Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures

In vitro shoot regeneration of gladiolus in three different culture systems, viz., semi-solid agar (AS), membrane raft (MR), and duroplast foam liquid (DF) cultures was evaluated following the kinetics of shoot multiplication and hyperhydricity at optimized growth regulator combinations. Compared to the AS system, matrix-supported liquid cultures enhanced shoot multiplication. The peak of shoot multiplication rate was attained at 18 days of incubation in the MR and DF systems, whereas the maximum rate in the AS system was attained at 21 days. An early decline in acceleration trend was observed in liquid cultures than the AS culture. The hyperhydric status of the regenerated shoots in the different culture systems was assessed in terms of stomatal attributes and antioxidative status. Stomatal behavior appeared to be normal in the AS and MR systems. However, structural anomaly of stomata such as large, round shaped guard cells with damage in bordering regions of stomatal pores was pronounced in the DF system along with a relatively higher K⁺ ion concentration than in the AS and MR systems. Antioxidative status of regenerated shoots was comparable in the AS and MR systems, while a higher incidence of oxidative damages of lipid membrane as evidenced from malondialdehyde and ascorbate content was observed in the DF system. Higher oxidative stress in the DF system was also apparent by elevated activities of superoxide dismutase, ascorbate peroxidase, and catalase. Among the three culture systems, liquid culture with MR resulted in maximum shoot multiplication with little or no symptoms of hyperhydricity. Shoots in the DF system were more prone to hyperhydricity than those in the AS and MR systems. The use of matrix support such as membrane raft as an interface between liquid medium and propagating tissue could be an effective means for rapid and efficient mass propagation with little or no symptoms of hyperhydricity.