The control of spontaneous lysis of protoplasts from Gossypium hirsutum anther callus

The isolation of cotton anther callus protoplasts is greatly enhanced when the amino acids arginine, serine or glycine, or the divalent cations Ca²⁺ or Mg²⁺ are included in the enzyme mixture. These compounds stabilize cotton protoplasts in the presence of RNase found in the cellulase enzyme mixture. The inhibition of RNase-induced lysis may involve cation or amino acid protection of critical membrane proteins during protoplast isolation. Using these protective agents, cotton protoplasts will give rise to macroscopic callus colonies after 3 weeks in culture.