Transgenic plants with beneficial traits
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:
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.
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.
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.
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.
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.
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 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.