Study On Tissue Culture And Rapid Propagation Of Ilex Asprella

Ilex asprella is a deciduous shrub.It is the main raw material for a variety of Traditional Chinese Medicines and herbal tea for its medicinal value and benefit to human health.In this research,it was studied that tissue culture and rapid propagation of Ilex asprella with explants of stems with axillary buds included explant induction,subcultures proliferation,rooting culture,hardening and transplanting,aseptic seedling stem and leaf callus induction and adventitious bud differentiation,to provide technical support and high quality seedlings for the development of this medicinal plants and the large scale production of seedlings.The main results were as follows:1.The best season for selecting explants and sterilization time of explantsThe best season for selecting material was August,using the stems with axillary buds as the explant.The optimizing time of disinfection was 8 minutes.The axillary bud germinating rate was 76.13%.The pollution rate was lower with the percent of 33.33%.2.Inducing axillary bud differentiation and subcultureThe optimized medium of inducing axillary bud differentiation was:MS+6-BA0.5mg/L +NAA0.05mg/L+GA30.2mg/L.The germination rate was 77.63%and the bud was so good that it grew to 1.57cm in length for 40 days,without edema and abnormal callus.The suitable medium for subculture of buds was MS+6-BA0.8mg/L+IBA0.4 mg/L.When the light intensity was 1000-15001x,the multiplication ratio was 3.51 and the average seedling height was 3.61cm for 30 days.3.The differentiation of adventitious buds from aseptic seedling leaf directlyThe leaves upward were stabbed,and then inoculated onto the culture medium in five plates.The optiming medium was WPM+6-BA3.0mg/L+NAA 0.1mg/L.After 50 days culture,the regeneration rate was 98.88%and the average number of buds per leaf reached 4.48 strains.4.The callus induction and proliferation differentiation(1)The optiming mediums for callus induction of aseptic seedling leaf and stems were MS+6-BA0.1mg/L+NAA 0.1mg/L and WPM+6-BA0.1mg/L+2,4D 0.5mg/L.After 60 days culture,the callus induction rate was 100%.The Callus were good,light green and yellowish-white,dense granular.(2)The optiming mediums for callus proliferation of aseptic seedling leaf and stems was WPM+6-BA1.0mg/L+NAA0.3mg/L.(3)The optiming mediums for redifferentiation of adventitious buds from aseptic seedling leaf callus was WPM+6-BA3.0mg/L+NAA0.3mg/L.After 60 days culture,the redifferentiation was 76.67%.The average number of buds percallus were 2.49 strains.The optiming mediums for redifferentiation of adventitious roots from aseptic seedling stem segment callus was WPM+6-BA1.0mg/L+NAA0.3mg/L.The redifferentiation was 21.36%.5.Subculture seedling and rooting at a timeThe optiming mediums of subculture seedling and rooting culture was MS+6-BA0.6mg/L+IBA0.2mg/L.After 50 days culture,new roots were observed from stem bark.The rooting rate was 80.25%.6.Rooting cultureThe optiming mediums of rooting was 1/2MS+NAA0.2mg/L+IBA1.0mg/L+AC1000mg/L.The rooting rate was 98.79%.Average number for root formation was 4.55.7.Rooting seedling hardening and transplantingUnder the natural light with intensity of 7000～10000lx,the plantlets werecultured for 45d and transplanted to the media(yellow subsoil:fine sand:peat=4:2:1),the survive rate reached up to 94.72%in 5-6 months.