Jul 30, 2019
Stable transformation of Nicotiana benthamiana
- Jana Ordon1,
- Hannelore Espenhahn1,
- Carola Kretschmer1,
- Johannes Stuttmann1
- 1Martin Luther University Halle/Saale
Protocol Citation: Jana Ordon, Hannelore Espenhahn, Carola Kretschmer, Johannes Stuttmann 2019. Stable transformation of Nicotiana benthamiana. protocols.io https://dx.doi.org/10.17504/protocols.io.sbaeaie
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: August 02, 2018
Last Modified: July 30, 2019
Protocol Integer ID: 14402
Keywords: Nicotiana, benthamiana, tabaccum, Agrobacterium, plant transformation
Abstract
Detailed protocol with pictures for the transformation of Nicotiana benthamiana. The same protocol will also work for transformation of Nicotiana tabaccum, but regeneration of transformed plants will take more time.
Attachments
Guidelines
Plant tissue culture has to be carried out under sterile conditions.
Materials
Required Material
YEB medium:
5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 5 g/l sucrose, 0.5 g/l MgCl2 [add 1.5 % agar for plate preparation)
Antibiotics:
Rifampicin: stock 100 mg/l in DMSO or DMF; 1000 x for bacterial selection
Gentamycin: stock 15 mg/l in H2O; 1000 x for bacterial selection
Kanamycin: stock 50 mg/l in H2O; 1000 x for bacterial, 500 x for plant selection
Spectinomycin: stock 50 mg/ml in H2O; 1000 x for bacterial selection
Carbenicillin: stock 100 mg/ml in H2O; 1000 x for bacterial selection
Induction medium:
YEB medium, 10 mM MES pH 5.7 – 5.8 [KOH], 20 µM Acetosyringone, antibiotics
MMA medium:
4.3 g/l MS salts including vitamins (Duchefa M0222), 20 g/l sucrose, 10 mM MES pH 5.7 - 5.8 [KOH]
MS medium:
4.3 g/l MS salts including vitamins (Duchefa M0222), 20 g/l sucrose, 10 mM MES pH 5.7 - 5.8 [KOH],
8 g/l Gelrite (Duchefa G1101). Verify pH, and adjust with KOH if required.
MSII medium:
MS medium, 1 mg/l 6-Benzylaminopurine (BAP), 0,1 mg/l Naphthalene acetic acid (NAA), 100 mg/l Kanamycin, 200 mg/l Cefotaxime (Claforan, Duchefa C0111)
MSIII medium:
MS medium, 100 mg/l Kanamycin, 200 mg/l Cefotaxime (Claforan, Duchefa C0111)
Tobacco plants:
We use 3-5 week-old, greenhouse-grown Nicotiana benthamianaplants (growth conditions: 16h light period, 60 % relative humidity at 24/20 °C (day/night), 130-150 µE m-2 s-1 light intensity ).
Preparation of Agrobacterium suspension
Preparation of Agrobacterium suspension
- Streak the Agrobacterium tumefaciens (A. tumefaciens; GV3101 pMP90) strain carrying the transformation construct on YEB plates containing required antibiotics (Rifampicin, Gentamycin + construct-specific antibiotic).
- Two days prior to transformation: Inoculate 5 ml YEB liquid culture (with antibiotics); grow for 24 h with shaking at 30 °C.
- One day prior to transformation: Inoculate 100 ml culture with 1 ml of the pre-culture in induction medium (containing antibiotics), shake over night at 30 °C.
- Day of transformation: Pellet bacteria by centrifugation (40 ml in Falcon tubes, 20 min, 4000 x g. Resuspend bacteria in MMA medium (without antibiotics) to an OD600=0.8 (50 ml).
Preparation of N. benthamiana leaf tissue for transformation
Preparation of N. benthamiana leaf tissue for transformation
Try to injure plant tissue as little as possible! All steps following the surface-sterilization have to be carried out under sterile conditions.
- Harvest the 2-3 youngest, but fully expanded leaves of 3-5 weeks-old Nicotiana benthamiana plants (3-5 leaves are required for each transformation).
- Cut leaves into pieces, omitting main veins.
- Incubate leaf cuts for 30 sec in 1.2 % NaOCl (+0.01 % Tween).
- Wash twice in H2O (+0.01 % Tween) to get rid of the bleach.
- Store leaf cuts in H2O.
Transformation
Transformation
- Further cut surface-sterilized leaves into smaller pieces (ca. 1x1 cm).
- Incubate leaf cuts in A. tumefaciens suspension: - Pour A. tumefaciens suspension in a square petri dish. - Place leaf cuts on the surface of the suspension. When the surface is covered, you will have more than enough leaf cuts for each transformation. - Incubate at least 30 min.
- Prepare a fresh square petri dish with a sterile piece of water-wetted Whatman paper.
- Transfer leaf cuts onto Whatman paper.
- Seal dishes with Leukopor tape and incubate for 2 days in the dark (24 °C, wrap in aluminium foil to protect from light).
Selection and shoot induction
Selection and shoot induction
- Prepare square dish with 50 ml H20 (containing Kanamycin (100 mg/l) and Cefotaxime (250 mg/l)) for washing of the leaf cuts.
- Pick up the Whatman paper with the leaf cuts using sterile forceps and place it upside-down onto the surface of the wash solution to release leaf cuts from the filter into the wash solution. Gently shake the petri dish, and incubate for > 3 min.
- Dry leaf cuts by gently swiping on a fresh, sterile Whatman filter.
- Place dried leaf cuts on shoot induction medium (MS-II). Use standard round petri dishes to minimize the risk of contamination. Place 8-10 leaf cuts on each plate.
- Seal plates with Leukopor tape. Incubate in a light cabinet until shoots occur (5-6 weeks under our conditions: 23°C, 24h light, intensity 105 – 125 µE m-2s-1).
- It may be necessary to transfer leaf cuts/ developing calli to new MS-II plates during the incubation if media start drying out.
Root induction and transfer to soil
Root induction and transfer to soil
Shoots start developing from calli on MS-II plates and need to be transferred to MS-III plates to induce rooting. Since calli do not develop homogeneously, shoots may be cut and transferred at different time points.
- Cut well-developed shoots with a sterile blade. Try to cut as low as possible, but avoid transferring any callus tissue, as this will prevent further development of the shoot.
- Stick shoots with the cut surface into MS-III medium, and incubate under the same conditions as before for further development of the shoot and rooting. Note: Red light will prevent rooting!
- We use 0.5 l Weck glasses for rooting and place max. 3 shoots in each jar.
- Shoots originating from a single callus are, in general, originating from the same transgenic event. Thus, to only sample independent transformants, we only transfer one single shoot per callus to rooting medium.
- Calli can be kept as a back-up on MS-II medium, as shoots will continue to develop.
- Shoots will further develop in MS-III media, and will eventually form roots (2-3 weeks).
- Transfer well-developed shoots to soil. Remove MS medium from shoots/ roots to avoid contamination. We transfer shoots directly into potting soil that had been passed through a sieve. Again, shoot development is not homogeneous, and some shoots will not start rooting in a timely manner. Indeed, even rather small shoots without any roots will, in many cases, develop well once transferred to soil.
- It is important to keep plants under high humidity after transfer to soil. Place respective plants in a tray covered with a lid for ~ 2 weeks. Once plantlets appear sufficiently vigorous, reduce humidity in the course of 2-3 days by first displacing the lid before completely removing it.
- Grow plants as usually to obtain seeds. In our hands (GV3101 pMP90, pVS1 origin for transformation constructs), most plants will contain multiple insertions of the T-DNA.