Study On Reproductive Biology Fundamental And Germplasm Resources Molecular Markers Research In Clivia Miniata

Clivia miniata which belongs to Clivia Lindl, the family Amaryllidaceae is a perennial herb. All parts of Clivia mimiata are used as medicine and it also has ornamental value. The genus Clivia Lindl includes six diploid species (2n=2x=22). Clivia miniata is cultivated varieties common in Northern China. In this paper, Clivia miniata was used as experimental materials. The different florescence androgenesis processes were observed and its pollen viability was determined. The stigma receptivity and self-incompatibility mechanism were studied and flowering habit were also observed. Meanwhile, the characteristics of sexual reproduction and asexual reproduction breeding technology were studied. The close relative relationship between the wild species and cultivars was analysesed by using microsatellite molecular marker technique. The main results are as follows:61test samples were processed by DNA amplification, of which,51Clivia miniata cultivated varieties,8Clivia nobilis wild types,2hybrid variety between Clivia miniata and Clivia nobilis. The clear amplicons appeared in the objective fragments from11pairs of primers of Clivia miniata (CM3, CM9, CM12, CM54, CM65, CM103, CM137, CM253, CM289, CM357, CM425) and3pairs of primers of Clivia nobilis (CN68, CN89, CN106). The detection results with denaturing polyacrylamide gel electrophoresis indicate that4pairs of primers (CM3, CM253, CM425, CN89) are singlet state and10pairs of primers are with polymorphism. The innovation is that screening10pairs of effective, reliable genetics and molecular marker by using microsatellite molecular marker technique firstly, which provides useful information for the phylogeny relationship and genetic diversity research of Clivia Lindl.The Clivia miniata stigma receptivity and self-incompatibility phenomenon was observed by using fluorescence microscopy technique which indicates that Clivia miniata stigma has a strong receptivity while Clivia miniata blossom2-3days and irregular callose plug appear in the Clivia miniata pollen tube affect the pollen germination.The callose also appeared in the stigma which result in the pollen tube can not enter the stigma normally. The concentration of NaCl solution1%-5%can effectively overcome the self-incompatibility;0.3-0.5%H3BO3solution in NaCl solution can increase the effect slightly. The innovation point is to solve the Clivia inbred breeding barriers, so as to establish the foundation work for overcoming self-incompatibility with hybrids.This paper divided the Clivia miniata bloom clearly and determines the morphological characteristics of different florescence. Also, each period of anther development characteristics and androgenesis process were studied. The results show the microspores remain at uninucleate stage or late-uninucleate when Clivia miniata in budding stage. Microspore in this period is suitable for explant material of haploid culture. The optimal determine method for pollen viability was screened and that is I2-KI staining method. The best medium of pollen germination is liquid MS medium+NAA0.5mg/L2,4-D0.1mg/L.The innovation point is laid the theoretical foundation for the microspore culture.The characteristics of Clivia miniata blossom and growth regularity of scape were also studied. The results show that scape growth exist "slow-fast-slow" circle and the growth trendline was "S" type. The concentration of GA3solution50-100mg/L can effectively promote the scape length growth and improve the quality of the flower. Innovation is to find the scape rapid growth time and to determine the best time to fertilizer and water management for cultivation so that to control the scape length growth.The Clivia miniata asexual propagation methods were studied in this experiment. The results show that "head disposed" and "scape broken" these two kinds of treatment methods can promote offshoot germination. The highest rate of explants form even is placenta+ovules in tissue culture of young ovaries and ovules of Clivia miniata; The best medium is MS+0.8%agarophyte+3%sucrose+2,4-Dlmg/L+KT2mg/L;The best culture method is liquid-solid double-layer culture. The most suitable sowing seed is a medium size vigor seed, and being warehoused no more than3months.