Agrobacterium and Constructs used in transformation experiments.
Agrobacterium-based experiments were performed using Agrobacterium tumefaciens strain GV3101 pMP90 (17). The following plasmids were used: pGFPGUSPlus (18); Addgene plasmid # 64401; contains CaMV35S-driven hygromycin resistance, EGFP reporter, and GUS reporter; pMYB113 (15); contains NOS promoter driven hygromycin resistance, CaMV35S:Arabidopsis MYB113; pPM-YB (19); contains plasma membrane localized YFP, Mannopine Synthase Promoter:phosphinothricin (Basta) acetyl transferase; pEZS-CL (20); a high copy plasmid containing CaMV35S:EGFP.
For the leaf infiltration experiments, we also included the RNA silencing suppressor, P19 in Agrobacterium strain GV 3101 (21). Agrobacterium and E. coli strains were maintained on LB media with appropriate antibiotics to select for the plasmids and Rifampicin 10 mg/L and Gentamycin 30 mg/L to select for Agrobacterium GV3101 pMP90 (17).
Agrobacterium mediated transformation and regeneration of common dandelion.
10 to 15 cm long, young leaves were excised from soil grown plants (Fig. 1a) and placed in 10% bleach with 0.04% Tween 20 for 10 minutes for surface sterilization, and then rinsed 4 times in autoclaved water. The Agrobacterium solution was prepared from overnight Agrobacterium cultures grown in 5mL LB media under antibiotic selection. These cultures were pelleted via centrifugation at 3000 x g for 10 minutes. The pellet was resuspended in induction solution (1XMS, 3% Sucrose, 1% Glucose, 0.2mM acetosyringone, pH 5.2) as a first wash, and then pelleted and resuspended a second time in 5 mL induction solution. The Agrobacterium in induction solution was then incubated at 30 oC with agitation for 1 hour. Leaves were cut into strips approximately 0.75 cm long spanning the width of the leaf (Fig. 1b). The leaf strips were placed into an empty petri dish, and the Agrobacterium induction solution was added to the leaf strips with gentle agitation for an incubation period of 5 minutes. These strips were gently tapped on an empty petri dish to remove excess agrobacterium induction solution, and placed adaxial side up on 10 cm petri plates containing cocultivation media (1XMS, 3% Sucrose, 1% Glucose, 0.1mM acetosyringone + 2mg/L BAP, 0.1 mg/L NAA, 0.9% agar pH 5.2). Petri dishes were sealed with micropore tape and leaf pieces and Agrobacterium were co-cultivated for 2 days in darkness at 26 oC. After co-cultivation, leaf pieces were moved to media containing 1XMS, 3% Sucrose, + 2mg/L BAP, 0.1 mg/L NAA, 300 mg/L Timentin, 0.9% agar pH 5.2 and sealed with parafilm. After 7 days, the leaf pieces were transferred to shoot induction media (1XMS, 3% Sucrose, 2mg/L BAP, 0.1 mg/L NAA, 0.9% agar, 300 mg/L Timetin, pH 5.7) containing the appropriate antibiotic or herbicide to select for transformed dandelion cells. We used 100 mg/L kanamycin, 50 mg/L hygromycin, or 3 mg/L BASTA (phosphinothricin), all from Sigma. Callus was observed to grow mostly from leaf veins, and leaf pieces were moved to new selection media approximately every 2 weeks (Fig. 1c). The timing of differentiation of shoots was variable but often occurred as early as 5 weeks. When leaves on shoots were approximately 1.5-2 cm long, they were transferred to the same media but with 0.1 mg/L NAA to elicit root differentiation (Fig. 1d, e). Root formation was also variable with some shoots rooting in 6 weeks and some shoots taking 4 months. Shoots with well-formed roots at least 1.5 cm long were transferred to soil (Promix BX; Hummert International) and placed in flats with transparent lids to keep the humidity high (Fig. 1f). Once new leaf growth was observed, the lids were gradually removed.
Genotyping of transgenic dandelion; inheritance of TDNA in progeny of primary transformants
PCR amplification of sequences on the Agrobacterium TDNA was used to assess whether potential regenerated transformants indeed contained the transgene. PCR primers and the size of the expected DNA fragments are listed in Table 1. Genomic DNA was isolated using the CTAB method (22), using 10-15 mg of tissue from leaves that emerged after the plants were transferred to soil or from tissue from the next generation seedlings. To test for markers in transgenic progeny, seeds were sown in 10 cm square pots on the soil surface (promix BX) and grown at 24o C under fluorescent lights with a 16/8 hour light/dark cycle. Seeds generally begin germination in 4 days.
