Engineered resistance and risk assessment associated with insecticidal and weeds resistant transgenic cotton using wister rat model

Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to the non-transgenic control plants. Evaluation of effect of temperature and soil nutrients availability on transgene expression in cotton plants was done at two different cotton growing regions, Multan and Lahore, Pakistan and results suggested that despite of higher temperature in Multan field, an increased level of Cry and cp4EPSPS proteins was recorded due to higher soil organic matter availability compared to Lahore field. Before commercialization of any transgenic variety its biosafety study is mandatory so, a 90 days biosafety study of the transgenic cotton plants with 40% transgenic cottonseeds in standard diet showed no harmful effect on wister rat model when studied for liver function, renal function and serum electrolyte.


Scientific Reports
| (2022) 12:2518 | https://doi.org/10.1038/s41598-022-06568-y www.nature.com/scientificreports/ Among Bacillus thuringiensis (Bt) genes such as cry1Ac + cry2Ab, cry1Ac + cry1F or cry1Ac, have been mainly used in genetically modified (GM) crops including cotton and maize [5][6][7][8] . There is no doubt that abiotic stresses, such as temperature, light, water scarcity, salinity, or nutrient deficiency, directly affect the quality and quantity of many crops production 9 . It has been reported that the expression of transgenes is affected by extreme environmentalfactors in GM crops such as water stress and nitrogen deficiency affects the Bt maize 10,11 . Chemical herbicides are used to control weeds but the differentiation between crop plants and weeds by herbicides is still a problem. Development of herbicide tolerant crops is the solution to this problem which is anticipated 4 . Synthetic herbicides have been used worldwide in agriculture to control weeds since 1940 12 . Registration of glyphosate-resistant soybean in (1996) and cotton in (1997) has made this technology for crops producers to accept more readily. Moreover, adaptation of Glyphosate resistant crop has generally reduced the opposing environmental and health influences of weed management 13 .
The first GM crop was introduced in 1994 and since then the concerns about the release of GM crops in the environment has increased tremendously 14 . Therefore, for the past few decades, the biosafety study of GM crops has become an area of intense interest for the idea of general acceptance of GM crops to consider them as safe to use as food and feed 15 . Risk assessment study is necessary before the release of any GM crop in environment for its commercial use and GM crop can only be considered as safe to use when, after risk assessment study, the GM crop is equivalent to the non-GM crop in terms genomic and phenotypic characteristics 16 .
In current study, a local transgenic cotton variety VH-289, harboring cp4EPSPS, Cry1Ac and Cry2A genes, was evaluated for protein expression and biosafety study. The effect of temperature, water and soil was also evaluated through plantation at two different cotton growing regions i-e Multan and Lahore. Local crop verities are preferred to use for the development of transgenic crops as they are best suited to the local environmental conditions. VH-289 is a local cotton variety. This variety was selected for the study as it has 95% germination rate and out of germinated embryos 98% plant formation rate which one of the highest germination and plant formation rates among various local cotton accessions 17 .

Materials and methods
Acquisition of Transgenic seed. The Transgenic cotton seeds were acquired from the Plant Biotechnology Lab of Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. It has been confirmed that the experimental samples of plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from authorities of Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan for collection of plant specimens. All methods for using rats in experiments were carried out in accordance with relevant guidelines and regulations of Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan and ARRIVE guidelines.

Molecular confirmation of transgenic cotton plants. The experimental protocol was approved by
Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan which are also similar as American Veterinary Medical Association (AVMA) Guidelines. Genomic DNA was isolated from leaves of transgenic cotton plants by following the same procedure 18  Soil nutrients, water composition and temperature analyses. The advanced generations of transgenic cotton plants of VH-289 were grown at two cotton growing regions i-e Multan and Lahore to compare the impact of abiotic factors such as soil nutrients, water composition and temperature on transgenes expression at protein level. The data regarding irrigation water and soil composition of both locations was analyzed from laboratory testing facility of agriculture department government of Punjab Pakistan (http:// www. agrip unjab. gov. pk/ labs), while temperature was recorded from the official website of AccuWeather (https:// www. accuw eather. com/). Bradford assay 19 was used for the quantification of total crude protein from young leaves of transgenic cotton plants of VH-289. The value of each sample was determined with the help of spectrophotometer (Bradford 1976). Protein expression of Cry1Ac, Cry2A and cp4EP-SPS was quantified through Enzyme Linked Immunosorbent Assay, (ELISA) using Envirologix Kit (Cat # 051) according to procedure provided by the manufacturer and quantification of proteins was done as ng per gram of fresh tissue weight.

