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Pinellia (Pinellia ternata Thunb.Briet) rhizomes contain of Pinellia proteins, tannins and alkaloids medicinal ingredients, is our proprietary medicinal plants. Pinellia species degradation caused due to a virus often leads to lack of seed source, the yield and quality of a serious decline, has become one of the main obstacles in Pinellia production, removal the Pinellia seedlings and Microtuber of viral culture through the apical meristem and other means of biotechnology is the way to resolve artificial seed production and increase production quality. The Pinellia ternata tuber tissue culture conditions induced metamorphosis stem its genetic stability, physiological and biochemical characteristics, no different from conventional tubers, Pinellia production research are especially great importance is unclear, but the formation mechanism of the test tube small tubers, production efficiency is not high, there are still a lot of difficulties in the application. This study was designed to add different endogenous hormones inhibitors, research Pinellia ternata tuber formation process endogenous hormone changes position with small tuber formation related gene expression patterns, and to explore the mechanism of regulation of tuber formation, for Pinellia Microtuber production provide theoretical and technical basis. The main findings are as follows: treatment by different concentrations of GA inhibitors (cycocel,, CCC) MS medium Pinellia ternata tuber expansion and endogenous hormone content. In the the Pinellia microtubers formation of the process, CCC can effectively increase tuber weight inhibit GA 3 , IAA, ZR of synthesis, and showing a trend of decrease with increasing pharmaceutical concentration. CCC can effectively influence the content of ABA and JA, was the \MS medium in different concentrations of ABA inhibitors (Norflurazon, NOR) treatment, of Pinellia ternata tuber expansion and endogenous hormone content had a significant effect. In Pinellia microtubers formation process, NOR can effectively reduce tuber weight, inhibit the ABA, IAA, JA Synthesis, JA content was \NOR can affect GA 3 and ZR content. MS medium in different concentrations of JA (salicyl hydroxamic acid, SHAM) inhibitor treatment, Pinellia ternata tuber expansion and endogenous hormone content. In the the Pinellia microtubers formation of the process, SH AM effectively delayed tuber formation time promote tuber the formation late GA 3 , IAA, ZR synthesis, but are lower than those in the control. SHAM effectively inhibit the synthesis of JA, while ABA affect the downward trend. From a tuber effective method of extracting high quality total RNA. Conventional guanidine isothiocyanate Plant Total RNA Isolation improvements have been made to establish a simple and practical method of total RNA was extracted from plant material rich in polyphenols and polysaccharides and other substances. The method mentioned in RNA A 260 / A 230 are greater than 2.0 A 260 / A 280 of value of 1.7 to 2.0, the electrophoretic bands RNA integrity. The Pinellia test tube using the method to extract small tubers of total RNA with high quality and purity, and the yield is high, fully satisfy the follow-up DDRT-PCR molecular biology research. Establish and optimize the conditions before and after the induction of the tuber silver staining mRNA differential display. Using orthogonal design, template, the Mg 2 , dNTPs, primers and DNA polymerase level of four of the five factors, screening and of Pinellia test-tube small tubers induced DDRT-PCR reaction systems. Obtain stable expansion of most strip suitable Pinellia vitro induction of small tubers DDRT-PCR reaction system, that is, 20μL of the reaction system containing dNTPs 150μmol / L, Taq enzyme 0.6 U the anchor primer 2μmol / L, Random of primer 1μmol / L Mg 2 2.5mmol / L, 2μL 10 × buffer and 2.5μg of cDNA template. The various factors that affect the degree of Taq enzyme concentration> cDNA template concentration> dNTPs concentration> the Mg 2 concentration> primer concentration. MRNA differential display to isolate and clone the gene fragments Pinellia test tube small tuber development. MRNA differential display (DDRT-PCR) method of comparative analysis of gene expression differences in the the Pinellia test tube during the development of small tubers. The results are separated into 15 of Pinellia tubes small tubers during development-specific expression of cDNA fragments, 6 homology found in GenBank no homologous sequence match; 3 fragment deduced amino acid sequences were compared with the true eukaryotic promoter, MADS-box proteins and ethylene signal transduction factors have a high degree of homology. Semi-quantitative RT-PCR confirmed that they exist, and to generate highly specific expression of the different number of days in Pinellia. Among them, the three fragments deduced amino acid associated with tuber development To explore the molecular mechanisms of tuber development to provide new information.
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