**2. Results and discussion**

To exemplify what degree of variation can be expected by sorghum varieties, a set of European sorghum lines was grown under sterile conditions in filter paper and in soil-filled rhizotrons. Grown in filter paper, the primary root and its lateral roots were identified easily, while no seminal roots were observed (**Figure 1**). In agreement with [28] 14 and 21 days after sowing (DAS), no seminal roots were observed in any of the varieties grown, while a varying number of crown roots was found. All root types of all the tested sorghum varieties did have root hairs and all those hairs were excreting sorgoleone, visible as droplets on each root hair tip. On soil-grown roots no sorgoleone was observed, but that might have been absorbed by the surrounding soil or washed away during the washing procedures at harvest.

#### **2.1 Diverse set of sorghum genotypes, but little aboveground diversity**

30 diverse sorghum genotypes, selected for variation in origin and breeding status, including physiological traits such as drought tolerance, and flowering time, (summarized in **Table 1**) were grown in soil-filled rhizotrons and their roots and shoots non-invasively phenotyped over three weeks. Although genotypic variation was large for most traits, the mean shoot height over all genotypes followed a linear increase (**Figure 2**) and its variation was stable in the last week of growth. Both, shoot height as well as shoot dry weight had a variation of ~2x and ~ 4x, meaning the largest genotype had a dry weight or shoot height twice or four times as large as the smallest genotype. At harvest, 21 DAS, the most contrasting lines had 35 cm compared to 60 cm high shoots and 0.23 g compared to 0.82 g shoot dry matter. Among the varieties with largest shoot height were 'Mace Da Kunya', 'SC35', and 'Mota Maradi', landraces described either as drought tolerant, post-flowering or preflowering drought tolerant, respectively. The shortest three varieties were 'Tx430', 'Tx631', and 'Tx436', all American feed-grade hybrids. Genotypes with large shoot height tended to also have higher shoot dry mass compared to genotypes with shorter


**Table 1.**

*Commercial sorghum parent lines and accessions.* Sorghum bicolor *lines selected for whole genome sequencing including diverse varieties from Africa, Striga-resistant lines from West Africa, and elite sorghum parent lines.*

shoots. A higher variation in shoot biomass compared to shoot height implicated additional factors influencing the first independent of the latter, such as leaf number, width and thickness. Given the highly diverse origin of these selected genotypes (**Table 1**) a high phenotypic variation above- and below ground was expected.

## **2.2 Diverse set of sorghum genotypes with much higher belowground diversity**

In contrast to the relatively small above ground variation in the rhizotrongrown sorghum lines, root dry matter varied much more after three weeks of *How Sorghum Root Traits Can Contribute to Cereal Yield Increase DOI: http://dx.doi.org/10.5772/intechopen.97158*

#### **Figure 2.**

*Variation in growth of 30* Sorghum bicolor *genotypes. Shoot height of 30 sorghum genotypes over 3 weeks grown in soil-filled rhizotrones are shown (A). Single genotype values are means over 4 replicates. At harvest, 21 DAS (example image in B), root and shoot dry weight was determined (C) and shown per genotypes as mean (n = 4) +/*− *SE. Per timepoint a one-way ANOVA Least Significant Difference (LSD) is depicted. Detailed information on genotypes can be found in Table 1.*

growth- almost 7x between the most extreme genotypes (**Figure 2**). At harvest the root dry weight varied between 0.22 g and 1.4 g. Again, the three largest root biomass varieties were observed as drought tolerant landraces ('Ajabsido', 'Segeolane', 'Mace Da Kunya'), while improved and hybrid varieties had lower root biomass ('Tx430', 'SC599', 'Wassa'). A similar wide range of variation (6-7x) was found for root length of all separated types- the primary root, nodal roots, and lateral roots (**Figure 3**), but it changed over time. One week after sowing the first emerging primary root showed the highest length and variation while crown and lateral roots were almost not detected. Primary root length reached a plateau between 14 and 17 DAS, both due to the physical rhizotron constraints and the

#### **Figure 3.**

*Variation in root growth of 30* Sorghum bicolor *genotypes. Root length of 30 sorghum genotypes grown for 3 weeks in soil-filled rhizotrones are shown. Single genotype values are means over 4 replicates. Visible and traced primary root (PR) length (A), nodal root (NR) length (B), and lateral root (LR) length (C) are depicted. Maximum intensity projections of all replicates per genotype are shown for the smalles LR and NR length (D, genotype 'SC599') and the largest LR and NR length (E, genotype 'Mota Maradi'). Coloured lines represent traced PR (green), NR (blue), and LRs (red). Per time point a one-way ANOVA Least Significant Difference (LSD) is depicted. Detailed information on genotypes can be found in Table 1.*

limited number of one primary root per plant. Two weeks after sowing, nodal root length varied from not detected to close to primary root length (50 cm), and just three days later their length doubled, and more than doubled again at harvest, 21 DAS. Lateral root length showed an even stronger increase in length over

time, the genotype with the longest LRs had 1,400 cm LR length at harvest, while the most contrasting genotype on the other end had only 250 cm LR length. With increase in NR and LR length over time, their variation among the tested genotypes also increased both in absolute and relative values. Although the genotypic ranking per investigated root type varied slightly, also over time, a general trend of stable ranking became visible. Since these plants were grown without nutritional, water, light, or biological stress this expresses their genetic potential to either form rather small or large root systems, often also with higher numbers of main axis. In all three root types, 'Tx436', a food-grade hybrid pollinator parent, and 'SC599' (**Figure 3D**), a post-flowering drought tolerant accession, were among the lowest ranking genotypes. Among the largest root systems were 'Mota Maradi' (**Figure 3E**), a pre-flowering drought tolerant landrace, and 'SK5912' and short 'Kaura', an improved open pollinated variety. Thus, previously drought tolerant described varieties did not show comparable root system developments in contrast to their early shoot development.

To gain more detailed knowledge about root morphology of these 30 sorghum genotypes, microscopic analyses were performed. Per root type (PR, NR, LRs) root diameter, root hair length, and root hair density were measured (**Figure 4**). When root hair density was plotted against root hair length per root type, a dependency became visible: roughly the more root hairs the longer they were (**Figure 4A**). All root types except for nodal roots showed significant correlations of root hair length and density. On all root types the genotypes 'Tx430', 'Tx631', 'Tx436', and 'Mace Da Kunya' formed the shortest and fewest root hairs. As root hairs are known to be instrumental for water und nutrient uptake [59] it is surprising to find 'Mace Da Kunya' in this list as it was also producing high root and shoot biomass. It should be noted that without nutrient and water limitation short and fewer root hairs were shown to be sufficient for plant growth [60, 61]. The longest and most root hairs were formed on roots of 'Segeolane', 'MR732', and 'Mota Maradi'. Since the latter, a pre-flowering drought tolerant landrace, also had the largest root system, it overall has the highest root surface area leading to the most soil contact for water and nutrient uptake. Like 'Mota Maradi', the genotypes with most root hairs also have the potential to excrete more sorgoleone into their soil environment compared to varieties with smaller root systems and fewer root hairs. Nodal roots had longer root hairs compared to all other root types, followed by primary roots, but their lateral roots did not differ from each other. Overall these soil-grown roots did produce many, but short root hairs of ~150 μm length; similar ranges of root hair formation have been reported for soil-grown rice varieties [60]. On the other hand, field-grown barley genotypes were reported to form longer root hairs from 400 μm [62] up to 700 μm [61]. Root hair formation in these studies varied with environmental conditions, be it nutrient or water supply, or other soil properties, therefore it is likely that sorghum root hairs could be longer in less optimal conditions then the one they were grown in here. Studies on rice root type-dependent root hair formation also showed a high dependency on the growth media used [63, 64].

The rhizotron-grown 30 genotypes showed root type-specific separation of root diameters (**Figure 4B**). For every genotype, nodal roots were not only thicker than primary roots, they did separate clearly with PRs ranging from ~300–550 μm and NRs from ~700–1,050 μm thickness. In contrast, their lateral roots had similar diameters, and both main roots (NR & PR) had 'thin' and 'thick' lateral roots, the first with ~100 μm diameter and little variation, the latter with higher variation from ~150–300 μm. In rice, distinct classes of lateral roots, S-type (thin) and L-type (thick) have been identified that are distinguishable by their diameter, but also branching ability [65, 66]. Recently those LR types have also been indicated to have

#### **Figure 4.**

*Root morphology of 30 Sorghum bicolor genotypes. Root morphological traits of 30 sorghum genotypes grown over 3 weeks in soil-filled rhizotrones were determined using a stereomicroscope and analysed with the software Image J. Each point represents a single genotype value which is a mean over 4 replicates. Shown are root hair length depending on root hair density per root type (A) and lateral root (LR) diameter depending on their main root diameter (B). Significant linear correlations are depicted. Detailed information on the genotypes can be found in Table 1.*

different functions in water and nutrient uptake and transport [67, 68]. If these different diameters do also indicate different LR functions in Sorghum would be interesting to investigate in future experiments, especially with resource limited conditions. Interestingly, while the primary root diameter did significantly correlate with the diameter of its lateral roots, this was not found for nodal roots and nodal root lateral roots. This may be due to the higher variation in lateral root diameter on nodal roots. The genotypes 'Tx436', 'Koro Kollo', and 'Tx631' had thin roots, while 'CSM-63', 'Feterita Gishesh', and 'Framida' were among the biggest root types. Interestingly, 'Kuyuma' had very thick PR and NRs, but very thin lateral roots, especially on NRs, while 'Ajabsido' behaved contrastingly. Overall, the thicker the root, the longer root hairs were measured (**Figure 4**), a trend that has also been observed in maize [69] and in rice [63].
