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Isolation of disease-tolerant cotton (Gossypium hirsutum L. cv. SVPR 2) plants by screening somatic embryos with fungal culture filtrate

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Abstract

We report an in vitro selection method that has led to isolation of Fusarium wilt and Alternaria leaf spot disease-tolerant plantlets in cotton (Gossypium hirsutum L. cv. SVPR2). Embryogenic callus was isolated from hypocotyl explants of cotton cultured on 5–50% Fusarium oxysporum culture filtrate-fortified callus induction medium. Somatic embryos tolerant to fungal culture filtrate (FCF) were isolated from this embryogenic callus on somatic embryo regeneration medium fortified with 40% FCF. Sixteen plantlets were selected as FCF-tolerant from 34 somatic embryos tested, which corresponds to about 47% success rate. The FCF-tolerant plants were analyzed for disease tolerance by challenging them with spores of F. oxysporum and Alternaria macrospora. Four plants were selected as F. oxysporum tolerant from a total of 24 plants tested. The selected plants showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1 × 105 spores/mL of F. oxysporum. From the FCF-tolerant plants, another nine randomly selected plantlets were challenged with spores of A. macrospora in order to test their tolerance to Alternaria leaf spot disease. The number of lesions per leaf significantly decreased from 8.2 to 0.9 and the lesion lengths were also reduced from 2.8 to 1.2 mm per leaf spot in these plants. Electrophoresis analysis of extracellular proteins from the FCF-tolerant plants showed enhanced secretion of proteins in the range of 24–36 kDa. Isozyme analysis by of FCF-tolerant plants by using native gels showed the presence of chitinase. Quantitative analysis showed that there was 13-fold increase in a chitinase activity in the selected FCF-tolerant plants compared to the control plants. Our results show that over-expression of chitinase enzyme leads to enhanced disease resistance against F. oxysporum and A. macrospora.

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Abbreviations

2iP:

Isopentenyl adenine

Kin:

Kinetin

GA3 :

Gibberellic acid

MS:

Murashige and Skoog medium

BAP:

Benzylaminopurine

B5 medium:

Gamborg medium

SE:

Standard error

2,4-D:

2,4-Dichlorophenoxyacetic acid

NAA:

Naphthaleneacetic acid

T.HCl:

Thiamin hydrochloride

Glu. NAc.:

N-acetyl-d-glucosamine

FCF:

Fungal culture filtrate

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Acknowledgments

The authors are grateful to Dr Steve Millam, Gene Expression, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK for language correction of this manuscript. We also thank, Dr N. Sivasamy, Cotton Research Station, Srivilliputhur, TN, India for providing SVPR 2 seeds of cotton.

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  1. Department of Plant Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620 024, India

    M. Ganesan & N. Jayabalan

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  1. M. Ganesan
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  2. N. Jayabalan
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Correspondence to N. Jayabalan.

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Ganesan, M., Jayabalan, N. Isolation of disease-tolerant cotton (Gossypium hirsutum L. cv. SVPR 2) plants by screening somatic embryos with fungal culture filtrate. Plant Cell Tiss Organ Cult 87, 273–284 (2006). https://doi.org/10.1007/s11240-006-9164-5

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