Sonication Assisted Agrobacterium-mediated Transformation (SAAT) is a very easy, low cost method to substantially enhance the efficiency of Agrobacterium-mediated transformation of low or non-susceptible plant species. For those who are currently using Agrobacterium, this technique involves one additional step: Sonication. The strength of this method is that the cavitation caused by sonication results in thousands of microwounds on and below the surface of the plant tissue. This wounding pattern permits Agrobacterium to travel deeper and more completely throughout the tissue than conventional microscopic wounding, increasing the probability of infecting plant cells. We have shown enhancement of transient expression in various explants and tissue types as well as in several species including soybean, cowpea, maize, wheat, and white spruce. Described below is a protocol that routinely gives our laboratory excellent transient expression parameters in immature cotyledon of soybean. In addition I have briefly outlined some important parameters which will need attention. It is very important to keep in mind that species and tissue type we have tried has a different optimum SAAT condition.

You will need to modify the conditions for your particular system.

PARAMETERS TO KEEP IN MIND

Sonicators- We use a Fisherbrand FS5 sonicator (discontinued, from Fisher, replaced by PC5 model, made by L&R Manufacturing Company in Kearny, NJ, now also discontinued). The sonication time is controlled by an electronic digital timer. We have used other sonicators including probe-type sonicators, however the bath sonicators gave us the best results. Our digital timer is manufactured by GraLab (GraLab505) but any timer will work as long as it times as low as 0.1 seconds.

SAAT medium- We SAAT our tissue in the same medium we use for co-culture.

SAAT vessels- The type of container used to SAAT-treat your tissue will influence the outcome of your experiments. Sonication will pass through denser material (glass and stainless steel) better than less dense material (polypropylene tubes) whereas the less dense material will buffer the effects of sonication. Sometimes it is beneficial to reduce the intensity of sonication by "buffering" the target tissue.

Duration of SAAT- This depends on several factors including sonicator frequency, wattage, and resiliency of tissue to name a few. The researcher needs to evaluate this parameter on their own. Using the FS5, the lowest timepoint I would recommend is 0.2 seconds. The internal electronics of the FS5 are not reliable at 0.1 seconds and below; sometimes it sonicates and other times it does not. Be aware that long durations of SAAT can cause a substantial heating to the tissue. For sensitive tissue (i.e. immature embryos) a duration of 0.2 to 5 seconds is a good place to start while less sensitive tissues may tolerate longer SAAT treatments (<1 minute).

Vir inducer- Vir inducers such as acetosyringone or constitutively expressed vir genes will increase the transformation rates and are recommended. We do not pre-culture our Agrobacterium with acetosyringone.

Addition of Bacteria- SAAT can be performed before the addition of or with the Agrobacterium. We see no statistical difference between these two treatments. We typically SAAT with Agrobacterium for convenience.

Bacterial concentration- We recommend an OD600nm between 0.1 to 0.2, depending on your sample.

Temperature- We have been very successful using 27°C for co-culture. We see no benefit from using lower temperatures with co-culture following SAAT of soybean cotyledons although co-culture at low temperatures may be useful for other systems.

Co-cultivation period- With our work, the longer you co-cultivate without the Agrobacterium overgrowing and killing your tissue, the better. In addition we have found that it can be difficult to remove Agrobacterium from overgrown cultures. However, we did not see a benefit from daily washes without antibiotics, allowing co-culture to proceed for 7 days. In most cases co-cultivation should be two days.

Standard protocol for transient expression for immature cotyledons:

1. Agrobacterium was grown overnight at room temperature (27°C) in LBS (10 g/l tryptone, 5 g/l yeast extract, 5 g/l sucrose, 5 g/l sodium chloride) shaking at 150 rpm.

2. Agrobacterium was centrifuged at 1500 g for 10 minutes and washed twice with D40 medium (MS salts; B5 vitamins; 40 mg/l 2,4-D; 6% sucrose; pH 7.0). After the second wash, the Agrobacterium was diluted to an O.D. 600nm of 0.2.

3. Immature cotyledons were excised from immature soybean seeds (4-6 mm in length) and placed on D40 medium + 0.2% Gelrite until enough cotyledons were harvested for SAAT treatment.

4. Approximately 10 cotyledons were placed in a 1.5 ml microcentrifuge tube along with 0.5 ml of the Agrobacterium suspension. Tissue was SAAT- treated for two seconds.

5. Approximately five minutes after SAAT-treatment, excess Agrobacterium was removed by blotting on a sterile filter paper. Immature cotyledons were then placed on D40 medium + 100 µM acetosyringone + 0.2% Gelrite and co-cultivated for three days.

6. Immature cotyledons were transferred to D40 medium + 500 mg/l Timentin + 0.2% Gelrite for two days and assayed for transient (GUS) expression.

In short, SAAT is another method for high efficiency delivery of Agrobacterium to the target tissue.

SAAT and untreated germinating maize

SAAT-treated cowpea seedling

SAAT-treated soybean cotyledon

For further information on SAAT, see the following references:

Trick HN, JJ Finer (1999) Induction of somatic embryogenesis and genetic transformation of Ohio buckeye (Aesculus glabra Willd.). In Vitro Cellular and Developmental Biology - Plant 35:57-60

Santarém ER, HN Trick, JS Essig, JJ Finer (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Reports 17:752-759

Trick HN, JJ Finer (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean (Glycine max [L.] Merrill) embryogenic suspension culture tissue. Plant Cell Reports 17:482-488

Trick HN, JJ Finer (1997) SAAT: Sonication Assisted Agrobacterium-mediated Transformation. Transgenic Research 6:329-336

A patent covering SAAT has been issued to The Ohio State University.
"Method for Transforming Plant Tissue by Sonication", John J. Finer and Harold N. Trick.  US Patent # 5,693,512, issued December 2, 1997. For licensing information, please contact Erin Bender at Technology, Licensing and Commercialization (TLC)