Background
The yield and quality of rice have been suffering the damages by lepidoptern pests in the most rice-producing countries in the world. Especially in the recent years, the major pests such as stemborers and leaffolders are damaging the rice more and more severely. For a long time, the methods to control the pests depended on poisonous chemical insecticides in large amounts, which would bring many negative effects such as the pollution to the environment, health hazard to human and animals, the increasing product costs and so on. During the last two decades, transgenic approaches that to introduce insecticidal crystal protein genes from Bacillus thuringiensis (Bt) into plants have got great development. Bt toxin protein is known as biopesticide to control the pests efficiently. First, the protoxins could identify and bind steadily to specific receptors of midgut after the solubilization under the alkaline conditions in the gut (Hofmann et al., 1988a; Hofmann et al., 1988b; van Rie et al., 1989; van Rie et al., 1990). Then, some parts of the toxins could induce the ion channels or nonspecific pores in the target apical membrane by the help of the receptors and specific proteases (Knowles, 1994; Schwartz et al., 1997; Gazit et al., 1998; Schnepf et al., 1998). Finally, the target pests would be killed because of the decomposition of the midgut membrane by the disruption of ionic equilibrium or ionic equilibrium. No doubt, using Bt crops could sharply not only decrease the amounts of poisonous chemical insecticides spray which would bring great burden to the environment and health hazard, but also lower the costs of planting. Therefore, Bt crops would greatly help us to efficiently control the rice lepidopteran pests and build a harmonious relationship between people and the environment.

As one class of Bt genes, crystal protein gene (cry) are used currently and widely in developing transgenic crops to control lepidopteran pests. There are threes classes of cry genes (cry1Ac, cry1C* and cry2A*) employed in this study, and there are also some related reports about the application of these three cry genes which could give their severe toxicity to the main lepidopteran pests. Taking the rice for an example, as the first generation of Bt gene, cry1Ac got good performance at the toxicity to the target pests, the contents of toxin protein got 11.09±0.35 µg/g leaf fresh weight, and possessed an overall soluble protein content of 0.02%. While cry2A* and cry1C* are considered as the second generation of Bt gene, the contents of their toxin protein separately reached 84.94±2.34 and 1.46±0.12 μg/g leaf fresh weight(Tang et al., 2006; Chen et al., 2005). They not only have severe toxicity to the target pests, but also have better target specificity to the pests without expressing high dose of the toxin protein, which would lower the burden of plant. Therefore, these Bt genes mentioned above have got excellent performances to the resistance. What’s more, the expression of the Bt toxin has extreme specificity in plants, only in the leaves and stems could the toxin protein be efficiently expressed, while in the seeds, the expression of Bt protein nearly could not be found even that using real-time to detect them. So the above three Bt genes are safe to be used in the application in the future.

In the 1950s, Bt insecticides as biopesticides became popular in America (Martin and Travers, 1989), and there were 182 sorts of Bt reagents which were registered in US Environmental Protection Agency (EPA) until 1995. In 1987, the first Bt transgenic plant was reported (Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). In 1995, the Bt transgenic plants got the commercialization for the first time in Canada. In 2004, the global area of Bt crops got 2240 hectare(James, 2004). Huazhong Agricultural University got the first safety certificate of cry1Ab/c transgenic crops, Huahui No.1 and Bt Shanyou 63. In this study, the three genes,cry1Ac, cry1C* and cry2A*, were introgressed to 9311 and Fuhui 838 from the donor parents respectively by molecular maker selection approach in order to supply the useful materials and potential theory basis for the application of Bt crops in the future.

1 Results
1.1 Homozygous lines identified by marker-assisted selection
All the three Bt genes were incorporated with bar genes which could be used as weedicide. The designed markers for homozygous line selection are dominant in this study. Therefore, both marker-assisted selection and assay of weedicide were used to get the homozygous lines. Only when all the 24 plants were positive by PCR detection and were resistant to the weedicide could we take them as one homozygous line. In this study, more than two homozygous lines with each Bt gene were found by the above ways. The part PCR detection figures of homozygous lines are as follows (Figure 1).

