Compositional analysis of transgenic Bt-chickpea resistant to Helicoverpa armigera

ABSTRACT

Transgenic chickpeas expressing high levels of a truncated version of the cry1Ac (trcry1Ac) gene conferred complete protection to Helicoverpa armigera in the greenhouse. Homozygous progeny of two lines, Cry1Ac.1 and Cry1Ac.2, had similar growth pattern and other morphological characteristics, including seed yield, compared to the non-transgenic counterpart; therefore, seed compositional analysis was carried out. These selected homozygous chickpea lines were selfed for ten generations along with the non-transgenic parent under contained conditions. A comparative seed composition assessment, seed storage proteins profiling, and in vitro protein digestibility were performed to confirm that these lines do not have significant alterations in seed composition compared to the parent. Our analyses showed no significant difference in primary nutritional composition between transgenic and non-transgenic chickpeas. In addition, the seed storage protein profile also showed no variation between the transgenic chickpea lines. Seed protein digestibility assays using simulated gastric fluid revealed a similar rate of digestion of proteins from the transgenic trcry1Ac lines compared to the non-transgenic line. Thus, our data suggest no unintended changes in the seed composition of transgenic chickpea expressing a trcry1Ac gene.

Introduction

The comparative compositional analysis of GM crops is required to ascertain that these crops are similar in the composition of nutrients to their non-GM counterparts. The seed composition assessment is carried out to determine that the changes made in the GM crops by introducing novel gene(s) to improve trait(s) are safe for humans and the environment.¹ Although molecular characterization of transgenic lines confirms the presence and expression of the transgene(s) but phenotypic analyses such a plant growth, yield and biochemical composition of transgenic plant are useful to select the best lines for further assessment such as integration of any plasmid sequences, site of insertion, novel protein expression and dietary exposure and other parameters.

To date, 27 crops, including genetically engineered cotton, maize, soybean, rice, papaya, have been commercialized after their substantial equivalence was established and widely adopted by the farmers since 1996.¹ Also, more than 80 peer-reviewed publications concluded that the compositional safety of many transgenic crops. A few reports are available stating that the introduction of a transgene may modify or silence an active gene, or it may induce a pleiotropic effect on the regulation of other genes due to insertion of a transgene.^2-9 The reports are not enough to support that such changes are common or frequent in GM crops,^10,11 however, useful to identify rare unintended changes at a very early stage of technology transfer in the field and help scientists or developers to improve. Therefore, in the present study, we performed compositional analysis of the transgenic chickpea seed expressing Bt gene resistant to pod borers to establish that there is no alteration in the seed composition of Bt-chickpea lines to due to the expression of trcry1Ac gene.

Chickpea (Cicer arietinum) is an important grain legume which is grown widely in India, Australia, Canada, Ethiopia, and the Mediterranean regions. Seeds of chickpea are a valuable source of protein for humans and their livestock. India is the largest producer and consumer of chickpea; to meet the domestic demand, India imports chickpea from Australia and Canada.¹² In the field conditions, one of the significant production constraints is pod borer, Helicoverpa armigera. Annually, considerable losses are caused by Helicoverpa infestation and under favorable conditions, yield reduction of >90% was recorded.¹³ Breeding for pod borer resistant chickpea is limited due to the lack of resistance sources within the gene pool. As a suitable strategy, we developed transgenic chickpea expressing high levels of the Bacillus thuringiensis Cry.^14,15 We generated transgenic chickpea lines expressing trcry1Ac¹⁵ gene and identified homozygous lines with high levels of resistance to pod borers in the glass-house. The plant growth and other morphological characteristics of the transgenic chickpea lines appeared similar to the non-transgenic counterpart. Also, no significant difference in the total seed yield per plant was observed. As a next step, we performed seed nutritional composition analysis of Bt-chickpea lines.

The compositional analysis of Bt-chickpea seeds is important because they are rich in carbohydrate (50–65%), protein, fiber, and several minerals. It is consumed as a cheap source of vegetable protein and often called as poor man’s meat. The seed proteins in chickpea are mainly globulins and albumins with smaller amounts of glutelins and prolamines. The two significant globulins of chickpea seeds are legumin (11 S) and vicilin (7 S). These seed proteins are composed of various essential amino acids, however, limited in sulfur-containing amino acids (methionine and cysteine). Seeds are a good source of polyunsaturated fatty acids (PUFA), linoleic acids, and oleic acid, which are higher in chickpea than any other legumes. The seeds are also rich in minerals such as potassium, calcium, sodium, magnesium, copper, iron, and zinc.¹⁶ The vitamins of chickpea seeds are folic acid, riboflavin (B₂), pantothenic acid (B₅), and pyridoxine (B₆). The seeds also contain several isoflavones such as biochanin A, formononetin, daidzein, genistein, and matairesinol. Many anti-nutritional factors, such as tannins, phytic acid, saponins, phenolic, and protease inhibitors (trypsin inhibitors and chymotrypsin inhibitors) lectins and antifungal peptides are also present in chickpea seeds.

