Root screening in thin chambers
Materials and methods
A full description of the experimental procedure is given in Price et al. 2002; Field
Crops Research 76 11-24). Here only a brief description is given in order to allow
much of the significance of the work to be appreciated without reference to the other
publication.
1.1 Plant material
A mapping population of 205 recombinant inbred (F6) seeds was produced from a
cross of varieties Bala and Azucena (original parental seed obtained from the
International Rice Research Institute) by single-seed descent as described in Price et
al. (2000) TAG 100: 49-56.
2.2 1997 experiment of Bala, Azucena and the mapping population
A total of 140 recombinant inbred lines and 5 replicates of Azucena and Bala were
grown in thin soil chambers under two treatment conditions: a long-term, early waterdeficit (WD0(97)) which started with fully saturated soil but received no more water,
and a well-watered treatment in which water was withheld after day 49 (during the
final, 8th week of the experiment) (WD49(97)). The chambers were made by taping
together two sheets of 4-mm-thick glass (1.2 x 0.3 m width x length) 15 mm apart and
filling the space with soil (sandy loam, pH 5.5). The chambers had a 5 mm diameter
drainage hole at the bottom of each side. Before sowing, each chamber was saturated
with Yoshida’s nutrient solution, pH 5.5 (Yoshida et al., 1976). 5 seeds were sown in
each chamber. After seedling emergence, the seedlings were thinned to two per
chamber. One set of chambers did not receive further water or nutrient (WD0). The
other chambers were given 1.0 l of nutrient solution three times a week for the first 4
weeks. Thereafter, they received 1.0 l nutrient every day, and after 6 weeks received
1.0 l of water in addition to 1.0 l of nutrient every day. After 7 weeks, no water or
nutrients were applied, giving a late water deficit (WD49) treatment for the final
week. Sheets of expanded polystyrene were placed behind all stacks of chambers to
prevent heat loss. Within each treatment, chambers were arranged randomly in stacks
of 6 chambers, but the two treatments were positioned next to each other. Plants were
grown for 8 weeks in a greenhouse (minimum temperature 25oC) in Bangor during
the Summer of 1997, under daylight supplemented by 150 mol m-2 s-1 PAR. Twice a
week, the position of the chambers within a stack was rotated to remove positionwithin-stack effects.
On a weekly basis and for both treatments, shoot growth was monitored as height
of the plant (length from the soil to the tip of the longest leaf) while the length of the
longest visible root and the number of roots past 25, 50, 75 and 100 cm was recorded.
At the end of the experiment (after 8 weeks), shoots were removed in a single day.
Over a 4-week period, each chamber was opened and soil and roots placed on a nailboard of 15 mm nails arranged 2 cm apart in a grid to allow the soil to be washed
from the roots by water spray. A short section of three of the thickest roots were
removed from each root system both near the base of the shoot and at 90 cm depth
and placed in 50% ethanol. These were stored at 4oC and subsequently used to
measure root thickness. The washed root systems were separated into 4 depth classes,
0-30 cm, 30-60 cm, 60-90 cm and 90-120 cm and dry weights were recorded.
2.3 1998 experiment of the mapping population
The experiment was repeated in 1998 with minor modifications. Both early water
deficit treatment (WD0(98)) and late water deficit treatment (WD49(98)) did not have
small drainage holes at the bottom of the chambers. In the WD49(98) treatment the
chambers received nutrient or water at a rate of only 800 ml per chamber.
2.4 Data handling
Data was tested for normality before statistical analysis. The means of the both the
parents and the F6 in 1997, and only the F6 in 1998 are presented in Table 1. Since
there was no replication for the F6 and both treatment and year had a significant and
interacting effect on most traits, it is not sensible to calculate broad-sense heritability
based on F values from analysis of variance. Broad-sense heritablity was therefore
calculated as 100 x (1 minus the ratio of the average variance of the non-segregating
genotypes over the variance of the F6 population). These are presented in Table 2. In
1997, the non-segregating genotypes were Azucena and Bala. In 1998, they were Bala
and 4 other Indian upland varieties that were included in the experiment (data not
shown).
Total plant mass was the sum of shoot and root dry weight after harvest on day 56.
Root to shoot ratio was the root dry weight divided by the shoot dry weight.
Maximum root length was calculated as the average of measurements of the longest
visible root on days 28 and 35. All traits were normally distributed except for relative
water content in 1997 for both WD0 and WD49 treatments, which were squared
before analysis, and dry weight of root below 90 cm in WD49(97) and WD49(98),
root thickness at both base and at 90 cm for WD0(98) and root to shoot ratio in all
treatments, for which the square root was used for QTL analysis.
QTLs were calculated for individual experiments and for the average of the two
treatment types and for the overall average, in order to identify regions with a broad
environmental stability from those that are perhaps environment specific. For
calculating average values (and for visible root lengths for each experiment) data were
first normalised by dividing the value for each individual by the population mean for
that trait, before a mean was calculated.