Study zone

The samplings were carried out in the Yaqui Valley, located in the south of the state of Sonora, in four different agricultural plots and where two varieties of genetically modified cotton were planted, during the 2014 cycle (Table 1).

A variety that does not express the Cry1Ac and Cry2Ab proteins was also planted in each agricultural plot, as a refuge.

Take and sample preparation

Samples of leaves were taken in six dates, in the first four dates samples of square were also taken and acorns were collected in the last two dates. The sample of vegetal tissue was taken at the height of the third node from top to bottom. For immunological detection, a total of 120 samples were taken, corresponding to 60 leaves, 40 squares and 20 acorns, of these 24 (12 of leaves, eight of squares and four of leaves) were from the refuge. For gene detection only samples were taken on three dates, taking five samples each time, from each field, which were processed independently.

For the case of the leaf, it was taken into account that it was completely unfolded and integrated, so that the square and acorn were careful not to have any alteration at the time of sampling. 30 mg of leaf tissue, 80 mg of square tissue and 9 mg of acorn tissue were taken, all fresh. For the immunological detection tests, the tissues were processed fresh with their respective extraction buffer and the maceration proceeded. For gene detection the tissues were placed in contact with liquid nitrogen and subsequently crushed with the help of a sterile micropystile.

Side flow strips

Before proceeding with protein and gene quantification, the presence of transgenic proteins was evaluated by the use of lateral flow strips, following the methodology suggested by the manufacturer (EnviroLogix, Portland, ME, USA). For this, a 1.5 mL tube was made of a sample composed of five portions of the different tissues of cotton, 0.5 mL of extraction buffer for leaf analysis and 1.5 mL for square and acorn were added. The sample was homogenized with the help of a micropystile. Subsequently, a strip was introduced into the tube and left for 10 min to observe and interpret the results. The samples that were positive were analyzed by the Elisa test and real time PCR.

Immunosorbent assay linked to enzymes (Elisa)

A ‘sandwich’ test was performed for enzyme-linked immunosorbent assay (Elisa) following the protocol of the QualiPlate AP 051 Kit of EnviroLogix. For the calibration curves, five standards were used in concentration of 3.13, 6.25, 12.5, 25 and 50 ng mL^-1 of protein Cry1Ac and Cry2Ab. The contents of the wells were mixed and then placed in incubation at room temperature for one hour. After incubation the contents of the wells were shaken, the wells were completely flooded with the washing buffer, then emptied. This washing step was repeated three times.

100 μL of p-nitrophenyl phosphate (substrate S1) was added to each well, mixed and incubated at room temperature for 30 min. It was read on the SpectrOstar Nano spectrum (BMG Lab Tech, Ortenberg, Germany) at 405 nm for the Cry2Ab protein. Four washes were made to subsequently add 100 μL of 3,3’, 5 5’-tetramethylbenzidine (substrate S2) to each well, incubated at room temperature for 30 min and read at 650 nm for Cry1Ac. The interpretation of the results was made based on the absorbances of each of the samples analyzed (Table 2).

Extraction of RNA from plant tissues

The plant tissue to be analyzed was placed in a 1.5 ml tube and 500 μL of PureLink^® Plant RNA Reagent (Life Technologies™, Carlsbad, California, USA) was added and subsequently homogenized. The samples were incubated for 5 min at room temperature keeping the tube horizontally for maximum extraction. They were centrifuged at 14 000 RPM for two min at 4 °C and the supernatant was transferred to a new sterile tube. 100 μL of 5 M NaCl was added to clarify. 300 μL of chloroform was added and mixed vigorously.

It was centrifuged at 14 000 RPM for 10 min at 4 °C and the supernatant was transferred to a new sterile tube. The RNA was precipitated with an equal volume of 100% cold isopropanol to the aqueous phase which was separated and mixed by inversion. It was incubated for 10 min at room temperature. It was incubated at room temperature for 10 min and subsequently centrifuged at 14 000 RPM for 10 min at 4 °C. The supernatant of the tube was decanted leaving the pellet of RNA, for the washing 500 μL of 75% ethanol was added, it was stirred and centrifuged at 14 000 RPM for five minutes at 4 °C, afterwards the RNA pellet was dried by approximately 5-10 min and resuspension was carried out with 50 μL of DEPC water mixing with the micropipette. It was left to resuspend at 4 °C and then proceeded to determine the concentration and purity of the extraction.

