I am planning to assess the total GAs (also want to assess GA3 seperately if possible within my circumstances) from inflorescence of grape berries and mature berries. I had stored samples at -80 since the day of harvesting. I found an enzymatic kit but didn't find any article related to this.....could you suggest me the best methods if you have experiences eg/ HPLC, kit or any others
The procedure for the extraction of endogenous gibberellins involve using 80% acetone.the extraction procedure repeated three times. the extraction should be done at 4c in darkness-refrigerator. then combine the extract-150ml-, and evaporate at45c using rotary evaporator to aqueous phase. the aqueous phase is made up to 40ml with distilled water.then basic lead acetate(25%w/v) -1ml- is added to the aqueous solution,and centrifuge to remove the precipitate.Excess lead is then removed by the addition of potassium oxalate, or sulfuric acid,and the the solution again centrifuged. the solution is now shaken three times with freshly distilled ether. the ether fraction is now evaporated to 2ml.this fraction is now analyzed by HPLC and total gibberellins are deter-mined on 205nm.GA3 is used as internal slandered. total endogenous gibberellins are estimated from the area under the peak in comparison with slandered GA3. It is not possible to deternmineGA3 alone. any way, total gibberellins determination is more than enough.
Please, see a sample of my published research on extraction and purification of endogenous gibberellins. the second paper on IAA, but the method is exactly the same, the difference is gibberellins are detected at 205nm, following HPLC.
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