Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

Rice Science, 2009, 16(3): 194–200

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Copyright © 2009, China National Rice Research Institute. Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(08)60079-7

Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene GAO Fang-yuan1, 2, 3, LU Xian-jun1, 3, HE Shu-lin3, 4, CHEN Xiao-juan3, 4, LU Dai-hua3, 4, SUN Shu-xia1, 3, LI Zhi-hua1, 3, LIU Guang-chun1, 3, ZHANG Yi-zheng2, REN Guang-jun1, 3 (1Institute of Crop Sciences, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China; 2College of Life Sciences, Sichuan University, Chengdu 610041, China; 3Innovation Center of Rice Regional Technology, Ministry of Agriculture, Chengdu 610066, China; 4Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China)

Abstract: Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer (SSB) in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer (YSB) under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile (CMS) lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions. Key words: hybrid rice; restorer line; Bt gene; insect resistance; Magnaporthe oryzae; seed germination; pollen yield

Lepidoptera insects are the main pests in rice growing areas. Pests such as striped stem borer (SSB), Chilo suppressalis (Walker) and yellow stem borer (YSB), Scirpophaga incertulas (Walker) cause a loss of about 10% of the total rice output in Asia. However, extensive utilization of chemical pesticides not only raises production costs but also pollutes the ecological environment. Introducing foreign anti-insect genes by means of transgenic technique is currently one of the main approaches to control rice pests. Many studies have been done to introduce the foreign Bt gene into indica or japonica rice, and some transgenic rice lines with high resistance to snout moth larvae were subsequently obtained [2-5]. However, it has been reported that the agronomic traits of these plants in the transgenic T0 and the subsequent self-crossed generations were Received: 10 September 2008; Accepted: 26 December 2008 Corresponding author: REN Guang-jun ([email protected]) This is an English version of the paper published in Chinese in Chinese Journal of Rice Science, Vol. 22, No. 4, 2008, Pages 353–358.

universally worse than those of their original parents, which restricts the direct use of transgenic rice. However, taking the transgenic material as donor and crossing it with some indica or japonica rice with good agronomic traits could improve the grain quality and yield. It has been proved to be an effective way to breed new rice lines by combining superior agronomic traits and good resistance to pests [6-8]. In this study, a Bt transgenic rice line, Bt Minghui 63 was used as the donor to introduce the Bt gene into an indica rice restorer line Chenghui 177, which has been characterized to have good grain quality and high blast resistance [9]. Subsequently, a new indica rice restorer line with good insect and rice blast resistances was obtained. Meanwhile, the degrees of resistance to insect and rice blast, the degree of heterosis, the seed germination rate and the pollen yield were compared between the recipient parent and the new rice restorer line. We hope that the data should serve as a useful reference for breeding and utilization of the Bt rice.

GAO Fang-yuan, et al. Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

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Test paper detection

MATERIALS AND METHODS Plant materials and field experiments In 2001, an indica rice restorer line Chenghui 177 was selected as a female parent to cross with a transgenic rice restorer line Bt Minghui 63, whose selective marker gene had been removed [10] (Bt Minghui 63 was provided by the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, China). The resultant F1 plants were backcrossed with Chenghui 177 to produce BC1F1 and then were repetitively self-crossed to generate BC1F7. In this study, the pedigree method was used to choose the comprehensive agronomic traits. Test papers, described below, were used to detect Bt gene expression, and the nursery inoculation was taken to identify blast resistance. The plants of all generations were planted in a greenhouse at the Sichuan Academy of Agricultural Sciences (SAAS), Chengdu, China. By testcrossing with two cytoplasmic male sterile (CMS) lines G46A and 23A, we preliminarily identified some single plants, selected from the offspring of Chenghui 177 and Bt Minghui 63, for their combining ability, restorability and the heading date of F1s. In 2006, we succeeded in developing a new rice restorer line Bt5198 with good transgenic insect-resistance. Its restorability and yield heterosis were tested by four different CMS lines (G46A, Zhong 9A, D62A and Chuanxiang 29A). Identification of insect-resistance The insect-resistance of the new rice restorer line Bt5198 and its hybrid combinations were identified through PCR analysis, test paper detection, detachedculm bioassay and field inoculation. PCR analysis DNA was extracted from fresh leaves of rice materials according to a modified CTAB method [11]. Bt gene detection was carried out as Tu et al [4] described, and a 1.83-kb fragment of the Bt gene was amplified. The forward primer was BTF, 5ƍ-TTCAGCCTCGAG TGTTGCAG-3ƍ, and the reverse primer was BTR, 5ƍ-ATGGACAACAACTGCAGG-3ƍ.

