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

Abstract

<jats:p>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<jats:sup>−1</jats:sup> activated charcoal and 500 mg humic acid in disposable fast-food takeout containers. When using vermiculite as a substrate, adding 2.0 g·L<jats:sup>−1</jats:sup> 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.</jats:p>

Recent Advances in Adventitious Root Formation in Chestnut

“…It is believed that such reduction will ease the exit of primordia from the stems as well as help in the growth of new roots, because full-strength media might inhibit rooting, though the response is a species-dependent and probably a genotype-dependent issue. Indeed, rooting rates of microshoots of C. sativa and C. dentata in the presence of full-strength media were satisfactory [ 98 , 112 ]. In addition, better rooting rates and mean number of roots were achieved in C. henryi microcuttings rooted in full-strength media than in half-strength media [ 100 ].…”

“…Low concentrations of AC, ranging from 0.2 to 1%, have been successfully used in the rooting of chestnut microshoots [ 33 , 97 ]. Depending on the genotype, the rooting frequencies were increased up to more than two fold in the presence of AC [ 93 , 98 ]. Nonetheless, as IBA was the hormone frequently used and it is believed to be more stable than IAA in culture media and under light conditions [ 131 ], the mode of action of AC in AR remains to be defined.…”

Micropropagation of Chinquapin (Castanea henryi) Using Axillary Shoots and Cotyledonary Nodes

“…With the appropriate cytokinin and auxin concentration, chinquapin shoots were strong enough for rooting and surviving after potting ex vitro. As the study on the American chestnut showed that in vitro shoots are recommended only if they have more than six leaves before rooting and are greater than 3 cm in height so they would be large enough to survive after potting (Oakes et al, 2016).…”

“…Micropropagation of Castanea species has been investigated on the American chestnut (C. dentata) (Oakes et al, 2013(Oakes et al, , 2016Xing et al, 1997Xing et al, , 1999, the Chinese chestnut (C. mollissima) (Lu et al, 2017;Qi-guang et al, 1986), the European chestnut (C. sativa) (Corredoira et al, 2003;Rodriguez, 1982;Sezgin and Dumano glu, 2014), the Japanese chestnut (C. crenata) (Tetsumura and Yamashita, 2004), and their hybrid chestnut (Cuenca et al, 2017;Vidal et al, 2015). Micropropagation of Castanea species in most of the reports was successful.…”

“…Producing woody plants from micropropagation is an extremely time-and laborintensive process, needing many steps and various growing media (Gonc xalves et al, 1998;Oakes et al, 2016;Pijut et al, 2010). Axillary shoot explants have been used to achieve regenerated plants in many species, including hybrid chestnuts (Cuenca et al, 2017;Vidal et al, 2015), Vaccinium sp.…”