Synthesis of the cDNA by reverse transcription

The retrotranscription was performed using the GoTaq^® Probe 2-Step RT-qPCR System kit (Promega, Madison, Wisconsin, USA) and was carried out in 0.2 mL PCR tubes containing the following mixture for each reaction: 2 μL RNA (200 ng), 1 μL of oligo (dT) 15 primers, 1 μL of random primers and 3 μL of free water of RNAse/DNase to complete a volume of 7 μl. RNA and primers were denatured in a heating block at 70 °C for 5 min, placed immediately on ice for 5 min and then centrifuged for 10 s.

For the alignment and synthesis of cDNA the following components were added: 4.9 μL of free RNAse/DNAse water, 4 μL of GoScript 5X reaction buffer, 1.6 μL of MgCl2, 1 μL of nucleotides, 0.5 μL of recombinant RNasin as inhibitor, 1 μL of GoScript reverse transcriptase, to complete a volume of 13 μL and add it to the 7 μL of the denaturation step. For the alignment they were placed at 25 °C for 5 min, for elongation at 42 °C for 45 min and to inactivate the reverse transcriptase at 70 °C for 15 min. It is proceeded to RT-PCR or were stored at -20 °C.

Real-time polymerase chain reaction (RT-PCR)

Real-time PCR amplification was carried out in 0.2 mL PCR tubes containing the following mixture for each reaction for the samples: 2 μL of RNA (200 ng), 7 μL of RNAse/DNase-free water, 10 μL of GoTaq^® Probe qPCR Master Mix (Promega, Madison, Wisconsin, USA) and 1 μL of the primer/probe mix (300 nM), finally obtaining a volume of 20 μL for each reaction.

To perform the calibration curves, the mixture for each reaction was as follows: 5 μL of each point on the curve, 4 μL of RNAse/DNAse-free water, 10 μL of GoTaq^® Probe qPCR Master Mix and 1 μL of the first mix/probe (300 nM), to obtain a final volume of 20 μL for each reaction. All samples were analyzed in the 7500 Fast Real-Time PCR system (Life Technologies™, Carlsbad, California, US) under the conditions shown in Table 3.

The primers used for the amplification in each of the reactions are shown in Table 3. For the quantification, a calibration curve was made by serial dilutions from an initial concentration of 200 000 copies per microliter (Table 4 y 5).

Analysis of results

The results obtained in protein detection were analyzed by means of an analysis of variance (Anova), based on a linear model of fixed effects for p< 0.05, from the means obtained in the analyzes. The dates were taken as a source of variation (six levels), without interaction with the varieties or the sites, elements that were only taken to increase the size of the sample. For the conformation of the calibration curves of the Cry1Ac and Cry2Ab proteins, a simple linear regression analysis was performed, presenting the regression equation and the coefficient of determination. The results obtained in gene detection were analyzed by means of the evaluation of medians in the Kruskal-Wallis test with p< 0.05. The statistical package Statgraphics Centurion XV (StatPoint Technologies, Inc.) was used.

Immunological detection

Side flow strips

Prior to the analyzes by ELISA and RT-qPCR, analyzes were performed by immunochromatography, with 96 of the 120 samples collected and presumably expressing the Cry1Ac and Cry2Ab proteins. ^{Dohare and Tank (2014)} detected the presence of the same proteins, which were sought in this study, by analyzing a total of three varieties of genetically modified cotton from the Bollgard II^® line. While ^{Larbouga et al. (2013)} used the same tool for a similar study five varieties of cotton from the same line.

This technology allows the immobilized and specific antibodies for each protein to be incorporated into a strip of nitrocellulose coupled to a molecule that will react in the presence of the proteins analyzed (^{Lipton et al., 2000}; ^{Wang et al., 2007}). That is why this tool is widely used today due to the speed and ease of obtaining results, either in the field or in the laboratory.