Rice leaves were ground in a 1.5-mL centrifugal tube, then extracted and diluted with distilled water. One piece of BT-Cry1Ab/1Ac colloidal gold strip (provided by Beijing Silver Land Bio-technology Co., Ltd, China) was immerged into the sample solution, and observed 3–5 min later. On the test paper, a detection line and a quality control line were positive (indicating the expression of Bt gene, i.e. the presence of the Bt protein), recorded as ‘+’; and only one quality control line was negative (indicating the absence of Bt protein), recorded as ‘–’. Detached-culm bioassay Eggs of SSB sampled from the paddy fields of Chengdu, China were used for tests after incubation. A total of five plants were taken from each rice line. A 6-cm long section was cut from the lower culms of each plant, and 30 larvae of SSB were then put on each section using a fine writing brush. After that, the culm sections were placed at the bottom of a small glass tube (15 mm × 15 mm) with 2 mm clear water and then sealed with filter paper. Five days later, the culm sections were peeled for observation. The numbers of live and dead larvae were recorded, and the larval death rate was calculated. Field inoculation SSB and YSB eggs were collected from the fields of Chengdu and Deyang, Sichuan Province, China by using light to attract moth mothers. Six test blocks with three replications were set for SSB and YSB, respectively. Therefore, a total of 36 test blocks were designed in a randomized complete block design. Each test block had an area of 1.5 m2 (1.5 m long and 1.0 m wide), in which 30 plants were planted (6×5), and one egg block was allowed to incubate on each plant. Plant incubation tested with SSB eggs started after the rice plants had recovered from transplanting. After the plants suffered from dead hearts in late June, an investigation on the quantity of dead hearts was conducted. YSB egg incubations were conducted in the middle of July, and the quantities of white heads for all test areas were investigated in late August. The ratios of dead hearts and white heads were then

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calculated for both egg treatments. Identification of rice blast resistance Spray inoculation with mixed strains was conducted on rice plants at the 4- or 5-leaf stage. The leaf blast incidence was investigated at the middle and late tillering stages. A countercheck was manipulated one week later. The investigation on neck blast was carried out at the yellow ripening stage. A countercheck was also executed one week later. The recording and appraisal of the paddy disease was conformed to the Standard for Appraisal of Paddy Diseases and Insects, a guidebook for crops in China. Investigation of agronomic traits In April 2006, the new rice restorer line Bt5198 and its hybrid combinations were planted in the greenhouse of SAAS, Chengdu, China. The plants were transplanted in a randomized complete block design with three replications. The growth duration (days from sowing to heading) was recorded. At maturity, ten plants from each plot were harvested to evaluate their agronomic traits, including plant height, number of effective panicles per hill, panicle length, seed setting rate, 1000-grain weight and single plant yield. Germination test The germination ability of the new restorer line Bt5198 and the recipient parent Chenghui 177 were measured in two ways, namely, by indoor germination and direct seeding. For indoor germination, 100 mature filled seeds were soaked for 48 h before being incubated overnight at 35ºC for pre-germination, and then put onto two layers of wet filter paper in a Petri dish. The germination rate was counted on the 3rd and 7th days after the pre-germination. The test was replicated three times. In the direct seeding test, 100 mature filled seeds were directly sown on the seedbed without soaking and pre-germination. The germination rate was counted 10 days later. The test was also repeated three times. Evaluation of pollen yield Fifty main panicles of Bt5198 and Chenghui 177 were randomly sampled and cut into 2–3 mm long

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fragments. Five hundred anthers randomly collected from 200 florets were put into a centrifugal tube (1.5 mL), mixed with 100 μL of sucrose solution (1 mol/L), and pounded to pieces. In order to induce pollen release from anthers, 900 μL of sucrose solution (1 mol/L) was added, then 100 μL of the mixture was transferred to another 1.5-mL tube, followed by adding 900 μL of sucrose solution (1 mol/L). After completely mixing, 5 μL mixture was dropped onto a glass slide and dyed with I2-KI. The pollen quantity was counted with the help of a 10× microscope, and the pollen concentration was calculated. Each test was repeated three times.

