Enhanced Tolerance to Multiple Abiotic Stresses in Transgenic Alfalfa Accumulating Trehalose

Abstract

ABSTRACTAlfalfa (Medicago sativaL.) is an important forage crop in many countries with high biomass production and the third‐most‐cultivated crop in the United States, with high protein content and potential use as biofuel. Here we describe the use of a chimeric translational fusion of yeast trehalose‐6‐phosphate synthase (EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (EC 3.1.3.12) genes to engineer stress tolerance in alfalfa. The gene fusion was overexpressed using either the constitutive35Spromoter or the stress‐regulatedrd29Apromoter. Transgene insertion in the genome was checked by polymerase chain reaction and transcript expression by reverse transcription polymerase chain reaction. Several independent transgenic plants were selected in the presence of kanamycin and further analyzed. Yeast gene encoding trehalose‐6‐phosphate synthase (TPS1)–yeast gene encoding trehalose‐6‐phosphate phosphatase (TPS2) gene expression under the35Spromoter led to plants with stunted growth and less biomass, whereasTPS1‐TPS2expression driven by therd29Apromoter improved growth and provoked a significant increase in plant biomass at the foliage level in the different transgenic lines. Trehalose accumulated in all the different lines at similar levels under stress conditions. Transgenic plants displayed a significant increase in drought, freezing, salt, and heat tolerance. This is the first time that genetic engineering of trehalose metabolism is reported in alfalfa and demonstrates that it can improve multiple stress protection in this crop. Thus, the yeast TPS‐TPP fusion protein represents a great potential for generating stress‐tolerant crop plants for agriculture.