Transgenic Research

, Volume 28, Issue 1, pp 33–50 | Cite as

Stability of expression of Cry1Ac and Cry2Ab2 proteins in Bollgard-II hybrids at different stages of crop growth in different genotypes across cropping seasons and multiple geographies

  • Anik Luke DhanarajEmail author
  • Alan Ray Willse
  • S. P. Kamath
Original Paper


Bollgard-II cotton expressing Cry1Ac and Cry2Ab2 insecticidal proteins has been commercially cultivated in India since 2006 to control bollworms. These genes were introgressed into parental germplasm of numerous hybrids. Therefore, it is imperative that these insecticidal proteins are expressed in sufficient quantities in different tissues, throughout the season irrespective of genetic background or environmental conditions for effective performance. Here, we document results of a comprehensive study on pattern of expression of Bt proteins across different stages of crop growth in > 2000 cotton hybrids (Gossypium hirsutum), across 12 cropping seasons tested in the Northern, Southern or Central zones in India, in terminal leaf, pre-candle square and boll epicarp tissues. Statistical analysis of variability using Linear mixed effect model was used to estimate factors contributing to variability in expression of Bt proteins. For Cry1Ac, variability was maximally contributed by genotype × season × plant growth stage effect in terminal leaves and boll epicarp, while season effect drove variability in pre-candle square. In Cry2Ab2, season effect drove variability in three tissue types. Pre-candle square tissue had most variability in expression of both proteins followed by terminal leaf and boll epicarp. Further, expression of Bt proteins in 234 G. hirsutum × G. barbadense hybrids showed similar expression patterns as intra specific hybrids though there was a significant difference in expression levels. Cry2Ab2 was expressed in significantly higher amounts when genes were in homozygous state. Bt proteins were also found to be expressed in varied amounts in different tissues and were expressed even when hybrids were grown at sub-optimal temperatures.


Bollgard-II Bt Cry1Ac Cry2Ab2 Mathematical model 



Bacillus thuringiensis


Gossypium hirsutum × Gossypium hirsutum


Gossypium hirsutum × Gossypium barbadense


Days after sowing

S1 to S6

Sampling timepoint 1 to Sampling timepoint 6


Multi location research trial


Kharif season



Over the year’s various team members from Mahyco Monsanto Biotech (MMB), ELISA lab from Monsanto Research Centre, Bangalore and various Monsanto teams from Technology Development, Regulatory, Trait Introgression, Biotech, from India and US were responsible for coordinating testing requirements of this project with technology partners. The project involved movement of plant tissues to the lab, protein estimation, analysis and data compilation. Douglas Sumerford made suggestions on modelling, data interpretation and presentation. Ponnuswamy Thillaichidamba generated the figures using ‘R’ or spotfire and Shiva Prakash helped with structuring, presentation and proof reading.

Author contributions

ALD contributed to generating protein data, analysis, reporting along with the team and collated, cleaned the data and wrote the paper. AW was responsible for the mathematical modelling and interpretation of results. KSP interfaced with Mahyco Monsanto Biotech (MMB) and external stakeholders for testing requirements and movement of tissues to the lab, interpretation and representation of results.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11248_2018_102_MOESM1_ESM.docx (481 kb)
Supplementary material 1 (DOCX 481 kb)


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Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Anik Luke Dhanaraj
    • 1
    Email author
  • Alan Ray Willse
    • 2
  • S. P. Kamath
    • 1
  1. 1.Monsanto Research CentreBangaloreIndia
  2. 2.Monsanto CompanySt. LouisUSA

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