BIOTECHNOLOGICAL CONTROL

Submitted To :- Submitted By :- Dr O. P. Chaudhary Vaibhav

BIOTECHNOLOGY

• The use of biological means to develop processes and products by studying organisms and their components.

• Biotechnology is any technique that uses living organisms or substances from these organisms to make or modify a product for a practical purpose. Biotechnology can be applied to all classes of organism - from viruses and bacteria to plants and animals.

Agricultural biotechnology

• Agricultural biotechnology is a collection of scientific techniques used to improve plants, animals and microorganisms. Based on an understanding of DNA, scientists have developed solutions to increase agricultural productivity.

Biotechnological approaches in Agriculture

Tissue culture techniques rDNA technology Development of transgenic crops.

Tissue Culture

• Technique of growing plant tissues on synthetic medium under controlled and aseptic conditions.

• German plant physiologist G. Haberlandit, is is known as the father of plant tissue culture.

• Plant tissue culture includes several specialized areas, like micro propagation, somaclonal variation, protoplast and anther culture.

rDNA technology

• One of the most significant breakthroughs in modern sciences is the development of techniques to transfer genes from unrelated sources into crop plants

• Recent advances in molecular biology have made it possible to introduce genes from diverse sources such as unrelated plants, bacteria, fungi. Insects and even from chemical synthesis.

• The whole process of introduction, integration and expression of the foreign gene(s) in the host is called gene transformation or transgenesis.

• Unlike conventional plant breeding, in this case only the cloned gene(s) of agronomic importance are introduced into the plants without the co-transfer of other undesirable genes from the donor.

• The recipient genotype is less desturbed.

Foreign gene can be transferred by two methods :• Direct gene transfer• Vector-mediated gene transfer

Direct gene transfer• Since the host range of agro bacterium has been largely

limited to dicotyledonous plant species, some other methods are used for monocotyledonous plants such as cereals have been developed.

• Methods used in direct gene transfer tech. are –Direct uptake of DNAElectroporationMicroinjectionMicroprojectile Bombardment

Direct uptake of DNA

• It is based on the ability of protoplast to uptake the foreign DNA from surrounding solution.

• An isolated plasmid DNA (vector) is mixed with protoplasts in the presence of polyethylene glycol (PEG), which inhance the uptake of DNA by protoplast.

• This method depends on the plant regeneration ability of the protoplast and has been successfully used to produce transgenic plants in brassica, strawberry, lettuce, rice, wheat and maize.

Electroporation

• Electroporation, or electro permeabilization, is a molecular biology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell.

• The cells recovered from the electric shock can be regenerated into whole plants.

• Electroporation has been successfully used for obtaining transgenes in tobacco, maize, rice, wheat and sugercane.

Microinjection• During microinjection, DNA is injected directly into the

cell, or even into the cell nucleus via an inserted cannula. The process is observed and controlled under the microscope. The DNA is then integrated into the plant genome – probably during the cell’s own DNA repair processes.

Micro injection into the cell of DNA inserted into onion cells a potato callus

Micro projectile Bombardment

• The Particle bombardment device, also known as the gene gun, was developed to enable penetration of the cell wall so that genetic material containing a gene of interest can be transferred into the cell.

• Today the gene gun is used for genetic transformation of many organisms to introduce a diverse range of desirable traits.

• The transgenic plants have been recovered in banana, barley, bean, cotton, maize, papaya etc.

Vector-mediated 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.

Transgenic crops

• Transgenic crops or Genetically modified crops (GMCs, GM crops, or biotech crops) are plants used in agriculture, the DNA of which has been modified using genetic engineering techniques. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in the species.

Development of insect-resistant transgenic crop cultivars has focused on two distinct approaches :-

Integration of bacterial genes from Bacillus thuringienesis (Bt)

Integration of plant genes for the production of enzyme inhibitor and suger binding lectins.

