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Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench]

Abstract

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301–bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 µM acetosyringone and co-cultivated for 3 days on a medium containing 100 µM acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days.

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Abbreviations

MS:

Murashige and Skoog medium

hpt II:

Hygromycin phosphotransferase

gus A :

β-Glucuronidase gene

CaMV 35S:

Cauliflower mosaic virus 35S promoter

YEP:

Yeast extract peptone medium

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Acknowledgments

The authors are thankful to Bharathidasan University, Tiruchirappalli, Tamil Nadu, India for providing laboratory facilities to carry out the present work. The corresponding author (Andy Ganapathi) is thankful to University Grants Commission (UGC), Govt. of. India, for providing fellowship under UGC–BSR scheme.

Author contribution statement

The authors have made the following declarations regarding their contributions; MM and AG conceived and designed the experiments. KS and IR performed the experiments. KS, MM, and DE collected and analyzed the data. KS performed the Southern blot hybridization experiments. KS and AG contributed to the writing of the manuscript. MM and AG read and evaluated the manuscript.

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Correspondence to Markandan Manickavasagam or Andy Ganapathi.

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The authors declare that they have no competing interests.

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Manickavasagam, M., Subramanyam, K., Ishwarya, R. et al. Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench]. Plant Cell Tiss Organ Cult 123, 309–320 (2015). https://doi.org/10.1007/s11240-015-0836-x

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Keywords

  • Abelmoschus esculentus (L.) Moench
  • Acetosyringone
  • In planta genetic transformation
  • Sonication
  • Vacuum infiltration