We're looking for our next director.
This is an open-rank professor position. Application review starts on September 2, 2024.
The Plant Transformation Core Research Facility can provide resources for the evaluation of transgenes in plant systems. The facility is a state-of-the-art laboratory with the expertise to deliver genetic constructs into both model plant systems and two target crops, wheat and soybean. Clients are provided with R0 plants or R1 seed derived from the primary transformants.
Director: Tom Clemente
N308 Beadle Center
402-472-1428
tclemente1@unl.edu
Clemente Lab
Plant Transformation Services (Option 1)
Full service transformations for the model plant species, Tobacco (Nicotiana tabacum and Nicotiana benthamiana) and Tomato (Solanum lycopersicum) is available upon request. Transformations are conducted using Agrobacterium-mediated transformation protocols. Clients outside the state of Nebraska will be required to obtain USDA/APHIS interstate movement permits for shipment of transgenic events. We will advise customers and collaborators on how to obtain the necessary permits.
The University of Nebraska’s Plant Transformation Core Research Facility has the capacity to conduct transformations for the major commodities, including maize, soybean, sorghum and wheat. All transformations are conducted using Agrobacterium-mediated protocols. Transformations with commodities are carried out on a project agreement basis and custom quotes will be provided for each project.
Plant Transformation Services (Option 2)
Transformations of the model crops, wheat and soybean, will be carried out via Agrobacterium-mediated protocols. Preliminary screens will be conducted to verify transformation events, by monitoring for the expression and/or integration of the selectable marker. Presence of the desired transgene will be guaranteed within the lines delivered, however, co-expression of the trait can not be assured.
The client may request the full service of the Facility, in which case we will initiate and maintain all the cultures and the client will be provided with the transformants. Clients wishing to conduct their own transformation may utilize the laboratory and have all the necessary supplies and growth chamber space provided. In the latter case the Facility's staff will be available for assistance and consultations.
Agrobacterium-mediated transformation will be conducted utilizing established protocols for the model crop systems, Arabidopsis and Tobacco. For the former plant species, transformations may be conducted by either the root transformation or by the vacuum infiltration of whole plants.
Soybean transformations will utilize a direct organogenic regime for plantlet differentiation coupled with Agrobacterium-mediated transformation for gene delivery into the crop.
Wheat transformations will be carried out using Agrobacterium-mediated transformation protocol using immature embryos as the target tissue. The plantlet formation will arise via an embryogenic pathway from the cultured explants.
The client will provide the Agrobacterium culture carrying the desired cassettes. Plasmid rescue from Agrobacterium and conformation of the plasmid's integrity must be conducted prior to cultures being handed off to the Facility.
Preliminary expression data must be ascertained prior to the constructs being delivered into soybean or wheat. Preliminary expression data may include information obtained from stable expression within model crop systems and/or transient expression data collected from a protoplast system.
Service Charges
Option 1
| Service | Fee |
|---|---|
| Tobacco Transformations | $450 per construct (10 events) for rooted plants in medium |
| Tomato Transformations | $750 per construct (10 events) for rooted plants in medium |
| Wheat, Soybean, Maize & Sorghum Transformations | Please contact Plant Transformation Core Facility Director |
Option 2
Due to large volume of transformation set ups across the plant species, the Plant Transformation Facility will be purchasing supplies in bulk quantities and therefore will be recovering a significant discount on the media additives. This savings in media costs will allow the Facility to provide services at rates well below those that would be accrued within individual laboratories conducting plant transformations on a small scale. The Facility will provide a detailed cost assessment for the transformation setups prior to the initiation of the project.
Price list of services
| Vector Constructions | $10 per vector |
| Tobacco Transformations | $7.50 per plant (10 plant minimum) |
| Arabidopsis Transformations | $3.50 per pot (4 pot minimum) |
| Tissue Culture/Transformation Service | Per liter of medium (at cost) |
| Wheat, Soybean, Maize & Sorghum Transformations | Setup as Research Collaboration or as an negotiated Research Agreement |
Plant Transformation Protocol
Soybean Cotyledonary-node Agrobacterium-mediated Transformation System
The protocol outlined below is a modification of that described by Hinchee et al. (Bio/Technology 1988. 6:915). The modifications have been previously reported. See Zhang et al., 1999. Plant Cell Tiss. Organ Cult. 56:37; Clemente et al. 2000. Crop Sci. 40:797, and Xing et al. 2000 In Vitro Cell Dev. Biol. Plant 36:456.
- Seed Sterilization Step
We currently use three genotypes A3237 (Asgrow Seed Company), Thorne (Ohio State University) and NE3001 (University of Nebraska).
Place seed in an open petri plate as a single layer. Place the petri plate into a standard size desiccator within a fume hood. Place a 250 ml beaker with 100 ml of Chlorox™ bleach in the center of the desiccator. Add 3.3 ml of 12 N HCl, dropwise, along the side of the beaker. Close the desiccator and let stand overnight. The next day cover petri plate with a sterile lid. The sterilization may need to be repeated depending on the seed lot.
