Practice question

Effective recovery of transformed roots requires stringent chemical selection distinguishing transgenic from non-transformed background. Hygromycin B, aminoglycoside antibiotic, inhibits protein biosynthesis by stabilizing tRNA-ribosome interaction causing misreading. Plant cells are highly sensitive, with lethal concentration around 20 mg per liter for many dicots. Transformation vectors carry hpt gene under NOS promoter encoding hygromycin phosphotransferase that phosphorylates antibiotic hydroxyl group, preventing ribosome binding. Co-transformation with Ri T-DNA and binary vector results in roots expressing both rol genes and hpt, growing on hygromycin containing basal medium while non-transformed roots bleach. Kanamycin sometimes fails due to endogenous resistance in some species, making hygromycin selection more reliable in hairy root protocols for tobacco, tomato, and Withania. This system ensures high frequency recovery of genuinely transformed events for downstream metabolic studies. Killing curve determined by exposing untransformed roots to 0 to 100 mg per liter hygromycin for 14 days. Selection maintained for two subcultures to eliminate chimeric sectors. Molecular confirmation via amplification of hpt cassette confirms integration. Use of hygromycin avoids cross-resistance issues observed with kanamycin in species containing endogenous nptII-like activity, making it preferred for many dicots.