Location: Floral and Nursery Plants Research2011 Annual Report
1a. Objectives (from AD-416)
Utilize genetic engineering to improve resistance of bulb crops to fungal diseases and plant parasitic nematodes. (a) Develop transformation technology for Easter lily; (b) test candidate genes to confer resistance to Fusarium in Gladiolus; and (c) determine whether dsRNA expressed in plants can target RNAi to control plant parasitic nematodes.
1b. Approach (from AD-416)
Transform a commercially important Gladiolus cultivar, Easter lilies, and Ornithogalum with genes for resistance to either cucumber mosaic virus or ornithogalum mosaic virus. Determine if the genetically engineered plants are resistant to these viruses. Complete experiments involving cucumber mosaic virus resistance in transgenic Gladiolus plants that contain either the CMV coat protein subgroup 1 or subgroup 2, CMV replicase, or single chain antibodies to the CMV coat protein. Optimize the transformation system for Easter lilies and Ornithogalum using either the gene gun or Agrobacterium. Develop a transformation system for roots of Easter lilies using Agrobacterium rhizogenes. Transgenic Gladiolus plants with D4E1 and cpo have been developed using the gene gun and are being grown for challenging with Fusarium in the greenhouse. Gladiolus will also be transformed with various chitinase genes for possible resistance to Fusarium. Evaluate effectiveness of genetically engineered resistance to Fusarium oxysporum in Gladiolus by comparison to biological control technologies. Develop Easter lilies that are resistant to the nematode Pratylenchus penetrans, the root lesion nematode. Genes involved in nematode development will be isolated and tested for their ability to kill Pratylenchus penetrans for affect its reproduction using RNAi.
3. Progress Report
Conditions have been optimized for transformation of lilies using the gene gun, and this has resulted in six lines of transgenic lilies with the cystatin gene for nematode resistance. Lilies are now being transformed with the gus gene under control of either the rolD or mas2 promoters using the gene gun. These promoters have been shown to express high levels in roots which is desired for engineering nematode resistance in lilies. Experiments are being done to determine the conditions for Agrobacterium-mediated transformation of lilies. Transient gus expression with A. tumefaciens has been acheived using bulb scales and basal plates. A. rhizogenes strains R1000 and K599 did not infect lilies. Fusarium oxysporum has been transformed with three fluorescent reporter genes. The cyan gene was found to be better than the green or yellow fluorescent genes for future studies on studying the interaction of Fusarium with transgenic Gladiolus roots because non-transformed Gladiolus roots fluoresce green. A comparison of transgene expression and resistance using plant extracts and in vitro-grown plants is being made between the transgenic lines of Gladiolus that have the synthetic antimicrobial peptide D4E1. The selected line will be used in a detailed study of the interaction between Fusarium and Gladiolus roots. It has been difficult acclimating in vitro-grown Gladiolus plants to grow in soil in a greenhouse. Several Metromix, sand, vermiculite, and perlite combinations have been tested for their effect on germination and growth of in vitro-grown corms.