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Enhanced Agrobacterium-mediated Transformation of Embryogenic Calli of Upland Cotton via Efficient Selection and Timely Subculture of Somatic Embryos

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Abstract

Agrobacterium-tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. After 48-h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse-granule EC. It indicated that efficiency of transient transformation was affected by EC morphology. And transient transformation efficiency was also improved by cocultivation on the medium adding 50 mg l−1 acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density was beneficial to production of more kanamycin-resistant (Km-R) calli lines. From an original 0.3-g EC, an average of 20 Km-R calli lines were obtained from a selection dish and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments and the GUS-positive rate of regeneration plants was about 72.60%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and uidA. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, has been integrated into the cotton genome.

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Abbreviations

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

AS:

acetosyringone

EC:

embryogenic calli

GUS:

β-Glucuronidase

IBA:

indole-3-butyric acid

Km:

kanamycin

Km-R:

kanamycin-resistant

KT:

cytokinin

MS:

Murashige and Skoog medium

MSB:

MS basal salts plus B5 vitamins

npt-II :

neomycin phosphotransferase II gene

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Acknowledgement

This work was financially supported in part by grants from the High-tech program 863 (2006AA100105), the Changjiang Scholars and Innovative Research Team in University of MOE, China (RT0432), and the Jiangsu Planned Project for Postdoctoral Research Funds (No. 0701018B).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China

    Shen-Jie Wu, Hai-Hai Wang, Fei-Fei Li, Tian-Zi Chen, Jie Zhang, Yan-Jie Jiang, Yezhang Ding, Wang-Zhen Guo & Tian-Zhen Zhang

  2. Cotton Research Institute, Shanxi Academy of Agricultural science, Yuncheng, 044000, China

    Shen-Jie Wu

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  1. Shen-Jie Wu
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Correspondence to Tian-Zhen Zhang.

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Shen-Jie Wu and Hai-Hai Wang should be considered as joint first authors

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Wu, SJ., Wang, HH., Li, FF. et al. Enhanced Agrobacterium-mediated Transformation of Embryogenic Calli of Upland Cotton via Efficient Selection and Timely Subculture of Somatic Embryos. Plant Mol Biol Rep 26, 174–185 (2008). https://doi.org/10.1007/s11105-008-0032-9

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