(a) How male sterility helps in heterosis breeding ? Discuss the application of barnase-barstar system in achieving heterosis…

(a)(i) Male Sterility in Heterosis Breeding

Male sterility—the inability of plants to produce viable functional pollen—serves as an indispensable tool in heterosis (hybrid vigour) breeding. In self-pollinated and bisexual crops, manual emasculation is tedious and economically unfeasible. Genetic Male Sterility (GMS) and Cytoplasmic-Nuclear Male Sterility (CMS) bypass manual emasculation, ensuring complete cross-pollination. In the classic three-line system, an A-line (male sterile), B-line (maintainer), and R-line (restorer carrying nuclear fertility restorer genes) facilitate hybrid seed production, utilized extensively in crops like pearl millet, sorghum, and hybrid rice (e.g., WA-CMS).

(a)(ii) Barnase-Barstar System in Heterosis Breeding

The Barnase-Barstar system, derived from the bacterium Bacillus amyloliquefaciens, provides a genetic engineering approach to overcome the limitations of CMS, such as cytoplasmic vulnerability and lack of natural restorer genes.

The male-sterile line is created by expressing the barnase gene (encoding a cytotoxic extracellular ribonuclease) under the strict control of a tapetum-specific promoter (such as TA29). During microsporogenesis, expression of barnase selectively lyses the nutritive tapetal cell layer, starving developing microspores and resulting in complete male sterility without affecting vegetative vigour.

To restore male fertility in the commercial F1 generation, the pollinator line is engineered with the barstar gene under the same TA29 promoter. Barstar acts as an intracellular inhibitor that binds to barnase with high affinity in a one-to-one stoichiometric complex, completely neutralizing its ribonuclease activity. In India, this technology was adopted in developing the transgenic hybrid mustard DMH-11 (by the Centre for Genetic Manipulation of Crop Plants, Delhi University) using a bar-barnase-barstar cassette to exploit heterosis between Indian and East European mustard lines. GEAC cleared its environmental release in October 2022, but a split verdict of the Supreme Court in July 2024 left the issue pending before a larger bench.

(b)(i) Steps in DNA Sequencing

DNA sequencing determines the exact order of nucleotides in a DNA molecule. In the classical Sanger chain-termination method, the process follows four key steps:

1. Template preparation and primer annealing: Target DNA is denatured into single strands, and a complementary oligonucleotide primer is annealed.
2. Enzymatic extension: DNA polymerase extends the primer in reaction mixtures containing standard deoxynucleotide triphosphates (dNTPs) along with limiting concentrations of four distinct fluorescently labelled dideoxynucleotide triphosphates (ddNTPs).
3. Chain termination: Because ddNTPs lack the 3'-OH group required for phosphodiester bond formation, their random incorporation terminates synthesis, generating fragments of variable lengths.
4. Electrophoresis and detection: Fragments are resolved by high-resolution capillary electrophoresis, where laser excitation of the fluorophores yields an electropherogram readout. Modern Next-Generation Sequencing (NGS) executes this massively in parallel via library preparation, clonal amplification (cluster generation), and sequencing-by-synthesis.

(b)(ii) Applications of DNA Sequencing Applications include whole-genome sequencing of crops (e.g., Indian initiatives in rice, chickpea, and pigeonpea), Marker-Assisted Selection (MAS) and QTL mapping, transcriptomics and functional genomics, forensic profiling, clinical diagnostics, and resolving phylogenetic lineages.

(c)(i) Meaning of Signal Transduction

Signal transduction is the biological cascade wherein an extracellular stimulus (hormone, ligand, physical stress) is recognized by a specific receptor, converted into an intracellular signal via secondary messengers and protein cascades, and ultimately translated into a cellular or genomic response.

(c)(ii) Types of Intracellular Signal Transduction Intracellular signal transduction proceeds through several major receptor mechanisms:

1. G-Protein Coupled Receptors (GPCRs): Ligand binding causes conformational shifts that activate heterotrimeric G-proteins, which trigger downstream effectors like adenylyl cyclase (producing cyclic AMP) or phospholipase C (yielding inositol 1,4,5-trisphosphate [IP₃] and diacylglycerol [DAG]), mobilizing cytosolic Ca²⁺.
2. Receptor Kinases and MAPK Cascades: Extracellular binding induces receptor dimerization and autophosphorylation (e.g., Receptor-Like Kinases in plants), activating mitogen-activated protein kinase cascades (MAPKKK → MAPKK → MAPK) that modulate target transcription factors.
3. Ion Channel-Linked and Intracellular Receptors: Ligand-gated channels alter membrane potential via ion fluxes, whereas lipophilic signals traverse the membrane to bind cytoplasmic or nuclear receptors acting directly as transcriptional regulators.

In plants, these pathways mediate phytohormone responses—such as Abscisic acid (ABA)-induced stomatal closure via PYR/PYL/RCAR receptors and Ca²⁺ flux, and Auxin signalling via TIR1/AFB ubiquitin-ligase degradation of Aux/IAA repressors—as well as Pathogen-Associated Molecular Pattern (PAMP)-triggered immunity (PTI) via surface pattern-recognition receptors.