Agrobacterium-Mediated Genetic Transformation of Larix kaempferi (Lamb.) Carr. Embryogenic Cell Suspension Cultures and Expression Analysis of Exogenous Genes

Abstract

A simple and efficient protocol for the genetic transformation of Japanese larch (Larix kaempferi (Lamb.) Carr.) was developed by altering the infection method and duration and the bacterial removal process. More than 600 hygromycin-resistant embryonal masses with the vector pCAMBIA1301 were obtained, with an average of 20 transgenic lines per gram of fresh weight. Nine hygromycin-resistant transformation events (designated P1–P9) were analyzed using PCR, quantitative real-time PCR, and histochemical β-glucuronidase (GUS) assays. The GUS transcript abundance in each transformed cell line ranged from 101 to 103 magnitudes, with a maximum abundance of 2.89 × 103. In addition, the pLaTCTP::GUS vector, which contains GUS under the control of the L. kaempferi LaTCTP promoter, led to constitutive expression of GUS in embryonal-suspensor mass and somatic embryos. The transcript abundance of the exogenous genes HPT and GUS, driven by the CaMV 35S or LaTCTP promoter, ranged from 101 to 104, which was equivalent to genes with moderate and low abundances in Japanese larch. The relatively low expression levels of exogenous genes in transformants might reflect the large genome of Japanese larch. Additional transgenic cell lines need to be screened to obtain transformants with higher expression levels of foreign genes for further functional research in Japanese larch.