Location: Floral and Nursery Plants Research2016 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 level of beta-glucuronidase (GUS) expression was determined for lilies transformed with the uidA gene under control of either the CaMV 35S, mas2, or rolD promoters. Levels of GUS expression were comparable for roots with either the CaMV 35S or mas2 promoters, and levels were generally very low for roots with the rolD promoter. GUS expression was high in leaves with the CaMV 35S promoter and low with the mas2 and rolD promoters. Hairy roots of soybean have been transformed with eight nematode metabolic genes, seven parasitism-related genes, and currently five putative effector genes. These plants were confirmed to contain the transgenes by PCR, and levels of expression determined by qPCR. Hairy roots with the highest levels of expression are being multiplied for future challenge with the root lesion nematode, P. penetrans. Lilies transformed with dsRNA of the replicase gene have been multiplied and planted in soil for future challenge. Expression of the transgene was determined using qPCR and PCR of the intron. Plants of Ulmus parvifolia, the Chinese elm, that resulted from regeneration or in vitro multiplication have been established in the greenhouse. The acclimatization rate was high (96-100%).
1. Characterization of four promoters for expression in roots of lilies. Successful genetic engineering of lilies for resistance to root lesion nematodes requires a promoter that directs high levels of expression in roots. It is desirable that the promoter direct expression specifically in roots rather than other plant organs. ARS scientists in Beltsville, MD compared expression in roots of lilies using either the CaMV 35S, rolD, rice root3, or mas2 promoters and determined that levels of expression were comparable with the CaMV 35S and mas2 promoters. Expression was low with the rolD promoter. Histological staining showed expression throughout the root with the CaMV 35S, rolD, and mas2 promoters, but expression was limited to the stele with the rice root3 promoter. The CaMV 35S promoter directed high levels of expression in leaves as compared to the low levels from the mas2 and rolD promoter. The mas2 promoter has been identified as a promoter that directs expression of gene expression primarily in roots rather than shoots making it potentially useful for genetic engineering lilies or other crops for resistance to the root lesion nematode that feeds on roots.
Kamo, K.K., Lakshman, D.K., Pandey, R., Guaragna, M.A., Okubara, P.A., Rajasekaran, K., Cary, J.W., Jordan, R.L. 2015. Resistance to Fusarium oxysporum f. sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes. Plant Cell Tissue And Organ Culture. 124:541.
Vieira, P., Eves-Van Den Akker, S., Verma, R., Wantoch, S., Eisenback, J.D., Kamo, K.K. 2015. Characterization of the Pratylenchus penetrans transcriptome including data mining of putative nematode genes involved in plant parasitism. PLoS One. 10(12):e0144674. doi: 10.1371/journal.pone.0144674.