Re-Programming Photosynthetic Cells of Perennial Ryegrass (Lolium perenne L) for Fructan Biosynthesis through Transgenic Expression of Fructan Biosynthetic Genes under the Control of Photosynthetic Promoters

Abstract

High molecular weight fructans are the main class of water-soluble carbohydrate used for energy storage in many temperate grass species including perennial ryegrass (Lolium perenne L.). As well as being important readily mobilisable energy reserves for the plant, fructans are also involved in stress tolerance. Fructans are also readily digested by grazing ruminants and hence are a valuable source of energy for sheep, beef and dairy production systems in temperate regions. This paper describes the re-programming of the expression of fructan biosynthesis genes through the transgenic manipulation of 6-glucose fructosyltransferase (6G-FFT) and sucrose:sucrose 1-fructosyl-transferase (1-SST) in perennial ryegrass. Transgenic events were developed with altered fructan accumulation patterns with increases in fructan accumulation and greatly increased accumulation of fructan in leaf blades as opposed to the traditional site of fructan accumulation in the pseudostem. This altered site of fructan accumulation has potential benefits for animal production as leaf blades form the major part of the diet of grazing ruminants. Some of the transgenic events also exhibited enhanced biomass production. This combination of high quality and enhanced yield is of great interest to forage plant breeders and whilst the expression of these phenotypes needs to be confirmed under field conditions, the identification and characterisation of the transgenic events described in this paper validate the potential for the manipulation of fructan biosynthesis in perennial ryegrass.

Development and Application of Droplet Digital PCR Tools for the Detection of Transgenes in Pastures and Pasture-Based Products

“…Clonal replicates of the genotype FLp418-20, were subjected to transformation using biolistic-mediated DNA delivery. A detailed description of the transgenic ryegrass plants generation is published in Panter et al (2017). The transgenic event 10 used in this experiment was hemizygous for the transgene, and the non-transgenic material was its null segregants (a genotype from perennial ryegrass plant FLP418-20).…”

“…Manual sequence analysis and primer design was performed using Sequencher version 5.0.1 (GeneCodes, Ann Arbor, MI, United States) and Primer3. 1 PCR primers and probes for the perennial ryegrass Cullin 4 gene, Lp Cul4 (Marín, 2009), the transgenic cassette containing modified fructan biosynthesis genes (1SST-6G-FFT; Panter et al, 2017), and the hph gene (Blochlinger and Diggelmann, 1984) were designed in this study and tested to generate amplicons shorter than 201 base pairs in length (Supplementary Table S3). The same primer and probe sets were used for both qPCR (SYBR Green I-based and probe-based) and ddPCR assays.…”

“…A transgenic high-energy perennial ryegrass was selected as case study for the evaluation and comparison of qPCR and ddPCR. A targeted up-regulation of fructan biosynthesis in the leaf blades of perennial ryegrass was obtained by re-programming the expression of fructan biosynthesis genes through the transgenic manipulation of 6-glucose fructosyltransferase (6G-FFT) and sucrose:sucrose 1-fructosyl-transferase (1SST) (Panter et al, 2017). During the transformation of high-energy ryegrass an antibiotic resistance factor, hygromycin B phosphotransferase ( hph ) gene, was introduced as a selectable marker.…”

Improving the Yield and Nutritional Quality of Forage Crops

“…Furthermore, the amounts of WSCs varies between varieties as well as within the environment; Lolium cultivars AberMagic, AberDart, and AberElite all had highest growth rates correlated to highest WSC concentration during spring/summer, corresponding to high N availability from the roots alongside optimal photosynthesis conditions ( Winters et al, 2010 ). Recent advances in the identification and manipulation of photosynthesis promoters for both Lolium perenne RBCS and Chlorophyll a/b Binding ( CAB ) ( Panter et al, 2017 ) has provided transgenic lines for assessing increases in yield, fiber and, more importantly for digestibility, the fructan concentrations in both pseudostem and leaf blades in field trials ( Badenhorst et al, 2018 ). Such work provides a platform for future studies to identify promotors important in other nutritional traits.…”

Safety Assessment of Genetically Modified Feed: Is There Any Difference From Food?

“…Perennial ryegrass plants with a high-energy trait have been also developed, using the transgenic technologies. A synthesized construct of perennial ryegrass 6-glucose fructosyltransferase (6G-FFT) and sucrose:sucrose 1-fructosyl-transferase (1SST) genes were introduced into the perennial ryegrass genome, aiming enhancement of fructan biosynthesis in the leaf blades (Panter et al, 2017). The transgenic plants showed a substantial increment in fructan accumulation in leaf blades, as well as enhanced biomass production.…”