Research On Somatic Protoplast Fusion Between Two Species In Liriodendron

In this experiment, explant materials were collected both from a 30years provenance testing of Liriodendron chinense and Liriodendron tulipifera on Xiashu Forest Farm of Nanjing Forestry University and on campus. New shoots, buds and flowers were taken as explants. The major results were as follows:1. Sterilization of ExplantsThe materials were young shoots, twigs, leaves, petioles, flower bud petals in 4⁵cm long. The new leaves from pure individual of Liriodendron chinense and Liriodendron tulipifera were collected 15 days after buds breaking in the spring. And the appropriate sterilization procedures are as fellows, for the shoot, wash with detergent for 15mins, rinse two hours, dealing with alcohol for 30 seconds, then mercuric chloride for 9 min, washed with sterile water for 4 to 5 times , inoculated into induction medium.2. Callus InductionThe MS medium was suitable for initiation to Liriodendron Chinese and Liriodendron tulipifera basically. The rate of callus induction on medium MS+ 2,4-D2.0mg/L+ 6-BA0.2mg/L+ sucrose30g/L, for young shoots, twigs, leaves, petioles is up to 100%, but, the callus induction medium for anther was 1/2MS+ 2,4-D2mg/L+6-BA0.5mg/L+ NAA2mg/L+ KT0.2mg/L+ GA0.2mg/L+ Gln200mg/L+ Asn200mg/L+ Pro100mg/L+ Arg200mg/L+ CH₃00mg/L.3. Establishment of Suspension Culture SystemThe callus cultured in the medium of MS+NAA 0.5mg/L+KT 0.5mg/L+6-BA 0.5mg/L+Vc 5mg/L+LH 500mg/L+pH5.8+SUC50g/L can be induced into embryogenic callus.4. Free and Culture of ProtoplastThe optimal enzyme combination for the protoplast isolation from mesophyll tissue is combination E i.e.Cellulase2%+ Hemicellulase1%+ PectolyaseY-23 0.2%. And the most suitable enzyme combination for the protoplast free with suspension is combination F, i.e.Cellulase2%+ Hemicellulase1%+ PectolyaseY-23 0.1%. For protoplast culture was KM8p, add mannitol of 0.6mol/L, and MES of 0.1%.5. Electro Fusion of ProtoplastThe optimum parameters for electro fusion: alternating current (AC) 110V/cm, duration 30s; direct current (DC) 1000V/cm, duration 40μs, 5 times. The rate of protoplast fusion can be reached to 30%.6. The Transformation of Exogenous DNA The mixture of exogenous plasmid DNA and protoplasts, then adding the solution of PEG (30%PEG-8000+0.3mol/LGlucose+10mmolCaCl₂) steeply, the GFP gene carried by plasmid DNA can be expressed transiently.