Quantitation of gibberellins and the metabolism of [(3)H]gibberellin A 1 during somatic embryogenesis in carrot and anise cell cultures

Abstract

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [(3)H]GA1 was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2-0.3 μg g(-1) dry weight) of polar GA (e.g. GA1-like), but much (3-22 times) higher levels of less-polar GA (GA4/7-like); GA1, GA4 and GA7 are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9-4.4 μg g(-1)), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [(3)H]GA1 very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [(3)H]GA1 slowly. The major metabolites of [(3)H]GA1 in anise were tentatively identified as GA8-glucoside (24%), GA8 (15%), GA1-glucoside (8%) and the Δ(1(10))GA1-counterpart (2%). Thus, high levels of a GA1-like substance and a reduced ability to metabolize GA1 are correlated with the absence of embryo development, while lowered levels of GA1-like substance and a rapid metabolism of GA1 into GA8 and GA-conjugates are correlated with continued embryo development. Exogenous application of GA3 is known to reduce somatic embryogenesis in carrot cultures; GA4 was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.