Modifying protein production in plants

A method for increasing yield of a heterologous protein in a Nicotiana plant per gram fresh leaf weight and per plant, the heterologous protein of interest comprising an influenza hemagglutinin (HA) protein, the method comprising:

• a) treating the plant to increase secondary leaf biomass, wherein the treating comprises cultivating the plant in the presence of 100 to 700 ppm of a cytokinin under growth conditions of 12 hours to 24 hours light per day and a light intensity of 60 μmol/m2·s to 350 μmol/m2·s to produce a treated plant;
• b) introducing one or more than one nucleic acid into the treated plant, the nucleic acid comprising a nucleotide sequence encoding the hemagglutinin (HA) protein, the nucleotide sequence operatively linked to a regulatory region that is active in the plant;
• c) incubating the treated plant under conditions that permit the expression of the nucleotide sequence encoding the hemagglutinin (HA) protein, thereby increasing yield of the hemagglutinin (HA) protein per gram fresh leaf weight and per plant compared with the yield of the hemagglutinin (HA) protein obtained from the same plant tissue of a similar plant that is grown under the same conditions, but that has not been treated to increase the secondary leaf biomass;
• and followed by
• d) harvesting one or more of S3 leaves S2 leaves and S1 leaves from the treated plant;
• wherein the step of treating, step a), is performed from 40 days prior to the step of introducing, step b), up to 9 days prior to the step of introducing, step b).