Novel Methodology for Delivering Putrescine into the Apple Explants Using Hormone Anchored Nanotube

doi:10.21203/rs.3.rs-681947/v1

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Novel Methodology for Delivering Putrescine into the Apple Explants Using Hormone Anchored Nanotube

https://doi.org/10.21203/rs.3.rs-681947/v1

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Multi wall carbon nanotubes have been successfully exploited as growth regulator for manipulation of plant development. Also, nanoparticles are gradually involved in target delivery systems as the carrier of hormones. Polyamines and their derivations play crucial roles in plant growth and development. Take the mentioned subjects into consideration, putrescine anchored carbon nanotube which had been labeled with fluorescein was synthetized in this study. A set of physiological and morphological parameters were assessed in an attempt to examine the usage potential of de novo synthetized nanotube in terms of plant in-vitro culture. For this purpose, the nanotube was applied onto the in-vitro plantlets of Malus niedzwetzkyana in three concentrations (0, 50 and 100 mg/l). Localization of the nanotube in the plantlets was accomplished using fluorescence microscopy. Bio-imaging of tissues indicated the existence of nanotube in nearly all studied organs. Application of the nanotube at both concentrations (50 and 100 mg/l) increased the rate of leaf formation and speeding up the plastochron. Also, proliferation of the plantlets was enhanced using the nanotube. The levels of the photosynthetic pigments, including chlorophyll a, b and carotenoids increased following application of the nanotube. Glutathione peroxidase activity was significantly affected by the nanotube. However, polyphenol oxidase and peroxidase were not influenced by the nanotube. Stomatal density was increased by treatment of the plantlets with the nanotube. Representing geometrical transformation of shape as a thin plate spline revealed that the nanotube effectively increased longitudinally of stomata and changes their aspect ratio.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

bio-imaging

Malus niedzwetzkyana

multi-wall carbon nanotubes

plastochron

putrescine

stomatal density

Plant development is known as the multistage complicated process at which various internal and external factors are involved (Goldberg 1988). Generally, plants receive many signals from under and above ground organs in regard to environmental condition which are converted to biochemical massages through signaling network (Mulligan et al. 1997; Foyer et al. 2003). Plants could figure out environmental and nutritional cues which beside their metabolic roles can act as signals for plant development (Gilroy and Trewavas 2001; Considine 2018). In this regard, signals are received by cell membrane occupied receptors specially protein kinases and other second messengers for activating transduction proteins and subsequently running transcription factors (Trewavas and Malhó 1997). Therefore, either of external or internal signals such as light and plant growth regulators (PGRs), control the numerous specific transcription factors and set into motion a cascade of biochemical events that ultimately evoke physiological response which finally determine the plant growth and developmental fate (Gaspar et al. 1996; Gilroy and Trewavas 2001; Jarad et al. 2020).

Many evidences have indicated that polyamines (PAs) and their derivations play crucial roles in plant developmental process (Allen 1983; Evans and Malmberg 1989; Galston and Sawhney 1990; Martin-Tanguy 1997; Weiger and Hermann 2014; Tiburcio and Alcázar 2018). Actually, PAs including putrescine, spermidine and spermine are low molecular and aliphatic nitrogen organic cation (Galston and Sawhney 1990) which are mainly derived from the amino acids such as ornithine and methionine via decarboxylation reaction (Shah and Swiatlo 2008). Putrescine, the major form of PAs, interferes in many plant developmental processes encompassing cell division, leaf growth, floral and inflorescence formation and morphogenesis (Slocum et al. 1984; Walden et al. 1997; Kakkar et al. 2000; Pal Bais and Ravishankar 2002; Baron and Stasolla 2008). On the other hand, many researches indicated the mediatory role of PAs especially putrescine in relation to environmental stress (Alcázar et al. 2010). Considering the PAs importance in regard to plant growth and development, demands for their usage in-vitro and in-vivo conditions as valuable PGRs have been increased (Walden et al. 1997; Pal Bais and Ravishankar 2002; Zhu and Chen 2005; Ioannidis and Kotzabasis 2007; Baron and Stasolla 2008; Cai et al. 2015; Tiburcio and Alcázar 2018; Khoshbakht et al. 2018).

Nowadays, nanoparticles are increasingly utilized in some field of plant sciences in particular tissue culture and in vitro procedures. Some studies have indicated that multi wall carbon nanotubes (MWCNTs) trigger the expression pathways of aquaporin water carrier proteins, PIP₁ and PIP₂, which can enhance plant growth and development. Moreover, CNTs could activate enzymatic systems related to cell cycle upon which cell division is profoundly amplified (Khodakovskaya et al. 2012). The interaction of CNTs with protein and polysaccharides generates a cascade of signals resulting in accumulation of compounds leading to cell wall thickening and subsequent growth (Burman and Kumar 2018). To that end, due to considerable roles of nanomaterials on plant and development, the compounds have been recently classified as plant growth regulators (Hermes et al. 2020).

Beside the biochemical characteristics of nanoparticles they are gradually exploited as the carrier for primary ingredients for target delivering in biological systems (Azizi et al. 2020; Sabzi et al. 2020). Undoubtedly, enrichment of nanoparticles with PGRs is a novel approach by which manipulation of plant morphogenesis would be possible and acceleration of plant growth and development could be achieved (Pereira et al. 2017; Pascoli et al. 2018). So, planning for production of the new generation of PGRs anchored nanoparticles will perhaps be a new challenge for bio-science researchers to overcome the human food safety in future.

This study, investigates the effect of the putrescine anchored MWCNT as the putrescine delivering composite on growth and development of the red-leaf apple, i.e., Malus niedzwetzkyana. Niedzwetzky's apple is a rare, difficult-to-propagate species which has been put on endangered plants list throughout its range due to human activities (Dzhangaliev 2003). In recent experiment in-vitro cultured apple plantlet were treated by synthetized nanoparticles and the range of their growth as well as development was tested.

Preparation of nanoparticles

The putrescine anchored MWCNT was synthetized for the first time using ingredients including MWCNTs, putrescine, thionyl chloride (SOCl₂) and fluorescein. The MWCNTs were preliminary purified and activated by multiple ultra-sonication in water bath using 10% HCl solution, ethanol (95%) and distilled water, separately. Small amount (0.5 gr) of the purified MWCNTs were then treated with 20 mL aqueous solution of HNO₃ (60%) in an ultrasonic bath for at least 20 min, and left under reflux condition for 24 h. Afterward, the precipitate was filtered and washed several times using distilled water to ensure the pH of the filtrate reaches to neutral point. Prepared oxidized MWCNTs (denoted as MWCNT-CO₂H) were stored under vacuum condition (2 atm., 60^°C) and dried up to 24 h (Kong et al. 2004).

