TITLE: Tomato Transformation
AUTHORS: Alex Rajewski, Joyce van Eck

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is slightly modified from the citatation below. All credit for this protocol should be directed toward those authors.</div><div class = "text-block">This protocol details how to transform tomato (Solanum lyycopersicum) with Agrobacterium and to regerneate the plants via tissue culture.</div></div>

[STEPS]
?. [Sterilize and Sow Seeds]
Surface sterilize seeds with chlorine gas according to the following protocol
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?. [Sterilize and Sow Seeds]
Sow the seeds on plates 1/2 MSO media and place in the growth room in the light. They can be sown fairly densely because they won’t spend long on this media.
?. [Prepare Agrobacterium]
About two days before the transformation (Step 8) and one day before the cotyledons are to be dissected (Step 4) inoculate a 25mL broth of selective LB with Agrobacteria and incubate for ~36 hours at 28ºC.
Selection will depend on the Agro strain and plasmid you are using. We find that when incopulating from a glycerol stock of Agrobacterium and using kanamycin selection, a 25mL broth will take approximately 36 hours to grow to the correct density. It is best to know how fast your Agro will likely grow beforehand so you can time the transformation well.
?. [Prepare Agrobacterium]
Early on the day of the transformation, check the OD600. The optimum is 0.6-07. If the OD is above 1.0, dilute it to below 0.5 and allow to grow for another hour. Once the optimum OD is reached, transfer the broth into a 50mL falcon tube and centrifuge at 8000rpm for 10 minutes.
?. [Prepare Cotyledons]
One day before the transformation (step 8), after the cotyledons have emerged but before the first true leaves appear (about 6-8 days after sowing for tomato), take the plates sterilely into the hood and (one at a time) place each seedling onto the lid of a new sterile petri dish. Using an autoclaved scalpel cut off each cotyledon at its petiole. Also cut off the tip of the cotyledon, and, if the cotyledon is larger than 1cm, cut it in half.
When dissecting a cotyledon, make sure to keep the lid of the seedling plate closed to prevent dessication, which will make dissection much harder.For other species (ie tobacco) the cotyledons will not be large enough to remove the tip without destroying the cotyledon.In tobacco, the petiole is often so short that the apical meristem is captured along with the cotyledon. Including this tissue leads to faster regeneration, but we have not throughly evaluated if this leads to untransformed plants in tomato.
?. [Prepare Cotyledons]
Place the excised cotyledons adaxial (top) side-down onto plates containing KCMS media, seal with micropore tape, and allow to grow for one day at 25ºC, 16-hour days.
?. [Transformation]
In the sterile hood, transfer the explants to the Falcon tube of Agrobacteria and incubate for 5 min with occasional shaking.
?. [Regeneration]
In the sterile hood, transfer the explants to selective 2Z media with their adaxial side up, seal with micropore tape, and culture at 25ºC and 16-hour days. Transfer the explants to new 2Z media twice weekly for two weeks.
Plant selection and concentration here will depend on the T-DNA being used for transformation.This media should also include timentin (300mg/L) in order to kill the Agrobacterium.We are currently experimenting with adding indole-3-butyric acid (1mg/L) to this media as well.
?. [Regeneration]
After two weeks, transfer the explants to selective 1Z media (~15 per container) and refresh on new selective 1Z media once every two weeks until shoots begin to form. Once shoots have formed (4-6 weeks), reduce the explants to 5 per container and continue to refresh as usual. (See note above on selection.)
?. [Regeneration]
Once shoots are at least 2cm long and have at least one node, excise them and place them singly into containers with selective Rooting Media. For tomatoes, roots should form within about 2 weeks.
?. [Regeneration]
For propagation of confirmed lines, transfer to nonselective Rooting Media. This allows the plants to grow faster and will let you check if Agrobacterium is still present. However, do not transfer to antibiotic-free media until the shoots have undergone at least 3 transfers onto media with antibiotics and selection.
?. [Prepare Agrobacterium]
Observe the volume of the supernatant, discard it, and resuspend the pellet with the same volume of liquid MS0-2% media. The inoculum is now ready to use.
?. [Transformation]
Pour the suspension of agrobacterium and cotyledons into an empty, sterile petri dish for better access. Transfer the cotyledons from this suspension to new KCMS plates (adaxial side-down again), seal with micropore tape, and culture for 48 hours in the dark.
For tomato, you can fit ~20 cotyledons on a single plate.Alternatively, you can pour the suspension onto sterile paper towels and then transfer the cotyledons onto KCMS plates. We have found that this infrequently introduces yeast contamination.