Seeds Symbiotic Germination Affecting Factors And Test-tube Seedlings Domestication Conditions Of Cremastra Appendiculata

In this study,the seeds of Cremastra appendiculata obtained by artificial pollination and Coprinellus disseminatus were used as experimental materials.Firstly,the culture conditions of C.disseminatus were optimized,and the factors influencing the symbiosis and interaction between C.disseminatus and C.appendiculata seeds to promote seed germination were preliminarily identified by screening the types of basic medium,components of medium and temperature and light in the culture room for the co-culture of seeds and fungi.Orthogonal experiment was used to optimize the culture conditions and finally determine the suitable conditions for the symbiotic germination of the fungi and C.appendiculata seeds.At the same time,through screening the seedling conditions of the protocorms obtained from the symbiotic culture of C.appendiculata seeds and the optimization of the domestication conditions of test-tube seedlings,it provides the basis for the construction of effective technology for the production of C.appendiculata seedlings.The main results are as follows:1.The optimization of culture conditions was carried out with C.disseminatus as the experimental material.With colony diameter and fresh weight of mycelium as the main measurement indexes,a series of single factor experiments were carried out in different culture temperature,photoperiod,initial p H value,nitrogen source,carbon source,etc.The results showed that the optimum culture temperature was 25℃,light condition was dark culture,carbon and nitrogen sources were maltose and peptone,p H value was about 7.0.On the basis of single factor experiment,the suitable medium for C.disseminatus was found to be potato 200 g·L^-1+maltose 20 g·L^-1+peptone 2 g·L^-1+agar 15 g·L^-1.2.The co-culture experiment of C.appendiculata seeds and C.disseminatus was carried out.The total seed germination rate and protocorm growth rate of C.appendiculata were set as the determination indexes.Single factor experiments were conducted to explore the effects of different basic medium,carbon source,nitrogen source,p H value,photoperiod,light intensity,temperature and other factors on the symbiotic interaction between the seeds of C.appendiculata and C.disseminatus.The orthogonal experiment of three factors（p H value,maltose concentration,peptone concentration）and three levels was designed to optimize the symbiotic germination medium.The results showed that the optimal medium formula was OMA medium+cellulose 10 g·L^-1+agar 8 g·L^-1,p H value was 6.0,and the appropriate culture room temperature and light were 20℃and dark culture,respectively,so as to construct the suitable culture system for the symbiotic germination of C.appendiculata seeds.3.In this study,the protocorms obtained from the symbiotic culture of C.appendiculata seeds and C.disseminatus was taken as the research object,the regulation effects on growth and development of the protocorms were explored under different basic medium and antibiotic types and dosages.The results showed that the proliferation coefficient of protocorm was relatively high when 1/4MS+cephalosporin 200 mg·L^-1was used as the base medium.Orthogonal experiment was conducted to determine the influence of sucrose,NAA and 6-BA concentrations on the seedling formation process.The optimal medium composition was 1/4MS+cephalosporin 200 mg·L^-1+activated carbon 0.5 g·L^-1+sucrose 20 g·L^-1+NAA 0.2 mg·L^-1+6-BA 1.5 mg·L^-1+agar 5.5 g·L^-1.4.A large number of test-tube seedlings of C.appendiculata obtained from the established in vitro culture system were used as experimental materials,and the factors influencing the adaptation to the external environment and normal growth of test-tube seedlings of C.appendiculata were preliminarily explored from the aspects of different mixed substrates,different application frequencies of water and the addition of C.disseminatus.The results showed that the survival rate of test-tube seedlings was significantly correlated with the aerated porosity of the substrate,and the mixture of humus+coconut bran+perlite=1:1:1was suitable for the acclimation and transplanting of test-tube seedlings.The appropriate water application frequency is once every 3 days,so that the relative humidity of the substrate reaches 40%,too high or too low water is not conducive to the growth of test-tube seedlings.In the experiment of co-acclimation culture of additional fungi and test-tube seedlings,it was found that the addition of C.disseminatus did not significantly increase the plant height,fresh weight,root length and other physiological indexes of test-tube seedlings during the domestication stage.