Compositional and morphological analysis of salt stress tolerant MTLD- transgenic potato plants

doi:10.21203/rs.3.rs-3113448/v1

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Compositional and morphological analysis of salt stress tolerant MTLD- transgenic potato plants

https://doi.org/10.21203/rs.3.rs-3113448/v1

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Undesired effects often occur in genetically modified (GM) plants, especially during metabolite engineering. Nevertheless, conducting a comparative study between GM and non-GM plants can identify the unintended alterations and facilitate the risk assessment of GM crops. This research compared the morphology and composition of a transgenic potato plant containing mannitol-1-phosphate dehydrogenase (MTLD), with its non-transgenic counterpart. The results indicated significant differences in plant height, number of leaves, length and width of leaves, as well as tuber number and weight between the transgenic and non-transgenic plants. However, compositional analysis revealed no significant differences in soluble protein, starch, total sugar, fructose, fiber, and ascorbate contents between MTLD-GM and non-GM potatoes. Nevertheless, sucrose and glucose levels were found to be higher in the transgenic potato tubers and leaves, respectively, when compared to the non-transgenic plants. In addition to ammonium, potassium, chloride, nitrite, and nitrate levels, significant differences were observed in the amino acids asparagine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, and valine between the GM and non-GM plants. Apart from the target gene product, mannitol, all the changes in chemical compositions observed in the transgenic potato plants fell within the ranges of normal variability for potato plants. Moreover, despite some phenotypical differences between the GM- MTLD potato and its non-GM counterpart, it is believed that this variation is a common phenomenon among potato varieties. In conclusion, the morphological and compositional analysis of the MTLD transgenic potato plant revealed substantial equivalence with its non-transgenic counterpart.

abiotic stress

mannitol-1-phosphate dehydrogenase

potato

transgenic

Genetic engineering is an innovative technology that accelerates plant breeding by incorporating desired genes from different genetic resources into crop genomes. This technology leads to the development of transgenic crops, which exhibit enhanced performance in terms of resistance to biotic and abiotic stresses and increased nutritional value and yield [1]. Despite rapid development of genetically modified (GM) crops, their approval and commercialization remain contentious among within scientists and the general public [2]. Many countries have implemented legal frameworks to address these challenges, regulating the assessment of risks, marketing, labeling, and traceability of transgenic crops. Risk assessment of GM foods involves various aspects, with a comparative safety assessment (CSA) approach serving as a crucial guiding principle for regulatory bodies [3]. In the CSA approach, multiple aspects of GM crops, including essential safety specifications related to food, feed, and the environment, are compared to their conventional counterparts. In the CSA process, the agronomic and phenotypic characteristics of GM crops are evaluated. Additionally, a compositional analysis is conducted to assess essential nutrients, anti-nutrients, toxic compounds, and allergens in food and feed derived from these crops [3, 4]. Therefore, CSA represents a practical approach to identifying any significant differences between GM crops and non-GM varieties and any biologically significant disparities undergo further analysis to assess their potential impact on human health and the environment [2].

Metabolite engineering is a powerful tool that can enhance plant tolerance to abiotic stress by introducing new pathways for synthesizing osmoprotectant molecules [5]. These low molecular weight, non-toxic organic compounds accumulate in plants at high concentrations, as an adaptive response to abiotic stresses [6, 7, 8]. However, due to the complex network of plant metabolic pathways, excessive production of osmoprotectants as a genetic engineering strategy for improving tolerance may affect the composition of plant metabolites and potentially limit plant yield and performance [9].

This study used a transgenic potato plant carrying a bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene to produce mannitol, a sugar alcohol osmoprotectant. Our previous research demonstrated that the transgenic potato plants with the mtlD gene exhibited higher salt stress tolerance than non-transgenic control plants [6]. The induction of mannitol biosynthesis in the transgenic plant may influence the accumulation of intermediate compounds produced within the pathway (Fig. 1). The biosynthesis of starch and sucrose, for instance, is closely connected to the intermediate products within the mannitol biosynthetic pathway. These components serve as the carbon skeletons for all plant compounds and have a crucial function in plant growth and development. Consequently, any disturbance in the levels of sucrose, starch, and other critical key molecules can impact the performance of the plant. Hence, the initial step in the CSA of GM foods is rooted in the concept of substantial equivalence, which entails comparing GM crops to their isogenic conventional counterparts while maintaining the same concept.

