Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 50 (2017)

Low polyphenol oxidase mutant induced by 12C6+ ion beam irradiation
to protoplasts of lettuce (Lactuca sativa L.)†R. Sawada,䠆1,䠆2 T. Hirano, 䠆3䠆4 K. Iimure,䠆1 T. Abe,䠆4 and Y. Ozaki䠆5Lettuce is one of the most important vegetables in Japanand has been established as a designated vegetable by theMinistry of Agriculture, Forestry and Fisheries. Lettuce isoften eaten raw in dishes such as salads, and the browningof the cut section is one of the causes of quality loss duringstorage. In the cut section, activated phenylalanine
ammonia-lyase (PAL) produces cinnamic acid, andpolyphenols are synthesized from the cinnamic acid. Thebrowning is due to the accumulation of pigments derived
from the oxidation of the polyphenols by polyphenoloxidase (PPO). Since PAL and PPO are key enzymes for thebrowning, the suppression of enzyme activity can preventthe browning.1) In the present study, the induction of PPO
gene mutation by heavy-ion beam irradiation was attempted
for the production of lettuce plants with a low browningproperty as a cut vegetable.
We used protoplasts as plant materials for the irradiation
to prevent of chimera mutants. Protoplasts of L. sativa‘Round’ were embedded at a density of 0.75 × 104/ml in
modified Murashige and Skoog medium3) supplemented
with 0.3% gellan gum and 100 mg/l cefotaxime sodium.
The protoplasts were stored overnight at 4°C in the dark.
Then, the protoplasts were transported at room temperatureand cultured at 25°C in the dark until irradiation. The
protoplasts were irradiated with 12C6+ ions (135MeV/nucleon, corresponding to 22.5 keV/µm linear energytransfer) at a dose of 0.5, 1.0, 2.0, and 5.0 Gy. The
irradiated protoplasts were cultured according to themethods described by Nishio et al. (Ref. 2). Twenty threedays after the irradiation, the number of colonies (> 0.1 mmin diameter) in each sample was counted, and colony
formation rates were calculated as percentages relative tovalues in unirradiated protoplasts. In the calli derived fromthe protoplasts, mutation in the PPO gene was detected by
the Targeting Induced Local Lesions IN Genomes(TILLING) method3) with Surveyor Mutation Detection kits
(Transgenomic, Inc).
The colony formation rates decreased with increasing
dose (Fig. 1); the colony formation rate was approximately
64% at 2.0 Gy and 26% at 5.0 Gy. A total of 869 calli
formed from the colonies were used for mutation detection
by TILLING, and two of them, 2Gy-3-36 from 2.0-Gy
irradiation and 5Gy-2-27 from 5.0-Gy irradiation, showed
positive fragments due to mutation in the PPO gene. The†Condensed from the article in Hort. Res. (Japan) 15, 347 (2016)
䠆1Kumamoto Prefectural Agricultural Research Center.
䠆2
Graduate School of Bioresource and Bioenvironmental Sciences,Kyushu University
䠆3
Faculty of Agriculture, University of Miyazaki.
䠆4
RIKEN Nishina Center
䠆5
Faculty of Agriculture, Kyushu University

mutation frequencies for 2.0-Gy and 5.0-Gy irradiation
were 0.41% and 1.10%, respectively.
We obtained regenerated M1 plants from the callus of2Gy-3-36, and their M2 progenies were produced byself-pollination. When PPO activity in the leaves wasmeasured according to the method of Dan et al. (Ref. 4)
with modification, the M1 plants show about half the PPO
activity of the control. In the M2 progenies, the segregationof individual genotypes was estimated by TILLING
analysis and PPO activity; homozygous dominant plants
showed no cleavage fragment in TILLING analysis andshowed similar or higher PPO activity compared with thecontrol, heterozygous plants showed the cleavage fragment
and similar or lower PPO activity, and homozygous
recessive plants showed no cleavage fragment andmarkedly lower PPO activity. These results suggest thatmutation in the PPO gene is cause of the PPO activity
decrease.
The reverse genetic approach combined with the carbon
ion beam has been thought to be effective in Arabidopsisthaliana.5) It is revealed in the present study that thisapproach is also useful in horticultural crops. The
homozygous recessive plants are promising as breeding
materials for low-browning lettuce.

Fig. 1 Effect of carbon ion irradiation on colony formation.
Data are represented as mean ± standard error of three
individual experiments.
References
1) H. Hashimoto et al., Biosci. Biotechnol. Biochem. 65, 1016
(2001).
2) T. Nishio et al., Japan. J. Breed. 38, 165 (1988).
3) B. J. Till et al., Genome Res. 13, 524 (2003).
4) K. Dan et al., Food Preser. Sci. 25, 209 (1999).
5) Y. Kazama et al., BMC Plant Biol. 11, 161 (2011).

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