Study On Transgenic Technology Of Pollen-Mediated By Magnetic Nanoparticles In Tree Peony

The genetic background of tree peony is complex and the cross breeding cycle is long that it takes more than 10 years to develop a new breed.Molecular breeding including transgenic breeding will be the main development direction of genetic improvement of tree peony.While the regeneration of tree peony tissue which no mature genetic transformation technology system has been established so far is difficult.In consequence,it is of great significance for tree peony to explore a simple fast and efficient method of genetic transformation to avoid tissue culture.We studied the pollen-mediated transgene of tree peony for the first time based on the transformation technology of Fe₃O₄magnetic nanoparticles and chose the magnetic nanoparticles poly MAG100 as gene carrier to construct MNPs/DNA complexes with different mass ratios.The ability of poly MAG100 binding to DNA and protecting DNA from digestive enzyme was analyzed by agarose gel electrophoresis.Under the magnetic field,we introduced the FT-EPSPS gene into tree peony pollen that use MNPs to adsorpt plasmid DNA and studied the effect of magnetic transfection on pollen viability by vitro culture of pollen.The sensitivity of FI seedlings of the three hybrid combinations of tree peony to glyphosate solution and the resistance to hygromycin solution was studied to explore the suitable screening system of tree peony.Exogenous gene molecular detection was performed on the positive seedlings and analyzed its integration in the genome.The conclusions are as follows:1 This study confirmed that poly MAG100 which is an ideal gene carrier can bind to DNA stably and protect DNA from digestive enzyme when the mass ratio of poly MAG100 to DNA is 1:1-1:10.2 The pollen germination rate of P.ostii‘Fengdan',P.ostii‘Jinghong'and P.rockii maintained above50%which had the same germination power as untreated pollens after the magnetic transfection.It showed that magnetic transfection did not cause significant damage to the pollens.3 The extent of damage to plant leaves was increased with the increase of glyphosate solution concentration.All the weed in the treated plot died,but the leaves of tree peony were only dry at the tip when glyphosate solution concentration ratio was in 1:100-1:25.The phenomenon of leaf desiccation and yellowing was obvious which is easy to be distinguished when glyphosate solution concentration ratio was going to 1:5.4 We screened 22 transgenic of PCR positive seedlings by using molecular detection of resistant plants without significant leaf color response after the screened by hygromycin solution of 2200mg/ml.This experiment adopted a new research method of pollen-mediated tree peony transgene.In the follow-up research work,CRISPR-Cas9 technology can be comprehensively used to further improve the study,so as to accelerate the cultivation of even more excellent varieties and promote molecular breeding of tree peony.