This research aims to compare the phenotypic characteristics and composition of an MTLD-transgenic potato with its conventional counterpart. The substantial equivalence analysis revealed that the MTLD-transgenic potato generally resembles its non-transgenic counterpart.

Plant Material

Transgenic potato plants harboring CaMV35S-mtlD gene for resistance to salt stress were developed at Agricultural Biotechnology Research Institute of Iran (ABRII) [6]. These genetically modified potatoes, due to enhanced level of mannitol in their cells, demonstrated increased tolerance to salt stress compared to non-transgenic potatoes [6]. Transgenic and non-transgenic potato plants were propagated as in-vitro culture and then grown in a controlled greenhouse conditions. Transgenic and non-transgenic potatoes were planted 0.3 m apart in rows. Each row contained ten plants, referred to as 'Reps.'

Morphological assessment

Morphological studies, including the number and diameter of stems, as well as the color, number, length, and width of leaves, and flower characteristics were conducted throughout the growing period. After a three-month growth period, the leaves and developed tubers of the potatoes were harvested and placed in paper bags at 4 ºC for storage at dark conditions. Following the assessment of morphological characteristics and determination of tuber number and weight, the tubers were freeze-dried and preserved at -70 ºC until further analysis [10].

DNA extraction and polymerase chain reaction (PCR)

The characterization of transgenic potato plants was conducted by PCR analysis. Genomic DNA was extracted from potato plant leaves using Magnan Plant DNA Extraction Kits (Gil NanoGene Biotech, Iran) according to the manufacturer's instruction.

PCR amplification was carried out in a mixture consisting 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 200 µM dNTPs, 50 pM of each primer pairs (F-mtld: 5'-gtcatagctatcaggatgt-3', R-mtld: 5'-cttgatcaatactgcaccactt-3' for mtlD and F-nptII; 5'-gctattcggctatgactgggcac-3', R-nptII: 5'-tcatcctgatcgacaagaccggc-3' for nptII genes) and 1 U Taq DNA polymerase (Roche, Germany). PCR reactions included an initial denaturation cycle of 94°C for 5 min, followed by 35 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 2 min) and a final elongation cycle of 5 minutes at 72°C. Finally, PCR products were electrophoresed in a 1% agarose gel and visualized under UV light, following staining with ethidium bromide

Compositional analysis

Total dry matter, ash, and crude fiber were determined using the method described by AOAC [11]. Soluble protein was determined based on the Bradford method [12], while soluble sugar and starch content were assessed by the phenol sulfuric acid method [13]. Soluble sugars were analyzed by High Performance Liquid Chromotograhy (HPLC) (Knauer, Germany) with an IR detector and Eurokat column, with a flow rate of 0.7 mL/min [4]. The extraction and analysis of mannitol content were carried out according to Rahnama et al. [6].

Ascorbate

Ascorbate (Vitamin C) was extracted using meta-phosphoric acid (5%) [14] and analyzed according to Roe and Kuether [15] using spectrophotometric analysis. Total ascorbate was measured following the reducing of dehydroascorbate (DHA) to ascorbic acid (ASC) using dichlorophenol indophenol (DCPIP). The DHA content was estimated as the difference between the total ascorbate and ASC [4].

Glycoalkaloids: Potato glycoalkaloids, specifically α-chaconine and α-solanine, were extracted from 0.5 g of freeze-dried samples. The extraction process involved adding 10 mL of methanol:acetic acid (95:5 v/v) to the samples and subjecting them to shaking at 60°C for 4 hours. Subsequently, the sample was centrifuged at 10000g for 15 min at 4°C. The resulting supernatants were then dried using a rotary concentrator at 30°C. The obtained pellets were dissolved in 1 mL of methanol:acetic acid (99:1 v/v) and subjected to analysis using HPLC (Knauer, Germany) with a C18 column (Phenomenex, Torrance, USA), following the method described by Dale et al. [16].

