Reducing recalcitrance of black pepper to Agrobacterium-mediated transformation: an efficient way through nucellar apomixis to establish transgenic and genome-edited plants at high frequency and scale-up through bioreactor

Abstract

Nucellar apomixis is truly clonal and is a powerful tool for broadening the genetic base of crops. Black pepper ( Piper nigrum L.), the ‘King of Spices’ is difficult to improve through conventional breeding. Although transgenesis and genome editing are prime strategies for rapid crop improvement, recalcitrance hinders genetic modifications. Here, we report a highly efficient Agrobacterium-mediated procedure for generating genetically modified black pepper plants using nucellar apomixis-derived embryos of the varieties Sreekara and Karimunda, with a 99% survival rate. Both Agrobacterium tumefaciens and Agrobacterium rhizogenes were efficient in transformation, and the AGL1 strain harboring the plasmid with mgfp achieved >90% frequency following 20 min in the infection medium, 30 s sonication, 10 min vacuum infiltration, and 4 days of cocultivation. Sugar type determined embryonal taproot development and soil establishment. Glucose-supplemented medium produced plantlets with well-developed root systems that displayed a high expression of PnPIN2. Transgenic plantlets survival ex vitro from glucose-supplemented liquid medium was 99%. The genome-editing efficiency of Pds using CRISPR/ Cas9 was 89%. Agroinfiltration of black pepper in this study is useful for high-throughput screening of disease resistance. Composite plants of black pepper generated at >60% efficacy is an easy strategy to develop plants expressing disease-resistant genes in roots to reduce yield loss, especially by root-rot. This study demonstrates that black pepper is an easy-to-transform crop, which reinforces speedy trait development through genetic modifications. Scale-up using temporary immersion bioreactors in this study fast-track high throughput accomplishment of untransformed/transformed/genome edited plants empower the market demand for black pepper.

Acknowledgements

We acknowledge the financial support of The Presidential Court, United Arab Emirates. This research project (MP 0824) was funded by The Presidential Court, United Arab Emirates.

Author contributions

Shina Sasi (Conceptualization, Writing-original draft, Validation, Methodology, Investigation, Formal analysis), Martin Kottackal (Conceptualization, Writing-original draft, Supervision, Analysis), Saranya Krishnan (Validation, Formal analysis, Review, Editing), and Khaled M.A. Amiri (Supervision-review & editing)

Data availability

All data supporting the findings of this study are included in this manuscript.

Conflicts of interest statement

The authors declare no conflicts of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

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