Location: Floral and Nursery Plants Research2018 Annual Report
Objective 1: Determine whether parasitic genes previously isolated from the root lesion nematodes associated with hairy roots of soybean can confer nematode resistance in lilies, and use the biolistic approach to transform lilies and develop cultivars that are resistant to this migratory nematode pest. [NP301, C1, PS1A, PS1B; C3, PS3B] Sub-objective 1.1: Optimize biolistic-mediated transformation of lilies to increase the frequency of transformation events. Sub-objective 1.2: Compare levels of uidA reporter gene expression in roots of lilies transformed with either the CaMV 35S, maize Ubi1, Arabidopsis UBQ3, or Gladiolus GUBQ1 promoters to determine the promoter that directs the highest levels of transgene expression in roots. Sub-objective 1.3: Transform Easter lilies with ds-FAR1 and ds-GPX genes and evaluate them for resistance to P. penetrans. Objective 2: Identify parasitic genes that can be used to confer burrowing nematode resistance in anthuriums using genomic analysis techniques, develop a system of delivery to incorporate the identified genes into resistant cultivars of anthuriums, and evaluate the genes. [NP301, C1, PS1A, PS1B; C3, PS3B] Sub-objective 2.1: Develop carrot hairy roots containing unc87 and FAR1 from R. similis as dsRNA constructs and screen the carrot hairy roots for resistance to R. similis. Sub-objective 2.2: Transform Anthurium with ds-unc87 and ds-FAR1 gene constructs and screen Anthurium lines for resistance.
Transgenic Easter lilies and anthuriums that contain dsRNA constructs of parasitic genes will be developed and plants screened for resistance to the migratory nematodes that infect them. The biolistic transformation system for Easter lilies will be optimized. Four promoters, CaMV 35S, maize Ubi1, Arabidopsis UBQ3, and Gladiolus GUBQ1, will be compared in Easter liies to determine which promoters express highly in roots where the migratory nematode Pratylenchus penetrans infects. Lilies will be transformed with either the parasitic gene ds-FAR1 or ds-GP, and transgenic plants will be screened for resistance to P. penetrans infection. Two genes, unc87 and FAR1, will be isolated from a transcriptome of Radopholus similis, the burrowing nematode, using BLAST to search for homology to these same genes in other migratory nematodes. Hairy roots of carrots and anthuriums containing dsRNA constructs of the R. similis unc87 and FAR1 will be developed and screened for resistance.
This is the first year of this new project, which began on 2/14/18. Additional information can be found in the report for the terminating project, 8020-21000-058-00D. The size of gold particles (0.6 µm), pressure (900 psi), and concentration of osmoticum (0.125 M mannitol) to be used for gene gun bombardment of lilies has been optimized. Lilies have been bombarded with four promoters (CaMV 35S, maize Ubi-1, Gladiolus UBQ1, and Arabidopsis UBQ3) controlling the gus gene to compare expression in roots. Two parasitism genes (ds-FAR1 and ds-GPX) have been used to bombard lilies for transient expression. Several cultivars of anthuriums are being multiplied in vitro. A pure culture of the burrowing nematode is being prepared by the ARS scientists (Hilo, Hawaii) for isolating gene sequences of interest.