In the past five years, no confined field trials were approved by the GEAC; therefore, the greenhouse-grown homozygous chickpea lines expressing a trcry1Ac gene were tested for seed compositional analyses. Based on the Biosafety guidelines outlined by the GEAC (2008),¹⁷ nutrient composition analyses of GE crop which includes proximates, fatty acid profile, micronutrient profile (vitamins and minerals), anti-nutritional factors, and predictable secondary metabolite profile on plants grown in the greenhouse, but preferably by collecting samples from confined field trials.

In the present study, we collected seeds from the greenhouse grown plants and both essential nutrients and anti-nutrients of transgenic and non-transgenic chickpea seeds were performed. Also, the amino acid content, seed storage proteins, and the digestibility of seed proteins were also assessed.

Materials and Methods

Plant Material

Transgenic chickpea lines expressing a trcry1Ac gene for resistance to pod borers were generated. The growth pattern and seed yield of these two transgenic lines were comparable with the non-transgenic control. We evaluated the seed compositional analysis of these two lines based on the guidelines of GEAC.¹⁷

The transgenic lines were multiplied by selfing for 10 generations (T₁₀) in the greenhouse. Progeny of T₁₀ generation was grown in the greenhouse in randomized blocks. Seeds were bulked from each block and used for analyses along with the non-transgenic counterpart. Plants were grown in soil mixed with perlite (2%) and compost (10%) and controlled conditions of 28 ± 2°C with 70% relative humidity. Seeds were harvested from the homozygous lines, stored at room temperature. Samples were grouped into three biological replicates and grounded into a fine powder. The fine powder of each sample was used for analysis of proximate, vitamins, amino acids, minerals, fatty acids, isoflavones, anti-nutrients, seed storage protein analyses, and in vitro digestibility of total seed protein. All the experiments were repeated twice with three technical replicates. Statistical analyses were carried out using a standard software and Microsoft Office Excel, 2007.

Nutritional Analysis

The major components that were estimated were proximate, fatty acids, isoflavones, minerals, vitamins, amino acids, and anti-nutrients. The methods used to determine those were described below.

Proximates

Starch and reducing sugar contents of both transgenic and non-transgenic chickpea samples were estimated as described, previously.^18,19 Flour from each sample was used to determine the starch content according to the Anthrone method¹⁸ and absorbance was recorded by spectrophotometry at 620 nm while reducing sugar was estimated by the colorimetric carbohydrate analysis method using dinitrosalicylic acid.¹⁹ Crude fat was extracted using petroleum ether in a Soxhlet apparatus for 6 hr.²⁰ Crude fat content was measured as the difference in weight between the dried crude fat extract and chickpea flour. Total nitrogen and crude proteins were analyzed by the Kjeldahl method and crude ash content by the dry ash method.²⁰ Carbohydrate levels of dry weight were estimated by the formula detailed below.

Estimation of Vitamins and Amino Acids

About 2 g of transgenic and non-transgenic chickpea seed flour samples was homogenized using extraction buffer (50 ml of acetonitrile with 10 ml of glacial acetic acid) and incubated at 70°C for 40 min. in a water bath. The extract was cooled down to room temperature, filtered using syringe filter, and vitamins were quantified after loading 30 µL of the extract on to reverse phase HPLC (Shimadzu, Model CBM 20 A).

Amino acids of chickpea seeds were estimated using the services of the Sandor Life Sciences Private Limited, Hyderabad, India. Chickpea seeds (about 2 g) were finely homogenized with metabolite extraction buffer. The extracts were treated with five volumes of SDS buffer with protease inhibitors, and 0.1% Tris-buffered phenol then subjected to organic solvent precipitation followed by centrifugation at 12,000 rpm. The pellet obtained after centrifugation was air-dried and dissolved in 50 mM ammonium bicarbonate buffer.

A total of 50 µL of the sample was digested with 2 ml of 6 N HCl and placed in a dry bath at 60°C under N₂ gas for 15 min. The temperature was then raised to 110° C and incubated for 24 hr. To the pellet 300 µL of sample diluent was added, vortexed, and incubated at 60°C for 10 min. After incubation, 7 µl of the sample was loaded onto an HPLC system for separation, and levels were quantified using standards (Sigma, Ltd).

