| Rosa roxburghii Tratt.is a fruit tree of the Rosa family of Rosaceae,and the fruit is rich in vitamin C,phenols,flavonoids,triterpenes and other nutrients and active substances.As the growth and development of the fruit is affected by the season and climate conditions Larger,the use of cell engineering to study the type and content of the target active substance has the advantages of short cultivation period and is not affected by natural conditions.In this study,the pear fruit was used as an explant to screen for suitable callus induction and proliferation and growth conditions,and on this basis,study the effects of different media and culture conditions on the content of important nutrients and active substances.The results of the research can establish new technologies for exploring and regulating the synthesis and accumulation mechanisms of these metabolites at the cellular level in the future method system.The main research results are as follows:1.The induction of callus of Rosa roxburghii fruit was affected by many factors.Fruit maturity,basal medium type and plant growth regulator concentration all had different effects on the callus of Rosa roxburghii.The results of single factor experiment indicated that the fruit of 20 days showed the highest browning degree and the callus was difficult to dedifferentiate after browning.However,the callus induction rate was the highest in fruits at 80 days after flowering,reaching 80.83%.In this study,1.5 mg.L-1 2,4-D combined with 0.1 mg·L-1 BA reached the maximum value of callus induction.Different culture medium types had significant effect on the induction rate,among which WPM was the most suitable for callus induction culture.The concentration ratio of plant growth regulators played a key role in callus induction.Based on the above factors,the conditions for callus induction of Rosa roxburghii fruit could be determined as follows:the healthy,pest free fruit of Rosa roxburghii at 80days after flowering can be inoculated to 0.1 mg·L-1 BA+1.5 mg·L-12,4-D,and the cultured callus of the callus under dark condition can produce better callus.2.The factors affecting the proliferation and growth of callus of Rosa roxburghii fruit and the determination of the optimum medium and culture conditions.The combination of plant growth regulators TDZ and NAA was beneficial to the growth of callus in Rosa roxburghii fruits.When the concentration of TDZ was 3.0 mg·L-1,the proliferation rate of callus was the fastest and the state is the best.The growth rate and the state of callus in the three basic media of MS,WPM and 1/2MS were quite different.and the growth state in MS medium was the best,and the growth rate was moderate.The growth rate of callus with glucose as carbon source was faster than sucrose on the whole,but the condition was slightly worse.The callus of sucrose concentration of 30g·L-1 is dense.Light and temperature significantly affected the proliferation rate and growth state of Rosa roxburghii callus,and the browning of Rosa roxburghii fruit was aggravated when the temperature was 30?,and the growth rate of the callus was slowed down when the temperature was 20?.The growth rate of callus under 12 h of light per day was faster and the callus was denser.According to this,the callus culture conditions of roxberry fruit were determined as follows:MS+3.0 mg·L-1 TDZ+0.1mg·L-1 NAA+30 g·L-1 sucrose,the incubation temperature was 25?,and the light time was suitable for 12 h of light per day.3.The influence of culture factors on the main active substances in the callus of Rosa roxburghii.The contents and yields of the main active substances in the callus were determined while the conditions of the subculture medium were screened.TDZ had a significant effect on the active substance content of the callus of the fruit of Rosa roxburghii.The contents of Vc,total phenols,total flavonoids and total triterpenes increased first and then decreased with the increase of TDZ concentration.When the concentration of TDZ was 3.0 mg·L-1,the content of active substances was the highest.The contents of Vc,total phenols,total flavonoids and total triterpenes were 45.12mg·100g-1,785.05 mg·100g-1,381.16 mg·100g-1 and 320.33 mg·100g-1respectively.MS medium was suitable for the accumulation of Vc,total triterpenes and total phenols in callus of Rosa roxburghii.WPM was suitable for accumulation of total flavonoids and total three terpenes in callus.When the concentration of sucrose or glucose reached 60 g·L-1,the accumulation of active substances was higher.High concentration sucrose was more favorable for Vc production than glucose.Light time was also an important factor affecting the content of main active substances in the callus of Rosa roxburghii fruit.The content of active substances in callus reached the highest level when irradiated at 12h every day.Different temperature had different effects on the content of main active substances in the callus of Rosa roxburghii.The culture temperature at 25?was most conducive to the accumulation of active substances in the callus of Rosa roxburghii. |
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