Quality parameters in Elisa

To verify the linearity and quantification of the transgenic proteins, calibration curves were made with five known concentrations at different levels. For the Cry1Ac protein R² was obtained higher than 0.99, the same for the Cry2Ab protein. The values of R² recommended to ensure the linearity of the method is ≥0.99, agreeing with the results obtained by ^{Shan et al. (2007)}, in the construction of curves for the quantification of the Cry1Ac protein in different cotton tissues. In the Figure 1 shows the linearities of the calibration curve system for the Cry1Ac and Cry2Ab proteins.

Figure 1 Calibration curves for the Elisa test of the proteins, a) Cry1Ac and b) Cry2Ab.  

The accuracy and precision of the method was evaluated by performing all the analyzes in duplicate of each of the quantified samples. ^{Karuri et al. (2013)} obtained coefficients of variation of less than 15%, similar to those obtained in this study, which reflects the quality of the results.

Quantification of Cry proteins through Elisa

The results of the analysis of the quantification of protein expression for Cry1Ac and Cry2Ab in the four farms analyzed, in six moments and two different cultivars, showed an expression rate which decreased over time, with highly significant differences (p< 0.0025 for Cry1Ac and p< 0.0045 for Cry2Ab), reaching levels to which it could not be detected, this for the square and acorn cases. The minimum level of Cry1Ac protein quantified in leaf (Table 5) was 0.3 μg g^-1, while in tables and acorns (Table 6 and 7) the trend occurred to zero, as it was undetectable in the field on February 5.

The maximum levels of expression occurred in leaves, with up to 8.5 μg g^-1 being found in early stages of cultivation, while in tables and acorns it was 2.5 μg g^-1. ^{Greenplate et al. in 2003}, in a similar study with the cotton variety MON-15985, they were able to determine the Cry1Ac protein; through the Elisa test, obtaining average values of 4.8 μg g^-1 in leaf tissue, results very similar to those obtained in this study.

In the analysis of the expression of proteins conferred to GM crops, it is important to bear in mind their direct relationship with the growth and development of the plant itself and that both are influenced by environmental factors. The temperature, light, salinity, water availability, nutritional status and environmental interactions (with biotic agents of their environment) are identified as the main environmental factors that affect the growing conditions and therefore the development of the plants and their composition ^{Sivasupramaniam et al. (2008)}; (^{Jamal, 2009}); ^{Knight et al. (2013)}.

According to ^{Kranthi et al. (2005)}, the Cry1Ac protein tends to vary between the different hybrids and the different tissues of the plant, finding the highest levels in terminal leaves, followed by square, acorns and finally the flowers. This variation was clearly observed throughout the samplings and analyzes carried out. A very similar behavior was observed and quantified with the Cry2Ab protein in all tissues analyzed. The highest levels were quantified in leaves with 74 μg g^-1 in the vegetative stage, while in later stages lower levels of up to 8.5 μg g^-1 were obtained, without reaching undetectable levels. In table and acorn, similar levels were obtained to leaf levels in early stages, quantifying up to 92.6 μg g^-1, while in late stages levels of up to 0.3 μg g^-1 were obtained, ^{Knight et al. (2013)}.

Extraction and determination of the concentration and purity of RNA from plant tissues

The absorbance at 260 nm and the A260/280 ratio gives us an indication of the amount of RNA and the degree of contamination of organic and inorganic impurities. RNA extracts were obtained from leaf, square and acorn tissues with A260/A280 ratios between 1.7-2, which indicates a high degree of purity of isolated RNA, according to ^{Kang (2012)}. The extraction yields from leaf were superior to 1 000 ng μL^-1, while for square and acorn they were between 50 and 340 ng μL^-1.

Quality parameters in gene detection

To verify the linearity and gene quantification of the transgenic proteins, calibration curves were made with six concentrations, in number of copies, known at different levels. For the protein Cry1Ac R² was obtained higher than 0.99 in most cases, the same for the Cry2Ab protein. The values of R² recommended to ensure the linearity of the method is ≥0.99. In the Figure 2 shows the linearities of the calibration curve system for the gene quantification of the Cry1Ac and Cry2Ab proteins.

