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Effects of Bleomycin on microspore embryogenesis in Brassica napus and detection of somaclonal variation using AFLP molecular markers

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Abstract

Bleomycin, a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus, has been demonstrated to be an effective mutagen in Arabidopsis thaliana. The objective of the present study was to determine the effect of bleomycin on embryo production and to assess the genetic variation of the doubled haploid (DH) populations by amplified fragment length polymorphism (AFLP). The effects of bleomycin on microspore embryogenesis and cell division were investigated using three concentrations of bleomycin in five semi-winter genotypes of Brassica napus viz. T8, T10, B409, P30, and DH1142. Inclusion of bleomycin in the culture medium at a concentration of 0.1 μg ml−1 for 30 min significantly improved embryo production and cell division in all five genotypes. Embryo production was induced at rates two- and four-fold higher than controls after bleomycin treatment. Fifty plants regenerated by microspore embryogenesis treated with bleomycin in addition to non-treated controls of T8, T10, and B409 were selected for AFLP analysis. The results suggest that microspore culture is capable of producing 0.095–0.114% genetic variation, and there was no effect of bleomycin treatment on genetic stabilisation of doubled haploid populations versus the non-treated control.

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Abbreviations

DH:

Doubled haploid

ALFP:

Amplified fragment length polymorphism

PCR:

Polymerase chain reaction

B5 medium:

Gamborg medium

NLN medium:

Nitsch & Nitsch medium

RAPD:

Random amplified polymorphic

STMS:

Sequence tagged microsatellite site

PCIB:

p-Chlorophenoxyisobutyric acid

PEG:

Polyethylene glycol

NIR:

Near Infrared Spectroscopy

CV:

Coefficient of variation

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Acknowledgments

We are grateful for the financial support of the national key basic research special foundation of China (No.2006CB101604 and No. 2007CB109006), the national high-tech research and development program (2009AA101105), the modern agriculture industrialisation system construction (nycytx-00501) and the national key technology R&D program (2008BAD97B04). We also thank Professor Shyam Prakash, Biotechnology Centre, Indian Agricultural Research Institute for critically evaluating this manuscript.

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement in Wuhan, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China

    Xinhua Zeng, Jing Wen, Zhengjie Wan, Bin Yi, Jinxiong Shen, Chaozhi Ma, Jinxing Tu & Tingdong Fu

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  1. Xinhua Zeng
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  2. Jing Wen
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  3. Zhengjie Wan
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Correspondence to Tingdong Fu.

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Zeng, X., Wen, J., Wan, Z. et al. Effects of Bleomycin on microspore embryogenesis in Brassica napus and detection of somaclonal variation using AFLP molecular markers. Plant Cell Tiss Organ Cult 101, 23–29 (2010). https://doi.org/10.1007/s11240-009-9658-z

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