Mineral elements

The composition of mineral element was determined according to the modified procedures outlined by Jiang et al. [17]. Briefly, 50 mg of dried matter was subjected to extraction using 10 ml HCl 0.1 N (or 10 ml dH₂O for anion extraction) at a temperature of 80°C for 4 hours. The extract was centrifuged for 10 min at 3000g, and the supernatant was analyzed using Ion chromatography (IC) with a Metrohm Professional IC 850 instrument from, Switzerland [4].

Fatty acids (FA)

Twenty-five mg freeze-dried sample was treated with methanol and sulphuric acid, followed by incubation at 80°C for 2 hours [18]. The extraction process was completed by adding 0.09% NaCl and N-hexane. Following centrifugation at 3000 g for 5 min at room temperature, the supernatant was analyzed by gas chromatography (GC) with a VARIAN model 4000 instrument (USA). The injector and detector temperatures were set at 240°C and the injection volume was 1 µL.

Amino acids: Freeze-dried samples (50 mg) were extracted using 1 ml 80% ethanol, followed by centrifugation at 14000g for 5 min at 4°C. The resulting supernatant was freeze-dried and stored at -20°C for subsequent analysis. The freeze-dried samples were then re-suspended in 1 ml dH₂O, and their derivation was conducted by adding O-phthaldialdehyde (OPA) reagent [4]. For HPLC analysis, 50 µL 0.5M HCl was added to the derivative samples, and 5 µl of the mixture was injected into the instrument (HPLC Knauer, Germany; HALO C18; fluorescent detector: Excitation 330, Emission 450 nM).

Statistical analysis

All data were reported as mean ± standard errors (SE) for the three replicates. Analysis of variance of the data was conducted using one-way analysis of variance (ANOVA) with the software SPSS (version 17.0, Inc. Chicago, US). The statistical significance of differences between means was determined by Duncan’s test at a significant level of P < 0.05.

Genetic changes as a common phenomenon have been observed in the potato tissue culture and plant regeneration process [19]. This changes and mutations caused by gene insertion or pleiotropic effects of recombinant proteins, may strengthen the unintended effects on the phenotypic and agronomic characteristics of transgenic potato events [20, 21]. Metabolic engineering due to changing the metabolites profile, may severely affects the morphological and compositional characteristic of transgenic potato plants.

Morphological Characteristics

The morphological analysis indicated noteworthy differences in particular traits between MTLD- GM and non-GM potato plants (Table 1). These differences were observed in plant height, number, length and, width of leaves, as well as the number and weight of tubers between MTLD-GM and non-GM potato plants. However, other characteristics of transgenic plant, such as flower morphology and tuber color and shape, were comparable to those of the non-transgenic control potatoes (Fig. 2). Morphological variations are a well-known phenomenon in in-vitro generated potato plants, resulting from somaclonal variations under tissue culture conditions [22]. Additionally, genetic transformation may cause 1–20% abnormalities in transgenic potato plants [23]. Conner et al [24] and Metry et al. [25] attributed the phenotypic changes in transgenic potato plants to epigenetic or genetic effects that happened during in-vitro culture in the genetic transformation process of plants. Recently, Gonzales-Romero et al. [26] demonstrated that inducing bezylglucozinolate production in transgenic potato plants led to reduced growth, and tuber production and altered leaf morphology compared to non-transgenic plants. However, except in one line, Cingel et al [27] observed that all other transgenic potatoes expressing rice cystatin genes (OCI and OCII) exhibited comparable morphological characteristics to non-transgenic plants. These phenotypical changes in in-vitro cultured plants can induce by different factors such as explants, medium composition, plant source and genotype, and the frequency of plant tissue sub-culturing [28]. Additionally, modifications in metabolic pathways may adversely affects the phenotypic characteristic of transgenic plants [29].