Mineral Content

In a muffle furnace, 2 g of chickpea seed flour from each sample was ignited at 600°C, then dissolved in 0.2 N HCl and filtered through a filter paper. Mineral contents were determined using atomic absorption spectroscopy.²¹

Levels of Fatty Acids and Isoflavones

Finely ground chickpea seeds (2 g) flour was homogenized with extraction Buffer (Methanol: Chloroform: Water in a ratio of 1:2.5:1) and centrifuged (10,000 rpm) for 20 min at 4°C. After centrifugation, the chloroform layer derivatization was performed by using a mixture of 10% boron trifluoride and methanol. The above extracts were vacuum dried and dissolved in 50 µL of 0.1% formic acid in water and centrifuged at 13000 rpm. After centrifugation, 10 µL of the supernatant was injected on the C18 UPLC column for separation of metabolites followed by analysis on the Q-TOF (Quadrupole time of flight mass spectrometry) instrument (Q-TOF SYNAPT G2 Mass) for MS and MS/MS. The raw data were uploaded to the MassLynx 4.1 WATERS software to get the complete integrated ions in the sample. The individual ion MS spectrum was matched to the available metabolite database (LIPIDSMAPS and KEGG Compound DB) for fatty acids.

Isoflavones were extracted from chickpea flour following the same method as described above for fatty acids. The individual ion MS spectra were matched to the metabolite database, and isoflavones identification was performed using the MZmine software, 2.23.

The data for both fatty acids and isoflavone were read based on the intensity of each compound identified significantly in the samples and represented in the ratio of each compound in trCry1Ac chickpea lines compared to the non-transgenic counterpart. A ratio above 2 indicated significantly higher levels in the trcry1Ac lines, while a rate of 0.5 showed significantly lower levels in transgenic lines compared to non-transgenic lines.

Antinutrients Analysis

Quantification of anti-nutrients such as phytic acid, protease inhibitors, and tannins in the transgenic and non-transgenic lines was performed by the methods described below.

Phytic Acid

Phytic acid content was determined by the method of Haug and Lantzsch.²² Samples were extracted with HCl (0.2 N) and heated with an acidic iron (III) solution of known iron content. The phytic acid content was measured at 510 nm along with 2,2 bipyridine and sodium phytate as standards.²³

Activity of Trypsin and Alpha-amylase Inhibitors

The activity of trypsin inhibitor (TI) was determined by extracting samples with 0.01 N NaOH following a modification of AOCS 2009.²⁴ Extracted samples were mixed with trypsin and benzoyl-DL-arginine-nitroanilide hydrochloride (BAPNA), and absorbance was recorded at 410 nm using a spectrophotometer. The mean values for TIA (trypsin inhibitor activity) were expressed as trypsin inhibitor units per milligram of the extracted sample. The calculation was done using the formula described below.

100 is the factor to convert 0.01unit Abs in TIU units;

D the dilution factor of supernatant,

V is the extraction volume,

X is the aliquot used in the assay, and

Y is the final reaction volume in the cuvette.

The alpha-amylase inhibitor activity was carried out using a modified procedure and was calculated as percentage inhibition applying the below formula of McCue and Shetty.²⁵

$$\mathrm{\%}\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{(\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t})}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

Quantification of Tannin

Tannin content was estimated by the Folin-Denis method described in the textbook of Analytical Techniques in Biochemist.²⁶ In brief, Folin-Denis reagent (2.5 ml) and 5 ml of a sodium carbonate solution were added to 0.5 ml of the chickpea flour extract (chickpea flour refluxed with water) and diluted with 10 ml of water. The solution was mixed well, and the absorbance was read at 700 nm.

Statistical Analysis

The transgenic and non-transgenic chickpea lines were analyzed in triplicates with two repeats. The means and standard deviation were calculated for each nutritional component. Statistical significance among the samples was obtained by the t-test. The ANOVA was used to determine whether there are any statistically significant differences between means of samples. The ‘post hoc’ analysis was carried out to get significant differences between the samples with a ‘p’ value of ≤0.05. The statistical analyses were carried out in the SPSS software.

Seed Storage Protein Analysis and in Vitro Protein Digestibility

Seed storage protein fractionation was carried out following the protein fractionation protocol.²⁷ Chickpea seed flour was defatted thrice with chloroform/methanol (2:1 v/v) then air-dried. Defatted chickpea flour was extracted with (1:10 w/v) 0.2 M borate buffer (0.2 M boric acid, 0.2 M borax) pH 8 containing 0.5 mol L⁻¹ NaCl and centrifuged (15000 rpm) for 45 min. at 4°C. The supernatant was retained, and the sediment was re-extracted. The extracts were combined, adjusted to a pH of 4.5 with glacial acetic acid, and centrifuged (15000 rpm) for 30 min. at 4°C. The sediment (1) or pellet was re-dissolved in borate buffer, followed by dialysis against distilled water to obtain legumins (11 S) fraction. The supernatant was also dialyzed extensively against distilled water and centrifuged. The fresh sediment (2) obtained was stored at −80°C as the vicilin fraction (7 S). The supernatant was then precipitated twice with 82% ammonium sulfate [(NH₄)₂SO₄] followed by centrifugation (12000 rpm, 30 min, 4°C). The sediment (3) was dialyzed extensively (72 h) against distilled water to obtain the albumin fraction and stored at – 80°C.

