Role of biosynthesized silver nanoparticles in mitigating hyperhydricity in micropropagated watermelon (Citrullus lanatus Thunb.) and assessment of genetic fidelity using RAPD and SCoT markers

Abstract

Watermelon (Citrullus lanatus) is one of the five most consumed fresh fruits and is a commercially important horticulture crop. The consumer demand for seedless watermelons has considerably increased due to their superior taste and ease of consumption. Seedless watermelon plants are triploids hence plant tissue culture technology which is the cultivation of plant cells, tissues, or organs on specially formulated nutrient media can facilitate the rapid multiplication of seedless watermelon varieties. However, hyperhydricity has been reported as a frequent problem for in vitro growth and development of watermelon. Hence, the present study highlights the application of silver nanoparticles biosynthesized from Parthenium hysterophorus (PHAgNPs) for reversal of hyperhydricity and enhancing in vitro shoot production in watermelon. The biosynthesized PHAgNPs was characterized using UV–vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis and Transmission electron microscopy. Cotyledonary nodes used as explant and cultured in three types of cytokinins (BAP, TDZ and KIN) became hyperhydric, losing their ability to regenerate at high cytokinin concentrations (> 1.5 mg/L) irrespective of the cytokinin type. The optimum concentration of BAP (1.0 mg/L) fortified with PHAgNPs (15.0 mg/L) exhibited an improved response of 91 % and induced a maximum of 32.33 shoots with a maximum shoot length of 5.66 cm after 6 weeks. The incorporation of PHAgNPs reduced the percentage of hyperhydricity from 29 % to 1.33 % in regenerated shoots, which was confirmed by the reduction in relative water content (RWC) from 93.33 % to 80.33. Silver ions reduced H2O2 content by 50 % and increased photosynthetic pigments (chlorophyll a, b and carotenoids) by 50 %, which contributed to the formation of healthy regenerated shoots, with opened stomata compared to closed in hyperhydric shoots. Compared with those of hyperhydric shoots, the gene expression patterns of 1-aminocyclopropane-1-carboxylase synthase (ACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1) decreased after the retroversion of shoots to 15 mg/L PHAgNP medium. The relative gene expression profiles of ACS1 and ACO1 in hyperhydric shoots were 8.8- and 1.8-fold greater than those in normal shoots, respectively. Profuse root growth was observed in MS basal media supplemented with IBA (1.0 mg/L) and PHAgNPs (10.0 mg/L), which resulted in the greatest number of roots (12.33), with an average root length of 4.33 cm and a response of 89.33 %. The genotoxic effect of PHAgNPs evaluated using RAPD and SCoT molecular markers, showed that the addition of low levels of silver nanoparticles to the medium did not cause any genetic variation. The efficient regeneration protocol standardized in this study provides a solution to solve the problem of hyperhydricity in watermelon regeneration.