The effects of catecholamines and ethylene on somatic embryogenesis from orchardgrass (Dactylis glomerata L.) leaf cultures

Catecholamines (CAs) are neurotransmitters in mammals and have been also detected in plants with no definite established function. They have been reported to decrease auxin catabolism and increase ethylene biosynthesis. Auxins and ethylene play important roles in somatic embryogenesis.

The objectives of this work were to (1) investigate the effect of the CAs epinephrine and norepinephrine on somatic embryogenesis and ethylene emanation from orchardgrass leaf cultures, (2) study the effect of stress ethylene produced by CAs in culture media, cold pretreatment and thigmomorphogenesis on in vitro cultured leaf segments, (3) make a vector construct carrying the bacterial gene encoding for 1-aminocyclopropane-1-carboxylic acid deaminase. (ACCdase), the β-glucuronidase (GUS) reporter gene (widA) and the selectable phosphinothricin acetyltransferase (bar) gene, and (4) produce transgenic plants expressing the ACCdase gene and therefore having reduced ethylene biosynthesis.

Epinephrine and norepinephrine at 10-100 µM stimulated somatic embryogenesis from leaf segments cultured on SH medium with 30 µ:M indole-3-acidic acid (IAA). Oxidation of [1-¹4;C]IAA included in the medium was reduced threefold by 10 µM epinephrine. Ethylene evolution was increased at epinephrine concentrations >25 µM. CAs enhanced somatic embryogenesis from leaves cultured on SH medium with 30 µM of the synthetic auxin dicamba. Ethylene was drastically increased at >500 µM epinephrine whereas somatic embryogenesis was decreased.

Ethylene emanation was decreased during 1-7 d cold (4°C) pretreatment of leaf cultures and was stimulated after transfer of the cultures to 21°C. Somatic embryogenesis also increased after transfer to 21°C. Mesh application on leaf segments, significantly increased ethylene accumulation in the petri dish headspaces. Ethylene biosynthesis inhibition with aminooxyacetic acid and cobalt did not increase somatic embryogenesis. Inhibition of ethylene action. by silver thiosulfate at 5 µM increased somatic embryogenesis 1.8fold.

To produce transgenic plants with reduced ethylene biosynthesis, the bacterial gene ACCdase was subcloned into PAHC25,a plasmid containing the bar gene for selection on medium containing bialaphos and the uidA gene encoding GUS histochemical assay. Leaf segments were bombarded with plasmid coated tungsten particles by a particle inflow gun. Transient GUSexpression was recorded 2 d after bombardment. Embryos showed transient GUS expression but were not uniformly stained. Putative Transgenic plants were obtained for reduced ethylene biosynthesis.