Protein Profiling and Identification by Mass Peptide Fingerprinting

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to compare the electrophoretic mobility of various protein fractions. About 40 µg of proteins was separated in a 4–12% linear gradient Mini-Protean TGX precast gels using Tris-glycine buffer system. A pre-stained molecular protein maker was also loaded to the gel. Electrophoretic bands were stained with Coomassie Brilliant Blue solution.

Protein identification was carried out following the protocol used in earlier studies.²⁸ Major protein bands of each fraction, as identified based on similar reports on chickpea,²⁹ were excised out from the gel. The destained gel pieces were dehydrated using acetonitrile and then incubated with iodoacetamide, followed by an ammonium bicarbonate solution. The samples were then digested with trypsin solution at 37°C and vacuum dried. TA (Tris-acetate) buffer was used to re-suspend the dried samples. Alpha-cyano-4-hydroxycinnamic acid (HCCA) was mixed with the peptides obtained in a 1:1 ratio and 2 µl of the mix was spotted onto the matrix-assisted laser desorption ionization (MALDI) plate. Samples were analyzed on the MALDI TOF/TOF ULTRAFLEX III instrument, and the FLEX ANALYSIS SOFTWARE was used for obtaining the peptide mass fingerprint. The masses obtained in the peptide mass fingerprint were submitted to the MASCOT search of the NCBI database to identify the protein. The parameters for protein identification were; a) peptide mass tolerance: ±380 ppm, b) taxonomy: Viridiplantae, c) fixed modification: carbamidomethylation of cysteine, and d) variable modification: methionine oxidation.

In Vitro Protein Digestibility

In vitro digestibility of transgenic and non-transgenic chickpea seed proteins was determined by transient pepsin hydrolysis (to mimic simulated gastric fluid) followed by trypsin (to mimic simulated intestinal fluid) hydrolysis according to the method described, previously.³⁰ Based on our previous report,¹⁴ the accumulation of the Bt protein in the dry seeds was negligible, which could be due to the green tissue-specific promoter. Total seed proteins and pepsin were mixed in the ratio of 100:1 (w/w) in 0.1 M HCl, respectively. The mixture was gently shaken at 37°C for 120 min. The solution was then neutralized with 1.0 M phosphate buffer (pH 8.0), followed by the addition of trypsin (100:1 ratio of substrate/enzyme ratio, w/w), and incubated at 37°C for 120 min. About 200 µL of aliquots was removed from each tube after 0, 10, 60, and 120 min. of incubation and mixed with sample buffer (4X SDS-PAGE loading) and boiled at 100°C for 5 min. followed by centrifugation at 10,000 g for 10 min. The digested samples (100 µg) were loaded on an SDS-PAGE along with pre-stained molecular weight markers (from 10 to 130 kDa). Quantitative analysis of protein digestibility was carried out using the multienzyme method.³¹ A multienzyme solution (1.6 mg trypsin, 3.1 mg chymotrypsin and 1.3 mg peptidase/ml of sterile water) was used as the simulated gastric fluid for digestion of aqueous protein suspension (6.25 mg protein/ml) which resulted in a rapid decline in pH. The digestibility was calculated using the following equation described below.

$$\mathrm{Y}=210.46-16.10\mathrm{X}$$

where Y = in vitro digestibility %

X = changes in pH after 10 min.

Results and Discussion

We selected two homozygous, Helicoverpa-resistant trCry1Ac lines for compositional analysis. Two lines Cry1Ac.1 and Cry1Ac.2 were found to be high expressing (>50 µg Cry1Ac protein/g of fresh weight of leaves), have a single copy of the transgene and complete (100%) mortality of the neonate larvae in bioassays. The phenotypes of the plants were compared in the glasshouse and found similar to the parental line.¹⁵ The seed composition analysis was performed to extend the phenotypic assessment of these lines and ascertain that the transgene insertion did not alter seed composition in transgenic chickpea lines. We performed the compositional assessment based on the guidelines of the GEAC, Government of India.

