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Adapting rice anther culture to gene transformation and RNA interference

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Abstract

Anther culture offers a rapid method of generating homozygous lines for breeding program and genetic analysis. To produce homozygous transgenic lines of rice (Oryza sativa L.) in one step, we developed an efficient protocol of anther-callus-based transformation mediated by Agrobacterium after optimizing several factors influencing efficient transformation, including callus induction and Agrobacterium density for co-cultivation. Using this protocol, we obtained 145 independent green transformants from five cultivars of japonica rice by transformation with a binary vector pCXK1301 bearing the rice gene, Xa21 for resistance to bacterial blight, of which 140 were further confirmed by PCR and Southern hybridization analysis, including haploids (32.1%), diploids (62.1%) and mixoploids (7.5%). Fifteen diploids were found to be doubled haploids, which accounted for 10.7% of the total positive lines. Finally, by including 28 from colchicine induced or spontaneous diploidization of haploids later after transformation, a total of 43 doubled haploids (30.7%) of Xa21 transgenic lines were obtained. We also generated two RNAi transgenic haploids of the rice OsMADS2 gene, a putative redundant gene of OsMADS4 based on their sequence similarity, to investigate its possible roles in rice flower development by this method. Flowers from the two OsMADS2 RNAi transgenic haploids displayed obvious homeotic alternations, in which lodicules were transformed into palea/lemma-like tissues, whereas identities of other floral organs were maintained. The phenotypic alternations were proved to result from specific transcriptional suppression of OsMADS2 gene by the introduced RNAi transgene. The results confirmed that OsMADS2 is involved in lodicule development of rice flower and functionally redundant with OsMADS4 gene. Our results demonstrated that rice anther culture could be adapted to gene transformation and RNAi analysis in rice.

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Authors and Affiliations

  1. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

    Chen Caiyan, Xiao Han, Zhang Wenli, Wang Aiju, Xia Zhihui, Li Xiaobing, Zhai Wenxue, Cheng Zhukuan & Zhu Lihuang

  2. Graduate School of the Chinese Academy of Sciences, Beijing, 100039, China

    Chen Caiyan, Xiao Han, Zhang Wenli & Wang Aiju

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  1. Chen Caiyan
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  2. Xiao Han
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  3. Zhang Wenli
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  4. Wang Aiju
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  5. Xia Zhihui
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  6. Li Xiaobing
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Correspondence to Zhu Lihuang.

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The authors contributed equally to this work.

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Chen, C., Xiao, H., Zhang, W. et al. Adapting rice anther culture to gene transformation and RNA interference. SCI CHINA SER C 49, 414–428 (2006). https://doi.org/10.1007/s11427-006-2013-2

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  • DOI: https://doi.org/10.1007/s11427-006-2013-2

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