Table 1

Comparison of the morphological characteristics of transgenic MTLD and non-transgenic control potato plants.
Character	Non-transgenic	MTLD
Plant height	110 ± 2.8^a	104.3 ± 3.1^b
Stem diameter	5.8 ± 0.44^a	5.97 ± 0.36^a
Stem color	green	green
Leaves number	21.4 ± 2.23^a	18.6 ± 2.38^b
Leaves length	20.7 ± 1.73^a	18.4 ± 1.70^b
Leaves width	18.8 ± 0.77^a	15.2 ± 1.42^b
Tubers number	11 ± 1.92^b	14.8 ± 1.2^a
Tubers weight (gr)	293 ± 19^a	232.8 ± 25^b
tuber skins color	Light brown	Light brown
Pulp color	Light yellow	Light yellow
Flowering	+	+
Petal number	5	5
Sepal number	5	5
Anthers number	5	5
Pistil number	1	1
Filament length (mm)	6	6
Pistil length (mm)	9	9

Molecular characterization

PCR analysis was used to validate the existence of mtld and nptII genes in the transgenic potato plants. Molecular analysis confirmed the stability of the transgenes throughout more than seven years of sub-culturing of transgenic plants under in-vitro conditions (Fig. 3).

P: Plasmid (as positive control), C: Non-transgenic potato plant (as negative control), T: Transgenic MTLD potato line. M: Molecular weight marker, 1 kb ladder.

Compositional analysis

Compositional analysis is more critical for food safety analysis, particularly in the case of genetically modified plants that have undergone metabolic engineering. The comparison of transgenic and non-transgenic potato plants revealed no significant differences in crucial components such as soluble protein, starch, total sugar, fructose, fiber, and total ascorbate (Table 2). Mannitol as a desired compound was detected in the MTLD-GM potato plants. While the sucrose content remained unchanged in potato leaves, it significantly increased in transgenic potato tubers. On the other hand, transgenic potato leaves exhibited a significant increase in glucose content compared to the tubers. The variation in glucose and sucrose levels in transgenic and non-transgenic potato tubers can be accounted for by the biosynthetic dependence of mannitol to glucose, and sucrose, which illustrated in Fig. 1 [30]. In our study, we observed significant variations in the levels of ammonium, potassium, chloride, nitrite, and nitrate between MTLD-GM potato plants and non-GM ones, while we did not find any notable differences in other ions (Table 3).

The fatty acid analysis showed similar profiles between MTLD-GM and non-GM potato plants, except for stearic acid (Table 4). Earlier studies by Shepherd et al. [31] and Rahnama et al. [4] reported similar fatty acid profiles for both types of the plants.

The amino acid composition varied considerably between transgenic and control plants (Table 5). Transgenic plants exhibited higher levels of several amino acids, such as asparagine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, and valine, than non-transgenic plants. The presence of recombinant proteins (MTLD and NPTII) and the expression of unidentified proteins resulting from the insertional effects of transgenes in the plant genome contribute to the increased amino acid levels [32]. Nevertheless, previous investigations [33, 34] failed to observe significant alterations in the amino acid composition when comparing transgenic and non-transgenic potato plants. Overall, in the present study, all the changes observed in chemical composition of transgenic potato plants fell within the ranges reported for regular potato plants [35].

Table 2

Comparison of the major components between transgenic (MTLD) and non-transgenic (control) potato plants. Significant differences between means are indicated by different letters. na: not analyzed
Component	Control		MTLD
	Leaves	Tubers	Leaves	Tubers
Soluble Protein (mg/g FW)	0.19 ± 0.015^a	0.20 ± 0.017^a	0.19 ± 0.02^a	0.21 ± 0.018^a
Starch (mg/g DW)	0.15 ± 0.072^cd	0.77 ± 0.121^a	0.16 ± 0.094^c	0.68 ± 0.180^ab
Total Sugar (mg/g DW)	0.33 ± 0.092^a	0.34 ± 0.097^a	0.31 ± 0.025^a	0.30 ± 0.018^a
Mannitol (µmol/g)	0	0	53.3 ± 2.36^a	48.9 ± 1.77^ab
Sucrose (µM/g DW)	8.1 ± 0.69^ab	4.5 ± 0.43^c	9.6 ± 0.77^a	6.9 ± 0.83^b
Glucose (µM/g DW)	5.3 ± 0.18^c	10.5 ± 0.43^a	7.3 ± 0.49^b	10.9 ± 0.71^a
Fructose (µM/g DW)	10.1 ± 1.22^ab	11.9 ± 1.10^a	11.2 ± 1.77^a	11.6 ± 0.93^a
Crude Fiber (%)	na	2.5 ± 0.31^ab	na	2.6 ± 0.53^a
Total Ascorbate (mg/kg FW)	na	266 ± 19.6^a	na	202 ± 11.8^b