Definite amount of the synthesized MWCNT-CO₂H (0.3 g) was suspended in 15 mL of SOCl₂ and then heated under reflux condition for 24 h. Then, remained SOCl₂ was distilled off and the obtained precipitate (MWCNT-COCl) was suspended in 10 mL of dry tetrahydrofuran (THF) using ultrasonic bath. Immediately, putrescine (0.5 g) was added to the suspension and stirred at room temperature for 24 h. Finally, the obtained MWCNT-Put was isolated by filtration and washing with THF as well as distilled water (three times) followed by drying under vacuum condition (2 atm, 60°C) for 24 h.

To provide the fluorescein labeled MWCNT-Put (MWCNT-Put-Fl), fluorescein methyl ester was at first synthetized. For this purpose, esterification of the fluorescein was accomplished via sulfuric acid catalyzed reaction in refluxing methanol to produce fluorescein methyl ester. Small amount of the prepared ester (0.05 g) was added to the suspension of the previously obtained MWCNT-Put in 10 mL of methanol (ultra-sonication in water bath for 30 min) and refluxed for 24 h. Subsequently, isolation of the MWCNT-Put-Fl was attained following filtration and washing with methanol and distilled water. In order to determine the functional groups on the prepared materials, Fourier transform infrared (FT-IR) spectra were collected over the range of 400–4000 cm^− 1 using a Win-Bomem spectrometer (version 3.04 Galactic Industries Corporation, USA).

All used chemicals and substances in the case of synthetizing nanoparticles were purchased from Millipore Sigma (formerly Sigma-Aldrich, Burlington, USA).

In-vitro Plant culture and treatment

In-vitro plant establishment was accomplished using common plant tissue culture procedures. For this purpose, dormant vegetative buds were collected from five years old Niedzwetzky's apple trees cultivated in the botanical garden, university of Tabriz, Iran (Khalat cord). The collected buds were dissected using a Nikon SMZ 1000 stereomicroscope (Nikon Corp. Tokyo, Japan) under a lamina hood. All plant materials were sterilized with 1.0% sodium hypochlorite for 10 min then immersed in ethanol 70% for 5 min and finally washed with sterilized distilled water for 20 min. The shoot apical meristems (SAMs) were excised and cultured in half strength Murashige and Skoog medium (Murashige and Skoog, 1962). The established meristems were subculture in the basal Murashige and Skoog medium as control and the same medium was supplemented with two concentration values of synthetized MWCNT-Put-Fl, i.e., 50 and 100 mg/L. Auxin (IAA) and benzyl amino purine (BAP) in low concentrations (0.2 mg/L for IAA and 0.5 mg/L BAP) were added to all treatments. The treated plantlets were maintained for 30 days in culture room (22℃/18℃, day/night). This study was performed in randomized design with two treatments and 5 replications.

Biochemical analysis and enzyme assay

Activity of some antioxidant enzymes including glutathione peroxidase (GPX), polyphenol oxidase (PPO) and peroxidase (POD) was assessed in the present study. Leaves were collected from in-vitro plantlets and weighted up. Certain amount of phosphate buffered saline (PBS) was added to incised samples (10 mL per 1 gr) and they thoroughly homogenized. The homogenates were centrifuged (6000 rpm, 20 min) and the supernatant were utilized for the assay of enzyme activity. The enzyme activity, i.e., the amount of the enzyme requires for catalyzing decomposition of one micromole (µmole) of substrate to product per minute, was determined using colorimetric procedures. For this purpose, a multi-mode micro plate reader Biotek Synergy HT (BioteK, Vermount, USA) equipped with UV-Vis absorbance detectors was exploited. Enzyme bioassay kits including GPX (CAT number: 353919) and POX (CAT number: MAK092) were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). The kit for PPO (CAT number: ARG82015) was provided from Arigo (Arigo Biolaboratories Corporation, Tiwan). All steps in sample preparation was fulfilled according to the given instructions in kits and absorbance was read at 412 nm, 410 nm and 570nm for GPX, PPO and POX, respectively. For every treatment, three replications were considered.

The contents of photosynthetic pigments were achieved by grinding fresh leaves in acetone 90% and recording the absorbance at 663 nm, 645 nm and 470 nm for chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car), respectively. For this purpose, a UV-Vis spectrophotometry (UV-1800 Shimadzu, Japan) was used. The pigment contents (Chl a, b and carotenoids) were calculated based on traditional method (Kocsanyi et al. 1988).

Electron microscopy

For studying the morphology and elemental analysis of the nanoparticles, they were coated with a thin layer of gold using routine methods and visualized using a Mira3 Tescan (Tescan, Czech Republic) scanning electron microscope (SEM) integrated with an energy depressive spectroscope (EDS) analyzer. To evaluate nanostructure and dimension of the synthetized nanoparticles, they were dispersed in doubled-distilled water and studied under a Zeiss EM-90 (Zeiss, Jena, Germany) transmitted electron microscope (TEM).

Wide angle X-ray diffraction (XRD) profiles of MWCNT-Put-Fl were collected using a Bruker D8 Advance diffractometer with wavelength, λ = 0.154059 nm (Cu Kα) at 30 keV.

Epi-fluorescence microscopy (EFLM)

Nanoparticles in the prepared samples were detected and localized exploiting EFLM techniques. For this purpose, five well-developed specimens including stems and leaves were randomly collected and carefully sectioned by hand microtome. Prepared samples were immediately mounted onto the glass slide and several drops of deionized water were added onto them. The samples were viewed through an Olympus BX51 fluorescence microscope equipped with a BX-RFA fluorescence illuminator, U-CA intermediate magnifying device and catadioptric UMPlanFL-BDP objectives (Olympus optical Co., Ltd. Tokyo, Japan). The samples were illuminated for fluorescence excitation using U-MWB3 (480–510 nm) and U-MWG3 (510–550 nm) mirror cube units. Z-stacks from successive focal plates were acquired utilizing a Hamamatsu C7780-10 cooled CCD (Hamamatsu photonic Co., Tokyo, Japan). Digital colour images were saved in TIFF format (12 bits per each channel) with resolution of 1344 x 1024 pixels.

Studying the stomata was carried out according to the techniques which had been previously used in detection of protodermal cells (Dadpour et al. 2011). Leaves from in-vitro plantlets were collected and fixed in FAA (5 parts formalin: 5 parts glacial acetic acid: 90 parts 50% ethanol) for one day. The samples were then immersed in glacial acetic acid up to 5 min and stained with saturated aniline blue (CAS number: 28631-66-5, Sigma-Aldrich, USA) for an overnight. Re-staining of the samples was performed using 0.2% (v/v) fuchsin acid (CAS number: 3244-88-0, Sigma-Aldrich, USA) up to several days. Excess stain was washed away by ethanol and studied utilizing the microscopic methodology which was described in the previous section. To avoid tissue crumpling, imaging process was done while the sample had been immersed under the absolute ethanol.

