Verification of Glyphosate Resistance, Lepidopteran Resistance and Wide Compatibility of Male Sterile Line E1C4008S in Rice

doi:10.1016/j.rsci.2020.04.004

Abstract

Abstract:

Weeds and insect pests are two important biotic stresses resulting in yield loss in rice, and wide compatibility is the essential characteristic of breeding inter-subspecific hybrid rice. This study focused on glyphosate resistance, lepidopteran resistance and wide compatibility as well as identification of molecular and some agronomic characteristics of transgenic male sterile line E1C4008S. The results indicated that glyphosate resistance gene Epsps^# and lepidopteran resistance gene Cry1ca^# were transferred into japonica wide compatibility male sterile line 4008S by Agrobacterium-mediated method, and four independent transformation events named E1C4008S-1, E1C4008S-2, E1C4008S-3 and E1C4008S-4 were obtained, in which E1C4008S-3 and E1C4008S-4 were of single copy insertion. The EPSPS protein contents of E1C4008S-3 and E1C4008S-4 in different organs were significantly different both in descending order of leaf > stem > root, and ranged from 300.58 to 1410.69 μg/g at the tillering stage. The glyphosate tolerable concentration (dosage) of E1C4008S-3 and E1C4008S-4 reached at least 16 g/L (54.42 kg/hm²), and the seeds of E1C4008S-4 can germinate normally on the medium containing 1 g/L glyphosate. The CRY1C protein contents of E1C4008S-3 and E1C4008S-4 in different organs were significantly different both in descending order of leaf > stem > root, and ranged from 0.62 to 2.43 μg/g at the tillering stage. The larvae mortalities of rice leaf rollers fed on leaves of E1C4008S-3 and E1C4008S-4 for 5 d were 95.35% and 97.77%, respectively, while the average mortalities of silkworms fed with protein extracts from leaves of E1C4008S-3 and E1C4008S-4 reached 94.55% and 83.64%, respectively. The results suggested that wide compatibility and evaluated agronomic traits of E1C4008S-4 were not significantly changed by insertion of the exogenous genes. Overall, a novel male sterile germplasm with glyphosate resistance, lepidopteran resistance and wide compatibility was verified to be developed in rice.

Key words: rice, glyphosate resistance, lepidopteran resistance, wide compatibility, male sterile line, bioengineered crop

Qiang Zeng, Lihua Deng, Wenbin Hu, Xingjian He, Qiucheng Meng, Jianghui Yu, Jinjiang Li, Lushui Weng, Guoying Xiao. Verification of Glyphosate Resistance, Lepidopteran Resistance and Wide Compatibility of Male Sterile Line E1C4008S in Rice[J]. Rice Science, 2020, 27(3): 215-226.

Figures/Tables 9

Fig. 1. T-DNA region of plant expression vector pC3300-Epsps-Cry1Ca. The Epsps# gene used as a selection marker was driven by a maize ubiquitin promoter with Ω enhancer and terminated by nopaline synthase (Nos) terminator. The Cry1Ca# gene was driven by a maize ubiquitin promoter and terminated by CaMV35S poly(A). LB, Left border of T-DNA region; RB, Right border of T-DNA region.

Table 1 Primers for PCR and Southern blot.

Primer	Forward (5′-3′)	Reverse (5′-3′)	Temperature (ºC)	Product size (bp)	Assay
Epsps	CGCCAAGTCTTTGTGGGTG	GTCCACGGTGACAGGGTTC	58	561	PCR, Southern blot
Cry1C	CGTCGCTCAACTGCCTAC	CCTGAACACGGGTCCATT	55	287	PCR

Fig. 2. Molecular identification of T1 transgenic lines. A, PCR detection. B, Southern blotting analysis. M, DNA molecular weight marker; ‘+’, Plasmid pC3300-Epsps-Cry1Ca; 4008S, Transformation receptor; 1, 2, 3 and 4, Transformants E1C4008S-1, E1C4008S-2, E1C4008S-3 and E1C4008S-4, respectively.

Fig. 3. Quantification of EPSPS and CRY1C proteins. A, Comparison of EPSPS protein contents between different transformants and different organs. B, Comparison of CRY1C protein contents between different organs and different transformants. E1C4008S-3 and E1C4008S-4 are two single-copy insertion events of T2 generation. Values represent Mean ± SD (n = 3). Different uppercase letters above the bars mean significant difference at the 0.01 level.

Fig. 4. Evaluation of resistance to rice leaf roller. A, Leaves gnawed by rice leaf rollers for 5 d. B, Mortality of rice leaf rollers in 5 d. E1C4008S-3 and E1C4008S-4 are of single-copy insertion events in T3 generation; 4008S is of wild type. Values represent Mean ± SD (n = 3). Different uppercase letters above the bars indicate significant difference at the 0.01 level.

Table 2 Injuries on plants caused by rice leaf rollers in the field.

Line	No. of injured plants	No. of injured tillers	No. of injured leaves
4008S (CK)	10.33 ± 1.53 A	29.00 ± 6.00 A	35.33 ± 6.43 A
E1C4008S-3	0.67 ± 0.58 B	0.67 ± 0.58 B	0.67 ± 0.58 B
E1C4008S-4	0.33 ± 0.58 B	0.33 ± 0.58 B	0.67 ± 1.15 B

Fig. 5. Toxicity evaluation of protein extracts from rice leaves on the second-instar silkworms. A, Bioassay of the second-instar silkworms fed on mulberry leaves painted with or without CRY1C protein extracts. B, Mortality of the second-instar silkworms fed on mulberry leaves coated with or without CRY1C protein extracts. E1C4008S-3 and E1C4008S-4 are of single-copy insertion events in T3 generation; 4008S is of wild type. Values represent Mean ± SD (n = 3). Different uppercase letters above the bars indicate significant difference at the 0.01 level.

Fig. 6. Identification of glyphosate resistance of transgenic rice. A, Spraying glyphosate at different concentrations on T3 generation transgenic plants. 0, 0.5, 1, 2, 3, 8, 10, 12, 14 and 16 g/L indicate different concentrations of glyphosate solution (these converted to dosage were 0, 1.70, 3.40, 6.80, 10.20, 27.21, 34.01, 40.82, 47.62 and 54.42 kg/hm2, respectively). B, Germination of T3 generation seeds on media containing different concentrations of glyphosate. 0, 34, 170, 340, 680 and 1000 mg/L indicate different concentrations of glyphosate in culture medium. E1C4008S-3 and E1C4008S-4 are transgenic lines; 4008S is wild type.

Table 3 Agronomic traits of male sterile lines and F1 hybrids from E1C4008S-4 and 4008S crossing with the same male parent.

Hybrid ^a	Plant height (cm)	Main panicle length (cm)	No. of effective panicles per plant	No. of grains per panicle	Seed-setting rate (%)	1000-grain weight (g)
4008S/Yunnan aromatic rice	77.22 ± 1.89 Aa	20.19 ± 0.43 Aa	12.6 ± 1.3 Aa	116.9 ± 10.7 Aa	92.1 ± 1.0 Aa	24.36 ± 0.40 Aa
E1C4008S-4/Yunnan aromatic rice	78.00 ± 1.25 Aa	19.54 ± 0.49 Aa	13.2 ± 1.3 Aa	101.1 ± 8.5 Ab	88.6 ± 2.2 Aa	24.37 ± 0.43 Aa
4008S/W017	67.99 ± 0.82 Aa	17.06 ± 0.57 Aa	13.1 ± 1.6 Aa	113.0 ± 13.4 Aa	89.0 ± 2.2 Aa	23.87 ± 0.19 Aa
E1C4008S-4/W017	68.68 ± 1.82 Aa	16.51 ± 0.49 Aa	13.8 ± 1.0 Aa	106.7 ± 9.0 Aa	88.6 ± 3.5 Aa	23.70 ± 0.30 Aa
4008S/Dalixiang	73.13 ± 1.09 Aa	18.53 ± 0.82 Aa	10.4 ± 1.3 Aa	87.1 ± 7. 8 Aa	87.3 ± 1.2 Aa	25.78 ± 0.14 Aa
E1C4008S-4/Dalixiang	72.54 ± 1.72 Aa	18.47 ± 0.37 Aa	10.7 ± 1.8 Aa	83.9 ± 9.3 Aa	86.1 ± 3.0 Aa	25.90 ± 0.19 Aa
4008S/Guanghui 390	80.03 ± 1.62 Aa	19.73 ± 0.48 Aa	11.3 ± 1.6 Aa	115.1 ± 10.7 Aa	73.3 ± 8.1 Aa	22.49 ± 0.30 Aa
E1C4008S-4/Guanghui 390	80.49 ± 2.33 Aa	18.94 ± 0.78 Aa	10.8 ± 2.2 Aa	107.7 ± 10.6 Aa	77.1 ± 2.6 Aa	22.20 ± 0.26 Aa
4008S/R2292	74.80 ± 1.53 Aa	19.42 ± 1.56 Aa	10.4 ± 1.4 Aa	96.8 ± 9.1 Aa	62.6 ± 6.8 Aa	22.73 ± 0.37 Aa
E1C4008S-4/R2292	75.14 ± 2.12 Aa	19.06 ± 0.93 Aa	10.7 ± 1.3 Aa	92.2 ± 7.2 Aa	58.7 ± 2.9 Aa	23.03 ± 0.60 Aa
4008S/R299	80.43 ± 1.62 Aa	18.46 ± 0.65 Aa	12.0 ± 1.6 Aa	100.4 ± 14.5 Aa	51.3 ± 5.7 Aa	23.01 ± 0.39 Aa
E1C4008S-4/R299	80.06 ± 1.73 Aa	18.42 ± 0.60 Aa	12.5 ± 1.1 Aa	94.4 ± 5.4 Aa	51.4 ± 1.9 Aa	23.22 ± 0.24 Aa
4008S	64.30 ± 2.42 Aa	18.03 ± 0.85 Aa	13.5 ± 1.2 Aa	85.3 ± 14.6 Aa	38.8 ± 4.0 Aa	24.86 ± 0.62 Aa
E1C4008S-4	66.39 ± 3.00 Aa	18.83 ± 1.03 Aa	10.7 ± 2.2 Ab	92.0 ± 13.3 Aa	42.4 ± 5.1 Aa	24.67 ± 0.30 Aa

