1. Introduction
The release of nanoparticles (NPs) into the environment has raised concern because of their toxic effects on the environment and human health
[1]. Moreover, the release of NPs into the environment could result in their entry and accumulation in agricultural soils from bio-solids impregnated with NPs through the application of sewage sludge for agricultural purposes
[2]. Thus, the application of NPs in plant tissue cultures is promising, as this technique is used to screen different aspects of plants’ growth and development, as well as to engage in genetic manipulation, bioactive compound production and plant improvement
[3]. It has been noted that NPs have a positive impact because of their reduced size, elevated reactivity, mass-to-area ratio and other physico-chemical properties, but the negative effects of NPs have also been noted, which mainly depend on the type of metal, dissolution power and plant species
[4][5].
2. Efficiency of Nanoparticles in Eliminating Contamination
The production of healthy plantlets is a prime concern behind the technique of plant tissue culture but microbial contamination is a common problem faced during this procedure. Conventionally, antibiotics are employed to eliminate microbes, but their frequent application can negatively affect plant tissue growth, e.g., antibiotics like carbenicillin and cefotaxime inhibit plant cell growth, organogenesis and embryogenesis
[6][7]. Reports suggest that streptomycin and chloramphenicol interact with protein synthesis, rifampicin hinders nucleic acid synthesis and penicillin inhibits cell-wall membrane synthesis
[8][9]. There is also the risk of a decreased genetic stability and lower regeneration capability of plants when a high level of antibiotics is used
[10]. Nanomaterials are an alternative because of their distinctive features, which have been shown to possess antifungal and antibacterial properties that restrict microbial growth in in vitro cultures resulting in the successful mass propagation of selected species
[11]. Silver nanoparticles (AgNPs) have been considered one of the better options, as the anchoring and penetration of Ag ions into microbes alter the cellular signals, via dephosphorylation, of key peptide substrates on tyrosine
[12][13]. Another study suggested that Ag
+ ions cause a reduction in DNA replication, as well as inactivate the thiol group in proteins, that ultimately reduces microbial growth
[14]. Similarly, Min et al.
[15] reported that AgNPs restrict the growth of sclerotium-forming phytopathogenic fungi and, hence, can become an alternative to pesticides. AgNPs have been employed to reduce contamination during in vitro cultures of
Olea europaea L.
[16],
Nicotiana tabacum L.
[17][18],
Gerbera jamesonii Bolus ex Hook.f.
[19],
Solanum tuberosum L.
[11], almond x peach (G x N15) hybrid rootstock
[20],
Rosa hybrida L.
[21],
Vitis vinifera L.
[22],
Vanilla planifolia Jacks. ex Andrews
[23], and
Phoenix dactylifera L. cv. Sewi and Medjool
[24]. In addition, combined treatment with nanosilver and nano-iron particles was reported to have a significant effect on decreasing the contamination rate in
Fragaria ×
ananassa L. cv. Roby Gem
[25]. Similarly, biosynthesized silver, chitosan, and selenium NPs were tested for their antimicrobial potential for the in vitro multiplication of three olive cultivars (Koroneiki, Picual, and Manzanillo). Of all the three NPs, AgNPs showed the best antimicrobial properties in all cultivars
[26]. However, some studies have also suggested that the concentrations of AgNPs played a crucial role in culture growth as higher concentrations might induce adverse effects on explant response
[2][11]. The phytotoxic effect of higher AgNPs has been observed in crop plants of
Phaseolus radiatus L. and
Sorghum bicolor (L.) Moench
[27]. Whereas in tomato and potato plants, it has been reported that lower concentrations of AgNPs with longer exposure time effectively reduced the contamination without hampering explant viability
[28].
Titanium dioxide (TiO
2) is another NP that has gained attention due to its antimicrobial potential, as it has photocatalytic properties to eliminate contamination from various sources, but its toxicity against microbial growth depends on the intensity and wavelength of light with concentration and particle size
[9]. TiO
2 reacts with water molecules and forms free radicals like OH, HO
2, and H
2O
2 which in turn results in the oxidation of bacterial cells, suggesting that the photo-activation of TiO
2 via UV irradiation retards the bacterial growth
[29][30]. It has been evaluated that the addition of TiO
2NPs in the Murashige and Skoog (MS)
[31] medium enhanced the microbial resistance during the micropropagation of tobacco
[17],
S. tuberosum [9], and
Hordeum vulgare L.