Image processing

Acquired consecutive images were imported to Image J 1.37 software (freely available from http://rsb.info.nih/ij/) to produce Z stack file (Peighambardoust et al. 2010). Then, image level was adjusted in entire of images stack. Subsequently, image sharpness was improved by applying the Unsharp mask filter at the moderate range (Gaussian radius = 5 and Mask weight = 0.5). Z-stack composition was done automatically using Z-function plug in to improve depth of focus (Forster et al. 2004). The final outputs were trimmed to proper size and scale bars were added based on optical magnification.

Estimating the density of stomata using image analysis procedure

Image analysis for estimation of stomatal density was fulfilled based on the previously described method with some modification (Albrechtová et al. 2014). Final fluorescence images of the stomata were used for providing local image with 500 × 500 micron area randomly collected from various portions of leaf (Fig. 4A). Extracted images were then imported to Image J 1.37 software and three gray images belonging to red, green and blue channels extracted by RGB channel splitting mode. Image of red channel with higher contrast was collected for setting threshold (Fig. 4B). Threshold adjusted image was then used for automated analysis of stomatal density (Fig. 4C). In this study 10 replicates including local images were utilized for estimating of stomatal density in each treatment. Using particles analysis mode, the software detected outlines of stomata and counted them (Fig. 4D). Obtained data from microscopic image analyzing were used for statistical assessment of stomatal density.

Geometric morphometric characteristics of stomta

A geometric morphometric study was conducted for evaluating the form of stomata and change in their features followed by application of the treatments. For this purpose, digital images of individual stomata were extracted from high magnified microscopic images. For every treatment including control, MWCNT₅₀-Put-Fl and MWCNT₁₀₀-Put-Fl, 20 images of stomata were provided. In this study, 100 semi-landmarks were digitized along the outline of stomata using the TpsDig software. Consensus image was created from obtained semi-landmarks of the replications in every treatment using TpsSuper software (Zelditch et al. 2012; Rohlf 2015). Realized datasets, representing average stomatal forms corresponding to treatments, were imported into the PAST 3.1 software. Semi-landmark configurations were optimally aligned, translated and rotated (excluding size factor) based on the Procrustes superimposition method. Testing the significance of shape differences among the treatments was accomplished using one-way analysis of covariance (ANCOVA). A Principal component analysis (PCA) was applied on datasets and similarity between groups was determined using UPGMA (Unweighted Pair Group Method with Arithmetic mean) method. Stomatal shape deformation as well as differences between treatments and control was graphically presented in transformation grids through thin-plate spline (TPS) analysis.

Preparation and characterization of MWCNT-Put-Fl

Synthetizing the putrescine anchored MWCNT has been schematically described in Fig. 1. As shown, MWCNT-Put was prepared in three stages. At first step, the surface of MWCNT was oxidized using HNO₃ aqueous solution to production of MWCNT-CO₂H. The product was subsequently converted to MWCNT-COCl using SOCl₂ under reflux conditions. The last stage in preparation of the nanotube was executed by manipulating the putrescine in dry THF at room temperature. As the result, linkage of the putrescine with the MWCNTs was occurred due to formation of amide. To provide the possibility of detecting the nanotube in the plant tissues by means of fluorescence microscopy, a few molecules of fluorescein, as fluorescence labels, were covalently bonded to MWCNT-Put. For this purpose, some free amine moieties of putrescine were linked to the MWCNTs with fluorescein methyl ester through amidation (Fig. 1).

The obtained MWCNT-Put-Fl was characterized using FT-IR, SEM, EDX and TEM analysis. XRD pattern of MWCNT-Put-Fl shows two distinct peaks at 2θ = 25.6° and 42.7°, corresponding to the (002) and (100) reflections, indicating that the crystallinity of MWCNTs is not lost during oxidation, and then functionalization with putrescine and fluorescing molecules, in comparison with reported XRD patterns in the literature (Fig. 2A). (Das et al. 2014; Abdulrazzak et al. 2019).

FT-IR spectra were measured to study the presence of functional groups along with chemical modification of MWCNTs. FT-IR spectrum of MWCNT-CO₂H exhibits peaks at 1013–1333, 1550–1643, 1709 cm^− 1 and a broad peak at 2570–3461 cm^− 1, corresponding to the C―O, C = C, C = O and O―H stretching vibrations, respectively. Putrescine shows two distinct peaks at 3290 and 3359 cm^− 1 associating with symmetric and asymmetric vibrations of NH₂ moiety, respectively. Symmetric and asymmetric vibrations of CH₂ are appeared at 2852 and 2921 cm^− 1, respectively. Peaks at 1601, 1461 and 1071 cm^− 1 are attributed to the bending vibrations of NH₂ and CH₂, and stretching vibration of C―N bond, respectively. In the FT-IR spectrum of the MWCNT-Put, peaks at 1013, 1200, 1556, 1712 and 2564–3079 (broad) cm^− 1 are shown, which are attributed to the stretching vibrations of C―O, C―N, C = C, C = O of unreacted carboxylic acids, and O―H bonds, respectively. In addition, the appearance of peaks at 2844, 2925 and 3402 cm^− 1, corresponding to the symmetric and asymmetric vibration of CH₂ and stretching vibration of N―H, along with peak of C = O of amides at 1698 cm^− 1 could be attributed to the covalently bonding of putrescine to MWCNT via amide formation. In the FT-IR spectrum of the MWCNT-Put-Fl, in addition to distinct peaks of MWCNT-Put, a new broad peak at 3170–3551 cm^− 1, corresponding to the stretching vibration of O―H of fluorescein, was appeared (Fig. 2B).

Figure 3 shows SEM and TEM images and EDX analysis of MWCNT-Put-Fl. SEM images were recorded in order to investigate the morphology of MWCNT-Put-Fl, which shows the irregular aggregation and agglomeration of MWCNTs (Fig. 3A). TEM images show an interwoven network of carbon nanotubes (Fig. 3B). Both SEM and TEM images exhibited that the structure of MWCNTs was remained unchanged during acidic oxidation and modification. Also, EDX analysis indicated the presence of C, O and N atoms in the structure of MWCNT-Put (C: 84.47, O: 13.29 and N: 2.24 wt%) and MWCNT-Put-Fl (C: 82.17, O: 15.92 and N: 1.91 wt%), approving the modification of MWCNTs surface by putrescine and fluorescein (Fig. 3C).

Localization of MWCNT-Put-Fl in the plantlets

Tracing of the fluorescein labeled nanotubes using fluorescence microscopy demonstrated high affinity for their absorption in the plantlets. Nearly in all studied tissues and cells, MWCNT-Put-Fl at two concentrations, was detected. However, localization of the nanotubes indicated that distribution in the vascular tissue and stomata is higher than other tissues. In the leaf epidermis, tendency for aggregation of the MWCNT-Put-Fl in cell walls (perhaps cell membrane) was higher than the other section (Fig. 5). High magnified microscopic images indicated that major amounts of the MWCNT-Put-Fl have been aggregated in the cellular microtubules and actins (Fig. 5, A). Also, localization of the nanotubes in stomata was higher than pavement cells, and high fluorescence signals were detected in the guard cells (Fig. 5, E).

