Abstract
Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.
Highlights
Subterranean clover (Trifolium subterraneum L.) is the most widely grown annual pasture legume in southern Australia, with an estimated area of 29.3 Mha [1]
Daliak, increasing the transformation efficiency from the previously reported 1% in other subterranean clover cultivars [10,11] to 5.2%
Subterranean clover differs from all other clovers in its underground seed development [1]
Summary
Subterranean clover (Trifolium subterraneum L.) is the most widely grown annual pasture legume in southern Australia, with an estimated area of 29.3 Mha [1]. It is welladapted to acidic soils, and there are subspecies adapted to neutral and alkaline soils, as well. It is mainly autogamous (inbreeding nature), diploid (2 n = 16), with a genome size of. In addition to the general benefits of legume pastures such as nitrogen fixation and soil structure improvement [4,5], it has three key additional features. It is highly tolerant to intensive grazing as the growing points of its prostrate growth habit are close to the ground It buries its seeds, protecting them from predators and grazing and allowing reliable self-regeneration. It has a high nutritional value for livestock [6,7]
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