by competing with crop plants for light, water and nutrients. Several biotechnological strategies for weed control are being used e.g. the over-production of herbicide target enzyme (usually in the chloroplast) in the plant which makes the plant insensitive to the herbicide. This is done by the introduction of a modified gene that encodes for a resistant form of the enzyme targeted by the herbicide in weeds and crop plants. Roundup Ready crop plants tolerant to herbicide-Roundup, is already being used commercially.
The biological manipulations using genetic engineering to develop herbicide resistant plants are: (a) over-expression of the target protein by integrating multiple copies of the gene or by using a strong promoter., (b) enhancing the plant detoxification system which helps in reducing the effect of herbicide., (c) detoxifying the herbicide by using a foreign gene., and (d) modification of the target protein by mutation.
Some of the examples are:
Glyphosate resistance - Glyphosate is a glycine derivative and is a herbicide which is found to be effective against the 76 of the world’s worst 78 weeds. It kills the plant by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it’s structural similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly with EPSPS and thus blocks the shikimic acid pathway.
Certain strategies were used to provide glyphosate resistance to plants.
(a) It was found that EPSPS gene was overexpressed in Petunia due to gene amplification. EPSPS gene was isolated from Petunia
and introduced in to the other plants. These plants could tolerate glyphosate at a dose of 2- 4 times higher than that required to kill wild type plants.
Herbicide tolerance
Weeds are unwanted plants which decrease the crop yields and by competing with crop plants for light, water and nutrients. Several biotechnological strategies for weed control are being used e.g. the over-production of herbicide target enzyme (usually in the chloroplast) in the plant which makes the plant insensitive to the herbicide. This is done by the introduction of a modified gene that encodes for a resistant form of the enzyme targeted by the herbicide in weeds and crop plants. Roundup Ready crop plants tolerant to herbicide-Roundup, is already being used commercially.
The biological manipulations using genetic engineering to develop herbicide resistant plants are: (a) over-expression of the target protein by integrating multiple copies of the gene or by using a strong promoter., (b) enhancing the plant detoxification system which helps in reducing the effect of herbicide., (c) detoxifying the herbicide by using a foreign gene., and (d) modification of the target protein by mutation.
Some of the examples are:
Glyphosate resistance - Glyphosate is a glycine derivative and is a herbicide which is found to be effective against the 76 of the world’s worst 78 weeds. It kills the plant by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it’s structural similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly with EPSPS and thus blocks the shikimic acid pathway.
Certain strategies were used to provide glyphosate resistance to plants.
(a) It was found that EPSPS gene was overexpressed in Petunia due to gene amplification. EPSPS gene was isolated from Petunia
and introduced in to the other plants. These plants could tolerate glyphosate at a dose of 2- 4 times higher than that required to kill wild type plants.
(a) By using mutant EPSPS genes- A single base substitution from C to T resulted in the change of an amino acid from proline to serine in EPSPS. The modified enzyme cannot bind to glyphosate and thus provides resistance.
(b) The detoxification of glyphosate by introducing the gene (isolated from soil organism- Ochrobactrum anthropi) encoding for glyphosate oxidase into crop plants. The enzyme glyphosate oxidase converts glyphosate to glyoxylate and aminomethylphosponic acid. The transgenic plants exhibited very good glyphosate ressitance in the field.
Another example is of Phosphinothricin resistance
Phosphinothricin is a broad spectrum herbicide and is effective against broad-leafed weeds. It acts as a competitive inhibitor
of the enzyme glutamine synthase which results in the inhibition of the enzyme glutamine synthase and accumulation of ammonia and finally the death of the plant. The disturbace in the glutamine synthesis also inhibits the photosynthetic activity.
The enzyme phosphinothricin acetyl transferase ( which was first observed in Streptomyces sp in natural detoxifying mechanism against phosphinothricin) acetylates phosphinothricin, and thus inactivates the herbicide. The gene encoding for phosphinothricin acetyl transferase (bar gene) was introduced in transgenic maize and oil seed rape to provide resistance against phosphinothricin.
of the enzyme glutamine synthase which results in the inhibition of the enzyme glutamine synthase and accumulation of ammonia and finally the death of the plant. The disturbace in the glutamine synthesis also inhibits the photosynthetic activity.
