# Liquid Overlay-Induced Donor Plant Vigor and Initial Ammonium-Free Regrowth Medium Are Critical to the Cryopreservation of Scrophularia kakudensis

## TL;DR

This paper presents a successful cryopreservation method for Scrophularia kakudensis shoot tips using specific in vitro culture and droplet-vitrification techniques.

## Contribution

The study introduces a systematic cryopreservation protocol with a novel two-step preculture and ammonium-free regrowth medium for Scrophularia kakudensis.

## Key findings

- A two-step preculture with sucrose improved cryopreservation outcomes.
- An ammonium-free regrowth medium was essential for normal plantlet development.
- Liquid overlay increased regeneration success by up to 80%.

## Abstract

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although the large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocols in wild flora is hampered by difficulties in vitro propagation and a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing an in vitro culture and droplet-vitrification cryopreservation procedure for shoot tips of Scrophularia kakudensis. The standard procedure includes a two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 30 min, cryoprotection with A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with growth regulators was essential for developing normal plantlets from cryopreserved shoot tips. Liquid overlay on the gelled medium two weeks after inoculation resulted in vigorous growth during subcultures. Moreover, liquid overlay increased LN regeneration by up to 80%, i.e., 23% higher than no liquid overlay.