Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2020 Aug 3;10(8):370. doi: 10.1007/s13205-020-02359-2

A novel, simple, and stable mesoporous silica nanoparticle-based gene transformation approach in Solanum lycopersicum

Zahra Hajiahmadi 1, Reza Shirzadian-Khorramabad 1,✉, Mahmood Kazemzad 2, Mohammad Mehdi Sohani 1, Jahangir khajehali 3
PMCID: PMC7399006  PMID: 32832330

Abstract

In this study, a novel and stable gene transformation system was developed under control of Maize Proteinase Inhibitor (MPI) as an inducible promoter using the Mesoporous Silica Nanoparticles (MSNs). The functionalized MSNs with a proper particle size were synthesized and attached to a recombinant construct (pDNA) containing cryIAb gene under the control of MPI promoter (pPZP122:MPI:cryIAb:MSN [pDNA: MSN]) following transformation of tomato plants through injection of the pDNA: MSN complex into tomato red fruit at early ripening stage and then, putative transgenic seeds were collected. As an initial selection, gentamicin-resistant seedlings of T1 (24.24%) and T2 (61.37%) plants were identified. The transgene integration and expression were confirmed through the PCR, RT-PCR, and western blot approaches in the selected seedlings. PCR analysis showed that transformation frequency was equal to 10.71% in T1 plants. Semi-quantitative RT-PCR analysis confirmed the transcript expression of cryIAb in all the T1 and T2 PCR-positive plants. Western blot analysis confirmed the existence of CryIAb protein in the leaves of T2 putative transgenic plants. Accordingly, the results demonstrated that the transgene has more likely integrated into the tomato genome through homologous recombination. Bioassay was carried out for further assessment of the plant responses to Tuta absoluta resulting in an enhanced tolerance of the plant. In conclusion, the MSN-mediated stable transformation system under the MPI as an inducible promoter can be used as a suitable alternative for conventional genetic transformation methods due to its biodegradability, biocompatibility, cost and time-effectiveness, and positive effect on the plant defense against pathogens and pests.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02359-2) contains supplementary material, which is available to authorized users.

Keywords: cryIAb gene, Msns, Stable transformation, Tuta absoluta, Wound-inducible promoter

Introduction

Nanotechnology has had a key role in many research areas (Du et al. 2020) including agriculture during the last 2 decades. The application of nanotechnology in the field of agriculture has basically been limited to the delivery of the pesticides and nutrients to the plants. Therefore, there is a need for more progression in other areas of research (Motyka et al. 2019). Nanomaterials can be used in plant protection and nutrition due to their small size (less than 100 nm) (Auffan et al. 2009; Ghormade et al. 2011) and are widely used in minimum amounts to deliver the pesticides to the plants, causing a decrease in leaching and evaporation of harmful chemicals (Zheng et al. 2005). Thus, environmental protection could be considered as one of the important advantages and reasons for utilizing the nanoencapsulated materials (Duhan et al. 2017). Consumption of the pesticides has been estimated about two million tons per year in 2014 (De et al. 2014) leading to an increase in the pest resistance, a decrease in the number of important soil microorganisms, and the reduction in the abundance of helpful insects and birds in the nature (Tilman et al. 2002). Accordingly, on the one hand, employing new strategies is necessary to overcome these shortcomings, and on the other hand, gene transformation through the nanoparticles might increase the pest resistance in the plants (Rai and Ingle 2012).

Pests are among the major threats to global agriculture. They destroy the crop production all around the world and influence the crop quality and quantity (Chakraborty and Newton 2011). Tuta absoluta is an oligophagous pest attacking many Solanaceae species especially the Solanum lycopersicum (tomato) (Cifuentes et al. 2011). Tuta causes extensive damage (50–100%) to the tomatoes in all stages of their life (Shahbaz et al. 2019). Leafminer damage in tomato was reported in Europe (2006) for the first time and later it was spread throughout the Middle East in 2010 (Baniameri and Cheraghian 2011; Campos et al. 2014; Tonnang et al. 2015). Given the negative and harmful effects of the pesticides on the environment, employing the alternative methods to control this pest is essentially reasonable. To date, various researches have been conducted on management of the Tuta, but none of them have succeeded to completely protect the tomatoes against T. absoluta (Camargo et al. 2016; Campos et al. 2014; Galdino et al. 2011; Hamza et al. 2018; Moreno et al. 2017; Pereira et al. 2014). Likewise, an increase has been reported in the Tuta resistance to the conventional pesticides (Campos et al. 2014). More importantly, the first instar enters the leaf tissues after hatching (Hamza et al. 2018), and thus, it comes in direct contact with the pesticides, such as spinosad (Campos et al. 2014). Hence, the development of Tuta-more resistant transgenic plants could be considered as an important goal in the plant breeding programs.

Agrobacterium-mediated transformation, biolistics, ultrasound, and microinjection are among the most commonly used methods for genetic transformation of the plants. Although these approaches have some disadvantages, such as low transformation efficiency in the biolistics and ultrasound methods, false-positive outcomes in the Agrobacterium-mediated transformation due to the existence of Agrobacterium in the transgenic plants, and the need for expensive equipment in the microinjection approach (Hao et al. 2013). Nanoparticle-mediated plant transformation provides a number of benefits including (1) rapid transformations, cost- and time-effectiveness (Pasupathy et al. 2008), (2) the ability to transfer various macromolecules, such as proteins, DNA, and RNA (Eggenberger et al. 2010), (3) the use of cheap and simple equipment for transferring the DNA into cells (Perez et al. 2001), (4) the ability to transfer gene into callus, embryo, and other plant tissues (Pasupathy et al. 2008), (5) protection of the transgene against degradation (Peer et al. 2007), and (6) the ability to transfer multiple genes simultaneously (Fu et al. 2012a). Despite aforementioned advantages, toxicity effects of some nanoparticles on the plant cells is also an important shortcoming (Eggenberger et al. 2010) requiring more attention. Interestingly, Mesoporous Silica Nanoparticles (MSNs) have attracted a great deal of attention compared to other nanomaterials due to their biocompatibility and biodegradability (Chang et al. 2013). Though there are some studies on the MSN-mediated gene transient transformation into the plant roots or protoplasts under in vitro and sterile conditions (Chang et al. 2013; Fu et al. 2015; Hussain et al. 2013), requiring grand costs and special conditions. Thus, in our previous study, we introduced a new MSN-mediated plant transient transformation approach under in vivo condition (Hajiahmadi et al. 2019), which is a simple and quicker approach and comprises some advantages, such as more safety, cost, time, and energy-effectiveness, over the other transient transformation methods. MSNs with particle size less than 40 nm were able to temporarily transfer the pDNA (plasmid) into the tomato plants through three methods including spraying the pDNA–MSN solution on the abaxial surface of the leaves, injection of the solution into the shoot, and the abaxial surface of the leaves (Hajiahmadi et al. 2019). Based on the results of the study on MSN-mediated gene transient transformation and the benefits of the MSNs, we subsequently sought to develop a new method for stable transformation system using the MSNs in tomato plants. Hereby, in the present study, a novel stable transformation system was developed using the MSNs in the tomato plants, and the potential tolerance of the putative transgenic tomatoes against Tuta was investigated.

