Two independent transgenic sorghum lines, featuring the pABS042 construct, for the targeted suppression of γ-kafirin-1 (27 kDa) and γ-kafirin-2 (50 kDa) were assessed in the present study. Previous analyses found complete suppression of the targeted kafirin proteins and a significant improvement (of up to 37%) in the in vitro protein digestibility of the transgenic grains (Grootboom et al., 2014). The transgenic lines represent the fifth generation of self-crossing progenies from T₀ sorghum transformants, which were originally produced using particle bombardment as described in Grootboom et al. (2014). Plants from each independent transgenic line (hereafter referred to as 42-1 and 42-2) and the parental control cultivar P898012 (hereafter referred to as WT), were grown in pots in a containment glasshouse located at the Centre for Scientific and Industrial Research (CSIR) Biosciences Division (Pretoria, South Africa). Environmental conditions were controlled (12-h photoperiod, 25°C/20°C day/night, ~50% humidity, 800 μE/s/m² irradiance), with a minimum of three biological replicates per line. At each occasion of watering, the plant arrangement in the glasshouse was randomized to counter any possible positional effects due to the microhabitat, and at anthesis, the panicle of each plant was bagged to prevent outcrossing. At full maturity, the grains were hand-harvested from each plant, and manually cleaned to remove all glumes, damaged grains, and other extraneous matter. For the physical characterization of the grain, whole kernels, separately derived from individual plants of each genotype were used to make up the sample. However, for the chemical analyses, the whole kernels had to be ground into fine flour. To reduce biological variation and to maximize the limited quantity of sample material available, equal portions of grain from individual plants from each genotype were pooled and homogenized to fine flour, using a standard laboratory mill. Milled grain flour was used immediately for the intended analysis or stored at −20°C in airtight containers until analyzed.

Unless otherwise stated, investigated parameters were analyzed in triplicate and the results expressed as the mean ± SD. Data were subjected to analyses of variance (ANOVA) and means compared by the Fisher Least Significant Difference (LSD) test (p < 0.05) using Statistica for Windows Version 13.0 (StatSoft Inc., USA).

The results pertaining to differences in the physical traits of grain from the two transgenic lines (42-1, 42-2) and their non-transgenic parental counterpart (WT) are presented in Figure 1. For the mean 100-grain weight, both transgenic lines were observed to have a lower weight in comparison to WT, but a statistically significant reduction could only be ascribed to the grains in line 42-2. In the case of the proportion of floury endosperm types and the percentage of floating kernels, both transgenic lines were found to have significantly increased levels of these characteristics in comparison to WT.

According to the literature, physical traits such as grain weight, density, and endosperm texture are important indicators of quality for sorghum because of their impact on grain hardness. Hard grains tend to be heavy (>2.0 g 100-grain weight) with a low percentage of floaters (<40%), and a corneous to intermediate endosperm texture (Gomez et al., 1997). For large scale production and processing, hard sorghum grains are generally preferred because of their association with reduced post-harvest losses (due to breakage and/or spoilage) and enhanced milling yields (Jambunathan et al., 1992; Simonyan et al., 2007).

The high percentage of floating grains amongst the transgenic lines (>96%) indicated a significant decrease in the grain density in comparison to the WT. The rationale informing the use of the sodium nitrate solution with a specific gravity of 1.25 g/mL is that this value is approximately equal to the average density of a wide range of sorghum kernels (Kirleis and Crosby, 1981). Thus, in this solution, kernels of lower density than average will float, while those of greater density will sink (Gomez et al., 1997). It is therefore clear that the transgenic grains are not only less dense than the WT, but are also less dense than the average known for sorghum grain. The reason for this decreased density is likely attributable to the significant increase in the percentage of floury endosperm types. The floury endosperm phenotype is associated with loosely packed starch granules that accommodate more air spaces (Rooney and Miller, 1982; Hoseney, 1994). It is therefore likely that the reduced density in the transgenic grain is attributable to the inclusion of more air spaces within the grain endosperm, which results in the characteristic opaque or floury visual appearance of this endosperm structure.

The increased percentage of floury endosperm phenotypes in the transgenic lines was commensurate with similar findings reported previously (Da Silva et al., 2011; Grootboom et al., 2014). Amongst the transgenic lines, a significant difference in the average number of floury endosperm types, was observed, with the highest percentage found in line 42-1 (85%), which was close to twice the value of that found in line 42-2 (47%). The floury endosperm phenotype is generally associated with improved nutritional qualities, such as improved protein digestibility and essential amino acid content (Singh and Axtell, 1973; Tesso et al., 2006; Da Silva et al., 2011). However, these nutritional gains are often overshadowed by the increased susceptibility of these softer floury grains to post-maturity losses and damage due to molding and insect infestation (Waniska and Rooney, 2002).

Morphological features of the grain protein bodies were studied using TEM analysis, the results of which are shown in Figure 2. In the earlier work of Grootboom et al. (2014), the protein bodies of the transgenic lines transformed with the ABS042 construct did not deviate much in terms of overall shape and size, as compared to the protein bodies found in the grain of the non-transgenic parent, P898012. This finding is consistent with similar studies featuring transgenic sorghum with targeted γ-kafirin suppression, such as the ABS 149 line (Da Silva et al., 2011) and the TX430 line (Kumar et al., 2012). In the present work, however, some important deviations in the morphology of the protein bodies in the transgenic lines were detected. Firstly in terms of size, no protein bodies of less than 0.5 μm diameter were found in the WT. In contrast, both transgenic lines were found to have protein bodies (of varying number) that were diminutive in size (<0.5 μm) in comparison to the WT. The reduced size of the protein bodies translate to a reduced surface to volume ratio, which would favor enhanced proteolytic digestion. Secondly in terms of shape, the most extreme change of morphology was evident in transgenic line 42-1. WT protein bodies conformed to the expected highly-ordered polygonal structure, with internal concentric rings, that is typical of normal sorghum. In contrast the protein bodies of line 42-1, were highly irregular in shape, with no internal concentric ring structures, and with deep invaginations present at the periphery. In line 42-2, the protein bodies were closer in morphology to the WT, exhibiting a regular overall polygonal shape, with some distinctive patterns of internalized concentric rings. The only features that served to differentiate the protein bodies of transgenic lines 42-2 from WT, were the small peripheral indentations that gave the boundary region a cracked appearance. The highly misshapen protein body morphology of line 42-1 is commensurate with the structural changes found in protein bodies of high-lysine Ethiopian landraces (Singh and Axtell, 1973; Oria et al., 2000) and transgenic lines featuring both γ- and α-kafirin suppression (Da Silva et al., 2011; Grootboom et al., 2014). It may therefore be speculated that the intended γ-kafirin suppression in line 42-1 manifests itself in a unique way that is aligned to the genetic programming in the floury endosperm phenotypes, but is distinctly different from other “related” transgenic lines with targeted γ-kafirin suppression only. This result warrants further investigation, particularly into the storage protein expression profile of 42-1 grain, which may provide vital clues into the determining factors that may underlie this “novel” phenotype.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.