Study On Leaf Regeneration And Genetic Transformation System Of Qiuzi Pear And ‘Conference’ Pear

Pear is an important fruit tree cultivated worldwide,with a long history of cultivation and abundant germplasm resources.However,due to its long fruit development period,high heterozygosity,and self incompatibility,traditional fruit tree breeding has a long cycle and low efficiency.With the continuous progress of modern biotechnology,plant genetically modified technology has been increasingly applied in the production of horticultural crops.Genetic transformation technology can improve the improvement effect and accelerate the breeding process,which has important theoretical and practical significance for pear production and breeding.Therefore,this experiment investigated various factors that affect the regeneration of pear leaf buds,and based on the establishment of a pear leaf regeneration system,explored the effects of different conditions on the transformation system of pear leaves,aiming to obtain a method for genetic transformation of pear using Agrobacterium mediated technology.The main research findings are as follows:1.Establishment of pear regeneration system:Using the leaves of Qiuzi pear and‘Conference’pear as explants,a set of efficient and stable methods for inducing adventitious bud formation was constructed by studying the effects of different regeneration conditions on leaf regeneration buds.The experiment found that under conditions such as medium type,hormone type and concentration,leaf blade parts,leaf cutting method,leaf placement direction,dark culture time,and seedling age,both types of pear leaves were subcultured with tissue culture seedlings for 30 days（d）.The whole leaf cutting method was used to make the distal surface of the leaves contact the medium,resulting in the highest regeneration rate.Through the effects of different hormone concentration combinations on the regeneration of tender and mature leaves of two pear varieties,it was determined that Qiuzi pear can use tender and mature leaves as explants and be placed in a regeneration medium of NN₆₉+3.0mg/L TDZ+0.3 mg/L IBA+30 g/L sucrose+7.5 g/L agar for 14 days,with the highest regeneration rate.And‘Conference’pear must choose tender leaves for regeneration and incubate in a regeneration medium of NN₆₉+1.0 mg/L TDZ+0.5 mg/L NAA+30 g/L sucrose+7.5 g/L agar for 28 days,with the highest regeneration rate.2.Screening of antibiotic concentrations in pear:In order to determine the effects of different antibiotic concentrations on pear leaf regeneration,resistance screening was conducted on kanamycin（Kan）,cefotaxime（Cef）,and timentin（Tim）in the leaves of Qiuzi pear and‘Conference’pear.The optimal screening concentrations for Kan and Cef in two pear leaves were 16 mg/L and 300 mg/L,respectively,while the optimal screening concentrations for Tim were 300 mg/L and 150 mg/L,respectively.3.Study on the Agrobacterium tumefaciens mediated transient genetic transformation system of pear:The leaves of Qiuzi pear and‘Conference’pear were pre-cultured for 2 days.The concentration of Agrobacterium tumefaciens liquid OD₆₀₀was 0.3-0.5,and the conditions of infection time,co-culture time,and delayed culture time of the leaves were transformed.The optimal co-culture time for two types of pear leaves was determined to be 2 days,while the infection time was 12 minutes and 8 minutes,respectively.When the delayed culture time was 2 days and 1 day,the GUS staining rate was the highest.4.The study of Agrobacterium rhizogenes mediated pear genetic transformation system:This experiment used Agrobacterium rhizogenes liquid to infect the leaves of Qiuzi pear.The regenerated root rate of the leaves was 46.67%,and the average number of regenerated roots in the leaves was 1.50,indicating a low regenerated root rate;However,puncture was performed on the stem tip of the‘Conference’pear tissue culture seedlings,and the regeneration root rate of the stem tip of the tissue culture seedlings reached 80.00%,with an average of 4.08 regenerated roots,indicating a relatively high regeneration root rate.By extracting the total DNA of hairy roots for PCR identification and analysis,it was determined to be a transgenic positive root.In summary,this experiment established leaf regeneration bud systems for Qiuzi pear and‘Conference’pear.By studying the leaf regeneration conditions of the two pear varieties,the leaf regeneration methods that obtained the highest frequency of regeneration buds were determined.At the same time,this experiment also conducted research on the transformation systems of Qiuzi pear and‘Conference’pear.After exploring the transient transformation experiment of two pear leaves by Agrobacterium tumefaciens,the optimal transformation conditions were obtained.In addition,transgenic roots were successfully obtained by puncturing the stem tips of‘Conference’pear using Agrobacterium rhizogenes.