Sep 12, 2025
Tragopogon (Asteraceae) transformation and regeneration
- Shengchen Shan1,2
- 1Florida Museum of Natural History;
- 2University of Florida
Protocol Citation: Shengchen Shan 2025. Tragopogon (Asteraceae) transformation and regeneration . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbo5zvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 02, 2025
Last Modified: September 12, 2025
Protocol Integer ID: 117586
Keywords: tetraploid tragopogon, genome duplication, polyploidy, asteraceae, immediate consequences of polyploidy, tragopogon, regeneration, regeneration protocol, whole genome, evolutionary model system, significant evolutionary force
Funders Acknowledgements:
National Science Foundation
Grant ID: IOS-1923234
National Science Foundation
Grant ID: DEB-2043478
Abstract
Polyploidy or whole-genome duplication is a significant evolutionary force. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. Here, we provide an improved transformation and regeneration protocol for diploid and tetraploid Tragopogon.
Materials
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Tragopogon transformation and regeneration protocol
Prepare Tragopogon material
Remove the pericarp (fruit wall) of Tragopogon achenes to expose the seed; sterilize the seeds in 20% (v/v) bleach (containing a few drops of Tween-20) for 20 min (shake slowly). Rinse the seeds 4 times with autoclaved deionized water to remove excess bleach on the seed surface. Blot the seeds on the autoclaved filter paper.
Grow the seeds on seed germination medium (also known as 1/2 MS medium) (2.22 g/L MS basal medium with vitamins, 30 g/L sucrose, 5 g/L agargellen, pH 5.8) in a sterile ice cream box in an incubator (25 °C, 14 h light/10 h dark, 52.5 μmol m-2 s-1; model CU36L4C8 from Percival 26 Scientific, Perry, IA, USA).
Cotyledons and true leaves are harvested approximately 2 weeks after seed germination for Agrobacterium-mediated transformation (Step 3.1).
Agrobacterium culture
Streak the Agrobacterium tumefaciens strain EHA 105 onto a selective LB plate (40 g/L LB agar, 50 mg/L kanamycin, 50 mg/L rifampicin). Incubate the plate at 28 °C for 2 days.
Pick Agrobacterium colonies and inoculate into 20 mL selective liquid TY medium (5 g/L tryptone, 3 g/L yeast extract, 50 mg/L kanamycin, 50 mg/L rifampicin, 0.1 mM acetosyringone, pH 5.5). Shake at 100 rpm, 28 °C for 24 h.
Tragopogon transformation and regeneration
Prepare leaf explants. Chop leaf tissues from Step 1.3 into 1-cm segments. To promote transformation efficiency, the surfaces of the leaf segments are slightly scored to create wounds using a sterile blade. The Tragopogon leaf explants are placed on the callus induction medium [4.44 g/L MS basal medium with vitamins, 30 g/L sucrose, 0.5 mg/L naphthaleneacetic acid (NAA), 1 mg/L 6-benzylaminopurine (BAP), 6 g/L gelrite, pH 5.8] for 2 days (in the dark at room temperature, at 23 °C) before transformation (a process referred to as “pre-culture”).
Prepare Agrobacterium suspension. Centrifuge TY medium from Step 2.2 at 2000 rpm for 10 min; remove the TY medium and re-suspend the Agrobacterium pellet with AAM infection medium (2.95 g/L KCl, 0.25 g/L MgSO4·7H2O, 0.15 g/L CaCl2·2H2O, 0.15 g/L NaH2PO4·H2O, 10 mg/L MnSO4·4H2O, 3 mg/L H3BO3, 2 mg/L ZnSO4·7H2O, 0.75 mg/L KI, 0.25 mg/L Na2MoO4·2H2O, 0.025 mg/L CuSO4·5H2O, 0.025 mg/L CoCl2·6H2O, 0.876 g/L L-glutamine, 0.266 g/L L-aspartic acid, 0.174 g/L L-arginine, 7.52 mg/L glycine, 0.1 g/L myo-inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine hydrochloride, 0.1 mg/L thiamine hydrochloride, 27.8 mg/L FeSO4·7H2O, 37.26 mg/L Na2EDTA, 0.5 g/L casamino acids, 68.5 g/L sucrose, 36 g/L glucose, 0.1 mM acetosyringone, pH 5.2). For each transformation experiment, prepare 20 mL AAM infection medium with OD600 = 0.1-0.2.
Co-culture of leaf explants and Agrobacterium. Immerse explants in 20 mL AAM infection medium for 30 min; explants are thoroughly blotted dry on autoclaved filter paper and transferred to co-cultivation medium (4.44 g/L MS basal medium with vitamins, 30 g/L sucrose, 0.5 mg/L NAA, 1 mg/L BAP, 6 g/L gelrite, 0.1 mM acetosyringone, pH 5.2). Place the plates in the dark for 3 days at room temperature (23 °C).
Transfer the explants to selective callus induction medium (4.44 g/L MS basal medium with vitamins, 30 g/L sucrose, 0.5 mg/L NAA, 1 mg/L BAP, 6 g/L gelrite, 300 mg/L timentin, 15 mg/L hygromycin, pH 5.8), and place the plates (covered by foil) inside the incubator (25 °C) for approximately 24 days. Callus will emerge from the cut edge of the explants. Successful transformants will survive on the selection medium and emit green fluorescence under blue light.
Shoot initiation. Calli of Tragopogon porrifolius (2x) and T. mirus (4x) are transferred to the selective COCO-1 [2.41 g/L Woody plant basal medium with vitamins, 30 g/L sucrose, 2.5 mg/L BAP, 8.5 g/L agargellan, 10% (v/v) coconut water, 300 mg/L timentin, 15 mg/L hygromycin, pH 5.7] and COCO-3 (identical to COCO-1, but with 15 g/L agargellan instead of 8.5 g/L)) shooting media, respectively. Place the plates inside the same incubator for approximately 5 weeks. The medium is renewed every 2-3 weeks.
Shoot elongation. Calli with tiny shoots are transferred to selective WPM-3 medium (2.41 g/L Woody plant basal medium with vitamins, 30 g/L sucrose, 8.5 g/L agargellan, 300 mg/L timentin, 15 mg/L hygromycin, pH 5.7) for approximately 2 weeks.
Rooting. The elongated shoots are gradually transferred (up to approximately 4 weeks on selective WPM-3 medium) to selective COCO-R medium [2.41 g/L Woody plant basal medium with vitamins, 30 g/L sucrose, 1.5 mg/L indole-3-butyric acid (IBA), 8.5 g/L agargellan, 300 mg/L timentin, 15 mg/L hygromycin, pH 5.7] for rooting.
After approximately 6 weeks, rooted shoots are gradually transferred to soil and continue their growth inside a growth chamber (14 h light/10 h dark, 25 °C; model AR-1110 from Percival Scientific). After approximately three months of growth in soil, the regenerated plants are subjected to a three-month cold treatment (8 h light/16 h dark, 7 °C). The plants are then moved back to the growth chamber (14 h light/10 h dark, 25 °C), and after approximately 3-4 weeks, most plants begin to bolt and flower.