International Journal of Molecular Sciences Article An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.) Fernando Perez Rojo , Sumedha Seth , William Erskine and Parwinder Kaur * UWA School of Agriculture and Environment/Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia; [email protected] (F.P.R.); [email protected] (S.S.); [email protected] (W.E.) * Correspondence: [email protected] 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 tech- nologies, 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. Addition- ally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone Citation: Rojo, F.P.; Seth, S.; Erskine, combinations were tested to analyse the best rooting media. Roots were obtained in a medium W.; Kaur, P. An Improved Protocol for µ Agrobacterium-Mediated supplemented with 1.2 M IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS Transformation in Subterranean gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transforma- Clover (Trifolium subterraneum L.). Int. tion. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, J. Mol. Sci. 2021, 22, 4181. https:// tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other doi.org/10.3390/ijms22084181 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 Academic Editor: Gian-Pietro Di improved transformation study presents an opportunity to increase the feeding value, persistence, Sansebastiano and nutritive value of the key Australian pasture. Received: 22 March 2021 Keywords: subterranean clover; Agrobacterium transformation; in vitro culture Accepted: 14 April 2021 Published: 17 April 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Subterranean clover (Trifolium subterraneum L.) is the most widely grown annual iations. pasture legume in southern Australia, with an estimated area of 29.3 Mha [1]. It is well- adapted 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 540 Mb (cv. Daliak) divided into eight pseudo-molecules [2,3], and smaller than many other legume species [4]. In addition to the general benefits of legume pastures such as nitrogen Copyright: © 2021 by the authors. fixation and soil structure improvement [4,5], it has three key additional features. Firstly, it Licensee MDPI, Basel, Switzerland. This article is an open access article is highly tolerant to intensive grazing as the growing points of its prostrate growth habit distributed under the terms and are close to the ground. Secondly, it buries its seeds, protecting them from predators and conditions of the Creative Commons grazing and allowing reliable self-regeneration. Thirdly, it has a high nutritional value for Attribution (CC BY) license (https:// livestock [6,7]. creativecommons.org/licenses/by/ However, subterranean clover has limitations such as the need for relatively high 4.0/). available soil P levels due to its poor capability to explore the soil for P compared to other Int. J. Mol. Sci. 2021, 22, 4181. https://doi.org/10.3390/ijms22084181 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 12 However, subterranean clover has limitations such as the need for relatively high Int. J. Mol. Sci. 2021, 22, 4181 available soil P levels due to its poor capability to explore the soil for P compared to2 other of 12 accompanying pasture grasses [8]. There is a huge scope for genetic improvement in sub- terranean clover to increase herbicide tolerance from inexpensive herbicide, reduce the accompanyingcapability of producing pasture grasses methanogenic [8]. There compou is a hugends for scope rumens, for geneticovercome improvement self-incompati- in subterraneanble fertilisation, clover increase to increase overall herbicide feeding tolerancevalue, persistence from inexpensive and nutritive herbicide, value reduce of the crop the capability[4]. Therefore, of producing it is required methanogenic to optimise compounds in vitro culture for rumens, conditions overcome for subterranean self-incompatible clover fertilisation,cv. Daliak. Only increase a few overall publications feeding value,are available persistence on the and use nutritive of in vitro value culture of the techniques crop [4]. Therefore,to transform it is this required species to [9–11]. optimise In inall vitro cases,culture direct conditions organogenesis, for subterranean using cut seeds clover as cv.ex- Daliak.plants, Onlywas used a few to publicationsestablish in vi aretro available culture plants. on the Additionally, use of in vitro Agrobacteriumculture techniques tumefa- tociens transform transformation this species needs [9– 11to ].be In optimised all cases, as direct a delivery organogenesis, method in using subterranean cut seeds clover, as ex- plants,as has was been used done to establish for Medicagoin vitro truncatulaculture plants. [12]. A. Additionally, tumefaciensAgrobacterium has largely tumefaciensbecome the transformationmethod of choice needs for togene be optimisedtransfer in ascrop a delivery improvement method [13–15]. in subterranean In this project, clover, we as aimed has beento optimise done for theMedicago in vitro truncatulaculture conditions[12]. A. tumefaciensand Agrobacteriumhas largely transformation become the for method subterra- of choicenean clover for gene cv. transfer Daliak. in crop improvement [13–15]. In this project, we aimed to optimise the in vitro culture conditions and Agrobacterium transformation for subterranean clover cv.2. Daliak.Results 2.1. In Vitro Culture Optimisation: Selection of Shooting and Rooting Media 2. Results The seeds, when grown according to the protocol of Ding et al. [10], showed contam- 2.1. In Vitro Culture Optimisation: Selection of Shooting and Rooting Media ination in 45.3 % of the cotyledons and in 57.7 % of embryo tips. However, the addition of 0.3%The chlorine seeds, and when 0.8 grownmg mL according−1 cefotaxime to thein the protocol seed sterilisation of Ding et step al. [10 reduced], showed contami- con- tamination in 45.3 % of the cotyledons and in 57.7 % of embryo tips. However, the nation—by comparison to 2%. In subsequent−1 experiments, this percentage was main- additiontained. of 0.3% chlorine and 0.8 mg mL cefotaxime in the seed sterilisation step re- duced contamination—by comparison to 2%. In subsequent experiments, this percentage was maintained. Regarding shoot regeneration, the medium RM73 worked appropriately for in vitro Regarding shoot regeneration, the medium RM73 worked appropriately for in vitro shoot regeneration with a frequency of explant regeneration of 80%. Additionally, it was shoot regeneration with a frequency of explant regeneration of 80%. Additionally, it was necessary to identify which rooting medium was most suitable for subterranean clover necessary to identify which rooting medium was most suitable for subterranean clover cv. Daliak.cv. Daliak. Three Three out out of the of the four four rooting rooting media media tested tested did did not not show show any any root root development develop- afterment two after months two months of culture. of culture. By contrast, By contrast as shown, as shown in Figure in Figure1, explants 1, explants cultivated cultivated into rootinginto rooting media media containing containing 1.2 µ M1.2 IAAµM IAA developed developed roots roots with with 16.6 16.6 % efficiency % efficiency after after two monthstwo months of culture. of culture. FigureFigure 1. 1.Explant Explant cultivated cultivated forfor twotwo monthsmonths inin rootingrooting medium containing 1. 1.22 µMµM IAA. Presence Presence of multiple roots coming out from the bottom part of the explant. Image captured under stereo- of multiple roots coming out from the bottom part of the explant. Image captured under stereo- microscope Leica MZ6®. microscope Leica MZ6®. 2.2. Antibiotic Sensitivity Testing 2.2.1. Kanamycin Concentration Test Treatment with kanamycin at 100 mg L−1 reduced the percentage of explant survival to 23.3% after 30 days of culture (Table1). Increasing the concentration of kanamycin in the solid media reduced explant survival (Figure S1). In some cases, all explant tissue died Int. J. Mol. Sci. 2021,