Adventitious shoot regeneration and genetic transformation of pumpkin ash (Fraxinus profunda) hypocotyls

Micah E Stevens, Purdue University

Abstract

The rapid increase in global trade beginning in the early 20th century has led to the inadvertent introduction of many non-native fungal, animal, insect, and plant species into the United States. These introductions, released from their native predators or pathogens, can quickly and severely impact the pre-existing flora and fauna. The emerald ash borer (EAB) ( Agrilus planipennis Fairmaire; Coleoptera:Buprestidae) is an exotic obligate insect pest of ash trees, genus Fraxinus. Since its first detection in Michigan in 2002, millions of ash trees have been killed. With little innate resistance in North American ash populations, the EAB continues to spread. Pumpkin ash (Fraxinus profunda) is a fine hardwood species integral to wetland communities where it is predominantly found. Because of its narrow habitat requirements, the threat of extirpation is further exacerbated by EAB, and pumpkin ash has been listed as threatened or endangered in a number of states. To mitigate this threat, the objective of this research was to develop an in vitro adventitious regeneration and transformation system that would allow for genetic modification of pumpkin ash for EAB resistance. Hypocotyls isolated from mature pumpkin ash seed were used as the initial explants for developing an in vitro regeneration protocol. Aseptically cultured hypocotyls produced adventitious shoots at a 43% frequency when cultured on Murashige and Skoog (MS) medium with Gamborg B5 vitamins plus 0.2 g l -1 glycine (B5G), and supplemented with 22.2 μM benzyladenine (BA) in combination with 4.5 μM thidiazuron (TDZ). Shoots were elongated on MS medium containing 10 μM BA and 10 μM TDZ. When exposed to woody plant medium (WPM) supplemented with 4.9 μM indole-3-butryic acid (IBA) pumpkin ash microshoots readily formed adventitious roots. Newly formed plantlets were then successfully acclimatized to ambient greenhouse conditions. Once this in vitro regeneration system was completed, the next step was to develop a genetic transformation protocol. Utilizing an Agrobacterium -mediated transformation system, a designed vector (pq35GR) containing the β-glucuronidase (GUS), green florescent protein (GFP), and neomycin phosphotransferase (nptII) genes, was incorporated into the genome of pumpkin ash. These transgenes permitted the visualization and selection of transgenic adventitious shoots. Hypocotyls were exposed to a suspension of Agrobacterium tumefacians (At) strain EHA105 carrying the pq35GR vector. At was introduced by vacuum-infiltration for 10 min after 90 s sonication, and then co-cultured with hypocotyls in the dark for 3 d, after which the hypocotyls transferred to selection medium. Transformed hypocotyls were selected and adventitious shoots regenerated on MS medium supplemented with 22.2 µM BA, 4.5 μM TDZ, 50 mg l -1 adenine hemisulfate (AS), 10% coconut water (CW), 400 mg l -1 timentin, and 20 mg l-1 kanamycin. A replicated factorial experiment showed 400 mg l-1 timentin, and 20 mg l-1 kanamycin to be optimal for controlling Agrobacterium growth and selecting transgenic plant material, respectively. Transformation was confirmed through polymerase chain reaction and GFP visualization. The development of this successful regeneration and transformation protocol will allow for the incorporation of genes imparting resistance to EAB, and provides an additional tool for managers who wish to keep pumpkin ash in the native landscape.

Degree

M.S.

Advisors

Pijut, Purdue University.

Subject Area

Plant biology|Forestry

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