Propagation Of Virus-free Mulberry Saplings Using Tissue Culture Technology

Mulberry mosaic dwarf viroid (MMDVd) disease is one of the main mulberry diseases.The pathogen of MMDVd is a viroid-like small RNA. MMDVd had caused serious harm tothe production of mulberry leaves and the preservation and utilization of mulberrygermplasm resources. The infection and spread of the disease is diffcult to prevent by thetraditional propagation method such as grafting.In this paper, the virus-free saplings were breeded by using the method of plant tissueculture which had been successfully used in some flowers and woody plants. Theestablishment of systems of mulberry virus-free tissue culture had great significance notonly on sericulture production but also for the preservation and utilization of mulberrygermplasm resources.Using the material of mulberry variety Yu71-1, the construction of virus-free systemwas carried out by the method of plant tissue culture. Growth conditions of explants werecompared by orthogonal experiment, and hormones and their concentrations were chose ininitial mediums. The optimized mediums for growth and rooting were MS+0.5mg/L2,4-D+1.5mg/L6-BA+1.0mg/L KT+0.1%PVP and1/2MS+0.25mg/L2,4-D+0.1%PVPrespectively.Primers were designed on the basis of MMDVd nucleotide sequences reported. Theleaves of cultured saplings were detected by RT-PCR, and the specific stripes of MMDVddid not be found in the result. Using the healthy segments or buds of mulberry whichinfected with MMDVd as explants, the virus-free saplings can be obtained through theestablished virus-free system. The virus-free tissue culture system established in this studyhad a certain reference to later experiments. This study had laid the foundation for theproduction of bulk virus-free saplings.In order to control the spread of the disease effectively, the breeding system ofmulberry virus-free saplings was established successfully by using the method of tissueculture. Different varieties of mulberry were cultured by using the method of tissue culture.The healthy segments or buds of mulberry infected mosaic dwarf disease were adopted asmaterials of explants. The result showed that the culture period of the mulberry varieties ofYu71-1, Husang199and Husang197were much shorter, and their growth status can metour requirements. Two leaves with no leaf curl and other adverse symptoms can be found inthese explants in15days. Our research will help to understand the growth condition of mulberry tissue culture and will lay a foundation for the MMDVd prevention and cure formulberry tree.