Efficiency of gene transformation by Agrobacterium tumefaciens in node tissue on nilegrass (Acroceras macrum)

Foreage grasses are critical to livestock industries throughout the world. Nilegrass (Acroceras macrum) is one of the most important tropical forage grass species. It is commonly used for pastures, hay production and silage. Soon after its release, Nilegrass has become one of the major forage crops grown in Taiwan. Dry yield and crude protein content in Nilegrass are found greater than Pangolagrass (Jeng et. al., 2004). Improvement of forage by conventional breeding is slow due to the genetic complexity of these species (Ha et. al., 1992). Genetic transformation is an alternative that permits direct introduction of useful genes into a plant's genome. Agrobacterium-mediated transformation allows stable integration of transgene into the plant genome (Hiei et. al., 2000). Agrobacterium-mediated transformation has been successfully used to transfer genes into a wide range of forage grasses plant species. The objective of the study, the efficient genetic transformation system for Nilegrass, using Agrobacterium-mediated TDNA delivery, is described herein. Sterilized nodes were infected and co-cultivated with 2 strains of Agrobacterium tumefaciens. EHA 105 and LBA 4404, which harbor the plasmids, PCAMBIA (1301) and pTOK (233), respectively and contain genes for hygromycin phosphotransferase (HPT) and beta-D-glucuronidase (GUS). Agrobacterium solution concentration of 0.8 O.D at 600nm were co-cultivation on solid LS medium, supplemented with 1 mg/l BA, 30 g/l sucrose and 2.6 g/l phytagel, 50 and 100 micro M acetosyringone, 3, 5, and 7 days of co-cultivation period. The efficiency of gene transformation was evaluated by determination of GUS expression. In this study, the highest percentage of transformation was 82.22% after transformation with EHA 105 at 50 micro M acetospringone for 7 days. Putative transgenic plants and control plants were chosen, analyzed by PCR analysis bands corresponding to the HPT gene and GUS gene were clearly shown in transgenic plants. Preliminary study of agrobacterium-mediated gene transformation in Nilegrass is a useful technique for generate of transgenic Nilegrass containing genes of agronomic importance and can be applied as a promising system to enhance transformation efficiency in Poaceae family.