Table 3

Cation and Anion contents (mg/g dw) in transgenic potato plants. Control: non-transgenic potato plant, MTLD: transgenic potato lines. Significant differences between means are indicated by different letters.
Ions		Control		MTLD
		Leaves	Tubers	Leaves	Tubers
Cations	Li⁺	0.04 ± 0.005^a	0.01 ± 0.003^b	0.04 ± 0.006^a	0.01 ± 0.001^b
	Na⁺	0.27 ± 0.002^b	0.20 ± 0.019^c	0.26 ± 0.014^b	1.15 ± 0.54^a
	NH₄⁺	0.71 ± 0.031^a	0.63 ± 0.037^b	0.35 ± 0.01^c	0.24 ± 0.013^d
	K⁺	7.1 ± 0.52^a	2.4 ± 0.14^c	8.1 ± 0.15^b	2.3 ± 0.08^c
	Ca²⁺	14.4 ± 1.19^b	1.4 ± 0.04^c	16.2 ± 0.67^ab	1.8 ± 0.07^c
	Mg²⁺	2.4 ± 0.19^a	0.16 ± 0.025^b	2.2 ± 0.35^a	0.16 ± 0.012^b
Anions	F⁻	0.05 ± 0.014^b	0.15 ± 0.007^a	0.05 ± 0.019^b	0.14 ± 0.048^a
	CH₃CO₂⁻	0.27 ± 0.003^c	0.76 ± 0.007^a	0.13 ± 0.007^d	0.61 ± 0.021^b
	Cl⁻	1.6 ± 0.23^b	4.5 ± 0.27^a	1.4 ± 0.39^b	5.4 ± 0.16^a
	NO₂⁻	0.04 ± 0.003^c	0.7 ± 0.11^a	0.03 ± 0.009^d	0.6 ± 0.095^b
	Br⁻	0.4 ± 0.09^a	0.39 ± 0.048^a	4.7 ± 1.56 ^a	0.42 ± 0.011^a
	NO₃⁻	0.46 ± 0.04^b	0.92 ± 0.079^a	0.34 ± 0.057^c	0.80 ± 0.089^a
	PO₄³⁻	2.7 ± 0.075^a	1.7 ± 0.22^b	2.5 ± 0.38^a	1.5 ± 0.12^b
	SO₄²⁻	0.12 ± 0.012^c	1.6 ± 0.098^a	0.11 ± 0.073^c	1.4 ± 0.063^b

Table 4

Fatty acid contents (%) in the transgenic potato plants. Control: non-transgenic plant, MTLD: transgenic line. Significant differences between means are indicated by different letters.
Fatty acid	Control		MTLD
Fatty acid	Leaves	Tubers	Leaves	Tubers
Myristic (14:0)	1.15 ± 0.55^a	0.82 ± 0.01^b	1.17 ± 0.19^a	0.81 ± 0.02^b
Palmitic (16:0)	25.4 ± 0.72^a	22.9 ± 0.14^b	26.2 ± 1.04^a	23.3 ± 2.48^b
Palmitoleic (16:1)	2.96 ± 0.47^a	1.34 ± 0.04^c	3.17 ± 0.12^a	1.97 ± 0.21^b
Stearic (18:0)	7.24 ± 0.03^b	6.76 ± 0.14^c	7.9 ± 0.35^a	6.79 ± 0.54^c
Oleic (18:1)	8.70 ± 0.61^a	8.81 ± 0.42^a	7.81 ± 1.1^ab	8.84 ± 0.84^a
Linoleic (18:2)	30.5 ± 1.9^b	34.7 ± 0.65^a	31.2 ± 0.52^b	33.5 ± 0.9^a
Linolenic (18:3)	20.9 ± 0.82^b	27.2 ± 0.71^a	20.5 ± 0.75^b	26.8 ± 1.1^a