Nutritional compositional analysis of transgenic crops is an important part of the safety assessment of food derived from genetically modified (GM) crops. In most cases, a comparative approach was used to evaluate that the transgenic and their non-transgenic counterparts are nutritionally equivalent.^32,33 It has also been reported that transgenic crops can be considered as safe as the conventional counterpart if their nutritional composition is within the range reported in the published literature.⁹

Nutritional Composition Analysis

We compared the levels of critical nutrients, anti-nutrients, and seed protein quality of chickpea trcry1Ac lines in comparison to their non-transgenic counterpart. We compared our data to available reports on commercial chickpea varieties of Jukanti and coworkers³⁴ as well as the USDA database on the National Nutrient Database for Standard Reference which was published on the 1^st of April 2018 (Software v.3.9.5.1_2018). Similar assessments were carried out on several other GM crops.^2,5,9,35

Proximates of Bt-chickpea Seeds

Chickpea is an excellent source of carbohydrates with a low glycemic index, and an inexpensive source of dietary protein for the people of developing countries. Analysis of proximates is an important to assess the nutritional aspects of a genetically modified organism; therefore, we studied dietary components such as carbohydrate, ash, crude fat, and crude protein. Seed proximate (Table 1) of transgenic and non-transgenic chickpea lines was within the range reported for several chickpea varieties.³⁴ Therefore, our results revealed that the proximate of chickpea seeds expressing a trcry1Ac gene was similar to their non-transgenic control.

Table 1.

Proximates and vitamins in the whole seed of transgenic chickpea and non-transgenic chickpea lines.

Proximates (gm/100 gm)	trcry1Ac-1 Line	trcry1Ac-2 Line	Control	Ref Range a
Starch	30.85 ± 1.54	27.83 ± 1.16	30.91 ± 2.73	25.90–53.90
Reducing sugar	0.41 ± 0.01	0.66 ± 0.08	0.44 ± 0.03	0.40–3.10
Crude Fat	4.40 ± 0.08	5.40 ± 0.07	5.00 ± 0.12	3.80–10.20
Nitrogen	4.15 ± 0.07	4.45 ± 0.21	3.97 ± 0.11	3.40–4.60
Crude protein	26.97 ± 0.45	28.92 ± 1.37	25.83 ± 0.68	21.70–28.90
Ash	4.90 ± 0.84	5.50 ± 1.41	4.95 ± 0.63	3.55–4.47
Vitamins (mg/100 gm)
Vitamin C	1.293 ± 0.04	1.472 ± 0.09	1.509 ± 0.07	1.3
Vitamin B9	0.521 ± 0.11	0.461 ± 0.05	0.515 ± 0.12	0.176
Vitamin B6	0.464 ± 0.01	0.444 ± 0.12	0.585 ± 0.12	0.53
Vitamin B2	0.207 ± 0.14	0.176 ± 0.01	0.160 ± 0.01	0.058–0.30

Values are mean±SD; n = 3

^aSource: (Jukanti et al.,2012)

^bSource: (USDA,2018)

Composition of Vitamins and Amino Acids in Bt-chickpea Seeds

Chickpea seeds contain several essential vitamins such as B2, B6, B9, and C, which were analyzed and listed in Table 1. We found that the composition of vitamin B6, B9, and C was significantly similar to the non-transgenic chickpea. Although the difference in the vitamin B2 (0.215 mg/100 g) was recorded; the mean values measured were within the range of normal variability (0.058–0.30 mg/g) reported for commercial chickpea varieties.³⁶

Total protein and amino acids are the essential nutritional contents of chickpea, which is designated by its protein-rich seeds. We evaluated the levels of various amino acids and observed that the transgenic and non-transgenic chickpea seeds exhibited nearly identical amino acid profiles (Table 2). Thus, the amino acid analysis demonstrated a similar profile of the parental line. Similar observations were also reported on transgenic rice harboring a cry1Ac gene and found to be substantially similar to other commercial varieties.³⁷

Table 2.

Levels of amino acids (gm/100 gm of dry weight) in transgenic chickpea lines when compared with parent (control).

