Study On Protoplast Isolation And Fusion Of Mulberry

As a perennial woody plant, mulberry has a long growth cycle, their breeding cycle islong as well. Though people can expand the mulberry cultivation area through cuttingpropagation, but still can’t guarantee mulberry will against virus infection and thecontinuation of some mulberry elite germplasm. With the rapid development of the“Engineering of Plant Cells”, this technology is become more and more obviously the trend ofimproved and strengthen the modern mulberry breeding research. Nowadays, the focus ofengineering research on mulberry cell is ‘Tissue Culture, Callus Induction Explants andTransgenic’. In contrast, the report of protoplast fusion technique in the mulberry breeding isparticularly rarely. In view of this situation, use “GUISANGYOU-12, NONGSANG-14andXINSANG-11” as materials, this paper discuss the mulberry protoplast isolation, Culture andintegration, initial screening of suitable conditions and methods of mulberry protoplastplasmolysis, purification, culture and hybrid selection. The purpose of this paper is to providetheoretical basis for the somatic hybridization of mulberry breeding research and for themulberry germplasm conservation. The main research results are as follows:1. This paper cleared the mulberry callus induction conditions, and also the acquisition ofembryo callus. Young mulberry stem segments are suitable for callus induction, the Mediumis MS+2,4-D2mg/L; Whatever, the subculture medium for callus is MS+6-BA1.0mg/L+2,4-D2mg/L; After3times subculture, we can obtain the pale yellow embryogenic calluswith crunchy structure.2. This paper established the mulberry cell suspension culture system through testedimpacts of training materials, culture medium, the initial inoculum size, rotation speed,concentration of sucrose, replace fluid volume, pH, subculture cycle and other factors ofmulberry suspension culture system, then given the best conditions of mulberry cellsuspension culture. By using guisangyou12young embryogenic callus as research material,in the liquid medium B5+6-BA1.5mg/L+NAA0.15mg/L, with sucrose40g/L, the liquidvolume of flasks is25mL/50mL, inoculated PCV volume is1mL per bottle, culture in theshaker, set the speed to175rpm, every fine days subculture one time, the replace fluidvolume is15mL. Cultured21days as above, the cells yield numbe of mulberry reach thehighest as98.1mg/L, besides, the cells grew fastest which up to183.3mg/（L·d）.3. This research also cleared that enzyme solution composition and concentration have greaty affection on mulberry protoplast isolation. Making mulberry embryogenic suspensioncell be the separate materials, using combinations enzyme solution as forllows:100U/mL ofcellulase R-10,150U/mL of pectinase Y-23and6U/mL of macerozyme R-10, which addedthe cell-protoplast washing buffer （CaCl₂·2H₂O1480.0mg/L, KH₂PO₄27.2mg/L）, withmannitol of0.6mol/L as the osmoregulate agent, enzymatic under28℃for6hours, theprotoplast yield is7.8×10⁶/g; Moreover, the protoplast vitality is highest that up to91.4%.4. This paper also optimized the basic conditions and methods of mulberry protoplastculture. Diluted the mulberry protoplast which was separated and purificated to1×10⁵/mLwith KPR liquid medium, then, on the KPR medium with added6-BA1.0mg/L,2,4-D0.2mg/L, glucose0.6mol/L as the penetrant, choose the low melting point agarose-embeddedcultures, on the5th or6th day, you can see the first cell division, a large number of celldivision occurs in13th to17th day, on the15th day, the mulberry protoplast divisionfrequency reached the highest, up to9.88%, and the plating rate is4.96%.5. Test induced by PEG-high Ca²⁺at high pH guisangyou12protoplast and nongsang14protoplast asymmetric fusion. Before fusion of donor and recipient were UV radiationtreatment and IOA treatment, UV radiation and the IOA protoplast division and growth of theinactivation, the results showed that the donor protoplast Guisangyou12, the radiationintensity of300μW/（cm²）, the processing time of6min is appropriate; Will receptorprotoplast nongsang14with16mmol/L of IOA for10min passivation. In the the mulberryprotoplast fusion process, the molecular weight and concentration of the inducer, theprocessing time and protoplast density has an impact on the protoplast. Test selection of PEG6000concentration of35%in the protoplast density of0.5×10⁵/mL of induced mulberryprotoplast aggregation rate of51.47%for15min, and protoplast fusion effect of the mulberry.The protoplast fusion cell can be observed protoplast aggregation of culture2h after, visiblecallus occurs in25th to30th day.