Abstract
We have transformed internode sections of the hybrid elm cultivar “Pioneer” to assess the possibilities of introducing significant genes into elms. Foreign DNA encoding beta-glucuronidase (GUS) was introduced into internode sections using microprojectile- or Agrobacterium tumefaciens-mediated transformation. Positive signals were detected by Southern analysis. Also, some enzymatic activity was detected in Agrobacterium transformed cultures using a fluorogenic assay.
Dutch elm disease (DED) is a devastating vascular wilt disease caused by the fungus Ophiostoma ulmi, which has nearly annihilated the American elm in North America. Approaches toward protecting susceptible elms from DED include introducing disease resistance genes (to deter the fungal pathogen) or insect resistance genes (to ward off the beetle vector). These goals, which utilize biotechnology, require: 1) the identification of a single-gene trait associated with the desired phenotype, 2) access to the DNA responsible for this phenotype, and 3) techniques for the transformation and regeneration of the plant of interest.
Progress in these three areas, with respect to DED, has been slow. Because of the complexity of plant resistance and defense mechanisms, identifying the isolating single gene products has not yet been possible, although work is currently underway to identify restriction fragment length polymorphisms which could be used as molecular markers for resistance (R.K. Hajela and M.B. Sticklen, unpublished observations). In addition, some work has been done to establish regeneration procedures for the American elm (2, 8, 9, 17, and M.G. Bolyard, C. Srinivasan, and M.B. Sticklen, unpublished observations).
We have attempted to introduce foreign genes into a variety of elm for which regeneration protocols have been developed. The regeneration of shoots from the hybrid elm “Pioneer” (16) was reported following treatment of shoot tips, leaves and internodes (4) and protoplasts (15). Foreign DNA was inserted into “Pioneer” elm internode sections via techniques that have been used previously for major crop species, including microprojectile bombardment (13) or Agrobacterium tumefaciens-mediated transformation (5).
Materials and Methods
Plant Material
“Pioneer” elms were obtained as rooted cuttings from the Agricultural Research Service, Delaware, OH. Rapidly growing stem sections of “Pioneer” elm were excised from greenhouse grown plants and surface sterilized with 20% commercial bleach (Chlorox) and 1 % Tween 20, for 20 minutes, then rinsed extensively with sterile distilled water. After two days of preculture on modified Murashige and Skoog medium (11) [Sigma #6899 supplemented with 100 mg/L casein enzymatic hydrolysate, 100 mg/L myoinositol, 3% sucrose, and 200 mg/L thiamine HCI, hereafter referred to as MS] to test for contamination, internode sections were excised for transformation.
Transformation Procedures
Co-cultivation of internode sections with a 1:10 dilution of Agrobacterium tumefaciens (strain LBA4404) containing the binary vector pBI121 (Clontech, 7) was carried out overnight. Incubated internodes were washed twice in liquid MS medium containing 500 mg/L carbenicillin, then cultured in petri dishes containing the same medium supplemented with 10 uM benzyl adenine (BA) and 6% phytagar (Gibco). Internodes obtained in the same fashion were bombarded with tungsten particles (1.2 urn, Sylvania GTE M17) coated with pBI121 or pActF-1 (10) plasmid DNA via the “Biolistic” PDS-1000 microprojectile bombardment device (DuPont). Bombarded internodes were plated on MS medium containing 0.5 mg/L pyridoxine, 0.5 mg/L nicotinic acid, 10 uM BA, and 6% phytagar.
DNA Analysis
DNA from callus of putatively transformed cultures was extracted by the method of Dellaporta et al. (1). Total DNA was analyzed by Southern analysis (14) following transfer to Nytran (0.45 urn, Schleicher and Schuell). Filters were hybridized with the appropriate probes which had been labeled with 32P (800 Cl/mmol, New England Nuclear) using the random priming method (3). Following hybridization, filters were washed at high stringency (12) and exposed to X-ray film (X-omat AR-5, Kodak).
