The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Ni...The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.展开更多
将1.4kb Class I patatin基因的5′侧翼区与GUS基因融合,构建了双元表达载体pPATIs(含patatin部分信号顺序)和pPATI(不含patatin部分信号顺序)。pPATI通过基因枪介导在块茎切片中获得了瞬间表达。以上建构物通过农杆菌介导转入了马铃薯...将1.4kb Class I patatin基因的5′侧翼区与GUS基因融合,构建了双元表达载体pPATIs(含patatin部分信号顺序)和pPATI(不含patatin部分信号顺序)。pPATI通过基因枪介导在块茎切片中获得了瞬间表达。以上建构物通过农杆菌介导转入了马铃薯品种Desiree。X-Gluc染色(PATIs不能染色)及PCR结果证实已获得转基因植株。利用离体块茎诱导系统,GUS的表达进一步用荧光进行定量检测,结果显示,PATI-GUS的转基因植株中GUS比活性均以块茎明显高于茎段,达10-20倍。蔗糖浓度的升高,PATI-GUS植株中的GUS比活性无明显变化,与前人报道有不同。此外,光照促进PATI-GUS的表达。展开更多
文摘The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.
文摘将1.4kb Class I patatin基因的5′侧翼区与GUS基因融合,构建了双元表达载体pPATIs(含patatin部分信号顺序)和pPATI(不含patatin部分信号顺序)。pPATI通过基因枪介导在块茎切片中获得了瞬间表达。以上建构物通过农杆菌介导转入了马铃薯品种Desiree。X-Gluc染色(PATIs不能染色)及PCR结果证实已获得转基因植株。利用离体块茎诱导系统,GUS的表达进一步用荧光进行定量检测,结果显示,PATI-GUS的转基因植株中GUS比活性均以块茎明显高于茎段,达10-20倍。蔗糖浓度的升高,PATI-GUS植株中的GUS比活性无明显变化,与前人报道有不同。此外,光照促进PATI-GUS的表达。
