Approximately 1 g of fresh lateral roots were excised
from each plant in order to quantify the degree of
mycorrhizal colonization (the dry weight of such roots
was estimated and added to the root mass). These sampled
roots were first order lateral roots emerging from
the upper half of the main tap root. The roots were kept
in FAA (ethanol 50%, acetic acid and formaldehyde
35% in the proportion of 9:0.5:0.5, v/v/v) for 3 days
and then transferred to an ethanol solution (70%) until
clearing and staining procedures were done. The technique
described by Phillips and Hayman (1970) was
adapted for A. angustifolia, because the original root
clearing procedure, which included heating the roots at
90 ◦C for about 1 h in 10% KOH, was not successful in
clearing the roots, and going beyond this time-period
resulted in excessive damage to the root tissues. The
adapted technique included the following procedures:
(1) autoclaving of the roots in a 10% solution of KOH
for a period of 20 min, followed by water-rinsing, (2)
immersion of the cooled roots in alkaline H2O2 for
20 min, followed by another water-rinsing, (3) root immersion
in a 10% solution of HCl for 10 min; and (4)
staining of the root samples with Trypan Blue in lactoglycerol
(0.05%) for a 1 h period. Quantification followed
the grid-intersection method (Giovannetti and
Mosse, 1980), using a 1.27 cm2 grid area.
Approximately 1 g of fresh lateral roots were excisedfrom each plant in order to quantify the degree ofmycorrhizal colonization (the dry weight of such rootswas estimated and added to the root mass). These sampledroots were first order lateral roots emerging fromthe upper half of the main tap root. The roots were keptin FAA (ethanol 50%, acetic acid and formaldehyde35% in the proportion of 9:0.5:0.5, v/v/v) for 3 daysand then transferred to an ethanol solution (70%) untilclearing and staining procedures were done. The techniquedescribed by Phillips and Hayman (1970) wasadapted for A. angustifolia, because the original rootclearing procedure, which included heating the roots at90 ◦C for about 1 h in 10% KOH, was not successful inclearing the roots, and going beyond this time-periodresulted in excessive damage to the root tissues. Theadapted technique included the following procedures:(1) autoclaving of the roots in a 10% solution of KOHfor a period of 20 min, followed by water-rinsing, (2)immersion of the cooled roots in alkaline H2O2 for20 min, followed by another water-rinsing, (3) root immersionin a 10% solution of HCl for 10 min; and (4)staining of the root samples with Trypan Blue in lactoglycerol(0.05%) for a 1 h period. Quantification followedthe grid-intersection method (Giovannetti andMosse, 1980), using a 1.27 cm2 grid area.
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