[32]. Zinc oxide nanoparticles (ZnONPs) have eliminated nine strains of bacteria (
Bacillus megaterium,
Cellulomonas uda,
C. flarigena,
Corynebacterium panrometabolum,
Erwinia cypripedii,
Klebsiella spp.,
Pseudomonas spp.,
Proteus spp., and
Staphylococcus spp.) and four fungal species (
Aspergillus spp.,
Candida spp.,
Fusarium spp., and
Penicillium spp.) which increased difficulties during banana micropropagation
[33]. Thus, it can be observed that although nanomaterials at higher concentrations have been proven as toxic for plant growth, they can be employed as disinfectant agents for the in vitro multiplication of various economically important crops. The majority of the reports used Ag, TiO
2, and Zn-based NPs for the inhibition of microbial growth during in vitro propagation, but new types of NPs should also be assessed. In this regard, various kinds of advanced nanomaterials like graphene, graphite, quantum dots, carbon nanotubes, polymer dendrimers, and atomic clusters will provide enough scope for the study; along with this, evaluations of concentrations, sizes, and types of NPs on various crop species and type of explant are also needed
[34].
3. Influence of Nanoparticles on Seed Germination
Seed germination is a crucial stage for crop development since young seedlings are more vulnerable to biotic and abiotic stresses
[35]. Therefore, lots of efforts to improve the efficiency of seed germination are published from time to time with new technological interventions. Studies to analyze the effect of NPs have been conducted during the last few years, and it was observed that genotype, variety, seed age, and environmental conditions determined the response to NPs
[36]. Yasur and Rani
[37] and Hatami
[38] suggested that the water uptake during seed germination is critical because seeds are relatively dry and requires a substantial amount of water to initiate cellular metabolism and growth. The positive effects of NPs on germination begin with the high capability of NPs to penetrate the seed coat and promote water uptake along with the absorption of nutrients in the seed
[39]. Mehrian et al.
[40] documented that NP treatment accelerated seed germination from better water uptake by the seeds during the initial days, whereas a decrease in germination efficiency was noted as time passed because of the breakdown of stored nutrients or alternations in permeability properties of the cell membrane. Similarly, Rizwan et al.
[41] noted that NPs can penetrate through the seed coat and affect the development processes of embryos through stimulation of the enzymes of metabolic processes. During the radicle appearance stage of seed germination, root apex tissues come in contact with NPs, which then move into the rhizodermis through the apoplast with endocytosis. In the root, they flow towards the plant secretory tissue using symplastic pathways and translocate to other plant organs. However, it has been noted that NPs at a high concentration result in a perforation of the cell wall and penetrate the protoplast and damage the root cell vacuoles. This triggers more production of reactive oxygenspecies (ROS) and it causes a blockage of electron transfer which induces oxidative stress. NPs also up-regulate the genes involved in cell division and carbon/nitrogen metabolism, and the negative effects observed in seedling growth are due to chromosomal aberrations and mitotic abnormalities. This leads to a decrease in cell division of the root meristem, hormonal imbalance, ROS over-production, and increased levels of lipid peroxidation
[42]. The increased oxidative stress, in turn, increases hydrogen peroxide (H
2O
2) contents, activities of malondialdehyde (MDA), catalases (CAT), peroxidases (POD), and superoxide dismutase (SOD), as well as the production of compounds having antioxidant activities like phenolics and flavonoids
[43]. Many studies have documented that NPs exert positive or negative influences on seed germination, seedling biomass as well as biochemical and metabolite contents. In the present research, researchers have taken only those examples where NPs were added into the media and not where seeds were placed on filter paper or water agar media after sonication treatment with NPs.
3.1. Silver Nanoparticles (AgNPs)
In the majority of the studies, NPs’ effect has been evaluated under in vivo conditions
[44], but few were tested under in vitro conditions on the culture media. It is also observed that most reports suggested the usage of AgNPs (
Table 1), e.g., Lee et al.
[27] recorded a negative effect of AgNPs on
P. radiates and
S. bicolour seedling growth. Similarly, the growth of
Physalis peruviana L. seedlings also decreased along with chlorophyll content, but biomass in terms of fresh (FW) and dry weights (DW) was increased. It was also revealed that the seedling growths were not much affected in soil as compared to the agar-based medium. This might be due to changes in the physico-chemical properties of NPs in the soil, as pore water harbours a range of electrolytes that increase the aggregation of AgNPs in soil. These aggregates were larger than the pore size of plant root cells and thus failed to pass through the cells. Greater aggregation may be the principal reason for the reduced phytotoxicity of AgNPs in soil. Thus, the relative germination index is extensively used as an indicator of phytotoxicity, and root growth is one of the sensitive biomarkers for the phytotoxicity assay
[45]. Zaka et al.
