Eficiência diferencial de transformação de variedades de arroz   <i>japonica</i> desenvolvidas no norte da China

A produção de arroz japonica no norte da China desempenha um papel importante na segurança alimentar para o mundo. Transferência de genes mediada por Agrobacterium está se tornando uma abordagem poderosa para gerar germoplasma e desenvolver variedades. No entanto, a eficiência transgênica das variedades de arroz japonica no norte da China tem sido completamente desconhecida, o que obstrui o desenvolvimento da criação de transgênicos e a exploração das funções dos genes. Neste estudo, as eficiências da transgenia de seis variedades de arroz japonica desenvolvidas no Norte da China são avaliadas. As taxas de indução de calo primário e secundário das variedades são semelhantes. Contudo, a eficiência transgênica e a capacidade de regeneração das variedades são bem distintas. Foi estabelecida uma plataforma para a transformação das variedades de arroz e proposta uma variedade adequada, SN9816, para transferência de genes. SN9816 pode ser aplicada como um germoplasma-elite para melhoramento transgênico e pesquisa básica de biologia molecular no norte da China ou em uma área na mesma latitude.

Transformação mediada por Agrobacterium; eficiência de transformação; arroz japonica; no norte da China; Shen Nong 9816

Autoria

Dan Li

Rice Research Institute, Key Laboratory of Northeast Rice Biology and Breeding, Ministry of Agriculture; Key Laboratory of Northern Japonica Super Rice Breeding, Ministry of Education; Shenyang Agricultural University, Shenyang 110866, ChinaRice Research InstituteChinaShenyang, ChinaRice Research Institute, Key Laboratory of Northeast Rice Biology and Breeding, Ministry of Agriculture; Key Laboratory of Northern Japonica Super Rice Breeding, Ministry of Education; Shenyang Agricultural University, Shenyang 110866, China

Hai Xu

Xiaoxue Sun

Zhibo Cui

Yuan Zhang

Yuguan Bai

Northeast Yucai School, Shenyang 110179, ChinaNortheast Yucai SchoolChinaShenyang, ChinaNortheast Yucai School, Shenyang 110179, China

Xiaoxue Wang ^**E-mail: xiaoxuewang6@163.com

Wenfu Chen

*E-mail: xiaoxuewang6@163.com

SCIMAGO INSTITUTIONS RANKINGS

Northeast Yucai School, Shenyang 110179, ChinaNortheast Yucai SchoolChinaShenyang, ChinaNortheast Yucai School, Shenyang 110179, China

Figuras

Figuras (6)

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Figure 1
A map of pCambia 1301 binary vector between the left border and right border. LB: left border repeat; polyA: CaMV 3' UTR polyA signal; hpt II: hygromycin resistance gene; 35S2: CaMV 35S promoter duplicated; MCS: multiple cloning site; Lac Z: Lac Z promoter and Lac Z alpha fragment; 35S: CaMV 35S promoter; gusA E1: gusA first exon; gusA Int: gusA intron; gusA E2: gusA second exon; nos T: nopaline synthase 3'UTR polyA signal; RB: right border repeat.

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Figure 2
Procedures of Agrobacterium-mediated transformation for japonica rice varieties in northern China. (A) Sterilization of Seeds. (B) Inoculation of seeds in N6D medium. (C) Formation of primary calli in N6D medium. (D) Induction of secondary calli in N6D medium. (E) Preculture of the secondary calli in N6D medium three days before transformation. (F) Infection of the preculture secondary calli with the Agrobacterium harboring pCambia 1301 vector. (G) Drying of the infected calli before co-cultivation. (H) Co-cultivation with the Agrobacterium for three days in the dark. (I) Proliferation of Hyg-tolerant calli on the secondary selection medium, N6D-S medium. (J) Regeneration of transgenic shoot in root generation medium, MS-HF. The white or black line is the scale bar, representing 2cm or 1cm as indicated.

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Figure 3
Callus formation of japonica rice varieties in northern China. (A) The 7-day-old primary calli of the varieties. (B) The rate of primary callus induction of the varieties. (C) The rate of secondary callus induction of the varieties. (D) The fresh weight of 100 callus cluster of secondary calli after 15 days of subculture. The white line is the scale bar, representing 1cm. The values are the mean and standard deviation (SD) from three independent biological experiments. SN9816: Shen Nong 9816; LX1: Liao Xing 1; SN265: Shen Nong 265. One asterisk (*) represents significant difference (P<0.05). Two asterisks (**) represent highly significant difference (P<0.01).

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Figure 4
Agrobacterium-mediated transformation efficiency of the varieties in northern China. (A) Transient expression of 35S:intron-GUS after 3 days of co-cultivation with Agrobacterium. (B) The rate of GUS stained calli of the varieties. (C) The proliferation of the Hyg-tolerant calli in secondary selection medium, N6D-S medium. (D) Transgenic efficiencies of the varieties in northern China after secondary selection. Three biological replicates were performed. Similar results were obtained. The results from two or three replicates are shown. The values are the mean and standard deviations from three independent biological experiments. One asterisk (*) represents significant difference (P<0.05). Two asterisks (**) represent highly significant difference (P<0.01). SN9816: Shen Nong 9816; LX1: Liao Xing 1; SN265: Shen Nong 265. White lines are the scale bar, representing 1cm or 2cm as indicated.