Amino Acids (gm/100 gm)	trcry1Ac-1 Line	trcry1Ac-2 Line	Control	Ref Range b
Aspartic Acid	3.550 ± 0.002	*3.38 ± 0.354	3.733 ± 0.239	2.422
Threonine	2.052 ± 0.071	*1.272 ± 0.129	1.902 ± 0.027	0.766
Serine	1.268 ± 0.231	1.120 ± 0.067	1.232 ± 0.125	1.036
Glutamic Acid	4.249 ± 0.084	5.589 ± 0.177	5.355 ± 0.527	3.603
Glycine	3.531 ± 0.352	*1.891 ± 0.199	2.593 ± 0.168	0.857
Alanine	*1.931 ± 0.121	2.479 ± 0.047	2.130 ± 0.127	0.882
Valine	2.033 ± 0.221	1.421 ± 0.544	1.273 ± 0.197	0.865
Methionine	0.656 ± 0.124	0.407 ± 0.015	0.377 ± 0.049	0.27
Isoleucine	0.745 ± 0.062	0.848 ± 0.180	0.931 ± 0.041	0.882
Leucine	4.550 ± 0.240	*2.142 ± 0.133	3.622 ± 0.515	1.465
Tyrosine	1.847 ± 0.082	1.716 ± 0.136	1.465 ± 0.067	0.512
Phenylalanine	1.811 ± 0.159	1.804 ± 0.026	1.279 ± 0.187	1.103
Histidine	*1.704 ± 0.099	*1.839 ± 0.074	1.258 ± 0.084	0.566
Lysine	2.727 ± 0.134	1.778 ± 0.192	2.695 ± 0.277	0.566
Arginine	3.369 ± 0.395	3.6248 ± 0.011	3.063 ± 0.108	1.939
Cystine	0.152 ± 0.020	0.446 ± 0.048	0.190 ± 0.0944	0.279

Values are mean ± S.D; n = 3: (*) = p value <0.05

^aSource: (Jukanti et al.,2012)

^bSource: (USDA,2018)

Minerals in Transgenic Bt-chickpea Seeds

Chickpea is also a source of dietary minerals that are essential for human physiological functions. Various minerals (K, Na, P, Fe, Zn, Mn, Cu, Ca, and Mg) were analyzed in transgenic chickpea and found to be statistically similar except for iron (Table 3). The mean value of calcium statistically deviates from the control (412 mg/100 gm) in both the trCry1Ac lines (trCry1Ac-1: 238 mg/100 gm and trCry1Ac-2: 275 mg/100 gm). However, the variation in Fe levels may not be considered as biologically significant because the Fe content in other commercial chickpea varieties varied from 0.5 to 2.20 mg/gm.³⁴ Thus, the data suggest that essential minerals in the trcry1Ac chickpea lines were comparable to the other commercial chickpea varieties. Similar results were also reported in genetically modified rice.⁹

Table 3.

Mineral and anti-nutrient composition of transgenic and non-transgenic chickpea.

Minerals (mg/100 gm)	trcry1Ac-1 Line	trcry1Ac-2 Line	Control	Ref Range b
K	480.00 ± 0.08	670.00 ± 0.10	500.00 ± 0.08	427.6–780
Na	25.60 ± 0.15	21.10 ± 0.14	27.50 ± 0.14	10–24
P	454.00 ± 0.05	461.00 ± 0.08	369.0 ± 0.001	276.2–553
Fe	1.63 ± 0.01	1.46 ± 0.03	0.87 ± 0.001	4.6–7.0
Zn	0.78 ± 0.00	0.89 ± 0.01	0.84 ± 0.01	2.8–5.1
Mn	2.95 ± 0.21	2.70 ± 0.49	2.80 ± 0.04	0.8–4.10
Cu	1.20 ± 0.14	1.23 ± 0.31	1.21 ± 0.02	0.5–1.40
Ca	*238.00 ± 0.04	*275.00 ± 0.16	412.00 ± 0.08	80.5–220.0
Mg	122.00 ± 0.01	124.00 ± 0.06	135.00 ± 0.01	119.0–177
Anti – nutrients
% alpha amylase inhibition	10.20 ± 2.43	11.25 ± 1.70	9.54 ± 1.15	0.50–15.00
Trypsin Unit Inhibitor/mg	16.24 ± 0.02	17.49 ± 0.03	15.33 ± 0.17	6.90–19.00
Tannin (mg/100 mg)	1.18 ± 0.01	1.18 ± 0.01	1.18 ± 0.01	Trace amount
Phytic acid (µg/100 mg)	314.71 ± 0.09	314.71 ± 0.26	314.71 ± 0.26	Trace amount
In vitro protein Digestibility %	87.65 ± 0.38	86.66 ± 0.50	87.71 ± 0.73	48.00–89.010

Values are mean ± S.D; n = 3: (*) = p value <0.05

^aSource: (Jukanti et al.,2012)

^bSource: (USDA,2018)

Concentration of Fatty Acids in Bt-chickpea Seeds

Individual fatty acids of the seeds of chickpea trcry1Ac lines were quantified and compared with non-transgenic chickpea seeds. In all, 15 fatty acids, which include both saturated and unsaturated fatty acids, were significantly similar in chickpea trcry1Ac lines compared to the non-transgenic counterpart (Table 4). Transgenic corn harboring cry genes also showed significant similar fatty acid composition when compared with their non-transgenic counterparts.³⁸

Table 4.

The relative concentration of isoflavone and fatty acid in transgenic chickpea seeds when compared with non-transgenic lines.

