A transgenic rice resistant to glyphosate and flazasulfuron

doi:10.3969/j.issn.1004-1524.20240340

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (9): 1957-1968.DOI: 10.3969/j.issn.1004-1524.20240340

• Crop Seience • Previous Articles Next Articles

A transgenic rice resistant to glyphosate and flazasulfuron

WU Haofeng¹(), LIN Zhaoyang¹^,², SHEN Zhicheng¹^,^*()

1. Instittute of Insect Sciences, Zhejiang University, Hangzhou 310058, China
2. The Rural Development Academy, Zhejiang University, Hangzhou 310058, China

Received:2024-04-12 Online:2024-09-25 Published:2024-09-30

Abstract

Abstract:

Weed control is an important aspect of rice production. In this study, a transgenic herbicide tolerant rice was created by expressing the glyphosate tolerant gene CP4-EPSPS and multiple-herbicide tolerant gene P450-N-Z1. The transformant event GF-9 was selected by screening with two herbicides, glyphosate and flazasulfuron. Field trial demonstrated that theGF-9 could tolerate combined herbicides at rate of 1 800 g·hm^-2 (based on acid equivalent) glyphosate and 30 g·hm^-2 (based on active ingredients) flazasulfuron. Southern blot and high-efficiency thermal asymmetric interlaced PCR results showed that the GF-9 was a single copy T-DNA insertion event and its insertion site was not inside a known or predicted functional gene. The field agronomic performance of GF-9 showed no significant difference compared to non-transgenic controls. In summary, the transgenic herbicide tolerant event GF-9 is a potential commercial event for development of herbicide tolerant rice.

Key words: rice, transgenic, herbicide tolerant

CLC Number:

S511

WU Haofeng, LIN Zhaoyang, SHEN Zhicheng. A transgenic rice resistant to glyphosate and flazasulfuron[J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 1957-1968.

Figures/Tables 18

Fig.1 Diagram of the T-DNA structure

Table 1 List of compound herbicide ingredients

除草剂浓度 Herbicide concentration	41%草甘膦异丙胺盐水剂用量 41% Glyphosate isopropylamine aqueous solution dosage	25%啶嘧磺隆水分散粒剂用量 25% Flazasulfuron water dispersible granules dosage
0(清水Water control)	0	0
1倍浓度Single concentration	5 mL·L^-1(900 g·hm^-2)	0.1 g·L^-1(15 g·hm^-2)
2倍浓度Double concentration	10 mL·L^-1(1 800 g·hm^-2)	0.2 g·L^-1(30 g·hm^-2)
4倍浓度Quadruple concentration	20 mL·L^-1(3 600 g·hm^-2)	0.4 g·L^-1(60 g·hm^-2)

Table 2 PCR primer sequences for the foreign genes

引物 Primer	引物序列 Primer sequences(5'→3')
CP4-F	ATGGCGGCGACCATGGCGTCCAACG
CP4-R	TCAAGCGGCCTTCGTGTCAGACAGTTC
N-Z1-F	ATGGATAAGGCCTACGTGGCCCTCC
N-Z1-R	TCAGAGCTCCTGCAAAACCTCACGC

Table 3 Primers sequences for high-efficiency thermal asymmetric interlaced PCR

引物Primer	引物序列Primer sequences(5'→3')
LB-SPI	TTTCTCCATAATAATGTGTGAGTAGTTCCC
LB-SP2a	ACGATGGACTCCAGTCCGGCCCTCATGTGTTGAGCATATAAGAAACCCTTAG
LB-SPIII	CTAAAACCAAAATCCAGTACTAAAATCC
RB-0b	CGTGACTGGGAAAACCCTGGCGTT
RB-1b	ACGATGGACTCCAGTCCGGCCCAACTTAATCGCCTTGCAGCACATC
RB-2b	GAAGAGGCCCGCACCGATCGCCCTT
LAD1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
LAD2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
LAD3	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
LAD4	ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
AC1	ACGATGGACTCCAGAG

Table 4 Boundary validation primer sequence

引物 Primer	引物序列 Primer sequences(5'→3')
L1	CTCCCCCATTCTGGCTTGGAA
L2	CATTGTGAAGCCCTCGAGTAATTGG
L3	ACCGGAGACATATAGTGCTGGTTT
R1	TGGGCGCTCTTCGACGGATT
R2	GGCCATCGCAGCCATTAAATCG
R3	TCCTCTGTGGGGCTTTGGAC

Fig.2 Glyphosate tolerance test results of transgenic rice events in greenhouse Water, Water control; Glyphosate, Spary glyphosate at a rate of 900 g·hm-2 (based on acid equivalent); TG, GF series transgenic rice events; NT, Non-transgenic control.

