A New Method to Prepare Root Tip Chromosomes in Alfalfa

Abstract

We used root tips of annual Medicago disciformis DC. (2n=2x=16), perennial M. sativa L. ‘Lahontan’ (2n=4x=32), and plants of Lahontan that were regenerated from anther calluses to test various enzyme treatments for root tip chromosome preparation. These Medicago entries responded similarly to the types of enzymes and duration of treatment. Treatment with an enzyme solution (5.0% pectinase and 2.5% cellulase) for a duration of 60 to 90 minutes at 37°C gave the best results. The reliability of this treatment and the number of chromosome-countable cells was high. Distribution of cells and spreading of chromosomes in a cell on a focal plane was easily achieved. Treatment periods of less than 1 hour resulted in incomplete digestion. When the digestion time exceeded 90 minutes, many cells were too fragile to be processed. Either pectinase or cellulase alone failed to completely digest the roots, even after treatment for 5 hours. A solution comprised of 5% pectinase and 5% cellulase was so drastic that controlling the treatment time became very critical and difficult.