# Induction of Embryogenic Callus, Protoplast Isolation, and PEG-Mediated Transformation Protocols in Eucommia ulmoides

## TL;DR

This study develops a reliable method for genetic transformation in Eucommia ulmoides, a Chinese tree species, using embryogenic callus and protoplasts for gene research and breeding.

## Contribution

The paper introduces a stable and efficient protoplast isolation and PEG-mediated transformation system for E. ulmoides.

## Key findings

- The optimal medium for embryogenic callus induction achieved a 97.50% callus induction rate and 86.30% embryogenic callus formation.
- Protoplast isolation yielded 1.82 × 10⁶ protoplasts/g FW with 91.13% viability under specific enzymatic and osmotic conditions.
- The highest transfection efficiency (53.23%) was achieved using 10 µg of plasmid and 40% PEG4000 for 20 minutes.

## Abstract

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, the optimal conditions for callus induction were identified, and formation of the embryogenic callus was confirmed by histological analysis. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using suitable embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5 + 1.5 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose + 7 g/L agar (pH = 5.8). In this medium, the induction rate of callus achieved 97.50%, and the rate of embryogenic callus formation was 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82 × 106 protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridisation and transient gene expression in this species.