Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Abstract

Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull-less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fiber component, (1,3;1,4)-β-glucan (mixed-linkage glucan), primarily present in starchy endosperm cell walls. Over-expression of the HvCslF6 gene, driven by an endosperm-specific promoter, produced lines where mixed-linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub-aleurone cells. This work provides proof-of-concept evidence that mixed-linkage glucan content in hull-less barley grain can be increased by over-expression of the HvCslF6 gene, but also indicates that hull-less cultivars may be more sensitive to attempts to modify cell wall composition.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development

“…Barley plants were grown following Lim et al (2018) under a day/night temperature regime of 23 °C/15 °C. Progeny of the T 3 generation from four independent transformed lines (15-3, 18-6, 25-5, and 16-5) overexpressing the HvCslF6 gene were selected; these lines were referred to as lines F6-15, F6-18, F6-25, and F6-16 in Lim et al (2018); in addition, Torrens [wild type (WT)] and WT plants regenerated from tissue culture [WT(tc)] were selected and analysed. Developing grains from individual plants were collected from the middle of the spike from 7 to 24 DAP at approximately mid-day, covering all phases of grain development.…”

“…The resulting vector MA032 (pAsGlo1:3xnlsYFP) was transformed into Agrobacterium tumefaciens strain AGL1 and used for transformation of the Hordeum vulgare cv. WI4330 following the protocol outlined in Lim et al (2018).…”

“…Pellets were washed with 100% ethanol (1 ml) at 97 °C for 10 min, centrifuged at 5000 rpm for 5 min, and supernatants removed. MLG content was measured using the Megazyme Mixed-linkage Beta-glucan Assay Kit (K-BGLU) (McCleary and Codd, 1991) as outlined in Lim et al (2018).…”

“…Morphological changes in these lines were detected in aleurone tissues that were on average three cell layers thick, with strongly defined walls and a pronounced cubic shape as well as deformed and, in places, completely missing transfer cells at the grain-filling phase (15 DAP). Mature transgenic grain had reduced size relative to control lines, a shrunken and darkened appearance, and increased brittleness (Lim et al , 2018). In this study, we used transgenic grains overexpressing the HvCslF6 gene as a model system to investigate the effects of increased MLG on carbohydrate metabolism, cavity formation, and cell identity during grain development.…”

A Genome Assembly of the Barley ‘Transformation Reference’ Cultivar Golden Promise

“…Most barley transformations are successfully conducted using Golden Promise as it usually achieves the best shoot recovery from callus (Hensel et al 2008). While many other cultivars have been tested and some successfully used, the transformation efficiency of Golden Promise is always superior (Murray et al 2004;Ibrahim et al 2010;Lim et al 2018) With the rise of the CRISPR-Cas9 genome editing technology, a potential Golden Promise reference assembly has already sparked wide interest in the barley community. The use of CRISPR-Cas9 ideally requires a complete and correct reference assembly for the identification of target sites (Karkute et al 2017).…”