Development of Transgenic North American White Ash (Fraxinus americana) Expressing a Bacillus thuringiensis Protein for Management of the Emerald Ash Borer

White ash (WA), Fraxinus americana, is an integral part of the hardwood forest ecosystem. Economically, WA provides wood for important products such as baseball bats, tool handles, and hardwood flooring. Ecologically WA provides cover and mast to support wildlife. The emerald ash borer (EAB) is a significant threat to all ash species because of a lack of native resistance in North American ash trees, its rapid spread, and the ineffectiveness and expense of control measures. EAB is a non-native beetle that consumes tree vascular tissue while in the larval stage. The development of an Agrobacterium-mediated transformation protocol may impart systemic resistance to EAB via the Cry8Da toxic protein, which is lethal to EAB larvae and naturally found in Bacillus thuringiensis SDS-502. Development of a transformation system for WA will allow this gene or others imparting resistance to be successfully incorporated into the WA genome. Embryos were extracted, transformed, and cultured on a selection-regeneration medium, killing any non-transgenic tissues while allowing for transgenic shoot formation. Transformed hypocotyls will be further cultured on a selection-elongation medium to allow for shoot development, after which shoots will be rooted and acclimatized to greenhouse conditions. Additional confirmation of transgenic plants will be conducted through quantitative measurements of the FLAG protein, polymerase chain reaction, and finally a feeding assay performed with EAB larvae. WA hypocotyls regenerated shoots after transformation at a 27.8% efficiency rate. The transformed hypocotyls received 6 weeks of exposure to kanamycin in the selection medium, allowing for positive results within the scope of this project. This protocol can be used to further genetically modify F. americana, allowing for preservation of this ecologically and economically important species

Isolation and characterization of a floral homeotic gene in Fraxinus nigra causing earlier flowering and homeotic alterations in transgenic Arabidopsis

A B S T R A C T Reproductive sterility, which can be obtained by manipulating floral organ identity genes, is an important tool for gene containment of genetically engineered trees. In Arabidopsis, AGAMOUS (AG) is the only C-class gene responsible for both floral meristem determinacy and floral organ identity, and its mutations produce sterility. As a first step in an effort to develop transgenic sterile black ash (Fraxinus nigra), an AG ortholog in black ash (FnAG) was isolated using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Analysis of the deduced amino acid sequence showed a typical MIKC structure of type II plant MADS-box protein with a highly conserved MADS-domain. Phylogenetic analysis revealed that FnAG had a close relationship with AG orthologs from other woody species. FnAG transcript was mainly expressed in reproductive tissues, but rarely detected in the vegetative tissues, consistent with the ABC model for floral development. A functional analysis was performed by ectopic expression of FnAG driven by the CaMV 35S promoter in transgenic Arabidopsis. Transformed plants showed homeotic conversions of carpeloid sepals and stamenoid petals. Curled leaves, reduced plant size, and earlier flowering were also observed in transgenic Arabidopsis. These data indicated that the FnAG functions in the same way as AG in Arabidopsis. These results provide the framework for targeted genome editing of black ash, an ecologically and economically important wetland species

In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols