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Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens

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Abstract

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pΔgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 108 cells ml−1 (OD600=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l−1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.

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Abbreviations

AS:

acetosyringone

EC:

embryogenic callus

IBA:

indole-3-butyric acid

MS:

Murashige and Skoog (1962) medium

MSB:

-MS basal salts plus B5 (Gamborg et al. 1968) vitamins

NAA:

α-naphthaleneacetic acid

npt-II:

neomycin phosphortransferase II

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070, Wuhan, Hubei, China

    Shuangxia Jin, Xianlong Zhang, Shaoguang liang, Yichun Nie, Xiaoping Guo & Chao Huang

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  1. Shuangxia Jin
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  2. Xianlong Zhang
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  3. Shaoguang liang
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  4. Yichun Nie
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  5. Xiaoping Guo
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Correspondence to Xianlong Zhang.

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Jin, S., Zhang, X., liang, S. et al. Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 81, 229–237 (2005). https://doi.org/10.1007/s11240-004-5209-9

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