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 use of these varieties. To overcome this, we have 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 fibre component, (1,3;1,4)-β-glucan (mixed-linkage glucan), primarily found 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 HvCslH1grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker colour, 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, includingaleurone 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.