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Figure 5
Regeneration ability of the varieties in northern China. (A) Regenerated shoots from the Hyg-tolerant tissues. (B) Plantlets with roots derived from Agrobacterium-mediated transformation. (C) Regeneration rates without Agrobacterium-mediated transformation of the varieties in northern China. (D) Regeneration rates with Agrobacterium-mediated transformation of the varieties in northern China. (E) The rate of root generation from the regenerated shoots without transformation. (F) The rate of root generation from the regenerated shoots with Agrobacterium-mediated transformation. Three biological replicates were performed. Similar results were obtained. The results from two or three replicates are shown. The values are the mean and standard deviationsof two or three independent biological experiments. One asterisk (*) represents significant difference (P<0.05). Two asterisks (**) represent highly significant difference (P<0.01). SN9816: Shen Nong 9816; LX1: Liao Xing 1; SN265: Shen Nong 265. White lines are the scale bar, representing 2cm or 4cm as indicated.

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Figure 6
Detection of transgenic plantlets with PCR. (A) A gel image of PCR products with the HygF/HygR primer pair on Hyg-tolerant transgenic plantlets. The number represents the individual transgenic plantlets. The asterisk (*) labeled 5 and 12 represents negative plantlets. (B) Transgenic efficiency detected by PCR based on the number of the rooted regeneration plantlets. (C) Transgenic plants in heading stages. The white line is the scale bar, representing 10cm.

Figure 1 A map of pCambia 1301 binary vector between the left border and right border. LB: left border repeat; polyA: CaMV 3' UTR polyA signal; hpt II: hygromycin resistance gene; 35S2: CaMV 35S promoter duplicated; MCS: multiple cloning site; Lac Z: Lac Z promoter and Lac Z alpha fragment; 35S: CaMV 35S promoter; gusA E1: gusA first exon; gusA Int: gusA intron; gusA E2: gusA second exon; nos T: nopaline synthase 3'UTR polyA signal; RB: right border repeat.

Figure 2 Procedures of Agrobacterium-mediated transformation for japonica rice varieties in northern China. (A) Sterilization of Seeds. (B) Inoculation of seeds in N6D medium. (C) Formation of primary calli in N6D medium. (D) Induction of secondary calli in N6D medium. (E) Preculture of the secondary calli in N6D medium three days before transformation. (F) Infection of the preculture secondary calli with the Agrobacterium harboring pCambia 1301 vector. (G) Drying of the infected calli before co-cultivation. (H) Co-cultivation with the Agrobacterium for three days in the dark. (I) Proliferation of Hyg-tolerant calli on the secondary selection medium, N6D-S medium. (J) Regeneration of transgenic shoot in root generation medium, MS-HF. The white or black line is the scale bar, representing 2cm or 1cm as indicated.

Figure 3 Callus formation of japonica rice varieties in northern China. (A) The 7-day-old primary calli of the varieties. (B) The rate of primary callus induction of the varieties. (C) The rate of secondary callus induction of the varieties. (D) The fresh weight of 100 callus cluster of secondary calli after 15 days of subculture. The white line is the scale bar, representing 1cm. The values are the mean and standard deviation (SD) from three independent biological experiments. SN9816: Shen Nong 9816; LX1: Liao Xing 1; SN265: Shen Nong 265. One asterisk (*) represents significant difference (P<0.05). Two asterisks (**) represent highly significant difference (P<0.01).

Figure 4 Agrobacterium-mediated transformation efficiency of the varieties in northern China. (A) Transient expression of 35S:intron-GUS after 3 days of co-cultivation with Agrobacterium. (B) The rate of GUS stained calli of the varieties. (C) The proliferation of the Hyg-tolerant calli in secondary selection medium, N6D-S medium. (D) Transgenic efficiencies of the varieties in northern China after secondary selection. Three biological replicates were performed. Similar results were obtained. The results from two or three replicates are shown. The values are the mean and standard deviations from three independent biological experiments. One asterisk (*) represents significant difference (P<0.05). Two asterisks (**) represent highly significant difference (P<0.01). SN9816: Shen Nong 9816; LX1: Liao Xing 1; SN265: Shen Nong 265. White lines are the scale bar, representing 1cm or 2cm as indicated.

Figure 5 Regeneration ability of the varieties in northern China. (A) Regenerated shoots from the Hyg-tolerant tissues. (B) Plantlets with roots derived from Agrobacterium-mediated transformation. (C) Regeneration rates without Agrobacterium-mediated transformation of the varieties in northern China. (D) Regeneration rates with Agrobacterium-mediated transformation of the varieties in northern China. (E) The rate of root generation from the regenerated shoots without transformation. (F) The rate of root generation from the regenerated shoots with Agrobacterium-mediated transformation. Three biological replicates were performed. Similar results were obtained. The results from two or three replicates are shown. The values are the mean and standard deviationsof two or three independent biological experiments. One asterisk (*) represents significant difference (P<0.05). Two asterisks (**) represent highly significant difference (P<0.01). SN9816: Shen Nong 9816; LX1: Liao Xing 1; SN265: Shen Nong 265. White lines are the scale bar, representing 2cm or 4cm as indicated.

Figure 6 Detection of transgenic plantlets with PCR. (A) A gel image of PCR products with the HygF/HygR primer pair on Hyg-tolerant transgenic plantlets. The number represents the individual transgenic plantlets. The asterisk (*) labeled 5 and 12 represents negative plantlets. (B) Transgenic efficiency detected by PCR based on the number of the rooted regeneration plantlets. (C) Transgenic plants in heading stages. The white line is the scale bar, representing 10cm.

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Eficiência diferencial de transformação de variedades de arroz <i>japonica</i> desenvolvidas no norte da China

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[1] *E-mail: xiaoxuewang6@163.com