Materials and methods

Construction of the pPZP122: MPI:cryIAb

In the current study, pPZP122 expression vector (Hajdukiewicz et al. 1994) was used due to its high homology with chromosome 11 of tomato (~ 800 bp), which potentially can lead to the homologous recombination (Electronic Supplementary Material). The Maize Proteinase Inhibitor (MPI) promoter (Accession number: KP793074) was taken out from the pTZ57R/T vector constructed in our previous study (Hajiahmadi et al. 2018). Two fragments of cryIAb and MPI were cut out separately from the pCIB4421 (Koziel et al. 1993) and pTZ57R/T using two sets of enzymes including BamHI/EcoRI (Thermo Fisher Scientific, Lithuania) and HindIII/BamHI (Thermo Fisher Scientific, Lithuania), respectively, to construct the pPZP122:MPI:cryIAb. Then, the fragments were cloned into the pPZP122 vector at EcoRI–BamHI and HindIII–BamHI sites following the transformation of Escherichia coli DH5α competent cell (Fig. 1a). Colonies containing pPZP122:MPI:cryIAb were selected using both colony PCR method and recombinant plasmid enzyme digestion approach. The PCR program for amplifying the MPI and cryIAb was as follows: 5 min at 95 °C followed by 30 cycles of 1 min 95 °C, 1 min at the appropriate primer annealing temperature (Table 1) and 1 min at 72 °C, with a final extension of 10 min at 72 °C.

Fig. 1.

Fig. 1

Open in a new tab

Recombinant pDNA and molecular analysis of the mesoporous silica nanoparticle-mediated putative transgenic tomato plants. a Construction of the pPZP122:MPI:cryIAb. LB left border, P nopaline synthase promoter, GmR gentamicin resistance, MPI Maize Proteinase Inhibitor promoter, PEPC In 9 Intron 9 of the maize-phosphoenolpyruvate carboxylase gene, T nopaline terminator, RB right border. b PCR confirmation regarding the presence of MPI promoter (886 bp) and the cryIAb gene (1194 bp) in the putative transgenic tomatoes (T2). L: 100 bp ladder, lanes 3–9: putative transgenic samples, lanes 1–2: negative controls (non-transgenic), c confirmation of the cryIAb gene expression (160 bp) through the semi-quantitative RT-PCR approach, lanes 2–7: putative transgenic samples (T2), lane 1: negative control (non-transgenic sample)

Table 1.

The primer sequences for amplification of the Maize Proteinase Inhibitor promoter and cryIAb gene

Primer name Primer sequence
F-MPI 3′-AGCTTTTTAGGTTCTACACAAAACCCTC-5′
R-MPI 5′-TCTAGACCGGACCAGTTGACGA-3′
F-cry1Ab 3′-GCGGCGAGAGGATCGAGA-5′
R-cry1Ab 5′-TCGGCGGGACGTTGTTGTTC-3′
RT-F-cryIAb 5′-CCGTGACCGACTACCACAT-3′
RT-R-cryIAb 5′-AGCGTACAAAAACCAGCAACT-3′
RT-F-Actin 5′-GCTCCTCAGTTGAGAAGAGC-3′
RT-R-Actin 5′-CCTTCCTGATATCCACGTCAC-3′
Open in a new tab

Synthesis and characterization of the positively charged MSNs

The functionalized MSNs were synthesized based on our previous report (Hajiahmadi et al. 2019) (Fig. 2). In brief, 3.71 g of Cetyltrimethylammonium Bromide (CTAB) was dissolved in 100 mL of the buffer solution (1.74 g of sodium hydroxide (NaOH), 10.2 g of monopotassium phosphate (KH2PO4) in 1.5 L of deionized water) at 30 °C and 550 rpm, followed by adding 1.86 mL of Tetraethyl Orthosilicate (TEOS) drop-wise at 550 rpm for 8 h. After centrifugation and removal of the excess TEOS, 3-aminopropyl triethoxysilane (APTES) was used to functionalize the synthesized MSNs. The functionalized MSNs were dispersed in the Phosphate-Buffered Saline (PBS) to create a positive charge on their surface at pH level of 7.4. Field Emission Scanning Electron Microscope (FE-SEM) (Mira 3-XMU, Czech Republic) with an accelerating voltage of 15 kV and the Transmission Electron Microscope (TEM) (Zeiss, Germany) with an accelerating voltage of 100 kV were used to assess the size and morphology of the MSN-APTES, respectively. Zetasizer 3000 HS (Malvern, UK) was used to investigate the zeta potential of the synthesized MSNs. BJH (Barrett–Joyner–Halenda) method was applied to assess the pore size distribution. Likewise, the Belsorp-Mini II, Gemini 2375 (BEL Japan Inc., Osaka, Japan) was used to calculate the specific surface area and the pore volume (Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods). Small-Angle X-ray Scattering (SAXS) was recorded using the PANalytical X’Pert MPD instrument operating at 40 kV and 40 mA with Cu Ka (k¼ 1.5406 Å) as X-ray source to identify the phase of the MSNs.

Fig. 2.

Fig. 2

Open in a new tab

General synthesis steps of the functionalized mesoporous silica nanoparticles. a Cetyltrimethylammonium Bromide (CTAB) was dissolved in the monopotassium phosphate (KH2PO4) buffer solution containing NaOH at 30 °C and 550 rpm. b 1.86 mL of Tetraethyl Orthosilicate (TEOS) was added at 550 rpm for 8 h. c CTAB acted as a structural template. d Aminopropyl Triethoxysilane (APTES) was added to the solution to functionalize the MSNs. e Ethanol and HCl were added to remove the excess template under constant stirring at 550 rpm and 60 °C for 16 h, and f the MSNs were dispersed in the PBS (pH 7.4) to get positive charge

Stable transformation of the plant using the MSNs carrying pPZP122: MPI:cryIAb

Seeds of Solanum lycopersicum var. falat were obtained from the Pakan Bazr Seed Company (Isfahan, Iran). Based on our previous study (Hajiahmadi et al. 2019), the best ratio for binding of the plasmid (pDNA) to MSNs is 1:100 (µg/µL) thus, it was used in the current research. Injection of the solution into red fruits was performed at early ripening stage (600 µL per fruit) to transfer the pPZP122:MSN into the fruits. In this study, fruit ripening stage was selected because in this stage, the cell walls are almost decomposed by the enzymes, which may improve the penetration of the MSNs. PBS (pH 7.4) containing only MSNs (without pPZP122:MPI:cryIAb plasmid) was also utilized as the negative control. Each treatment (injection of pDNA:MSNs and empty MSNs) was administered on the three fruits from separate plants in two biological replicates. The treated plants were incubated at 23 °C in the growth chamber with a photoperiod of 16 h. Seeds were collected after fruit maturity (10 days after injection). T2 plants were obtained from self-pollination of the T1 plants.

