Optimization Of Vector Backbone-free And Selectable Marker-free Transformation System For Maize (Zea Mays L.)

The rapid development of transgenic technology in agroculture has given rise to various transgenic crops and their products,which have an important role in tackling the problems currently facing human being such as the crisis of environmental pollution,food shortage and population explosion.However,Genetically Modified(GM) crops have caused biosafe issues, which have restricted progress in large scale production of transgenic crops.The issue of transgenic biosafety particularly concerns the unexpected problem of biosafety caused by vector backbone sequence and marker genes.Therefore,generation of vector backbone-free and selectable maker-free transgenic plants is an important advantage in both plant biotechnology research and commercial development of GM crops.At present,the pollen-tube pathway could be used to introduce linear gene cassettes into the genome of plants and directly generate vector backbone-free and selectable maker-free transgenic lines. However,this method still has its limitation due to its low transformation frequency and reproducibility.Maize(Zea mays L.) is one of the most important cereal crops and is used world wide for human and animal consumption.In this study,the ovary-drip method for directly generating vector backbone-free and selectable marker-free transgenic plants was described,in which a linear gene cassette(Promoter-Gene-Terminator) flanked by 25bp T-DNA borders was transformed into maize.The key features of this method centered on the complete removal of the styles,and the subsequent application of a DNA solution directly to the ovaries.Based on the preliminary experiment of observing the growth of pollen tubes,transformation was performed at least 16h after artificial pollination was initiated.The movement of the exogenous DNA was monitored using FITC(fluorescein isothiocyanate) labeled-DNA,which showed that the time taken by the exogenous DNA to enter the ovaries was shortened, compared to that of the pollen-tube pathway.The 0.05%surfactant Silwet L-77+5%sucrose used as a transformation solution played an important role in transporting DNA molecules into the ovules.PCR analysis showed that the most suitable maize variety was 9818,the optimal transformation time was 18-20h after artificial pollination and the optimum transformation solution was 0.05%Silwet L-77 + 5%sucrose.Under these conditions,the positive transformation frequency as obtained by PCR was 6.47%.We further analyzed the integration pattern,copy number,expression,and inheritance of the transgene.Southern blot analysis showed that transgenic plants had low transgene copy number and simple integration patterns(1-3 hybridization bands on Southern blot).Northern blot analysis showed that the GFP gene was correctly expressed in the transgenic plants. Green fluorescence was observed in roots and immature embryos of transgenic plants by fluorescence microscopy.Progeny analysis showed that GFP insertions were inherited in T₂ generation with normal expression.Both Mendelian and non-Mendelian inheritances were observed in T₁ progenies of transgnic maize,and non-Mendelian inheritances were observed in T₂ progenies.To validate the feasibility and reproducibility of the ovary-drip method,the linear GUS gene cassette that was constructed from GUS expression cassette flanked by T-DNA borders or LKR/SDH(lysine-ketoglutarate reductase/saccharopine dehydrogenase) gene sequences as borders(5' LKR/SDH gene sequences - 35S CaMV -GUS- nos -3' LKR/SDH gene sequences) was transferred into maize genome.The positive transformation frequency as obtained by PCR was 7.2%and 11.5%.The results showed that the addition of LKR/SDH gene sequences at both ends of the linear gene cassettes improved the transformation frequency of the ovary-drip method.Besides,the ovary-drip method provided better reproducibility and higher transformation frequency than that obtained with existing pollen-tube pathway method.The integration,expression and inheritance of the linear LKR/SDH-GUS gene cassette were also analyzed.Southern blot analysis showed that transgenic plants had simple integration pattern (1-2 hybridization bands on Southern blot).RT-PCR analysis showed that the GUS gene was expressed in the transgenic plants.Histochemical staining showed that GUS was expressed in roots,leaves and immature embryos of transgenic plants.Further progenies of 4 transgenic lines were identified by histochemieal staining and PCR analysis.The results showed that the GUS gene was stably inherited by the progenies in a Mendelian fashion.To optimize the vector backbone-free and seleetable marker-free transformation system on maize,a linear GFP gene cassette flanked by 25bp T-DNA borders was transformed into maize via the pollen-tube pathway and a simple method to screen selectable marker-free transgenic plants was established.After 17-18h of artificial pollination,styles were cut flush with the top of the spike-stalk and linear GFP gene cassette solution were applied to the cut styles evenly using a micropipette.The forepart of each maize ear was divided into five segments(segmentsâ… toâ…¤) at an interval of two rows of kernels.The segments of maize ear that were most likely to contain transgenic kernels were then quickly identified by monitoring GFP expression in the immature embryos 10 days after transformation.Transgenic kernels were primarily identified in segmentsâ…¢andâ…£(located at 5-8 rows of kernels of the maize ears).The transgenic plants were screened from segmentsâ…¢andâ…£by visual detection of GFP and PCR analysis.Six out of 121 plants(4.96%) showed the presence of the GFP gene cassette.Southern blot analysis showed the transgenic plants had simple integration patterns. Northern blot analysis showed the GFP gene was correctly expressed in the transgenic plants.In this study,the ovary-drip method giving higher transformation frequency and reproducibility was established for maize.In addition,a simple method to screen selectable marker-free transgenic maize obtained from transformation with a linear GFP gene cassette via the pollen-tube pathway was also established.These two methods complemented each other and form the basis of a complete vector backbone-free and selectable marker-free transformation system for maize.The validity of the ovary- drip method of transformation has been established by a series of experimental study including integration pattern,copy number, gene expression,and progeny analysis.