Effects of the MWCNT-Put on the growth and development of in-vitro plantlets

Effects of the putrescine anchored MWCNT onto the growth and development of in-vitro apple plantlets was evaluated. For this purpose so called leaf number, plastochron and proliferation (the rate of lateral outgrowth) were estimated and statistically analyzed (Fig. 6). The effect of time on the rate of leaf formation and plastochron was significant (p < 0.05). However, at the t₀, t₁ and t₂, any significant differences between treatments was observed. Comparison of treatments was performed in every time independently from the other dates. Obviously, treatment of in-vitro explants with the MWCNT-Put, exhibited positive effect on leaf formation at the last dates of study (t₃ and t₄). Two concentrations of the MWCNT-Put, with any statistically difference, had significant effect (p < 0.05) on leaf formation at the mentioned dates in compare to control (Fig. 6A). As shown in Fig. 6B, the trend of leaf formation in all treatments was nearly similar up to 3rd week. Distinct ascending feature in leaf emerging appeared in 4th weak in relation to MWCNT₅₀-Put and MWCNT₁₀₀-Put comparing with the control.

Plastochron, i.e. the time interval at which new leaf primordium is initiated, was obviously influenced by treatments (Fig. 6). During the first two time intervals including t₀-t₁ and t₁-t₂, no significant differences were observed between treatments in corresponding dates (Fig. 6C). After this period, however equal descending trends regarding to plastochron were obtained for all treatments, while the factor was significantly lower in the case of MWCNT-Put treatments than the control (Fig. 6D). Obviously, in the control plantlets, plastochron nearly reached to steady state at the last time interval. However, descending trend continued in the case of treated plantlets with the both concentrations of the MWCNT-Put (Fig. 6C). In contrast to first moiety of the experiment, MWCNT₅₀-Put and MWCNT₁₀₀-Put similar together, had significant (p < 0.05) effect on reduction of plastochron comparing to control at the t₂-t₃ and t₃-t₄ time intervals (Fig. 6C).

Proliferation of the plantlets presented as the number of lateral shoots (Fig. 7A), significantly (p < 0.05) increased due to application of the MWCNT₅₀-Put and MWCNT₁₀₀-Put (Fig. 7B). According to the counted shoots per primary explant after 4 weeks period of culture, MWCNT₅₀-Put and MWCNT₁₀₀-Put treatments presented statistically similar effect on proliferation. In the same way, treatment of plantlets with MWCNT-Put led to formation of shoots with longer internodes comparing to control (Fig. 8AB).

Effects of the MWCNT-Put on the biochemical characteristics of in-vitro plantlets

Data obtained from biochemical analysis indicated that photosynthetic pigments, including chlorophyll a, chlorophyll b and carotenoids, were significantly (p < 0.05) affected by MWCNT-Put in all concentration in compare to control. Whereas, any difference between MWCNT₁₀₀-Put and MWCNT₅₀-Put treatments was observed in this regard (Fig. 9A).

Study of the enzymatic activity in the treated plantlets revealed that only GPX has been positively influenced by the MWCNT-Put treatments. Differences between MWCNT₁₀₀-Put and MWCNT₅₀-Put were not statistically significant (Fig. 9B). While, any significant effects of treatments were found in relation to activity of either PPO or POX in comparison to control.

Effects of the MWCNT-Put on the stomatal density and form

Data extracted from image analysis of microscopic observations and their statistically comparing demonstrated that the density of stomata was significantly affected by MWCNT-Put treatment (p < 0.05). Any symptom of stomatal patterning was observed in the adaxial surface of the studied leaves in all treatments. In contrast, considerable density of stomata was found in the abaxial surface of leaves. Stomatal density in leaves of the MWCNT-Put treated plantlets was significantly (p < 0.05) higher than control (Fig. 10).

Studying the abaxial surface of the leaves with more accuracy was shown the treated plantlets have considerable amount of stomata over the leaf veins. These types of stoma were longitudinally arranged over the leaf veins and parallel to their directions (Fig. 11).

The form of stomata was remarkably affected by MWCNT-Put treatment. Geometrical analysis of semi-landmarks revealed a distinct transforming in stomatal form as the result of MWCNT-Put application. The higher the concentration of the nanotube, the more change from circularity to elliptically in stomatal form occurred. The XY graph of the consensus images of stomata in relation to treatments showed significantly change in aspect ratio (length/width) from 0.9 to 1.4 in regard to control and MWCNT₁₀₀-Put, respectively (Fig. 12A). In other words, circularity in stomatal form in control with nearly 90% deceased to 71.5% in MWCNT₁₀₀-Put treatment. Evaluation the similarity of stomatal form between treatments has been shown in Fig. 12B. As it appears, more similarity was achieved for MWCNT₁₀₀-Put and MWCNT₅₀-Put than control (Fig. 12B).

Demonstrating stomatal shape transformation as a thin plate spline surface described the similarities and differences in geometrically reciprocal characters. The contrast between the treated and control plantlets in terms of the digitized semi-landmarks used to quantify stomatal forms. As per morphometric convention, the configuration of the target (the treated plantlets with MWCNT₅₀-Put and MWCNT₁₀₀-Put) versus reference (the control) specimen was plotted along with the spline grids. The highest deformation of stomatal form was shown in regard to MWCNT₁₀₀-Put versus control. Arrows accentuated areas of the largest differences which are concentrated around the vertices and co-vertices (Fig. 13A). At the same manner, slightly low changes in the stomatal form were observed regarding MWCNT₅₀-Put versus control (Fig. 3B). The lowest deformation belonged to the MWCNT₁₀₀-Put versus MWCNT₅₀-Put (Fig. 13C) which confirmed validation of stomatal shape clustering (Fig. 12B).

Demand for the application of nano-materials in the field of biology, medicine and food sciences has been recently increased (Azizi et al. 2020; Salama et al. 2021; Panahirad et al. 2021). Many reports revealed that nano-materials could directly influence the biological systems as promoter. In fact, nano-particles could somewhat act as carriers for conjugated substances by which target delivery could be possible. In this way, probability of degradation of conjugated substance would be considerably decreased. The main challenge in producing substance-anchored nanoparticle, would be adopting the appropriate synthetizing procedure at which the considered substance is mounted onto the special nanoparticle. Many studies have demonstrated the amplified effects of substance anchored nanoparticles compared to individual treatment of substance or nanoparticle.

