pekinensis (Lour) Olsson in Vitro

The promotive effect of AgNO3 and aminoethoxyvinylglycine (AVG) on in vitro shoot regeneration from cotyledons of Brasska campestris ssp. peklnensls in relation to endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, and ethylene production was investigated. AgNO3 enhanced ACC synthase activity and ACC accumulation, which reached a maximum after 3 to 7 days of culture. ACC accumulation was concomitant with increased emanation of ethylene which peaked after 14 days. In contrast, AVG was inhibitory to endogenous ACC synthase activity and reduced ACC and ethylene production. The promotive effect of AVG on shoot regeneration was reversed by 2-chloroethylphosphonic acid at 50 micromolar or higher concentrations, whereas explants grown on AgNO3 medium were less affected by 2-chloroethylphosphonic acid. The distinctive effect of AgNO3 and AVG on endogenous ACC synthase, ACC, and ethylene production and its possible mechanisms are discussed. Plants usually exhibit enhanced ethylene production when wounded or under environmental stresses or pathogen attack (31). Ethylene biosynthesis in plants occurs via SAM' and ACC intermediates using methionine as a precursor (31), but the molecular mechanism of its action is not clear. It has been known for some time that cultured plant cells evolve ethylene (1 1). However, the role of ethylene in plant cells and tissues grown in vitro is not well understood. In recent years there has been increasing evidence that the occurrence of morphogenesis in cultured plant cells may be associated with ethylene. The presence of ethylene was found to be important for embryogenesis from anther cultures of Hordeum vulgaris (6) and flower bud formation from thin-layer explants of Nicotiana tabacum (28). In contrast, shoot regeneration from callus cultures of Helianthus annuus (18) and N. tabacum (10) was inhibited by ethylene. The use of EIs, e.g. AgNO3, norbornadiene, and CoCl2, promoted shoot regeneration from callus cultures of Nicotiana plumbaginifolia and Triticum aestivum (23) and enhanced somatic embryogenesis of Daucus carota (25). In the genus Brassica, cells and tissues of B. campestris ' Abbreviations: SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-l-carboxylic acid; Els, ethylene inhibitors; AVG, aminoethoxyvinylglycine; CEPA, 2-chloroethylphosphonic acid. have been known to be recalcitrant in culture (17), although tissue culture systems for other species, e.g. Brassica alboglabra (19, 22), Brassica napus and Brassica oleracea (33) have been well developed. Recently, the use of EIs promoted embryo production from anther culture (2) and shoot differentiation from callus culture (26) of B. oleracea. We previously reported enhanced shoot organogenesis and plant regeneration from cultured cells and tissues of several recalcitrant genotypes in Cruciferae including B. campestris and Brassica juncea using AgNO3 and/or AVG (21). In this study, we extend our investigation and show the capacity of de novo shoot organogenesis from cotyledons of B. campestris ssp. pekinensis in vitro in relation to ethylene, with respect to the production of endogenous ethylene, ACC synthase activity, and ACC. MATERIALS AND METHODS Plant Tissue Culture Cotyledonary explants excised from 3-d-old aseptically germinated seedlings of Brassica campestris ssp. pekinensis cv Shantung and cv Wong Bok were used. Because our preliminary investigations showed that explants cultured in 50-mL Erlenmyer flasks and 100x 25-mm Petri dishes exhibited the similar capacity of shoot regeneration, both culture containers were used in this study. Explants were cultured either in a flask containing 25 mL medium or in a Petri dish containing 30 mL medium consisting of all constituents of Murashige and Skoog's medium (16) except the growth regulators which were replaced by 8.8 gM benzyladenine and 5.4 ,gM naphthaleneacetic acid (NlB2 medium), either in the presence or absence of 30 gM AgN03 or 5 ,AM AVG. The flask was sealed with a rubber serum stopper for ethylene measurement, and the Petri dish was sealed with two to three layers of Parafilm. The choice of the type and concentration of growth regulators and EIs for use in this study was based on their efficiency in promoting in vitro shoot differentiation of B. campestris in a previous study (5). For assays of endogenous ACC synthase activity and ACC, explants were grown for 3, 7, 10, and 14 d on medium with or without AgNO3 or AVG in a Petri dish, after which they were harvested, frozen in liquid nitrogen, and stored at -80'C until homogenization. The effect of exogenously supplied ethylene on shoot regeneration was investigated by culturing explants in a Petri dish containing NIB2 medium supplemented with 30 jAM AgNO3