applications of transgenic plants

APPLICATIONS OF TRANSGENIC PLANTS:

APPLICATIONS OF TRANSGENIC PLANTS K.B SHASIKIRAN M PHARM (PHARMACOGNOSY) G.PULLA REDDY COLLEGE OF PHARMACY

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TRANS ENIC PLANTS

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CONTENTS : Herbicide resistance Insect resistance Viral resistance Fungal resistance Stress resistance

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PATTERN OF STUDY : Definition 2. Effects 3. Strategies 4. Examples

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HERBICIDE RESISTANCE: What is herbicide in relation to its effects on plants? A herbicide, commonly known as a weed killer, is a type of pesticide used to kill unwanted plants Atrazine Sulphonyl ureas Glyphosate

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What are the unwanted effects of herbicides? The unwanted effects are: Inhibition of lipid synthesis Inhibiting enzymes Inhibition of amino acid synthesis Inhibition of photo systems by reducing electron flow FEATURES: Here mainly photo synthesis and bio synthesis of amino acids is effected TOBACCO PLANT FEATURES: Here mainly photo synthesis and bio synthesis of amino acids is effected TOMATO PLANT

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Strategies to provide herbicide resistance – to prevent inhibition of enzymes There are three main strategies: 1. Herbicide target modification : Here the target is protein. Herbicide binds to protein and inhibits photosynthesis or amino acid biosynthesis. Strategy used here is to modify protein. 2. Target enzyme over production: Here the target is enzyme, herbicide binds to enzyme and inhibits it. Strategy used here is to over produce the enzyme. This over production of enzyme can be done by transferring genes, which code for the enzyme from other herbicide tolerant transgenic sources to the plant that is under treatment. 3. Enzyme detoxification: Here the strategy is to detoxify the herbicide before binding to target protein and inhibiting it. Gene encoding an enzyme responsible for the detoxification is introduced into the plant.

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1. Target modification: Inhibition of photosynthesis: Herbicide binds to Qb protein present in thylakoid membrane and blocks electron transport at photo system 2. So here the target Qb protein is modified by substituting serine with glycine at position 264 in the Qb protein resulting in decreased binding . This modified Qb protein is encoded by a psbA gene which is taken from other transgenic source providing resistance to the plant . Atrazine Nicotiana tabaccum Arabidopsis thaliana

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1. Target modification: Inhibition of amino acid bio synthesis: Herbicide binds to acetolactate (ALS ) synthase enzyme which is essential in amino acid biosynthesis. Binding is decreased by altering one of the amino acid in the ALS. This modified ALS is encoded by gene taken from wild type plants like arabdopsis Sulphonyl urea Beta vulgaris Arabidopsis thaliana

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2. Target enzyme over production: Here the target is enzyme, herbicide binds to enzyme and inhibits it. Strategy used here is to over produce the enzyme. This over production of enzyme can be done by transferring genes, which code for the enzyme from other herbicide tolerant transgenic sources to the plant that is under treatment. EPSP synthase is essential in aminoacid biosynthesis. Glyphosate Nicotiana tabaccum Petunia hybrida E. coli

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Glutamine synthase : phosphothricin Nicotiana tabaccum Alfalfa Glutamine synthase

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3. Enzyme detoxification: Here the strategy is to detoxify the herbicide before binding to target protein and inhibiting it. Gene encoding an enzyme responsible for the detoxification is introduced into the plant. Phosphinothricin inhibits glutamine synthase which prevents the accumulation of ammonia. Accumulation of ammonia in cell is toxic to cells phosphothricin Beta vulgaris Streptomyces hygroscopius Resultant product is phophothricin acetyl transferase which acetylates free NH 2 group.

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Nicotiana tabaccum Alcaligens eutrophus 3. Enzyme detoxification: Here the strategy is to detoxify the herbicide before binding to target protein and inhibiting it. Gene encoding an enzyme responsible for the detoxification is introduced into the plant. 2,4-D is a synthetic auxin . Uncontrolled, unsustainable growth ensues, causing stem curl-over, leaf withering, and eventual plant death. Resultant product is 2,4 Dichlorophenoxyacetate mono oxygenase which degrades 2,4 D.

