Cultivate Maker-Free Transgenic Rice Of Zhonghua 11 And N5088S With Bt Gene By Agrobacterium-Mediated Method

Insect damage is the major cause of yield loss. Lots of studies showed that there are no resources in rice for resisting rice lepidopteran insect species (yellow stem borer and striped stem borer, et al.). For a long time, people used chemical insectcide to control the damage of insect pest. However, chemical insectcide is not completely efficient because stem borer larvae remain only for a short time on the outer surface of the rice plant before they penetrate the stem.One efficient method for controling these insects damage is to develop genetic modified (GM) insect-resistant rice cultivars. Bt crops can efficiently reduced the use of chemical insecticides. It not only cut down the cost for the growers, but also is friendly to environment and human health.World-wide growing of transgenic crops brought about the issue of biosafety. The environmental safety of transgenic crops is a very important and subtle aspect. One of the most important problems is that all the transgenic plants almost contain the selectable maker genes such as antibiotic and herbicide resistance. Maker-free is a rapidly developing method which can eliminate the safety concern of transgenic food.In this study we utilized the method of mixture of A. tumefaciens strains (two-strain /two-plasmid) to obtain marker-free N5088S and zhonghua 11 transgenic rice with cry1C^* gene. We have generated marker-free transgengnic rice with high insect resistance. The main results were as follows:1 A high efficient marker-free expression vector pC130 harboring with cry1C^* have been constucted. The construt and PCAMBIA1300 were co-transformed into the rice calli by the concentration of 3:1. 865 N5088 independent transformants and 178 ZH11 independent transformants were obtained respectively.2 PCR analysis for cry1C^* showed that 99 N5088S and 11 ZH11 were positive cry1C^* transgenic plants.3 Based on the field performance, 40 T₀ trangenic plants of N5088S and ZH11 were selected, the mian agronomic traits of which had no significant difference with wild type plants.4 PCR analysis of the selected T₁ lines showed LZ52-21 was marker-free plant deriving from ZH11.5 Negtive selection and Southern blot analysis validated LZ52-21 was a marker-free plant. 6 The Bt protein concentration of four cry1C^* single-copy T₂ lines was quantified by ELASA assay. The results showed that Bt toxin protein cry1C^* in the LN22-21 transgenic line is at the level as 0.82±0.35μg/g tissue weight, LN24-9 transgenic line at the level as 0.90±0.1μg/gt, LZ52-21 at the level as 0.78±0.36μg/g, and LZ62-4 at the level as 0.32±0.23μg/g.7 Three T₁ plants with no significant phenotypic changes were selected from three cry1C^* single-copy T₀ plants respectively. The seeds of the three T₁ plants and LN52-21 were reproduced in Hainan.8 Through PCR analysis of the selected T₂ lines with hph and cry1C^* and agronomic traits selection, We obtained 19, 21, 14, 24 marker-free and enhanced insect-resistance transgenic plants from 4 target lines respectively9 The major morphological and agronomic traits of 4 Bt lines were tested in the field, and they all showed variation at some extent by compared with control.