Synthetizing MWCNT-Put-Fl

As mentioned earlier, features of the MWCNT-Put-Fl were characterized by various methodologies including FT-IR, EDX, SEM and TEM analysis. Obviously, EDX analysis indicated the characteristic peak only for carbon, oxygen and nitrogen approving the modification of MWCNTs surface by putrescine and fluorescein. It means high purity of the synthetized nanotube and removal of the unreacted elements from the nanotube during the synthesis. Our results are in conformity with the opinions of the researchers which had been used EDX analysis for elucidation of nanoparticle purity (Ramachandran et al. 2013; Gohari et al. 2020a). Likewise, XRD is known as valuable tool for demonstrating the microstructure of synthetized nanotubes (Das et al. 2014). In the case of the MWCNT-Put-Fl, XRD pattern indicated that the microstructure of MWCNT remain unchanged during oxidation and functionalizing with putrescine and fluorescein. Also, the robust diffraction peaks indicates that the MWCNT-Put-Fl possess proper crystallinity which is in agreement with the results reported by other researchers (Ramachandran et al. 2013; Subbenaik 2016; Yang et al. 2018; Azizi et al. 2020). Also, line widening of the XRD peaks indicates that the size of the MWCNT-Put-Fl reduces in the domain of nano range (Ramachandran et al. 2013). So, we presumed that MWCNT retains own functionality while acts as carrier for putrescine. Alternatively, FT-IR spectra have been successfully utilized determining the presence of conjugated groups along with chemical modification of MWCNTs (Gohari et al. 2020b, a). In the FT-IR spectra of the MWCNT-Put, peak of C = O at 1698 cm^− 1 depicts bonding of putrescine to MWCNT via amide formation. In this way, another broad peak could be seen at 3170–3551 cm^− 1, which is due to O―H stretching vibration of fluorescein. (Fig. 2B). The results confirmed the successful linkage of putrescine and fluorescein on the surface of MWCNTs. Current study is in accordance with the works indicating the potential of FT-IR in detection of chemical groups onto the nanoparticles (Jelvehgari et al. 2014; Azizi et al. 2020).

Supplemented to chemical analysis, SEM images show the irregular aggregation and agglomeration of MWCNTs (Fig. 3A). Furthermore, TEM images demonstrate an interwoven network of carbon nanotubes (Fig. 3B). Both SEM and TEM images depicted that the structure of MWCNTs was remained unchanged during acidic oxidation and modification.

In this study we described an ordinary methodology for production of putrescine anchored MWCNT for the first time. The results obtained from various tests, including electron microscopy and chemical analysis confirmed the successful linkage of putrescine on the surface of MWCNTs.

Bio-imaging and localization of the MWCNT-Put-Fl

Divers methods are used for bio-imaging and localization of nanoparticles in tissue or cells (Hauser et al. 2017). Epi-fluorescence microscopy (EFLM) is a good choice for this purpose, because it known as low cost and easy to use in respect to other procedures (Peighambardoust et al. 2010). The technique has been successfully utilized for detecting nanoparticles in plants. The approach for indirectly detection of non-fluorescence nanoparticles created based on the fact that they could be visible if their background, i.e., tissue or cell, stained with suitable fluorchrome (Ghafariyan et al. 2013). Our results indicated that labeling the nanotube with fluorescein as a fluorescence dye, provides opportunity for direct detection of nanotube using easy-to-use conventional fluorescence microscopy.

Effects of MWCNT-Put on the growth and development of plantlets

Leaf formation and plastochron are good indicators for assessment of apple growth and development (Foster et al. 2003; Dadpour et al. 2011). The rate of leaf initiation is in close relation with the organogenesis of shoot apical meristem (SAM) and its activity. Leaf primordia are formed at the peripheral zone of SAM subsequent to generous cell division. Therefore, leaf formation is known as dependent process to cell division from organogenesis point of view (Fleming 2005). Also, plastochron which defines the leaf formation as the function of time is directly tied with the rate of cell division (Dadpour et al. 2011). Many reports indicated that cell growth and division is promoted by nanotubes (Khodakovskaya et al. 2012; Khorrami et al. 2020; Samadi et al. 2020). Alternatively, polyamines could enhance cell division (Maki et al. 1991), act as effective regulator for shoot morphogenesis (Walden et al. 1997; Yadav and Rajam 1997; Zhu and Chen 2005) and increase seedling height (Farooq et al. 2009). As well, polyamines has decisive role in regard to cell structure by effect on actin formation and microtubules assembling (Oriol-Audit 1978; McCormack et al. 1994; Savarin et al. 2010; Sánchez-Elordi et al. 2019). In the current study, enhanced growth and development of the in-vitro plantlets could be associated with stimulatory effects of both components of the MWCNT-Put, i.e., MWCNT and putrescine, which is confirmed by the above mentioned literatures. Furthermore, well organized microtubules in treated cells by MWCNT-Put which were appeared in microscopic studies (Fig. 5A), could elucidate the effectiveness of putrescine in this regard. Ability of the putrescine in adjusting the water contents of a cell could attributes to plant growth and performance (Farooq et al. 2009). Similarly, evidence indicated MWCNTs can trigger the expression of PIP₁ and PIP₂ (water carrier proteins) which enhance plant growth and development through improving the water status of tissue (Khodakovskaya et al. 2012). In the current study, inimitable growth of the plantlets is in agreement with the mentioned reports and may be related to accumulative effects of MWCNT and putrescine which have been assembled in the individual particle.

Effects of MWCNT-Put on the biochemical characteristics

Indisputably, photosynthesis is the most important process which influenced by nanoparticles. Some studies has been depicted that the levels of photosynthetic pigments could increase in response to application of nanotubes (Sharma et al. 2019; Gohari et al. 2020b). Also, the substances could enhance photosynthetic activity, carbon assimilation and biomass (Khodakovskaya et al. 2012; Ghafariyan et al. 2013; Banerjee and Kole 2016). Likewise, polyamines including putrescine, are now being deliberated as a specific PGR which could modulate photosynthesis (Farooq et al. 2009; Yuan et al. 2015; Abd Elbar et al. 2019). Enrichment of photosynthetic pigments in the present study may correlates with the nature of the MWCNT-Put. The outstanding role of putrescine in plants would be its implication on chloroplast bioenergetics by means of motivating ATP synthesis. Also, putrescine can increase light energy utilization through stimulation of photophosphorylation (Ioannidis et al. 2006; Ioannidis and Kotzabasis 2007). So, putrescine could enhance the growth of plants by providing more ATP as the common source of energy. In this study, intensive growth which plantlets were experienced after treatment by MWCNT-Put would be explained from bioenergetics point of view. In other respects, increased level of the GPX in our study, as the enzymatic system which plays array of physiological roles in protection of photosynthetic apparatus against oxidative damages (Herbette et al. 2002; Dayer et al. 2008), could explicates the ascending trends of Chl a and b in the case of treated plantlets with MWCNT-Put.

