Mist culture for mass harvesting of root border cells: aluminum effects

Root border cells, which form a cell layer around the root tip, seem to play multiple roles in the rhizosphere of the apical root. As these cells (species-dependent dozens to several thousand per root tip) are rapidly sloughed off in water, studies in hydroponic culture fail to elucidate their role in most conventional physiological studies. The common method for harvesting these cells consists in germination of seeds in a humid atmosphere (usually a Petri dish), but labor and time constraints allow to yield only very limited amounts of uniform cells. We thus developed a low-cost mist-culture method, where intact border cells can be collected in the range of several grams. We applied this technique in a preliminary experiment where the influence of aluminum (Al) supply on calcium (Ca) release and viability of this cell type was studied. Purified detached border cells of pea were incubated with 0, 50, and 500 mmol m(-3) AlCl3 solution (pH 4.5) for 90 min at a ratio of 3 x 10(5) cells (4 mL)(-1). After incubation, cells contained 4.27 and 13.28 mg Al g(-1) C at 50 mmol m(-3) and 500 mmol m(-3) AlCl3, respectively, while their total Ca content decreased correspondingly. Cell viability of border cells as tested by fluorescein diacetate-propidium iodide (FDA-PI) fluorescence yielded unexpected results: the test exhibited significantly lower vitality at 50, but not at 500 mmol m(-3) AlCl3. Assessing mitochondrial activity by 3-(4,5)-dimethylthiazol-2-yl-2,5 diphenyl-tetrazolium-bromide (MTT) reduction showed that viability decreased in a dose-dependent manner with increasing Al concentrations. This apparent contradiction is attributed to the formation of dense mucilage around border cells at high Al concentrations, which likely inhibits the access of the dye PI or may chemically inactivate this compound and thus wrongly suggest higher viability. Mist culture allows harvesting selectively large amounts of homogeneous border cells quickly and to study their physiological reactions separated from the root tip.