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TRANSGENIC PLANTS INTRODUCTION Culture of plant cells IN VITRO generates a considerable amount of genetic variation called somaclonal variation. Fusion of protoplast of selected plant species constitute somatic hybridization. Transfer of constructed genes through various transformation techniques constitutes GENETIC ENGINEERING. The plants obtained through genetic engineering contain a gene or genes from unrelated organisms. Such genes are called TRANSGENES and the plants containing transgenes are called as TRANGENIC PLANTS APPLICATIONS OF TRANSGENIC PLANTS 1. Valuable Tools in Studies 2. Resistance to Abiotic Stresses 3. Resistance to Biotic Stresses 4. Produce Quality 5. Production Of Biochemical 6. Expression of Antigenic Proteins VECTORS FOR PRODUCTION OF TRANSGENIC PLANTS • A Vector is a circular DNA molecule capable of independent existence and replication. • Eg. Plasmids, viruses • In case of Plants , Agrobacterium plasmids Ti and Ri are commonly used. • • • Both plasmids have a T-region which contains gene for opine metabolism and phytohormone independence This T- region is transferred into the plant cells and is integrated into their genomes Another region called vir region , produces an endonuclease essential for the excision transfer of the T-region into the plant cells • DNA viruses like caulimoviruses and gemini viruses are used for gene amplification. • The phenomenon of placing viral genome within T-DNA of Ti or Ri plasmid is called AGROINFECTION. PLASMID VECTORS 1) Plants cells do not have any endogenous plasmids. 2) Plant vectors used for cell transformation are based on :- pTi (tumour- inducing plasmid ) of A.tumefaciens pRi ( root-inducing plasmid ) of A.rhizogenes. A.tumefacians causes crown . A.rhizogenes causes hairy root. MOLECULAR BIOLOGY OF Agrobacterium INFECTION The process of infection by A.tumefaciens culminates in the transfer of a small part of pti into plant cell genome. This DNA sequence is called T-DNA (transferred DNA). The infection is governed by both chromosomal and plasmid borne genes. Firstly Agrobacterium cells attaches to plant cell. This step determines the host range of bacterium. PROPERTIES OF CROWNGALL CELLS Infection by A.rhizogenes give rise to “hairy roots”. These are capable of growing in culture on a growth regulator (GR) The crowngall and hairy root cells also synthesize unique nitrogenous compounds called opines (not used and produced by normal cell). Agrobacterium use opines as their carbon and nitrogen source. It is generally present in intercellular spaces of crowngall. Opine produced depend upon the bacterial strain. When pTi is introduced into Rhizobium trifolii , it gains the ability to produce galls and to utilize opine. THE Ti PLASMID It is a large conjugative plasmid or megaplasmid of about 200kb. pTi is lost when Agrobacterium is grown above 28 C such cured bacteria do not induce crowngalls i.e. become avirulent. The pTi and pRi are unique bacterial plasmids because :- 1) They contain some genes which have regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences.as a result, the former is expressed only in plant cells, while the latter ae expressed only in bacterium. 2) These plasmids naturally transfer their T-DNA into the host plant genome , which makes Agrobacterium a natural genetic engineer. DISARMING = The deletion of genes governing auxin and cytokine production (the oncogenes) from T-DNA of a Ti plasmid. FEATURES OF Ti PLASMID Ti plasmids are classified into different types based of opine produced. Opines can be octopine , nopaline , succinamopine and leucinopine. Basically they are of two types differing only in organization of their T-DNAs :- 1. Octopine type 2. Nopaline type Some functional regions of octopine and nopaline type pTi :I. Opine catabolism region II. T-DNA region III. vir region IV. Conjugative transfer region V. Origin of Replication TRANSFORMATION TECHNIQUES Uptake of foreign DNA or transgenes by plant cells is called transformation. Three broad categories are :- A. Agrobacterium mediated – Coculture with tissue explants & In Planta Transformation B. Agroinfection C. Direct gene transfer – Chemical Methods , Electroporation , Particle Gun Method , Lipofection , Microinjection , DNA Delivery via Growing Pollen Tubes ELECTROPORATION Introduction of DNA into cells by exposing them for very brief periods to high voltage electrical pulses , which induce transient pores in the plasma lemma. Its steps :- I. Plant protoplasts suspended in a suitable ionic solution containing linearized recombinant plasmid DNA. II. The electroporation mixture is then exposed to the chosen voltage- pulse combination for desired number of cycles. III. Protoplast are then cultured to obtain cell colonies and plants. IV. High voltage –short pulses give high rates of stable transformation. Example = tobacco , petunia , maize , rice , wheat PARTICLE GUN METHOD TRANSGENE ACTION Transgene expression can be exploited in several ways :- A. The transgene protein product itself may be a important biomolecule B. The transgene protein itself may generate a valuable phenotype C. The transgene product may trigger a pathway endogenous to the plant D. The transgene product may extend an existing pathway E. A transgene encoding the rate limiting enzyme F. The transgene may create a novel pathway G. The transgene may introduce a new branch in an