Tissue Culture Of Catalpa Bungei C.A. Mey And Its Active Flavonoid Composition In Callus

Catalpa bungei C.A.Mey is a deciduous tree of the genus Catalpa bungei in the family Bignoniaceae.It has high industrial,ornamental and medicinal values,and has been known as the "King of Wood" since ancient times.In recent years,research on the active ingredients of Catalpa has received more and more attention,and the active ingredients in Catalpa bungei play an important role in anti-inflammatory,antibacterial and antiviral activities.Due to the self-flowering and unaffinity of Rowan tree,fruiting is scarce,the difficulty of reproduction in real life,the reproduction coefficient is low,and the supply in the market exceeds the demand.To alleviate the contradiction in the supply of rowan seedlings and to improve the output of active ingredients,this study was carried out on the rapid propagation of rowan trees in vitro,induction and redifferentiation of callus,and optimization of the culture of flavonoid active ingredient content in callus,to provide a scientific basis for rowan tree factory nursery.The main research results are as follows.1.A rapid propagation system of Catalpa bungei with shoot stem segments as explants was established.The most suitable sterilization method for stem segments with buds was soaking in 75%alcohol for 30s,rinsing in sterile water 3-4 times and then sterilizing with 0.1%mercury ascending for 8 min and rinsing in sterile water 4-5 times,with a survival rate of 46.67%.The most suitable primary medium formulation was MS+0.20 mg·L-16-BA+0.01 mg·L-1 NAA+30 g·L-1 sucrose+6.8g·L-1 agar,and the axillary bud induction rate was 66.67%;the suitable proliferation medium formulation was MS+3.0 mg·L-16-BA+0.10 mg·L-1 IBA+30 g·L-1 sucrose+6.8 g·L-1 agar,with a proliferation coefficient of 5.67;the suitable rooting medium was WPM+0.3 mg·L-1 NAA+0.5 mg·L-1 IBA+20 g·L-1 sucrose+6.8 g·L-1 agar,the rooting rate was 75.56%;after transplanting after refining,the suitable substrate composition ratio was grass charcoal soil:vermiculite(1:1),and the survival rate was 66.67%.2.Suitable formulations for the induction of callus in different explants of Catalpa bungei and the optimal medium formulations for the redifferentiation of petiole callus were screened.The suitable formulation for leaf induction of leaf callus of Catalpa bungei sterile seedlings was MS+1.0 mg·L-1 6-BA+0.05mg·L-1 NAA with white irradiation light quality;the suitable formulation for petiole induction of callus was MS+1.0 mg·L-16-BA+0.10 mg·L-1 NAA with white irradiation light quality,and the medium formulation for adventitious shoot differentiation was DKW+0.2 mg·L-1 TDZ+0.01 mg·L-1 NAA,irradiated with blue light;the suitable formula for stem segment induction of callus was DKW+1.0 mg·L-1 6-BA+0.05 mg·L-1 NAA,irradiated with white light.The suitable medium formulation for the induction of callus in leaves of Catalpa bungei potted seedlings was DKW+2.0 mg·L-1 6-BA+1.0 mg·L-1 NAA(based on callus induction rate),with a callus induction rate of 71.11%;DKW+2.0 mg·L-1 6-BA+0.5 mg·L-1 NAA(based on total callus flavonoid content),with a total flavonoid content of 101.376 mg/g.(30 g·L-1 sucrose and 6.8 g·L-1 agar were added to the above medium)3.The main flavonoid active components of the callus were clarified,and the callus culture formulations for pro-flavonoid synthesis were optimized.A total of 340 flavonoid metabolites were detected in the leaves and healing tissues of Catalpa bungei by flavonoid broadly targeted metabolomic techniques,including 247 differential metabolites between the callus and wrinkled bark leaves and 226 differential metabolites between the callus and light bark leaves.The results of the differential metabolites showed that geranoside had the shortest retention time,was efficiently detectable,and was significantly higher in the healing tissues than in the leaves.Among the elicitors treatments,10 μmol·L-1 salicylic acid(SA)had a greater effect on the induction rate of healing tissues,and the healing rate obtained with the DKW+2.0 mg·L-1 6-BA+1.0 mg·L-1 NAA+30 g·L-1 sucrose+6.8 g·L-1 agar+10μmol·L-1 SA formulation culture could be increased by 29%compared with the control;the addition of different concentrations of salicylic acid(SA)and yeast extract(YE)could both increase the total flavonoid content in the callus,where the total flavonoid content was significantly increased under the treatment of 100 mg·L-1 yeast extract(YE),by 41.65%and 59.20%,respectively,compared with the control group;the geranoside content was the highest in the J-YE3 group,at 8.595 mg/g;it increased by 24.63%compared with the control group,and the medium formulations were:DKW+2.0 mg·L-16-BA+0.5 mg·L-1 NAA+30 g·L-1 sucrose+6.8 g·L-1 agar+200 mg·L-1 YE.