Fig.3 Flazasulfuron tolerance test results of transgenic rice events in greenhouse Water, Water control; Flazasulfuron, Spary flazasulfuron at a rate of 15 g·hm-2 (based on active ingredient); TG, GF series transgenic rice events; NT, Non-transgenic control.

Fig.4 Images of weed control effects in paddy fields using compound herbicides A, Occurrence of weeds in rice fields 7 days after application of a mixed herbicide;B, Occurrence of weeds in rice fields 28 days after application of a mixed herbicide; TG, GF series transgenic rice events; NT, Non-transgenic control; Single, Single concentration compound herbicide treatment; Double, Double concentration compound herbicide treatment; Quadruple, Quadruple concentration compound herbicide treatment.

Fig.5 The damage situation of GF-9 after 28 days of treatment with different concentrations of herbicide TG, GF series transgenic rice events; NT, Non-transgenic control; Single, Single concentration compound herbicide treatment; Double, Double concentration compound herbicide treatment; Quadruple, Quadruple concentration compound herbicide treatment.

Table 5 Plant height for GF-9 under different herbicide concentrations

除草剂浓度 Herbicide concentration	GF-9	秀水134 XS-134
0(清水Water control)	83.57±2.44 Aa	86.16±3.67 A
1倍浓度Single concentration	82.03±2.38 a	—
2倍浓度Double concentration	78.50±2.36 b	00.00±0.00
4倍浓度	78.20±2.57 b	—
Quadruple concentration

Table 6 Agronomic performance of GF-9 under different concentrations of herbicide

试验材料 Material	除草剂浓度 Herbicide concentration	穗长 Panicle length/cm	分蘖数 Tiller number	百粒重 Hundred-grain weight/g	结实率 Setting rate%
GF-9	0(清水Water control)	14.82±0.72 Aa	9.00±1.18 Aa	2.536±0.027 Aa	68.29±4.60 Aa
	1倍浓度Single concentration	15.84±0.81 a	10.20±2.35 a	2.575±0.025 a	71.01±7.52 a
	2倍浓度Double concentration	15.72±1.05 a	10.20±2.30 a	2.519±0.048 a	69.12±5.49 a
	4倍浓度Quadruple concentration	15.47±1.21 a	8.58±2.50 a	2.254±0.042 b	55.20±2.60 b
秀水134 XS-134	0(清水Water control)	14.32±1.57 A	8.50±1.35 A	2.543±0.051 A	65.00±5.70 A

Fig.6 Panicle length and fruiting rate of GF-9 under different concentrations of herbicide NT, Non-transgenic control under water treatment; Single, Single concentration compound herbicide treatment; Double, Double concentration compound herbicide treatment; Quadruple, Quadruple concentration compound herbicide treatment.

Fig.7 PCR detection results of the transgenes in GF-9 A, PCR results of CP4-EPSPS gene; B, PCR results of P450-N-Z1 gene; M, DNA Marker; +, Positive plasmid; -, Non-transgenic control; 1, GF-9 T0 generation sample genome; 2-4, GF-9 T1 generation sample genome; 3-7, GF-9 T2 generation sample genome.

Fig.8 Boundary verification results of the event GF-9 A, PCR validation results of the left border of GF-9; B, PCR validation results of the right border of GF-9. M, DNA Marker; L1-L3, PCR results of the verification primers for three different distances outside the left border of T-DNA, respectively with LB-SPI; R1-R3, PCR results of the verification primers for three different distances outside the right border of T-DNA, respectively with RB-0b; -, Negative rice control.

Fig.9 Integration model of the foreign T-DNA in GF-9 and verification results of the boundary sequencing L1-L3, Primers for validating the left border of T-DNA.; R1-R3, Primers for validating the right border of T-DNA.

Fig.10 Schematic diagram of GF-9 restriction enzyme and CP4 probe location and expected hybridization signal band size Probe, Gene sequence used to make probe.

Fig.11 Southern blot hybridization of GF-9

Fig.12 Western blot results of CP4-EPSPS protein M, Protein pageruler; +, CP4-EPSPS protein expressed in prokaryotes; -, Non-transgenic rice control group; 1-7, Seven independent plants of T1 generation transgenic line GF-9.

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A transgenic rice resistant to glyphosate and flazasulfuron

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