Figure 1 PCR detection results of cry1C*, cry2A* and cry 1Ac homozygous lines

1.2 Field resistance of the improved lines to lepidopteron insects
It is clear from table 1 that the most of the improved 9311 and Fuhui 838 lines which separately have cry1Ac, cry1C* and cry2A* genes got excellent resistance to the target leaffolders, and only 9311 (cry2A*) got a little damage. Especially in the improved lines Fuhui 838 (LX111), Fuhui 838 (LX183) and Fuhui 838 (LX124), there were no damaged situations in the whole growth period. The above results bring into correspondence with the reports (Tang et al., 2006; Chen et al., 2005).While, the controls suffered some damage about 10 percents by the leaffolders, though the natural pests were not too much in Wuhan of centered China in 2009.

Table 1 The damage investigation of leaf folders in the different lines (natural infestation in Wuhan, China, 2009)

1.3 The agronomic performance of the selected homozygous lines
The improved homozygous lines got good performance in the agronomic traits, and many improved lines have more advantages than the negative controls in the different agronomic traits, such as LX83(Fuhui 838/1C* ), the line got some improvements comparing to the controls in yield related traits, such as grains per plant, yield per plant, grains per tillers (Table 2).

Table 2 Comparison of agronomic traits of the different improved lines under field conditions (Wuhan, China, 2009)

2 Discussion
Rice germplasm 9311 and Fuhui 838 are two elite parent lines widely used for two and three-line indica hybrid rice in China respectively; both of them have excellent combining ability. While the lepidopteran pests are causing increasing damages to rice, which have negative effects to their performance. In this study, three Bt genes, cry1Ac, cry1C* and cry2A*, were introgressed into 9311 and Fuhui 838. Some new improved lines which both had excellent resistance to the target pests and good agronomic traits were obtained by marker-assist selection and pheotyping assays.

It is obvious from assays of insect-resistant in the field that the resistance of the improved lines to the target pests got great improvement after the three Bt genes were integrated into the plants. The lines with cry1Ac and cry1C* had strong resistance, whereas, the lines with cry2A* got a little damage which would not bring any obvious negative effects. The above results show no differences with the previous reports (Tang et al., 2006; Chen et al., 2005). Therefore, the three Bt genes could be used in the future application for Bt hybrid rice.

Regarding the agronomic performance, the basic agronomic traits did not show any differences with the controls, 9311 and Fuhui 838. In the natural infestation, the agronomic traits of many improved lines had some advantages comparing to the negative controls, but some lines did not show any better performance in many agronomic traits. As this result, we confer that there are two followed reasons. One is that, the weather in Wuhan, 2009 was cooler than normal years, so the natural infestation was not severe in Wuhan, 2009; The other is that the main harm period of the leaffolders was after the heading stages, and many agronomic traits, especially the yield related traits, showed no obvious differences between the improved lines and negative controls.

This study is aim to evaluate and apply the three Bt genes which were constructed by the National Key Laboratory of Crop Genetic Improvement. At the same time, it could increase the efficiency of breeding by marker-assist selection. In 2009, the group of Qifa Zhang brought up the strategy of super green rice, which requires that on the premise of continued yield increase and quality improvement, Green Super Rice should possess resistances to multiple insects and diseases, high nutrient efficiency, and drought resistance, promising to greatly reduce the consumption of pesticides, chemical fertilizers, and water (Zhang, 2009). This study is the first step to realize the strategy of super green rice, which exploits the resistant genes to improve resistances of rice to the target insects by transgenic techniques and marker-assist selection. In addition, Huazhong Agricultural University got the first GMO safety certificates of cry1Ab/c transgenic plants, Huahui No.1 and Bt Shanyou63. cry1Ab and cry1Ac are known as the first generation Bt genes, and cry1C* and cry2A* are considered as the second generation Bt genes which have better resistance and specificity to the target insects. Therefore, we believe that the improved lines in the study would supply many useful and promising materials in the future for Bt hybrid rice breeding programF.

