The impact of sucrose and 6-benzylaminopurine on shoot propagation and vitrification in Aroniamelanocarpa (black chokeberry)

Abstract

Vitrification is one of the most significant issues encountered in plant tissue culture applications. It diminishes the quality of in vitro plants, causing their leaves and stems to appear watery and translucent, and it may impede the success of the acclimatization step. In this respect, this study investigates the impact of sucrose and 6-Benzylaminopurine (BAP) concentrations on shoot regeneration and vitrification development in Aronia melanocarpa, known for its high antioxidant content and health benefits. Initially, the presence of BAP, in combination with varying sucrose concentrations, leads to a substantial increase in shoot number, and the largest number (7 shoots per nodal explant) was observed in the Murashige and Skoog (MS) medium containing 3% sucrose and 5.0 mg/L BAP. Furthermore, sucrose concentration plays a crucial role in shoot growth, with higher concentrations promoting more extensive shoot development. However, when 3% sucrose was combined with higher BAP (from 1.0- to 5.0 mg/L), an increased incidence of vitrification was observed over time. Interestingly, lower sucrose concentrations (1% or 2%) combined with 0.5 mg/L or 2.5 mg/L BAP initially delayed vitrification but eventually led to its occurrence. Microscopic analysis of leaf samples with varying levels of vitrification indicates significant differences in the density of stomata, further confirming the detrimental impact of vitrification on cellular structures and physiological processes. The recovery of vitrified plants was evaluated using different growth media combinations. The absence of BAP in the medium led to higher recovery percentages (min 96%) without necrosis, while the addition of 0.5 mg/L BAP promoted shoot growth but potentially inhibited root development. It has been found that media with 1 mg/L BAP and either 10 g/L or 20 g/L sucrose, as well as media with 30 g/L sucrose and 0.5 mg/L BAP, are the most suitable for efficient shoot regeneration with minimal vitrification risk. However, increasing BAP levels for faster shoot regeneration also raises the risk of vitrification. During acclimatization, vitrified plants exhibited stunted shoot growth, shorter and narrower leaves, reduced root numbers and lengths, and decreased survival rates, particularly under lower humidity conditions. The cultivation period required for the recovery of the crop was determined to be 6 weeks under greenhouse conditions for a sustainable plant propagation.