Identification of the putative transgenic plants using the MS medium containing gentamicin

Ripened fruits (T1 and T2) were harvested and then, their seeds were air-dried on the blotting paper. Next, the seeds were sterilized using 3% sodium hypochlorite (NaOCl) and 1% Triton for 20 min and were rinsed three times using the distilled water. Different concentrations of gentamicin including 25, 50, 75, 100, 125, and 150 µg/mL were applied to determine the lethal threshold in the control plants. Accordingly, no control plant could survive at 100 µg/mL gentamicin. Therefore, 40 and 44 seeds of T1- and T2-treated plants were inoculated on the half-strength Murashige and Skoog medium (Murashige and Skoog 1962) containing 100 µg/mL of gentamicin, respectively (Table 2). Subsequently, the number of survived seedlings was recorded after 40 days and considered as putative transgenic seedlings that were subsequently transferred to the growth chamber.

Table 2.

Transformation efficiency of the tomato fruits using the mesoporous silica nanoparticles (MSNs)-mediated gene transformation approach

Generation Sample Number of putative transgenic line Seed Germinated seed (%) Gentamicin-resistance plantlets (%) PCR+ plantlets (%) Transformation frequency
Number of seeds Average number of seed in each line
T1 Treated plants – 40 – 28 (70%) 7 (25%) 3 (42.8%) 10.71%
Negative Control – 40 – 37 (92.5%) 0 (0%) – –
T2 Treated plants 3 44.66 ± 8.81 29.33 ± 5.54 (63.85%) 18 ± 3.21 (61.37%) 11.66 ± 2.02 (64.77%)
Open in a new tab

The molecular investigation of the gentamicin-resistant plants by the PCR, semi-quantitative RT-PCR, and western blot analysis

Genomic DNA was extracted from gentamicin-resistant plants using the modified CTAB method proposed by Murray and Thompson (Murray and Thompson 1980). Presence of the MPI promoter and cryIAb gene in the gentamicin-resistant plants was investigated through their specific primers and PCR amplification (Table 1). Based on their specific primers, 1194 and 886 bp bands were expected for MPI and cryIAb magnification, respectively.

First, the leaves of 3-week putative transgenic (after transferring the gentamicin-resistant plants to the growth chamber) and non-transgenic tomatoes were exposed to wounding and, after 24 h, total RNA isolation was performed based on the manufacturers’ instructions (RNX-Plus, Sinaclon, Iran). DNase treatment was applied on the RNA solution before cDNA synthesis to remove any DNA contamination. Subsequently, complementary DNA was synthesized using cDNA synthesis kit (Pishgam, Iran). Non-transgenic tomato samples were also used in the experiment as the negative control plants. The semi-quantitative RT-PCR was conducted using the specific primers of cryIAb gene. The Actin gene was utilized as the endogenous reference gene in the semi-quantitative RT-PCR amplification (Table 1). The PCR program was as follows: 5 min at 95 °C followed by 30 cycles of 45 s at 95 °C, 30 s at 60 °C and 45 s at 72 °C, with a final extension of 5 min at 72 °C. Based on the applied specific primer for cryIAb gene, amplification of a 160 bp band was expected.

For further confirmation regarding the presence of cry1Ab in the putative transgenic plants, western blot analysis was also employed. Therefore, 1 g of leaf sample was taken from various plants including putative transgenic plants, non-transgenic (as the negative sample), and also Bacillus thuringiensis (Bt)-transgenic rice plants expressing a 67 kDa CryIAb protein [as the positive control (Ghareyazie et al. 1997)]. Then, the leaf from each sample was grounded and mixed with 2 ml of QB extraction buffer containing 100 mM potassium phosphate (KPO4) (pH 7.8), 1 mM EDTA, 1% Triton X-100 and 10% glycerol, and 1 mM Dithiothreitol (DTT). Total protein concentration for each sample was calculated by the dye-binding method (Bradford 1976) and, therefore, the supernatants were used after being centrifuged at 12,000 rpm for 15 min at 4 °C. About 100 μg of total protein from each sample was electrophoresed by the Sodium Dodecyl Sulfate (SDS)-Polyacrylamide gel electrophoresis. After electrophoresis, the proteins were electroblotted to the Polyvinylidene Difluoride (PVDF) membrane (Cyto Matin Gene Immune Company, Iran) by a wet trans-blotting device (Payapajouh Company, Iran). TBST buffer (1X Tris buffer saline with 0.1% Tween-20) containing 5% non-fat dried milk was used to block the blots. Then, the PVDF membrane was incubated with the rabbit polyclonal anti-Cry1 antibody (Ghareyazie et al. 1997) at a dilution of 1:2000 for 6 h at 4 °C. The membrane was washed in the TBST buffer five times, each time lasting 5 min. Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma Chemical Co., St Louis, USA) was used as the secondary antibody at a dilution of 1:5000 for 2 h at room temperature and then, was washed five times for 5 min each using the TBST buffer. The color reaction was done using the 3,3′-diaminobenzidine (DAB) substrate (Sigma-Aldrich Company, USA) in the PBS buffer (pH 7.4) within 10–15 min.

Insect bioassay

The tomato leaves infested with Tuta larvae were collected from several greenhouse tomatoes in Isfahan and Qazvin provinces, Iran. Then, the collected larvae together with leaves of tomatoes (as substrate) were placed on Petri dishes at room temperature to feed the larvae and obtain the adult insects (Campos et al. 2014). Next, the adult insects were allowed to oviposit on the tomato leaves for 24 h. Larvae at three stages including first, second, and third instar larvae (L1, L2, and L3) were collected afterwards. The age of larvae was determined by their size, i.e., the length of the L1, L2, and L3 was in the sequence of 0.5–0.9 mm, 1.4–1.8 mm, and 3.5–4.5 mm, respectively. Three healthy leaves from 1-month-old single plants including putative transgenic (T2) and non-transgenic in two replicates were employed to assess the tolerance of putative transgenic tomatoes against Tuta and then, five larvae per leaf were placed on the investigating plants. In this experiment, the bioassays were carried out on the intact tomato plants meaning that each leaf together with larvae was placed in a sealed round Petri dish; therefore, the larvae had no way out. The bioassay conditions included the temperature of 27 ± 2 °C, photoperiod of 16 h light, and relative humidity of 60–80%. Mortality and leaf area damage were recorded on the 4th, 7th, and 10th days after infestation (based on the larvae age). The level of leaf area damage was calculated by the ImageJ software (Rasband 1997). Bioassay data were analyzed using the SPSS software ver. 17 (SPSS Inc., Chicago, Ill., USA).

Results and discussion

Current stable gene transformation methods have several limitations which we can overcome by utilizing the nanoparticles-based genetic transformation approaches. Accordingly, in the present study, a new MSN-mediated stable genetic transformation technique was introduced in the tomato plant. The increase in the experiment speed due to the reduction of transformation steps is the most important advantage of the novel introduced method compared to the Agrobacterium-mediated gene transformation.

Moreover, generation of the transgenic plants carrying a proper promoter is among the important criteria in the plant genetic engineering. In many studies, constitutive promoters, such as CaMV35S, have been already used (Ghareyazie et al. 1997; Selale et al. 2017; Yasmeen et al. 2009) leading to the significant changes in the plant metabolic pathways even in the absence of abiotic and biotic stresses (Zhang et al. 2013). On the other hand, constitutive expression of the insecticidal genes can lead to the death of non-target insects, and thus, the use of inducible promoters, such as wound-inducible promoters, might be very helpful because expression of the foreign genes only happen when the plants are attacked by the pests (Hajiahmadi et al. 2018). Inducible- or tissue-specific promoters offer considerable advantages, such as saving the plant energy, and expression of the transgene after pest attack (Hajiahmadi et al. 2018). Results of our previous study showed that the MPI promoter is sufficient for expression of the transgene after pest attack compared to the CaMV35S, confirming the capability of this promoter in saving the energy and reducing the adverse effects on the non-target organisms (Hajiahmadi et al. 2018). Thus, herein, the MPI-inducible promoter together with cryIAb gene was used through the MSN-mediated stable genetic transformation method to investigate its efficiency and performance against. T. absoluta.

Investigation of the functionalized MSNs

The results of FE-SEM and TEM showed that the MSNs were synthesized with the size of 40 nm, porous structure, and spherical shape (Fig. 3a, b). The size of the MSNs is an important parameter influencing their toxicity on the cells. MSNs larger than 2500 nm or smaller than 20 nm are more toxic (Napierska et al. 2009), thus, the size of the MSNs selected as carriers to transfer the macromolecules into the plant cells must be between 20 and 2500 nm. Therefore, the MSNs with the average particle size of 40 nm were synthesized that had no toxic effects on the tomato plants. The MSNs with the zeta potential of + 9.8 mV were found to be positively charged in comparison with the uncharged samples with the zeta potential of − 10.3 mV. The mean surface area (990 m2/g), pore volume (2.57 cm3/g), and pore size (2.58 nm) were calculated using the BET-BJH method. Given the large surface area, it was confirmed that the surface of the synthesized MSNs should be very porous. According to the SAXS findings, the peak observed at 2θ = 1.82 was assigned to the (100) plane of the MSNs (Fig. 3c).

Fig. 3.

Fig. 3

Open in a new tab

Investigating the characteristics of the mesoporous silica nanoparticles (MSNs) by the FE-SEM (a), TEM (b), and c XDR pattern of the fabricated MSNs with the particle size of smaller than 40 nm. The peak observed at 2θ = 1.82 indicated the (100) plane of the MSNs

Identification of the gentamicin-resistant putative transgenic tomatoes

The seeds of the completely ripened fruits (T1 and T2 generations) were collected, dried and then, were screened on the half-strength Murashige and Skoog medium (Murashige and Skoog 1962) containing 100 µg/mL of gentamicin (Table 2). Among the MSN-mediated putative transgenic tomatoes, the average percentage of the gentamicin-resistant plants in T1 and T2 generations was equal to 25 and 61.37%, respectively. Hajiahmadi et al. (2018) applied the Agrobacterium-mediated transformation in the tomato plant through the in planta approach. Their results indicated that the average percentage of the gentamicin-resistant plants was equal to 24.24 and 77.27% in T1 and T2 plants, respectively (Hajiahmadi et al. 2018). Although, the newly introduced system was less efficient than the conventional Agrobacterium system, it provided valuable advantages, such as high levels of safety for both the researchers and their working environment, biocompatibility and biodegradability, cost- and time-effectiveness, and positive effect on the plant defense against pathogens and pests, over the conventional gene transformation methods (Fauteux et al. 2005; Ma and Yamaji 2008). The efficiency of gentamicin marker is high enough to select the transgenic dicot plants (Fig. 4a, b). However, high amounts of gentamicin can lead to acetylation of 3-amino group in the 2-deoxystreptamine ring causing a decrease in the plant growth (Hayford et al. 1988). Angenon et al. (1994) illustrated that the gentamicin is more efficient compared to kanamycin and hygromycin as selectable antibiotics (Angenon et al. 1994). Moreover, Mendelian inheritance of the gentamicin-resistant plants (T2 seedlings) was also evaluated using the Chi-square test. Evaluation of the T2 putative transgenic tomato lines (line numbers of Z1, Z2, and Z3) on the ½ MS medium containing 100 µg/mL of gentamicin demonstrated that the numbers of germinated seeds were equal to 38, 31, and 19, while the numbers of the gentamicin-resistant plantlets were equal to 23, 19, and 12, respectively. Therefore, the inheritance analysis confirmed the Mendelian segregation (3:1; p < 0.05, X2 = 2.19), implying that a single locus of the transgene has been integrated into the plant genome. According to the Chi-square results, the gentamicin-resistant gene has been properly segregated as a dominant character in T2 generation and, therefore, it has more likely integrated into the tomato genome.

Fig. 4.

Fig. 4

Open in a new tab

Gentamicin resistance and western blot verification of the mesoporous silica nanoparticles (MSN)-mediated putative transgenic and non-transgenic tomatoes. a, b Screening of the gentamicin-resistance in non-transgenic and T2 MSN-mediated putative transgenic plants. c Immunoblotting assessment of the CryIAb protein in the MSN-mediated putative transgenic tomatoes. Lanes 1–2: negative control (non–transgenic) and positive control (Bt-rice), respectively. Lanes 3–4: T2 putative transgenic lines. 67 kDa band was recognized in the putative transgenic lines demonstrating that cryIAb gene was integrated into the tomato genomes. L: Pre-stained protein marker (11–245 kDa)

Molecular confirmation of the gentamicin-resistant plants containing pPZP122: MPI:cryIAb

The integration of cryIAb gene and MPI promoter in the putative selected transgenic plants was evaluated in the gentamicin-resistant putative transgenic plants of both T1 and T2 generations through the PCR approach. An 1194-bp band and one 886-bp DNA fragment associated with the presence of cryIAb and MPI promoter were amplified from the gentamicin-resistant putative transgenic tomatoes (Fig. 1b), respectively. The average percentage of PCR+ samples was equal to 10.71 (3 PCR+ plants per 28 germinated seeds) and 39.75% (11.66 PCR+ plants per 29.33 germinated seeds) in T1 and T2 plants, respectively.

