Construction Of Rapid Propagation System Of Alpinia Mutica Roxb. Based On Tissue Culture Technology

Alpinia mutica Roxb.is a perennial herb of the genus Zingiberaceae(Alpinia),itsstyle can exhibit a typical curve called Flexistyly.The in-depth study of Flexistyly is important to understand the origin of the dichogamy mechanism and the evolution of the reproductive system.At present,anatomical,physiological tests,genetic tests,bioinformatics analysis and other methods have been carried out to comprehensively study the Flexistyly of Alpinia mutica Roxb.,but the molecular mechanism of its developmental regulation is still unclear.In order to verify the gene function of regulating the Flexistyly,it is necessary to construct a transgenic plant system,and the establishment of the transgenic system is based on plant tissue culture technology to establish a rapid propagation system of plants.Therefore,this study used Alpinia mutica Roxb.as a test material,using plant tissue culture technology,using different explants,disinfectant use,plant growth regulator types and ratios as the starting point,to build a rapid propagation system of Alpinia mutica Roxb..The research was carried out and the main findings are as follows:1.Established the the sterile seedling system of Alpinia mutica Roxb.:The soil matrix suitable for transplanting the seedlings ofAlpinia mutica Roxb.was obtained;the optimal aseptic methods for the seeds,tuber buds and roots of Alpinia mutica Roxb.were obtained.The results show that the concentration of disinfectant and the length of disinfection are important factors in determining the disinfection effect.The increase of NaClO and HgCl2 concentration and the increase of disinfection time can reduce the degree of pollution and increase the degree of browning of explants.The increase of NaClO concentration and the increase of disinfection time can promote seed germination.The combination of alcohol and NaCIO can obtain better seeds disinfection effect,but can not get better tuber buds disinfection effect.The combination of alcohol and HgCl2 is an effective method for disinfecting the tuber buds and roots.A separate treatment of alcohol before and after the HgCl2 treatment can obtain a better effect disinfection for tuber buds.2.Established the rapid propagation of the buds of Alpinia mutica Roxb.:The base of stem of the sterile shoots of Alpinia mutica Roxb.were used as explants,the optimal medium for the induction of shoot buds was MS + 1.0 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose,and the number of the best propagation shoots were is 2.96(70 d).The suitable species and ratio of plant growth regulators are important factors affecting the propagation of shoot buds.The combination of 6-BA and NAA can promote the propagation of the shoots of Alpinia mutica Roxb..The combination of 6-BA and IBA can not promote the propagation of shoots but it can romote seedling rooting.3.Callus induction culture of the roots of Alpinia mutica Roxb..Tthe optimal induction medium of the callus from the roots of Alpinia mutica Roxb.was MS + 0.25?0.75 mg/L 6-BA+ 1.0 mg/L 2,4-D + 30g/L sucrose and MS + 1.0 mg/L 6-BA + 0.25-0.75 mg/L 2,4-D + 30g/L sucrose,The type and ratio of suitable plant growth regulators are important factors in inducing callus.The explants treated by 6-BA or NAA alone would gradually brown and die;the combination of 6-BA and NAA can induce adventitious roots and cause browningand death of explants.The combination of different concentration of 6-BA,2,4-D and 6-BA,NAA,2,4-D can induce different types of callus of explants.4.Callus induction culture of the stems of Alpinia muticaRoxb.The optimal induction medium of the callus from the stems of Alpinia mutica Roxb.is MS 1.0 mg/L 6-BA +0.5?2.0 mg/L 2,4-D + 30 g/L sucrose.The ratio of suitable plant growth regulators are important factors in inducing callus.The combinations of different concentration of 6-BA and 2,4-D can induce different types of callus and cause browning and death of explants.The study explored the construction of rapid propagation system of Alpinia mutica Roxb.,obtained the optimal disinfection method of the its seeds,tuber buds and roots,and obtained the optimal induction medium of callus of roots and stems.It filled the blank of the research on the tissue culture of Alpinia mutica Roxb.,laid a fundation for establishing the genetic transformation system of Alpinia mutica Roxb.to study the molecular mechanism of Flexistyly by transgenic means,carried out the technical discussion,and provided technical support for the production of the virus-free seedlings and the large-scale cultivation of the plants.It has also created preliminary conditions for the development and utilization as extracting valid medicinal ingredients,producing secondary metabolites,etc.