The enzyme phosphinothricin acetyl transferase ( which was first observed in Streptomyces sp in natural detoxifying mechanism against phosphinothricin) acetylates phosphinothricin, and thus inactivates the herbicide. The gene encoding for phosphinothricin acetyl transferase (bar gene) was introduced in transgenic maize and oil seed rape to provide resistance against phosphinothricin.
Other abiotic stresses
The abiotic stresses due to temperature, drought, and salinity are collectively also known as water deficit stresses. The plants produce osmolytes or osmoprotectants to overcome the osmotic stress. The attempts are on to use genetic engineering strategies to increase the production of osmoprotectants in the plants. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding the key enzymes have been isolated. E.g. Glycine betaine is a cellular osmolyte which is produced by the participation of a number of key enzymes like choline dehydrogenase, choline monooxygenase etc. The choline oxidase gene from Arthrobacter sp. was used to produce transgenic rice with high levels of glycine betaine giving tolerance against water deficit stress.
Scientists also developed cold-tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperature. By introducing the coordinating gene (it encodes a protein which acts as transcription factor for regulating the expression of cold tolerant genes), expression of cold tolerant genes was triggered giving protection to the plants against the cold temperatures.
Scientists also developed cold-tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperature. By introducing the coordinating gene (it encodes a protein which acts as transcription factor for regulating the expression of cold tolerant genes), expression of cold tolerant genes was triggered giving protection to the plants against the cold temperatures.
Insect resistance
A variety of insects, mites and nematodes significantly reduce the yield and quality of the crop plants. The conventional method is to use synthetic pesticides, which also have severe effects on human health and environment. The transgenic technology uses an innovative and eco-friendly method to improve pest control management.About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants
A variety of insects, mites and nematodes significantly reduce the yield and quality of the crop plants. The conventional method is to use synthetic pesticides, which also have severe effects on human health and environment. The transgenic technology uses an innovative and eco-friendly method to improve pest control management.About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants
The first genes available for genetic engineering of crop plants for pest resistance were Cry genes (popularly known as Bt genes) from a bacterium Bacillus thuringiensis. These are specific to particular group of insect pests, and are not harmful to other useful insects like butter flies and silk worms. Transgenic crops with Bt genes (e.g. cotton, rice, maize, potato, tomato, brinjal, cauliflower, cabbage, etc.) have been developed. This has proved to be an effective way of controlling the insect pests and has reduced the pesticide use. The most notable example is Bt cotton (which contains CrylAc gene) that is resistant to a notorious insect pest Bollworm (Helicoperpa armigera).. There are certain other insect resistant genes from other microorganisms which have been used for this purpose. Isopentenyl transferase gene from Agrobacterium tumefaciens has been introduced into tobacco and tomato. The transenic plants with this transgene were found to reduce the leaf consumption by tobacco hornworm and decrease the survival of peach potato aphid.
Certain genes from higher plants were also found to result in the synthesis of products possessing insecticidal activity. One of the examples is the Cowpea trypsin inhibitor gene (CpTi) which was introduced into tobacco, potato, and oilseed rape for develping transgenic plants. Earlier it was observed that the wild species of cowpea plants growing in Africa were resistant to attack by a wide range of insects. It was observed that the insecticidal protein was a trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera, Orthaptera etc. Cowpea trypsin inhibitor (CpTi) has no effect on mammalian trypsin, hence it is non-toxic to mammals.
Virus resistance
There are several strategies for engineering plants for viral resistance, and these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in a number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. The induction of virus resistance is done by employing virus-encoded genes-virus coat proteins, movement proteins, transmission proteins, satellite RNa, antisense RNAs, and ribozymes. The virus coat protein-mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV.
The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, peanut, sugar beet, alfalfa etc. The viruses that have been used include alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), potato virus X (PVX) , potato virus Y (PVY) etc.
There are several strategies for engineering plants for viral resistance, and these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in a number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. The induction of virus resistance is done by employing virus-encoded genes-virus coat proteins, movement proteins, transmission proteins, satellite RNa, antisense RNAs, and ribozymes. The virus coat protein-mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV.
