| Fritillaria cirrhosa D.Don is the first base source plant included in the Medicinal Materials of Fritillariae Cirrhosae Bulbus(FCB)in the 2020 edition of the Pharmacopoeia of the People’s Republic of China.Its dry bulb has the functions of clearing heat and nourishing the lung,resolving phlegm and relieving cough,dissipating nodules and eliminating carbuncle,and so on.It is widely used in the prevention and treatment of respiratory diseases.Due to the harsh growing environment,low natural reproduction rate,and long-term over-harvesting,the wild resources of Fritillaria cirrhosa were nearly exhausted.At present,there is a significant market demand and a severe shortage of resources.Although artificial cultivation of Fritillaria cirrhosa has been successful,the seed source has limited the expansion of production scale,and the amount of commercial medicinal materials that can be provided is still tiny.In this study,the bulb of Fritillaria cirrhosa was used as explant material,mainly focusing on the tissue culture and rapid propagation system,cell suspension culture system,and artificial seed technology of Fritillaria cirrhosa,in order to provide an experimental basis for the establishment of the artificial provenance production technology system,the preservation of germplasm resources,and the industrial seedling cultivation of Fritillaria cirrhosa.The main research results are as follows:(1)The technology system of in vitro culture of Fritillaria cirrhosa was established.The bulb of Fritillaria cirrhosa was used as the explant,and the sterilization method of the explant was determined.By optimizing the culture conditions,the medium for embryogenic callus induction was MS + 30 g/L sucrose +1.0 mg/L Picloram,with an induction rate of 84.93%.The callus redifferentiation medium was MS + 30 g/L sucrose + 1.0 mg/L 6-BA + 0.2 mg/L NAA,the differentiation rate reached 77.92%,and the average number of buds differentiated was 6.70.The medium for bulblets induction was MS + 30 g/L sucrose + 0.3 mg/L6-BA + 1.0 mg/L 2,4-D.The medium for one-step seedling formation from explants was MS + 30 g/L sucrose + 0.5 mg/L KT + 0.5 mg/L 2,4-D.(2)A liquid culture environment suitable for embryogenic transformation of Fritillaria cirrhosa cells was constructed.The effects of different combinations of plant growth regulators,basal medium concentration,initial inoculation amount,and carbon source concentration on the growth,and development of suspension culture cells were investigated with loose callus as material and the dynamic growth curve of cells during suspension culture was established.The optimal suspension culture conditions for the growth and proliferation of Fritillaria cirrhosa cells were as follows: MS as the basic medium,callus inoculation amount was 50 g/L,sucrose concentration was 30 g/L,hormone composition was 1.0 mg/L 6-BA + 0.3 mg/L2,4-D,shaking speed was 110 rpm,subculture period was 18 days.Liquid medium containing 1.0 mg/L TDZ + 0.3 mg/L 2,4-D or 1.0 mg/L Picloram was favorable for the embryogenic transformation of Fritillaria cirrhosa cell masses.(3)The technical conditions for domestication and transplantation of tube bulbs of Fritillaria cirrhosa were established.The bulblets obtained in vitro culture were used as the artificial provenances,which was in accordance with the production technology characteristics of reproducing with bulbs in the traditional production of Fritillaria cirrhosa.Low-temperature treatment was helpful for tube bulbs to break dormancy,the induced bulblets were seeded in the mixed soil substrates at 4℃ for 45 days and then transferred to a 16℃ light incubator for further culture for 30 days,and the germination rate could reach 42.8%.(4)The production method of the artificial seed of Fritillaria cirrhosa was determined.Artificial seeds were produced by embedding the regenerated bulblets as propagules and the basic components of the artificial endosperm were 3% SA + 3%sucrose + MS + 2.0 mg/L 6-BA + 0.5 mg/L NAA.By investigating the effects of the addition of artificial coats and other substances such as preservatives on the physical properties of artificial seeds,the results are obtained by adding preservatives to the artificial endosperm after one embedding reaction and placing them in the outer seed coat after a second embedding.The maximum germination rate of artificial seeds produced under aseptic conditions was 83.77% and 61.33%.(5)Technical conditions for the production of artificial seeds of Fritillaria cirrhosa with the addition of endophytic bacteria were preliminarily established.Regenerated bulblets were used as propagators,3% SA + MS + 10 g/L sucrose + 2.0mg/L 6-BA + 0.5 mg/L NAA + 5% bacterial suspension as the base component of the artificial endosperm.After an embedded reaction,the artificial seeds were prepared by direct contact between the endophytic bacteria of Fritillaria cirrhosa seeds and propagules.The germination rate reached 25.1% when the artificial seeds were sown in mixed soil substrate at 4℃ for 45 days and then transferred to a 16℃ light incubator for 30 days.To sum up,this study focused on the research objective of large-scale production of artificial provenances of Fritillaria cirrhosa.It carried out a series of studies on in vitro regeneration culture,suspension cell culture,and artificial seed technology of Fritillaria cirrhosa.The results laid the experimental foundation for the industrial production of artificial provenances of Fritillaria cirrhosa.It is helpful to promote the production of sterile tissue culture seedlings back to the field.It also provides technical support for producing secondary metabolites,cell fusion,and genetic transformation using Fritillaria cirrhosa suspension cell lines.It is of great significance for the protection,development,and utilization of Fritillaria cirrhosa. |