Table 5

Comparison of amino acid contents (µmol/mg fresh weight) between transgenic (MTLD) and non-transgenic (Control) potato plants. Significant differences between means are indicated by different letters.
Amino acid	Control		MTLD
Amino acid	Leaves	Tubers	Leaves	Tubers
Ala	73.9 ± 2.0^b	106.6 ± 2.5^a	74.5 ± 3.2^b	106.6 ± 4^a
Arg	107 ± 2.5^b	150 ± 2.1^a	108 ± 4.9^b	149 ± 7.6^a
Asn	56 ± 2.9^b	58.1 ± 1.8^b	65.5 ± 0.4^a	63.7 ± 1^a
Asp	288 ± 2^bc	248.4 ± 2.3^d	332.2 ± 3.6^a	293.7 ± 2.2^b
Cys	30.0 ± 2.3^b	48.1 ± 2.6^a	31.2 ± 2.8^b	47 ± 1.8^a
Glu	301 ± 1.6^cd	298 ± 1.9^d	356 ± 8.4^a	349 ± 1.2^b
Gln	93.2 ± 2.1^a	80.1 ± 1.7^b	92.4 ± 1.3^a	79.1 ± 1.9^b
Gly	98.7 ± 2.8^a	86.6 ± 2.09^b	100.3 ± 3.5^a	87.2 ± 1^b
His	51.6 ± 1.7^a	41.6 ± 0.84^b	49.9 ± 1.4^a	40.7 ± 0.8^b
Ile	87.1 ± 3.3^b	61.7 ± 2.32^d	99 ± 5.3^a	74.6 ± 0.7^c
Leu	225.7 ± 3.1^d	247 ± 4.6^c	279.3 ± 7.1^b	326.7 ± 5^a
Lys	211.6 ± 1.5^b	177 ± 3.8^c	247 ± 2.4^a	212.8 ± 2.1^b
Met	38.3 ± 2.4^b	44.5 ± 1.2^a	37.8 ± 1.1^b	43.8 ± 1.7^a
Phe	95.4 ± 2.9^b	121.6 ± 2.5^a	96.6 ± 8.6^b	120.4 ± 3^a
Pro	115 ± 4.4^c	173.7 ± 1.3^ab	114.6 ± 6.2^c	164.3 ± 1.4^b
Ser	120.7 ± 2.7^b	81 ± 1.9^d	135.9 ± 2.9^a	94.5 ± 2.5^c
Thr	118 ± 3.2^b	177 ± 1.2^a	116.6 ± 6.4^b	178.9 ± 2.9^a
Trp	23.3 ± 2.7^b	51 ± 3.2^a	25.0 ± 1.5^b	48.6 ± 1.5^a
Tyr	122.4 ± 3.3^b	160.4 ± 2.6^a	118.3 ± 6.3^b	158.9 ± 6.8^a
Val	137 ± 4.4^c	161.3 ± 2.9^b	160.6 ± 2.9^b	180.8 ± 1.1^a

Finally, in the present study, significant differences were observed in some compositions and morphological characteristics between MTLD-GM potatoes and non-GM counterparts. However, all of these differences fall well within the range of known variations for potato varieties. In conclusion, we concluded that the transgenic MTLD potato plants are substantially equivalent with the non-GM control plants regarding their nutritional composition and morphological characteristics.

Ethics Approval Not applicable

Consent to Participate Not applicable

Consent for Publication Not applicable.

Conflict of Interest The authors declare no conflicts of interest.

Author’s Contribution H. Rahnama: Conceptualization, Supervision, Methodology, Investigation, Writing, reviewing and editing of the manuscript; A.B. Moradi: Investigation and Methodology; F. Moradi: Methodology, Data Curation, Formal Analysis; N. Noormohamadi: Investigation

Data Availability: All data generated or analyzed for this study are included in this manuscript

Funding This research received funding from the Agricultural Biotechnology Research Institute of Iran, Project number: 7-05-05-91001

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No competing interests reported.

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Compositional and morphological analysis of salt stress tolerant MTLD- transgenic potato plants

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Abstract

Figures

Introduction

Materials and Methods

Plant Material

Morphological assessment

DNA extraction and polymerase chain reaction (PCR)

Compositional analysis

Statistical analysis

Results and Discussion

Morphological Characteristics

Molecular characterization

Compositional analysis

Declarations

References

Additional Declarations

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