Cell suspensions for kinetic studies of inhibitor action

Because of uniformity and small distances for transport, cell suspensions offer a system for rapid measurements of initial reactions of phytotoxic compounds. We had previously shown that a growth regulator, dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-L-gulonate) inhibits amino acid incorporation into proteins. Using Solanum nigrum suspension cultures, it was found that dikegulac rapidly inhibits amino acid uptake into cells, before inhibiting incorporation, with time points starting at a few minutes, and kinetics that can be extrapolated back to time zero. With more rapid kinetics this compound induces leakage of a preloaded dye. The rate of leakage was less with stationary cells in suspension, reiterating that they are more resistant to the effects of this compound. It was thus concluded that at the concentrations used, the first effect of dikegulac (or one very close to the first effect) is on the cell membrane.Abbreviation FDA fluorescein diacetate     

Cell cultures vs. whole plants for measuring phytotoxicity II. Correlations between phytotoxicity in seedlings and calli

The effects of a broad range of compounds were assessed usingboth seedlings and callus cultures of five species. An experimentallyjustified ranking procedure was used to facilitate the comparisonof the inhibitory characteristics at multiple concentrations.When the effects on seedlings of Lycopersicon esculentum, Solanumnigrum, Chrysanthemum segetum and Cirsium arvense were comparedto those on white calli of the same species, some positive correlationswere obtained; photosynthesis inhibitors affected seedlingsmuch more than calli and some compounds affected calli muchmore than seedlings. Better correlations were obtained betweenthe effects on Rumex obtusifolius seedlings and on green Rumexcalli; the only exceptions being those compounds affecting mainlythe calli. Thus callus cultures, especially green ones, havea potential for use to assess possible phytotoxicity as wellas to detect potential toxicity of compounds not penetratinginto or being translocated in whole plants. (Received January 13, 1977; )     

Tissue culture of the alternate hosts of wheat,rye and oat rusts and their response to in vitro inoculation with the rust pathogens

Alternate host plants of cereal rust fungi are necessary for studying the rust sexual cycle and pathogenicity. These plants are usually difficult to propagate through cloning, while seed-propagated plants may have variable responses to the pathogen. To overcome these obstacles, tissue culture, under controlled and aseptic conditions, was utilized for clonal propagation and in vitro inoculation of the following species: Rhamnus palaestinus Boiss., the alternate host of oat (Avena spp.) crown rust (Puccinia coronata Corda); Thalictrum speciosissimum L., the alternate host of brown leaf rust of wheat (Puccinia recondita f. sp. tritici Eriks. & Henn.); and Lycopsis arvensis L., the alternate host of rye (Secala spp.) leaf rust (Puccinia recondita f. sp. recondita Rob. & Desm.). Shoot culture procedures for initial establishment and proliferation were developed for all three alternate host species. Shoot cultures were multiplied at rates ranging from 0.3 to 1.7 shoots/week. Successful infection following inoculation with teliospores of the corresponding rust fungi was obtained for R. palaestinus and T. speciosissimum but not for L. arvensis. The hardening and acclimatization efficiency of rooted T. speciosissimum and L. arvensis was of 80–90%. The propagation efficiency for R. palaestinus was not successful because of the low rate and poor quality of its rooting. It is concluded that the in vitro system might be used as an alternative method for inoculation and multiplication of alternate hosts of cereal rusts, although more experimentation is needed to define accurately the appropriate conditions for the proper infection response. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of the plant cell inhibitor propachlor (alpha-chloro-N-isopropyl-acetanilide) on the cell cycle of L1210 cells as evaluated by flow cytometry

Previously it was reported that the herbicide propachlor (alpha-chloro-N-isopropyl-acetanilide) has a strong inhibitory effect on the proliferation of L1210 mouse leukemia cells. It is now demonstrated that propachlor treatment causes L1210 cells to accumulate in the G1 phase as determined by flow cytometric analysis. This effect of propachlor is dose-dependent with more than 90% of G1 cells accumulating at 10 microM. Kinetic experiments demonstrated that the accumulation of cells in G1 starts in about 10 hours, and increased for up to about 44 hours of incubation with 10 microM propachlor. Treated cells can be revised to a normal DNA distribution by removing propachlor.     

Differential inhibition by dikegulac of dividing and stationary cells in in vitro cultures

Dikegulac sodium 2,3:4,6 di-O-isopropylidene-2-keto-L-gulonate) is a translocatable agent used for chemically pinching terminal buds to break apical dominance. It does not inhibit mature leaves. Cell culture systems were developed and used to ascertain whether or not it differentially inhibits green tissue and whether or not it differentially inhibits stationary cells at the concentrations used. A differential inhibition of dividing cells was found.Abbreviation FDA fluorescein diacetate     

Cell cultures vs. whole plants for measuring phytotoxicity III. Correlations between phytotoxicities in cell suspension cultures, calli and seedlings

The effects of a range of compounds were assessed using cellsuspension cultures of three species. The results were comparedwith previous data on seedlings and calli of the same species.The effects on seedlings of Lycopersicon esculentum, Solanumnigrum and Cirsium arvense were compared with those on cellcultures of the same species. There was a significant correlationbetween the suspension cultures and the calli from which theywere derived. When the results for seedlings were compared withthose for suspension cultures, the major deviations from positivecorrelations were compounds inhibiting the cells more than theydid the whole plants. Thus cell suspension cultures can be usedto assess possible phytotoxicity as well as to detect potentialtoxicity of compounds not penetrating into or being translocatedin whole plants. They are most amenable for use in rapid toxicitytests and metabolic studies. (Received February 3, 1977; )     

Cell cultures vs. whole plants for measuring phytotoxicity I. The establishment and growth of callus and suspension cultures; definition of factors affecting toxicity on calli

Callus tissue has been isolated from 11 species, of 8 whichare ‘weeds’ from which callus isolation has notbeen previously described. Continuous suspension cultures offour species have been started. Growth conditions were optimizedfor high yield. Logarithmic phase and stationary phase cellsreact differently to some inhibitors. The larger the inoculumsize, the greater the concentration of inhibitor that must beused to achieve equal inhibition. Hormonal constitution of themedium affects inhibition by ‘auxin’ type herbicides.Growth kinetics, delayed response and recovery must be consideredin assessing phytotoxicity. Highly uniform and controlled proceduresare required for interspecific comparisons of many compounds. (Received January 7, 1977; )     

Multistage disruption of protoplasts by dikegulac

Dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-I-gulonate) is a growth regulator used to differentially kill terminal apices, and it analogously inhibits basic metabolic functions in dividing cells, but not stationary cells, in suspension culture. This report demonstrates an analogous situation in isolated tobacco protoplasts. At the lowest concentrations, dikegulac partially suppresses division of the protoplasts. Higher concentrations are required to produce visual cytoplasmic damage to the protoplasts, which probably first occurs at the level of the plasmalemma, as the vacuoles can be released intact. Later, tonoplast disruption occurs.Abbreviation FDA fluorescein diacetate