3 Materials and methods
3.1 Materials of donor and receptor parents
In this study, the donor parents are Minghui63 (cry1Ac), Minghui63 (cry1C*) and Minghui63 (cry2A*) which has already been constructed by National Key Laboratory of Crop Genetic Improvement (Tang et al., 2006; Chen et al., 2005). The receptor parents are excellent hybrid lines in China, 9311 and Fuhui 838, which are introduced separately from Lixiahe Research Institute of Agriculture Sciences and Sichuan sc-nuclear technique institute.

3.2 Technical route by maker-assist selection
Bt genes were introgressed into 9311 and Fuhui 838 by crossing them with the donor Minghui63 (cry1Ac), Minghui63 (cry1C*) and Minghui63 (cry2A*), followed by three generation of backcrossing and one generation of selfing. In this scheme, the progeny of each backcross was detected for the presence of the Bt genes both by PCR detection and field assays for insect-resistant. Then, the selected individuals were self-pollinated to produce homozygote for each of the three Bt genes, thus completing the whole breeding procedure.

3.3 Cultivation in the field 
The selected Bt lines which followed by three generation of backcrossing and one generation of selfing randomly planted in the Experimental Farm of Huazhong Agricultural University, Wuhan, China, in the summer of 2009, all are 80 lines. Each line planted 24 individual, and the distance between plants within a row was 16.5 cm and the rows were 39.6 cm apart. During the whole growth period, there were no any insecticides spray in the experiment field.

3.4 Marker-assisted selection
The primers for three different Bt genes are as follows:
cry1C*-F (5’-ttctactggggaggacatcg-3’); cry1C*-R (5’-cggtatctttgggtgattgg-5’).
cry2A*-F (5’-cgtgtcaatgctgacctgat-3’); cry2A*-R (5’-gatgccggacaggatgtagt-5’).
cry1Ac-F (5’-tcgagacgttagcgtgtttg-3’); cry1Ac-R (5’-gaggaaaggtaaactcgggc-5’). 

All the three primers are dominant makers which could only detect the positive plants (Tang et al., 2006; Chen et al., 2005).

PCR selection and agarose gel electrophoresis: Total cellular DNA of the plants was isolated from fresh leaf tissue by the methods of CTAB (Murray and Thompson, 1980).The PCR reaction of all the three Bt gene was the same and carried out as follows: one cycle at 94℃ for 50 s; one cycle at 57℃ for 50 s, one cycle at 72℃ for 55 s, the above steps run for 32 cycles, followed by extension at 72℃ for 7 min. The products were then checked by agarose gel electrophoresis (3%, 250 V, 30 min).

3.5 Assays of insect-resistant and the agronomic performance
Assays of insect-resistant: To assay the insect resistance of the improved lines and controls to the leaffolders in the natural field in Wuhan, 2009, and investigate all the 24 plants of each line.
Assays of agronomic performance: The agronomic traits of the improved lines which were homozygous were measured, and five plants were chosen randomly in each line and investigated plant height, heading stages, tillers per plant, grains per plant, 1000-grain weight, yield per plant, spikelet fertility, and grains per tillers. 

Authors’ contributions
LX conducted the major part of this study including experimental design, maker assisted selection, and manuscript preparation. YZ contributed the donor materials of Minghui 63 with genes of cry1C*, cry1Ac and cry2A*. GGJ’s work is field management. LYJ is the person who have the patents of the genes of cry1C*,and cry2A. Both ZXP and YJY are help to do marker selection. HYQ’s contribute are experimental design and manuscript preparation. All authors read and approved the final manuscript.

Acknowledgements
This study was jointly supported by a grant from the National Natural Science Foundation of China, a grant from the National Program of High Technology Development of China, and a grant from the National Program on Research and Development of Transgenic Plants.