[46] compared AgNPs, gold nanoparticles (AuNPs), and copper nanoparticles (CuNPs) for
Eruca sativa Mill. and observed that AgNPs increased seed germination, shoot and root lengths, and seed vigour index, whereas the other two adversely affected these parameters (
Table 1). Further evaluation unveiled that all the NPs affected the biochemical milieu of the plants differently (
Table 2). In another study, green synthesized AgNPs using
Curculigo orchioides Gaertn. were found to exert a positive influence on seedling growth and biomass of
Oryza sativa L. cv. Swarna. When the germinated seedlings were biochemically analyzed, an increase in chlorophyll, flavonol contents and enzymes (POD, SOD, CAT, APX, and GR) activities, and a decrease in phenolics, flavonoids, H
2O
2, and MDA contents were observed. The gene expression analyses revealed that the SOD gene was down-regulated, whereas genes for CAT and ascorbate peroxidase (APX) were up-regulated after AgNP treatment
[47]. Similarly, increased seed germination, seed vigour index, shoot and root lengths, and fresh and dry biomass in
Pennisetum glaucum (L.) R. Br. after the addition of AgNPs in the medium was reported
[48]. The maximum germination was recorded at 40 ppm; at this concentration of AgNPs, mild activities of 2,2-Diphenyl-1-picrylhydrazyl (DPPH), SOD activities and proline content were recorded that significantly increased at higher dose of AgNPs. On the contrary, phenolic contents were higher at optimum germination concentration (40 ppm) and lower at higher concentration, whereas flavonoids were lower at 40 ppm and increased at high levels. AgNPs positively influenced the germination and seedling traits of
Brassica oleracea L. var.
sabellica ‘Nero di Toscana’ and
Raphanus sativus L. var.
sativus ‘Ramona’, whereas these traits were decreased in
Solanum lycopersicum L. ‘Poranek’. One of the reasons behind decreased growth
S. lycopersicum might be due to the presence of AgNPs in plasmodesmata, precluding the transport of nutrients that led to a reduction in plant biomass
[49]. Recently, Tomaszewska-Sowa et al.
[50] observed the effect of AgNPs and AuNPs on
Brassica napus L., and revealed that application of both NPs decreased shoot and root lengths of seedlings irrespective of treatment time. However, total chlorophylls, carotenoids, anthocyanins, free sugars, and H
2O
2 contents were higher, but no major change in phenolics was found. The seed germination of
N. tabacum was carried out using CTAB- and PVP-coated AgNPs, and coating with CTAB showed a positive influence whereas coating with PVP failed to show any positive effect on germination rate and biomass
[51]. Similarly, positive influences of AgNPs have been also documented in
Brassica juncea (L.) Czern. var. pusajaikisan
[52],
Hylocereus undatus (Haw.) Britton and Rose
[53], and
P. vulgaris [54] (
Table 1).
3.2. Other Metal and Metal Oxide Nanoparticles
Apart from AgNPs, other metal NPs are also used for seedling germination under in vitro conditions; Dehkourdi and Mosavi
[59] utilized TiO
2NPs and documented a positive influence on seed germination as well as on chlorophyll synthesis in
Petroselinum crispum (Mill.) Fuss, whereas Nair et al.
[61] observed that the application of copper oxide nanoparticles (CuONPs) on
Vigna radiata L. decreased seedling growth in terms of length and biomass. They have also reported that CuONPs decreased chlorophyll and increased proline contents, whereas it increased H
2O
2 and MDA contents in the root; however, no change in carotenoid, H
2O
2, and MDA contents in the shoot and increased lignification of root cells were detected (
Table 2). The negative effect of CuONPs on seedlings of
Cicer arietinum L. was also documented where decreased growth and biomass have been recorded at all the tried concentrations (50–500 mg/L), and elevated H
2O
2 generation, MDA level, and POD activity along with increased lignifications in roots were observed. Further expression analysis revealed that
CuZn-SOD,
CAT, and
APX genes were up-regulated in roots but no change was found in shoots
[56]. Similarly, CuONPs, when used for the treatment of
Brassica nigra (L.) K. Koch, delayed the germination of seedlings and decreased plantlet length and biomass significantly
[55]. ZnONPs in the media containing seeds of the same plant negatively influenced seedling growth, shoot FW, and reduced stem diameter as the NP amount increased in the media. However, the treatment increased free radical scavenging activity, total antioxidant capacity, total reducing power, phenolics, and flavonoid contents in the shoot and root of the seedling (
Table 2)
[55]. Moreover, in seeds of
Linum usitatissimum L. cv. Barbara, different concentrations of ZnONPs (1, 10, 100, 500, and 1000 mg/L) were tried, and 100 mg/L concentrations proved beneficial in terms of shoot and root lengths as well as seedling biomass, further higher concentrations adversely affected seedling growth
[58]. In another study, treatment with multi-walled carbon nanotubes (MWCNTs) showed a positive influence on germination, seedling lengths, as well as biomass in
Glycine max (L.) Merr. hybrid S42-T4,
H. vulgare hybrid Robust, and
Zea mays L. hybrid N79Z 300GT
[57]. Unlike the spherical shapes of other NPs, MWCNTs are the allotropes of carbon that are arranged in an elongated, tubular manner with many rolled sheets. Its unique features like functional group, diameter, length, and solubility make its penetration inside the seed coat convenient and it is efficiently translocated in plants
[62]. Similar observations have been well documented previously where MWCNTs improve germination, plant growth, and agronomic traits by penetration, and increasing the water and nutrient uptake
[63][64].