Expression analysis of transgenic cotton plants.
Temporal Bt and cp4EPSPS protein expression in transgenic cotton plants grown at different locations. The leave samples of transgenic and control cotton plants were collected from two locations Multan and Lahore in the two inner rows/replicate plots. Terminal leaves from the middle part of the plants were taken at 15 days interval starting from 15 days after sowing (DAS) upto 120 days after sowing of transgenic cotton plants from both locations. The collected samples were stored in liquid nitrogen and brought to the laboratory for protein extraction. The crisped-dried samples from three plants per plant were placed in a 1.5 mL tubes. Quantification of Cry1Ac, Cry2A and cp4EPSPS protein was done using method available with enzyme-linked immunosorbent assay (ELISA) kits (EnviroLogix). Five milligrams ground samples were processed for analysis. www.nature.com/scientificreports/ Absorbance readings of the samples were taken at 595 nm. A correlation of Bt protein expression with vegetative and reproductive stages of transgenic cotton crops at different temperature and time was calculated in this way.

Insect bioassay of Bt protein. The endotoxins efficacy against targeted insect pest i-e American Boll-
worms Heliothis armigera (2nd instar) larvae was evaluated through laboratory biotoxicity assays. The 60 days old leaves from upper, middle and lower portion of transgenic cotton plants were taken and placed in perti plate containing moist filter papers. Three 2nd instar larvae of Heliothis were subjected to each leave for 3 days. Mortality rate was observed and recorded after 2-3 days by the following formula: Herbicide spray assay. For the evaluation of expression of cp4EPSPS gene, leaves of transgenic cotton plants were subjected to glyphosate spray assay at both locations respectively. Roundup™, commercially available herbicide Glyphosate, was used for this assay. Expanded leaves of 45 days old field grown transgenic and control cotton plants, at both locations (Multan and Lahore), were sprayed in concentration 1900 ml/80 L water/0.404 hac. Transgenic cotton plants and control plants were grown in separate lines and the effect of glyphosate spray on weeds and transgenic along with control cotton plants were observed regularly up to 15 days as described by 3 .

Liver function test (LFT), renal function test (RFT) and serum electrolyte test for biosafety studies.
To determine the effect of potential unintended effects of transgenic proteins, an additional animal study using wister rat model was conducted. Briefly, 30 male rats of about 5-6 weeks age were selected and randomly divided into three groups namely G1, G2 and G3. Rats in group G1 were reared on standard diet while rats in G2 and G3 were respectively reared on a diet containing non-transgenic and 40% of transgenic cotton seeds (expressing Cy1Ac, Cry2A and cp4EPSPS) or it's near isogenic one. Animals were kept for 90 days under standard housing conditions 20 . At the end of the study blood samples were collected from all the animal groups in a serum separator tubes and sent to university of veterinary and animal science (diagnostic lab) for liver function test (LFT), renal function test (RFT) and serum electrolyte.
Statistical analysis. Statistical analysis were performed using Ghaphpad Prism 7.0 software. Asterisks, *, **, *** and **** were used for denoting statistically significant values as p-values ≤ 0.05, 0.01, 0.001 and 0.0001 respectively for the comparison between values of transgenic and non-transgenic control, while "ns" symbol was used for indicating non-significant values. Soil, water and temperature analyses. Before starting expression analysis, data of temperature was recorded from Multan and Lahore fields. Multan region found to be 2 °C to 3 °C hotter than Lahore ( Fig. 2A).

Results
The results of soil analysis showed that the organic matters, available phosphorus, available potassium and soil saturation percentage were higher in Multan soil as 24%, 22.39 mg/kg, 14.93 mg/kg and 5.3% respectively as compared to Lahore soil components (Fig. 2B), while water analysis revealed that both areas water was favorable for irrigation considering the water quality guidelines for irrigation in Pakistan 21 (Fig. 2C).

