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

摘要

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: beta -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 micro M, 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.