An effective enzyme-linked immunosorbent assay-based method for quantification of gibberellic acid in pecan (Carya...

Enzyme-linked immunosorbent assay (ELISA) is a reliable and low-cost method for quantifying gibberellic acid (GA) in plants but applying this method to certain plant tissues is challenging due to tissue hardness and/or contents rich in metabolites. Pecan (Carya illinoinensis) tissues in the bud and bark are examples of such tissues. This study aimed to establish an effective ELISA-based GA quantification method for these pecan tissues. We tested how different amounts of the tissues and the weight ratios of a tissue to the solvent methanol affected the detection of GA. We also tested whether heat-drying and freeze-drying and long-term storage of the fresh materials in -80 °C affected the detection of GA. Our results showed that using 0.0125± 0.0005 gram (g) of heat-dried or freeze-dried tissues and a relatively low tissue-to-methanol ratio consistently produced high GA concentrations in ELISA detection, and the levels of GA were stable in pecan samples stored long-term at -80 °C. Our results also showed that GA concentrations in buds were higher than in the bark. These findings indicate that our method is effective in detecting GA in small amounts of pecan tissues, and heat-drying of either fresh or freezer-stored samples can produce satisfying results of GA quantification. Moreover, the GA concentrations in the wood and bark were found to be positively correlated with the GA concentrations in the bud, which help estimate most part of the variability of the GA concentration in the bud based on the GA concentration in the bark, as it may be challenging to experimentally determine the GA concentration in a small bud at an early developmental stage. The final spike-recovery test with GA (From ELISA kit) as spike and sample to sample spike confirmed that our sample extracts exhibited acceptable recovery percentages in low and high spike with range of 72-102% of recovery indicating that matrix effects and phenolic interference did not significantly affect the results.