Temporal expression of Bt and cp4EPSPS protein.
Correlation analysis for the evaluation of Cry and cp4EPSPS protein expression, sampling from T2 generation after every 15 days intervals for at-least 120 days after the date of sowing from cotton plants experimental area, Multan and from Lahore fields displayed variations. A higher trends of protein values of Cry1Ac, Cry2A and cp4EPSPS were recorded in cotton plant samples taken from Multan field as compared to Lahore field (Fig. 3 A-C).

Insect bioassay. Lepidopteran insect bioassay was used to check the efficacy of Bt genes against American
Bollworms Heliothis larvae (2nd instar) in transgenic cotton plants. Mortality rate was evaluated after 2-3 days of insect assay on fresh cotton leaves of transgenic and non-transgenic control plants placed on wet filter paper in petri plates. On 2nd day 98.5 and 3rd day a 100% mortality rate of Heliothus larvae was found on transgenic cotton plants leaves with almost no damaged to leaves as compared to non-transgenic cotton plant leaves which were heavily damaged by bollworms larvae and the at third day and almost completely damaged at fifth day of insect assay ( Fig. 4A-C).
Herbicide spray assay. The efficacy of cp4EPSPSgene in transgenic cotton plants was confirmed through Glyphosate spray assay. Initially, the transgenic cotton plants in fields, compared to non-transgenic control plants and weeds, showed no effect of glyphosate spray (1900/acre) however, necrosis started at third day and leads to complete death of non-transgenic plants along-with weeds until fifth day (Fig. 5A-C).

