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).