- Seed Germination Step
Germinate the sterilized seeds on germination medium (GM) for 5 days at 24°C (18/6) light regime) in 100 X 25 mm petri plates (Place 10-15 seed per plate). Stack the plates 5 high and place in a clear plastic bags with 4 to 5 slices in the bag.
GM medium
Gamborg’s B5 medium supplemented with 2% sucrose, pH 5.8, solidified with 0.8% agar.
- Agrobacterium Inoculum Preparation
The protocol outlined below is a modification of that described by Hinchee et al. (Bio/Technology 1988. 6:915). The modifications have been previously reported. See Zhang et al., 1999. Plant Cell Tiss. Organ Cult. 56:37; Clemente et al. 2000. Crop Sci. 40:797, and Xing et al. 2000 In Vitro Cell Dev. Biol. Plant 36:456. Pellet 35 ml of the A. tumefaciens culture at 3,000 to 5,000 rpm for 10 min. Suspend the bacterial pellet in co-cultivation medium to a final OD650 of 0.6 to 1.0.
Co-cultivation Medium
1/10th Gamborg’s B5 medium supplemented with 1.67 mg/l BAP, 0.25 mg/l GA3, 3% sucrose, 200 µM acetosyringone, 20 mM MES, pH 5.4. Filter sterilize all growth regulators and acetosyringone.
- Explant Preparation
Place 25 ml of Agrobacterium inoculum in to a petri plate. Add the 30 to 40 prepared explants; be sure explants are completely covered with the inoculum. The inoculation step should be carried out for 30 min., with occasional agitation. Place the explants on co-cultivation plates (5 per plate), adaxial side down.
- Co-cultivation Plates
Co-cultivation medium solidified with 0.5% purified agar overlay a piece of sterile filter paper on the top of each plate. Wrap the plates with parafilm and place at 24°C, 18/6 light regime. Allow the tissue to co-cultivate for 3 days. Following the co-cultivation period briefly wash the explants in liquid shoot initiation medium supplemented with 0.25 mg/l GA3. Pellet 35 ml of the A. tumefaciens culture at 3,000 to 5,000 rpm for 10 min. Suspend the bacterial pellet in co-cultivation medium to a final OD650 of 0.6 to 1.0.
Shoot Initiation Medium
Gamborg’s B5 medium supplemented with 1.67 mg/l BAP, 3% sucrose, 50 mg/l ticarcillin, 50 mg/l cefotaxime, 50 mg/l vancomycin; 3 mM MES, pH 5.6. Solidify medium with 0.8% purified agar. If using the bar gene as a marker use 5 mg/l glufosinate. Filter sterilize all growth regulators, vitamins, antibiotics and selection agent.
- Shoot Initiation Step
Culture the explants adaxial side up, with the junction of the hypocoyl/cotyledon just below the surface. Wrap the plates (100 X 25 mm) with porous tape and culture at 24°C, 18/6 light regime.
Transfer the tissue to fresh shoot initiation medium after 14 days. At the 124-day period cut the hypocoyl region flush to the developing node. Orient the tissue so that the freshly cut surface is imbedded in the medium, with the differentiating region flush to the surface. Allow the tissue to culture for an additional two weeks under the same environmental conditions.
- Shoot Elongation Step At the end of the shoot initiation period only carry on the differentiating explants and discard the rest. Approximately 80% of the explant should be differentiating at this point. Remove the cotyledon from the explants, make a fresh cut at the base of the developing node (horizontally) and transfer the tissue to shoot elongation medium. Culture at 24°C (18/6 light regime).
Shoot Elongation Medium
MS salts/Gamborg’s vitamins supplemented with 1 mg/l zeatin riboside, 0.1 mg// IAA, 0.5 mg/l GA3, 3% sucrose, 100 mg/l pyroglutamic acid, 50 mg/l asparagine, 3 mM MES (pH 5.6) solidified with 0.8% purified agar. Maintain the levels of ticarcillin, cefotaime and vancomycin to that used during shoot initiation. If using the bar gene as a marker use 3 mg/l glufosinate. Filter sterilize all growth regulators, vitamins, antibiotics and selection agent.
Transfer the tissue to fresh shoot elongation medium every two weeks. At each transfer make a fresh horizontal slice at the base of the tissue. Once a shoot has elongated to approximately 1 cm in size transfer the isolated elongated shoot to a cup with elongation medium for an additional two weeks.
- Rooting Step
Root elongated shoots (> 3 cm) on rooting medium without further selection.
Rooting medium
1/2 MS salts full strength Gamborg’s vitamins, 2% sucrose, 0.5 NAA, 50 mg/l asparagine, 100 mg/l pyroglutamic acid, 3 mM MES (pH 5.6). Solidify the medium with 0.8% purified agar. Do not supplement the medium with antibiotics used to counter select A. tumefaciens. This will drastically impede root development. Root the shoots in cups. Rooting usually takes between 2 to 4 weeks. Acclimate the rooted shoots in Metro Mix 360 prior to establishing in the greenhouse.