Somatic embryogenesis in artificially pollinated seed families of 2nd generation plus trees and cryopreservation of embryogenic tissue in Cryptomeria japonica D. Don (Sugi)

Abstract

Cryptomeria japonica D. Don (common name is Sugi or Japanese cedar) is the most important forestation tree species in Japan, and 2nd generation plus trees with superior traits have been selected by breeding projects. Biotechnological approaches such as genetic transformation and genome editing are expected to accelerate to add useful traits (e.g., no-pollen traits) to superior trees in short time. To develop a platform for genetic transformation and genome editing of C. japonica superior trees, this study investigated the embryogenic potential of 2nd generation plus trees and obtained good cell lines with high embryogenic potential, which could be useful material for adding new and useful traits to superior trees by genetic transformation. However, the maintenance of embryogenic cell lines is laborious, and prolonged subculture leads to a loss of embryogenesis potential. Therefore, cell lines need to be cryopreserved for long without subculture. Therefore, in this study we made a simple cryopreservation protocol suitable for most C. japonica cell lines. We showed that cryopreserved cells using this protocol formed somatic embryos, which were then converted to plantlets. Transgenic cells produced from cryopreserved cells expressed transgene, gfp. These results indicated that our cryopreservation protocol can be used for prolonged storage of genetic transformation target materials in C. japonica.