RESULTS Breeding process of the new rice restorer line Bt5198 Chenghui 177, a restorer line developed by SAAS, Chengdu, China, is the progeny of a middle restorer line Mianhui 502 (containing multiple resistance genes, with high combining ability and wide adaptability) with an American commercial variety Lemont (having a wide compatibility and high light utilization efficiency) [9]. The male parent, Bt Minghui 63, was obtained in 2000 by introducing the Bt gene into Minghui 63 with the genetic transformation technology [4], and removing the selectable marker gene [10]. The Bt gene contained in the individuals of F1, BC1F1 and BC1F2 was detected through PCR analysis and test paper, and the test paper was only used to identify the Bt gene in BC1F3 and later generations. Furthermore, the insect-resistance was identified through artificial inoculation in both the laboratory and field. For BC1F2 and later generations, selection was emphasized on the single plant or lines with good leaf shape, good lodging resistance, medium or late maturity and high seed setting rate. Rice blast resistance was screened in the BC1F2 and BC1F4 as well. The testcrosses were conducted between a medium or high generation and CMS lines such as G46A and Jin 23A. Consequently, the superior restorer line Bt5198 integrating insect resistance, rice blast resistance and high restorability was obtained.

GAO Fang-yuan, et al. Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

Characteristic analysis of the new restorer line Bt5198 Insect resistance The Bt gene in Bt Minghui 63, Chenghui 177, F1, BC1F1 and BC1F2 was identified by PCR analysis, and the Bt protein was detected by test paper. A 1.83-kb fragment could be amplified in Bt Minghui 63 and F1 (Bt Minghui 63/Chenghui 177), but not in Chenghui 177. In addition, Bt Minghui 63 and F1 showed two bands (positive) on the test paper whereas Chenghui 177 showed only one band (negative) (Fig. 1-A, B). According to the F2 test of 48 BC1F1 plants, the positive and negative plants were segregated equally. Of 300 BC1F2 plants, the positive and negative plants had a ratio of 219:81, fitting to the segregation ratio of 3:1, which suggests that the foreign Bt gene is a single dominant gene and meets the Mendelian rule for gene segregation. Moreover, the results of PCR assay corresponded with those of the test paper. Therefore, the test paper method was selected to trace and detect the Bt gene from BC1F3 and later generations, due to its greater convenience compared with PCR analysis. From BC1F3, the self-crossed plants of each generation were artificially infested with the larvae of SSB and YSB in the laboratory and fields on the basis of test paper detection. The plants with 100% larval mortalities in the laboratory and without any dead hearts nor white heads in fields were further used for self-crossing. Combined with the analysis of combining

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ability, a new restorer line Bt5198, integrating insectresistance and high combining ability, was developed in BC1F7. In 2006, a detached-culm bioassay was performed to test the SSB-resistance of Bt Minghui 63, Chenghui 177, Bt5198 and the hybrid rice combinations from the two restorer lines and four CMS lines. The results showed that lots of frass and large larvae with strong activity were found in the culms of Chenghui 177, whereas frass was hardly found in the culms of Bt Minghui 63, Bt5198 and the hybrid rice combinations, and their larval mortalities were 100% (Table 1). These results indicate that Bt5198 and its combinations showed high resistance to SSB larvae under artificial inoculation in the laboratory. We infer that both homozygous and heterozygous Bt gene have improved the insect-resistance. Under the field infestation, the dead heart and the white head ratios of the control line Chenghui 177 were 17.5% and 18.2%, respectively, whereas those of Bt Minghui 63, Bt5198 and most of their combinations were zero, indicating a high resistance of the tested lines to SSB and YSB. Furthermore, very few plants of the combination Zhong 9A/Bt5198 suffered from dead heart and white head (Table 1). Rice blast resistance The generations showing good insect resistance in the preliminary selection (BC1F2 and BC1F4) were subjected to spray inoculation with the mixed strains

Fig. 1. PCR analysis of total genomic DNA (A) and test paper examination of Bt protein (B). M, DNA marker ladder (DL2000); Lane 1, Chenghui 177; Lane 2, Bt Minghui 63; Lane 3, F1 (Bt Minghui 63/Chenghui 177).

