Silver nitrate reduces hyperhydricity in shoots regenerated from the hypocotyl of snapdragon cv. Maryland Apple Blossom

Abstract

We developed an efficient protocol for shoot regeneration from hypocotyl explants in snapdragon (Antirrhinum majus L.) cv. Maryand Apple Blossom by plant growth regulators (PGRs), followed by ethylene synthesis inhibitor Aminoethoxyvinylglycine (AVG), ethylene precursor 1-Aminocyclopropane-1-carboxylate (ACC), and ethylene action inhibitor silver nitrate (AgNO3). The combination of benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) did not induce shoots, whereas naphthaleneacetic acid (NAA) and Zeatin (Zt) induced shoots. The combination of 0.05 mg/L NAA and 2.5 mg/L Zt resulted in the maximum shoot regeneration percentage and a reasonable number of shoots per explant. However, the regenerated shoots exhibited hyperhydricity and failed to develop into normal plants when introduced into plant growth media. Addition of ACC and AVG to the media containing 0.05 mg/L NAA and 2.5 mg/L Zt inhibited shoot regeneration, while AgNO3 (2.0 mg/L) marginally improved shoot regeneration and successfully reduced hyperhydricity. Shoots grown in AgNO3-containing media showed a reduction in ethylene related gene expression and reactive oxygen species (ROS) accumulation; the leaves showed reduced intercellular space as well as an increase in the number of open stomata under AgNO3 treatment. Moreover, shoots regenerated from AgNO3-containing media easily developed into normal plants in plant growth medium. This study highlights the importance of specific PGRs in shoot regeneration, as well as the role of AgNO3 in reducing the hyperhydricity of shoots regenerated in vitro. These findings will contribute to a better understanding of the hyperhydricity mechanism. We expect this protocol to be useful for in vitro shoot regeneration and genetic transformation of the snapdragon cultivar used in this study.