A New Rapid Propagation System Of Pleione Bulbocodioides (Franch.) Rolfe

Objective Using seeds and test-tube plantlets of Pleione bulbocodioides（Franch.）Rolfe as materials,we established a new type of rapid propagation System.Methods1.To determinine the vitality of the seeds of Pleione bulbocodioides（Franch.）Rolfe,we used the TTC staining method and study the optimum conditions of the TTC staining method.2.We used the NiKon ECLIPSE Ti fluorescence inverted microscope to observe the microstructure of the seeds of Pleione bulbocodioides（Franch.）Rolfe.3.We chose the seeds of Pleione bulbocodioides（Franch.）Rolfe as explants to investigate the effects of different plant growth regulators and natural additives on non-symbiotic germination and cultivation of its seeds.4.We chose the bulb stems of Pleione bulbocodioides（Franch.）Rolfe germinated in1/2MS liquid medium as explants to investigate the effects of different adsorbents and antioxidants,different plant growth regulators,different natural additives and combination formula on the rooting of seedlings.5.We chose the seeds of Pleione bulbocodioides（Franch.）Rolfe grown on 1/2MS solid medium as explants to investigate the effects of different plant growth regulators,different natural additives and the combination formula on the rooting of seedlings.6.We chose the seeds of Pleione bulbocodioides（Franch.）Rolfe as explants to investigate the effects of different plant growth regulators and natural additives on the induction and proliferation of protocorms.7.We chose the test tubes of Pleione bulbocodioides（Franch.）Rolfe as explant to investigate the effects of different plant growth regulators and natural additives on the induction,proliferation and differentiation of protocorm-like bodies of seedlings.Results1.The optimum testing conditions for TTC staining method:the temperature is 40℃,the concentration of TTC is 0.05%and the time for staining is 48 hour.2.The seeds of Pleione bulbocodioides（Franch.）Rolfe appeared elongated or spindle-shaped,slender at both ends of an irregular twist,with a reticulate pattern on the surface.Outer seed coats appeared light yellow and inhomogeneously alternated with white.Inner seed coats close to the endosperm are hard to separate.The embryos are nearly elliptical with embryonic length 1301.080±211.039μm,embryonic width 699.106±108.525μm and embryonic area 738435.478±208845.269μm²,accounting for 46.6±10.2%of the total seed cell area.3.The different plant growth regulators and natural additives had significant differences on non-symbiotic germination of the seeds of Pleione bulbocodioides（Franch.）Rolfe.The optimum medium for seed germination is 1/2MS+0.1 mg/L KT+100 ml/L Potato juice.The germination rate was 95.90±0.24%.The germination of bulb stems was consistent and the color was bright green.4.Bulb stems cultured in a medium containing 1g/L activated carbon had the lowest browning rate of only 2.60%.5.The different plant growth regulators and natural additives had significant differences on the seeding rooting of bulb stems of Pleione bulbocodioides（Franch.）Rolfe.Compared with IBA,2,4-D and TDZ,NAA and KT were more beneficial to seedling rooting of bulb stems.Compared with banana juice and coconut juice,potato juice was more beneficial to seedling rooting of bulb stems.On these basises,orthogonal experiments were carried out.The optimum medium for seedling rooting of bulb stems is MS+0.1 mg/L NAA+0.1～0.3mg/L KT+50～100 ml/L potato juice+1.0 g/L activated carbon.6.The different activated carbon content,auxin and natural additives had significant differences on rooting of seedlings.When 1.0 g/L activated carbon was added to the medium,the rooting number of seedlings was 2.43±0.53,and the seedling plants were growing well and the leaves were dark green.Compared with IBA,NAA at the same concentration was more beneficial to the rooting of seedlings.When 0.2 mg/L NAA was added to the medium,the rooting number of seedlings was 2.57±0.53.When potato juice was added to the medium,the seedling plants were growing well and the leaves were dark green.When 100 ml/L potato juice was added to the medium,the rooting number of seedlings was 2.71±0.49.On these basises,combination formula experiments were carried out.The optimum rooting medium was MS+0.2 mg/L NAA+100 ml/L potato juice+1.0 g/L activated carbon.7.Compared with 6-BA and TDZ,2,4-D and KT were more beneficial to induce the protocorms.The induction rate of protocorms in each treatment group which added potato juice and banana juice to medium was significantly lower than that of the treatment group which added coconut juice to the medium.At the same time,the low concentrations of AgNO₃ was beneficial to the induction of protocorms.On these basises,orthogonal experiments were carried out.The optimum medium for inducing protocorms is 1/2MS+2.0mg/L 2,4-D+2.0 mg/L KT+2.0 mg/L AgNO₃+50 ml/L coconut juice.In the protocorm proliferation experiment,the optimum medium was MS+2.0～3.0 mg/L 6-BA+1.0～1.5 mg/L NAA+1 g/L activated carbon.8.The different plant growth regulators and natural additives had significant differences on the induction of protocorm-like bodies from test-tube plantlets.Compared with NAA and2,4-D,IBA and KT were more beneficial to induce the protocorm-like bodies.At the same time,it has found that when the content of natural additives was the same,the induction rate of potato juice added to the medium was significantly higher than that of the culture medium added coconut juice.On these basises,combination formula experiments were carried out.The optimum medium for inducing protocorm-like bodies was MS+0.3 mg/L IBA+0.3 mg/L KT+100 ml/L potato juice+1 g/L activated carbon.In the protocorm-like bodies proliferation experiment,the optimum medium was 1/2MS+2.0 mg/L 6-BA+0.5～1.0mg/L KT+100 ml/L potato juice+1 g/L activated carbon.Protocorm-like bodies were fully dark green and undifferentiated.In the protocorm-like bodies differentiation experiment, the optimum medium was 1/2MS+0.2 mg/L NAA+2.0～2.5 mg/L 6-BA+1 g/L activated carbon.Conclusion Using seeds and test-tube plantlets as materials,We succeeded in establishing a new type of propagation System of Pleione bulbocodioides（Franch.）Rolfe.