Bt Endotoxins

• The use of genes encoding endotoxins from Bacillus thuringiensis is now a well-established technology for producing transgenic plants with enhanced resistance to the larvae of lepidopteran insect pests.

• Bt cotton was first released for commercial production in the USA in 1996 and subsequently grown in several countries including Argentina, Australia, China, Colombia, Indonesia, Mexico, South Africa, and India.

• Since then other transgenic crop species producing Bt toxins have been commercialized including maize, tomato and potato.

• The adoption of Bt crop varieties by farmers has been rapid reflecting the benefits of these crops such as reduced insecticide use, lower production costs and higher yields.

• B. thuringiensis, a Gram-positive soil bacterium, produces a proteinaceous parasporal crystalline inclusion during sporulation.

• There are two main categories of Bt toxins: Cry and Cyt.

• These two groups are classified further by a detailed nomenclature system that describes groups Cry1 to Cry55 and Cyt1 to Cyt2.

• The larvae of insect orders primarily affected by Bt toxins are Lepidoptera (butterflies and moths), Diptera (mosquitoes) and Coleoptera (larval and adult beetles).

• However, Bt toxins are not toxic to people, wildlife, or most beneficial insects and therefore the opportunities for biological control are great.

• The effect of Bt toxin on a range of lepidopteran insects has been studied including:

Bombyx mori, Helicoverpa armigera, Heliothis virescens , Manduca sexta, Ostrinia nubilalis,

Plutella xylostella, Sesamia nonagrioides,Spodoptera exigua, Spodoptera frugiperda and Spodoptera littoralis .

MECHANISM OF ACTION

• The Bt toxin mechanism of action is described by two models:

1. The pore formation model and2. the signal transduction model.

The initial steps of both models are the same. Upon ingestion by insects the crystalline inclusion is

solubilised in the midgut . Most target insects have a high gut pH that is crucial for the

efficacy of Bt toxins since Most Bt-protoxins are only soluble above pH 9.5. The 130

kDa protoxins are activated by insect gut proteases. In the pore formation model the activated toxins bind to the primary receptors in the brush border membrane of the

midgut epithelium columnar cells .

The major receptors for Cry toxins in lepidopterans are cadherin-like proteins.

The toxins then interact with secondary receptors in the midgut larval membrane.

Following secondary receptor binding, the toxin inserts into the membrane creates pores.

These pores lead to the disruption of membrane integrity and cause an electrolyte

imbalance that ultimately leads to death by starvation or septicaemia.

• An alternative model for the Bt toxin mechanism of action proposes that Cry toxins trigger a signalling cascade pathway. This model differs from the pore formation model in that it does not involve secondary receptors or the formation of pores in the membrane. Instead, in this model, binding to the cadherin receptor initiates a Mg2+ dependent signal cascade pathway that includes a guanine nucleotide-binding protein, adenylyl cyclase, and protein kinase A which ultimately results in cell death.

Protease inhibitors• Protease inhibitors are one component of a plant’s natural

defence mechanism against herbivores and pathogens.• Plants protect themselves directly by constitutively expressing

protease inhibitors and by inducing protease inhibitors in response to mechanical wounding or insect attack.

• They may also release volatile compounds after insect damage that function as potent attractants for predators of insect herbivores.

• The release of volatile compounds after wounding, such as methyl jasmonate also triggers the production of proteinase inhibitors in neighbouring unwounded plants essentially prearming the local population against insect attack.

examples :-Protease inhibitor

Protease family

Proteases inhibited

Transformed plant

Insect species used

bioassayEffect of PI on larval growth

Barley trypsin inhibitor [BTI]

