Influence of polyamines on hyperhydricity reversion and its associated mechanism during micropropagation of China pink ( L.).

Hyperhydricity (HH) is a physiological disorder that frequently occurs in plant tissue cultures, affecting healthy growth and development of clonal plants. The primary cultures raised in Murashige and Skoog (MS) medium supplemented with 2.5 µM N-benzyladenine (BA) produced normal microshoot (6.3 shoots/ culture) with least HH. However, the third subculture onwards, HH becomes a major problem. The role of ethylene on HH induction through stomatal closure mechanism were proved by the supplementation of ethephon (5 µM) in the culture medium containing 2.5 µM BA. In the present study, the application of polyamines (putrescine, spermidine, or spermine) to minimize the HH was examined. Supplementation of 5 µM spermine in MS medium significantly reduced the percentage of HH to 0.33%, in contrast to control (100%), while a maximum number of healthy reverted shoots (11.0) were observed in 5 µM spermidine treatment. The addition of polyamines effectively reduced HO content (50%) characterized by increased chlorophyll content with proper stomatal morphology. The relative gene expression profile of ethylene biosynthesis genes, 1-Aminocyclopropane-1-carboxylase synthase (ACS1) and 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO1) at 5 µM spermine added medium was 1.09 and 1.3 over normal (1) or HH cultures (1.93 and 2.53) respectively, and thus directed restoration of normal morphology of shoots. The present finding in brief, forward a novel method to regulate HH in terms of endogenous ethylene by adopting polyamines exposure and the procedure can be applied to many other plants facing similar HH problems.