The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, peanut, sugar beet, alfalfa etc. The viruses that have been used include alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), potato virus X (PVX) , potato virus Y (PVY) etc.
Resistance against Fungal and bacterial infections
As a defense strategy against the invading pathogens (fungi and bacteria) the plants accumulate low molecular weight proteins which are collectively known as pathogenesis-related (PR) proteins.
Several transgenic crop plants with increased resistance to fungal pathogens are being raised with genes coding for the different compounds. One of the examples is the Glucanase enzyme that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed.
Several transgenic crop plants with increased resistance to fungal pathogens are being raised with genes coding for the different compounds. One of the examples is the Glucanase enzyme that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed.
Delayed fruit ripening
The gas hormone, ethylene regulates the ripening of fruits, therefore, ripening can be slowed down by blocking or reducing ethylene production. This can be achieved by introducing ethylene forming gene(s) in a way that will suppress its own expression in the crop plant. Such fruits ripen very slowly (however, they can be ripen by ethylene application) and this helps in exporting the fruits to longer distances without spoilage due to longer-shelf life.
The most common example is the 'Flavr Savr' transgenic tomatoes, which were commercialized in U.S.A in 1994. The main strategy used was the antisense RNA approach. In the normal tomato plant, the PG gene (for the enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme polygalacturonase which is involved in the fruit ripening. The complimentary DNA of PG encodes for antisense mRNA, which is complimentary to normal (sense) mRNA. The hybridization between the sense and antisnse mRNAs renders the sense mRNA ineffective. Consequently, polygalacturonase is not produced causing delay in the fruit ripening. Similarly strategies have been developed to block the ethylene biosynthesis thereby reducing the fruit ripening. E.g. transgenic plants with antisense gene of ACC oxidase (an enzyme involved in the biosynthetic process of ethylene) have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in the fruit ripening.
The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC and removes amino group) has been transferred and expressed in tomato plants which showed 90% inhibition in the ethylene biosynthesis.
The gas hormone, ethylene regulates the ripening of fruits, therefore, ripening can be slowed down by blocking or reducing ethylene production. This can be achieved by introducing ethylene forming gene(s) in a way that will suppress its own expression in the crop plant. Such fruits ripen very slowly (however, they can be ripen by ethylene application) and this helps in exporting the fruits to longer distances without spoilage due to longer-shelf life.
The most common example is the 'Flavr Savr' transgenic tomatoes, which were commercialized in U.S.A in 1994. The main strategy used was the antisense RNA approach. In the normal tomato plant, the PG gene (for the enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme polygalacturonase which is involved in the fruit ripening. The complimentary DNA of PG encodes for antisense mRNA, which is complimentary to normal (sense) mRNA. The hybridization between the sense and antisnse mRNAs renders the sense mRNA ineffective. Consequently, polygalacturonase is not produced causing delay in the fruit ripening. Similarly strategies have been developed to block the ethylene biosynthesis thereby reducing the fruit ripening. E.g. transgenic plants with antisense gene of ACC oxidase (an enzyme involved in the biosynthetic process of ethylene) have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in the fruit ripening.
The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC and removes amino group) has been transferred and expressed in tomato plants which showed 90% inhibition in the ethylene biosynthesis.
Male Sterility
The plants may inherit male sterility either from the nucleus or cytoplasm. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility. In tobacco plants, these are created by introducing a gene coding for an enzyme (barnase, which is a RNA hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production. The restoration of male fertility is done by introducing another gene barstar that suppresses the activity of barnase at the onset of the breeding season. By using this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce etc. with male sterility have been developed.
The plants may inherit male sterility either from the nucleus or cytoplasm. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility. In tobacco plants, these are created by introducing a gene coding for an enzyme (barnase, which is a RNA hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production. The restoration of male fertility is done by introducing another gene barstar that suppresses the activity of barnase at the onset of the breeding season. By using this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce etc. with male sterility have been developed.
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:
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.
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.
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.
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.
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
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 engineeredArabidopisis 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 likePopulus, where PHB can be extracted from leaves.
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 engineeredArabidopisis 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 likePopulus, 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.
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:
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.
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.
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.
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:
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.
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