Agrobacterium-mediated transformation of Medicago truncatula cell suspension culture provides a system for functional analysis

Abstract

Over the past decade, Medicago truncatula has been adopted as a model legume species for a range of “omic” studies. The availability of different transformation techniques has greatly advanced functional genomic studies in this species. In the present work, an efficient procedure for Agrobacterium-mediated transformation of M. truncatula cv. “Jemalong 2HA” through a cell suspension culture was developed. This procedure resulted in transformed single cells or cell clusters, giving rise to stable transgenic plants within 4 mo. Transformation experiments were performed with a vector carrying two marker genes: β-glucuronidase (GUS) and green fluorescent protein (GFP) under the control of endogenous gene promoters from LIKE AUX1 3 (LAX3) and GRAS transcription factor (named after GD3BERELLIC ACID INSENSITIVE [GAI], REPRESSOR OF GA1 [RGA], and SCARECROW [SCR]), as well as with a binary destination vector for overexpressing the cyclin-like F-box gene fused to GFP. Maximum transformation efficiency was achieved under the following experimental conditions: acetosyringone at a concentration of 25 μM, bacterial suspension with an optical density of 0.3 at 600 nm, inoculation under agitation at 100 rpm for 24 h, co-cultivation periods of 48 h, and an uninterrupted selection with 50 mg/L kanamycin. Selection of positive transformation events was imposed early in the regeneration stage (after 48 h co-cultivation), following a large-scale screening for GFP activity. Histochemical GUS and GFP reporter activity was detected in single cells, embryogenic zones, emerging embryos, in vitro plantlets, and T1 progeny seedlings. The transgenic nature of transformed plants was further confirmed by nptII-specific PCR amplification of T0 and T1 plant lines. The transgenic plants grown under standard greenhouse conditions displayed a wild-type phenotype and the obtained progeny segregated in a classical Mendelian manner. The fundamental steps in the transformation procedure are outlined and discussed.