Fungi, are especially destructive crop pathogens and the search for new fungalresistance genes forms a valuable

part of plant biotechnology research. Recent progress in approaches that exploit proteins and molecules that either kill fungi or kill infected plant cells. Fungi are responsible for a range of serious plant diseases such as blight, grey mould, bunts, powdery mildew, and downy mildew. Crops of all kinds often suffer heavy losses. Fungal plant diseases are usually managed with applications of chemical fungicides or heavy metals. In some cases, conventional breeding has provided fungus resistant cultivars. Besides combating yield losses, preventing fungal infection keeps crops free of toxic compounds produced by some pathogenic fungi. These compounds, often referred to as mycotoxins, can affect affect the immune system and disrupt hormone balances. Some mycotoxins are carcinogenic.

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. 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.

Fungus resistant GM plants
Genetic engineering enables new ways of managing fungal infections. Several approaches have been taken: Introducing genes from other plants or bacteria encoding enzymes like chitinase or glucanase: These enzymes break down chitin or glucan, respectively, which are essential components of fungal cell walls. Introducing plant genes to enhance innate plant defense mechanisms (e.g. activing phytoalexins, proteinase inhibitors, or toxic proteins). Invoking the hypersensitive reaction: Plants varieties that are naturally resistant to specific types of fungal diseases are often programmed to have individual cells quickly die at the site of fungal infection. This response, known as the hypersensitive reaction, effectively stops an infection in its tracks. Genetic engineering can help plant cells 'know' when a fungus is attacking.

Several new approaches have been applied to use fungal proteins like Ag-AFP, from Aspergillus giganteus, ribosome inactivating protein (RIP), thaumatin-like protein (PR-5) and human lyzosome in plants to combat fungal diseases. Chitinase and AFP increase resistance in wheat. However better results were obtained with integration of novel cDNA for acidic chitinase and beta 1,3 glucanase as these enzymes were more effective. Introduction of infection related chitinase and rice thaumatin like protein in rice gave stable resistance against sheath blight. Studies on carrot transformation with human lyzosome which can cleave beta 1, 4 glycosidic bond of bacterial cell wall and chitin in fungal cell wall is found to be very effective against both pathogen. Alfa-alfa antifungal peptide defensin from seeds of Medicago sativa was shown to have significant activity against Verticillium dahliae. The transgenic potato expressing the peptide showed to reduce area of infection.

Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring chitinase, (ChiC) isolated from Streptomyces griseus. The transgenic plants demonstrated enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early blight). By introduction of ribosome inactivation protein (RIP) gene from the seeds of barley, produced tobacco plants with enhanced resistance to the fungal pathogen R. Solani.

HERBICIDE RESISTANCE Excessive weed growth forces crops to compete for sunlight and nutrients, often leading to significant losses. Because herbicides cannot differentiate between plants that are crops and plants that are weeds, conventional agricultural systems can only use 'selective' herbicides. Such herbicides do not harm the crop, but are not effective at removing all types of weeds. If farmers use herbicide resistant crops, 'non-selective' herbicides can be used to remove all weeds in a single, quick application. This means less spraying, less traffic on the field, and lower operating costs. 'Non-selective' herbicides: Not always useful 'Broad-spectrum', or non-selective herbicides are effective at killing a wide range of weeds. The problem is, they can also kill valuable crops. Therefore, broad-spectrum herbicides are only useful before seedlings emerge or in special cases like fruit orchards, vineyards, and tree nurseries.

everal biotechnological strategies for weed control are being used e.g. the overroduction of herbicide target enzyme in the plant which makes the plant insensitive o the herbicide. This is done by the introduction of a modified gene that encodes for resistant form of the enzyme targeted by the herbicide in weeds and crop plants. oundup Ready crop plants tolerant to herbicide-Roundup, is already being used ommercially.

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 d) modification of the target protein by mutation.

he biological manipulations using genetic engineering to develop herbicide esistant plants are:

Examples of herbicide are: glyphosate, chlorsulfuron, imazapur, DL-phosphinothricin, romoxynil, atrazine.

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 3phosphate 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. (b) 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.

c) 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 resistance 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 disturbance 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.

Herbicide resistant crops are changing weed management Several crops have been genetically modified to be resistant to non-selective herbicides. These transgenic crops contain genes that enable them to degrade the active ingredient in an herbicide, rendering it harmless. Farmers can thereby easily control weeds during the entire growing season and have more flexibility in choosing times for spraying. Herbicide resistant crops also facilitate low or no tillage cultural practices, which many consider to be more sustainable. Another advantage is that farmers can manage weeds without turning to some of the more environmentally suspect types of herbicides. Critics claim that in some cases, the use of herbicide resistant crops can lead to an increase in herbicide use, promote the development of herbicide resistant weeds, and damage biodiversity on the farm. Extensive ecological impact assessments have been addressing these issues.

Among the field trials conducted on herbicide resistant crops, studies in the United Kingdom have shown that different herbicides and different herbicide application practices can affect the amount of wild plants on the farm. In comparison with conventional cropping systems, weed and animal populations were negatively affected by herbicide tolerant sugar beet and rapeseed, but biodiversity was increased with the use of herbicide tolerant maize.

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