Improving Rooting and Shoot Tip Survival of Micropropagated Transgenic American Chestnut Shoots

Many hardwood tree species are being threatened by exotic pests, and for some, only genetic engineering can offer a solution before functional extinction occurs. An example of how genetic engineering can be a useful tool for forest restoration is the transgenic american chestnuts, which contain a wheat oxalate oxidase gene conferring resistance to the chestnut blight. Many hundreds of these trees are needed for field trials and eventual restoration plantings throughout its natural range, but production is bottlenecked because of the difficulty of making hardwood trees produce roots through micropropagation. The presence of roots and living shoot tips precede successful acclimatization of tissue culture-produced american chestnut plantlets. In these experiments, we attempted to improve the post-rooting stage of our american chestnut propagation protocol. We examined vessel type, hormone, and activated charcoal concentrations, and using a vermiculite substrate. For plantlets with the best combination of roots and living shoot tips we recommend using semisolid post-rooting medium containing 2 g·L activated charcoal and 500 mg humic acid in disposable fast-food takeout containers. When using vermiculite as a substrate, adding 2.0 g·L activated charcoal to post-rooting medium without a gelling agent was the preferred treatment. Improving the survival rates of the american chestnut plantlets will benefit the american chestnut restoration project by providing more plant material for both ecological studies and eventual restoration, since pursuit of a nonregulated status for these transgenic trees will require extensive field testing. These procedures may also be applicable to other difficult-to-root hardwood trees in transgenic programs, such as american butternut, white oak, and black walnut. The american chestnut was once a cornerstone of hardwood forests as far north as Maine and Ontario and as far south as Georgia and Mississippi, and once covered 200million acres of land (Saucier, 1973). Not only did this tree have an extensive range, but it also grew to huge proportions, reaching heights of 60–90 ft and diameters of 6–7 ft (Brooks, 1937; Saucier, 1973). American chestnuts comprised up to 25% of the timber volume in Appalachian forests and the versatile and rotresistant wood was used for telegraph and telephone poles, mine props, railroad ties, rail fences, shingles, and barn construction (Brooks, 1937). Chestnut seed fed turkeys, deer, black bears, and local residents every autumn and were an important cash crop for theAppalachian region (Freinkel, 2009; Hepting, 1974). The once massive canopy tree is now relegated to a rare understory shrub. The fungus that causes the chestnut blight, Cryphonectria parasitica, arrived in New York in the late 1800s aboard a shipment of imported Asiatic chestnut trees (Griffin, 2000). The disease was first documented on dying american chestnuts in the Bronx Zoo in 1904 and spread rapidly throughout the Eastern United States over the next 30 years, wiping out billions of mature canopy trees (Murrill, 1906). The fungus secretes oxalate, which kills the cambium cells and lowers the pH to 2.8 at the canker margins (McCarroll and Thor, 1978), making it impossible for the tree to sequester the infection. The canker grows unchecked in the cambium, eventually girdling the stem and killing the tree (Cessna et al., 2000; Schlarbaum et al., 1997). The trees can resprout from the root collar, but are killed back by blight before growing old enough to reproduce. Genetic engineering is poised to offer a solution: blight-resistant american chestnut trees. The addition of one gene, oxalate oxidase, can protect the american chestnut by breaking down the oxalic acid secreted by the fungus (Zhang et al., 2013). The fungus still infects wounds in the bark, but after the initial infection the tree halts the spread behind a wound periderm and fungus cannot spread further (Newhouse et al., 2014). Many oxalate oxidase–expressing lines of transgenic american chestnuts are currently in production, developed by an Agrobacteriummediated transformation (Polin et al., 2006). Many trees from each line are needed for field trials and ecological impact studies to assess their safety and nontarget environmental effects, such as mycorrhizae formation (D’Amico et al., 2014). Wild-type american chestnuts derived from tissue culture are important controls and are also needed in large numbers. Producing hardwood trees from tissue culture is an extremely timeand laborintensive process, requiring many steps and different growing media (Gonc xalves et al., 1998; Gonzalez Padilla et al., 2003; Pijut et al., 2010). The main production bottleneck for micropropagated american chestnuts are the rooting and acclimatization stages, where shoots are excised from callus, dipped in a rooting hormone, placed in a low-salt medium in the dark, moved to a postrooting medium on a light bench, and finally potted in soil in high humidity bins. Plantlets that survive this process are moved to individual pots and have a high survival rate in high-humidity growth chambers and finally the greenhouse. Sometimes over 50% of shoots are lost in each stage, with acclimatization success as low as 10% of all shoots initially entering the rooting process. Our research has been addressing this bottleneck by investigating improvements to our rooting protocol. A previous study found that plantlet survival in the growth chambers could be dramatically improved by shortening the time in rooting medium from 8–10 d to 3–4 d, and also by placing plantlets in the dark during that time (Oakes et al., 2013). In addition, acclimatization success can generally be improved by increasing the number of adventitious roots produced in culture and keeping the shoot tip of the plantlet alive (Vieitez et al., 1989; Xing et al., 1997). It is also advised to only root larger plantlets with more leaves to ensure acclimatization success, as leaves produce important rooting cofactors (Macdonald, 2006; Oakes et al., 2013). There are numerous factors which can affect rooting of micropropagated shoots. Activated charcoal is commonly used in plant tissue culture to darken the medium, but has the undesired effect of binding growth regulators and rendering them unavailable to the plant (Nissen and Sutter, 1990). It is generally added after auxin has been applied because of its inhibitory properties (Dumas and Monteuuis, 1995; Le Roux and Staden, 1991; McCown and McCown, 1987). We examined whether a concentration of activated charcoal lower than our standard 2 g·L would still enhance root production. Vessel type can have an impact on culture growth as well, by changing the rate of gas exchange and amount of oxygen available for respiration (Bateson et al., 1987). For three woody plant species, larger container size was found to increase rooting (McClelland and Smith, 1990). Ventilation helps reduce the relative humidity of the culture container to below 100%, encouraging transpiration (Gribble, 1999). We chose to examine whether using large clear plastic takeout containers in place of vented Magenta GA-7 Received for publication 4 Sept. 2015. Accepted for publication 14 Oct. 2015. Corresponding author. E-mail: adoakes@syr.edu. HORTSCIENCE VOL. 51(2) FEBRUARY 2016 171 vessels would influence root formation and shoot tip survival. The addition of vermiculite, a textured substrate, to rooting medium was found to improve root induction in multiple hardwood species, including walnut (Vahdati et al., 2004), papaya (Kaity et al., 2009), and pear (Tetsumura et al., 2011). Vermiculitecontaining medium also enhanced root morphogenesis in ginger and ginseng (Inden et al., 1988; Malamug et al., 1991; Shoyama et al., 1995), and improves rooting and root quality in specific apple cultivars (Druart, 1997; Navatel and Bourrain, 1994). We examined whether a gelling agent (phytagel) or activated charcoal were necessary when using vermiculite for rooting american chestnut shoots. Humic acids have been investigated as an additive to plant tissue culture because of their auxin-like activity (Cacco and Dell’agnola, 1984; Dell’Agnola and Nardi, 1987; Muscolo et al., 1999, 2007), chelating abilities (Ouni et al., 2014), and influence on root architecture (Trevisan et al., 2010). The addition of these compounds to growing medium has been shown to increase leaf and shoot growth in young seedlings (Piccolo et al., 1993) and stimulate cell growth and development (Nardi et al., 2002). This series of experiments focused on improving root production and shoot tip survival by modifying the post-rooting medium. We tested activated charcoal concentrations, vessel type, humic acid concentration, and parameters for using a vermiculite substrate.