References
Barton K.A., Whiteley H.R., and Yang N.S., 1987, Bacillus thuringiensis δ-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects, Plant Physiol., 85: 1103-1109 doi:10.1104/pp.85.4.1103

Chen H., Tang W., Xu C.G, Li X.H., Lin Y.J., and Zhang Q., 2005, Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against rice lepidopteran pests, Theor. Appl. Genet., 111: 1330-1337 doi:10.1007/s00122-005-0062-8

Fischhoff D.A., Bowdish K.S., Perlak F.J., Marrone P.G., McCoormick S.M., Niedermeyer J.G., Dean D.A., Kusano K.K., Mayer E.J., Rochester D.E., Rogers S.G., and Fraley R.T., 1987, Insect tolerant transgenic tomato plants, BioTechnology, 5: 807-813 doi:10.1038/nbt0887-807

Gazit E., la Rocca P., Sansom M.S.P., and Shai Y., 1998, The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis δ-endotoxin are consistent with an “unbrella-like” structure of the pore, Proc. Natl. Acad. Sci. USA, 95:12289-12294 doi:10.1073/pnas.95.21.12289

Hofmann C., Luthy P., Hutter R., and Pliska V., 1988b, Bingding of the delta-edotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae), Eur. J. Biochem., 173:85-91 doi:10.1111/j.1432-1033.1988.tb13970.x

Hofmann C., Vanderbruggen H., Hofte H., Van Rie J., Jansens S., and Van Mellaert H., 1988a, Sepcificity of Bacillus thuringensis δ-endotoxins is correlated with the presence of high-affinity binding sites in brush-border membrane of target insect midgets, Proc. Natl. Acad. Sci., USA, 85: 7844-7848 doi:10.1073/pnas.85.21.7844

James C., 2005, Global status of commercialized biotech/GM Crops: ISAAA

Knowles B.H., 1994, Mechanism of action of Bacillus thuringiensis insecticidal proteins, Adv Insect Physiol, 24: 275-308 doi:10.1016/S0065-2806(08)60085-5

Martin P.A.W., and Travers T.S., 1989, Worldwide abundance and distribution of BT isolates, Appl. Environ. Microbiol., 55: 2437-2442

Murray M.G., and Thompson W.F., 1980, Rapid isolation of high molecular weight plant DNA, Nucleic. Acids. Res., 8:4321-4325 doi:10.1093/nar/8.19.4321

Schwartz J.L., Juteau M., Grochulski P., Cygler M., Prefontaine G., Brousseau R., and Masson L., 1997, Restriction of intramolecular movements within the Cry1Aa toxin molecules of Bacillus thuringensis through disulfide bod engineering, FEBS Lett., 410: 397-402 doi:10.1016/S0014-5793(97)00626-1

Schnepf E., Crickmore N., Van Rie J., Lereclus D., baum J., Feitelson J., Zeigler D.R., and Dean D.H., 1998, Bacillus thuringiensis and its pesticidal crystal protein, Microbial Mol. Biol. Rev., 62: 775-806

Vaeck M., Reynaerts A., Höfte H., Jansens S., Beukeleer M.D., Dean C., Zabeau M., Montagu M.V., and Leemans J., 1987, Transgenic plants protected from insect attack, Nature, 328:33-37 doi:10.1038/328033a0

Van Rie J., Jansens S., Hofte H., Degheele D., and Van Mellaert H., 1989, Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects, Eur. J. Biochem., 186: 239-247 doi:10.1111/j.1432-1033.1989.tb15201.x

Van Rie J., McGaughey W.H., Johnson D.E., Barnett B.D., and Van Mellaert H., 1990, Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis, Science, 247: 72-74 doi:10.1126/science.2294593

Tang W., Chen H., Xu C.G., Li X.H., Lin Y.J., and Zhang Q.F., 2006, Development of insect-resistant transgenic indica rice witha synthetic cry1C* gene, Mol. Breeding, 13: 301-312

Zhang Q.F., 2007, Strategies for developing Green Super Rice, Proc. Natl. Acad. Sci., USA, 104: 16402-16409 doi:10.1073/pnas.0708013104