Location: Floral and Nursery Plants Research2014 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.
In FY2014, the project had significant progress in the areas of evaluation of transgene expression and evaluation. Lilies and soybean hairy roots transformed with a cystatin, a gene that confers resistance to nematode feeding, showed a reduction in the number of nematodes. Transgenic lilies also showed an increase in plant weight. This correlation shows that we should be able to use the much faster soybean hairy root system as a model for testing anti-nematode activity of genes before spending the long time needed to transform lilies. Anti-nematode genes that appear to confer resistance in the soybean hairy root system will be used to transform lilies. Following a transcriptome analysis of Pratylenchus (nematode) infection with soybean, several genes have been selected for testing in soybean. These studies could lead to the identification of an effective gene that can be used to confer nematode resistance in lilies. Cucumber mosaic virus (CMV) affects lilies, and a portion of the CMV replicase gene has been subcloned and used to transform lilies. If successful, this gene may result in lilies that are resistant to CMV. Ulmus parvifolia, elm, has been established in vitro and optimal micropropagation parameters are being determined. This will lead to a method to multiply select clones or taxa of these elms for future conservation or distribution.
1. Transgenic nematode resistant lilies created. Lilies are an important floral crop sold as cutflowers, pot plants, or grown in gardens. Attack by the root lesion nematode is a serious problem when growing lilies in the field, as it leads to reduced growth and increased susceptibility to attack by fungal pathogens. There are no commercially important lilies resistant to nematodes that can be used in breeding. ARS researchers in Beltsville, MD have developed lily plants that contain cystatin, an anti-nematode gene. Lily plants with the cystatin gene were challenged with nematodes in culture, and there was a decrease in the number of nematodes. These plants are now being grown under field conditions in the Pacific Northwest where root lesion nematodes are a major problem.
Kamo, K.K. 2014. Transgene expression of lilies grown in the greenhouse and outdoors. Scientia Horticulturae. 167:158-163.
Ozel, C.A., Kamo, K.K. 2014. Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White). Acta Horticulturae. 1002:231-236.