Low Polyphenol Oxidase Mutant Induced by ¹²C⁶⁺ Ion Beam Irradiation to Protoplasts of Lettuce (Lactuca sativa L.)

Abstract

The induction of polyphenol oxidase (PPO) gene mutation through ion beam irradiation of protoplasts was attempted for the production of lettuce plants with a low browning property as a cut vegetable. It was clarified that the optimum protoplast density was 0.63–0.75 × 10⁴ mL^–1 for shoot regeneration from protoplasts of lettuce ‘Round’. Effects of ¹²C⁶⁺ ion beam irradiation on the growth of protoplasts were investigated, and the percentage of colony formation decreased as the irradiation dose increased. The dose leading to 50% cell growth as compared with the untreated control was approximately 3.3 Gy. Two out of 869 calli irradiated with ¹²C⁶⁺ ion beams were found to be PPO gene mutations by the TILLING method. PPO activities of the regenerated plants from the two mutant calli named 2Gy-3-36 and 5Gy-2-27 were preliminarily assayed, and lettuce plants from 2Gy-3-36 exhibited half of the PPO activity as compared with the non-irradiated controls. A part of the M₂ population from 2Gy-2-36 exhibited the property of no band of heteroduplex cleavage by Cel I, with markedly low PPO activity as the control.