(a) Describe various methods of gene transfer in plants. 20 (b) Give an account of Operon model for regulation of gene activity…

(a) Gene transfer methods in plants Methods are indirect (vector-mediated) or direct. Agrobacterium-mediated transfer: Agrobacterium tumefaciens carries a Ti plasmid. Wound phenolics (acetosyringone) activate the vir genes, which nick the T-DNA at its border repeats and move it with VirD2 into the plant nucleus, where it integrates. In disarmed binary vectors the tumour genes are removed and replaced by the gene of interest with a selectable marker. Leaf discs or explants are co-cultivated and regenerated on selection medium. It is efficient for dicots (tobacco, cotton, brinjal) and now for rice and wheat. Direct methods: biolistics (gene gun) fires DNA-coated gold or tungsten microprojectiles into cells and works on cereals and organelles (chloroplast transformation); electroporation applies brief high-voltage pulses to protoplasts to open transient pores; PEG-mediated uptake in protoplasts; and microinjection delivers DNA with a fine needle into single cells or embryos. Others include silicon carbide whiskers, sonication and floral dip (Arabidopsis). Applications: Bt cotton, Golden Rice, herbicide-tolerant soybean, and CRISPR/Cas9 genome-edited crops. Direct methods avoid host-range limits but give higher multi-copy insertion than Agrobacterium.

(b) Operon model Jacob and Monod (1961) proposed the operon for coordinated gene regulation in prokaryotes. An operon has a regulator gene (producing a repressor), a promoter, an operator, and linked structural genes transcribed as one polycistronic mRNA. Inducible lac operon of E. coli: the repressor (lacI) binds the operator and blocks RNA polymerase. Lactose, as allolactose, binds the repressor and releases the operator, so lacZ (β-galactosidase), lacY (permease) and lacA (transacetylase) are transcribed. High glucose lowers cAMP, so cAMP–CAP cannot activate the promoter (catabolite repression); the operon is fully on only with lactose present and glucose low. Repressible trp operon: the repressor is inactive alone; tryptophan acts as co-repressor, activating it to bind the operator and switch off the operon. Attenuation by the leader sequence adds finer control. Eukaryotes mainly differ: they lack operons and use enhancers, transcription factors and chromatin states.

(c) Cell signalling Cells communicate through signal molecules (ligands) that act on receptors, producing a response in three stages. Reception: a ligand (hormone, peptide, neurotransmitter) binds a specific receptor, either on the plasma membrane (G-protein-coupled receptors, receptor tyrosine kinases, ion channels) or inside the cell (steroid and nuclear receptors) for lipid-soluble signals. Transduction: the receptor changes shape and starts a cascade of relays. Protein kinases and phosphatases add or remove phosphates, and second messengers such as cAMP, cGMP, IP3, diacylglycerol and Ca2+ amplify the signal. Example: adrenaline binds a GPCR, G protein activates adenylyl cyclase, cAMP activates protein kinase A and glycogen breakdown follows. Response: gene expression, enzyme activity, cytoskeletal change, growth or death. Plants use similar logic: auxin binds TIR1 and releases Aux/IAA repressors; ethylene and brassinosteroids use receptor kinases. Signalling can be autocrine, paracrine or endocrine; defects cause diseases such as diabetes and cancer.