Fluorogenic Assay
The fluorogenic assay was performed as described by Jefferson (6), using the substrate 4-methyl umbelliferyl beta-D-glucuronide (MUG). Briefly, approximately 50 mg of callus material was extracted in microcentrifuge tubes containing 500 ul GUS extraction buffer. 50 ul of extract was then added to 50 ul of assay buffer containing 1 mM MUG. After 60 minutes of incubation at room temperature, samples were analysed by ultraviolet irradiation and photographed.
Results
Callus cultures were established following bombardment of “Pioneer” elm internodes by culturing the explants on modified MS medium supplemented with 10 uM BA (4). Most callus cultures (Figure 1) did not regenerate shoots, although shoots were regenerated from cultures following a mock bombardment (Figure 2). After approximately six months of culture, the callus was pooled according to the genes used for transformation, and total DNA was extracted.
Callus culture from pioneer elm internode section cultured on MS medium supplemented with 10uM BA, photographed after approximately 3 months in culture following bombardment.
Shoot regenerated from an Internode following a mock bombardment with tungsten particles.
DNA from callus cultures bombarded with one of two plasmids containing a gene encoding betaglucuronidase (GUS), pBI121 and pActF-1 (10), as well control cultures, was probed with a 32P labeled Bam Hl-Eco Rl fragment from pBI121. As shown in Figure 3, a positive signal can be observed in each of the duplicate lanes containing pBI121 (lanes B and E), but no signal was observed in the control lanes (lanes A and D) or lanes containing pActF-1 (lanes C and F).
Southern analysis of total DNA isolated from control (Lanes A and D), pBI121 (lanes B and E), or pActF-1 (lanes C and F), following hybridization with a 32P labelled fragment from the GUS gene In pBI121.
Total DNA was also isolated from callus cultures initiated from internode sections which had been co-cultivated with Agrobacterium containing PBI121. These cultures also failed to produce shoots, although some organization was detected (data not shown), which was later covered with callus. DNA was extracted from ten pools of callus material for Southern analysis (14). Six of the ten samples showed a signal when probed with a portion of pBI121 (data not shown). In addition, part of the callus material was used in a fluorogenic assay which is routinely used to detect betaglucuronidase activity in transgenic plants (6). One of the extracts, which gave a positive signal by Southern analysis, showed fluorescence (Figure 4, tube F).
Microcentrifuge tubes containing pioneer elm callus extracts treated with buffer containing the substrate MUG. The tubes are arranged on a UV light box at a wavelength of 302 nm. Letters denote tubes containing extracts from different callus cultures.
Discussion
We have demonstrated the transformation of Pioneer elm internodes using the microprojectile and Agrobacterium methods. Southern analysis revealed bands from samples transformed with pBI121, while no signal has been detected from material treated with pActF-1, each following transformation by microprojectile bombardment. In addition, plants were regenerated from internodes following a mock bombardment, indicating that the physical stress to the internodes caused by bombardment with tungsten particles was not sufficient to prohibit regeneration. However, it is not clear why the transformed callus cultures did not produce shoots.
Also, fluorescence activity and positive signals following Southern analysis have been detected from samples co-cultivated with Agrobacterium. However, none of the callus cultures produced shoots, indicating that perhaps co-cultivation with Agrobacterium, culture on media containing carbenicillin, or the lower levels of vitamins in the media could have interfered with regeneration.
These experiments provide the first evidence of elm transformation. It is to be hoped that these results will be the beginning of more investigations into the applications of the tools of molecular biology for the improvement of elms, particularly in the area of increasing resistance to Dutch elm disease, including the fungal pathogen and the beetle carriers, in order to maintain the American elm as part of the North American landscape.
Acknowledgements
The authors wish to thank the International Society of Arboriculture Trust (Grant #144) and the Department of Interior (CA-3040-9-8001) for supporting this research. We also thank Warren Masters of the Agricultural Research Service, Delaware, OH, for his assistance in helping us obtain plant material, Drs. C. Srinivasan and J. Cheng for reviewing our manuscript, and N. Hajela, N. Patel, B. Paxton, and M. Sandborn for their excellent technical assistance.
- © 1991, International Society of Arboriculture. All rights reserved.
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