Three second-instar S. littoralis larvae were attached in the second leaf of 14-day-old wheat seedlings by the clip cage and fed for 24 hours. After 24 hours, caterpillars were removed from the plants. Small leaf segments adjacent to the feeding sites were collected at several time points over a period of 7 days. After weighing, leaf segments were stored in the lysis tube containing ceramic beads and flash frozen with liquid nitrogen. The benzoxazinoid extraction buffer was added into the lysis tube (1 mg, 10 μl). Plant tissue was ground thoroughly by SpeedMill Plus (Analytik Jena, Germany) and then centrifuged at 13,200g for 20 min at 4°C twice to remove the pellet. Benzoxazinoids were analyzed as described above. To determine benzoxazinoid induction by aphids, five S. avenae adults were placed in cages (2-cm diameter) on the leaves of 14-day-old wheat plants. After 7 days, all aphids were removed. Aphid-infested leaf segments were collected from four to six different plants per line and analyzed as described above.

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