| Lonicera macranthoides Hand.-Mazz. Yulei No.1belongs to Caprifoliaceae, Lonicera japonica Thunb. In2009, L. macranthoides Hand.-Mazz. Yulei No.1was identified as a novel species with the improved variety ID of "Chongqing S-SV-LM-005-2008", and is the first new pharmaceutical variety. With the accumulation of information about the mechanism and components of Yulei No.1, new products have been launched continuously, and the market has grown rapidly. However, the growth of demand has outstripped the supply of seedlings in recent years. At the present time in Chongqing, the major propagation approaches of Yulei No.1are grafting and cuttage. However, drawbacks of these traditional methods including low survival rate, poor propagation percentage, and pathogenic bacteria infection become the bottlenecks of commercial application. Therefore, an effective rapid micropropagation procedure is a challenge to meet the strong demand from seedlings market.In this study, we focused on the in vitro rapid micropropagation of L. macranthoides Hand.-Mazz. Yulei No.1. By optimizing the corresponding media composition and cultivation conditions such as sterilization processes, temperature, humidity and lighting, a fine procedure was established. The entire scenario includes explant establishment, callus induction and multiplication using leaves, multiple shoots inducing and multiplying, rooting influenced by extra hormones, transplanting and measurement of secondary metabolites content.This thesis deals with the results of media optimization (standard MS+exogenous hormones) and secondary metabolites measuring, studied in the cultivation of Yulei No.1in vitro.Explant Selection And Sterilization Condition Establishment:Explants were originated from stem fragments and leaves of Yulei No.1. Preparation conditions were optimized in our experiments:stem segments were treated by75%alcohol for about25s, then were disinfected using0.1%HgCl2for2-3min; Leaves were treated by75%alcohol and0.1%HgCl2for25s and1-2min, respectively.Induction Of Group Buds And Callus:The optimized medium for group buds induced from stem fragments was MS+6-BA1.0mg/L (the unit of hormones mentioned below are the same), with the germination rate is70%. In addition, the optimal medium for leaf callus was MS+6-BA2.0, and the induction rate could approximate90%.Multiplication Of Group Buds And Callus:The optimal media for group buds and callus were MS+6-BA1.0+NAA0.2and MS+6-BA1.0+NAA1.5, respectively. The propagation coefficient is3.70while the rate of increase is18.4%.Rooting And Transplant:For rooting of tube plantlets, Murashige and Skoog (MS) media, supplemented with IBA and NAA was used, i.e.,1/2MS+IBA1.0+NAA0.2. The rooting percentage is93.3%. To transplant seedlings into natural condition, a fine procedure was established. Tube Seedlings with fine root system were planted in mixed medium containing peat and pearlite (1:1), humidity was maintained by covering PE membrane. Rational watering, lighting and temperature are required in follow-up stages. The survival rate could reach90%.Secondary Metabolite Analysis:Cholorogenic acid is one of the particular secondary metabolites, and also a pharmaceutical material. In this investigation, secondary metabolites content in leaves was analyzed. HPLC analysis reveals that there is no significant difference in Cholorogenic acid content between cultured plantlet and wild type Honeysuckle.Our investigation indicates that cultivars are identical to the wild type, and are highlighted in pharmic applications. Additionally, Tissue culture, due to its high propagation percentage, is a powerful tool or mechanism used to speed up the proliferation of certain plants, provides an opportunity to produce high quality seedlings, and might play a promising and significant role in large scale industrial production. |
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