Form and distribution of Stomata

Definitely, stomata are important tissues due to their controlling role in simultaneous exchange of carbon dioxide and water with the atmosphere (Webb and Baker 2002). Consequently, formation and spacing pattern of the stomata are considered as determining factors in relation to carbon assimilation, transpiration and plant performance. In overall, stomata are distributed on a leaf based on the one-cell-spacing rule. According to this hypothesis, separating two adjacent stomata from each other by at least one intervening pavement cell, is critical for achieving an optimal balance between carbon assimilation and water exhaust (Larkin et al. 1997; Bergmann and Sack 2007). Moreover, obligatory dependency of guard cell functioning, gas exchange, and ion transport with stomatal spacing have been proven (Torii 2007; Papanatsiou et al. 2016).

Therefore, changes in stomatal spacing pattern have significant impact in terms of physiological approaches (Casson and Gray 2008). In the present study, treatment of in vitro apple plantlets with the MWCNT-Put significantly increased the density of the stomata in comparison to the control (Fig. 10G). Our results are in contract with some other studies indicating positive effects of CNTs on the stomatal density (Joshi et al. 2018a, b; Khorrami et al. 2020). Studying the effects of CNTs on tobacco callus culture revealed that expressions of genes responsible for encoding cell division (CycB) and tobacco aquaporin (NtPIP₁) are enhanced in treated cells compared to the controls (Khodakovskaya et al. 2012). From the morphogenetic point of view, stomatal distribution and frequency are the main factors which could directly affect carbon assimilation and subsequently plant productivity (Shpak et al. 2005; Casson and Gray 2008; Torii 2015). Generally, formation of stomata is known as a complicated process at which numerous physiological and molecular factors involve (Shpak et al. 2005; Martin and Glover 2007; Bergmann and Sack 2007; Casson and Gray 2008; Serna 2009; Kim et al. 2012; Rudall et al. 2013; Franks and Casson 2014; Torii 2015; Castorina et al. 2016; Muir 2018). However, stomata is formed through a series of asymmetrical and symmetrical cell divisions being known as cell cycle dependent process (Croxdale 2000; Nadeau and Sack 2002; Torii 2007). It has been elucidated that the E₂Fa–DPa transcription factor together with the D cyclins and Cyclin Dependant Kinases (CDKs) are crucial components regulating cell division prior to formation of stomata (De Veylder et al. 2002; Bergmann 2004). However, E₂Fa is the main known factor for asymmetrical cell division in terms of stomatal spacing (Bergmann and Sack 2007; Kajala et al. 2014). Moreover, membrane-bound receptor-like kinases (RLKs) in pairing with two members of the epidermal patterning factor like (EPFL) protein family, namely, EPF₁ and EPF₂, have been recently stated to be essential for regulation of stomatal patterning. Noticeably, the EPF₁-ERL pair appears to enforce the one-celled spacing rule responsible for stomatal density (Lau and Bergmann 2012). On the other hand, the main molecular mechanism involving in the formation of stomata, MUTE protein, seems to activate another member of D Cyclins (CycD_5;1) proteins which causes the guard mother cells (GMC) to divide. Taking all into account, polyamines have been determined as the effective factor for enhancing cell division which is critical for stomata formation (Kono et al. 2007; Yamashita et al. 2013).

The present study indicated that application of MWCNT-Put increases the frequency of stomata, meaning low spacing between stomata and high ratio of guard per pavement cells. Accordingly, it seems that MWCNT-Put is effective on asymmetrical division of mesophylls with spacing division pattern. Research on the tomato plants treated with saline water, indicated some significant morphological and physiological changes (Romero-Aranda et al. 2001). They reported that reduction in stomatal density leads to attenuating stomatal conductance and decreasing net CO₂ assimilation which indirectly confirms our results. Salt stress could decrease CDK activity and could profoundly suppress the expression levels of the cyclins genes including A and B Cyclin which leads to extensive inhibition in both S and G₂/M phase (Qi and Zhang 2020). As it was mentioned above, salt stress can reduce stomatal density with suppressing cell cycle process. Another experiment conducted on the barley under salt stress, revealed that the stomata index is reduced by application of PAs (Çavuşoğlu et al. 2007). Our data, however, contradicts their results indicating negative effect of PAs in regard to stomata formation. In their work stomatal density was determined in terms of the stomata index, i.e., the ratio of stomata per total mesophyll cells. Also, in treated plants, total number of mesophyll cells was increased while their size diminished. Therefore, higher total mesophyll cells may lead to lower stomata index. The discrepancy between our data and the data of Çavuşoğlu et al. (2007) may be due to different methodologies which were exploited for calculating stomatal density. In our study, stomatal density was calculated as the observed stomata per distinct field of view, while they described the ratio of the number of stomata per surrounding cells.

The subsidiary effect of MWCNT-Put which was detected in our study is the formation of stomata over the main leaf vein in the case of nanoparticle application (Fig. 11). In general, stomata rarely develops over major veins with an exception, i.e., Selaginellaceae family (Brown and Lemmon 1985; Martin and Glover 2007). It seems the lack of stomata over leaf major veins mainly helps to prevent excess transpiration. As the control in-vitro plants conformed to the mentioned rule and stomata only appeared over the vein in regard to treated plants, it seems the MWCNT-Put is responsible for such change in the development of the studied leaves. We reported this phenomenon based on several microscopic observations and no statistical analysis was accomplished for this purpose. So, further studies are required for clarification of this morphological event.

The change in shape and form is largely a matter of importance to biologists (Cooke and Terhune 2015; Klingenberg 2016). Mostly, differentiation in the cellular levels could be demonstrated by anisotropy in the form of cell (Coen et al. 2004). The polyamines, especially putrescine, as well as nanotubes are well known for their implication in the developmental process. Geometric morphometric analysis in this study elucidated substantial anisotropic transformation of the stomatal form in the case of MWCNT-Put treatments in particular at the vertices area. So, it seems the synthetized nanotube might be involved in differentiation of the stomata.

Synthetizing hormone anchored carbon nanotubes is an optimistic approach that has great potential and flexibility for application towards manipulating plant growth and development. The new developed MWCNT-Put-Fl nanotube with profound effects in terms of plant morphological, physiological and biochemical characteristics could enhances performance of the in-vitro plantlets. Exploiting the synthetized nanotube in the micropropagation of the Niedzwetzky's apple demonstrated the positive effects of the composite on growth and development of the plantlets. Importantly, cumulative leaf formation, short plastochron and intensive shoot proliferation by treating the MWCNT-Put depicted its positive impact in respect to plant growth and development. MWCNT-Put application additionally led to increase the contents of photosynthetic pigments and GPX activities as the chlorophyll protective antioxidant enzyme. Interestingly, stomatal shape and density were profoundly affected by application of the nanotube. Consequently, the new developed MWCNT-Put might act as a specific PGR which able to stimulate plant growth and development, ultimately enhancing plant performance under in-vitro cultures and thus acting as a promising growth promoting substances.

Funding: This study was financially supported by grant for fulfillment of PhD course (no. 9401) from Vice Chancellery for Research and Technology, University of Tabriz, Tabriz, Iran.

Conflicts of interest/Competing interests: The authors declare no competing financial interest.

