# An efficient and reproducible cryopreservation protocol for sustainable conservation of Jojoba (Simmondsia chinensis)

## TL;DR

This paper introduces a reliable cryopreservation method for jojoba, a valuable plant species, to help conserve its genetic resources for long-term use.

## Contribution

The study presents the first cryopreservation protocol for jojoba germplasm, enabling long-term conservation.

## Key findings

- A droplet vitrification-based protocol achieved up to 85.7% post-thaw survival in jojoba shoot tips.
- The protocol showed reproducible regrowth rates of 50-76.13% across eleven jojoba accessions.
- The method provides a technical platform for establishing cryobanks of jojoba genetic material.

## Abstract

Simmondsia chinensis, commonly known as jojoba, is an important renewable source of liquid wax esters valued for its unique seed oil that is used in several industries. Elite jojoba germplasm needs to be conserved owing to its overexploitation, climate variability and excessive dependence of selected cultivars. This study presents a straightforward and efficient droplet vitrification-based cryopreservation protocol in jojoba. Shoot tips isolated from eight-week-old cultures were cultured for two days on high sucrose (0.3 M) enriched medium and then treated with loading solution containing 0.4 M sucrose and 2 M glycerol, followed by PVS2 exposure for 30 min at room temperature. Vitrified shoot tips were then directly frozen in liquid nitrogen by placing them on aluminium foil strips. Frozen shoot tips were rewarmed in an unloading solution containing 1.2 M sucrose for 15 min, and then cultured on regeneration medium consisting of Murashige and Skoog medium (MS) supplemented with 4.97µM benzyl-amino purine (BAP) and 0.28 µM gibberellic acid (GA3). The protocol was optimized in one accession, where as high as 85.7% post-thaw survival and 76.1% regrowth were observed. The developed protocol was then tested for its efficacy and reproducibility on eleven other accession, and high post-thaw regrowth, ranging from 50 to 76.13% was observed. This study presents a broad spectrum, reproducible and efficient protocol for the conservation of jojoba genetic resources. This is the first report on cryopreservation of jojoba germplasm for its long-term conservation, providing a technical platform to set up cryobanks of valuable material of this important commercial crop.