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INSECT RESISTANCE: What is an insect in relation to its effects on plants? Insect is an organism which cuts things into sections or parts . Manducta sexta Pectinophora gossypelia

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Strategies to provide insect resistance – to kill insects. Use of viral or bacterial toxin gene 2. Use of proteinase inhibitor gene

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Strategies to provide insect resistance – to kill insects. 1. Use of viral or bacterial toxin gene Here gene is used which codes for toxin protein, this toxic protein when ingested by insects gets cleaved by the proteases in the intestine resulting in conversion of protein into active toxin. Solubilized toxins form pores in the midgut epithelium of susceptible larvae. This toxin impaires digestion and mid gut paralysis as a result insect stops feeding and ultimately dies. Manducta sexta Lycopersion isculentum B. Thuringensis Pectinophora gossypellia Gossypium hirusutum B. Thuringensis

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Strategies to provide insect resistance – to kill insects. 2. Use of proteinase inhibitor gene: Protease is an enzyme that inhibits metabolism of protein. Trypsin is a protease which hydrolyses proteins by cleaving peptide chains mainly at carboxylic side of amino acids like lysine or arginine . Here the strategy is to introduce gene into the plant that inhibits the trypsin activity in the gut of insect. As a result there is indigestion of the leaves eaten by the insect. Manducta sexta Nicotiana tabaccum Vigna unguiculata

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VIRAL RESISTANCE: What is virus in relation to its effects on plants? Virus is a small disease causing agent which consists of genetic material and can replicate only in living cells of organisms like plants. Cucumber mosaic virus Tobacco mosiac virus

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What are the unwanted effects of viruses? Cytopathic effects like: 1. Cell lysis 2. Alteration of cell’s surface membrane 3. Programmed cell death ( apoptosis ) Tmv infected tobacco Tobacco ringspot

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Strategies to provide viral resistance – to prevent replication of viruses. Coat protein mediated protection Non – structural viral gene protection 3. Satellite RNA modification 4. Anti sense RNA resistance

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Strategies to provide viral resistance – to prevent replication of viruses. 1. Coat protein mediated protection: Strategy used here is based on the fact that virus replicates only if the protein surrounding the genetic material is uncoated. So, first mild virus is allowed to infect the cell. As a result of infection excessive amount of coat protein is produced which remains in a free state. When aggressive virus infects the plant cell, already present free protein inhibits uncoating of RNA subsequently delaying or preventing the RNA expression and replication. Virus Species modified Coat protein source Product AlMV Lycopersicon esculentum Mild strain of AIMV Coat protein TMV Nicotiana tabaccum Mild strain of TMV , CaMV 35-S promotor Coat protein

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Strategies to provide viral resistance – to prevent replication of viruses. 2. Non – structural viral gene protection: Non – structural gene is a gene that codes for a regulatory protein as in comparision with the structural gene which encodes for other proteins except regulatory protein. These proteins regulate the way that the information in genes is turned into gene products like proteins . Strategy used here is to introduce non structural genes which code for protein that play an important role in regulation of protein production. Virus Species modified Gene source Product TMV strain U1 Nicotiana tabaccum CaMV 35 S promoter and TMV- open reading frame coding region 54KD protein

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Strategies to provide viral resistance – to prevent replication of viruses. 3. Satellite RNA modification: Word satellite means an object that accompanies or orbits something else. A satellite is a subviral agent composed of nucleic acid that depends on the co-infection of a host cell with a helper or master virus for its multiplication Satellite RNA are the small molecules encapsulated in plant viruses . Presence of satellite RNA modify or ameliorate plant disease symptoms. Strategy used here is to introduce genes into plants that express satellite RNA. Risks associated here are : Satellite RNA which are ameliorative in one species may be lethal (death causing ) in another. Satellite RNA mutate and single nucleotide change introduced can make ameliorative satellite necrogenic ( origin of dead matter ). Virus Species modified Satellite RNA source Product TRV, CMV Nicotiana tabaccum TRV, CMV CaMV promoter Satellite RNA

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Strategies to provide viral resistance – to prevent replication of viruses. 4. Anti sense RNA resistance: Here the strategy is to inhibit translation process of m-RNA. Inhibition is by introducing antisense RNA which is complementary to m-RNA. As a result, base pairing occurs between antisense RNA and mRNA forming duplex RNA molecules which thereby cause blocking of translation. Virus Species modified Antisense RNA source Product CMV, TMV Nicotiana tabaccum CMV, TMV Antisense RNA

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FUNGAL RESISTANCE: What is fungus in relation to its effects on plants? Fungi are the organisms which have major role in decomposition of organic matter . Alternaria langipes Rhizoctonia solani

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Strategies to provide fungal resistance – killing fungi – damaging cell wall or producing defence related proteins Phytoalexins 2. Chitinase 3. Ribosome inhibiting proteins

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Strategies to provide fungal resistance – killing fungi – damaging cell wall or producing defence related proteins 1. Phytoalexins ( fungistatic ) : Here the strategy is to use the gene that codes for a secondary metabolites like terpenoids , glycosteroids and alkaloids called phytoalexins which have broad spectrum antimicrobial activity. They may puncture the cell wall, delay maturation, disrupt metabolism or prevent reproduction of the pathogen . The expression of this gene in the plant may be due to elicitors produced by host and fungal cell wall break down or by abiotic agents such as mechanical injury, ultra violet irradiation and heavy metals. Organism Species modified Gene source Product Fungal pathogen Nicotiana tabaccum Peanut stillbene Phytoalexin – resveratol