Availability of data and material: The authors confirm availability of data and material

Code availability: Not applicable

Authors' contributions:

M. Abdolalipour, B. Eftekhari-Sis, A. Motalebiazar and M. Dadpour accomplished the experiment. M. Abdolalipour established in vitro cultures and contributed to data collection. B. Eftekhari-Sis synthesized the hormone anchored nanomaterial and chemical analysis. M. Dadpour contributed in bio-imaging, fluorescence microscopy and image analysis. M. Abdolalipour and A. Motalebiazar performed statistical analysis. B. Eftekhari-Sis and M. Dadpour supervised the experiments. B. Eftekhari-Sis and M. Dadpour wrote the draft. M. Dadpour edited and finalized the manuscript.

Ethics approval: This study does not include any human or animal participants

Consent to participate: Not applicable

Consent for publication: Not applicable

Abd Elbar OH, Farag RE, Shehata SA (2019) Effect of putrescine application on some growth, biochemical and anatomical characteristics of Thymus vulgaris L. under drought stress. Ann Agric Sci 64:129–137. https://doi.org/10.1016/j.aoas.2019.10.001
Abdulrazzak FH, Alkiam AF, Hussein FH (2019) Behavior of X-ray analysis of carbon nanotubes. In: El-Din Saleh H, Mohamed El-Sheikh SM (eds). IntechOpen, Rijeka, p Ch. 7
Albrechtová J, Kubínová Z, Soukup A, Janáček J (2014) Image analysis: Basic procedures for description of plant structures. In: Žárský V, Cvrčková F (eds) Plant Cell Morphogenesis. Methods in Molecular Biology (Methods and Protocols). Humana Press Inc., pp 67–76
Alcázar R, Altabella T, Marco F, et al (2010) Polyamines: Molecules with regulatory functions in plant abiotic stress tolerance. Planta 231:1237–1249
Allen JC (1983) Biochemistry of the polyamines. Cell Biochem. Funct. 1:131–140
Azizi M, Valizadeh H, Shahgolzari M, et al (2020) Synthesis of self-targeted carbon dot with ultrahigh quantum yield for detection and therapy of cancer. ACS Omega 5:24628–24638. https://doi.org/10.1021/acsomega.0c03215
Banerjee J, Kole C (2016) Plant nanotechnology: An overview on concepts, strategies, and tools. In: Plant Nanotechnology: Principles and Practices. Springer International Publishing, pp 1–14
Baron K, Stasolla C (2008) The role of polyamines during in vivo and in vitro development. Vitr. Cell. Dev. Biol. - Plant 44:384–395
Bergmann DC (2004) Integrating signals in stomatal development. Curr. Opin. Plant Biol. 7:26–32
Bergmann DC, Sack FD (2007) Stomatal development. Annu. Rev. Plant Biol. 58:163–181
Brown RC, Lemmon BE (1985) Development of stomata in Selaginella: Division polarity and plastid movements. Am J Bot 72:1914–1925. https://doi.org/10.1002/j.1537-2197.1985.tb08465.x
Burman U, Kumar P (2018) Plant response to engineered nanoparticles. In: Tripathi DK, Ahmad P, Sharma S, et al. (eds) Nanomaterials in Plants, Algae, and Microorganisms. Concepts and Controversies: Volume 1. Elsevier Inc., pp 103–118
Cai G, Sobieszczuk-Nowicka E, Aloisi I, et al (2015) Polyamines are common players in different facets of plant programmed cell death. Amino Acids 47:27–44. https://doi.org/10.1007/s00726-014-1865-1
Casson S, Gray JE (2008) Influence of environmental factors on stomatal development. New Phytol. 178:9–23
Castorina G, Fox S, Tonelli C, et al (2016) A novel role for STOMATAL CARPENTER 1 in stomata patterning. BMC Plant Biol 16:172. https://doi.org/10.1186/s12870-016-0851-z
Çavuşoğlu K, Kılıç S, Kabar K (2007) Some morphological and anatomical observations during alleviation of salinity (NaCI) stress on seed germination and seedling growth of barley by polyamines. Acta Physiol Plant 29:551–557. https://doi.org/10.1007/s11738-007-0066-x
Coen E, Rolland-Lagan AG, Matthews M, et al (2004) The genetics of geometry. Proc Natl Acad Sci U S A 101:4728–4735. https://doi.org/10.1073/pnas.0306308101
Considine MJ (2018) Oxygen, energy, and light signalling direct meristem fate. Trends Plant Sci. 23:1–3
Cooke SB, Terhune CE (2015) Form, Function, and Geometric Morphometrics. Anat Rec 298:5–28. https://doi.org/10.1002/ar.23065
Croxdale JL (2000) Stomatal patterning in angiosperms. Am J Bot 87:1069–1080. https://doi.org/10.2307/2656643
Dadpour MR, Movafeghi A, Grigorian W, Omidi Y (2011) Determination of floral initiation in Malus domestica: A novel morphogenetic approach. Biol Plant 55:243–252. https://doi.org/10.1007/s10535-011-0035-5
Das R, Hamid S, Ali M, et al (2014) Carbon nanotubes characterization by X-ray powder diffraction – A review. Curr Nanosci 11:23–35. https://doi.org/10.2174/1573413710666140818210043
Dayer R, Fischer BB, Eggen RIL, Lemaire SD (2008) The peroxiredoxin and glutathione peroxidase families in Chlamydomonas reinhardtii. Genetics 179:41–57
De Veylder L, Beeckman T, Beemster GTS, et al (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor. EMBO J 21:1360–1368. https://doi.org/10.1093/emboj/21.6.1360
Dzhangaliev AD (2003) The wild apple tree of kazakhstan. In: Horticultural Reviews. John Wiley & Sons, Inc., pp 63–303
Evans PT, Malmberg RL (1989) Do polyamines have roles in plant development? Annu Rev Plant Physiol Plant Mol Biol 40:235–269. https://doi.org/10.1146/annurev.pp.40.060189.001315
Farooq M, Wahid A, Lee DJ (2009) Exogenously applied polyamines increase drought tolerance of rice by improving leaf water status, photosynthesis and membrane properties. Acta Physiol Plant 31:937–945. https://doi.org/10.1007/s11738-009-0307-2
Fleming AJ (2005) The control of leaf development. New Phytol 166:9–20. https://doi.org/10.1111/j.1469-8137.2004.01292.x
Forster B, Van De Ville D, Berent J, et al (2004) Complex wavelets for extended depth-of-field: A new method for the fusion of multichannel microscopy images. Microsc Res Tech 65:33–42. https://doi.org/10.1002/jemt.20092
Foster T, Johnston R, Seleznyova A (2003) A morphological and quantitative characterization of early floral development in apple (Malus x domestica Borkh.). Ann Bot 92:199–206. https://doi.org/10.1093/aob/mcg120
