The overall goal of this procedure is to describe how endogenous phytohormone levels change during adventitious shoot formation in ipecac. This method can help answer key questions related to tissue regeneration in the plant tissue culture wheel. The main advantage of this technique is the endogenous level of Indole-3-acetic acid and cytokinines can be simply analyzed with high sensitivity.
The implication of this technique extend throughout establishment of plant tissue degeneration because changes in endogenous phytohormone level trigger adventitious shoot formation. To begin, prepare a phytohormone-free B5 medium and add 0.2 percent gellan gum. Then, autoclave the medium to sterilize it.
Using a surgical scalpel on a sterile acrylic plate, slice eight millimeter internodal segments of ipecac plantlets and place them on the medium. Then, culture the ipecac segments at 24 degrees Celsius under 15 micromole per square meter seconds of light in a 14-hour light, 10-hour dark cycle for five weeks. After culturing the ipecac segments, identify regions one through four in each segment.
Once a week, use a microscope to count the number of adventitious shoots in each region. First, place a five millimeter zirconia bead into four two-milliliter sample tubes. Then, use a surgical scalpel on a sterile acrylic plate to cut the ipecac segments into regions I through IV.Then collect eight segments from each region into one sample tube.
Weigh each of the samples and freeze them in liquid nitrogen. Next, use a bead-based homogenizer to crush the frozen samples. Use a vortex mixer to suspend the samples in one milliliter of the acetonitrile with 500 picograms of each internal standard of oxen and CKs.
Home the samples at four degrees Celsius for one hour. And then centrifuge the samples at 3500 G for five minutes at room temperature. Wash the pellet in 80 percent acetal nitrile with one percent acetic acid.
Then repeat the centrifugation and use a disposable class tube to combine the supernatants. Add 600 microliters of water with one percent acetic acid to the combined supernatant. Then use a vacuum concentrator to evaporate the acetal nitrile.
Load the sample solutions into individual equilibrated HLB cartridges. And wash the cartridges with one milliliter of water with one percent acetic acid. Then allude all of the hormones with two milliliters of 80 percent acetyl nitrile with one percent acetic acid in a glass tube.
Using a vacuum concentrator, evaporate the acetal nitrile to obtain extract in water with one percent acetic acid. Load the sample solutions into individual equilibrated MCX cartridges and wash the cartridges with one milliliter of water with one percent acetic acid. Then allude IAA in a glass tube, with two milliliters of 30 percent acetal nitrile with one percent acetic acid.
Then wash the cartridges with two milliliters of 80 percent acetal nitrile with one percent acetic acid. Next, wash the cartridges with two milliliters of water and one milliliter of water with five percent aqueous ammonia in a draft chamber. Allude the CKs in a glass tube with two milliliters of 60 percent acetal nitrile with five percent aqueous ammonia in a draft chamber.
Then use a vacuum concentrator to evaporate the solvent of each hormone fraction. Stir the samples at minus thirty degrees Celsius until LCMSMS analysis. First, dissolve each hormone extract in a tube with 600 microliters of methanol.
Then transfer the solution to a screw-neck total recovery vial and use a vacuum concentrator to evaporate the solvent. Dissolve the IAA fraction in 20 microliters of 30 percent acetal nitrile and the CK fractions in 20 microliters of water with one percent acetic acid. Next use a triple quadrupole MS system equipped with an HPLC system to analyze the samples in positive ion mode.
Finally quantify endogenous IAA in CK levels against a standard curve of the ration of unlabeled to deuterium label standards. In this protocol endogenous oxidant cytokinin in adventitious chutes was measured via LCMSMS for five weeks. At the first week, no adventitious chutes had formed and the first small chutes appeared during the second week.
During the third and fourth weeks, the number of chutes increased mostly in regents one and two. During the fifth week the number of chutes was approximately seven in region one, and five in region two. Before culture, the IAA level was slightly higher in region one than in regions two, three, and four.
During the first week, the IAA level increased greatly in region four, and decreased slightly in regions one, two, and three. By five weeks of culture an IAA concentration gradient emerged. With levels increasing from region one to region four.
Before culture, there were only trace levels of most of the CKs. At the first week, the tZR level increased to 13.8 picograms per milligram in region two. And 18.1 picograms per milligram in region three.
In contrast the levels of tZ, iP and iPR changed only slightly during culture. While attempting this procedure it's important to remember that endogenous part hormone are stable. After it's developed man, this technique paves the way for researchers in the field of plant tissue culture.
To explore adventitious formation in other organisms. After watching this video, you should have a good understanding of how to analyze endogenous Indole-3-acetic acid and cytokinines.