Table 2. Biochemical changes in seedlings and cultures after NP treatment.
Plant |
Nanoparticle (NP) Treatment and Culture Type |
Biochemical Changes |
Reference |
Brassica juncea var. pusa jaikisan |
AgNPs, shoots |
Increased chlorophyll and decreased MDA, H2O2, and proline content, increased CAT, GPX, and APX activities |
[52] |
Brassica napus |
AgNPs/AuNPs, shoots |
Increased chlorophylls, carotenoids, anthocyanins, free sugars, H2O2 contents, no change in phenolic content |
[50] |
Brassica nigra |
ZnONPs, shoots and roots (seedling), callus |
Increased free radical scavenging activity, total antioxidant capacity, total reducing power, phenolic, and flavonoid contents |
[55] |
Brassica nigra |
CuONPs, seedling and roots (from leaf and stem derived callus) |
Seedlings increased free radical scavenging activity, total phenolic, and flavonoid content, decreased total antioxidant and reducing potential; Roots increased free radical scavenging activity, total antioxidant and reducing potential, total phenolic, and flavonoid contents |
[4] |
Brassica oleracea var. sabellica ‘Nero di Toscana’ |
AgNPs, leaves |
Decreased chlorophyll, carotenoid, and anthocyanin contents, no change in phenolic, protein contents and SOD activities, increased GPOX activity |
[49] |
Campomanesia rufa |
AgNPs, shoots |
No significant difference in SOD activity |
[65] |
Caralluma tuberculata |
AgNPs, callus |
Increased PAL and free radical scavenging, SOD, POD, CAT, APX activities, total phenolics, and flavonoid contents |
[66] |
Cicer arietinum |
CuONPs, seedling |
Increased H2O2 generation, MDA content, POD activity, and lignification in roots |
[56] |
Cichorium intybus |
Fe2O3NPs, hairy roots |
Increased hairy root growth, total phenolic, and flavonoid contents |
[67] |
Corylus avellana cv. Gerd Eshkevar |
AgNPs, cell suspension |
Increased CAT, APX, H2O2, PAL activities, decreased SOD and POD activities, and total soluble phenol content |
[68] |
Corylus avellana cv. Gerd Eshkevar |
AgNPs, cell suspension |
Increased MDA, total phenolic, anthocyanin, and flavonoid contents |
[69] |
Cucumis anguria |
AgNPs, hairy roots |
Increased total phenolic and flavonoid contents, and antioxidant activities |
[70] |
Eruca sativa |
AuNPs, CuNPs, and AgNPs, seedling |
AuNPs decreased total antioxidant capacity, total phenolic and flavonoid contents, increased DPPH, SOD and POD activities, no change in protein content; CuNPs decreased total antioxidant capacity, DPPH activity, protein content, increased total phenolic, and flavonoid contents, SOD and POD activities; AgNP decreased total antioxidant capacity, DPPH activity, decreased total phenolics and flavonoid contents, POD activity, increased SOD activity, no change in protein |
[46] |
Fragaria × ananassa cv. Queen Elisa |
FeNPs, shoots |
Increased chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, total carbohydrates, total protein, and total free proline and iron contents, decreased H2O2 and MDA content, higher SOD and POD activities |
[71] |
Linum usitatissimum cv. Kerman Shahdad |
ZnONPs/TiO2NPs, cell suspension |
Increased PAL and CAD activities, and total phenol content |
[72] |
Linum usitatissimum cv. Barbara |
ZnONPs, seedling and callus |
Increased ROS production, membrane lipid peroxidation, protein carbonylation and 8-oxo guanine formation, SOD, POD, radical scavenging activities, total phenolics, and flavonoid contents |
[58] |
Maerua oblongifolia |
AgNPs, shoots |
Higher chlorophyll, total protein and proline contents, and increased activities of antioxidant enzymes |
