# A Cryopreservation and Regeneration Protocol for Embryogenic Callus of Larix olgensis

## TL;DR

This paper presents a successful method to cryopreserve and regenerate embryogenic callus in Larix olgensis, preserving its ability to grow into new plants.

## Contribution

A novel and effective cryopreservation protocol for L. olgensis embryogenic callus that maintains embryogenic potential and regenerative capacity.

## Key findings

- A 24-hour preculture with specific sucrose concentrations and cryoprotectants achieved a 100% recovery rate of cryopreserved embryogenic callus.
- Cryopreserved callus successfully underwent somatic embryogenesis, germination, and rooting.
- The protocol is suitable for long-term storage as cryopreservation duration did not affect cell viability or proliferation.

## Abstract

Larix olgensis is a valuable timber species in northern China, typically propagated through somatic embryogenesis (SE). However, long-term subculture can lead to a loss of embryogenic potential. This study aimed to establish a simple and stable protocol for the cryopreservation and regeneration of L. olgensis embryogenic callus (EC) that preserves its SE potential and regenerative capacity. The slow-freezing method was employed for cryopreservation. A cryopreservation protocol for L. olgensis EC was developed by optimizing the preculture duration and conditions, cryoprotectant composition and thawing temperature. The results showed that optimal outcomes were achieved using a 24 h stepwise preculture on medium containing 0.2 and 0.4 mol∙L−1 sucrose, followed by cryoprotectant treatment with 0.4 mol∙L−1 sucrose, 2.5% (v/v) dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 6000 (PEG6000), and thawing at 37 °C. EC cryopreserved using this protocol achieved a 100% recovery rate. Moreover, the cryopreserved recoverable EC successfully underwent SE, progressing through germination and rooting. Cryopreservation duration (storage duration in liquid nitrogen) did not affect cell viability and proliferation rate, confirming the protocol’s suitability for long-term cryopreservation of L. olgensis EC. This study provides a valuable reference for the cryopreservation and regeneration of L. olgensis EC, with potential applications for other coniferous species. It establishes a robust foundation for the large-scale propagation of conifers.