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Table 1. Resistance reactions of the new rice restorer line Bt5198 and its hybrid rice combinations to C. suppressalis and S. incertulas. Material Bt Minghui 63 Chenghui 177 Bt5198 G46A/Bt5198 D62A/Bt5198 Zhong 9A/Bt5198 Chuanxiang 29A/Bt5198

Test paper examination (+:–) a 20:0 020 200 200 200 200 200

Dead heart rate b (%) 0.0 (11.6) 17.5 (10.5) 0.0 (11.6) 0.0 (10.5) 0.0 (9.8) 0.0 (11.3) 0.0 (10.9)

Dead larvae rate (%) 100 12 100 100 100 100 100

White head rate c (%) 0.0 (9.4) 18.2 (9.4) 0.0 (9.4) 0.0 (9.2) 0.0 (9.2) 1.2 (8.3) 0.0 (9.3)

a

Test paper examination based on 20 individual plants. Numbers of rice tillers and dead hearts recorded on 30 June, 2006. Dead heart rate = Number of dead hearts/Number of tillers per plant×100%. Data in brackets are the average number of tillers per plant in 90 plants in three replications. c Numbers of rice panicles and white heads recorded on 20 August, 2006. White head rate = Number of white heads/Number of panicles per plant × 100%. Data in brackets are the average numbers of panicles per plant in 90 plants in three replications. b

at the seedling stage, taking Bt Minghui 63 and Chenghui 177 as controls. The lines with leaf blast and neck blast less than or equal to grade 4 after three years identification were selected for further testing of their combining abilities. A new restorer line, Bt5198, which integrated rice blast resistance, insect resistance and high combining ability, was obtained from the generation BC1F7. The identification of rice blast resistance in 2005 and 2006 showed that the rice blast resistance of Bt5198 was similar with that of Chenghui 177, but better than that of Bt Minghui 63 (Table 2). Heterosis of the combinations The new restorer line Bt5198 showed quite good restorability. The seed setting rates of its combinations with the four CMS lines ranged from 76.2% to 81.2% with an average of 78.6%, which was similar to that of II You 838 (CK) (Table 3). The average single plant yields of the four combinations were similar to or better than Table 2. Resistance reactions of the new restorer line Bt5198 to M. oryzae. Material

Leaf blast (Grade)

Neck blast (Grade)

2005

2006

2005

2006

Bt Minghui 63

9

9

9

9

Chenghui 177

4

3

3

2

Bt5198

2

3

3

3

that of the control (II You 838), suggesting that the Bt gene had no negative impact upon restorability and heterosis. As for days from sowing to heading, the combinations of Bt5198 with G46A, Zhong 9A and D62A were similar to that of the control. The heading date of the combination Chuanxiang 29A/ Bt5198 was 5.5 days later than that of the control. Chuanxiang 29A/Bt5198 had a very small yield increase due to its later heading date, inadequate light and relatively low temperature during the grain filling stage in Chengdu. However, it is possible for this combination to realize a higher yield in the paddy fields at the middle and lower reaches of the Changjiang River, China. Other characteristics Germination ability is one of the most important criteria for seed vitality. Under the appropriate laboratory conditions, the germination rates of Bt5198 after 3 and 7 days soaking were 95.3% and 95.7%, respectively, which suggests that the restorer line possessed great seed vitality as Chenghui 177. Under direct seeding cultivation, the germination rates of Bt5198 and Chenghui 177 showed no remarkable difference (Table 4). It is therefore clear that the introduction of the Bt gene through hybridization produces no notable changes in seed vitality.

Table 3. Major agronomic traits of the F1 hybrids derived from the new restorer line Bt5198. Combination G46A/Bt5198 D62A/Bt5198 Zhong 9A/Bt5198 Chuanxiang 29A/Bt5198 II You 838 (CK)

No. of panicles Plant height Panicle length per hill (cm) (cm) 9.2±2.2 9.0±1.9 8.7±1.5 9.5±1.8 8.4±1.2

120.8±2.5 118.5±1.8 122.5±2.2 121.2±2.0 121.7±2.2

25.6±1.0 26.8±0.8 26.2±0.7 26.8±0.4 26.8±0.9

Seed setting rate 1000-grain weight Yield per plant Days from sowing (%) (g) (g) to heading (d) 81.2±2.5 77.5±3.0 79.5±3.2 76.2±2.2 77.6±2.1

27.9±0.6 28.3±0.4 28.1±0.6 30.0±0.5 30.2±0.5

36.5±2.7 34.3±2.5 33.6±2.9 32.7±4.3 32.4±3.0

118.7±0.0 120.0±0.5 118.3±0.0 123.7±0.0 118.2±0.0

GAO Fang-yuan, et al. Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene Table 4. Germination rates of the new rice restorer line Bt5198. % Material

Indoors 3d

7d

Direct seeding

Chenghui 177

96.3

97.7

88.2

Bt5198

95.3

95.7

87.9

The pollen yield of the restorer line directly determines the seed production yield for hybrid rice. According to the evaluation of pollen quantity, the average pollen yield for each anther of Bt5198 was 1116.2 grains, similar to that of Chenghui 177 (1121.2 grains), suggesting that the amount of pollen of the new restorer line Bt5198 might have no negative effect on the yield of seed production in hybrid rice.