Basta herbicide resistance assay
For basta herbicide resistance assays, seeds were sown as above and 10 to 14 day old wildtype or basta resistant seedlings grown in 2.5 inch pots were treated with 25 ml of 3 mg/l basta, results were collected 7 days after exposure.
Agrobacterium mediated leaf-infiltration for transient gene expression.
The bacteria grown as above and were harvested by centrifugation for 3 min at 3,000 x g. The pellets were rinsed by resuspension in the same volume of infiltration buffer (10 mM MgCl2, 10µM acetosyringone) and centrifuged again for 3 min at 3,000 x g. Pellets were again resuspended in infiltration buffer. The OD600 was measured and each strain was diluted to OD600 of 0.1 with infiltration buffer. Each infiltration experiment contained two Agrobacterium strains: one strain contained the transient gene expression construct and the other contained the RNA silencing suppressor, P19 (21).
Young dandelion plants approximately four weeks of age were used for infiltration. The bacteria suspension was infiltrated into the abaxial leaf surface using a 1 ml tuberculin syringe without needle in the method of Vaghchhipawala et al. (16). Briefly, the syringe tip is held tightly to the abaxial leaf surface and a gloved finger is held on the opposite adaxial side while the syringe plunger is gently but firmly pushed forcing the Agrobacterium solution into the leaf interior. 3-5 days post infiltration the leaf was excised from the plant and transient gene expression was tested by either GUS staining according to Jefferson et al. (23), observing YFP fluorescence under a fluorescent microscope, or visual inspection for anthocyanin accumulation in the infiltrated area.
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Protoplast preparation
Two incubation regimes were tested, 30o C for 3 hours and 22o C for 15 to 17 hours. For the 30o C treatment, the enzyme solution was prepared as follows, 20 mM MES (pH 5.7), 0.5 M mannitol, 20 mM KCl, cellulase R10 1.0% wt/vol (Yakult Pharmaceutical Industry Co., Ltd), macerozyme R10 0.5% wt/vol (Yakult Pharmaceutical Industry Co., Ltd). This enzyme solution was heated to 60o C for 5 minutes and cooled to room temperature. CaCl2 was added to 10 mM and BSA to 0.1%. The 22o C treatment used the same solutions except for 0.45% wt/vol cellulase R10, 0.2% wt/vol macerozyme R10.
Protoplasts were prepared from the 3rd to 5th leaves (approximately 5 to 6 cm long) from 3 to 4-week old dandelion plants. Dandelion leaves were detached from the plants and lightly scratched with sandpaper (3M 413Q, 220 grit) on the abaxial side before immediate submersion in 10 ml of enzyme solution. They were incubated at either 22o C overnight or 30o C for 3 hours in the dark in 10 cm petri dishes. Sterilization is not necessary for transient expression assays. After the incubation period, 10 ml of W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl) was then added to the enzyme solution to stop the reaction. The 20 ml reaction mix was filtered through a 100 µm cell strainer (Fisher Scientific., Cat# 22-363-549) and split into two 15 ml round bottom tubes. The protoplasts were collected by centrifuging for 2 minutes at 1000 x g. The supernatant was removed by pipetting as much liquid as possible while leaving the pellet intact in the tube. The green pellets were resuspended by adding 2 ml of W5 solution and the tubes were incubated on ice for 30 minutes. Healthy protoplasts could be easily resuspended in the W5 solution at this step.
The cell number was counted using a hemocytometer. We typically obtained 4-6 x 105 cells per 0.3 gm fresh weight of the leaves. The protoplasts were resuspended at 5 x 105 cells/ml in MMG solution (4 mM MES pH 5.7, 0.4 M mannitol, 15 mM MgCl2) prior to transformation.
Free DNA delivery to protoplasts
A total of 10 µg of plasmid DNA (pEZS-CL) at 1.5 to 3 µg/µL concentration was added to 100 µl of MMG resuspended protoplasts and this was gently mixed before adding 100 µl of PEG transformation solution (30% wt/vol PEG4000 (Sigma Aldrich., Cat# 95904), 0.2 M mannitol, 100 mM CaCl2) for 5 mins. The transformation reaction was stopped by adding 20 ml of W5 solution and the protoplasts were collected by centrifugation for 2 min at 1000 g. Transformed protoplasts were incubated at 22o C for 15 to 17 hours in WI solution (4 mM MES pH 5.7, 0.5 M mannitol, 20 mM KCl) before the GFP signal was detected. We also tested protoplast transformation using these same conditions but replacing 30% PEG with 20% or 40% PEG.
Observation of Fluorescent Protein or GUS expression
YFP or GFP protein fluorescence was observed on an Olympus BX53 microscope with a YFP or GFP filter at 10x with a 1 second exposure. GUS staining was done according to Jefferson et al. (23).