Biology subjects

Klak, T.

Publications and source records attributed to Klak, T..

2 recordsLinked to original sources

bioRxiv2025

Two-Year Field Trial of Genetically Engineered American Chestnut Reveals Greater Fungal Blight Tolerance Compared to Wild-Type Full-Sibling Trees

The American chestnut (Castanea dentata [Marsh.] Borkh.) was a foundational forest canopy species in eastern North America until an accidentally imported fungal blight (caused by Cryphonectria parasitica [Murr.] Barr.) rendered it functionally extinct across its native range. Biotechnological approaches, such as the bioengineered Darling 54 line, have potential for future restoration of American chestnut, but field-based evaluations of blight tolerance have been limited. Progress has been slowed by the many years it takes for seedlings to grow to saplings, then to full-fledged trees. Current regulatory restrictions also constrain the testing of transgenic chestnuts to within permitted orchards. This research reports on a two-year field trial of Darling 54, their non-transgenic wild-type siblings and Chinese chestnut (Castanea mollissima Blume) controls deployed a randomized block design to test for blight tolerance. In the two years, three replicates each of 261 trees were branch-inoculated with EP-155, a highly virulent isolate of the fungal blight. Based on canker length, Darling 54 trees consistently outperformed their non-transgenic wild-type siblings and Chinese chestnut. To our knowledge, this is the first report of a multi-year field trial of fungal blight inoculations comparing advanced generation Darling 54 families. This field-based evaluation suggests that reintroduction programs using Darling 54 American chestnuts, which can commence after federal approval, may offer a promising path to success.

synthetic biology↗

bioRxiv2025

Speed breeding transgenic American chestnut trees toward restoration

The American chestnut (Castanea dentata) was a dominant, foundational forest canopy tree in eastern North America until an imported chestnut blight (caused by Cryphonectria parasitica) rendered it functionally extinct across its native range. Biotechnological approaches have the potential to help restore the species, but field-based breeding advances are hampered by long generation times, [≤]50% transgene inheritance, and regulatory restrictions on outdoor breeding of transgenic trees. Self-incompatibility and flowering phenology further limit generational advances and field testing of chestnuts. Our work here demonstrates that long generational times and field constraints can be circumvented by producing both male and receptive female flowers in controlled indoor environments. Additionally, we developed an embryo rescue protocol for both indoor and field conditions, in which developing embryos can be extracted and micropropagated from immature seeds between 6- and 8-weeks post pollination. These advances have enabled production of the first homozygous transgenic American chestnuts, which have produced pollen that was used for outdoor controlled pollinations and yielded nearly 100% transgene inheritance by offspring. This work also provides event-specific DNA markers to differentiate transgenic chestnut lines and identify homozygous individuals. We demonstrate that an obligate outcrossing forest tree can reach sexual maturity rapidly in controlled, indoor environments. When coupled with genomic analyses and other biotechnological advances, this procedure could facilitate the reintroduction of this iconic species.

plant biology↗