Study On Tissue Culture And Srap Genetic Diversity Of Cymbidium Goeringii ’HuRui’

As one of the five traditional Chinese orchids,Cymbidium goeringii have long history of cultivation and rich resources,with aesthetic,cultural and economic values,it also be one of the top ten famous flowers in Chinese tradition.The development of C.goeringii has been restricted by difficult seed germination,low reproductive coefficient and long breeding period.In recent years,the germplasm resources of C.goeringii have been destroyed by field excavation and collection.C.goeringii‘Hu Rui’is very rare and precious,which has won many awards in orchid exhibitions,but is quite difficult in tissue culture.In order to improve the efficiency of seedling breeding,the rhizomes of‘Hu Rui’were used as materials in this research to study the optimal tissue culture system.Moreover,the genetic diversity analysis were carried out by means of SRAP-PCR.It provides scientific and technical basis for large-scale production practice and rational utilization of germplasm resources.The main results are as follows:1.The tissue culture and rapid propagation system of C.goeringii was established and optimized.The various stages of proliferation,differentiation and rooting into seedlings of rhizomes were studied,and the optimal medium for each stage of tissue culture was explored.The most suitable basic medium for rhizomes proliferation was Hyponex NO.1.When supplemented with NAA 1.0 mg/L+6-BA 0.5mg/L+AC 2.0 g/L,the medium was suitable for rhizomes proliferation with proliferation coefficient 4.20.Moreover,when the medium was sterilized at 121℃for15 min,the effect of rhizome proliferation was the best.And the suitable density for the proliferation of rhizomes was 5～10 rhizomes per bottle.The best plant regulatory combination for differentiation was NAA 0.2 mg/L+6-BA 2.0 mg/L,with the differentiation rate was 146.67%and the buds height reached 2.04 cm.The optimal medium for rooting and seedling development was the basic medium with NAA 0.5mg/L+6-BA 2.0 mg/L+AC 4.0 g/L.2.The medium composition,culture steps and light conditions were simplified to reduce the cost of tissue culture.The results showed that when the carbon source was 25 g/L white sugar,the rhizomes proliferation coefficient was the highest.White sugar could be used to replace sucrose to reduce the cost in factory production.Tap water and pure water are both suitable for water quality,tap water can be used instead of pure water in production.The light intensity of 36μmol·m^-2·s^-1（two fluorescent tubes）is more suitable for the proliferation of rhizomes of C.goeringii,one lamp or natural scattered light can be used for cultivation in production.When the concentration ratio of 6-BA to NAA was 1-1.3,it was better for the realization of proliferation and differentiation in step.The most suitable medium for one-step seedling formation was NAA 0.75 mg/L+6-BA 1.0 mg/L+AC 2.0 mg/L,with a proliferation coefficient of 3.65 and a differentiation rate of 17.5%,which could realize simultaneous proliferation and differentiation.3.The optimized SRAP-PCR reaction system was established to analyze the genetic diversity of C.goeringii.The results showed that the optimal SRAP-PCR reaction system of C.goeringii‘Hu Rui’is that 60 ng DNA,0.3 mmol/L d NTPs,3.0mmol/L Mg²⁺,0.5 U Ex Taq and 0.5μmol/L primers.Fourteen pairs of primers with clear and large numbers of bands was selected to analysis of genetic diversity.A total of 219 clearly identifiable bands were amplified,with an average of 13.69 bands per pair of primers,the polymorphism ratio was 65.30%,indicating that the polymorphism of molecular markers was pretty good.The average genetic similarity coefficient between parents and offspring was 0.8167,and the average genetic similarity coefficient between offspring was 0.8145,indicating that the degree of variation is similar,C.goeringii‘Hu Ruit’genetic similarity is high,the genetics are relatively stable.This study established the tissue culture and rapid propagation system of‘Hu Rui’,which laid a foundation for further research on the establishment of precious varieties tissue culture and large-scale factory production.