Transformation Of Lolium Perenne L. With Fructan: Fructan 1-fructosyltransferase Gene From Agropyron Cristatum And Enhancement Of Drought Tolerance In Transgenic Plants

Perennial ryegrass (Lolium perenne L.) is an important cool-season grass; it can be turfgrass and forage. But the weak drought tolerance limits its application in production. Isolation of resistance drought-related genes by biological methods and breeding new varieties through transgenic methods is an effective way to improve the drought resistance of ryegrass. In this study, Ac-1-FFT (fructan: fructan 1-fructosyltransferase) cloned from Agropyron cristatum (L.) Gaertn was transformed into embryogenic callus ryegrass (Lolium perenne L. ) Carter by Agrobacterium mediated method and particle bombardment. We obtained significantly enhanced drought resistance of transgenic plants.The main results are as follows:1. Ac-1-FFT (fructan: fructan 1-fructosyltransferase) was transformed into perennial ryegrassAc-1-FFT gene cloned from Agropyron cristatum (L.) Gaertn, the length of cDNA sequence is 1950 bp.We construct a plant expression vector Ac-1-FFT-pCambia3301, it contains Ac-1-FFT gene, the maize ubiquitin-1 gene as promoter and bar gene as selectable marker. Ac-1-FFT transformed into ryegrass through Agrobacterium infection and particle bombardment method.We totally obtained 122 independent lines of regenerated plants, 89 of which was by Agrobacterium infection and 33 of which was by particle bombardment method.2. Molecular identification of transgenic plantsTransgenic plants were analyzed by 5‰basta spraying, 18 strains showed normal growth and green leaves, the other plants and control palnts'leaves turned yellow, and even death. The 18 strains were identified on the molecular level through PCR, and semi-quantitative PCR. PCR analysis showed that Ac-1-FFT gene's specific fragment could be amplified, and RT-PCR analysis showed that Ac-1-FFT gene expressed strongly in transgenic Lolium perenne plants whereas didn't in control plants. The expression was different between the different strains.3. Drought resistance of transgenic plantsUnder drought stress, transgenic plants showed good drought resistance. After 6 days drought stress, the leaves of transgenic plants remained green, few leaves began to wither, in the same time the control plants showde withered, and even death. Rehydration experiments showed that the control plants lost the ability to recover while transgenic plants recovered rapidly. Although few leaves were still yellow, the whole plants were doing well.4. Physiological and biochemical determination of transgenic plantsThe results showed that water soluble carbohydrate and fructan contents in Ac-1-FFT transgenic ryegrass are significantly higher than those in control plants. Relative water content and chlorophyll content are significantly higher, while electrolyte leakage and malondialdehyde (MDA) content are significantly lower in Ac-1-FFT transgenic ryegrass than those in control plants 6 days after drought treatment. Relative water content, chlorophyll content, electrolyte leakage and MDA content restored to its original level in Ac-1-FFT transgenic plants rather than in control plants 7 days after rewatering, suggesting that the damages in Ac-1-FFT transgenic plants are reversible but not in control plants. These results indicate that Ac-1-FFT expression and fructan biosynthesis might play an important role in the improved drought tolerance in Ac-1-FFT transgenic ryegrass.