Cereal trypsin inhibitor

Trypsin

Tobacco Spodoptera exigua

29% reduction in survival

Wheat Sitotroga cerealella

No effect on growth or mortality

Soybean Bowman-Birk trypsin inhibitor

Bowman-Birk

Trypsin, chymotrypsin Sugarcane Diatraea

saccharalisGrowth severely retarded

PIS IN TRANSGENIC PLANTS : SUCCESS AND FAILURE

• The main problem of inadequate levels of PI expression is best exemplified by studies with P. xylostella, the diamondback moth. When larvae of the diamondback moth consumed transgenic plants expressing the chymotrypsin and trypsin specific potato type II proteinase inhibitor, Pot II, they suffered lower growth rates. However, this did not confer an advantage to the plants because the larvae consumed more tissue to compensate for their decrease in metabolism. As a result, the insects maintained population growth rates similar to those of larvae on non-transgenic plants.

Lectins

• These are the plant derived proteins that binds to oligo and polysaccharides, cause agglutination and cell aggregation.

• Lectins have been isolated and characterized from a wide variety of plants such as pea, rice, wheat, caster, soybean, garlic, tobacco and chickpea.

• A no. of plant lectins exhibits insecticidal property.• The precise mode of action of of insecticidal

lectins is unknown.

Transgenic crop transgene origin Target insect pest

Rice

GNA Snow drop Nilaparvata lugens,Nephotettics virescens

SFII and GNA Snow drop and spider (Segestria florentina)

N. legens

Wheat GNA Snow drop Sitobion avenae

Potato GNA Snow drop Lecanobia oleracea, Myzus persicae

Mustard WGA Wheat Lipaphis erysimi

Insect resistant transgenic crops expressing plant lectins :-

Alarm Pheromone

• Alarm pheromones are defined as chemical substances, produced and released by an organism, that warn or alert another of the same species of impending danger.

• This is common in the social Hymenoptera; for example, the honeybee, Apis mellifera (Hymenoptera: Apidae), and many ant species respond aggressively to their alarm pheromones.

• Many aphid species produce the sesqiuterpene , (E)-β- fernesens (EFB) as a component of alarm pheromone.

• Scientists at Rothemsted Research (UK) have developed GM wheat, by transferring EFB synthase gene from peppermint, to the genome of a spring wheat strain.

• Lab trials have shown that EBF emmiting wheat not only repels aphids, but also attracts their natural enemies.

• This is the world first crop which repels insects instead of killing them.

RESISTANCE OF LEPIDOPTERAN INSECTS TO BT TOXINS

• More recently there have been reports of field resistance to Bt crops in pink bollworm (Pectinophore gosspiella, cotton bollworm (Helicoverpa spp, armyworm (Spodoptera frugiperda and western corn rootworm Diabrotica virgifera virgifera.

• A decrease in field performance of Bt corn against S. frugiperda was observed in Puerto Rico and against Busseola fusca in South Africa. In southeastern US problems with control of H. zea on Bt cotton have also been reported.

MANAGEMENT OF RESISTANCE TO BT CROPS• There are two main strategies for management of insect resistance to

Bt crops: Refuge and pyramiding.

• The main approach for delaying evolution of resistance to Bt crops is the refuge strategy.

• Farmers are mandated to maintain an abundance of host non-Bt crops as a refuge surrounding their Bt crops.

• The theory behind this strategy is that any Bt resistant larvae that arise on the Bt crops will mate with susceptible individuals from neighbouring non-Bt crops.

• As long as inheritance of resistance remains recessive the offspring will be susceptible to Bt crops.

• The other major strategy to combat the evolution of Bt resistance is gene pyramiding.

• For example, the development of second generation Bt cotton that has at least two Bt toxins such as the Monsanto Bollgard II cotton variety, but up to six Bt toxins.

• Another resistance management strategy which is still in the research phase of development is the use of insecticidal genes with completely different modes of action such as proteinase inhibitors. The success of combining multiple Bt genes for resistance management is contingent on the individual toxins having different targets to prevent cross resistance developing.

• This information can be used to design combinations of Cry toxins that complement each other to delay the development of resistance to Bt crops.