Moreover, semi-quantitative RT-PCR was carried out to investigate the expression of cryIAb gene in the PCR+ plants. All the PCR+ plants (100%) showed the desired band (160-bp) in T1 and T2 samples. As shown in Fig. 1c, a 160-bp band associated with cryIAb expression was amplified in the T2 putative transgenic plants suggesting that cryIAb gene was inherited and expressed in the next generation. In fact, it means that the cryIAb must be first integrated into the tomato genome and subsequently expressed in the T2 plants. Inheritance of cryIAb gene in the second generation (T2) was also assessed using the media culture containing gentamicin showing that the segregation has happened based on the Mendelian ratio (3:1). Considering the obtained results and also the transformation frequency of tomato plants (number of PCR+ plants per total number of germinated seeds) in T1 generation (10.71%), the novel stable gene transformation method for tomato plant using the MSNs can be a good alternative approach to the Agrobacterium-mediated transformation (transformation frequency of 15.15%) (Hajiahmadi et al. 2018). Fu et al. (2015) successfully delivered the positively charged MSNs (500 nm) containing GUS gene into the tobacco by the gene gun bombardment approach. They developed stable transgenic tobacco plants with 47.11% of efficiency (the percentage of the PCR+ transgenic plantlets accounted for the kanamycin-resistant plantlets) (Fu et al. 2015). In this study, the percentage of PCR+ putative transgenic plants among the gentamicin-resistant ones was equal to 42.85% in T1 generation. Therefore, our newly introduced method is more efficient than the one used in the study by Fu et al. (2015) due to the elimination of the expensive and special equipment. However, it is suggested to use the transposon vectors to increase the homologous recombination efficiency that would result in high transformation frequency.

Our results showed that the integration of the desired gene into the tomato plant genome has successfully occurred more likely due to the homologous recombination between pPZP122 and chromosome 11 in tomato genome (Fig. 6f). Foreign DNA fragments containing a sufficient homology with the host genome can be integrated into the new genome through the homologous recombination process (Weeks et al. 2016). Homologous recombination allows the exchange of DNA fragments between the plant chromosome and inserted plasmids. If the transmitted plasmid contains a homologous fragment with a special region in the host chromosome, thus, a single crossover can occur resulting in the integration of recombinant plasmid into the plant chromosome (Primrose and Twyman 2013). Therefore, the presence of 90% homology between the 800 bp in the vector pPZP122 and chromosome 11 of tomato could be one of the important possible reasons for integration of the pPZP122 into tomato genome (Fig. 6f). In parallel with the results presented in the study, Inbar et al. (2000) reported that this length of homology is sufficient enough for local recombination (Inbar et al. 2000). Therefore, this approach can be considered as an efficient stable genetic transformation method in the tomato plants. Moreover, further research is needed to increase the efficiency of the MSN-mediated transformation method including the use of transposon vectors or CRISPR/Cas9.

Fig. 6.

Fig. 6

Open in a new tab

Schematic representation of the mesoporous silica nanoparticles (MSNs)-mediated genetic transformation system. a, b Functionalized MSNs, and the MSNs containing pPZP122:MPI:cryIAb (pDNA), respectively. c The pDNA-MSN injected to the red fruit before ripening stage, d pDNA-MSNs passing through the nuclear pore (about 70 nm), e the release of the pDNA from the MSNs in the nucleus and f integration of the pDNA into the plant genome has most likely occurred due to the homologous recombination (green boxes) between pPZP122 (red circle) and chromosome 11 of tomato (blue line), and g Putative transgenic plants identified on the gentamicin -based selective media

Verification of the cryIAb protein in the putative transgenic tomato plants

The results of western blot analysis showed a 67-kDa CryIAb protein band in the MSN-mediated T2 putative transgenic plants (Fig. 4). Interestingly, the same protein band was also found in the transgenic Bt-rice plants expressing cryIAb, as the positive control samples in this experiment (Fig. 4). Therefore, the results confirmed that the inheritance of cryIAb to the next generation (T2) has occurred in association with the CryIAb protein expression implying that the promoter, transgene, and terminator have been most probably integrated into the tomato genome. The presence of smaller detected fragments in the putative transgenic lines could be attributed to the degradation of Bt protein during the protein extraction (Ghareyazie et al. 1997). The previous studies have indicated that (Hajiahmadi et al. 2019), the MSNs can enter the tomato cell nucleus through the nuclear pore, and subsequently express and translate the cryIAb gene under the control of CaMV35S promoter. It has been shown that the MSNs smaller than 50 nm can enter the cell membrane by endocytosis and pinocytosis before passing through the plant nuclear pores (Chang et al. 2013). Consistent with a number of previous studies, the size of the wall pore in the plant cell is diverse and depends on the plant species. For instance, copper (50 nm), gold (10–50 nm), silver, and silica NPs (14, 50, and 200 nm) have been reported to pass through the cell wall of different plant species (Nair et al. 2011; Slomberg and Schoenfisch 2012; Wang et al. 2012). Fu et al. (2012a, b) transferred the ZnS nanoparticles (3–5 nm) containing the pBI121 vector into the Nicotiana tabacum plants through the ultrasound. Their results revealed the stable transformation of GUS gene into the tobacco plants (Fu et al. 2012b). However, their method requires special equipment and conditions leading to the reluctance in its application. Torney et al. (2007) successfully transferred the gold-capped MSNs (100–200 nm) containing the pER8-GFP vector into the tobacco protoplasts as well (Torney et al. 2007). However, protoplast isolation and plant regeneration are difficult processes. Generally, compared to the conventional methods, the MSN-mediated gene transformation method can be performed easier, with higher efficiency and no need for special equipment.

