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Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L.

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Abstract

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA3), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO3, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.

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Fig. 1a, b
Fig. 2a–h
Fig. 3a–c
Fig. 4a–f
Fig. 5a–c
Fig. 6a–e
Fig. 7a, b
Fig. 8a–i
Fig. 9a–c

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Abbreviations

BA:

Benzyladenine

GA3 :

Gibberellic acid

IAA:

Indole-3-acetic acid

MS:

Murashige and Skoog

NAA:

α-Naphthaleneacetic acid

PGR:

Plant growth regulator

TIBA:

2,3,5-Triiodobenzoic acid

TRIA:

Triacontanol

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Acknowledgments

The authors are grateful to the Department of Science and Technology, Government of India, for the research grant. R.P. is thankful to the Council of Scientific and Industrial Research, New Delhi, India, for the research fellowship.

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Authors and Affiliations

  1. Plant Cell Biotechnology Department, Central Food Technological Research Institute (a Constituent Laboratory of the Council of Scientific and Industrial Research), Mysore, Karnataka, 570 020, India

    Rangan Parimalan, Akshatha Venugopalan, Parvatam Giridhar & G. A. Ravishankar

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  1. Rangan Parimalan
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  2. Akshatha Venugopalan
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  3. Parvatam Giridhar
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  4. G. A. Ravishankar
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Correspondence to Parvatam Giridhar.

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Parimalan, R., Venugopalan, A., Giridhar, P. et al. Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L.. Plant Cell Tiss Organ Cult 105, 317–328 (2011). https://doi.org/10.1007/s11240-010-9870-x

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