Establishment of a transient transformation system for embryogenic callus tissue of Pinus koraiensis

HE Xin, XU Xiuyue, KANG Yimin, YANG Ling

Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 2026, Vol. 50 ›› Issue (1) : 12-22.

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南京林业大学学报（自然科学版）
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Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 2026, Vol. 50 ›› Issue (1) : 12-22. DOI: 10.12302/j.issn.1000-2006.202501010

Establishment of a transient transformation system for embryogenic callus tissue of Pinus koraiensis

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Abstract

【Objective】 This study aims to optimize a transient transformation system for Pinus koraiensis embryogenic callus mediated by Agrobacterium tumefaciens. 【Method】Using the ten-day cultured embryonic callus of P. koraiensis as the receptor material, transient transformation was carried out with the pCambia1300 vector carrying RUBY. The optimal concentration of infection solution, infection time, co-cultivation time, and recovery culture time were screened based on the expression level of RUBY. The vectors promoterPkDof5.6-EGFP, promoterPkSHR-RUBY, 35S-PkDof5.6-EGFP, 35S-PkSHR-EGFP, and 35S-PkSHR-RUBY were respectively transfected into the embryonic callus of P. koraiensis to verify the conditions of the transient transformation system.【Result】After transient transformation, RUBY expression was significant. When the concentration of the inoculum (A600) reached 0.4, with an infection time of ten minutes and co-cultivation for one day without recovery culture period, the copy number of the RUBY fragment was at its highest. Genes and promoters specific validation of the quantitative and semi-quantitative results of transient transformation indicates that this condition can achieve efficient transformation of mature callus from P. koraiensis. 【Conclusion】This study successfully established a simple, rapid, efficient and low-cost Agrobacterium-mediated transient transformation system for embryogenic callus of P. koraiensis, and optimized the main factors of this system. The best transformation conditions were found to be: an infection bacterial suspension concentration (A600) of 0.4, infection for ten minutes, co-cultivation for one day, with no recovery culture period. This system is of great significance for large-scale gene functional analysis of the somatic embryogenesis mechanism in P. koraiensis and accelerates the breeding and large-scale propagation of high-quality P. koraiensis varieties.

Key words

Pinus koraiensis / transient transformation / somatic embryogenesis / molecular breeding

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HE Xin , XU Xiuyue , KANG Yimin , et al. Establishment of a transient transformation system for embryogenic callus tissue of Pinus koraiensis[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 2026, 50(1): 12-22 https://doi.org/10.12302/j.issn.1000-2006.202501010

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