Nanoparticles are able to interact with biomolecules, creating functional nanosystems for transportation within in vivo cells, and leading to the study of their potential applications in the field of plant biotechnology. The refore, the aim of this research was to determine the growth and rooting effect of functionalized (SWCNTs-COOH) and non-functionalized nanoparticles with iron residue inner particles (SWCNTs-Fe) in blackberry (Rubus adenotrichos) in vitro plants. Two types of SWCNTs were used, both of them characterized in a solid sample through Raman spectroscopy (λ = 532 nm) showing differences in the G band between SWCNT + Fe and SWCNT + COOH. The in vitro plants (approximately 15 mm len gth) were inoculated in a rooting medium. Six treatments were established: 4, 8, 12 μg/ml for each type of SWCNTs and a control without nanotubes. The assessed variables consisted of the average number of days for root emergence, average number of roots per plant, average root length per plant and the average stem length. This study determined that, in general, the SWCNTs-COOH promoted the growth of the in vitro plants under this assay, when compared to the SWCNTs-Fe trials. The lowest SWCNTs-COOH dose evidenced the best results for the assessed variables. Additionally, the histological analysis also evidenced that the plants treated with SWCNTs-COOH nanotubes (4 μg/ml) increased their cellular metabolism when compared to the control group.
Generally, the effect of nanoparticles in plants varies according to their composition, size, physical and chemical properties, as well as the plant species the nanoparticles interact with; producing enhancing or inhibitory effects on their growth in the different development stages [
Among nanoparticles, carbon nanotubes (CNTs) present different physical and chemical characteristics, including length, diameter, atomic configuration, impurities, defects and functionality, which allow them to have wide-ranging conductivity, tension strength, flexibility, and chemical reactivity properties. The CNTs structures can consist of a single layered cylinder, also known as single walled carbon nanotubes (SWCNTs); it can be comprised of two layers, being double-walled carbon nanotubes (DWCNTs); or it may contain multiple layers, which are called multi-walled carbon nanotubes (MWCNTs) [
These physical and chemical features of carbon nanomaterials have been used in agriculture to increase the crop yield, mainly in the germination process, root growth, and photosynthesis. However, many other plant interaction mechanisms exist, including absorption, accumulation, transport, or rejection of the nanoparticles; which have not been fully studied. Hence, despite the rapid progress in the development of this technology, the results obtained from its application have been very divergent [
Therefore, the aim of this research was to determine the effect of functionalized (SWCNTs-COOH) and non-functionalized (SWCNTs with iron residues) nanoparticles in the growth, rooting, and histology of blackberry (Rubus adenotrichos) in vitro plants.
The study was performed with two types of nanotubes, Single Walled Carbon Nanotubes functionalized with a carboxyl group (SWCNTs-COOH) (90wt% 1 - 2 nm outer diameter, 0.5 - 2.0 μm length Cheaptubes Inc.) and non-functionalized Single Walled Carbon Nanotubes with iron residues in their interior (SWCNTs-Fe) (Obtained by HiPCO 90wt%, 3.5 - 4.0 nm outer diameter, previously used by Flores et al., [
Blackberry in vitro plants, with a length of approximately 15.0 mm and reproduced at Centro de Investigación en Biotecnología (CIB, for its acronym in Spanish) were used for this study. The plants were inoculated in a rooting culture medium developed by Flores et al. [
The media were dispensed in 150 ml glass flasks (20 ml of medium/flasks) and the number of plant segments was of two nodal sections per flask. The in vitro plants were incubated at 16 hours of light photoperiod and a temperature of 22˚C ± 2˚C.
The variables evaluated were the average number of days for root emergence, the average number of roots per plant, the average root length per plant, and the average length of the stem. The sample consisted of 45 explants per treatment and the information generated was analyzed through the General Linear Model (GLM) (p = 0.05) for the analysis of variance and Welch’s ANOVA (p = 0.05), using Minitab16® statistical software.
In order to analyze and compare the ultrastructure of the plants’ vascular system, five 0.3 mm stem samples of different control plants and another five plant samples from the best resulting SWCNTs treatment were used after a month of acclimation. In both cases, the samples were taken from the stem’s mid-section.
The procedure described by Çağlayan et al. [
It was determined that, the SWCNTs-COOH favored the in vitro plant growth when compared to the SWCNTs-Fe assay; however, the statistical analysis established that there were no significant differences for the interaction (p > 0.05) between the type of nanotube and the evaluated concentrations. In regards to the Main Factor Analysis, the treatment with the best values for the evaluated variables was SWCNTs-COOH in the lowest dose (4 µg/ml). Under this treatment, the roots developed in the shortest time (24.32 ± 6.09 average days) being statistically different from the rest of the treatments (p < 0.05), including the control. Moreover, this treatment presented the highest number of roots per plant (8.60 ± 5.75 roots), being comparable only to the treatment with 12 µg/ml of SWCNTs-COOH and the control (
In general, the application of SWCNTs-COOH was beneficial to the growth parameters evaluated. It was possible to prove that the effect is dependent on the concentration, where plants dosed with low concentrations of CNTs can have positive effects on seed germination, root development and water transport within the plant, or no evidence of phytotoxicity; contrary to the negative effects that can be produced by high dose trials [
rot, cucumber, lettuce, onion, and tomato seeds to concentrations ranging between 56 and 1750 mg/L of functionalized and non-functionalized SWCNTs, establishing that the root elongation was promoted by the lowest concentration of SWCNTs and inhibited at the highest doses, obtaining the best results with non-functionalized SWCNTs. On the other hand, Khodakovskaya et al. [
In
higher toxicity effects (Cañas et al. [
The characteristics of CNTs will influence absorption, translocation, and interaction with the plant’s cell. The CNTs absorption depends on their dispersion within the experimental medium in which they interact [
For instance, studies related to the SWCNTs dispersion within an ionic liquid medium have determined that they can interact through weak Van der Waals forces [
Additionally, the nanoparticles can be introduced in the plant by their attachment to carrier proteins, through aquaporins, using ionic channels, by endocytosis, and through their merge with organic compounds. In the particular case of CNTs, their entrance is associated mainly to the creation of new pores in the cells [
In the research carried out by Liu et al. [
Once inside the plant, it is likely that the SWCNTs are transported for long distances through its vascular system; however, more evidence is necessary to support this statement [
The CNTs singularity of transporting and releasing biomolecules has been proven in numerous research studies related to proteins and nucleic acids, where cell processes interfere, allowing the molecules transported by the CNTs to be available for their transfer into particular organelles [
The histological analysis performed through TEM evidenced ultrastructural differences between the control plant tissue and the tissue obtained from plants under the best treatment (
In the control tissue samples, the presence of vascular cambium and the initial differentiation of the xylem tissue is evident; as well as the absence of phloem tissue (
It has been considered that the plants rooted with SWCNTs must be monitored for a long term, in order to determine whether it is safe to use CNTs for plant biotechnology application; since the literature reports phytotoxic effects at high concentrations in other species [
The SWCNTs-COOH treatments were beneficial for plant growth and rooting. The SWCNTs-COOH assay at a 4 μg/ml concentration presented the shortest average time for root emergence and the plants also had the greatest stem growth.
Furthermore, the TEM analysis evidenced an enhancement in cell metabolism with the stem tissue of plants under the 4 μg/ml SWCNTs-COOH treatment.
The authors would like to thank the Vicerrectoría de Investigación y Extensión of the InstitutoTecnológico de Costa Rica for funding this research.