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

Arun K Pandey,Pusuluri Madhu,Basrur Venkatesh Bhat

doi:10.3389/fnut.2019.00122

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 T3. In T3 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.

Highlights

The sorghum crop [Sorghum bicolor (L.) Moench] is grown worldwide in 45.38 million hectares and accounts for the production of 6.37 million tons of grains [1]
The synthesis of a small quantity of the dhurrin may be desirable as a defense against insects [49, 50]
The antisense RNA approach was used to down-regulate dhurrin synthesis

Summary

Introduction

The sorghum crop [Sorghum bicolor (L.) Moench] is grown worldwide in 45.38 million hectares and accounts for the production of 6.37 million tons of grains [1]. 80% of the cultivated area lies in Asia and Africa. It is the fifth major cereal crop in the world, mainly cultivated in semi-arid regions. Sorghum is an important grain and forage crop of semi-arid regions due to its high adaptability and suitability to rain-fed low input agriculture. Development of Cyanogen Safe-Sorghum limiting the utilization of sorghum forage, especially till the flowering stage, is the production cyanogenic glycoside, i.e., dhurrin, which is toxic to the feeding livestock [2]. Hydrocyanic acid (HCN) toxicity due to hydrolysis of dhurrin from sorghum forage in the rumen of the cattle causes their death. Other plants produce HCN, but in lesser amounts, whereas in sorghum it is produced in larger amount which is hazardous to the animal species [3]

Methods

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Discussion

Conclusion