CHAPTER 2. MATERIALS AND METHODS 36
2.3 Analysis of root system architecture and leaf growth 44
experiments with a Celco 30 cm hardened stainless steel ruler because it is labeled from the edge. The measurements were started at 10 am, which was equivalent to four hours after the beginning of the photoperiod (6 am), and the plants were measured in
sequential order each day. The roots were out of liquid media for a maximum of five minutes while the primary root lengths were measured and the number of lateral roots was counted. The photographs presented were taken with a hand-held Sony DSC-H55 digital camera. The settings were determined automatically by the camera and are recorded in Appendix VI. The whole plant fresh weight was recorded after the final destructive harvest, and the roots and upper plant parts were weighed separately, to calculate the root to shoot ratios and estimated specific root length (SRL) (refer Table 3).
Leaf measurements and terminology
The newly emerged leaves on the main stolon were counted as they cleared the sheath that surrounds the unemerged leaves at the stolon apex (A) as a measure of leaf emergence and development (Figure 2.2a).
Figure 2.2. Petiole length and root system architecture measurements
a. Image of 45-14 grown in P-sufficient media for 23 days (post excision). The leaves are labeled from the apex (A). L1 is the first fully expended leaf (FFEL), and L3 is the oldest leaf. The unfolded leaf (U) was not used in the analysis. The primary roots emerge from either node three or node four. The lateral roots are visible in the branching zone. The newly emerged lateral roots (> 10 mm) were counted separately from the elongated lateral roots (> 10 mm). b. Stolons with the dominant primary root at node 3 (left) or node 4 (right). c. Schematic diagram of the branching zone that contains visible lateral roots, and the elongation zone without visible lateral roots as a proportion of the primary root length. d. Microscopic image showing the emerging lateral roots (top) and the region containing root hairs which is visible to
U L1 L2 L3
Node 3
Node 4
Elongation
zone
New leaf branches Elongated lateral roots > 10 mm ABranching
zone
Newly emerged lateral roots < 10 mm2a Dominant primary roots (DPR)
Node 3 DPR Node 4 DPR
Newly emerged lateral roots <10 mm 2b
2d
Elongation zone
Branching zone Proportion of primary root
with lateral roots 2c
Primary root length
The leaves were labeled relative to the position from the apex at harvest (Figure 2.2a). The youngest (first) fully expanded leaf grown on the main stolon is designated L1 and leaf 7 (L7) the oldest (Figure 2.2a). The difference in petiole length between treatment groups was calculated and is shown as Figure 3.4 for experiment I together with the final petiole length at harvest. In Figure 3.4, (b, d and f) a positive value represents a longer petiole in the P-sufficient group when compared with the P-stressed group and a negative value represents a longer petiole in the P-stressed plants. The data for petiole lengths in experiments II and III is not shown. The leaves on the lateral branches extending from the main stolon were only measured as part of the final fresh weight during the destructive harvest at the end of each experiment.
Root measurements and terminology
The lengths of the primary roots that grew from either node three or four were measured and the number of lateral roots on each of the primary roots was counted during the course of the experiment. The total primary root length was calculated for each day that the plants were measured by adding the lengths of all of the primary roots. The relative growth rate of the primary root length was calculated during the course of the
experiment by dividing the difference in primary root length (growth between days) by the primary root length on the current day of measurement (Table 3). The estimated specific root length (SRL) was calculated after the final destructive harvest by dividing the harvested fresh weight by the total primary root length at harvest (Table 3).
The specific root length is only estimation because the length of the lateral roots was not taken into account, but contributed to the total fresh weight.
All of the genotypes had a dominant primary root, which was used to represent lateral root development in response to limited P or S-supply (Figure 2.2b). The other primary roots are referred to as the “supporting primary roots” and contributed to the entire root system architecture (RSA) of the plant. The data for the number of lateral roots on the supporting primary roots is not shown. The lateral roots are in the designated branching zone, and absent from the lateral root formation zone designated ‘elongation zone’ in this study (Dubrovsky and Forde, 2012). An enlarged area between the branching and elongation zones was visible to the naked eye (because it contains a clump of root hairs), and was used to define the boundary between the two zones (Figure 2.2c).
The branching zone was determined by subtracting the elongation zone length from the primary root length and expressed as a percentage (of the primary root length).
The number of visible lateral roots less than 10 mm were counted separately from the number of lateral roots greater than 10 mm, and the total for the two parameters were added together to give the total. The length of 10 mm was chosen arbitrarily for this study to assist with quantifying lateral root emergence (<10 mm) and elongation (>10 mm). In experiment II, the longest lateral root was also measured and the
emergence of the tertiary roots was recorded (refer Figure 3.17). The lateral root density was calculated for the dominant primary root (Table 3), where larger value represents less lateral roots in the branching zone compared with a smaller value that represents a greater number of lateral roots in the branching zone (refer Figure 3.12).
Table 3. Measurements and calculations for root system architecture
Abbreviations: Primary root (PR), lateral root (LR), fresh weight (FW).
#
The term ‘growth of primary root’ refers to the primary root length on current day of measurement minus the previous day’s primary root length.
Measurement Calculation
Total primary root length Length of dominant and supporting primary roots Relative growth rate of PR length (%) #Growth of PR (mm)/PR length (mm)
Branching zone (%) PR length (mm) – elongation zone (mm)
LR density (LR/mm) PR length (mm) -elongation zone (mm)/total LRs Estimation of specific root length (cm.mg-1) Harvested root FW (mg)/total PR length (cm)