Efficient Regeneration-based Agrobacterium-Mediated Transformation of an Asexual Amphibious Brassicaceae Species, Rorippa aquatica

We address the lack of a simple, scalable agrobacterium transformation pipeline for asexual plants, enabling molecular analyses of gene functions. Recently, whole genome and physiological gene expression studies introduced rorippa aquatica as a model for heterophilic. It is useful in studying the molecular basis of land to water adaptation.

To begin, select two month old stock plants that exhibit healthy leaves with entire margins. Prepare a plant propagation tray, measuring 20 by 30 centimeters, with two layers of autoclave paper towels. Fold the paper towels neatly and saturate them with sterile water until a thin layer of water remains on the surface.

Ensure that the laboratory bench is equipped with 100 to 500 milliliters of double distilled water, sterile containers suitable for the anticipated volume of resuspended inoculum, a micro pipette, and appropriate pipette tips. Now, quickly excise 10 leaves at the petal base from the selected stock plants using scissors, and place them into a Petri dish containing sterile water. After 2.5 hours, pipette 50 microliters of surfactant per liter of resuspension solution to achieve a 0.005%surfactant concentration.

Gently swirl the flask to mix the solution and ensure a prepared propagation tray is accessible for explant placement after inoculation. Now place a leaf with the abaxile side facing upward. Using a scalpel, cut perpendicularly along the leaf's mid rib into pieces measuring one centimeter in length, and place them in a 50 milliliter tube suitable for use in a rotating tube mixer to serve as inoculation explants.

Pour the agrobacterium inoculum into the tube at a volume sufficient to fully saturate all explant material. Place the tube onto a rotary mixer and agitate at room temperature for 10 minutes to perform inoculation. Then using forceps, remove all inoculated leaf explants from the solution, and directly place them in the prepared propagation tray.

Separate and spread the soaked explants evenly so that they are partially immersed but not fully submerged in the thin layer of water atop the saturated paper towels. Next, seal the propagation tray with plastic wrap. Place the trays in a dark environment and incubate at an ambient temperature of 22 degrees Celsius for three days.

After the completion of the dark incubation period, confirm that the trays remain sealed with transparent plastic wrap. Then add double distilled water to the trays as needed to maintain moisture levels. Transfer the inoculation trays to a growth chamber and maintain them under vegetative propagation conditions.

Visually inspect regenerated rosette plantlets for transplant suitability. Identify ideal by confirming the presence of an intact chute with multiple leaves and roots measuring at least two to three millimeters in length. Then, excise intact transgenic rosette plantlets using forceps.

Transplant the excised plantlets under the same conditions used for wild type clonal regenerations. Excise a rosette plant lit while carefully avoiding contact with any transgenic leaves. Trim excess foliage immediately surrounding the transgenic leaf and transplant a single plantlet per pot.

After fertilizing each transplanted plant, water them thoroughly, mist the foliage, and place the plants under the appropriate conditions. Finally, return explant containing transformants that lack shoots and roots or appear too small for transplantation to the propagation tray. Continue screening these explants and transplant them once they have grown sufficiently.

The majority of one month old, our one T one seedlings, exhibited chimeric ruby expression patterns, while a small number showed entirely ruby expressing leaves. Maintained mature six month old, our one T one seedlings, expressed chimeric ruby expression. Chimeric ruby pigmented leaf sectors excised from our one T one seedlings were capable of asexual propagation.

Excised chimeric leaf pieces regenerated into either fully ruby expressing or chimeric, our one T one rosette plantlets within two to four weeks after excision with visible root formation during regeneration. After one month, most our two T one seedlings displayed predominantly ruby leaves. Our protocol bypasses sterile tissue culture steps and provides a transformation regeneration pipeline for asexual vegetatively propagated plants lacking seeds or flowers.

Our studies on ethylene pathway activation in rorippa aquatica under submergence provide key insights into terrestrial plant submergence responses. Our method opens the door to investigating how light and hormone pathways enable this amphibious plant to metamorphose underwater.