European Journal of Scientific Research - EuroJournals

409 Ahmed Malkawi
photoperiod on the endogenous levels of GA3 in potato plants grown in inducing and non-inducing
conditions. To our knowledge, time-course determination of endogenous GA3 absolute levels during
the whole developing tuber induction process has not been reported.
Knowledge of quantitative changes of GA3 content in potato plants may have important
implications not only for our understanding of regulation of tuber induction and elucidation of its
molecular mechanism, but also for the design of effective strategies for yield improvement.
Materials and Method
Plant material and growth conditions
Seed tubers of Solanum tuberosum cv. Russet Burbank were sowed in 0.8-L plastic pots filled with a
pre-moistened mixture of garden soil and sand (1:1 v/v). This was an appropriate growth medium for
the plants studied. The plants were grown in a growth chamber under 18 hours of light (at 30ºC) and 6
hours of darkness (at 26ºC). Temperatures were controlled to ±0.5ºC. Flourescent strip light was used
during the light period providing a photonsynthetic photon flux of 140 µmol m -2 s -1 at top of plant
canopies. Tubers were watered daily with tap water and fertilized weekly with 0.1% Miracle-Gro (Port
Washington, NY). When plants reached approximately 55-60 cm height, half of the 8-week old plants
were randomly selected and transferred to a second growth chamber adjusted to tuber-inducing
conditions (24ºC day, 12ºC night and a 10-hour photoperiod). Plant harvest began 2 days later and
continued for a period of 10 days at 2-day intervals. Each time three samples were randomly selected
from each chamber, removed from growth medium, washed with distilled water, separated into aerial
and underground portions, freeze-dried, ground with a mill to a fine powder and stored in glass bottles
at -20 C until analysis.
Extraction of gibberellic acid
One gram of ground tissue sample was mixed with 40 ml of ethyl acetate containing 500.0 ng of 7hydroxycoumarin-4-acetic
acid, Figure 1, as an internal standard for quantification. The resulting
mixture was placed in an ultra-sonic bath for 2 hours in ice-cooled water. The solution was vacuum
filtered and the supernatant was filtered through Nylon-66 filters (0.45 micron). Gibberellic acid was
separated from the ethyl acetate when the filtrate was partitioned 3 times with 15 ml of 1.0 M NaHCO3
solution. The combined aqueous extract was adjusted to pH 4.0 with 1.0 M H2SO4. Thereafter, this
aqueous phase was partitioned 3 times with 15 ml volumes of ethyl acetate. The combined ethyl
acetate fractions were evaporated to dryness under reduced pressure and the residue was reconstituted
in 1.0 ml ethanol for subsequent chromatographic analysis.
Preparative HPLC Analysis
A Hewlett-Packard HPLC system Model 1090 equipped with a photodiode array detector was used.
Chromatographic separation was performed with a Spherisorb ODS-2 C18 reversed-phase column (25
cm long x 4.6 mm i.d.; 5 µm packing; 80 Ao pore size; Waters Corporation, Taunton, MA) and a
Spherisorb ODS-2 C18 pre-column (7.5 mm x 4.6 mm; 5 µm packing; Alltech, MA). Prior to injection,
the standard and extract samples were filtered through Nylon Acrodisc syringe filters (13 mm
diameter, 0.2 micron pore size; Gelman Sciences, MI). All solvents were filtered through MSI Magna
Nylon membrane filters (Micron Separations, Westboro, MA; 47 mm diameter / 0.45-µm pore size).
A solvent system of acetonitrile and water (containing 0.1% acetic acid) was used for isocratic
elution with 80% acetonitrile over 10 minutes and solvent flow rate of 0.9 mL/min. Gibberellic acid
was detected by UV absorbance at 230 nm. Fractions at the retention time of GA3 (5 minutes) were
collected and combined and the solvent was evaporated and the residue was re-dissolved in 0.5 ml of
dichloromethane for subsequent GC-MS analysis.