Isoflavones	trcry1Ac-1 vs Control	trcry1Ac-2 vs Control
Biochanin A	0.55	0.55
Formononetin	1.79	0.68
Daidzein	0.71	0.55
Genistein	0.45	0.70
Matairesinol	0.66	0.76
Fatty Acids
Lauric Acid	0.70	0.77
Myristic Acid	0.73	0.53
Palmitic Acid	0.64	0.49
Palmitoleic Acid	0.71	0.55
Stearic Acid	1.10	0.49
Oleic Acid	1.62	0.66
Lineloic Acid	0.87	0.54
Linolenic Acid	4.54	5.00
Arachidic Acid (Eicosanoic)	0.53	0.54
Gadoleic (Eicosenoic Acid)	0.88	2.00
Eicosadienoic acid	1.10	0.80
Behenic Acid	1.43	1.49
Erucic Acid	1.49	1.40
Lignoceric Acid	0.56	1.79
Nervonic Acid	1.15	1.50

(*) = significant difference

Isoflavones Content in Chickpea Seeds Expressing Trcry1Ac Gene

The Isoflavones are produced mostly by the members of the Fabaceae. Daidzein and other isoflavones in plants act as signal carriers, and defense responses to pathogenic attacks.^39,40 Isoflavones are mainly generated in response to stimulation by biotic and abiotic stresses.⁴¹ We analyzed five major isoflavones such as daidzein, genistein, biochanin A, formononetin, and matairesinol in the trCry1Ac lines and found no significant variation in the levels of these isoflavones when compared with the non-transgenic counterpart (Table 4).

Composition of Antinutrients in Transgenic Chickpea Seeds

Chickpea seeds contain diverse anti-nutritional factors, such as trypsin inhibitor, alpha-amylase inhibitor, phytic acid, and tannins. Also, high levels of anti-nutrients in chickpea seeds reduce their nutritional value as well as digestibility of protein.⁴² Phytic acid is a chelator of many minerals such as calcium, magnesium, iron, and zinc, and can contribute to mineral deficiencies.⁴³ Similarly, tannins can create a complex with vitamin B12, thereby leading to a decrease in its absorption. Trypsin inhibitors in seeds are likely protector molecules against attack by predators. Trypsin inhibitors can have a significant impact on the nutritional value as they interfere with protein digestion. Therefore, the quantification of the anti-nutrients present in chickpea is required to ensure the bioavailability of other nutrients. We compared the seed phytic acid, tannins, trypsin inhibitor, and alpha-amylase inhibitor in trcry1Ac lines and found no alteration in their compositions when compared with the parental line (Table 3). The levels of these anti-nutrients were within the range reported for chickpea.³⁴

Analyses of Seed Storage Protein in Transgenic Chickpea Seeds

In chickpea, the major seed storage proteins are globulins, glutelins, albumins, and prolamines.⁴⁴ The determinants of the seed storage proteins are albumins and globulins. Therefore, we compared the electrophoretic mobility of seed proteins, eluted the major protein fragments and confirmed the protein composition by the mass peptide fingerprinting. In the trcry1Ac lines, electrophoretic bands of the seed proteins appeared similar to the non-transgenic counterparts (Figure 1). All the major seed storage proteins were observed in both the transgenic and non-transgenic chickpea lines. Seed storage proteins were also fractioned as 11 S (legumin-type), 7 S (vicilin-type), and 2 S (albumins). These major protein bands (11 S, 7 S, and 2 S albumins) were eluted from the gel based on size,^29,45 and identified by the MS/MS.

Figure 1.

Comparative seed storage protein fractions of chickpea trcry1Ac lines and the non-transgenic parent. Total seeds proteins (40 µg) were extracted with borate buffer and fractionated based on pI and solubility. In figure a, each lane indicates the electrophoretic band of probable molecular weight of 25 kDa for albumins fractions, while figure bis the gel showing electrophoretic bands of various peptides of vicilin fractions (MW ~70.2, ~50.7, ~35.0, ~33.6, ~18.9 and~15.5 kDa). Figure c shows bands of peptides with a similar molecular weight of legumin α- subunits (MW~ 40.6, ~39.5 kDa) and legumin β- subunits (MW~ 23.5, ~22.5 kDa). Arrow indicates the electrophoretic band used for mass peptide fingerprinting. In the gel figure abbreviations were used to mark molecular weight (MW), total protein (p), legumin (l), vicilin (v), and albumin (a) fraction.

