Location: Floral and Nursery Plants Research2017 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.
Lilies transformed with the rice cystatin gene showed resistance to root lesion nematodes in vitro, and bulbs of the transformed lilies were larger and weighed more than lilies lacking the rice cystatin gene. These results in vitro showed that the rice cystatin may be useful for nematode resistance if lilies with this gene show resistance to a high population of nematodes under field conditions. Successful genetic engineering for nematode resistance requires expression of the transgene in roots where root lesion nematodes feed. Expression of the GUS reporter gene under the control of either the CaMV 35S, mas2, rice RPC1, or rolD promoters was examined because a promoter that expresses at high levels in roots is needed for effective resistance to root lesion nematodes. Levels of GUS expression were highly expressed throughout roots with the CaMV 35S and mas2 promoters indicating the possibility of using the mas2 promoter for expression in roots of lilies. GUS expression was significantly lower with the rolD promoter, and expression was only in the stele of roots with the rice RPC1 promoter, so these two promoters are not useful when engineering lilies for root lesion resistance. In Objective 2, eight metabolic and seven parasitic genes were identified from a transcriptome of the root lesion nematode and used as dsRNA gene constructs to transform soybean hairy roots. Soybean hairy roots showed resistance to nematodes with several of these targeted genes using an RNAi approach. Under Objective 3 we developed lilies with a dsRNA antiviral gene for Cucumber mosaic virus and screened the lilies for resistance to the virus. Expression of the transgene was verified in lilies, and experiments to challenge the lily lines with virus are in progress. Under Objective 4, we found that meta-topolin is better for micropropagation and regeneration of Chinese elm trees from leaves and callus as compared to two other cytokinins tested (thidiazuron and benzyladenine). Both micropropagated and regenerated plants grown in the greenhouse appear phenotypically normal.
1. Improved production of Easter lilies. Easter lilies are a valuable commodity to the floral industry, but are increasingly difficult to grow because of their susceptibility to a variety of pathogens. Many of the pesticides used to control these pathogens have been banned, and others are expected to be removed from the market in the future, so growers are looking for methods to produce quality lily plants with decreased pesticide use. An ARS scientist in Beltsville, Maryland, created pathogen-free Easter lily plants in tissue culture and sent them to the Easter Lily Foundation in Brookings, Oregon, where they were grown for three seasons in the field. The pathogen-free lilies from tissue culture were much more vigorous than the controls. Growers are now planning to put lily bulbs through tissue culture in order to create pathogen-free planting stock to increase yield and quality of lily plants.
Vieira, P., Lakshman, D.K., Pandey, R., Slovin, J.P., Kamo, K.K. 2017. Symptom development in response to combined infection of in vitro grown Lilium longiflorum with the root lesion nematode Pratylenchus penetrans and soilborne fungi collected from diseased roots of field-grown lilies. Plant Disease. 101:1-8.
Vieira, P., Kamo, K.K., Eisenback, J.D. 2017. Plant-mediated silencing of the fatty acid- and retinoid-binding Pp-far-1 gene can reduce development of the root lesion nematode, Pratylenchus penetrans. Plant Pathology. 66:1214-1224.
Vieira, P., Mowery, J.D., Kilcrease, J., Eisenback, J.D., Kamo, K.K. 2017. Cytological changes of Easter lily (Lilium longiflorum) upon root lesion nematode (Pratylenchus penetrans) infection. Plant Pathology. 49:1-11.
Wade, E.H., Kamo, K.K. 2016. Gladiolus diseases. Handbook on Florist Crop Diseases. 2-19.