Uptake and biochemical analysis of 2,4-D in cultured zygotic embryos of Zea mays L.

In order to determine the influence of 2,4-D on the regeneration of zygotic embryos of maize, the uptake and metabolism of 2,4-D by immature embryos were compared in an embryogenic inbred line (A188) and a non-embryogenic inbred line (A632), cultured on induction medium with 2 mg/L 2,4-D that was partly C-14-labelled. Uptake of 2,4-D under exhaustive conditions, i.e. without subculture, was analyzed from the onset of culture until 14 days of culture. During the so called shock response period, i.e. the first 24 h of culture, uptake of 2,4-D was observed in both lines, with a higher uptake in the embryos of A632. The availability of 2,4-D in the medium became a limiting factor for uptake from 3 days of culture onwards. Then the concentration of 2,4-D per gram fresh weight was up to 125 times higher than the concentration in the induction medium for both inbred lines. Differences in uptake between the two lines were observed until 5 days of culture. In this period a lower concentration of 2,4-D per gram fresh mass was found in A188. After 5 days of culture, however, the concentrations of 2,4-D per gram fresh mass were comparable in both lines. Therefore, the difference in embryogenicity is likely not caused by differences in uptake of 2,4-D. Supplementing TIBA to the induction medium under exhaustive conditions caused a drop in the uptake of 2,4-D in both inbred lines, but TIBA neither inhibited the uptake completely nor prevented the induction of callus. As killed embryos only showed a residual uptake, not influenced by TIBA, it is concluded that immature embryos of the two inbred lines do accumulate the greater part of the 2,4-D in an active manner. The C-14 labelled 2,4-D that had accumulated in cultured embryos was analyzed biochemically. Up to 70 % of the radioactive 2,4-D accumulated in A188 embryos was present as free 2,4-D after 24 h of culture, and 37 % in A632 embryos. Conjugation of 2,4-D to sugars and amino acids started after 16 h of culture. Metabolization of 2,4-D was observed from 2 h onwards and increased with ongoing culture. As compared with A188, A632 embryos showed a higher metabolization rate of 2,4-D. It: might well be that the higher levels of free 2,4-D in A188, together with the higher metabolization of 2,4-D in A632, do cause differences in the influence of 2,4-D on the 2 inbred lines, i.e. the different response on tissue culture. Thus, it is concluded that the developmental differences found in cultured immature embryos of embryogenic and non-embryogenic inbred lines might be caused by differences in levels of free 2,4-D and metabolization of 2,4-D. This could be regulated by genetic differences between the two inbred lines; however, other genetic factors might determine the sensitivity of the embryos to 2,4-D as well.