Discussion
Cotton as a backbone of Pakistan's economy has been facing many challenges in the form of insect pest and weeds. According to an estimate upto 40% of the total losses of cotton crop is due to the weeds, 30% losses occur due to insect pest which are also major limiting factor for various other economically important crops 2,4,22 . In past insects were controlled by spraying pesticide and the expenditure alone in USA has reached to 40 billion USD annually 23 . Biotechnology holds promising solution for these types of problems. The use of genes responsible for adequate quantity production of toxin cry protein for insects and alternate EPSPS production through genetic modification of cotton with synthetic cp4EPSPS gene can be the possible measures against such type of problems opted in current study.    www.nature.com/scientificreports/ In current study the effort has been made to evaluate the protein expression and conduct biosafety study of transgenic cotton variety VH-289 harboring two insecticidal genes Cry1Ac and Cry2A genes in combination with a weedicide cp4EPSPS gene and to find out the effect of environmental factor such as temperature, soil nutrients and water quality on gene expression level. Based on the background genetically modification of cotton plants for resistance against insects and weeds through incorporation of the Cry1Ac & Cry2A and cp4EPSPS genes into Pakistan's local variety of cotton has been cemented.
Molecular approaches such as PCR were used for confirmation of transgene in advanced generation of cotton variety VH-289 as was done by 17,24 . Transgenic cotton plants successfully amplified the DNA fragments of 1.7 kb, 585 bp and 358 bp for Cry1Ac, Cry2A and cp4EPSPS respectively (Fig. 1). To Different transgenic cotton plants showed varied concentration of Cry1Ac, Cry2A and cp4EPSPS proteins at different time intervals when subjected to temporal expression as was the case with Bakhsh et al., (2010) having variable temporal expression of Cry proteins in different transgenic cotton plants. The protein expression for both Cry and cp4EPSPS gradually increased from 15 DAS up to 60 DAS and started decreasing until 120 DAS. The maximum expression of Cry1Ac, Cry2A and cp4EPSPS was recorded at 60 DAP where Cry1A was found to be 25.6 ng/g and 23.11 ng/g, Cry2A 6.37 ng/g and 4.71 ng/g and cp4EPSPS 25.73 ng/g and 23. 03 ng/g in transgenic plants of Multan and Lahore respectively (Fig. 3A-C).
As the expression of Bt genes vary with temperature as described by Chen et al., (2005), where expression of Cry1Ac gene significantly reduced at 37 °C but in contrast to this study, Multan region which is on average 2 °C to 3 °C hotter than Lahore as per data recorded ( Fig. 2A), it was found that protein expression of Cry1Ac, Cry2A and cp4EPSP in Multan region was higher than Lahore region right from the beginning till end of the growing season (Fig. 3A-C) this higher protein expression can be attributed to the higher soil organic matters, phosphorus, potassium and saturation level in Multan field as 24%, 22.39 mg/kg, 14.93 mg/kg and 5.3% respectively as compared to soil of Lahore field (Fig. 2B). Similar results were reported by 25 they got elevated level of Bt endotoxin expression by 14% on increasing dose of N fertilizer.
The bio-toxicity leaf assay was used to evaluate the efficiency of the transgenic proteins Cry1Ac and Cry2A in cotton plants compared to non-transgenic control plants using Heliothis (2nd instar larvae). The larvae were freed in each petri-plate with a transgenic cotton plant leaf, along with a control, non-transgenic cotton plant in a separate plate, died completely at day 3 of bio-toxicity assay (Fig. 4A). These results are in accordance with the results reported by 26 . The study of transgenic endotoxin protein level is very crucial, as it must be in a sufficient quantity at the time of insect infestation to protect the crop against lepidopterons, especially the boll worms 27 . A 100% insect mortality was recorded in petri plate of transgenic cotton plants compared to non-transgenic control cotton leaves where insects survived and grow with passage of time. Similar results were obtained by 27 .
A glyphosate-herbicide (1900 mL/acre) was sprayed to the field growing transgenic cotton plants to determine the herbicide resistance. The non-transgenic control cotton plants along-with weeds first showed the necroticspot and finally started dying as compare to the healthy transgenic plants (Fig. 5). These results are parallel with other similar studies including 28 . In view of the above results, it can be concluded that transgenic cotton plants that contain the cp4EPSPS gene have considerable resistance against weedicide. www.nature.com/scientificreports/ Before release of any GM crop in the environment for its commercial use it is very important to evaluate it for its any possible toxicity, therefore the biosafety study of GM crops has become area of interest for the past few decades 29 . The cottonseed are used as a protein source for the livestock and also used as a rich source of vegetable oil. Biosafety studies of many transgenic plants expressing insecticidal protein such as Cry1Ac and Cry2A especially in cotton plants has been done thoroughly 30,31 . This study was designed to evaluate transgenic cotton variety VH-289, containing Cry1Ac, Cry2A and cp4EPSPS for its expression analysis and biosafety study for the safe release in environments. Similar studies have also been reported previously [32][33][34] . It has been proven by the previous studies that the Cry1Ac and Cry2A proteins are safe to use in GM crops and cause no harm to the mammals because of their acidic environments in stomach 35 . Our biosafety study on wister rats, which involved using 40% GM cottonseed as a diet in (G3) group and 30% Non-GM cottonseed as diet in (G2) group, showed no comparable change of harmful effect on LFT, RFT and serum electrolyte when compared to the standard diet control group (G1). These results are consistent with the previous studies 20, 36 .
The study utilizes the novel idea of co-expression of three gene including two Bt genes and one weedicide resistant gene for the exploration of the factoring affecting their expression in local cotton variety VH-289. The expression of Cry1Ac and Cry2A in VH-289 has been found very effective against chewing insects (Fig. 4A-C) and the results of expression of cp4EPSP has also been very encoring for the control of weeds (Fig. 5A-C). Previous studies have shown that the success of the control of chewing insects including cotton bollworm, pink bollworm, spotted bollworm and armyworm depends on constantly higher expression of transgenes 27,37 . Similarly, higher expression of weedicide resistant gene (cp4EPSPS) is necessary for the effective control of weeds 3 . The study has also given the insight into exploring the external factors such as temperature, soil nutrients and water quality on the transgenic cotton variety VH-289. The study also evaluated the risk assessment associated with the locally developed transgenic variety VH-289 and concluded that no harmful effects were found on the wister rat model (Tables 1, 2, 3) and it is the safe to release for the commercial use in the field.