| With the rapid development of turfgrass industry in our country, more and more attentions are paid to the genetic breeding of turfgrass. Biotechnology has a great application potential in varietal improvement of turfgrass, mainly focused on the progress in turfgrass genetic engineering of the type and target gene of the genetic turfgrass. Drought and salinity become major limited factors of the growth of turfgrass. Expression of drought- and salt- resistant genes in turfgrass can contribute to improving stress resistance of turfgrass.Arabidopsis Vacuolar H~+-Pyrophosphatase (AVP1) controls auxin transport and consequently auxin-dependent development by regulating the proton gradients of vacuolar membranes, and results in the adaptability of salt and water stress tolerance. Arabidopsis rd29A (Responsive to Dehydration. rd29A ) promoter can be induced by cold, drought, salt and ABA. In this paper, AVP1 gene driven by rd29A promoter from Arabidopsis thaliana was introduced into creeping bentgrass by Agrobacterium tumefaciens. The physiological function of AVP1 transgenic plants were studied under dehydration and salinity condition, and possible mechanisms in the development of drought and salt tolerance were also discussedThe main results were summarized as follows:1. A protocol of tissue culture system for callus induction was developed in three speices of turfgrass. The results showed that embryogenic callus could be induced from seeds of creeping bentgrass and tall fescue at high frequency of 53.0±8.7% and 60.7±7.9%. And their callus growth rate could reach the level of 206.0±43.7% and 159.3±34.4%. Differently, the callus induced from seeds of burmudagrass was non embryogenic, with a very low induction rate of 14.7±4.0%. And its callus growth rate was only 78.4±23.2%. Embryogenic callus of burmudagrass could only be induced from nodal segments, while this callus would turn to non-embryogenic callus after several generations.2. The rd29A promoter and AVP1 gene, cloned from Arabidopsis thaliana, were placed into pCambia1302 vector to establish plant expression vectors. The AVP1 gene driven by rd29A promoter was transformed into creeping bentgrass mediated by Agrobacterium tumefaciens. The transformed callus were selected by 50, 75 and 100 mg/L hygromycin, and the selective ratios of transformant were 64.1±5.0%, 81.5±1.5% and 33.3±3.8%, respectively. After one and a half months of selection, the positive percentage of hpt-PCR analysis of transformant in 50 mg/L hygromycin was only 0.9 %, while it was as high as 96.7% in 75 mg/L hygromycin. So 75mg/L hygromycin was the best selective pressure for the transformed callus of bentgrass in our experiments.3. Transgenic creeping bentgrass was analyzed by PCR. Of 90 putative transformed plants recovered from selection medium, 96.7% were positive for integration of hph gene, while 14.4% were positive for AVP1 gene.4. Expression of A VP1 gene under the control of rd29A promoter was analyzed by RT-PCR. In transgenic callus of creeping bentgrass, AVP1 gene was expressed under the condition of drought (dehydration for 48 h), low temperature (4℃, 24h) and high salinity (200mM NaCl), while it was expressed at very low level in untreated condition. In wild type callus, no expression of AVP1 had been detected in either treated or untreated condition. It indicated that the rd29A promoter activity was very low in non-stress condition, yet it was obviously increased under drought, low temperature and high salinity treatment. The RT-PCR results confirmed that the expression level of AVP1 gene in transgenic creeping bentgrass could be increased by drought, low temperature and high salinity treatment.5. Dehydration and NaCl tolerance in transgenic creeping bentgrass callus were studied. The growth rates of transgenic callus after 2-or 4-day- dehydration were 645% and 543%, while those of wild type were 423% and 135%. It was obvious that transgenic creeping bentgrass were more drought-tolerant than wild type. The growth rates of transgenic callus were higher than wild type under various concentration of NaCl, especially in 50 mmol/L and 200 mmol/L NaCl treatments. Therefore, it was suggested that the salt tolerance was enhanced in transgenic creeping bentgrass. 6. Drought tolerance in transgenic creeping bentgrass plants was analyzed. Water content in transgenic plants was similar to that in wild type under normal condition. After a 9-day soil water deficit experiments, the leaves of transgenic plants exhibited still green while wild type became yellow and wilted. The water content of transformant was 82.6%, which was much higher than 77.5% in wild type. It was illuminated the transgenic plants exhibited the greater tolerance to drought than wild type. Root system biomass was also measured. The root dry weight of transgenic plants was 0.17±0.02g, while it was only 0.07±0.01g in wild type. It was concluded that increasing A VP1 expression in transgenic plants enhances root system development and thus increased root growth have the potential to increase the resistance to water deficit stress.
|
PDF Full Text Request