3. Roots for at least five independent mycorrhizal plants should be
harvest and analyzed. It is important to include also negative
controls without AM fungal inoculation to discard the presence
of contamination.
4. For a faster processing, roots can be also cleared by boiling in
10% KOH for 10 min.
5. Longer incubation times (2–4 h) results in better staining.
Therefore mycorrhizal colonization can be assessed easier
under de microscope.
6. Elimination of excess ink can be done also by rinsing the roots
with 2% acetic acid. This treatment will keep samples in better
condition for longer when stored at 4
C.
7. The dimension of the grid squares is important for the measurement of total root length (colonized or noncolonized), but
not for measuring the percentage of colonized roots, since the
gridlines only act as a device for the systematic selection of
observation points. Grids of 1 cm
2 are normally used.
8. Fresh material can be also used. If this is the case, at least
200 mg of material is required. However, larger quantities
give better signals in the chromatography, and therefore better
results.
9. Roots for at least five independent mycorrhizal plants should be
extracted and analyzed. Although the apocarotenoids C 13
α-ionol glycosides (blumenols) and C 14 mycorradicin derivatives are specific of mycorrhizal plants, it is important to include
also negative controls without AM fungal inoculation to compare the different profiles.
10. Ribitol is used as internal standard to normalize extraction
efficiency of apocarotenoids. Ribitol is a crystalline pentose
alcohol (C 5 H 12 O 5 ) formed by the reduction of ribose.
11. A total of three extractions are required to ensure the obtaining
99% of the polar compounds.
12. Pure KOH (pellets) is used to adjust (increase) the pH of the
samples previous their analysis by HPLC. It allows an easier
detection of polar compounds.
13. The software for the analysis of the HPLC data is normally
supplied by the company of the equipment.
14. The integrated values are normalized to the mean value of the
internal standard ribitol calculated for the whole set of experiments per extract.
298
Juan M. Garcı ´a et al.
harvest and analyzed. It is important to include also negative
controls without AM fungal inoculation to discard the presence
of contamination.
4. For a faster processing, roots can be also cleared by boiling in
10% KOH for 10 min.
5. Longer incubation times (2–4 h) results in better staining.
Therefore mycorrhizal colonization can be assessed easier
under de microscope.
6. Elimination of excess ink can be done also by rinsing the roots
with 2% acetic acid. This treatment will keep samples in better
condition for longer when stored at 4
C.
7. The dimension of the grid squares is important for the measurement of total root length (colonized or noncolonized), but
not for measuring the percentage of colonized roots, since the
gridlines only act as a device for the systematic selection of
observation points. Grids of 1 cm
2 are normally used.
8. Fresh material can be also used. If this is the case, at least
200 mg of material is required. However, larger quantities
give better signals in the chromatography, and therefore better
results.
9. Roots for at least five independent mycorrhizal plants should be
extracted and analyzed. Although the apocarotenoids C 13
α-ionol glycosides (blumenols) and C 14 mycorradicin derivatives are specific of mycorrhizal plants, it is important to include
also negative controls without AM fungal inoculation to compare the different profiles.
10. Ribitol is used as internal standard to normalize extraction
efficiency of apocarotenoids. Ribitol is a crystalline pentose
alcohol (C 5 H 12 O 5 ) formed by the reduction of ribose.
11. A total of three extractions are required to ensure the obtaining
99% of the polar compounds.
12. Pure KOH (pellets) is used to adjust (increase) the pH of the
samples previous their analysis by HPLC. It allows an easier
detection of polar compounds.
13. The software for the analysis of the HPLC data is normally
supplied by the company of the equipment.
14. The integrated values are normalized to the mean value of the
internal standard ribitol calculated for the whole set of experiments per extract.
298
Juan M. Garcı ´a et al.