Creation of a <i>Pseudotsuga menziesii</i> nucellus cell suspension culture system to produce proteins involved in reproduction

Abstract Premise Cell suspension cultures of Douglas‐fir ( Pseudotsuga menziesii ) nucellus were created to overcome phenological limitations to the in situ study of bioactive proteins within and secreted by nucellar cells. Methods Proteins isolated from the cell suspension culture medium were analyzed using mass spectrometry, as were Douglas‐fir post‐pollination prefertilization drops and nucellus tissue excised from the ovule at the time of drop production. Results A total of 280 proteins ranging in size from 5 to 224 kDa were found in the three sample types. Identical proteins were found in the post‐pollination prefertilization drop and in the nucellar suspension culture medium or the nucellus. Secretory proteins isolated from the culture medium were either carbohydrate metabolism‐related or defense proteins, consistent with previous proteomic studies of pollination drops. Intracellular proteins were found in all three sample types and were consistent with previous proteomic studies of pollination drops, implying similar metabolic processes are at work in the gymnosperm nucellus at the time of drop production. Discussion Cell suspension cultures of Douglas‐fir nucellus are able to produce secretory proteins that are biologically relevant during pollination. This is potentially a tractable system that can overcome barriers to in situ studies presented by gymnosperm reproductive phenology.