GENERATION OF A SUSTAINABLE PINUS RADIATA CELL SUSPENSION CULTURE AND STUDIES OF CELLULAR NITROGEN NUTRITION

A Pinus radiata D. Don cell suspension culture was developed from excised embryos using a Schenk and Hildebrandt medium modified by addition of 2.6 mM ammonium phosphate and 10 mM arginine hydrochloride. Initiation of the culture involved selection of initially rare meristematic cells from the dominant mass of differentiating cells with limited mitotic capacity. The chromosomally normal culture was found to be capable of long-term culture and amenable to quantitative growth experiments using dry-weight yields of suspension-cultured cells. Responses to nitrogen nutrients provided growth contour plots illustrating the interaction between ammonium and nitrate nutrients, with optimal growth in the vicinity of 3mM ammonium and 15 mM nitrate ions. Arginine was able to replace ammonia entirely, nitrate was necessary for good growth. Glutamine and asparagine were also growth effective, whereas little benefit was found with lysine, ornithine, or glutamate, and urea yielded an intermediate response. Higher levels of lysine (3mM), glutamate (10 mM), or casein hydrolysate (lOmM amino acids) resulted in growth reduction. The effectiveness of beneficial organic nitrogen supplements is ascribed to endogenous supply of reduced nitrogen for general cellular bio-synthesis, and such supplements are considered unnecessary when inorganic nitrogen is optimised