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Maternal haploid induction in African marigold (Tagetes erecta L.) through in vitro culture of un-fertilized ovules

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Abstract

A protocol for production of haploids from two heterozygous male sterile lines of Tagetes erecta using un-pollinated ovules as explant was standardized. Various factors affecting the in vitro gynogenic response viz., growth regulators, ovule developmental stage, basal media, sucrose concentration, photoperiod and cold shock duration were assessed in Tagetes erecta. Direct induction of parthenogenic embryos occurred in cultivar ‘Local Orange’ when the un-pollinated ovules were cultured in EMS (MS medium enriched with coconut water, AgNO3, PVP etc.) medium supplemented with 2 mg l−1 BAP and 0.5 mg l−1 NAA or 2 mg l−1 BAP along with 2 mg l−1 2,4-D. The developmental stage of the flower buds was vital in embryo induction from the excised ovules. The ovules collected from half-open flower buds were more responsive to gynogenesis as compared to ovules collected from un-open and fully open flower buds. EMS basal medium was found best for gynogenesis over the other commercially available basal media. Cold pre-treatment of flower buds at 4 ºC had no stimulatory effect and negatively affected the gynogenesis. The dark culture condition was imperative for direct parthenogenic embryo induction than 16 h light duration. The ploidy levels of 17 regenerants were determined by cytological studies, revealed 47.06% as diploids, 41.18% as haploids and 11.76% as mixoploids. These results were reconfirmed with flow cytometry analysis. The determination of ploidy level by counting the number of chloroplasts in stomatal guard cells of marigold was also established for rapid screening of haploids. The resultant haploids were successfully diploidised and are being utilized in the hybrid breeding programme at our institute. The standardized protocol for doubled haploids (DHs) production by un-pollinated ovule culture paved the way for Tagetes F1 hybrid breeding.

Key message

This is the first successful report of in vitro gynogenesis in Tagetes spp for production of doubled haploids (DHs). The protocol for obtaining high frequency parthenogenic embryo induction from unpollinated ovules was standardized and validated for rapid production of homozygous lines.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BAP:

Benzylamino purine

EMS:

Enriched Murashige and Skoog medium

NAA:

1-Naphthalene-acetic acid

SO:

Seracole Orange

LO:

Local Orange

PMS:

Petalous male sterile

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Acknowledgements

Authors sincerely acknowledge the ICAR-Indian Agricultural Research Institute, New Delhi for providing the research facilities and Dr. YSR Horticultural University, Andhra Pradesh for grant of deputation to the first author to carrying out this research work.

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Authors and Affiliations

  1. Horticultural Research Station, Kovvur, Dr.Y.S.R. Horticultural University, West Godavari, Andhra Pradesh, 534350, India

    K. Ravindra Kumar

  2. Division of Floriculture and Landscaping, ICAR-Indian Agricultural Research Institute, Pusa, New Delhi, 110012, India

    Kanwar Pal Singh, Reeta Bhatia & Sapna Panwar

  3. ICAR-Directorate of Floriculture Research, Pune, Maharashtra, 411005, India

    D. V. S. Raju

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  1. K. Ravindra Kumar
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  2. Kanwar Pal Singh
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  3. D. V. S. Raju
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  4. Reeta Bhatia
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  5. Sapna Panwar
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Contributions

KRK: He is responsible for conducting all the tissue culture related experiments, characterizing the ovule derived regenerants and writing manuscript. KPS: He has planned the experiments and provided the basic research facility for conducting these experiments. RB: She has planned the experiments, assisted in conducting the ploidy analysis and correcting the manuscript. DVSR: He has assisted in planning and data analysis of this study. SP: She has contributed in collecting, raising and maintenance of the marigold cultivars and ovule derived regenerants.

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Correspondence to K. Ravindra Kumar.

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Kumar, K.R., Singh, K.P., Raju, D.V.S. et al. Maternal haploid induction in African marigold (Tagetes erecta L.) through in vitro culture of un-fertilized ovules. Plant Cell Tiss Organ Cult 143, 549–564 (2020). https://doi.org/10.1007/s11240-020-01940-0

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