Stress responses in alfalfa (Medicago sativa L.) IV. Expression of defense gene constructs in electroporated suspension cell protoplasts

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Abstract
We have investigated conditions for the uptake and expression of chimeric genes in protoplasts of alfalfa (Medicago sativa L.). Constructs containing the bacterial reporter gene chloramphenicol acetyltransferase (CAT) under the control of either the cauliflower mosaic virus 35S promoter or a bean chalcone synthase (CHS) promoter were introduced into protoplasts by electroporation in the presence of polyethyleneglycol. The extent of expression in the absence of added inducers depended on the conditions for isolation, electroporation and subsequent culture of the protoplasts. Expression of the CHS promoter construct was increased on exposure of the protoplasts to a fungal elicitor or reduced glutathione. The relative levels of induced expression in relation to either basal expression or the type of elicitor used depended on the age of the suspension cultures from which the protoplasts were isolated. Electroporation of protoplasts with a construct from which bean CHS antisense transcripts were synthesized under the control of the 35S promoter resulted in the inhibition of appearance of elicitor-induced endogenous alfalfa CHS activity. The suitability of the alfalfa protoplast system for analysis and potential identification of defense response genes is discussed.