Marker-free Transgenic Insect-resistance Indica Rice Obtained By The Vector Carrying Two Separate T-DNAs

The selection of transgenic cell is a key technique in the plant genetic transformation.The acquisition of transgenic plants requires the use of selectable marker genes. But selectable marker genes are usually unnecessary once transgenic plants are produced. The marker genes in established transgenic plant are not only unnecessary, but they can also create problems. First, the safety of marker genes must be considered. Second, a different selectable marker gene must be used when additional foreign genes are introduced into a transgenic plants, but the number of available marker genes is limited. Thus the demand for marker free transgenic plants is one of the most popular tendency.Several techniques to eliminate marker genes have been developed. The co-transformation system appear to be the simplest among them. In this system, a marker gene and genes of interest are placed on separate transfer DNA (T-DNA), and the T-DNAs are introduced into plant genomes as unlinked fragments. Consequently, the non-selectable genes separate from the marker genes in the progeny.The objective the present study was to develop a super-binary vector system suitable for the co-transformation of Indica Rice. This system involved unique plasmid that carried two T-DNA segments, one T-DNA carried sck gene, the other carried hpt gene. 2788 embryogenic calli derived from immature seeds of minghui86 were co-cultivated with Agrobacterium tumefaciens. 261 Hyg resistant calli were obtained with the frequency of 9.36%. 214 Hyg-resistant calli obtained transgenic plants from 261 Hyg-resistant calli which had the percentage of 81.99% in the total Hyg-resistant calli.It was indicated that 39 Hyg-resistant calli occurred co-transformation in 61 Hyg-resistant calli. The percentage of co-transformation with hpt and sck were 63.93%. 166 marker free transgenic plants were obtained by PCR from 13 of lines which developed from TO plants. The marker free transgenic plants had thepercentage of 17.46% in the 951 TI plants stemmed from 13 lines.We had obtained 15 lines from 67 lines of marker free transgenic plants which had the complete positive reaction by PCR. MH24-A-12,24,38,53,55,63,78; MH17-H-6,45,71; MH18-D-87; MH08-H-10,14,49,60 were confirmed the homozygous marker free transgenic plants. Three plants had been further confirmed in TS lines of their Progenies.Transgenic lines were highly resistant to the stem borers(Chilo suppressalis) in the field test and in the detached-leaf bioassay. T0 plant which had the interested gene showed good resistance to the stem borers besides a few of susceptible plants in the field test. The effect of insect resistant of transgenic plants was slow. The feeding amounts of stem borers were significantly lower than those on the check variety (non sck transgenic) lines in six days, the mortalities in the sixth day after inoculation were up to 100%. A high resistant level had been reached in the eight days.