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Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv. JRC 321 using shoot tip explants

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Abstract

We have developed a reproducible method of Agrobacterium tumefaciens mediated stable genetic transformation of white jute (Corchorus capsularis cv. JRC 321) utilizing the shoot organogenesis potential of the shoot tip apical meristem. A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 was used in the transformation experiments. The explants were subjected to varying durations of preculture and cocultivation with A. tumefaciens in the presence of acetosyringone in order to optimize the conditions conducive for the highest expression of transgene. A schedule of 1 day preculture of shoot tips followed by 3 days cocultivation was optimized for Agrobacterium mediated stable genetic transformation of C. capsularis cv. JRC 321. The optimized lethal doses of the antibiotic hygromycin B for shoot tips (12 mg/L) and for 5 days old seedlings (14 mg/L) were employed in efficient selection of the transformed tissues. This method of transformation resulted in a mean transformation efficiency of 4.09 %. Stable expression of the intron harbored gusA transgene was observed in mature organs of the transformed plants and their progenies. Genomic integration and inheritance of the hpt transgene was further confirmed by Southern blot analysis. The transformed plants exhibited normal morphology and most of them produced viable progenies, many of which segregated in a 3:1 ratio following Mendelian inheritance for a single dominant locus. However, strong P value support for 3:1 segregation ratio was obtained in case of two lines of independent transformants. Nevertheless, the method of transformation mentioned in this protocol could be effectively implemented in genetic transformation of many other cultivars of jute due to the genotype independent regeneration potential of the shoot tip explants.

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Abbreviations

ACM:

Agrobacterium culture medium

BAP:

N6-Benzylaminopurine

BM:

Basal medium

CCM:

Cocultivation medium

GA3 :

Gibberellic acid

GM:

Germination medium

GUS:

β-Glucuronidase protein

gusA :

β-Glucuronidase gene

hpt :

Hygromycin phosphotransferase gene

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

LB:

Luria Bertani

MS:

Murashige and Skoog (1962)

OD600 :

Optical density at a wavelength of 600 nm

PCM:

Preculture medium

PCR:

Polymerase chain reaction

RM:

Rooting medium

SEM:

Shoot elongation medium

SGM:

Shoot growth medium

SM:

Shooting medium

vir :

Virulence genes of Agrobacterium tumefaciens

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Acknowledgments

We thank University Grants Commission (UGC) for funding in the form of UGC/UPE scheme at university of Calcutta, Department of Biotechnology (DBT), Government of India and RKVY project under Government of West Bengal, and Ministry of Agriculture, Government of India. Comments and suggestions on the manuscript from two anonymous reviewers are gratefully acknowledged.

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Authors and Affiliations

  1. Plant Molecular Biology and Biotechnology Laboratory, Department of Botany, University of Calcutta, Kolkata, 700019, West Bengal, India

    Prosanta Saha, Karabi Datta, Shuvobrata Majumder, Chirabrata Sarkar, Sailendra N. Sarkar & Swapan K. Datta

  2. Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India

    Shyamsundar P. China

  3. Biotechnology Unit, Division of Crop Improvement, Central Research Institute for Jute and Allied Fibres (CRIJAF), Barrackpore, Kolkata, 700120, West Bengal, India

    Debabrata Sarkar

  4. Indian Council of Agricultural Research, Krishi Bhawan, New Delhi, 110001, India

    Swapan K. Datta

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  1. Prosanta Saha
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  2. Karabi Datta
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  3. Shuvobrata Majumder
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  4. Chirabrata Sarkar
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  5. Shyamsundar P. China
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  6. Sailendra N. Sarkar
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Correspondence to Karabi Datta.

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Saha, P., Datta, K., Majumder, S. et al. Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv. JRC 321 using shoot tip explants. Plant Cell Tiss Organ Cult 118, 313–326 (2014). https://doi.org/10.1007/s11240-014-0484-6

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