Biotechnological applications in agriculture

BIOTECHNOLOGICAL APPLICATION IN AGRICULTURE

Food production can be enhanced by

(1) Agro-chemical based Agriculture

(2) Organic agriculture

(3) Genetically engineered crop-based agriculture

Green revolution resulted in increasing the food supply almost three times.

Green revolution is the great increase in the production of food grains (especially wheat and rice) that resulted in large part from the introduction of new, high yielding varieties begining in the mid 20th century.

Its early dramatic success was Maxico and the Indian subcontinent.

The new varieties required large amount of chemical fertilizers and pesticides to produce high yields, raising concern about cost and potentially harmful environmental effects.

This demands an alternate pathway that can result in maximum yield from the fields but the chemicals and fertiliser use is minimum i.e., harmful effects on the environment are reduced.

Genetically modified organisms or GMO can be the plants, bacteria, fungi and animals whose genes have been alteres by genetic manipulation.

(A) Genetically Modified Crops :

A transgenic crop is a crop that contains and expresses a transgene.

A popular term for transgenic crops is genetically modified crops or GM crops.

The techniques used for the production of transgenic crops offer the following two unique advantages: (i) any gene (from any organism or a gene synthesised chemically) can be used for transfer, and (ii) the change in genotype can be precisely controlled since only the transgene is added into the crop genome.

In contrast, breeding activities

(i) Can use only those genes that are present in such species that can be hybridised within them. In addition,

(ii) Changes occur in all those traits for which the parents used in hybridisation differ from each other.

When a transgene is introduced into the genome of an organism, it can achieve one of the following :

(i) Produces a protein that is the product in which we are interested.

(ii) Produces a protein that on its own produces the desired phenotype.

(iii) Modifies an existing biosynthetic pathway so that a new end-product is obtained.

(iv) Prevents the expression of an existing native gene.

Hirudin is a protein that prevents blood clotting. The gene enconding hirudin was chemically synthesised. This gene was then transferred into Brassica napus, where hirudin accumulates in seeds. The hirudin is purified and used as medicine. In this case, the transgene product itself is the product of interest.

A simplified representation of the production of

hirudin from transgenic Brassica napus seeds

The tomato variety 'Flavr Savr' presents an example where expression of a native tomato gene has been blocked.

Expression of a native gene can be stopped by many different methods.

Fruit softening is promoted by the enzyme polygalacturonase which degrades pectin.

Production of polygalacturonase was blocked in, the transgenic tomato variety 'Flavr Savr'.

Therefore, fruits of this tomato variety remain fresh and retain their flavour much longer than do the fruits of normal tomato varieties. In addition, the fruits have a superior taste and increased total soluble solids these are unexpected bonus.

(B) Genetically Modified Food:

The food prepared from the produce of genetically modified (= transgenic) crops is called genetically modified food or, in short, GM food. GM food differs from the food prepared from the produce of conventionally developed varieties mainly in the following aspects.

Firstly, it contains the protein produced by the trans-gene in question, e.g., Cry protein in the case of insect resistant varieties.

Secondly, it contains the enzyme produced by the antibiotic resistance gene that was used during gene transfer by genetic engineering.

Finally, it contains the antibiotic resistance gene itself.

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Biofortification is a method of breeding crops to increase their nutritional value.

This can be done either through conventional selective breeding, or through genetic engineering.

Biofortification differs from ordinary fortification because it focuses on making plant foods more nutritious as the plants are growing, rather than having nutrients added to the foods when they are being processed.

This is an improvement on ordinary fortification when it comes to providing nutrients for the rural poor, who rarely have access to commercially fortified foods.

As such, biafortifications seen as an upcoming strategy for dealing with deficiencies of micronutrients in the developing world.

It has been argued that the above features of GM foods could lead to the following problems when they are consumed.

Firstly, the transgene product may cause toxicity and/or produce allergies.

Secondly, the enzyme produced by the antibiotic resistance gene could cause allergies, since it is a foreign protein.

Finally, the bacteria present in the alimentary canal of the human could takes up the antibiotic resistance gene that is present in the GM food.

These bacteria would then become resistant to the concerned antibiotic.

As a result, these bacteria could become difficult to manage.

The scientists involved in the production of transgenic crops are addressing to these concerns.

Efforts are being made to use other genes in place of antibiotic resistance genes.

The toxic and allergenic actions of the trans-gene product can be adequately examined by detailed assays using suitable animal models.

GM PRODUCTS: BENEFITS AND CONTROVERSIES

Benefits

(1) Crops

(i) Enhanced taste and quality

(ii) Reduced maturation time

(iii) Increased nutrients, yields, and stress tolerance

(iv) Improved resistance to disease, pests, and herbicides

(v) New products and growing techniques

(2) Animals

(i) Increased resistance, productivity, hardiness, and feed efficiency

(ii) Better yields of meat, eggs, and milk.

(iii) Improved animal health and diagnostic methods

(3) Environment

(i) "Friendly" bioherbicides and bioinsecticides

(ii) Conservation of soil, water, and energy

(iii) Bioprocessing for forestry prroducts

(iv) Better natural waste management

(v) More efficient processing

(4) Society

Increased food security for growing populations

Controversies

Safety: Potential human health impact: allergens, transfer of antibiotic resistance markers, unknown effects Potential environmental impact: unintended transfer of transgenes through cross-pollination, unknown effects on other organisms (e.g., soil microbes), and loss of flora and fauna biodiversity.

