Plant regeneration from embryogenic callus derived from shoot apices and production of transgenic plants by particle inflow gun in dwarf napier grass (Pennisetum purpureum Schumach.)

• Published in: Plant Biotechnology Vol. 34; no. 3; pp. 143 - 150
• Main Authors: Gondo, Takahiro, Umami, Nafiatul, Muguerza, Melody, Akashi, Ryo
• Format: Journal Article
• Language: English
• Published: Japan Japanese Society for Plant Cell and Molecular Biology 2017; Japan Science and Technology Agency
• Subjects: 2,4-D; Benzyladenine; Bialaphos; bioenergy plant; Biofuels; Bombardment; C4 tropical grass; Callus; Callus culture; Copper sulfate; Dichlorophenoxyacetic acid; dwarf napier grass; Ethanol; Forage; Genetic transformation; Herbicide resistance; Inflow; Millet; Original Paper; Particle bombardment; particle inflow gun; Pennisetum purpureum; Plant breeding; Plants (botany); Regeneration; Reporter gene; Sulfates; Tissue culture; Tissues; Transgenic plants; particle inflow gun; dwarf napier grass; genetic transformation; C4 tropical grass; bioenergy plant
• ISSN: 1342-4580; 1347-6114
• DOI: 10.5511/plantbiotechnology.17.0623a

Napier grass (Pennisetum purpureum Schumach.) is a highly productive C4 tropical forage grass that has been targeted as a potential bioenergy crop. To further increase the efficiency of bioethanol production by molecular breeding, a reliable protocol for genetically transforming napier grass is essential. In this study, we report the creation of transgenic napier grass plants derived from embryogenic callus cultures of shoot apices. Embryogenic callus was initiated in three accessions of napier grass and a napier grass×pearl millet hybrid using Murashige and Skoog (MS) medium supplemented with 2.0 mg L－1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L－1 6-benzylaminopurine (BAP) and 50 µM copper sulfate (CuSO4). Of the accessions tested, a dwarf type with late-heading (DL line) had the best response for embryogenic callus formation. Highly regenerative calli that formed dense polyembryogenic clusters were selected as target tissues for transformation. A plasmid vector, pAHC25, containing an herbicide-resistance gene (bar) and the β-glucuronidase (GUS) reporter gene was used in particle bombardment experiments. Target tissues treated with 0.6 M osmoticum were bombarded, and transgenic plants were selected under 5.0 mg L－1 bialaphos selection. Although a total of 1400 target tissues yielded nine GUS-positive bialaphos-resistant calli, only one transgenic line that was derived from target tissue with the shortest culture term produced four transgenic plants. Thus, the length of time that the target tissue is in callus culture was one of the most important factors for acquiring transgenic plants in napier grass. This is the first report of successfully producing transgenic napier grass plants.