Differential transformation efficiency of <i>Japonica</i>rice varieties developed in northern China

The production of japonica rice in northern China plays an important role in food security for the world. Agrobacterium-mediated gene transfer is becoming a powerful approach to generate germplasm and develop varieties. However, the transgenic efficiency of the japonica rice varieties in northern China has been completely unknown, which obstructs the development of transgenic breeding and the exploration of gene functions. In this study, the transgenic efficiencies of six japonica rice varieties developed in northern China are evaluated. The rates of primary and secondary callus induction of the varieties are similar. However, transgenic efficiency and the regeneration ability of the varieties are greatly different. The results have established a platform for transformation of the rice varieties and proposed a suitable variety, SN9816, for gene transfer. SN9816 can be applied as an elite germplasm for transgenic breeding and basic research of molecular biology in northern China or an area in the same latitude.

Agrobacterium-mediated transformation; transformation efficiency; japonica rice in northern China; Shen Nong 9816

Authorship

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

Figures

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