Location: Floral and Nursery Plants Research
2019 Annual Report
Objectives
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.
Approach
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.
Progress Report
Easter lilies were transformed with the dsFar and dsGP genes from Pratylenchus penetrans. Two independently transformed lines were confirmed by PCR to have the dsFar gene and six with the dsGP gene. These plants are being challenged in vitro with P. penetrans to determine if they show resistance to this nematode.
A promoter that expresses at high levels primarily in roots rather than shoots would be useful when genetic engineering lilies for P. penetrans resistance. Four constitutive promoters - CaMV 35S, Gladiolus ubiquitin, Arabidopsis ubiquitin, and maize ubiquitin - were tested in transgenic lilies containing the GUS reporter gene under each promoter. Both the CaMV 35S and Gladiolus ubiquitin promoter showed higher levels of GUS expression than the other two promoters. Leaves with the CaMV 35S promoter showed more GUS expression than leaves with the Gladiolus ubiquitin promoter.
We tested the effects of several factors on lily transformation efficiency. We found that bombarding cells once with the gene gun is comparable to bombarding twice. We also found that increasing mannitol concentration (which protects cells from bursting) did not enhance the transformation frequency. We found that bulb scales that were preconditioned on BA and NAA with a low level of picloram had a lower rate of transformation, but higher regeneration, than bulb scales that were preconditioned with higher levels of picloram alone. Finally, we found that using smaller gold particles did not improve transformation frequencies.
In a collaborative project with ARS scientists in Hawaii, two transcriptomes were made using RNA from burrowing nematodes. These nematodes have some of the same parasitism genes as P. penetrans that affect plant roots, but also have unique genes that may serve as targets for specifically controlling burrowing nematodes.
Hairy roots of carrot containing ds-PAT, ds-unc, ds-pas, and ds-VAP were made using sequences from the burrowing nematode transcriptome. These transformants will be checked by PCR to verify presence of the entire transgene and the most promising lines will be sent to ARS collaborators in Hawaii.
Accomplishments
Review Publications
Vicente, C.S., Nemchinov, L.G., Mota, M., Eisenback, J.D., Kamo, K.K., Vieira, P. 2019. Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans. PLoS One. 14 (2):e0212540. https://doi.org/10.1371/journal.pone.0212540.
Kamo, K.K., Thilmony, R.L., Bauchan, G.R. 2019. Transgenic Lilium longiglorum plants containing the bar-uidA gene controlled by the rice RPC1, Agrobacterium rolD, mas2, and CaMV 355 promotors. Plant Cell Tissue and Organ Culture. 13(2):303-312.
