Localization of GFP in protoplasts
Protoplasts were prepared according to Yoo et al . (2007), with minor modifications. The digitate shape of Medicago’s leaves and the high density in venation imposed to cut between venation. We were able to get a yield of about 2.106 protoplasts from 0.5 g of leaves. Transfection with a 35S:ERF75:GFP construct were observed with a LSM 880 – confocal microscope (Zeiss, Germany) using laser wavelengths excitation for GFP (Laser Argon 458, 488 et 514 nm, 25 mW) and for chlorophyll autofluorescence excitation (Laser He-Ne 633nm, 5 mW). The pAVA vector (von Arnim et al., 1998) was used as a 35S:GFP control. Images were analyzed with ZEN 2 software (Zeiss, Germany).