Development of Transgenic Black Ash (Fraxinus nigra ) for Resistance to the Emerald Ash Borer, and Genome Editing for Reproductive Sterility
Black ash (Fraxinus nigra Marsh.) is valued for commercial hardwood products such as cabinets, paneling, flooring, and veneer, and for food and habitat for wildlife, specifically in riparian areas. The wood is preferred by Native Americans for making splints for basketry. The emerald ash borer (EAB), an aggressive exotic wood-boring beetle from Asia, is threatening all North American ash species. Since the first EAB infestation in the United States was confirmed near Detroit, Michigan in 2002, it has spread rapidly. In order to manage the EAB and conserve Fraxinus spp., there is a need to develop ash trees with resistance to the EAB. The goal of this research was to optimize the genetic transformation and shoot regeneration system for black ash hypocotyls, develop transgenic black ash for EAB resistance and reproductive sterility, and develop an adventitious shoot regeneration protocol using black ash leaves. An in vitro system for plant regeneration from leaf explants of black ash was successfully developed. Leaf explants were transversally cut across the midrib and cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators. MS medium supplemented with 22.2 µM 6-benzylaminopurine combined with 31.8 µM thidiazuron produced callus formation (100%) and adventitious shoot bud induction (28.8%). The frequency of regeneration response was significantly higher with compound leaves than single leaflets. Whole plants were acclimatized to the greenhouse. An Agrobacterium-mediated transformation system for black ash hypocotyls was optimized based upon a previously developed protocol in our lab. Sonication for 90 s followed by vacuum-infiltration for 10 min in suspension of Agrobacterium strain EHA105 (concentration at OD600 = 1.0) was found to be optimal. Silwet L-77 did not make a significant difference on transformation efficiency, but it had a negative effect at high concentration. Using this optimal transformation condition, three independent transgenic lines expressing Bacillus thuringiensis ( Bt) Cry8D2 gene were obtained. The integration of the full length, intact Cry8D2 gene was confirmed by polymerase chain reaction (PCR), and its expression in mRNA and protein level was detected by qPCR and Western blot analysis, respectively. All three transgenic lines contained two copies of the transgene. An AGAMOUS (AG) homolog of black ash (FnAG) was isolated and characterized as a potential target gene for achieving transgene containment. A 729-bp coding region of FnAG was obtained by reverse transcription PCR and rapid amplification of cDNA ends. Deduced amino acid sequence showed a highly conserved MADS-domain. Phylogenetic analysis confirmed that FnAG belongs to the clade of C-lineage AG subfamily genes. Expression of FnAG transcript was detected in the reproductive tissues (female and male flowers), but rarely detected in the vegetative tissues (leaves). Transgenic Arabidopsis thaliana plants overexpressing FnAG showed ap2-like phenotypic alteration and early flowering, indicating FnAG functions in the same way as AG. To achieve transgene containment, reproductive sterility can be produced by disrupting genes involved in development of floral organs such as AG. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) was used to induce mutations at three target sites within FnAG. A total of 50 transgenic lines harboring Cas9 expression cassette were obtained. Of these, only two showed one nucleotide substitution in the target site. Cas9 transgene silencing in some transgenic lines with no mutations resulted in the low mutagenesis rate.
Pijut, Purdue University.
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