Development of genetic transformation systems in creeping bentgrass (Agrostis palustris Huds.)

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Virginia Tech

As a first step toward improving creeping bentgrass (Agrostis palustris Huds.) via genetic engineering, this study was conducted to develop genetic transformation systems in creeping bentgrass.

Establishment of embryogenic cell cultures is a prerequisite for crop improvement via genetic engineering. A protocol for initiating and maintaining embryogenic callus and suspension cultures in creeping bentgrass was developed by substantially modifying and combining a few existing protocols. A high frequency of plant regeneration was obtained following this protocol.

Several factors affecting electroporation efficiency were studied using transient expression assay of the reporter uuid gene encoding B-glucuronidase (GUS). Increases in plasmid DNA resulted in increases in GUS activity. Maximal GUS activity was observed at field strength of 950 V/cm, protoplast density of 2 x 10⁶/ml, and KCl concentration of 125 mM in the electroporation buffer. Information obtained from this study facilitated optimization of electroporation conditions.

To identify a 5’ regulatory sequence conferring a high level of transgene expression in creeping bentgrass, the effect of six different 5’ regulatory sequences on transient gene expression was studied in electroporated creeping bentgrass protoplasts. The cauliflower mosaic virus (CaMV) 35S promoter was least active; whereas the rice actin 1 gene 5’ sequence was most active among the six sequences tested. Ranked in order of activity (high to low), the other four 5’ sequences were: 1) the CaMV 35S promoter plus the maize alcohol dehydrogenase 1 gene (Adh1) intron 6; 2) the 5’ sequence of the maize ubiquitin gene (Ubi-1), 3) the maize Adh1 promoter and its intron 1, and 4) the 35S promoter plus the Adh1 intron 1.

Stable transformation of creeping bentgrass was conducted via particle bombardment and electroporation using a plasmid, pZO1052, containing the reporter B-glucuronidase (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene under the control of CaMV 35S promoter plus the maize Adh1 intron 6. Putative transformants were selected by culturing cells on medium containing hygromycin. Transgenic plants and calli were obtained following particle bombardment. The frequency of putative transformants was 4.6 hygromycin-resistant colonies per bombardment. Integration of the transgenes was confirmed by Southern blot hybridization. A high frequency of escapes, however, occurred in the transformant selection following electroporation, which resulted in inefficient transformant recovery.

In this study, efficient genetic transformation systems using particle bombardment were established. Use of these systems will facilitate the improvement of creeping bentgrass.