A Fruit-Pulp-Derived Callus-Level Agrobacterium-Mediated Transformation Platform for Ziziphus jujuba

The jujube (Ziziphus jujuba Mill.) is a significant economic fruit tree, valued for its nutritional and medicinal properties. However, advances in functional genomics are hindered by the lack of an efficient transformation system. To overcome the limitations of conventional explant, we established a fruit-pulp-derived, callus-based Agrobacterium-mediated transformation system using fruit-pulp harvested 50 days after pollination. Through orthogonal experimental design, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid were identified as key regulators for inducing high-quality, friable callus in two jujube genotypes, 'JZ60' and 'LWCZ'. This system revealed significant genotype-specific variation in auxin requirements for callus proliferation and in differential antibiotic sensitivity. Transformation efficiency, as evaluated by fluorescence screening, was primarily determined by acetosyringone concentration and the binary vector architecture. The results revealed that the compact pCY (kanamycin resistance) vector achieved higher transformation efficiency (up to 77.8%) than pCAMBIA1301, whereas the pCAMBIA1301 (hygromycin resistance) vector enabled more uniform transgene expression. Integration and expression of the ZjCBF3 transgene were confirmed by polymerase chain reaction (PCR), reverse transcription quantitative PCR, and green fluorescent protein fluorescence assays. This study established a fruit-pulp-based callus transformation system for jujube, providing a rapid platform for its functional genomic studies.