endogenously pathway H. The transgene either by itself or through its RNA / protein product blocks a endogenous pathway HERBICIDE RESISITANCE Development of transgenic plants resistant to certain bio gradable herbicides - Major Achievement e.g. Glycophosphate , glufosinate , sulfonylureas GLYPHOSATE ACTION It is a broad spectrum herbicide that inhibits enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) Enzyme EPSPS involves in aromatic amino acid biosynthesis in plants. Thus the killing action of glyphosate results primarily from starving the cells of aromatic amino acid , which disrupts their protein synthesis. INSECT RESISTANCE Insect resistance transgenic plants contain either a gene from the bacterium B. thringiensis. It is used to control insect pests . CRY PROTEIN-: The cry gene of B. THURINGIENSIS produces a protein, which forms crystalline inclusion. Cry protein divides into – Cry1, Cry2 ,Cry3, Cry4. Cry 5, Cry6 are nematicidal in action. Cry protein – a parasporal inclusion protein from B. thringiensis that exhibit some verifiable toxic effect to a target organism. These protein are solubilized in alkaline environment of insect midgut. Cry I proteins are insecticidal to LEPIDOPTERANS. Cry II B & Cry IV is insecticidal to DIPTERA. Cry II A is active against both Lepidoptera and Diptera. Cry III protein are active against Coleoptera species. Cry V & Cry VI are active against Nematodes. TOXIC ACTION OF Cry PROTEINS Steps followed :- a) Cry protein ingested by insects , dissolves in alkaline juices present in midgut lumen. b) Gut proteases process them hydrolytically to release core toxic . c) Toxic fragments of Cry proteins :- d) Domain I = Pore and Ion Channel Formation e) Domain II = Receptor Recognition f) Domain III = Binds the Receptor g) The toxic fragments attaches themselves with brush border cells h) As a result , It develops pore , pertaining influx of water and ions i) Thus causes Swelling and eventual lysis. MODIFICATION OF STARCH QUALITY Starch , polymer of glucose – Chief Storage Polysaccharide (Plants) Strain grains contain amylose ( linear ) and amylopectin ( branched ). Properties :- Chain length Types of bonds linking the glucose units Degree of branching ENZYMES :- 1. AGPase 2. SSs 3. SBEs 4. SDBEs MODIFICATION OF STARCH BY GENETIC ENGINEERING ENZYMES FOR STARCH MODIFICATION GRANULE – BOUND STARCH SYNTHASE I ( GBSS I) :Main enzyme of amylose biosynthesis Increased activity of GBSS I increases amylose content , while suppression enhance amylopectin content. Suppression of GBSS I activity in Potato yielded an amylose free or “waxy” starch reported n 1991. SOLUBLE STARCH SYNTHASE (SSS) :Main enzyme of amylopectin biosynthesis It has two isoforms called SSS I and SSS II A freeze thaw stable potato starch ha s been created using antisense RNA technology. SYNERESIS is the separation of starch gel and water phases. STARCH BRANCHING ENZYME (SBE) :Required for amylopectin synthesis. Increased SBE activity expectedly increases branching. Its suppressions reduces the amount of amylopectins. MODIFICATION OF OIL QUALITY Oil Quality may be defined as types and proportions of different fatty acids present in given oil. Various Strategies used for modifying fatty acids :- Introduction of a novel enzyme or an acyl-ACP thioesterase , acyl – ACP desaturase Suppression of an enzyme activity Site directed mutagenesis to alter the specificity of an enzyme Creation of hybrid genes to generate novel enzyme activities EXAMPLE= Increased lauric acid content of B . napus MODIFICATION OF SEED PROTEIN QUALITY CEREAL proteins are deficient in LYSINE PULSES proteins are deficient in SULPHUR ( Methionine and tryptophan ) APPROACHES TO ACHIEVE THE OBJECTIVE :- o Introduction of an appropriate transgene o Modification of the endogenous protein encoding gene o Expressing a suitably designed synthetic gene o Separation of genes encoding a less desirable protein fraction GOLDEN RICE Vitamin A deficiency causes blindness among children and may even death . Rice grain does not contain provitamin A i.e. β- Carotene Genes transferred into rice by Agrobacterium mediated transformation Phytoene synthase Zeta- carotene desaturase Lycopene cyclase Condensation of geranyl-pyrophosphase -- Phytoene -- zeta carotene-Lycopene Cyclisation to form β carotene = Vitamin A Iron Deficiency = High Iron content Rice Phytate = Inhibit iron absorption , storage of phosphase in seeds ENZYME ACTIVITY FUNCTION GOLDEN RICE Phytoene synthase ( a plant gene ) Produce Phytoene by condensation of true molecules of geranyl-geranyl pyrophosphatase , the last precursor of β carotene biosynthesis found in rice endosperm * Phytoene denaturase Convert Phytoene into zeta- carotene ζ -carotene desaturase Lycopene β- cyclase ( a plant gene ) Convert zeta – carotene into lycopene Convert lycopene into β- carotene , the cyclic C40 carotene which is commonly known as provitamin A. ENZYME ACTIVITY FUNCTION HIGH IRON RICE Ferritin gene from Phaseolus A metallothionin – like gene from Oryza A mutant phytase gene from Aspergilus fumigatus encoding a thermostable enzyme. Endosperm iron content increase by a factor of 2.5 Endosperm cystein content increased 7fold; cystein enhances iron absorption in intestine. Hydrolyses Phytate to inorganic phosphate and myoinositol. The mutant enzyme refolds to 80% activity after 20 min at 100c, but the phytase expressed in rice, for some reason, failed to do so.
