When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of ,B-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of ,B-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1 -naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating 0-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced ,B-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 x 10-5 molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco ,Bglucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.

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Culture conditions, allowing the unlimited growth and maintenance in shaken suspensions of a kinetin dependent strain of tobacco pith cells, were determined. Cell clusters from 1 to 25 cells were selected from the cultures to study the cell multiplication after addition of specific growth factors to the basal medium. Cell division was found to be strictly dependent upon the presence in the medium of both kinetin and auxin. In complete medium the generation period was 45 to 49 hours. Kinetics of the total protein synthesis in the cell suspensions have been measured either by estimation of protein nitrogen or by 35S incorporation into the proteins. After 3 days of culture, growth was exponential, as expressed by cell number or protein synthesis, which varied proportionally. Evidence was also found that the initial incorporation rate of radioactivity into the proteins was more rapid than expected from the increment of the protein net weight. It seems therefore reasonable to assume that a significant amount of protein turn over does exist during the initial period of growth. This phenomenon was observed even when no cell division occurred. Kinetin significantly activated protein synthesis, whether or not auxin was present in the medium.

In conventional culture conditions without auxin, somatic embryos arising from suspension cultures of grapevine rootstock 41B (Vitis vinifera cv. Chasselas x Vitis berlandieri) are arrested at the heart stage of development. Starting from indications that inhibitors excreted in the culture medium could be responsible for this arrest, new culture conditions based on daily subculturing embryos in fresh medium have been successfully used to obtain full embryo development. From this technique, a microassay was devised for screening small amounts of extracellular molecules as potential inhibitors of embryonic development. Our results show that extracellular macromolecules of molecular weight higher than 10 kDa are likely involved in the inhibition of caulinary meristem initiation. However, other factors obviously cooperate to inhibit embryo development in conventional culture conditions.Abbreviations: CH76-cv. Chardonnay clone 76 (Vitis vinifera), NOA-2-naphthoxyacetic acid