The agrobacterium-mediated transformation is an easy method for producing genetically-modified potato plants. In order to understand specific gene functions and their contribution to organ physiology. Transformation by agrobacterium rhizogenes is a robust tool for rapidly assessing gene functioning roots, as similar results can be obtained by agrobacterium tumefaciens transformation.
Demonstrating this procedure will be Sandra Fernandez-Pinan, a post-doc, and Jennifer Lopez, a maste5r's student from our lab. Begin by growing a single agrobacterium colony overnight in five milliliters of yeast extract broth, or YEB medium, supplemented with antibiotics in a 50 milliliter centrifuge tube at 28 degrees Celsius, at 200 rotations per minute. For a-tumefaciens transformation, measure the optical density the next morning.
When the optical density at 600 nanometers, or OD 600 reaches 0.6 to one, centrifuge one milliliter of the agrobacterium culture and resuspend the pellet in one milliliter of fresh YEB medium without antibiotics. After the second wash, resuspend the pellet in fresh YEB medium without antibiotics, to the appropriate concentration to obtain a final OD 600 of 0.8, and place the bacterial cells on ice. For plant transformation using A.rhizogenes, carefully transfer a donor plant from a 2MS medium culture to a 120 by 120 millimeters square plate, and use a surgical needle to inject three microliters from an A.rhyzogenes culture to different stem internodes as possible.
Then immediately transfer the entire plant to a new square plate containing solid MS medium, supplemented with 0.1 millimolar acetosyringone. After placing a second plant onto the same plate, transformed in the same manner, seal the plate with surgical tape, and place the plate vertically inside a growth cabinet for four days. Transfer the plant to a square plate with MS medium supplemented with cefotaxime sodium to kill the A.rhizogenes and place the sealed plate vertically inside a growth cabinet for four days.
After 10 to 12 days, new hairy roots will appear. Their transformation can be confirmed by fluorescent stereo microscope. At this time, excise the native roots of each plant, and transfer the plants to a new square plate with MS medium, supplemented with cefotaxime sodium to kill the A.rhizogenes.
To obtain a composite plant, let the transgenic hairy roots grow for another three to four weeks in the MS medium, supplemented with cefotaxime sodium. For plant transformation using A.tumefaciens. Cut and place a leaf from a three to four week old plant in a petri dish, and use a scalpel to make one to three transverse cuts from the center of the leaf to the edges without cutting the pieces off of the leaf.
and to exclude the petiole. Immediately place the leaf into a petri dish containing 10 milliliters of fresh 2MS liquid medium, abaxial side up, and cover the plate. Quickly add 80 microliters of the A.tumefaciens culture to the liquid medium and gently stir the medium for one minute to homogeneously distribute the bacterial solution.
Then carefully seal and cover the plate with aluminum foil for a two-day incubation in a 24-degree Celsius chamber. At the end of the transformation period, transfer the leaves abaxial-side up, to the callous-inducing medium, scraped with tweezers for a one-week incubation in the growth cabinet. At the end of the incubation, transfer the leaves, abaxial-side up, onto tweezer-scraped shoot inducing medium, and incubate the leaves in a growth cabinet.
When the emerged chutes are about two centimeters tall, cut three emerging chutes from each callous. Place up to five different chute transformation events into individual culture flasks containing MG medium supplemented with cefotaxime sodium to allow rooting, and label the subset as appropriate for a three to four week incubation in the growth cabinet until the chutes are vigorous. At the end of the incubation, select the most vigorous plant of each event, and cut the apical segments of the chute with three to four internodes from each plant.
Place the segments into a culture flask containing 2MS medium supplemented with cefotaxime sodium and grow the plants until they develop vigorous chutes and roots. To perform a beta-glucuronidase or GUS histochemical reporter gene assay, fix the roots from a two to three week in-vitro plant culture with 90%chilled acetone for 20 minutes on ice. At the end of the fixation, wash the roots two times in distilled water and treat the roots with fresh GUS-staining solution under vacuum for 20 minutes.
At the end of the vacuum treatment, place the roots at 37 degrees Celsius in the dark for about four hours. When a blue color is visible, wash the roots two times with 70%ethanol, and visualize the staining under a bright field microscope. Transformed hairy roots exhibit a red florescence when illuminated with green light, while negative control untransformed agrobacterium demonstrate no such florescence, indicating the suitability of the DS red transformation marker for identifying transgenic hairy roots.
Here, GUS staining in roots of transgenic plants obtained by A.tumefaciens and transgenic hairy roots obtained by A.rhizogenes are shown. As observed, roots transformed with A, tumefaciens and grown in-vitro, demonstrate blue staining in the endodermis, a cell layer between the cortex and the stele. In more developed roots, the blue labeling is patchy in the external layer, corresponding to the exodermis.
In transformed hairy roots obtained by A.rhizogenes, and grown in hydroponics, the GUS marker is specifically located within the endodermis in the emergence of lateral roots in the wounded areas and in the exodermis. In addition to being a fast method, the transgenic roots obtained with agrobacterium rhizogenes also carried the DNA from the root-inducing plasmid, which may deregulate some directly-controlled processes. The transgenic hairy roots obtained with agrobacterium rhizogenes can be maintained while they shoot, but alternatively can be on-site until propagated in-vitro for massive transit production.
The potato transformation provides a good tool to check the gene function and promoter activation, and also to produce methodological rules in a species of high agronomic interest. The genetically modified organism should be discarded safely after use to avoid environmental contamination. Also, gas solution contains toxic cyanide derivative, so wear protection and discard the solution safely.
