Cry toxin expression in different plant parts of Bt cotton at different phenological stages

Compared to Bollgard-I, the utilization of Cry2Ab protein in Bollgard-II cotton cultivars enhances insect control. Field and laboratory studies demonstrated reduction in the numbers of bollworm-infested terminals, squares, and bolls in Bollgard-II cotton lines when compared to both Bollgard-I and non-Bollgard cotton cultivars. This indicates that the combination of Cry2Ab with Cry1Ac increased the overall expression of proteins. The expression of Cry protein varied across different plant parts, such as leaves, bracts, squares, and bolls. As the season progresses, the expression of Cry protein decreased in these plant parts. Leaves exhibited the highest levels of Cry protein expression, followed by squares, flowers, and bolls. Variation in the expression levels of delta endotoxins in different plant parts was one of the contributing factors to the survival of pest populations on Bt cotton. Using a commercially available QL 96 ELISA plate kit, the concentration of delta endotoxin in various plant parts at different phenological stages was determined in twelve BG-II cotton hybrids, namely Ajeet-155, JKCH-2245, RCH-3863, NCS-866, MRC-7373, JKCH-99, MRC-7387, NCEH-21, ANKR-3324, NCSI-1904, and NCHB 9902. Cry1Ac and Cry2Ab protein levels were determined from samples of flowers and fruiting parts (Rind, locule, seed, locule wall and seed) at 40, 75, 100 and 125 days after sowing (DAS) over two consecutive years 2018–2019 and 2019–2020. Cry1Ac protein content and expression was the highest at 100DAS in locule, seed and rind; followed by rind and locule wall and seed in green bolls at 125 DAS; followed by seed, locule, rind and flowers at 75 DAS; followed by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry2Ab protein content and expression was the highest at 125DAS in locule wall and seed and rind, followed by seed, locule and rind in green bolls at 100 DAS, followed by seed, locule, rind and flowers at 75 DAS, then by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry1Ac protein expression was less in comparison with Cry2Ab. The research findings indicate that the locule of BG-II cotton plants exhibited the highest expression levels of Cry1Ac and Cry2Ab proteins, followed by the seeds, rind, locule wall, and flowers. These results provide valuable insights into the distribution of Cry protein expression in different plant parts, which can contribute to a better understanding of insect control in Bt cotton cultivars.


Background
Bacillus thuringiensis (Bt) is a gram-positive, sporeforming, facultative, aerobic soil bacterium (Bacillaceae: Eubacteriales) from the silkworm Bombyx mori (Linnaeus L).The bacterium produces crystalline inclusion bodies in the course of sporulation and releases spores, Cry (Crystal) toxins or δ-endotoxins.Using the molecular biology tools, scientists have introduced genes from B. thuringiensis bacterium into cotton plants.These genotypes are referred to as transgenic Bt-cotton.The transgenic cotton containing Cry genes responsible for crystalline δ-endotoxin production in soil bacterium, B. thuringiensis var.kurstaki (Berliner) were transferred to cotton via Agrobacterium with CaMV 35S promoter.On ingestion in the midgut (having the alkaline condition) of susceptible insects Cry protein is activated by insect gut proteases, bind to receptors and get inserted into the microvillar brush-border membranes leading to disruption of osmotic balance, lysis of epithelial cells, starvation and ultimate death of insect (http:// www.lifes ci.sussex.ac.uk/ Home/ NeilC rickm ore/ Bt/).In India, the cultivation of Bt cotton expressing the Cry1Ac gene (BG I) has resulted in the effective control of all bollworms, including Helicoverpa armigera (Hubner), Erias vitella (Hubner) and Pectinophora gossypiella (Saunders) since 2002 (Bambawale et al. 2004).To maintain Bt cotton susceptibility and delay bollworm resistance to BGI, second-generation Bt cotton (BGII) expressing two Cry toxins (Cry1 Ac+Cry2Ab) was commercialized in the USA (2003) and India (2006), replacing more than 95 per cent of traditional cotton cultivation in India.For a long time (nearly ten years), Bt cotton was able to shield the crop from bollworms, reducing pesticide use from 46 per cent to less than 21 per cent.Pink bollworm has been more aggressive in the last three to four years since it has developed insecticide resistance (Kranthi 2016).However, for the Bt-transgenic cotton technology to be viable, the toxin expression levels must be present in sufficient amounts in appropriate plant parts at the appropriate time of the season to provide protection against major target insect pests, especially bollworms.The variance in overall Cry1Ac expression levels among Bollgards has been linked to the survival of various lepidopteran pests, suggesting that cultivars do not have the same degree of control during the crop season.Those Bt cotton hybrids which were being used by farmers contain adequate Bt toxin or not, period [at different phenological stages of cotton plant, i.e., (on leaf, square, bolls)] of Bt toxin expression is the major question.Considering the above objective, the present research aimed to study Cry toxin expression in different plant parts of Bt cotton at different phenological stages.

Methods
Cry1Ac and Cry2Ab protein was estimated from replicated samples of flowers and fruiting parts (Rind, locule, seed, locule wall and seed) at 40, 75, 100 and 125 days after sowing (DAS) in two consecutive years 2018-2019 and 2019-2020 from plant samples collected from different BG II cultivars from different districts of Vidarbha (research farms of DR.PDKV, Akola).