DISCUSSION Identification methods for the transgenic Bt gene In the breeding processes of crossing and backcrossing with transgenic materials, it is of great importance to establish quick and easy methods for foreign gene identification. Although PCR analysis, DNA molecular hybridization, protein immunoassay and other techniques can be used to select insectresistant plants, the technical requirements are rather high. According to some studies, detecting the marker gene closely linked to and having coordinated expression with the Bt gene can be a useful means for quick selection of transgenic insect-resistant plants. The gus gene and hygromycin resistance gene (hpt) have been the universally applied marker genes [12-16]. The marker gene in the transgenic rice Bt Minghui 63 was removed [10], and thus the insect-resistant single plant can only be determined by detecting the foreign Bt gene. The BTCry1Ab/1Ac colloidal gold strip used in the test paper examination was developed by a national scientific and technical key program during the Tenth Five-Year Plan period (2001–2005) in China. This strip can quickly and qualitatively detect the availability of Bt-Cry1Ab or Bt-Cry1Ac proteins in plant samples as short as 3–5 min, without the help of any other equipment. Therefore, the test paper detection is the easiest and quickest method in fields where test equipment is insufficient or unavailable. Moreover, inoculation tests in the laboratory and field are the most direct and reliable methods for

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detecting the insect resistance of Bt gene plants. In this study, artificial incubation both in laboratory and fields was conducted to plants showing a positive band on the test paper. The results showed that most of these plants were highly resistant to SSB and YSB. Of course, very few single plants or lines suffered from SSB or YSB, probably owing to the low concentration of Bt protein in plants or that some older larvae on non-Bt plants transferred to the Bt plants later and ate out the culms. These reasons need to be confirmed. Insect-resistant stability of the transgenic rice In the introduction of Bt gene into rice through transgenic techniques, insect resistance is one of the key problems currently concerned. Some researchers have observed a decline in insect-resistance of Bt transgenic rice in subsequent backcross generations [17]. However, a majority of studies have proved that introducing the foreign Bt gene into rice can remarkably improve the insect-resistance to Lepidoptera and be transferred to later generations [14, 18-19]. In this study, insect-resistance experiments in the laboratory and field showed that the new restorer line Bt5198, which is derived from one backcross and seven selfpollinations, still had high resistances to SSB and YSB, so did the hybrid rice combinations. These results proved the stable insect-resistance of the new restorer line obtained in this study. Development of new rice restorer lines with the transgenic Bt gene With the help of molecular marker-assisted selection and backcross breeding, some poor agronomic characters of Bt transgenic rice can be improved. It has been proved that some new rice lines with high stem borer resistance, high yield and good grain quality have been developed by using Bt transgenic rice [6-8]. In this way, complex genetic transformation can be omitted, and hybrid parent materials with high quality are provided for breeding insect-resistant hybrid rice. In this study, Bt Minghui 63 was used as donor to cross with an extensively applied restorer line, and a new restorer line with high resistances to SSB and YSB, good restorability, strong seed vitality and an amount of pollen quite similar to its recipient parent Chenghui 177 was developed. This new restorer line will be directly

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used in breeding three-line hybrid rice combinations with high insect-resistance.

ACKNOWLEDGEMENT

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Lu X J, Ren G J, Li Q M, Gao F Y, Lu D H, Zhang C, Liu G C. Effects of blast resistance of parents on that of their F1 hybrids in rice. Hybrid Rice, 2007, 22(2): 18–21. (in Chinese with English abstract)

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Tu J M, Datta K, Oliva N, Zhang G, Xu C, Khush G S, Zhang Q, Datta S K. Site-independently integrated transgenes in the

This study was supported by the grant from the National Research and Development Project of Transgenic Crops of Ministry of Science and Technology of China (Grant No. JY03-B-11).

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