Location: Floral and Nursery Plants Research2015 Annual Report
This project applies biotechnology to effectively mitigate the impact of pests and pathogens on priority floral crops and develops systems that can be manipulated to preserve select hardwood tree taxa for future genetic engineering applications. Genetic engineering of floral crops for pest and pathogen resistance contributes to sustainable production. There has been little research done on migratory nematodes such as Pratylenchus, and this project addresses the use of transgenes that may be effective in controlling Pratylenchus. Viruses are always a problem for propagated plants, particularly flower bulb crops that are propagated from the same bulb for many years. This project determines if an RNAi approach is effective for control of Cucumber mosaic virus (CMV), an economically important virus that affects numerous crops. In vitro manipulation of plants through genetic engineering allows for rapid clonal propagation, ploidy level manipulation, and preservation of germplasm and cryopreservation. Hardwood trees have been difficult to manipulate in vitro, and there are few reports of research done with North American hardwood tree taxa. This project will explore the possibilities of regenerating plants from select oak, elm, or maple taxa for future applications by using either embryogenesis or organogenesis and maintaining plants in vitro by micropropagation. Objective 1: Characterize the interaction between Pratylenchus and transgenic Easter lilies transformed with currently available anti-nematode genes for resistance response. (NP301; C1, PS 1B) Objective 2: Identify genes involved in the metabolic activities of Pratylenchus, using an RNAi approach, and determine if they are effective in enhancing resistance to Pratylenchus, using a soybean hairy root system. (NP301; C1, PS1B) Objective 3: Develop and evaluate lilies containing an antiviral gene for resistance to Cucumber mosaic virus. (NP301; C1, PS 1B) Objective 4: Develop a regeneration system from embryogenic callus for priority hardwood tree species in the tree improvement program at the U.S. National Arboretum. (NP301; C3, PS 3B)
Genetic engineering will be used to introduce genes into Easter lily for nematode and cucumber mosaic virus resistance. Genes that will be tested for nematode resistance are two Bt genes and a cystatin genes. Two genes targeting nematode movement will be used in an RNAi approach against the root lesion nematode. Seeds and cuttings from select hardwood tree species will be cultured in vitro and used for micropropagation and induction of callus. Treatments will be used to determine if plants can be regenerated from the callus.
The soybean hairy root system was used to test the effectiveness of two cystatin genes (OCI and OCII) and two Bt genes in conferring resistance to the root lesion nematode (Pratylenchus). These four genes were not as effective as the modified rice cystatin gene that has a deletion. Additional biological replicates have been examined for the transcriptome analysis of Pratylenchus infection of soybean. This provides information about the metabolic pathways and genes that are up- or down-regulated during infection of soybean roots by Pratylenchus. Several Pratylenchus genes were identified and are being subcloned for transformation of soybean hairy roots. An RNAi strategy will be evaluated for its effectiveness in decreasing infection of soybean roots by nematodes. The GUS gene was subcloned under either the rolD, mas2, or rice root3 promoters and used to transform lilies. The three promoters resulted in expression primarily in the roots, although there was some expression in the shoots. These promoters should be useful when transforming lilies with anti-nematode genes because the nematode infects roots. Lilies have been transformed with a dsRNA construct that contains a portion of the replicase gene from a lily isolate of Cucumber mosaic virus with the intent of creating virus-resistant lilies. Currently these plants are being multiplied for future challenge with Cucumber mosaic virus. A system for micropropagation and regeneration of Ulmus parvifolia has been defined. Meta-topolin added to Driver Kuniyaki Walnut medium resulted in regeneration of plants from leaves. Micropropagation required either benzylaminopurine or meta-topolin.
1. Transcriptome analysis of Pratylenchus penetrans, the root lesion nematode. Worldwide crop losses due to plant-parasitic nematodes have been estimated at $118 billion annually, with root lesion nematodes, Pratylenchus spp., ranking third in terms of economic loss. The molecular mechanisms of pathogenicity of root lesion nematodes are poorly understood, and this may be attributed to the limited knowledge of the functional analysis of their genes. ARS researchers in Beltsville, Maryland, have completed an overall analysis of the transcriptome of P. penetrans. A subset of genes, potential effector genes, has been identified and will be the subject for future research. Understanding the interactions between P. penetrans and its host plant at the molecular level will provide a basis for controlling this nematode species.
Kamo, K.K., Lakshman, D.K., Bauchan, G.R., Rajasekaran, K., Cary, J.W., Jaynes, J. 2015. Expression of a synthetic antimicrobial peptide, D4E1, in Gladiolus plants for resistance to Fusarium oxysporum f. sp. gladioli. Plant Cell Tissue And Organ Culture. 121:459-467.
Kamo, K.K., Lakshman, D.K., Rathore, K. 2014. Disease resistance: Molecular mechanisms and biotechnological applications. Plant Science. 228:1-2.