Figure 2 Calibration curve for the Cry1Ac (A) and Cry2Ab (B) proteins in RT-Qpc.  

The efficiency of the method (E) was measured, which is the adequate duplication of the sequence of interest in each cycle. The efficiencies obtained were 107.63% for Cry1Ac and 109.72% for the Cry2Ab protein. An adequate reaction yields efficiency with values of 90-110%. For ^{Kavanagh et al. (2011)} the slope should be another quality parameter evaluated in the polymerase chain reaction tests, those obtained in this study were -3.1515 for Cry1Ac and -3.109 for Cry2Ab, which fall within those recommended by bibliography that they should oscillate between -3.58 and -3.1.

The results obtained in the validation meet the criteria established in the minimum information guide for the publication of quantitative real-time PCR experiments (MIQE), published by ^{Bustin et al. (2009)} and used by ^{Dinon et al. (2011)}, for the development and validation of transgenic protein detection methods by real-time PCR.

Real-time polymerase chain reaction (RT-qPCR)

Once the complementary DNA was synthesized, we proceeded to quantify the genes coding for the Cry1Ac and Cry2Ab proteins expressed in the aforementioned tissues. Due to the dispersion obtained from the data, we proceeded to perform the Kruskal-Wallis test analyzing the median of the data to conclude whether or not there was a statistically significant difference. These results showed that there is no significant difference, with respect to time, in the expression of the protein Cry1Ac (p= 0.84) and Cry2Ab (p= 0.446) in leaves; if there is a difference in tables and acorns for both proteins (p= 0.0004 for Cry1Ac, p= 0.001 for Cry2Ab).

It was observed in each of the fields that, as the analysis was carried out at later stages, the quantification of the gene was lower, in some cases tending to zero (Figures 3 and 4), but there was always expression. In a similar study conducted by ^{Adamczyk and Meredith in 2004}, gene expression of the Cry1Ac protein tends to vary between different varieties and parts of the plant throughout the season. ^{Adamczyk et al. (2009)}, correlated the amounts of the Cry1Ac protein with the mRNA transcripts, finding differences in the varieties studied.

Figure 3 Quantification of the genes of the Cry1Ac and Cry2Ab proteins in GM cotton leaf tissues.  

Figure 4 Quantification of the genes of the Cry1Ac and Cry2Ab proteins in GM cotton square and acorn tissues.  

The order of the dates for the properties from left to right is: Field 1, 5 de febrero, Guamuchil and Field 9.

The date corresponding to the acorn analysis is that of 07/29/2014, the rest corresponds to tables. The order of the dates for the properties from left to right is: Field 1, 5 de febrero, Guamuchil and Field 9.

Olsen et al. (2005); ^{Xia et al. (2005)}, concluded that the mechanism is to some extent unknown. ^{Adamczyk and Gore (2004)}; ^{Kranthi et al. (2005)}; Olsen et al. (2005); ^{Wan et al. (2005)}, concluded that the expression of the Cry1Ac gene may decrease the effectiveness of the technology in crops due to the levels of expression observed in late stages, recommending constant monitoring to establish the baselines of each insect problem and avoid with it the development of resistance in white insects.

For the case of the Cry2Ab protein in leaf, acorns and squares the results show that there is variation and difference of expression in the tissue analyzed between the different dates and fields studied. ^{Bakhsh et al. (2012)} observed that the expression of the Cry2Ab gene undergoes variations among the varieties studied, as well as in the different tissues that make it up. ^{Monsanto (2011)} indicated that the variations of gene expressions depend on the periods of production, physiology of each plant, genetic variability of each plant, environmental conditions and agricultural practices; in addition, there may be variations between plants of the same batch and that cellular conformation may compromise the expression of proteins added to the plant genome.

Wessel et al. (2001) suggested that the activity of the promoter region of the transgene can be characterized only by the distribution of the different levels of expression in the plant, at each level (or tissue) a different behavior occurs. The temporal and spatial expression characteristic of a transgene in different independent transgenic lines must be intrinsic to the activity of the promoter. Therefore, the promoter who is the one who directs the expression of the transgene has different behaviors in each cell type in the plant, defining the spatial regulation of the expression (^{Head and Greenplate, 2012}).