Bt COTTON

DNA technology makes it possible to locate the genes that produces Bt proteins lethal to insects and transfer the gene into crop plants.

First scientists identify a strain of Bt that kills the targeted insect.

Then they isolate the gene that produces the lethal protein.

That gene is removed from the Bt bacterium and a gene conferring resistance to a chemical (usually antibiotic or herbicide) is attached that proves useful in later steps.

The Bt gene with the resistance gene-attached is inserted into plant cells.

These modified or genetically transformed cells are then grown into complete plant by tissue culture.

The modified plant produces the same lethal protein as produced by the Bt bacteria because plants now have the same gene.

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B. thuringiensis was first discovered in 1902 by Japanese biologist Shigetane Ishwatari.

In 1911, B. thuringiensis was rediscovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars.

In 1976, Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation.

B. thuringiensis is closely related to B.cereus, a soil bacterium, and B.anthracis, the cause of anthrax: the three organisms differ mainly in their plasmids.

Like other members of the genus, all three are aerobes capable of producing endospores. Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal -endotoxin (called crystal proteins of Cry proteins), which are encoded by cry genes.

In most strains of B. thuringiensis the cry genes are located on the plasmid.

Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), diptera (flies and mosquitoes), coleoptera (beetles), hymenoptera (wasps, bees, ants and sawflies) and nematodes.

Thus, B. thurengiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops.

When insects ingest toxin crystals, the alkaline pH of their digestive tract activates the toxin.

Cry inserts into the insect gut cell membrane, forming a pore. The pore results cell lysis and eventual death of the insect.

B. thuringiensis forms protein crystals during a particular phase of their growth.

These crystals contain a toxic insecticidal protein.

Why does this toxin not kill the Bacillus? Actually, the Bt toxin protein exists as inactive protoxin but once an insect ingests the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilises the crystals.

The activated toxin binds to the surface of midgut epithelial cells and creates pores that cause cell swelling and lysis and eventually cause death of the insect.

Bt is not harmful to humans, other mammals, birds, fish or beneficial insects.

Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into the several crop plants such as cotton.

The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific.

The toxin is coded by a gene named cry. There are a number of them, for example, the proteins encoded by the genec cry I Ac and cry II Ab control the cotton bollworm, that of cry I Ab controls corn borer.

Although Bt genes have been introduced into tobacco, tomatoes, cotton, and other broadleaf Plants, gene transfer technology for corn is a recent achievement.

The development of corn plants expressing Bt proteins requires substantial changes in the Bt genes, including the creation of synthetic versions of the genes, rather than the microbial Bt gene itself.

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There are several advantages in expressing Bt toxins in transgenic Bt crops:

The level of toxin expression can be very high thus delivering sufficient dosage to the pest:

The toxin expression is contained within the plant system and hence only those insects that feed on the crop perish.

The toxin expression can be modulated by using tissue-specific promoters, and replaces the use synthetic pesticides in the environment: The latter observation has been well documented Worldwide.

PEST RESISTANT PLANTS

Root-knot nematodes are the most economically important group of plant-parasitic nematodes worldwide. They attack nearly every food and fiber crop grown, about 2,000 plant species in all.

The nematode invades plant roots, and by feeding on the roots' cells, they cause the roots to grow large form galls, or knots, damaging the crop and reducing its yields .

The most cost-effective and sustainable management tactic for preventing root-knot nematode damage and reducing growers' losses is to develop resistant plants that prevent the nematode from feeding on the roots.

Because root-knot nematode resistance doesn't come naturally in most crops, bioengineering is required.

Four common root-knot nematode species (mainly Meloidegyne incognitia) account for 95 percent of all infestations in agricultural land.

By discovering a root-knot nematode parasitism gene that's essential for the nematode to infect crops, the scientists have developed a resistance gene effective against all four species.

Using a technique called RNA interference (RNAi), the researchers have effectively turned the nematode's biology against itself.

They genetically modified Arabidopsis, a model plant, to produce double-stranded RNA (dsRNA) to knock out the specific parasitism gene in the nematode when it feeds on the plant roots.

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Long double-stranded RNAs (dsRNAs; typically > 200nt) can be used to silence the expression of target genes in a variety of organisms and cell types (e.g.; worms, fruit flies and plants).

Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway.

First, the dsRNA get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer (initiation step).

Then, the complexes (RISCs), unwinding in the process.

The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step).

Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand.

In mammalian cells, introduction of long dsRNA (>30 nt) initiates a potent antiviral response, exemplified by nonspecific inhibition of protein synthesis and RNA degradation.

The mammalian antiviral response can be bypassed. however, by the introduction or expression of siRNAs.

RNAi takes place in all eukaryotic organisms as a method of cellular defense.

This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing).

The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate.

Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant.

The introduction of DNA was such that it produced both sense and anti-sense RNA in the host cells.

These two RNAs being complementary to each other formed a double stranded RNA that initiated RNAi and thus, silenced the specific mRNA of the nematode.

The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA.

The transgenic plant therefore got itself protected from the parasite.

This knocked out the parasitism gene in the nematode and disrupted its ability to infect plants.

"No natural root-knot resistance gene has this effective range of root-knot nematode resistance."

The efforts have been directed primarily at understanding the molecular tools the nematode uses to infect plants.

This is a prerequisite for bioengineering durable resistance to these nematodes in crop plants.