[73] |
Momordica charantia |
AgNPs, cell suspension |
Increased MDA, H2O2, total phenolics and flavonoid contents, and antioxidant activity |
[74] |
Musa paradisiacal cv. Grand Nain |
ZnNPs and ZnONPs, shoots |
Higher proline, chlorophyll, and antioxidant enzymes activities |
[33] |
Musa spp. |
AgNPs, shoots |
Increased chlorophyll content |
[75] |
Nicotiana benthamiana |
CH-ZnO, callus |
Increased chlorophyll, carotenoid, proline contents and PAL and AO activities, decreased MDA and H2O2 levels |
[76] |
Nicotiana tabacum cv. Bright Yellow-2 |
ZnONPs, cell suspension |
Decreased dehydrogenase, oxidoreductase SOD, POD and APX activities, increased GR, PAL, protease, caspase-like and acid phosphatases activities, and total phenolic content |
[77] |
Oryza sativa cv. Swarna |
AgNPs, seedling leaves |
Increased chlorophyll and flavonol contents and POD, SOD, CAT, APX and GR activities, decreased phenolics, flavonoids, H2O2 and MDA contents |
[47] |
Oryza sativa cv. IR64 |
AgNPs, shoot |
Decreased MDA, proline and H2O2 levels |
[78] |
Pennisetum glaucum |
AgNPs, seedling |
Increased DPPH, proline, SOD, POD, and CAT activities, total phenolics and flavonoid contents |
[48] |
Phoenix dactylifera |
MWCNTs, shoots |
Increased flavonoid, chlorophylls and carotenoid, nutrient contents, decreased phenolics and tannin contents, SOD, GPOX, and GR activities |
[79] |
Phoenix dactylifera cv. Hayani |
AgNPs, somatic embryos |
Increased chlorophyll content |
[80] |
Physalis peruviana |
AgNPs, seedling derived shoots and shoots |
Seedling derived shoots- increased CAT and APX activity, and decreased chlorophyll content, SOD and MDA activities;Shoots- no change in SOD, APX and MDA levels, decreased CAT activity |
[60] |
Raphanus sativus var. sativus ‘Ramona’ |
AgNPs, leaves |
Increased carotenoid, phenolic contents, and SOD activity, decreased chlorophyll, anthocyanins, protein contents, and GPOX activity |
[49] |
Saccharum spp. cv. Mex 69-290 |
AgNPs, leaves |
Increased N, Ca, Mg, Fe, Cu, Zn, Mn, and decreased P, K, and B content, higher total phenolics, ROS and lipid peroxidation contents, and antioxidant activity |
[81] |
Simmondsia chinensis |
MWCNTs, shoots |
Increased total tannin content and antioxidant activities, decreased phenolics and flavonoid contents |
[82] |
Solanum lycopersicon |
Fe3O4NPs, shoots |
Increased proline content and osmotic potential |
[83] |
Solanum lycopersicum |
ZnONPs, callus |
Increased Na, N, P, K, and Zn ionic, protein contents, SOD and GPX activity |
[3] |
Solanum lycopersicum var. Poranek |
AgNPs, leaves |
Increased chlorophyll, anthocyanins, phenolics, protein contents and SOD and GPOX activities, decreased carotenoid content |
[49] |
Solanum tuberosum |
SiO2NPs, leaves |
Increased antioxidant enzymes activity and expression of proteins |
[84] |
Solanum tuberosum cv. White Desiree |
AgNPs, shoots |
Increased total chlorophyll, carotenoids, proline, total flavonoids, phenolics, lipid peroxidation and H2O2 contents, decreased anthocyanins |
[85] |
Vanilla planifolia |
AgNPs, shoots |
Higher chlorophyll, increased elements like N and B, no change in P, Ca and Mg, and decreased K, Fe, Cu, Zn, Mn, and B contents, higher total phenolics, ROS and lipid peroxidation contents, and antioxidant activity |
[23] |
Vigna radiata |
CuONPs, seedling |
Decreased chlorophyll and increased proline contents, H2O2 and MDA contents in root, no change in carotenoid, H2O2 and MDA contents in shoots |
[61] |