[Establishment and optimization of sliver staining differential display of microtubers from Pinellia ternata in vitro].
OBJECTIVE
In this study, orthogonal design was used to optinize DDRT-PCR amplification system on Pinellia ternata microtubers in vitro in five factors four levels respectively.


METHODS
P. ternata stems and microtubers in vitro were selected as explants. The effects of five kinds of factors were studied by orthogonal design method including emplate cDNA, Mg2+, dNTPs, primers and Taq DNA polymerase, and in order to establish the optimum DDRT-PCR system of P. ternata microtubers in vitro.


CONCLUSIONS
A satisfactory DDRT-PCR technique system for P. ternata microtubers in vitro with desirable repeatability and polymorphic bands was established. In a total volume of 20 microL DDRT-PCR system, it contained 10 x buffer, 150 micromol L(-1) dNTPs, 2 micromol L(-1) anchor primer, 1 micromol L(-1) arbitrary primer, 2.5 mmol L(-1) Mg2+, 0.6 U Taq DNA polymerase and 2.5 microg template cDNA. The effect of the five factors was in sequence of Taq DNA polymerase > template cDNA > dNTPs > Mg2+ > Primers. The optimum DDRT-PCR system will provide scientific reference basis for studying effecting character of P. ternata microtubers associated with genes expression.
Xue
Jian-Ping
JP
Anhui Key Laboratory of Plant Resources and Biology, School of Life Science, Huaibei Coal Industry Teachers' College, Huaibei 235000, China. xuejp2000@yahoo.com.cn
Huang
Yue-Qin
YQ
Xu
You-Ming
YM
Tian
Zheng-Dong
ZD
D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D030021:Pinellia / Q000235:genetics*; D035281:Plant Tubers / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D016622:Silver Staining; D019914:Taq Polymerase / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D019914:Taq Polymerase