TITLE: Stable transformation of Nicotiana benthamiana
AUTHORS: Jana Ordon, Hannelore Espenhahn, Carola Kretschmer, Johannes Stuttmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Detailed protocol with pictures for the transformation of </span><span style = "font-style:italic;">Nicotiana benthamiana</span><span>. The same protocol will also work for transformation of </span><span style = "font-style:italic;">Nicotiana tabaccum</span><span>, but regeneration of transformed plants will take more time.</span></div></div>

[STEPS]
?. [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]
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]
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]
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]
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.