Establishment Of Tissue Culture Regeneration System And Genetic Transformation System Of ’Orah’

Citrus occupies a large proportion in the fruit industry and is a very important cash crop.Because of its unique flavor,it is loved by consumers.Plant genetic transformation technology is widely used in improving crop quality and verifying gene functions,and it is increasingly used in citrus research.There are few reports on the regeneration and genetic transformation experiments of some common citrus varieties in the market,and the regeneration and transformation operations of citrus are complex,with long cycles and low transformation efficiency,and there are significant differences between different genotypes As a representative late-maturing citrus variety in the market,’Orah’ has high economic benefits and good flavor.In this experiment,the epicotyl of ’Orah’ was used as an explant to establish a regeneration system for ’Orah’,and on this basis,a genetic transformation system for ’Orah’ was established.Ruby1 gene was introduced into ’Orah’ and identified.The purpose is to provide technical support for the subsequent research on genetic transformation of’Orah’ and other citrus varieties.The main experimental results of the study are as follows:(1)Establishment of the Regeneration System of ’Orah’This experiment was conducted to establish a regeneration system of ’Orah’ using the epicotyl of ’Orah’ seed culture as an explant.Screening out the best disinfection method suitable for ’Orah’ seeds: soaking with 75% ethanol for 30 seconds,and then disinfecting with 3% sodium hypochlorite(Na Cl O)for 15 minutes,which can ensure that the disinfected seeds have a low contamination rate and a high germination rate.After culturing the hypocotyl under sterile conditions,cut it into about 1 cm of stem segments,and screen the regeneration medium.The best regeneration medium was MT+1mg/L 6-BA+0.1mg/L NAA,with a regeneration rate of 91.67%.The cutting method of the epicotyl was optimized.The best cutting method was oblique cutting into a trapezoidal incision,with a regeneration rate of 88%.The effects of three light qualities,white light,red light,and blue light,on the growth of regenerated buds were compared.White light culture was the best,with a regeneration rate of 87%.The optimal rooting medium for regenerated buds was 1/2MT+0.5 mg/L IBA+0.5 mg/L AC,with a rooting rate of 86.67% and an average root length of 4.67 cm.(2)Establishment of a Genetic Transformation System for ’Orah’The Ruby1 gene was introduced into ’Orah’ by Agrobacterium tumefaciens mediated method.The effects of kanamycin concentration,seedling age of tissue culture seedlings,bacterial concentration,infection time,and co culture time on transformation efficiency were compared.The kanamycin resistance test showed that50 mg/L screening had the best effect,and this concentration was suitable for subsequent genetic transformation tests.The results showed that the best effect of inducing resistant buds was to inoculate the hypocotyls of ’Orah’ at the age of 30 days in a 0.6 concentration bacterial solution for 30 minutes,and then co culture them for 3days before transferring them to a screening medium.(3)Verification of transgenic plantsThe expression of Egfp gene was observed,and transgenic plants showed a green fluorescence phenomenon under excitation light.It was also observed that transgenic plants exhibited a purplish red phenotype of anthocyanin accumulation.After RT-PCR detection of the last 12 resistant buds,six specific target bands carrying Ruby1 gene were amplified.Through RT-q PCR analysis,it was found that Ruby1 gene was overexpressed in the positive buds,and the expression level was significantly higher than that of the wild type,indicating the success of the transformation.