The Establishment Of Walnut Genetic Transformation System By Pollen Tube Pathway

Establish a stable and efficient genetic transformation system is a basis for carrying outthe walnut molecular breeding research. However, there are some difficulties, such as tissueculture regeneration of walnut, plantlets rooting, establishment of genetic transformationsystem and import of exogenous gene. Both at home and abroad have been unable to get agood solution. The pollen tube pathway transformation is a germplasm system transformationmethod which is easy for an exogenous DNA integration. Importing the exogenous DNA intothe embryo sac through the natural pollen tube pathway formed after plant pollination, in orderto achieve the purpose of genetic transformation.In this study, Superior walnut varieties as test materials, first, we determined the perfecttransformation period, and then selected the appropriate conversion method, finally found asuitable filtrate and save method. An integrated walnut pollen tube pathway genetictransformation system was established. The main results are as follows:1. The pistillate flower flowering process was divided into six stages: markable flowering,the early opening stage, V word period, small ‘八’ characters stage, large ‘八’ characters stageand overturned period, according to the record morphological changes of ovary size, stigmashape and stigma opening angle changes during stigma pollination and fertilization.2. The peroxidase activity of the stigmas was studied by benzidine-H2O2method. Theresults showed that the stigma receptivity period was ranging from the stigma slightly open toit with brown stripes. The periods of small ‘八’ characters and large ‘八’ characters havestrongest receptivity.3. The period and conditions for keeping highest pollen viability were detected by MTTstaining method. The pollen viability was the highest when the walnut staminate flowerblossoming, reaching95%. The pollen can be stored for96hours under natural conditions and the viability decreased quickly to33%after4hours. Pollen viability could be reinforced at4℃and the average viability was63.3%after120hours.4. The internal structure of walnut pistillate flower was observed by paraffin sections. Wefound the appropriate period for pollen tube pathway importing was2～4d after flowering andcontacted the external morphology to determine overturned period is suitable for ovaryinjection method and large ‘八’ characters stage is proper for the dropping method.5. Import the E.coli plasmid DNA into walnut pistillate flower using different methodsand conversion time and concentration of plasmid. The results showed that there are obviousdifferences between different import methods and plasmid concentration on the fruit settingrate The conversion rate of ovary injection is lower than the dropping method and achieved asignificant level. The result showed non-significant level between different conversion time.Higher level of plasmid concentration due to lower fruit setting rate. It is easy for importingplasmid DNA into embryo sac using the cut stigma dropping method.6. Import the agrobacterium LBA4404carried vector pCAMBIA3301into walnutpistillate flower using different methods. The result indicated the difference of the receptorcontemporary seed rate was remarkable between different methods. The seed rate of ovaryinjection method was lower than that of dropping method and achieved a significant level. Itcould ensure the seed rate using cut stigma dropping method.7. The determination of gus gene using the histochemical GUS assay of fruitlet fromtransgenic walnut. The untransformed tissue growth was restrained, gradually browning inmedium containing kanamycin100mg/L, while the transformed organization would not beaffected. Different PPT concentration on the rate of phytotoxicity achieved a significantlevel.40mg/L PPT concentration is better to use determing bar gene in3-month geneticwalnut-tree. The untransformed tissue growth was restrained, gradually browning in mediumcontaining kanamycin100mg/L, while the transformed organization would not be affected. 8. Increasing FeSO4·7H2O concentration in DKW medium properly can reduce the yellowphenomenon of leaves on micro-branches. It can be effectively restored the growth potential ofmicro-branches by removing the tops, micro-branches degradation characteristics.