Vertical and horizontal placements of the roots in the soil influence plant performance through acquisition of below ground resources like water and nutrients, plant anchorage and intra- and inter-plant competition.1–4 Therefore the architecture of the root system plays important roles in regulating plant growth and yield, especially under abiotic stresses.5 As a seedling grows to become a mature plant, the root architecture develops continuously in response to various cues e.g., genotypic, environmental, hormonal, etc. Therefore studies of root architecture of plants of different ages are important for understanding the influence of these cues in regulating plant growth.

The root scaffold of a plant is comprised of different types of roots with different functions. A mature common bean (Phaseolus vulgaris L.) plant has root system consisting of primary, adventitious, lateral and basal roots. Among these, the basal roots are typically the earliest emerging secondary roots from the hypocotyl6 forming a major part of the mature root system. We have recently demonstrated important differences in architectural traits of the basal roots of common bean in the early seedling stage between two contrasting class of genotypes and how auxin-ethylene interplay regulates these traits.7 While this study of basal roots at a fixed time allows assessment and comparison of root development up to that point of time, investigation of the temporal events of emergence and growth of the basal roots is important and complementary to the understanding of their architectural traits. Therefore in the present study, we examined the detailed developmental patterns of basal roots through time lapse imaging in two genotypes.

We chose two bean genotypes with contrasting basal root growth angles (BRGA) relative to the gravity—B98311 producing basal roots of smaller BRGA (41.7° ± 14°) and TLP19 having roots of larger BRGA (56.4° ± 18°).8 The germinated seedling with 2–3 cm radical was transferred to the blue germination paper (Anchor Paper Co., St. Paul, MN), which was suspended in nutrient solution7 inside a growth chamber (ACMAS Technocracy Limited, Delhi, India) maintained at 25 ± 1°C. Time lapse photography was carried out for 48 h at 30 min intervals using Nikon D200 digital camera fitted with a macro lens to obtain high resolution digital images of the roots. Imaging started from the visibility of the protrusions of emerging basal root along the root-shoot interface. A computer program was developed in Matlab^® 7.8 (Mathworks, Natick) to analyze the images semi-automatically. From every image the computer program identified the basal roots using contrast of color between the roots (mostly white) and the germination paper (blue). Root midlines were determined following the methodology of Miller et al.9 and smoothed using the method of overlapping polynomials. Length of the midline is root length. The angle between gravity and the line connecting the root tip to the base is BRGA.7 The vertical distance of the root tip from the base of the lowest emerging root along the gravity vector is tip depth. From the midline, root curvature was also determined using the equation (1) $\kappa =\frac{x^{\prime }{y}^{″}-y^{\prime }{x}^{″}}{{\left({x^{\prime }}^2+{y^{\prime }}^2\right)}^{3/2}},$(1) where [x(x), y(s)] is coordinate of any point along the root midline, s is normalized distance along the midline, and the primes denote derivatives with respect to s. Here positive curvature signifies bending upward and vice versa.

Spatio-Temporal Development of Basal Roots

The temporal development of architectural trait of four basal roots each from a B98311 plant and a TLP19 plant is shown in Figure 1. For both plants, all basal roots emerged together but they grew at different growth rates which accounted for their differences in length (Fig. 1A). Similar observation was made for other Phaseolus plants of same and different genotypes as well. This result points to a marked difference in emergence patterns between basal roots compared with other types of secondary roots. For example, it has been reported that lateral roots of Arabidopsis emerge with specific temporal rhythm.10 Sequential emergence of seminal and adventitious roots have also been reported in grass.11 But our results show that the emergence of basal roots in common bean is almost simultaneous and therefore the heterogeneity of lengths of basal roots due to genotypic differences and position of origin reported in Basu et al.7 is primarily dependent on variations in growth rate. Initially the growth rate was slower and after 12–18 h the growth rate accelerated as indicated by the change in slope of the root length vs. time lines. Although in majority of the cases the growth rate was nearly maintained, a few roots also showed deceleration of growth rates. Furthermore the basal roots of TLP19 had higher growth rate compared to B98311, and lower basal roots of both genotypes grew faster than the upper ones resulting in corresponding variations in root length.7

The growth angles of these eight basal roots fluctuated by a greater extent initially and then tended to stabilize with time (Fig. 1B). As a result, any comparison of BRGAs at a fixed time is likely to be dominated by these highly transient fluctuations for the first 24 h. After the initial 24 h, although fluctuations of BRGAs tend to subside, the BRGAs continue to change as the basal roots show plagiogravitropic growth. It is at this time that the influence of genotype and position of origin tend to appear in the patterns of BRGA. It is also interesting to note that BRGA vs. time plots show both increasing and decreasing trends at 48 h which is an outcome of curvature production in the basal roots during their growth as illustrated later in Figure 2.

Combined effects of changing root length, curvature and growth angle are visible in tip depths (Fig. 1C). In spite of relatively large variations in BRGA during the initial 24 h, tip depths did not show much variability. Therefore as mentioned in Basu et al.7 tip depths represent a more robust and direct measure of vertical placement of the roots even during the very early stage of seedling development when the BRGAs fluctuate rapidly. The tip depths also show that the TLP19 plant produced more vertically spread out root system compared to B98311 at any stage of development.

Root Curvature

Figure 2 shows gray scale map of midline curvature of two example basal roots each from a B98311 and a TLP19 plant as a function of time and distance along the root midline. The temporal development of BRGAs of these four roots is indicated by black and gray asterisks in Figure 1B. The brighter shades indicate upward curvature (positive values) and darker shades show downward curvature (negative values). In each of the roots, gray shades change rapidly both in time as well as along the root midline during the initial 18–24 h indicating rapid oscillatory growth patterns of basal roots during early development. But after that, the fluctuations in gray shades tend to subside. During 24–48 h, the upper roots in both plants (Figs. 2A and B) tended to grow nearly straight as the shades are almost mid-gray albeit with very gentle changes along the root length. But the lower root of B98311 showed downward curvature (darker shade) near the root tip and slight upward curvature near root base (brighter shade) during 30–48 h (Fig. 2C). The lower root of TLP19 showed the opposite curvature patterns (Fig. 2D).

A comparison of Figure 1B with Figure 2C shows that the BRGA of the lower root of B98311 (marked with gray asterisk in Fig. 1B) started to drop around 36 h and the darker gray shade (i.e., downward curvature) near the root tip also began to arise at the same time. On the other hand, Figures 1B and 2D show that the BRGA of the lower root of TLP19 (marked by black asterisk in Fig. 1B) reduced till 30 h, but became nearly constant beyond that. The dark gray shade (i.e., downward curvature) between 0.2–0.8 cm lightened starting from 18 h, while the lighter shade (i.e., upward curvature) near the root tip started to appear around 30 h. As a result, between 24–42 h the lower root of TLP19 had smaller BRGA compared with lower root of B98311, but beyond 42 h the lower root of B98311 started to have smaller BRGA. Therefore these results indicate that instead of unidirectional bending, balance of both upward and downward bends along the root length underlies gravitropic response of the basal roots. Initially the basal roots bend rapidly both along root length and time resulting in fluctuations in BRGA. Later on as the curvatures of the roots stabilize, the BRGAs also stabilize.