Enhancement of tolerance in the putative transgenic tomato plants against the (T2) T. absoluta

Tuta is among the important pests causing a significant damage to tomato plants. The pest larvae attack at all parts of the tomato plants except roots, and the damage can lead to 100% of yield loss if no control strategy is adopted (Shahbaz et al. 2019). Therefore, in the current study, a different approach in genetic transformation was used to enhance the tolerance of tomato against T. absoluta. To confirm this, insect bioassay was carried out on 1-month-old intact putative transgenic T2 plants. Thus, the percentage of leaf area growth per day was considered in the calculations. The increase in the percentage of leaf area growth was equal to 5.77, 11.75, and 30.45% after 4, 7, and 10 days, respectively (Fig. 5). The putative transgenic lines demonstrated a relative tolerance to Tuta compared to the control non-transgenic plants. Although, most of the larvae in the non-transgenic plants reached the last developmental stage, the ones fed on the putative transgenic plants could not develop properly (Fig. 5e). The results demonstrated that the presence of CryIAb protein in the putative transgenic plants can induce more tolerance to the larvae of T. absoluta at L1 stage (Leaf area damage of 24.63 and 21.49%) and less tolerance to the larvae at L3 (Leaf area damage of 31.91 and 34.39%) (Fig. 5a and Table 3). Moreover, the mortality rate was equal to 94.44, 86.66, and 73.33% in different aged larvae including L1, L2, and L3, respectively. As a result, the CryIAb protein was efficient to increase the Tuta mortality, but was not able to completely prevent the damage to the leaf surface thus, it is also recommended to investigate the other Bt variants to obtain better results in further reducing the leaf area damage. According to Table 3, the variation in the mortality rate and amount of leaf area damage between the putative transgenic lines could be attributed to the differences in toxin expression level due to the epigenetic changes. Incomplete protection of the putative transgenic tomato plants against Tuta can be related to the resistant nature of the pest in response to CryIAb toxicity. Camargo et al. (2016) utilized the RNAi system to silence the Vacuolar ATPase (V-ATPase) and Arginine Kinase (AK) genes in the tomato plants to elevate their tolerance against Tuta. Their results revealed that the expression of both genes significantly reduced leading to an increase in the larval mortality (50% in V-ATPase mutants and 43% in AK mutants) and a reduction in the leaf damage (Camargo et al. 2016). Hamza et al. (2018) developed the transgenic tomato plants using the barley serine and cysteine proteinase inhibitor genes, and they showed that only 56% of larvae reached the adult stage with deformed wings and low fertility (Hamza et al. 2018). Likewise, Selale et al. (2017) developed the transgenic tomato plants using the cryIAc gene, and found that Tuta mortality rate increased up to 100% and minimum leaf area damage (less than 10%) was observed along with small scars on the treated leaves (Selale et al. 2017). Considering the advantages of the MSN-mediated gene transformation method and also the ability of cryIAc gene in significantly reducing the Tuta damage in the transgenic tomato plants, it is suggested to develop the Tuta-resistant transgenic tomato plants using the MSNs containing the cryIAc gene under the control of the MPI promoter, which can be a reasonable alternative compared to the conventional Agrobacterium-mediated transformation system.

Fig. 5.

Fig. 5

Open in a new tab

Bioassay of the mesoporous silica nanoparticles-mediated putative transgenic plants against T. absoluta. a–c Leaf damage of T2 1-month-old plants after infestation with the first, second, and third instar larvae (L1, L2, and L3), respectively. Each leaf was placed in a sealed round Petri dish and leaf area damage was recorded on the 4th, 7th, and 10th days after infestation (based on larvae age), d leaf area damage in the non-transgenic plant, e1 and e2) Alive larvae collected from the non-transgenic (developed to fourth instar) and putative transgenic plants. f Leaf area damage after infestation with the neonate, second and third instar larvae. T and N) Putative transgenic and non-transgenic plants. As can be seen, putative transgenic plants are more resistant against the neonate instar larvae compared to the second and third instar larvae

Table 3.

Bioassay of the putative transgenic tomato plants (T2) against T. absoluta

Sample Putative transgenic line Number of larva Leaf area damage (%) Mortality (%)
Larvae 1 (L1) Larvae 2 (L2) Larvae 3 (L3) Larvae 1 (L1) Larvae 2 (L2) Larvae 3 (L3)
Putative transgenic Putative transgenic 1 15 24.63 ± 1.63b 33.33 ± 0.88b 31.91 ± 1.11d 93.33 ± 4.21a 83.33 ± 6.14a 76.66 ± 3.33a
2 15 21.49 ± 1.21b 21.49 ± 1.21c 34.39 ± 0.71d 96.66 ± 3.33a 90.00 ± 4.47a 70.00 ± 4.47a
Non-transgenic 1 15 83.42 ± 0.71a 95.50 ± 0.79a 92.72 ± 0.91b 3.33 ± 3.33b 0.00 ± 0.00b 0.00 ± 0.00b
2 15 84.90 ± 1.41a 94.73 ± 0.97a 98.25 ± 0.49a 0.00 ± 0.00b 3.33 ± 3.33b 0.00 ± 0.00b
Open in a new tab

Means ± SE, difference between means in each columns followed by different letters are significantly different (p < 0.05) in Duncan multiple range test

Schematic representation of pDNA transformation into tomato genome using the functionalized MSNs

Herein, a schematic diagram was presented to display all viewpoints of the MSN-mediated genetic transformation (Fig. 6). In a nutshell, the functionalized MSNs with particle size of about 40 nm were synthesized and then, the constructed recombinant pDNAs (pPZP122:MPI:cryIAb) were attached to the MSNs (Fig. 6a, b). The MSNs containing the pDNA solution were injected into a red tomato fruit at early ripening stage (Fig. 6c). Subsequently, the recombinant pDNA complex moved through the nuclear pore (about ~ 70 nm, Fig. 6d). Then, pDNA was delivered to the nucleus and integrated into the plant genome due to local homologous recombination (Fig. 6e). As mentioned before, the first and foremost reason for the integration could be the homologous recombination event, which more probably happens between the 800 bp sequence in the pPZP122 expression vector with a segment in chromosome 11 of tomato genome (those segments showing 99% of homology in a sequence) (Fig. A1). Finally, the plant RNA polymerase recognized the CaMV35s promoter of gentamicin resulting in the development of the gentamicin-resistant plants (Fig. 6f).

Conclusion

In the current study, a new stable transformation method was developed using the MSNs for the first time in the tomato plant. Compared to the other conventional methods, the proposed method is convenient, affordable, and rapid. In the new stable transformation method, integration of the transgene to the tomato genome more likely happened due to homologous recombination between the pPZP122 expression vector with a segment in chromosome 11 of tomato genome. However, more molecular analyses are needed to confirm the integration of transgene in chromosome 11 of putative transgenic tomato plants. Moreover, the MSNs are probably accumulated in the plant chloroplasts and remain in the treated plants till the end of growth and development; therefore, they cannot to be transferred to the next generation. Hence, further research should be performed on the stability of the MSNs in the target plants. The transposon vectors, instead of pPZP122 might be used as an appropriate alternative to apply the similar transformation process in other plants. Regarding the benefits of the MSN-mediated gene transformation system, we are going to optimize this system in various plant species using the transposon vectors or the CRISPR/Cas9 system, in the future. CRISPR/Cas9 might be efficient enough to develop the transgenic plants using the MSNs because it is based on the homology of sgRNA, and the target gene can lead to knock-in or knockout of many plant species.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOC 725 kb) (725KB, doc)

Acknowledgements

The authors would like to acknowledge Reza Sayyad from University of Tehran and Dr. Ghadamyari from University of Guilan for their scientific and technical advices, and express our gratitude to Zahra Shirzadian for grammatically revising our manuscript.

