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Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis Megumu Takahashi Toshiki Uji Naotsune Saga Koji Mikami* *Corresponding author Financial support: This study was supported in part by a grant from the Sumitomo Foundation (to KM) and by Grants-in-Aid for Scientific Research (C; no. 21580213) and a National Project "Knowledge Cluster Initiative" (Innovative Stage, "Hakodate Marine Bio Industrial Cluster-Green Innovation of UMI") from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to NS). Keywords: allantoin, protoplast, P. yezoensis, regeneration, transient gene expression.
Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis, we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a β-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades. |