Protoplast isolation and transient transformation system for Ginkgo biloba L.

Abstract

Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full understanding of the comprehensive functions of ginkgo genes. At present, heterologous expression of genes in model plants is the primary method used in ginkgo-related research; however, these distant plant model relatives limit reliable interpretation of the results for direct applications in ginkgo breeding. To overcome these limitations, in this study, an efficient isolation and transient expression system for ginkgo protoplasts was established. A large number of intact and homogeneous ginkgo mesophyll protoplasts were isolated using 2% cellulase and 0.25% pectinase in 0.4 M mannitol. The activity of these protoplasts remained above 90% even after 24 h. Furthermore, when the concentration of the polyethylene glycol 4000 solution was 30%–40% (w/v), the transformation efficiency of the protoplasts reached 40%. Finally, the reliability of the system was verified using subcellular localization, transient overexpression, and protein interaction experiments with ginkgo genes, thereby providing a technical platform for the identification and analysis of ginkgo gene functions. The proposed method partially compensates for the limitations associated with the lack of a genetic transformation system and provides technical support to expand research on elucidating the functions of ginkgo genes.

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

“…The parameters of the transient transformation system set by Du et al [ 55 ] were as follows: the concentration of protoplasts is 5 × 10 5 cells/mL, exogenous DNA concentration of 500 µg/mL, PEG4000 final concentration of 40%, and transformation time of 15 min. Han et al [ 56 ] demonstrated that when the concentration of PEG4000 was 30–40%, the transformation efficiency of Ginkgo biloba protoplasts reached 40%. We successfully studied the localization of PsnMLP328 protein in the nucleus and cytoplasm by optimizing these factors and validated the feasibility of the instantaneous transformation system of the protoplasts of Populus simonii × P. nigra .…”

Genome-wide identification, expression analysis, and potential roles under low-temperature stress of bHLH gene family in Prunus sibirica

“…The homolog of ICE1 gene, PsbHLH42 , was used to verify its function in resisting low temperatures. Transient genetic transformation assays can rapidly identify gene function ( Han et al., 2023 ). Because the lack of mature genetic transformation and regeneration systems in P. sibirica , we used the ideal model tree plant P. ussuriensis as the genetic transformation material ( Jansson and Douglas, 2007 ; Movahedi et al., 2014 ).…”

Overview and Recent Progress on the Biosynthesis and Regulation of Flavonoids in Ginkgo biloba L.

“…Moreover, as a gymnospermous plant, ginkgo has a large genome, a long juvenile stage, and no effective genetic transformation system, which hinders the comprehensive understanding of the functions of genes related to flavonoid biosynthesis. An efficient ginkgo protoplast isolation and transient expression system has recently been established [89], and its reliability was initially verified through subcellular localization, transient overexpression, and protein interactions. However, a stable genetic transformation system still needs to be developed to verify the function of ginkgo flavonoids.…”