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

Black Walnut

Black walnut (Juglans nigra L.), also known as eastern black walnut or American walnut, is a fine hardwood species in the family Juglandaceae, section Rhysocaryon (Manning 1978). In general, J. nigra will not cross with species in the sections Cardiocaryon or Trachycaryonv, but J. nigra will cross with J. ailantifolia (Cardiocaryon) (Williams 1990). Juglans nigra will also hybridize to some extent with other Juglans species (Dioscaryon and Rhysocaryon), and one hybrid is recognized: J. nigra × J. regia = J. x intermedia Carr. (USDA-NRCS 2004). Native to the deciduous forests of the eastern United States (USA), from Massachusetts to Florida and west to Minnesota and Texas, and occurring naturally in southern Ontario, Canada, black walnut is seldom found in pure stands, but rather in association with five mixed mesophytic forest cover types: sugar maple, yellow poplar, yellow poplar – white oak – northern red oak, beech – sugar maple, and silver maple – American elm (Williams 1990). Black walnut is a large tree and on good sites may attain a height of 30 to 38 m and diameter of 76 to 120 cm and can exceed 100 years of age (Williams 1990; Dirr 1998; USDA-NRCS 2004). Black walnut is shade intolerant, and control of competing vegetation is especially important in new plantations for the first 3 to 4 years. Black walnut grows best on moist, deep, fertile, well-drained, loamy soils, although it also grows quite well in silty clay loam soils or in good agricultural soils without a fragipan (Williams 1990; Cogliastro et al. 1997). These sites include coves, bottomlands, abandoned agricultural fields, and rich woodlands. Black walnut forms a deep taproot, wide-spreading lateral roots, and has been cultivated since 1686. A toxic chemical ‘juglone’ (5- hydroxy-1, 4-naphthoquinone), naturally occurring in the leaves, buds, bark, nut husks, and roots of black walnut, is a highly selective, cell-permeable, irreversible inhibitor of the parvulin family of peptidylprolyl cis/trans isomerases (PPIases) and functions by covalently modifying sullfhydryl groups in the target enzymes (Henning et al. 1998; Chao et al. 2001). Certain plants, especially tomato, apple, and several conifer species, are adversely affected (allelopathy; foliar yellowing, wilting, and even death) by being grown near the roots of black walnut trees (Goodell 1984; Dana and Lerner 1994). Horses can contract acute laminitis, an inflammation of the foot, when black walnut wood chips or sawdust is used for stall bedding or stables and paddocks are located too close to walnut trees (Galey et al. 1991). Historically, the bark of black walnut was used by several Native Americans, including the Cherokee, Delaware, Iroquois, and Meskwaki, in tea as a cathartic, emetic, or disease remedy agent, and chewed or applied for toothaches, snake bites, and headaches (Moerman 1998, 2003). Caution: the bark should be used cautiously inmedicine because it is poisonous. TheCherokee, Chippewa, and Meskwaki also used the bark to make a dark brown or black dye (Moerman 1998, 2003). The Comanche pulverized the leaves of black walnut for treatment of ringworm, the Cherokee used leaves to make a green dye, and the Delaware used the leaves as an insecticide to dispel fleas (Moerman 1998, 2003). The nut meats were also a food source for Native Americans, and the nuts are still consumed today by people and are an important food source for wildlife

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