The electrophoretic banding pattern showed the expected 7 S fraction of molecular weight (MW) ~70.2, ~50.7, ~35.0, ~33.6, ~18.9 and ~15.5 kDa. Similarly, legumin fractions comprised α- subunits (MW ~40.6 and ~39.5 kDa) and β- subunits (MW~ 23.5 and ~22.5 kDa) and albumin fraction of ~25 kDa was observed in both transgenic and non-transgenic chickpea lines. The mass peptide fingerprinting concluded that the major bands in both transgenic and non-transgenic chickpea are legumin (band 1- ~40.5 kDa), vicilin (band 1of ~15.5, band 2 of ~35, and band 3 of ~70.2 kDa) and albumin proteins of ~25 kDa (Supplementary material 1). Thus, the study revealed no alteration in the storage proteins, legumin, vicilin, and albumins in the trcry1Ac lines. Similar results were also reported on genetically modified corn.³⁸

In Vitro Protein Digestibility of Transgenic Chickpea Lines

The in vitro digestibility of transgenic chickpea lines and their non-transgenic line was important to know that trCry1Ac protein (60–65 kDa) was digested by simulated gastric fluid and seed protein digestibility was not altered. We evaluated protein digestibility by using the pepsin–trypsin digestive system at various time points, followed by SDS–PAGE to confirm that the seed proteins were digested and there is no resistant trCry1Ac protein fragment after digestion. Pepsin digestion of chickpea seed proteins indicated that all polypeptides were hydrolyzed within 10 min of pepsin digestion resulting in the production of polypeptides of lower molecular weight (Figure 2(a)) and later, trypsin treatment further cleaved the polypeptides to small peptides of <15-10 kDa (Figure 2(b)) in both transgenic and non-transgenic chickpea lines. Similar results were reported in chickpea.⁴⁶ The results revealed that the order of the degree of hydrolysis (digestion) of the proteins of trCry1Ac chickpea seeds was like the non-transgenic chickpea. Even residual polypeptides observed after digestion had the same banding pattern in both transgenic and non-transgenic chickpea lines. Thus, the results suggest no potential unintended effects of seed storage protein digestion in trcry1Ac chickpea.

Figure 2.

In vitro digestibility of seed proteins of trcry1Ac chickpea transgenic and non-transgenic lines. Proteins from chickpea seeds were extracted and digested with pepsin and trypsin, sequentially, and loaded on to the gel. Proteins digested with pepsin at varying (0,10, 60, 120 min.) time interval were loaded in the gel (a), while samples digested with trypsin at different (0,10, 60,120 min.) time interval were loaded in another gel (b). The gel image was marked with M for protein ladder, and 1, 2, 3, 4 represented the digested protein samples at 0,10, 60, and120 min.

Although the Bt protein concentration was negligible in the seeds of chickpea (Appendix Table A1), we performed western blot to see the digestion pattern of the trCry1Ac protein. No cross reactivity of the Cry1Ac antibody was found in the undigested as well as in the digested samples (data no shown). It appears that the green tissue-specific Arabidopsis Rubisco small subunit (ats1A) gene promoter, which was used to regulate trCry1Ac gene is less active during the seed development process.

Conclusion

The nutritional composition analysis was carried out between transgenic chickpea lines expressing a trcry1Ac gene and the non-transgenic counterpart. Evaluation of the critical nutrients such as proximate, amino acids, minerals, vitamins, and anti-nutrients revealed no significant differences between the trcry1Ac chickpea lines and the non-transgenic chickpea. Furthermore, no significant alteration was observed in the seed storage protein profile and in vitro protein digestibility between the transgenic trcry1Ac chickpea and non-transgenic lines. Thus, our data suggest no change in seed protein quality and the trcry1Ac lines are compositionally similar to non-transgenic seeds.

Appendix.

Table A1.

The concentrations of Cry1Ac protein in the various organs of chickpea lines.

	Cry 1Ac Protein ($\mu$g/g FW
Tissue Samples	Cry1Ac.1	Cry1Ac.2
Leaf	88	120
Stem	23	35
Flower	94.5	126
Pod	23	15
Immature Cotyledons	41	62
Root	0.22	0.54
Dry seeds	1.1a	3.15a

^aThe concentration in the dry seed is calculated as micrograms per gram dry weight.

Funding Statement

The authors acknowledge the Indo-Swiss Collaboration in Biotechnology (ISCB), Australia, India Strategic Research funds (AISRF) and Department of Biotechnology, Govt of India for funding to generate transgenic chickpea lines. The work was funded by the Indian Council of Agriculture Research (ICAR) and Department of Biotechnology (DBT), Govt of India.

Author Contributions

The study was conceptualized and designed by SA and BKS, transgenic chickpea lines were generated by SA. RG performed composition analyses with the help from AMB, SA and BKS. RG wrote the draft of the manuscript; SA edited the final manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

Disclosure Of Potential Conflicts Of Interest

No potential conflicts of interest were disclosed.

Supplementary Material

Supplemental data for this article can be accessed on the publisher’s website.