2.2 HPLC
Quantification of
Molecular Markers
(Apocarotenoids)
This method is more accurate than the previous one, but it is also
more expensive and requires more specific analytical equipment.
The methodology is based on the protocol described by Fester et al.
[13]. All solutions should be prepared using deionized water and
analytical grade reagents., and they should be stored at room
temperature.
1. 80% (v:v) methanol (MeOH).
2. 2 mg/ml ribitol.
3. Potassium hydroxide (KOH), pure (pellets).
4. 1.5% phosphoric acid (H 3 PO 4 )
5. Acetonitrile (MeCN), pure.
3 Methods
3.1 Histochemical
Quantification
1. Rinse harvest roots with tap water to remove solid particles
from the substrate (see Notes 2 and 3).
2. Dry roots with filter paper, cut them into 1 cm long pieces and
place them in a 50 ml Falcon tube.
3. Incubate the roots with 40 ml 10% KOH for 2 days at 4
C in
darkness (see Note 4).
4. Rinse the samples three times with 40 ml deionized water.
5. Acidify the roots with 2% acetic acid for 5 min at room
temperature.
6. Dye the roots by immersion in the staining solution for at least
30 min (see Note 5).
7. Remove excess ink by rinsing the roots three times with 40 ml
deionized water (see Note 6).
8. Roots can be stored in deionized water or 70% ethanol at 4
C
for few months.
9. Mark a grid of lines of 1 cm on the bottom of a 90 mm Petri
dish (see Note 7).
10. Randomly disperse the stained roots (about 200) in the Petri
dish with gridlines using forceps and a dissecting needle.
11. Assess mycorrhizal colonization under a dissecting microscope
by following all horizontal and vertical lines.
12. Count intersects with roots and mycorrhiza (positive) separately. Do, at least, two sets of quantification, recording
100–200 root/gridline intersections.
13. Calculate the colonization percentage by counting all the positives contacts. Divide this number by the total number of
contacts (positives and negatives), and multiply by 100.
296
Juan M. Garcı ´a et al.
Quantification of
Molecular Markers
(Apocarotenoids)
This method is more accurate than the previous one, but it is also
more expensive and requires more specific analytical equipment.
The methodology is based on the protocol described by Fester et al.
[13]. All solutions should be prepared using deionized water and
analytical grade reagents., and they should be stored at room
temperature.
1. 80% (v:v) methanol (MeOH).
2. 2 mg/ml ribitol.
3. Potassium hydroxide (KOH), pure (pellets).
4. 1.5% phosphoric acid (H 3 PO 4 )
5. Acetonitrile (MeCN), pure.
3 Methods
3.1 Histochemical
Quantification
1. Rinse harvest roots with tap water to remove solid particles
from the substrate (see Notes 2 and 3).
2. Dry roots with filter paper, cut them into 1 cm long pieces and
place them in a 50 ml Falcon tube.
3. Incubate the roots with 40 ml 10% KOH for 2 days at 4
C in
darkness (see Note 4).
4. Rinse the samples three times with 40 ml deionized water.
5. Acidify the roots with 2% acetic acid for 5 min at room
temperature.
6. Dye the roots by immersion in the staining solution for at least
30 min (see Note 5).
7. Remove excess ink by rinsing the roots three times with 40 ml
deionized water (see Note 6).
8. Roots can be stored in deionized water or 70% ethanol at 4
C
for few months.
9. Mark a grid of lines of 1 cm on the bottom of a 90 mm Petri
dish (see Note 7).
10. Randomly disperse the stained roots (about 200) in the Petri
dish with gridlines using forceps and a dissecting needle.
11. Assess mycorrhizal colonization under a dissecting microscope
by following all horizontal and vertical lines.
12. Count intersects with roots and mycorrhiza (positive) separately. Do, at least, two sets of quantification, recording
100–200 root/gridline intersections.
13. Calculate the colonization percentage by counting all the positives contacts. Divide this number by the total number of
contacts (positives and negatives), and multiply by 100.
296
Juan M. Garcı ´a et al.