Table 3 Agronomic traits of male sterile lines and F1 hybrids from E1C4008S-4 and 4008S crossing with the same male parent.

Hybrid ^a	Plant height (cm)	Main panicle length (cm)	No. of effective panicles per plant	No. of grains per panicle	Seed-setting rate (%)	1000-grain weight (g)
4008S/Yunnan aromatic rice	77.22 ± 1.89 Aa	20.19 ± 0.43 Aa	12.6 ± 1.3 Aa	116.9 ± 10.7 Aa	92.1 ± 1.0 Aa	24.36 ± 0.40 Aa
E1C4008S-4/Yunnan aromatic rice	78.00 ± 1.25 Aa	19.54 ± 0.49 Aa	13.2 ± 1.3 Aa	101.1 ± 8.5 Ab	88.6 ± 2.2 Aa	24.37 ± 0.43 Aa
4008S/W017	67.99 ± 0.82 Aa	17.06 ± 0.57 Aa	13.1 ± 1.6 Aa	113.0 ± 13.4 Aa	89.0 ± 2.2 Aa	23.87 ± 0.19 Aa
E1C4008S-4/W017	68.68 ± 1.82 Aa	16.51 ± 0.49 Aa	13.8 ± 1.0 Aa	106.7 ± 9.0 Aa	88.6 ± 3.5 Aa	23.70 ± 0.30 Aa
4008S/Dalixiang	73.13 ± 1.09 Aa	18.53 ± 0.82 Aa	10.4 ± 1.3 Aa	87.1 ± 7. 8 Aa	87.3 ± 1.2 Aa	25.78 ± 0.14 Aa
E1C4008S-4/Dalixiang	72.54 ± 1.72 Aa	18.47 ± 0.37 Aa	10.7 ± 1.8 Aa	83.9 ± 9.3 Aa	86.1 ± 3.0 Aa	25.90 ± 0.19 Aa
4008S/Guanghui 390	80.03 ± 1.62 Aa	19.73 ± 0.48 Aa	11.3 ± 1.6 Aa	115.1 ± 10.7 Aa	73.3 ± 8.1 Aa	22.49 ± 0.30 Aa
E1C4008S-4/Guanghui 390	80.49 ± 2.33 Aa	18.94 ± 0.78 Aa	10.8 ± 2.2 Aa	107.7 ± 10.6 Aa	77.1 ± 2.6 Aa	22.20 ± 0.26 Aa
4008S/R2292	74.80 ± 1.53 Aa	19.42 ± 1.56 Aa	10.4 ± 1.4 Aa	96.8 ± 9.1 Aa	62.6 ± 6.8 Aa	22.73 ± 0.37 Aa
E1C4008S-4/R2292	75.14 ± 2.12 Aa	19.06 ± 0.93 Aa	10.7 ± 1.3 Aa	92.2 ± 7.2 Aa	58.7 ± 2.9 Aa	23.03 ± 0.60 Aa
4008S/R299	80.43 ± 1.62 Aa	18.46 ± 0.65 Aa	12.0 ± 1.6 Aa	100.4 ± 14.5 Aa	51.3 ± 5.7 Aa	23.01 ± 0.39 Aa
E1C4008S-4/R299	80.06 ± 1.73 Aa	18.42 ± 0.60 Aa	12.5 ± 1.1 Aa	94.4 ± 5.4 Aa	51.4 ± 1.9 Aa	23.22 ± 0.24 Aa
4008S	64.30 ± 2.42 Aa	18.03 ± 0.85 Aa	13.5 ± 1.2 Aa	85.3 ± 14.6 Aa	38.8 ± 4.0 Aa	24.86 ± 0.62 Aa
E1C4008S-4	66.39 ± 3.00 Aa	18.83 ± 1.03 Aa	10.7 ± 2.2 Ab	92.0 ± 13.3 Aa	42.4 ± 5.1 Aa	24.67 ± 0.30 Aa

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