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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Enhanced Tolerance to Multiple Abiotic Stresses in Transgenic Alfalfa Accumulating Trehalose

Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca Mor. 62209, Mexico

^* Corresponding author (iturri{at}buzon.uaem.mx)

Alfalfa (Medicago sativa L.) 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 constitutive 35S promoter or the stress-regulated rd29A promoter. 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 the 35S promoter led to plants with stunted growth and less biomass, whereas TPS1-TPS2 expression driven by the rd29A promoter 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.

Abbreviations: HPLC, high-performance liquid chromatography • MS, Murashige and Skoog • PCR, polymerase chain reaction • RWC, relative water content • SGWC, soil gravimetric water content • T6P, trehalose-6-phosphate • TPP, trehalose-6-phosphate phosphatase • TPS, trehalose-6-phosphate synthase • TPS1, yeast gene encoding TPS • TPS2, yeast gene encoding TPP