Down-Regulation of CYP79A1 Gene Through Antisense Approach Reduced the Cyanogenic Glycoside Dhurrin in [Sorghum bicolor (L.) Moench] to Improve Fodder Quality

Abstract

A major limitation for the utilization of sorghum forage is the production of the cyanogenic glycoside dhurrin in its leaves and stem that may cause the death of cattle feeding on it at the pre-flowering stage. Therefore, we attempted to develop transgenic sorghum plants with reduced levels of hydrogen cyanide (HCN) by antisense mediated down-regulation of the expression of cytochrome P450 CYP79A1, the key enzyme of the dhurrin biosynthesis pathway. CYP79A1 cDNA was isolated and cloned in antisense orientation, driven by rice Act1 promoter. Shoot meristem explants of sorghum cultivar CSV 15 were transformed by the particle bombardment method and 27 transgenics showing the integration of transgene were developed. The biochemical assay for HCN in the transgenic sorghum plants confirmed significantly reduced HCN levels in transgenic plants and their progenies. The HCN content in the transgenics varied from 5.1 to 149.8 μg/g compared to 192.08 μg/g in the non-transformed control on dry weight basis. Progenies with reduced HCN content were advanced after each generation till T 3 . In T 3 generation, progenies of two promising events were tested which produced highly reduced levels of HCN (mean of 62.9 and 76.2 μg/g, against the control mean of 221.4 μg/g). The reduction in the HCN levels of transgenics confirmed the usefulness of this approach for reducing HCN levels in forage sorghum plants. The study effectively demonstrated that the antisense CYP79A1 gene deployment was effective in producing sorghum plants with lower HCN content which are safer for cattle to feed on.

Agronomical and breeding approaches to improve the nutritional status of forage crops for better livestock productivity

“…The ideal forage ideotype includes delayed flowering (Schwartz & Amasino, 2013), high leaf area and photosynthetic capacity (Uauy et al, 2006), delayed senescence (Thomas & Ougham, 2014), increased reproductive tillers with short intermodal distance, amended cross-stress tolerance (Guo & Gan, 2014), upgraded root functions, augmented nitrogen assimilation and lower chlorophyll degradation (stay-green traits; Yang & Udvardi, 2018;Zhou et al, 2011; Figure 1). The desired forage traits can be achieved by improved agronomic management practices (Jat & Ahlawat, 2006) and plant breeding (Pandey et al, 2019), including conventional and advanced approaches (Figure 2). The molecular breeding and transgenic approaches primarily target to engineer the cell wall enzymes (Ruckle et al, 2017;Sakiroglu & Brummer, 2017), macromolecules (fructose and oligosaccharides; Badenhorst et al, 2018) and bio-fortified genes (Zhu et al, 2009).…”

Metabolomic Study of Sorghum (Sorghum bicolor) to Interpret Plant Behavior under Variable Field Conditions in View of Smart Agriculture Applications

“…In fact, it was proved that dhurrin biosynthetic enzymes are induced by nitrate availability. 14 A high content of dhurrin is toxic to animals 37,38 because of the release of HCN. Considering the fact that, together with grain, leaves and stems of sorghum are also generally used as forage, it is important to monitor and keep the level of this metabolite low in these organs also.…”

The application of antisense technology for crop improvement: A review

“…Upon exposure to enzymes and gut flora within the human intestine, it decomposes into a toxic chemical compound (Mansoor et al, 2006). Linamarin is synthesized through two cytochrome P450 enzymes, CYP79D1 and CYP79D2, in leaves and transported to roots (A K. Pandey et al, 2019). The leaf-specific inhibition of these enzymes lowered the linamarin content of the roots in transgenic plants by 99% (Meena et al, 2017).…”

“…The molecular methods of crop improvement currently under use include hybridization, mutation, tissue culture, and antisense technology (Kim et al, 2007;Ismail & Horie, 2017;Bailey-Serres et al, 2019, Pandey et al, 2019. Antisense technology is the most convenient and novel technology employed by crop breeders for the development of various crop species/varieties.…”