European Journal of Scientific Research - EuroJournals
European Journal of Scientific Research - EuroJournals
European Journal of Scientific Research - EuroJournals
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
411 Ahmed Malkawi<br />
The results obtained for all samples had a deviation below 3% suggesting a high precision in<br />
the quantification. The percentage mean recovery from extracts spiked with authentic GA3 was 94.0 ±<br />
2.0 % determined in 6 runs. All measured amounts were corrected for the % recovery.<br />
Method validation<br />
7-Hydroxycoumarin-4-acetic acid, which was not detected in potato extract, was selected as internal<br />
standard for endogenous GA3 because it contains structural features that are similar to those present in<br />
GA3 and thus its extraction behavior resembles that <strong>of</strong> GA3. Consequently, the recovery <strong>of</strong> this<br />
compound is indicative <strong>of</strong> the recovery <strong>of</strong> GA3 during the analytical procedure.<br />
The composite calibration graph was used to determine GA3 in synthetic samples <strong>of</strong> known<br />
concentrations to further determine the accuracy and precision <strong>of</strong> the method. On the other hand, to<br />
assess the effect <strong>of</strong> the sample matrix on accuracy and precision, known amounts <strong>of</strong> GA3 spiked in<br />
potato tissues were separately determined. Results are shown in Table 1. Each result was obtained from<br />
3 independent determinations (triplicate samples per day X 3 injections per sample).<br />
Table 1: Precision (as RSD %) and accuracy (as relative Error %) <strong>of</strong> the proposed method for GA3 analysis<br />
Matrix Spiked amount (ng) Determined amount(ng) RSD (%) Error (%)<br />
Solvent 100.0 98.0 3.0 -2.0<br />
Solvent 150.0 147.6 1.3 -1.6<br />
Solvent 200.0 202.8 2.9 1.4<br />
Spiked tissue 100.0 98.7 3.2 -1.3<br />
Spiked tissue 150.0 146.6 1.5 -2.3<br />
Spiked tissue 200.0 203.6 3.1 1.8<br />
Mean <strong>of</strong> 3 independent analyses in triplicate over 3 days (one replicate X 9 injections each day)<br />
RSD % = relative standard deviation <strong>of</strong> the mean<br />
The precision expressed as the relative standard deviation (RSD %) and the accuracy (% error)<br />
<strong>of</strong> the method were less than 3% for all samples. Such relatively low deviations and errors indicate a<br />
high precision and accuracy in the quantification method.<br />
The determined amount <strong>of</strong> GA3 in potato tissues was directly obtained from the measurement<br />
<strong>of</strong> its peak area and the calibration equation. The amount <strong>of</strong> GA3 spiked in the tissue extract just before<br />
GC-MS analysis was obtained from the total determined amounts <strong>of</strong> GA3 after spiking minus the<br />
determined original amount <strong>of</strong> GA3 in the tissue. Peak purity by GC-MS showed peak homogeneity<br />
thereby excluding the possibility <strong>of</strong> the presence <strong>of</strong> interfering components and demonstrating the<br />
specificity <strong>of</strong> the method.<br />
Results and Discussion<br />
I. Levels <strong>of</strong> gibberellic acid in potato tissues<br />
In this study we measured the levels <strong>of</strong> GA3 in potato (Solanum tuberosum cultivar “Russet Burbank”)<br />
plants grown differentially under tuber inducing and non-inducing conditions.<br />
We specifically chose to analyze GA3 for two reasons. First, it is the most widely known GA<br />
with inhibitory effect on tuberization established by experimentation with exogenous application to<br />
plant systems. Secondly, it is reported that GA3 promotes root growth in plants (Tanimoto 2005) and at<br />
the same time gibberellins are thought to be responsible for stolon growth in potato plants (Smith and<br />
Rappaport, 1969; Kumar and Wareing, 1972). Consequently, a screening <strong>of</strong> such hormone for its<br />
endogenous levels in the whole plant system within a time course experiment may be advisable.<br />
The effect <strong>of</strong> induction on endogenous levels <strong>of</strong> GA3 as influenced by photoperiod and<br />
temperature, induction duration, and tissue location (aerial and underground) can be inferred from the<br />
data listed in Table 2. Our data support the hypothesis that day length and temperature control the