Author contributions

ZH did the research experiments and data analysis, RSH conducted and supervised the research project. MK supervised the nanoparticles synthesis and assessments. MMS and JKH worked in order as advisor of gene transformation and insect bioassay experiments, respectively. All authors have read and approved the manuscript.

Funding

This work was partially funded by University of Guilan and also in part by Iran National Science Foundation (Grant no: 93033715).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Angenon G, Dillen W, Van Montagu M. Antibiotic resistance markers for plant transformation. In: Gelvin SB, Schilperoort RA, editors. Plant molecular biology manual. Dordrecht: Springer; 1994. pp. 125–137. [Google Scholar]
  2. Auffan M, Rose J, Bottero J-Y, Lowry GV, Jolivet J-P, Wiesner MR. Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat Nanotechnol. 2009;4(10):634. doi: 10.1038/nnano.2009.242. [DOI] [PubMed] [Google Scholar]
  3. Baniameri V, Cheraghian A (2011) The current status of Tuta absoluta in Iran. In: International symposium on management of Tuta absoluta (Tomato Leafminer), Agadir, Moracco, pp 16–18
  4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1–2):248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Camargo RA, Barbosa GO, Possignolo IP, Peres LE, Lam E, Lima JE, Figueira A, Marques-Souza H. RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum) PeerJ. 2016;4:e2673. doi: 10.7717/peerj.2673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Campos MR, Rodrigues ARS, Silva WM, Silva TBM, Silva VRF, Guedes RNC, Siqueira HAA. Spinosad and the tomato borer Tuta absoluta: a bioinsecticide, an invasive pest threat, and high insecticide resistance. PLoS ONE. 2014;9(8):e103235. doi: 10.1371/journal.pone.0103235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chakraborty S, Newton A. Climate change, plant diseases and food security: an overview. Plant Pathol. 2011;60:2–14. [Google Scholar]
  8. Chang F-P, Kuang L-Y, Huang C-A, Jane W-N, Hung Y, Yue-ie CH, Mou C-Y. A simple plant gene delivery system using mesoporous silica nanoparticles as carriers. J Mater Chem B. 2013;1(39):5279–5287. doi: 10.1039/c3tb20529k. [DOI] [PubMed] [Google Scholar]
  9. Cifuentes D, Chynoweth R, Bielza P. Genetic study of Mediterranean and South American populations of tomato leafminer Tuta absoluta (Povolny, 1994) (Lepidoptera: Gelechiidae) using ribosomal and mitochondrial markers. Pest Manag Sci. 2011;67(9):1155–1162. doi: 10.1002/ps.2166. [DOI] [PubMed] [Google Scholar]
  10. De A, Bose R, Kumar A, Mozumdar S. Targeted delivery of pesticides using biodegradable polymeric nanoparticles. Berlin: Springer; 2014. [Google Scholar]
  11. Du J, Tang J, Xu S, Ge J, Dong Y, Li H, Jin M. ZnO nanoparticles: recent advances in ecotoxicity and risk assessment. Drug Chem Toxicol. 2020;43(3):322–333. doi: 10.1080/01480545.2018.1508218. [DOI] [PubMed] [Google Scholar]
  12. Duhan JS, Kumar R, Kumar N, Kaur P, Nehra K, Duhan S. Nanotechnology: the new perspective in precision agriculture. Biotechnol Rep. 2017;15:11–23. doi: 10.1016/j.btre.2017.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eggenberger K, Frey N, Zienicke B, Siebenbrock J, Schunck T, Fischer R, Bräse S, Birtalan E, Nann T, Nick P. Use of nanoparticles to study and manipulate plant cells. Adv Eng Mater. 2010;12(9):B406–B412. [Google Scholar]
  14. Fauteux F, Rémus-Borel W, Menzies JG, Bélanger RR. Silicon and plant disease resistance against pathogenic fungi. FEMS Microbiol Lett. 2005;249(1):1–6. doi: 10.1016/j.femsle.2005.06.034. [DOI] [PubMed] [Google Scholar]
  15. Fu Y-Q, Li L-H, Wang P-W, Qu J, Fu Y-P, Wang H, Sun J-R. Delivering DNA into plant cell by gene carriers of ZnS nanoparticles. Chem Res Chin Univ. 2012;28:672–676. [Google Scholar]
  16. Fu Y-Q, Li L-H, Wang P-W, Qu J, Fu Y-P, Wang H, Sun J-R. Delivering DNA into plant cell by gene carriers of ZnS nanoparticles. Chem Res Chin Univ. 2012;28(4):672–676. [Google Scholar]
  17. Fu Y, Li L, Wang H, Jiang Y, Liu H, Cui X, Wang P, Lü C. Silica nanoparticles-mediated stable genetic transformation in Nicotiana tabacum. Chem Res Chin Univ. 2015;31(6):976–981. [Google Scholar]
  18. Galdino TVdS, Picanço MC, Morais EGFd, Silva NR, Silva GARd, Lopes MC. Bioassay method for toxicity studies of insecticide formulations to Tuta absoluta (Meyrick, 1917) Ciência e Agrotecnologia. 2011;35(5):869–877. [Google Scholar]
  19. Ghareyazie B, Alinia F, Menguito CA, Rubia LG, de Palma JM, Liwanag EA, Cohen MB, Khush GS, Bennett J. Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA (b) gene. Mol Breed. 1997;3(5):401–414. [Google Scholar]
  20. Ghormade V, Deshpande MV, Paknikar KM. Perspectives for nano-biotechnology enabled protection and nutrition of plants. Biotechnol Adv. 2011;29(6):792–803. doi: 10.1016/j.biotechadv.2011.06.007. [DOI] [PubMed] [Google Scholar]
  21. Hajdukiewicz P, Svab Z, Maliga P. The small, versatilepPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 1994;25(6):989–994. doi: 10.1007/BF00014672. [DOI] [PubMed] [Google Scholar]
  22. Hajiahmadi Z, Shirzadian K, Reza KM, Sohani M. Expression of cryIAb driven by a wound inducible promoter (MPI) in tomato to enhance resistance to Tuta absoluta. J Plant Genet Res. 2018;4(2):1–16. [Google Scholar]
  23. Hajiahmadi Z, Shirzadian-Khorramabad R, Kazemzad M, Sohani MM. Enhancement of tomato resistance to Tuta absoluta using a new efficient mesoporous silica nanoparticle-mediated plant transient gene expression approach. Sci Hortic. 2019;243:367–375. [Google Scholar]
  24. Hamza R, Pérez-Hedo M, Urbaneja A, Rambla JL, Granell A, Gaddour K, Beltrán JP, Cañas LA. Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta. BMC Plant Biol. 2018;18(1):24. doi: 10.1186/s12870-018-1240-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hao Y, Yang X, Shi Y, Song S, Xing J, Marowitch J, Chen J, Chen J. Magnetic gold nanoparticles as a vehicle for fluorescein isothiocyanate and DNA delivery into plant cells. Botany. 2013;91(7):457–466. [Google Scholar]
