Seed Germination Of Polygonatum Sibiricum Red.and Establishment Of Tissue Culture System

Polygonatum sibiricum Red.is a perennial herb,belonging to Polygonatum Liliaceae.It is widely distributed in the whole country,mainly in Hebei,Inner Mongolia,Shaanxi Province and other provinces.Its rhizome was used as Chinese medicine,which has the function of tonifying and nourishing for body,strengthening spleen and tonifying kidney.It has received extensive attention from researchers.Under natural conditions,the seed of yellow essence has long breeding period and low germination rate,all this caused the wild resources of Polygonatum sibiricum Red.to be unable to satisfy the market demand,Therefore,artificial culture has become one of the channels to obtain it.The artificial culture mainly used by asexual reproduction of rhizome,in the process of cultivation,the quality of rhizome decreased,and the efficacy weakened.Tissue culture can preserve quality of germplasm resources,avoid loss of good genetic genes,and increase the active ingredient content of plant.Stratification treatment plays an important role in breaking seed dormancy,To explore the physiological changes of the material inside the seed during the process of stratification,which can provide data support and basis for the release of seed dormancy.Based on above,this thesis used Polygonatum sibiricum Red.as experimental material,which is collected from Liaoning Province,The author studied the tissue culture and seed germination physiology of Polygonatum sibiricum Red..The main findings are as follows: Seed germination physiology:(1)The germination rate of Polygonatum sibiricum Red.seed was investigated by exogenous hormone treatment,chemical reagent treatment,physical method and stratification treatment.It is found out that,Na OH,KNO3,salicylic acid and concentrated sulfuric acid had no significant effect on germination of seed.While the influence of gibberellin and 6-benzylaminopurine on germination were obvious.Moreover the most favorable concentration seed of GA3 for germination is 150 mg/L,the concentration of 6-BA is 100 mg/L.The seeds was treated with a low-temperature stratification treatment and 10.0 mg/L gibberellin GA3 for 14 weeks,which could greatly promote seed germination.The effect was the most significant,helping theseed to break the dormancy state.It lays a stable foundation for artificial seeding and cultivation.(2)The seed of Polygonatum sibiricum Red.belongs to starch seed,which has the high content of starch and sugar.During the process of stratification,the content of starch was decreased first and then increased,while the soluble sugar was increased first and then decreased,at the same time,the amylase was increased first and then decreased then increased.This is because the hydrolysis of macromolecular compound starch into soluble sugar under the action of amylase.The changes of starch hydrolysis process not only creates conditions for seed germination,but also saves energy for seed germination.On the other hand,the content of soluble protein increased continuously,and crude fat decreased in the process of stratification.This results showed that stratification contributed to the protein metabolism of the seed,and the decrease of crude fat content provided nutrition for seed development.Tissue culture:(1)Seeds of Polygonatum sibiricum Red.were treated with gibberellin and low temperature stratification and then germinated to get the seedlings who were treated as explants to induce callus.It is found that,the induction callus ability of different tissues is related to the differentiation ability of cells.The induction ability was sorted as rhizome bud > seed seedling > rhizome > stem > leaf;Compared with the half-injection type,the flat-out type explant access is more beneficial to the callus induction;And the induction rate of callus of Polygonatum sibiricum Red.was related to the type of medium and the induction rate is different.The induction rates were in order as: MS > 1/2MS > B5>N6> White;25 ℃,24 h/d dark culture for callus are the optimal culture temperature and duration of illumination.For single hormone culture,suitable culture concentration are 0.20 mg/L NAA and 1.5 mg/L 6-BA.The highest induction rate of callus tissue was at the condition of that the concentration of NAA is 0.2mg/L and the concentration of 6-BA is 1.5 mg/L.(2)Based on the above experimental results,we explored how to optimize the conditions for the proliferation of Polygonatum sibiricum callus.The results of the experiment is shown that : 0.9 g/L callus were connected to MS 0.5mg L-1 NAA 1.5mg L-1 6-BA,medium with sucrose concentration of 25 g/L,p H value was 6.0,and cultured in 12 h/d light culture artificial climate box.The best proliferative tissue can be obtained.The optimum medium for inducing bud of Polygonatum sibiricum Red.was MS 1.00mg/L 6-BA 0.70mg/L IBA;optimal medium for proliferating bud,the MS 1.00mg/L 6-BA 0.50mg/L IBA,p H value was 6.0,the temperature was 26℃,and the illumination was 12 h/d.