Methodology adopted
The qualitative estimation of Cry protein in Bollgard-II hybrids was performed at Biotechnology center, Dr PDKV, Akola and Mahabeej, Akola using commercially available QL 96 enzyme-linked immunosorbent assay (ELISA) plate kits (Table 1) of Design, Agrisure Diagnostics LLP, Jalna-Aurangabad as per manufacturer's protocol.
Samples of 50 mg were collected in a 1.5-ml microfuge tube.One ml of ice-cold sample extraction buffer was added and the mixture was macerated at 4000 rpm for 5 min at room temperature with a motor-driven pestle.It was then chilled on ice for 10 min before being macerated for 30 s. Finally, the sample was centrifuged for 10 min at 10,000 rpm.The supernatant was then extracted and stored at 4 °C.
The positive and negative standards were diluted with standard buffer before loading the sample extracts onto the plate.After that, the respective antibody-coated plates were opened.In each well of the ELISA plate, 50 µl of conjugate is added.Then, in each plate, 50 µl of negative and positive control were added.Then, in each ELISA plate, 50 µl of supernatant sample was added.After loading, the plates were incubated at 37 °C for 1.5 h in a humid environment and in the dark, after which the samples were discarded and the plates were washed twice with 200 µl wash buffer to remove traces of wash buffer.Plates were dried on paper towels before 100 µl of substrate solution (dark reaction) was added and plates were gently vibrated for 2-3 min.Plates were incubated in the dark for 15 min at 37 °C.Following the incubation period, 100 µl of stop solution was added.For color development, the plate was incubated at room temperature for 30 min.The plates were then run through an ELISA reader.

Data analysis
The qualitative analysis of Cry protein was estimated using the Gen5 3.04 EPOCH/2 program based on absorbance values of the plate at 405 nm,were depicted and the results were reported as optical density values.Optical density (OD) values were used to determine the presence or absence of a specific antigen or antibody in a sample.
The OD of the wells is then measured using a microplate reader at a specific wavelength, typically 450 nm for most ELISAs.The OD value is a measure of the intensity of the color developed in each well.

Flowers
The
Among all the hybrids, the Cry protein level was highest in ANKR-3066 cultivar at 100 DAS (3.943 OD).The lowest Cry2Ab protein concentration was recorded in NCS-866 (2.138 OD) at 75 DAS.The Cry2Ab protein content in rind remained slight increase from 75 to 100 DAS and increased from 100 to 125 DAS in twelve BG-II cotton hybrids.

Locule
The Cry2Ab protein content was estimated in locule at 75 and 100DAS.The Cry2Ab protein in locule at  2013), the level of Cry1Ac in squares decreased at the start of the sampling period before increasing the level in locule higher than other plant parts (seed, rind, locule wall).The current findings are consistent with the findings of Mahon et al. (2004) who found a good evidence for variable expression of Cry genes not only for one gene but also for two genes in a pyramid case, as expression of Cry2Ab in Bollgard-II were the highest in squares, followed by leaves and then the entire plant.Suji et al. (2013) quantified the Cry1Ac toxin during different stages of the cotton crop and discovered a typical pattern of toxin expression with maximum expression at the beginning of the vegetative phase, a decrease in concentration at the beginning of the reproductive stage, a gain in the middle of the reproductive stage and a decrease at the end of crop.Ramanjali et al. (2015) discovered the highest level of Cry1Ac toxins at 75 days after sowing, followed by decreasing toxins at 60 and 90 DAS.However, Zaman et al. (2015) discovered an increasing trend in Cry1Ac concentration from 60 to 90 DAS, with a peak value at 90 DAS.Cry1Ac toxin showed a decreasing trend after 90 days, with the lowest value at 150 DAS.Cry protein content was significantly high in locules of all BG-II cotton hybrids.The current findings were consistent with Srinivasa and Arjuna (2008) who reported higher toxin content in locules, rind and seeds of bolls than flower (anthers and pollen), square bracts and square buds.Cheema et al. (2015) reported that a variety of internal and external factors reduced transgene expression at both the transcriptional and translational levels.The same type of event was observed in all genotypes, with significant variation in toxin level, emphasizing the role of genetic background in transgene expression.The Cry toxin protein was abundant in early vegetative and mid reproductive stage of the crop and then decreased as the crop aged.The decline in Cryprotein expression in Bt cotton was caused by the parental background and a reduction in mRNA production levels, Apart from the reduction in Cry2Ab toxicity, the interference of condensed tannins with Cry1Ac toxicity was especially pronounced (Olsen and Daly 2000).Toxin expression was significantly high in all plant parts at 90 DAS and gradually declined to very low levels by 125 DAS in different hybrids of transgenic Bt cotton.Soujanya (2008) reported that the high toxin content at early to mid-reproductive stages of crop growth compared to later stages.

Conclusion
The highest levels of Cry1Ac and Cry2Ab expression were found in the locule of BG-II cotton plants, followed by the seeds, rind, locule wall and flowers.

Table 2
Cry1Ac protein content was estimated in flowers at 40 and 75 DAS during study period.The Cry1 Ac protein in flowers at 40 DAS in descending order was 2.795, 2.77, 3863, ANKR-3066, JKCH-99,NCSI-1904, MRC-7387,  NCHB-9902 and ANKR-3324, respectively.Among all the hybrids, Ajeet-155 recorded the highest Cry1Ac protein content and ANKR-3324 had the lowest Cry1Ac protein content.Among all hybrids Cry protein level was the highest in Ajeet-155 cultivar at 75 DAS (2.43 OD).The lowest Cry1Ac protein concentration was recorded in Ajeet-155 (2.048 OD) at 40 DAS.The Cry protein content in flowers remained uncertain with advance in the age of the crop from 40 to 75 DAS in twelve BG-II cotton hybrids.Cry protein (Cry1Ac) content in BG-II cotton hybrids (optical density)(2018-2019 and 2019-2020 pooled)*DAS days after sowing S.