  26. Hayford MB, Medford JI, Hoffman NL, Rogers SG, Klee HJ. Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases. Plant Physiol. 1988;86(4):1216–1222. doi: 10.1104/pp.86.4.1216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hussain HI, Yi Z, Rookes JE, Kong LX, Cahill DM. Mesoporous silica nanoparticles as a biomolecule delivery vehicle in plants. J Nanopart Res. 2013;15(6):1676. [Google Scholar]
  28. Inbar O, Liefshitz B, Bitan G, Kupiec M. The relationship between homology length and crossing over during the repair of a broken chromosome. J Biol Chem. 2000;275(40):30833–30838. doi: 10.1074/jbc.C000133200. [DOI] [PubMed] [Google Scholar]
  29. Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Nat Biotechnol. 1993;11(2):194. [Google Scholar]
  30. Ma J, Yamaji N. Functions and transport of silicon in plants. Cell Mol Life Sci. 2008;65(19):3049–3057. doi: 10.1007/s00018-008-7580-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moreno SC, Silvério FO, Picanço MC, Alvarenga ES, Pereira RR, Santana Júnior PA, Silva GA. New pyrethroids for use against Tuta absoluta (Lepidoptera: Gelechiidae): their toxicity and control speed. J Insect Sci. 2017;15(5):1–6. doi: 10.1093/jisesa/iex072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Motyka O, Štrbová K, Olšovská E, Seidlerová J. Influence of nano-ZnO exposure to plants on l-ascorbic acid levels: indication of nanoparticle-induced oxidative stress. J Nanosci Nanotechnol. 2019;19(5):3019–3023. doi: 10.1166/jnn.2019.15862. [DOI] [PubMed] [Google Scholar]
  33. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–497. [Google Scholar]
  34. Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8(19):4321–4326. doi: 10.1093/nar/8.19.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nair R, Poulose AC, Nagaoka Y, Yoshida Y, Maekawa T, Kumar DS. Uptake of FITC labeled silica nanoparticles and quantum dots by rice seedlings: effects on seed germination and their potential as biolabels for plants. J Fluoresc. 2011;21(6):2057. doi: 10.1007/s10895-011-0904-5. [DOI] [PubMed] [Google Scholar]
  36. Napierska D, Thomassen LC, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, Martens JA, Hoet PH. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small. 2009;5(7):846–853. doi: 10.1002/smll.200800461. [DOI] [PubMed] [Google Scholar]
  37. Pasupathy K, Lin S, Hu Q, Luo H, Ke PC. Direct plant gene delivery with a poly (amidoamine) dendrimer. Biotechnol J Healthc Nutr Technol. 2008;3(8):1078–1082. doi: 10.1002/biot.200800021. [DOI] [PubMed] [Google Scholar]
  38. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol. 2007;2(12):751. doi: 10.1038/nnano.2007.387. [DOI] [PubMed] [Google Scholar]
  39. Pereira RR, Picanço MC, Santana PA, Jr, Moreira SS, Guedes RN, Corrêa AS. Insecticide toxicity and walking response of three pirate bug predators of the tomato leaf miner Tuta absoluta. Agric For Entomol. 2014;16(3):293–301. [Google Scholar]
  40. Perez C, Sanchez A, Putnam D, Ting D, Langer R, Alonso M. Poly (lactic acid)-poly (ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA. J Control Release. 2001;75(1–2):211–224. doi: 10.1016/s0168-3659(01)00397-2. [DOI] [PubMed] [Google Scholar]
  41. Primrose SB, Twyman R. Principles of gene manipulation and genomics. New York: Wiley; 2013. [Google Scholar]
  42. Rai M, Ingle A. Role of nanotechnology in agriculture with special reference to management of insect pests. Appl Microbiol Biotechnol. 2012;94(2):287–293. doi: 10.1007/s00253-012-3969-4. [DOI] [PubMed] [Google Scholar]
  43. Rasband WS. IImageJ. Bethesda: US National Institutes of Health; 1997. [Google Scholar]
  44. Selale H, Dağlı F, Mutlu N, Doğanlar S, Frary A. Cry1Ac-mediated resistance to tomato leaf miner (Tuta absoluta) in tomato. Plant Cell Tissue Organ Cult (PCTOC) 2017;131(1):65–73. [Google Scholar]
  45. Shahbaz M, Nouri-Ganbalani G, Naseri B. Comparative damage and digestive enzyme activity of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) on 12 tomato cultivars. Entomol Res. 2019;49(9):401–408. [Google Scholar]
  46. Slomberg DL, Schoenfisch MH. Silica nanoparticle phytotoxicity to Arabidopsis thaliana. Environ Sci Technol. 2012;46(18):10247–10254. doi: 10.1021/es300949f. [DOI] [PubMed] [Google Scholar]
  47. Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S. Agricultural sustainability and intensive production practices. Nature. 2002;418(6898):671. doi: 10.1038/nature01014. [DOI] [PubMed] [Google Scholar]
  48. Tonnang HE, Mohamed SF, Khamis F, Ekesi S. Identification and risk assessment for worldwide invasion and spread of Tuta absoluta with a focus on Sub-Saharan Africa: implications for phytosanitary measures and management. PLoS ONE. 2015;10(8):e0135283. doi: 10.1371/journal.pone.0135283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Torney F, Trewyn BG, Lin VS-Y, Wang K. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol. 2007;2(5):295. doi: 10.1038/nnano.2007.108. [DOI] [PubMed] [Google Scholar]
  50. Wang Z, Xie X, Zhao J, Liu X, Feng W, White JC, Xing B. Xylem-and phloem-based transport of CuO nanoparticles in maize (Zea mays L.) Environ Sci Technol. 2012;46(8):4434–4441. doi: 10.1021/es204212z. [DOI] [PubMed] [Google Scholar]
  51. Weeks DP, Spalding MH, Yang B. Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnol J. 2016;14(2):483–495. doi: 10.1111/pbi.12448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yasmeen A, Mirza B, Inayatullah S, Safdar N, Jamil M, Ali S, Choudhry MF. In planta transformation of tomato. Plant Mol Biol Rep. 2009;27(1):20–28. [Google Scholar]
  53. Zhang S, Lian Y, Liu Y, Wang X, Liu Y, Wang G. Characterization of a maize Wip1 promoter in transgenic plants. Int J Mol Sci. 2013;14(12):23872–23892. doi: 10.3390/ijms141223872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zheng L, Hong F, Lu S, Liu C. Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach. Biol Trace Elem Res. 2005;104(1):83–91. doi: 10.1385/BTER:104:1:083. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOC 725 kb) (725KB, doc)

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES