Abstract
This study was conducted to establish a rapid method for barley (Hordeum vulgare L.) protoplast isolation to provide an easy-to-use procedure for the transformation and primary investigation of new gene constructs by transient gene expression assays. Protoplasts were successfully isolated from the chopped embryo and scutellum parts of mature barley seeds by digesting three hours with an enzyme mixture. Isolated protoplasts were washed in W5 washing solution, sieved through plastic meshes and then cleaned on sucrose gradient. The suitability of these directly from embryo-scutellum complexes derived protoplasts for transient gene expression studies was determined by transforming the protoplasts using the PEG (polyethylene glycol) method. Plasmid pAct1-F containing the rice Act1 promoter linked with the gus coding sequences and the nos polyadenylation signal was used in the transformation. After the PEG treatment protoplasts were cultured on KPR culture medium and the transient gus expression was assayed 24-36 hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts with X-gluc. The results of this study show that the protoplasts isolated directly from dissected mature barley scutellum-embryo complexes could be used to investigate transient gene expressions in barley. This procedure requires negligible time prior the transformation experiment and so can be done in a very short time compared to the protoplast system based on a suspension culture.
Highlights
During the last decade much interest has been focused on the culture of plant protoplasts and their potential in producing transgenic plants as well as in assaying the expression of recombinant gene constructs in transformed cells
Considerable progress has been made in protoplast culture of important monocot species and the first protoplast derived regeneration in cereals was reported in rice (Oryza saliva L.) by Fujimura and his group (1985)
Protoplast transformation resulting in stable integration of foreign genes into the target cells has been reported in other cereals such as rice (Shimamoto et al 1989), maize (Mörocz et al 1990) and barley
Summary
During the last decade much interest has been focused on the culture of plant protoplasts and their potential in producing transgenic plants as well as in assaying the expression of recombinant gene constructs in transformed cells. Succesful protoplast-plant regeneration systems have been reported in wheat (Vasil et al 1990, Chang et al 1991, Yang et al 1993, Ahmed and SAgi. 1993, Pauk et al 1994), maize (Mörocz et al.1990) and barley (Jähne et al 1991). The transformation of protoplasts and the subsequent regeneration of transgenic rice plants have become a routine procedure (Jenes et al 1992). Protoplast transformation resulting in stable integration of foreign genes into the target cells has been reported in other cereals such as rice (Shimamoto et al 1989), maize (Mörocz et al 1990) and barley (Lazzeri and Lörz 1990, Lazzeri et al.1992)
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