Begin by collecting seeds of wild soybean, S.Americanum, and the local variety of pumpkin. Sow the seeds in vermiculite at a depth of one centimeter and water them thoroughly. Cultivate them in a growth chamber at approximately 70%relative humidity.
Remove the A.rhizogenes strain K599 from the 80 degrees Celsius freezer and activate it on a solid LB medium at 28 degrees Celsius for 48 hours. Then select a single clone of strain K599 and culture it in one milliliter of liquid antibiotic containing LB medium for 12 hours. Spread 500 microliters of the bacterial suspension evenly on a solid antibiotic containing LB medium and incubate at 28 degrees Celsius for 24 hours.
After seven days, when seedling cotyledons are unfolded, use a sterilized sharp scalpel to cut approximately 0.5 to one centimeter of the hypocotyl. Discard the primary root and a portion of the hypocotyl. Coat the hypocotyl incision with K599 bacteria.
Plant seedlings in moist vermiculite and infect 30 plants of each species using A.rhizogenes-mediated hairy root transformation. Then water the plants with five milliliters of resuspended K599 bacterial suspension in quarter-strength Gamborg B5 basic medium. Cover the pots with a transparent plastic bag and place them in a growth chamber.
Grow the plants for approximately 12 days post-inoculation. At this time, new hairy roots should be generated at the incision site, typically reaching lengths of two to five centimeters. Using a CAMI luminescence imaging system with green excitation light at 540 nanometers and emission at 600 nanometers, detect the expression of the reporter gene DsRed2.
Roots of composite plants of wild soybean, S.Americanum, and pumpkin under natural and green excitation light are visualized.
