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Isolated leaves were incubated for 1 h in staining solution (1 vol. 2.5 mg ml−1 trypan blue in lactophenol, 2 vol. ethanol) after boiling. To detect cell death, stained material was incubated in chloral hydrate solution (2.5 g ml−1 chloral hydrate in water) for 3 days (ref. 45).
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