Development and assessment of onion germplasm engineered to resist Allium white rot attack

Abstract

Allium white rot (AWR), caused by the soil-borne fungus Sclerotium cepivorum (Berk.), is the predominant disease of onion crops worldwide. This disease is difficult to control using conventional methods due to the resilience and longevity of soil borne sclerotia. No naturally occurring source of genetic resistance has been identified in onion. Therefore, the development of onions, which express traits with the potential to confer resistance to pathogenic microbes such as Sclerotium cepivorum, would be a major advance for onion breeding. Two genes with the potential to confer such resistance were integrated into the onion genome using genetic transformation. The first gene (mgd) encoded a magainin peptide derived from the African Clawed Frog (Xenopus laevis), which exhibits antimicrobial activity against numerous fungal and bacterial plant pathogens through membrane destabilisation and cell lysis. The second gene (oxo) encoded the oxalate oxidase enzyme, which degrades the fungal toxin, oxalic acid, produced during S. Cepivorum infection and enhances plant disease resistance through the concomitant production of hydrogen peroxide. These genes were transferred to onions through genetic transformation. Over 24,000 embryos were transformed with the mgd and oxo gene constructs over four seasons. Transgenic sectors were selected through their ability to grow on selective growth medium containing 15 g/L mannose or 20 mg/L geneticin and positively identified through the expression of the gfp visual reporter gene. Two magainin expressing lines (0215 2 and 0309 30) and one oxalate oxidase expressing line (0407 46) were confirmed as transgenic through molecular analyses. A bioassay using hydroponically grown roots inoculated with pre-germinated S. Cepivorum sclerotia detected significant differences in hyphal abundance and lesion size on the transgenic mgd line (0309 30) and oxo onion line (0407 46), respectively, compared to a non-transgenic onion line.

Promoter regions from a highly expressed root-specific transcript, identified from an onion cDNA library, were isolated using TAIL-PCR. Three putative promoter sequences (AD1, AD5 and AD9) were identified in association with three proline-richgenes (AD10, AD57 and AD92). Bombardment of full-length versions of the putative promoters (613 bp, 770 bp and 673 bp, respectively) fused to the gfp visual reporter gene identified non-specific promoter activity in both bulb and root tissue in the AD5 and AD9 promoters. Truncation of these promoters at approximately -500 bp, 300 bp, -200 bp and -100 bp revealed elevated levels of promoter activity in the -200 bp truncations and the maintenance of promoter function in the -97 bp truncations. Levels of gfp expression directed by the -197 bp truncation of the AD9 promoter were not significantly different to those directed by the viral CaMV 35S promoter.

Analysis of the transgenic plants in the field is expensive and highly regulated. Therefore, an in vitro screening protocol was developed to detect resistance to AWR in seedlings. Preliminary experiments demonstrated the protocol to be sufficiently robust to detect significant differences in resistance between different Allium species.