Establishment Of Vector-free And Marker-free Transformation System On Maize And Breeding Of Transgenic Salt-tolerance Lines

It has been of great concern that transgenic plants could effect food and environment safety. In this study, we established a vector-free and marker-free transformation system, the minimum linear exogenous gene was introduced into maize by pollen-tube pathway method, and it mainly solved the biosatety issue of transgenic plants.To achieve the vector-free and marker-free transformation, the BADH gene, which was cloned from Suaeda liaotungensis Kitag, was introduced into maize by pollen-tube pathway transformation method, using the principle of minimum linear length of the transformation cassette, which was composed of only the functional gene, expression regulatory sequence (35S CAMV promoter, NOS terminator), and T-DNA border sequence at both sides. We explored the feasibility of establishing a vector-free and marker-free transformation system in the study. Twenty-seven of 2076 transformed samples were positive in PCR amplification and the PCR positive rate of T₁ generation was 1.3ï¼…. Parts of positive samples were examined with further Southern blotting and RT-PCR detection, the results indicated that BADH gene was integrated into maize genome in three transgenic lines (Shen 137-19, Shen 137-20 and Shen 137-21) and expressed.The progeny plants of the three transgenic lines were examined for tolerance to salt by induced salt stress with diluted artificial seawater (NaCl content is 1.25ï¼…). After two-week treatment, 62.5ï¼…seedlings of the Shen 137-19 transgenic line survived and grew evenly, while most wild-type seedlings wilted and showed loss of chlorophyll after stress. Only 8.0ï¼…of the wild-type plants survived after salt stress. So the plants of this transgenic line were selected as the materials for the following research. The survival transgenic plants after two-week salt stress were examined with PCR detection. It has been confirmed that exogenous BADH gene could be inherited into progeny plants. Parts of positive transgenic progeny were examined with further systematic physiological detection. The results showed that the glycinebetaine concentration in transgenic progeny was 15.7-19.5μg g^-1 fresh weight under salt stress. Transgenic maize plants accumulated 15-to 19-fold more glycinebetaine in their leaves than wild type plants; the transgenic progeny had higher relative water content than wild type; and the activity of SOD of transgenic progeny was 1.2-to 1.5-fold higher than that of wild type. The activity of POD of transgenic progeny was 1.4-to 1.9-fold higher than that of wild type; the relative electrical conductivity of the transgenic was only 69-71ï¼…of that in the wild type under salt stress; and the transgenic progeny had much lower MDA content than wild type. It indicated that the cell membrane of transgenic could be protected well under salt stress; Transgenic progeny had 1.12- to 1.16-fold more chlorophyll content in their leaves than wild type plants; The photosynthetic rate of transgenic progeny was 22.2-29.5μmolCO₂m^-2s^-1 with salt stress, but that of wild type was only 18.1μmolCO₂m^-2s^-1; The transpiration rate of the transgenic progeny was lower than wild type, and the difference was significant. It showed that the photosystem of the transgenic progeny was much more stable than wild type. The results of physiological detection indicated that BADH gene was expressed in the transgenic progeny in field salt-stress trail. It also laid a foundation for the breeding of BADH transgenic homozygous lines.Another key mechanism of plant salt tolerance is ionic compartmentalization in vacuole by antiporter. Na⁺/H⁺ antiporter gene (AlNHX1) was transformed into maize by pollen-tube pathway through the vector-free and marker-free plant transformation system. The results of PCR and Southern blot detection indicated that AlNHX1 was integrated into maize genome. The feasibility of vector-free and marker-free transformation system established in the study was further confirmed.To increase the transformation rate, we brought up a new plant transformation method on the basis of pollen-tube pathway transformation-ovary dipping method. This method had been preliminarily researched in this study.In this research, the minimum linear vector-free and marker-free BADH gene was introduced into maize by pollen-tube pathway method. The biosafety of two BADH transgenic lines had been confirmed by Biosafety Management Office of Agricultural Transgenic of People's Republic of China, and the pilot-scale production is in process.