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Strategies to provide fungal resistance – killing fungi – damaging cell wall or producing defence related proteins 2. Chitinase : Chitinase is an enzyme which damages the cell walls of the fungus by breaking glycoside bonds ( bonds between carbohydrate and other nitrogen contaning group). This enzyme is very stable, resistant to heat and inhibits fungal growth. Here the strategy used is to introduce a gene into the plant that codes for chitinase enzyme. Fungal pathogen Species modified Gene source product Alternaria longipes Nicotiana tabaccum Serratia marcescens Chitinase Rhizoctonia solani Brassica nappus Phaseolus vulgaris Bean endo chitinase chitin chitinase

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Strategies to provide fungal resistance – killing fungi – damaging cell wall or producing defence related proteins 3. Ribosome inhibiting proteins: Seeds of various cereals contain proteins that are toxic to some fungal pathogens. These proteins inhibit the ribosome function by changing its chemical structure by enzymatic hydrolysis of the N - glycosidic bond at A 4324 of rRNA and thus inhibiting peptide elongation and consequently growth of fungi. Strategy used here is to introduce gene into the plants which code for ribosome inhibiting proteins. Fungal pathogen Species modified Gene source Product Rhizoctonia solani Nicotiana tabaccum Barley – produces glycosylase protein which cleaves a glycosol from 60 S sub unit of ribosome . thus preventing peptide elongation glycosylase

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STRESS RESISTANCE: What is stress in relation to its effects on plants? Stress refers to any change in the environmental conditions that might reduce or adversely change a plants growth or development. Plants depend on internal mechanisms for tolerating variations in the external environment.

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Water deficit stress and Strategy to provide tolerance 1. Drought 2. Heat and cold 3. Salinity All the above stresses are caused due to the water deficit caused by a number of environmental conditions like salinity, heat, chilling conditions and drought. When there is a water deficit in the soil, there arises a deficit between the supply of water from root and loss of water from leaf by transpiration. This may lead to dehydration and death

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Idea here is to cause the accumulation of solutes ( osmoprotectants ) which have property of providing stress tolerance to the plant. Osmoprotectants are the solute molecules that accumulate in cells and balance the osmotic difference between the cell's surroundings and the cytosol . As a result, water moves slowly from root to aerial parts like leaves. Different types of osmoprotectants used are Amino acids like proline Quaternary ammonium compounds like glycine betaine Polyols and sugars like mannitol .

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So, the strategy is to introduce the genes into the plants that code for the production of above osmoprotectants , which provide the ability to tolerate water deficit stresses. 1. Proline : D1-pyrroline-5-carboxylate synthase has an essential role in the synthesis of proline in the plant. When genes encoding for the above enzyme are introduced into the plant, result is enhanced production and accumulation of proline in the cells. Example : Gene P5CS from V.aconitifolia into tobacco cultivar xanthi .

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So, the strategy is to introduce the genes into the plants that code for the production of above osmoprotectants , which provide the ability to tolerate water deficit stresses. 2. Glycine betaine : This compound is prepared by two step oxidation of choline in plants. Enzymes involved in the above processes are choline monooxygenase and betaine aldehyde dehydrogenase . When genes encoding for these two enzymes and exogenous sufficient betaine aldehyde are introduced into the plant, result is enhanced production and accumulation of gylcine betaine in cells. Example : Transgenic rice plant

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So, the strategy is to introduce the genes into the plants that code for the production of above osmoprotectants , which provide the ability to tolerate water deficit stresses. 3. Mannitol : Genes encoding for the enzymes which have essential role in mannitol synthesis are introduced into the plant. Result is enhanced production and accumulation of mannitol in the cells Example : Bacterial gene for mannitol 1- phos phate dehydrogenase introduced into tobacco plants – resistance to salt stress .

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CAUSE FOR DISEASE DISEASE RESISTANCE GENE SOURCE OF GENE TRANSGENIC CROP glyphoshate, sulphonylurea herbicides leaf discoloration gene eroa and mutant als gene e.coli and arabidopsis tobacco bromoxynil herbicide leaf discoloration bxn gene klebsiella tomato manducta sexta eaten leaves bt2 toxin b.thuringiensis tobacco heliothis virescens eaten leaves cpti gene cow peas tobacco tmv light green coloration between veins coat protein gene tmv tobacco tswv brown spots and inward cupping of leaves n gene tswv tobacco ringspot virus chlorotic ring spots satellite rna from tobrv ringspot virus tobacco alternaria langipes brownspot chitimase gene soil bacterium tobacco phytopthora infestans late blight cosmotin gene potato potato

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REFFERENCES: RECENT PROGRESS IN MEDICINAL PLANTS. ENVIRONMENTAL BIOTECHNOLOGY. PLANT BIOTECHNOLOGY ( OXFORD PRESS ) www.youtube.com www.wikipedia.org WEBSITE OF UNIVERSITY OF NEBRAKSA.