Concentration and potency experiments of growth hormone analogs determined by the bud sheath elongation method
Concentration and potency experiments of growth hormone analogs determined by the bud sheath elongation method
Growth hormone (indoleacetic acid) promotes elongation of oat germ sheath cells. In the case where the source of growth hormone is cut off from the apical shoot sheath cuttings, the elongation of the cuttings is linearly related to the logarithm of the concentration of the applied growth hormone over a certain range; therefore, the shoot sheath cuttings can be incubated with a series of growth hormone solutions of known concentration and plotted as a growth hormone concentration versus shoot sheath elongation curve in order to characterize the growth hormone content in the last-known samples.
Operation method
Concentration and potency experiments of growth hormone analogs determined by the bud sheath elongation method
Principle
Growth hormone (indoleacetic acid) promotes elongation of oat germ-sheath cells. In the case where the source of growth hormone is cut off from the apical shoot sheath cuttings, the elongation of the cuttings is linearly related to the logarithm of the concentration of the applied growth hormone over a certain range; therefore, the shoot sheath cuttings can be incubated with a series of growth hormone solutions of known concentration and plotted as a growth hormone concentration versus shoot sheath elongation curve in order to characterize the growth hormone content in the last-known samples.
Materials and Instruments
Wheat Oat seed Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
IAA solution Phosphoric acid-citric acid buffer
Petri dishes Filter paper Fine glass wires Tweezers Simple cutters Graduated pipettes Glass plates with chevron paper Thermostat Enameled trays Dark room Simple cutters
Wheat or oat seeds
Petri dish, filter paper, fine glass wires, forceps, simple cutter, graduated pipette, glass plate with chequered paper, thermostat, enameled dish, dark room.
Simple cutters can be made of wood, steel or Plexiglas and two double-sided blades with a distance of 6 mm between them.
10-3 M IAA solution: accurately weigh 17.5 mg of indoleacetic acid, dissolve it in phosphate-citrate buffer containing 2% sucrose, and volume the solution to 100 ml .
Phosphoric acid-citric acid buffer containing 2% sucrose (pH 5.0): Take 41.794 g of K2HPO, 1.019 g of citric acid, and 20 g of sucrose, and dissolve in 1000 ml of distilled water.
Experimental steps
Selected varieties of pure and sound wheat or oat seeds 100, immersed in saturated bleach solution, a few hours after the removal of distilled water to wash the seeds in a clean filter paper or quartz sand with a lid of the enameled tray, in order to make the embryonic sheath grows straight, can be aligned with the seed, the seed embryo upward and uniformly towards one side, will be placed diagonally into the tray 45?angle, so that the embryo tilt down, the tray is appropriate to add water and add a cover, placed in a dark room to grow, the room The temperature is maintained at 25°C and relative humidity at 85%.
Three days after sowing, when the embryo sheaths were about 25-35 mm long, 50 seedlings with uniform length of the shoot sheaths were selected, and the shoot sheaths were removed from the base with forceps, and 50 segments of the cut sections (see Fig. 8), which were 3 mm away from the tip of the shoot sheaths and 5-6 mm in length, were cut with a pest cutter on a glass plate with squares, and were immediately placed in a 2% sucrose-containing phosphate buffer (0.01 M , pH 4.5) for 1-2 h to wash away endogenous growth factors.
Five sets of Petri dishes were washed and dried, numbered, and 9 ml of phosphate buffer containing 2% sucrose was added to each dish. Then to ① was added 10 -3 M indoleacetic acid buffer 1 ml, shaking well, that is, the buffer containing indoleacetic acid 10-4 M; then from ① sucked out 1 ml injected into ②, shaking well, that is, the solution of 10-5 M, and so on to ④ dish, the preparation of 10-7 M solution, and sucked out 1 ml and discarded. No indoleacetic acid was added to ⑤ as a control.
Remove the cut segments of the germ sheath from the buffer, gently absorb them with filter paper, thread the cut segments onto glass wires, put 10 segments of the germ sheath into each Petri dish, cover them, and incubate them in a 25℃ warm box. All the above operations should be carried out under weak red light.
Remove them after 24 hours, aspirate the solution, and measure their lengths with a meter stick, accurate to 0.1 mm, to find out the average length of each treatment, and then compare the lengths in the growth hormone solution with the lengths in the control (L/L), and use this value as a longitudinal ordinate, and use the logarithm of the concentration of indoleacetic acid as a transverse ordinate to draw a standard curve.
If there is a growth hormone extract or other similar growth hormone solution with unknown concentration, and it is necessary to measure its concentration or potency, it can follow the above method to find the L/L. Then check the standard curve, and it can be obtained.