Preliminary Exploration Of DNA-free Gene Editing System In Poplar Based On CRISPR/Cas

CRISPR/Cas has quickly become the preferred technology for genome editing because of its simplicity,efficiency and versatility.Plant genome editing mainly depends on transmission in the form of DNA vectors,which will introduce genes such as antibiotics into the genome.Because of the long period of forest tree breeding,it is difficult to separate foreign genes by self-crossing or hybridization.Therefore,it is very important to establish a non-transgenic gene editing system suitable for forest trees.In this study,the haploid Qu-1 cell line of Populus simonii × P.nigra was used as material to explore the non-transgenic gene editing system of poplar by means of protoplast transfection and particle bombardment.1.In order to establish DNA-free gene editing system by protoplast transformation,we established protoplast cell wall regeneration system,single cell callus regeneration system and Qu-1 cell line protoplast transient transformation system.By adjusting different carbon sources and plant hormones in the culture medium and counting cell division,the conditions of protoplast culture and cell wall regeneration of Qu-1 cell line were determined.By gradually reducing the osmotic pressure,the single cell restoring the cell wall was successfully induced to recover into active callus again.2.Through the comparison of the effects of different enzymatic hydrolysis methods and different enzymatic hydrolysis time,it was determined that the enzymatic hydrolysis method combined with low-speed mixing and vacuum treatment could effectively increase the protoplast yield,and the most suitable enzymatic hydrolysis time was 4 h.By regulating different osmotic pressures with double distilled water and mannitol,it was determined that the most suitable osmotic pressure for protoplasts of Qu-1 cell line was 0.4 M.The cells and impurities that could not be completely hydrolyzed could be effectively removed by purifying protoplasts with 21% sucrose solution.Through the observation of the conversion efficiency by transforming p UC19-GFP,it is determined that the best concentration of PEG is 40%,and the conversion efficiency is about 40%.3.The DNA-free gene editing system of haploid Qu-1 cell line of Populus tomentosa mediated by particle bombardment was established.By bombarding p UC19-GFP plasmid and counting the transfection efficiency under different conditions,the best condition of particle transfection by particle bombardment was determined.The gene editing vector p Eg P237-Psn PDS-2A-GFP of Phytoene desaturase(PDS)gene was bombarded,the albino buds of gene editing were obtained,and the system of gene bombardment transfection of DNA for haploid Qu-1 cell line of P.simonii × P.nigra was established.Bombarding GFP protein to determine the parameters of protein transfer into Qu-1 cell line without affecting protein activity.4.PDS was selected as the target gene,sg RNA was designed and transcribed in vitro,and the purified Cas9 protein was assembled into ribonucleoprotein complex(RNPs)in vitro.Its activity was verified by in vitro cutting experiment and protoplast transfection experiment.RNPs was encapsulated by gold powder and transferred into Qu-1 cell line by particle bombardment,and non-transgenic gene editing albino buds were obtained.In this study,the main steps of the CRISPR/Cas9RNPs-mediated non-transgenic gene editing system of haploid Qu-1 cell line of P.simonii × P.nigra include the preparation of RNP,functional verification of RNP,encapsulation and transmission of RNP,cell line induction and mutant identification.The whole experimental method has a cycle of 44-56 days.5.Through this method,other sg RNA in PDS gene and sg RNA in acetolactate synthase(ALS)gene were selected,and non-transgenic gene edited calli were also obtained,indicating that this method can be applied to the editing of other gene loci.To sum up,in this study,a non-transgenic gene editing method which does not involve foreign gene insertion and can cause target gene mutation was established in haploid Qu-1 cell line of P.simonii × P.nigra.Through the continuous improvement of this method,it will promote the application of gene editing technology in forest molecular breeding.