Research On The In Vitro Multiplication,Cell Suspension Culture And Flavonoid Accumulation Ability In Cell Suspension Of Stahlianthus Thorelii Gagnep

Stahlianthus thorelii Gagnep is one of the valuable medicinal herbs in the Zingiberaceae family commonly known as"Suoshitutianqi"which is used to cure cancer,pneumonia,diarrhea,and inflammation in both Vietnamese and Chinese folk medicine.The tubers of Stahlianthus thorelii Gagnep contain biologically active substances mainly flavonoids,coumarins,polysaccharides,and essential oils.The cultivation of Stahlianthus thorelii Gagnep is essentially spontaneous without a master plan and valorization of the products.In particular,currently,the main cultivation method of Stahlianthus thorelii Gagnep is vegetative propagation.This method has a limited number of seedlings and a small multiplication factor,ranging from 0.99 to 2.0 times.Flavonoids are abundant secondary metabolites in plants,fruits,and seeds responsible for color,aroma,and flavor characteristics.In humans,these compounds are associated with a wide range of health benefits arising from their bioactive properties,such as anti-inflammatory,anti-cancer,anti-aging,cardioprotective,neuroprotective,immunomodulatory,antidiabetic,antibacterial,and antiparasitic properties.and antivirals.Stahlianthus thorelii Gagnep in the wild are dwindling in number,making it difficult to have an abundant and stable source of raw materials.The use of in vitro and cell suspension cultures of Stahlianthus thorelii Gagnep in the production of flavonoids promises high efficiency.The purpose of this work was to identify the right medium for the in vitro culture of Stahlianthus thorelii Gagnep callus and cell suspensions.The obtained results are described below:（1）The Reproduction of In Vitro Stahlianthus thorelii Gagnep:When sterilizing samples,the combination of 70⁰alcohol for 20 seconds and 0.1%Hg Cl₂alcohol for 20 minutes is the most effective for sterilizing samples.It was found that a basal MS growth medium with BAP（5.0 mg/L）or kinetin（4.0 mg/L）showed the correct number of shoots/explant,5.45±0.59 and 5.48±0.87,respectively.In vitro shooting basal media with 2 mg/L and 0.5 mg/L of BAP and NAA showed7.45±0.79 shoots/explant.The most significant rooting development（26.17±1.5 roots/shoot）occurred in the MS medium containing 0.5 mg/L（BAP）and 0.5 mg/L（IBA）.Survival was maximum（100%）and the tree height was 29.40±3.59 cm when plants（tissue-cultured）were acclimated in soil with sand and compost（1:1:1）.（2）Callus cells of Stahlianthus thorelii Gagnep were most bactericidal when combined with Johnson’s solution 10%for 20 minutes and Hg Cl₂0.1%for 15 minutes.The best results were obtained using the MS media consisting of 3.0 mg/L of 2.4-D and 3.0 mg/L（BAP）,in which48.49±0.44%of the samples developed callus.The MS media having 1.0 mg/L of 2.4-D and 1.0mg/L（BAP）had a maximum callus biomass with a proliferation efficiency of 0.58±0.009 mg.（3）Cell suspension cultures with a sample volume of 3 g cells and a shaking speed of 120 rpm gave the highest growth results.The MS media having 1.5 mg/L of 2.4-D and 1.0 mg/L（BAP）was the most suitable to achieve a cell mass of 8.31±0.11 g fresh（0.74±0.11 g dry）.A saccharose content of 30g/L favored the growth of Stahlianthus thorelii Gagnep cells.Under appropriate conditions,cell growth peaked at day 14 with a fresh weight of 15.97±0.89 g（dry weight 0.85±0.09 g）and greater flavonoid accumulation than Stahlianthus thorelii Gagnep tubers.Gainep in nature,the highest is 3.73±0.1%on the 14th day after implantation.The oil content of Stahlianthus thorelii Gagnep cell suspension reached its maximum at 10-16 days of culture,which was 0.22%to 0.26%higher than that of Stahlianthus thorelii Gagnep natural tubers（0.11%）.