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GA and amylase experiment

John Hewitson John_Hewitson at compuserve.com
Sat Feb 14 03:54:07 EST 1998


Here is a quick copy and paste of the protocol we use for this experiment. 
There is a problem that modern wheat varieties are GA tolerant (ie they do
NOT respond to GA so that they produce short straw).  To get reliable
results, you need to find a long-straw variety.  Some univerisites test
batches of seed to find the best for this experiment and then keep a stock
of the best from year to year.
The pattern for the petri dishes, at the end of this email, won't be spaced
right.  The protocol also mentions a diagram - you'll have to DIY!


| Dr. John Hewitson                                               |
| Berrystead Barn                   +44 (0)1832 272 209 phone/fax |
| Oundle,                                                         |
| Peterborough, PE8 4DY, UK.  e-mail John_Hewitson at compuserve.com |
(Oundle School)


 The Germination and Structure of Wheat and Barley Grains

Equipment:-     Per Class       100cm3 0.1% Tetrazolium Salt Soln.
                Wheat grains (soaked for 3 hours)
                Barley grains (with husks)
                Millon's reagent        Iodine solution (very dilute)
                Boiling water bath      Plasticine      Microscope slides

                Per Pair                Scalpel         Forceps        
Mounted needles
                Glass Petri dish        2 staining dishes
                Sterile water   Dilute bleach solution
                Test tube       Clean matchstick
                1 large plate Starch (0.2%) Agar (1%) + 20mM Calcium
                1 large plate Starch (0.2%) Agar (1%) + 20mM Calcium
chloride + GA (1ppm)
                2 dehusked barley grains (Soak for 3.5 hours in 50%
sulphuric acid;
                         rinse vigorously with shaking in several changes
of water; air dry.
                         This removes lemma and palea.)
                2 endosperm halves for aleurone samples (Endosperm halves
of dehusked barley grains
                        placed in damp, sterile sand at 25oC for 2 to 3

1) The Site of Respiration in Germinating Seeds

Fully soaked grains of wheat are cut longitudinally and placed in
Tetrazolium salt solution in a petri dish at room temperature for about 30
minutes in the dark (the salt is unstable in light).  5-10 grains are
sufficient.  Tetrazolium acts as a hydrogen acceptor in respiration and is
changed to an insoluble red dye which is deposited at the sites of
After about 30 minutes, mount the cut grain in a small piece of plasticine
on a microscope slide and examine the cut surfaces using the low power
objective and reflected light.  Report on the distribution of actively
respiring tissue by  making a diagram and shading it in at the end of this
This technique is used as a standard viability test for seeds.  What is the
percentage viability of your sample of seed?

2) Site of Protein and Starch in Seeds.

Cut some seeds in half longitudinally and some transversely.  Warm the cut
seeds with Millon's reagent (CARE - poison).  Use about 2cm3 of water and 3
drops of Millon's in a test tube, in a boiling water bath.  Examine the cut
surfaces using low power objective and reflected light.  Report on the
distribution of protein, by shading in the diagram at the end of this
Treat other seeds with VERY DILUTE iodine solution to locate the site of
starch storage.  Examine and report as before.

3) Structure
The grains of wheat and barley are true fruits (the product of a fertilised
ovary), the seed coat being the fused pericarp (ovary wall) and testa
(integuments).  In some cases (eg. barley and oat) there may also be the
remains of the floret or spikelet.
The spikelet has outer scales called glumes which enclose the rest of the
spikelet.  This consists of slender tapering structures each composed of
two scales, smaller than the glumes.  The outer scale of each pair is the
lemma and the shorter, inner scale the palea.  Within the palea is the

4) The Effect of Gibberellic Acid on Amylase Production
To investigate the production of amylase by various parts of the fruit,
each part is placed on starch agar and incubated for at least 24 hours. 
After incubation, the plates are flooded with dilute iodine solution and
zones where amylase have hydrolysed the starch are shown up as cleared
 The starch agar with GA incorporated in it will show (by comparison with
the plain starch agar) the effects of GA on the various parts with regard
to amylase production.
Before placing the parts on agar, they are surface sterilised by first
washing in dilute bleach solution and then thoroughly rinsed in sterile
  Why is it necessary to sterilise each item before it is placed on the
starch agar?
  Matchsticks are treated in a similar way to check that the clearing of
the starch is a function of the living material and not any simple chemical
or physical effect.

a) Obtain one petri dish of plain starch agar and one dish of starch agar
with 1ppm GA added.
b) Cut prepared barley grains in half transversely.  Wash in dilute bleach
and rinse in sterile water.  Place on each agar in the pattern below.
c) With sterile forceps, remove portions of aleurone layer from the treated
embryo-less halves and place on each agar in the pattern below.  Place
sterilised pieces of matchstick on the agars as controls.
d) Incubate at 25oC for about 48 hours.
e) Flood the plates with very dilute iodine solution and report your
observations by drawing the cleared areas on the plans below.
f) Measure the diameters of the cleared areas, tabulate your results and
comment fully on these data.

A = Half fruit with embryo              B = Half fruit without embryo
C = Aleurone layer                      D = Matchstick control

        Starch Agar             Starch Agar + GA

        A       A                       A       A

        B       B                       B       B

        C       C                       C       C

        D       D                       D       D

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