Optimizating Subculture And Regeneration Of ’Huang Guan’ And ’Zao Su’

Pear is one of the three major fruits in China,playing the important roles in overcoming poverty and earning foreign exchange.’Huang Guan’,’Zao Su’ and ’Cui Guan’are all important pear varieties in china.These pear varieties,which are loved by consumers at home and abroad,have a long history of cultivation and high commercial value.But they also face the problem of low efficiency of traditional breeding like other pears.Genetic transformation technology can be used to cultivate pear with highly quality stress-resistant varieties.A stable and high-frequency system for subculture and regeneration is the premise of genetic transformation of pear.Growth regulators determine the growth and differentiation of plants,and their addition is the most critical for the subculture and regeneration of pear.In this study,the common pears of ’Huang Guan’,’Zao Su’ and ’Cui Guan’ were used as experimental materials and their subculture and regeneration were optimized by selecting the categories and proportions of plant growth regulators.Besides,the addition of antibiotics was modified,which benefits the contamination control during subculture and regeneration.The main research contents are as follows:1.The stem segments of ’Huang Guan’ and ’Zao Su’ was used as test materials to germinate pear buds in primary culture.Then,12 different ratios of 6-BA and IBA were screened by full factor design and variance analysis.The ratio of 6-BA and IBA in subculture has been optimized.The main and interactive effects of 6-BA/IBA were analyzed at propagation coefficient and plantlet height.Moreover,in view of the negative effects on pear plantlets caused by the accumulation of 6-BA in the subculture process,an improvement scheme was put forward that the subculture plantlets with different plant height were inoculated in the medium with reduced 6-BA concentration and no hormone addition.At the basic medium of MS supplemented with 30 g/L sucrose and 7.5 g/L agar,the combination of 1.5 mg/L 6-BA+0.3 mg/L IBA is most suitable for the subculture of’Huang Guan’ and 2.0 mg/L 6-BA+0.3 mg/L IBA for ’Zao Su’.2.Using the leaves of ’Huang Guan’,’Zao Su’ and ’Cui Guan’ as experimental materials,27 different ratios of 6-BA,TDZ and NAA were screened by full factor design and analysis of variance.Ultimately,the ratio of 6-BA,TDZ and NAA in the regeneration system was optimized,and the main and interactiveeffects of them on regeneration frequency and regenerated buds number of the three varieties were analyzed.The basic medium,carbon source and coagulant were also studied.When NN69 was used as the basic medium,supplemented with 40 g/L sorbitol and 3 g/L plant gel,1.0 mg/L 6-BA+1.5 mg/L TDZ+0.1 mg/L NAA was the most suitable for ’Huang Guan’ leaf regeneration,3.0 mg/L6-BA+1.5 mg/L TDZ+0.5 mg/L NAA was the most suitable for ’Zao Su’ leaves,and 3.0mg/L 6-BA+1.0 mg/L TDZ+1.0 mg/L NAA was the most suitable for ’Cui Guan’ leaves.In addition,for ’Huang Guan’ leaves,there was regeneration on NN69 medium but no response to MS;sorbitol carbon source was better than sucrose,and the optimum concentration range was 30 g/L-40 g/L;plant gel was more suitable as coagulant than Agar,and the optimum concentration range was 3.0 g/L-4.0 g/L.3.In view of the contamination in the subculture and leaf regeneration of pear,the addition of antibiotics was optimized by ultrasound assistance,surface coating and setting protective layer.The contaminated leaves were treated with ultrasound for 60 s at a frequency of 40 KHz in the aqueous solution containing 100 mg/L timentin.After standing for 15 min,it was transferred to the medium containing 200 mg/L timentin and coated with50μL mother liquid of temetine(100 mg/m L)for 30 days.Such measures can greatly reduce the use of antibiotics and ensure the healthy of explants,while reducing the pollution rate to 22.13%.Furthermore,the contaminated plantlets were inoculated in the culture medium with a protective layer at the top,which did not contain any nutrients and was added with 500mg/L temetine.This also makes the pear plantlets grow healthily and keeps the pollution rate at 0%.