在IAA和ZT16种不同浓度组合的培养基中,对番茄(L.esculeutum Miller C.V.402)离体叶组织培养和植株再生的最适条件作了初步研究。在改良MS基本培养基附加IAA 0.4mgL^(-1)和ZT 1mgL^(-1)条件下,接种后的叶圆片在一周后,开始发生愈伤组织...在IAA和ZT16种不同浓度组合的培养基中,对番茄(L.esculeutum Miller C.V.402)离体叶组织培养和植株再生的最适条件作了初步研究。在改良MS基本培养基附加IAA 0.4mgL^(-1)和ZT 1mgL^(-1)条件下,接种后的叶圆片在一周后,开始发生愈伤组织;25天后,直接从愈伤组织边缘发生幼芽,分化频率达100％。其中,20％幼芽能发育为正常芽。正常幼芽转移到改良的1/2 MS基本培养基中,6天后生根、并发育为完整小植株。展开更多
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
We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated...We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13?and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.展开更多
During vascular development, procambial and cambial cells give rise to xylem and phloem cells. Because the vascular tissue is deeply embedded, it has been difficult to analyze the processes of vascular development in ...During vascular development, procambial and cambial cells give rise to xylem and phloem cells. Because the vascular tissue is deeply embedded, it has been difficult to analyze the processes of vascular development in detail. Here, we establish a novel in vitro experimental system in which vascular development is induced in Arabidopsis thaliana leaf-disk cultures using bikinin, an inhibitor of glycogen synthase kinase 3 proteins. Transcriptome analysis reveals that mesophyll cells in leaf disks synchronously turn into procambial cells and then differentiate into tracheary elements. Leaf-disk cultures from plants expressing the procambial cell markers TDRpro：GUS and TDRpro：YFP can be used for spatiotemporal visualization of procambial cell formation. Further analysis with the tdr mutant and TDIF （tracheary element differentiation inhibitory factor） indicates that the key signaling TDIF-TDR-GSK3s regulates xylem differentiation in leaf-disk cultures. This new culture system can be combined with analysis using the rich material resources for Arabidopsis including cell-marker lines and mutants, thus offering a powerful tool for analyzing xylem cell differentiation.展开更多
Human growth hormone (hGH) gene has been inserted into the plasmid pLGV1103 to give the recombinant plasmid pLB-9. It has been introduced into the agrobacterium containing plasmid pGV3850. The recombinant Ti plasmid p...Human growth hormone (hGH) gene has been inserted into the plasmid pLGV1103 to give the recombinant plasmid pLB-9. It has been introduced into the agrobacterium containing plasmid pGV3850. The recombinant Ti plasmid pGL198(hGH) has been obtained by homologous recombination. The monocotyledon Caladium bicolor has been transferred with pGL198 (hGH) with the leaf-disk co-cultivation method, and transgenic plants have been regenerated. The results of nopaline analysis, NPT II detection Southern blot and Western blot show that the hGH gene was integrated into the genome of Caladium bicolor, and a 22-kD protein was synthesized in the transgenic plants.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We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13?and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.
文摘During vascular development, procambial and cambial cells give rise to xylem and phloem cells. Because the vascular tissue is deeply embedded, it has been difficult to analyze the processes of vascular development in detail. Here, we establish a novel in vitro experimental system in which vascular development is induced in Arabidopsis thaliana leaf-disk cultures using bikinin, an inhibitor of glycogen synthase kinase 3 proteins. Transcriptome analysis reveals that mesophyll cells in leaf disks synchronously turn into procambial cells and then differentiate into tracheary elements. Leaf-disk cultures from plants expressing the procambial cell markers TDRpro：GUS and TDRpro：YFP can be used for spatiotemporal visualization of procambial cell formation. Further analysis with the tdr mutant and TDIF （tracheary element differentiation inhibitory factor） indicates that the key signaling TDIF-TDR-GSK3s regulates xylem differentiation in leaf-disk cultures. This new culture system can be combined with analysis using the rich material resources for Arabidopsis including cell-marker lines and mutants, thus offering a powerful tool for analyzing xylem cell differentiation.
文摘Human growth hormone (hGH) gene has been inserted into the plasmid pLGV1103 to give the recombinant plasmid pLB-9. It has been introduced into the agrobacterium containing plasmid pGV3850. The recombinant Ti plasmid pGL198(hGH) has been obtained by homologous recombination. The monocotyledon Caladium bicolor has been transferred with pGL198 (hGH) with the leaf-disk co-cultivation method, and transgenic plants have been regenerated. The results of nopaline analysis, NPT II detection Southern blot and Western blot show that the hGH gene was integrated into the genome of Caladium bicolor, and a 22-kD protein was synthesized in the transgenic plants.