Foyer CH, Parry M, Noctor G (2003) Markers and signals associated with nitrogen assimilation in higher plants. J Exp Bot 54:585–593. https://doi.org/10.1093/jxb/erg053
Franks PJ, Casson S (2014) Connecting stomatal development and physiology. New Phytol 201:1079–1082. https://doi.org/10.1111/nph.12673
Galston AW, Sawhney RK (1990) Polyamines in plant physiology. Plant Physiol. 94:406–410
Gaspar T, Keveks C, Penel C, et al (1996) Plant hormones and plant growth regulators in plant tissue culture. Vitr. Cell. Dev. Biol. - Plant 32:272–289
Ghafariyan MH, Malakouti MJ, Dadpour MR, et al (2013) Effects of magnetite nanoparticles on soybean chlorophyll. Environ Sci Technol 47:10645–10652. https://doi.org/10.1021/es402249b
Gilroy S, Trewavas A (2001) Signal processing and transduction in plant cells: The end of the beginning? Nat. Rev. Mol. Cell Biol. 2:307–314
Gohari G, Mohammadi A, Akbari A, et al (2020a) Titanium dioxide nanoparticles (TiO2 NPs) promote growth and ameliorate salinity stress effects on essential oil profile and biochemical attributes of Dracocephalum moldavica. Sci Rep 10:1–14. https://doi.org/10.1038/s41598-020-57794-1
Gohari G, Safai F, Panahirad S, et al (2020b) Modified multiwall carbon nanotubes display either phytotoxic or growth promoting and stress protecting activity in Ocimum basilicum L. in a concentration-dependent manner. Chemosphere 249:. https://doi.org/10.1016/j.chemosphere.2020.126171
Goldberg RB (1988) Plants: Novel developmental processes. Science (80- ) 240:1460–1467. https://doi.org/10.1126/science.3287622
Hauser M, Wojcik M, Kim D, et al (2017) Correlative super-resolution microscopy: New dimensions and new opportunities. Chem Rev 117:7428–7456. https://doi.org/10.1021/acs.chemrev.6b00604
Herbette S, Lenne C, Leblanc N, et al (2002) Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities. Eur J Biochem 269:2414–2420. https://doi.org/10.1046/j.1432-1033.2002.02905.x
Hermes PH, Gabriela MP, Ileana VR, et al (2020) Carbon nanotubes as plant growth regulators: Prospects. In: Nanotechnology in the Life Sciences. Springer Science and Business Media B.V., pp 77–115
Ioannidis NE, Kotzabasis K (2007) Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro. Biochim Biophys Acta - Bioenerg 1767:1372–1382. https://doi.org/10.1016/j.bbabio.2007.10.002
Ioannidis NE, Sfichi L, Kotzabasis K (2006) Putrescine stimulates chemiosmotic ATP synthesis. Biochim Biophys Acta - Bioenerg 1757:821–828. https://doi.org/10.1016/j.bbabio.2006.05.034
Jarad M, Antoniou-Kourounioti R, Hepworth J, Qüesta JI (2020) Unique and contrasting effects of light and temperature cues on plant transcriptional programs. Transcription 11:134–159
Jelvehgari M, Barghi L, Barghi F (2014) Preparation of chlorpheniramine maleate-loaded alginate/chitosan particulate systems by the ionic gelation method for taste masking. Jundishapur J Nat Pharm Prod 9:39–48. https://doi.org/10.17795/jjnpp-12530
Joshi A, Kaur S, Dharamvir K, et al (2018a) Multi-walled carbon nanotubes applied through seed-priming influence early germination, root hair, growth and yield of bread wheat (Triticum aestivum L.). J Sci Food Agric 98:3148–3160. https://doi.org/10.1002/jsfa.8818
Joshi A, Kaur S, Singh P, et al (2018b) Tracking multi-walled carbon nanotubes inside oat (Avena sativa L.) plants and assessing their effect on growth, yield, and mammalian (human) cell viability. Appl Nanosci 8:1399–1414. https://doi.org/10.1007/s13204-018-0801-1
Kajala K, Ramakrishna P, Fisher A, et al (2014) Omics and modelling approaches for understanding regulation of asymmetric cell divisions in arabidopsis and other angiosperm plants. Ann. Bot. 113:1083–1105
Kakkar RK, Nagar PK, Ahuja PS, Rai VK (2000) Polyamines and plant morphogenesis. Biol. Plant. 43:1–11
Khodakovskaya M V., De Silva K, Biris AS, et al (2012) Carbon nanotubes induce growth enhancement of tobacco cells. ACS Nano 6:2128–2135. https://doi.org/10.1021/nn204643g
Khorrami SK, Jamei R, Darvishzadeh R, Sarghin SH (2020) The multi-walled carbon nanotubes induced anatomical and morphological changes in root and shoot of two cultivars of Okra (Hibiscus escolentus L.) seedlings. Journal of Plant Process and Function
Khoshbakht D, Asghari MR, Haghighi M (2018) Influence of foliar application of polyamines on growth, gas-exchange characteristics, and chlorophyll fluorescence in Bakraii citrus under saline conditions. Photosynthetica 56:731–742. https://doi.org/10.1007/s11099-017-0723-2
Kim TW, Michniewicz M, Bergmann DC, Wang ZY (2012) Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature 482:419–422. https://doi.org/10.1038/nature10794
Klingenberg CP (2016) Size, shape, and form: concepts of allometry in geometric morphometrics. Dev. Genes Evol. 226:113–137
Kocsanyi L, Haitz M, Lichtenthaler HK (1988) Measurement of the laser-induced chlorophyll fluorescence kinetics using a fast acoustooptic device. In: Lichtenthaler HK (ed) Applications of Chlorophyll Fluorescence in Photosynthesis Research, Stress Physiology, Hydrobiology and Remote Sensing. Springer Netherlands, pp 99–107
Kong H, Gao C, Yan D (2004) Controlled functionalization of multiwalled carbon nanotubes by in situ atom transfer radical polymerization. J Am Chem Soc 126:412–413. https://doi.org/10.1021/ja0380493
Kono A, Umeda-Hara C, Adachi S, et al (2007) The Arabidopsis D-type cyclin CYCD4 controls cell division in the stomatal lineage of the hypocotyl epidermis. Plant Cell 19:1265–1277. https://doi.org/10.1105/tpc.106.046763
Larkin JC, Marks MD, Nadeau J, Sack F (1997) Epidermal cell fate and patterning in leaves. Plant Cell 9:1109–1120. https://doi.org/10.1105/tpc.9.7.1109
Lau OS, Bergmann DC (2012) Stomatal development: A plant’s perspective on cell polarity, cell fate transitions and intercellular communication. Dev 139:3683–3692. https://doi.org/10.1242/dev.080523
Maki H, Ando S, Kodama H, Komamine A (1991) Polyamines and the cell cycle of Catharanthus roseus cells in culture. Plant Physiol 96:1008–1013. https://doi.org/10.1104/pp.96.4.1008
Martin-Tanguy J (1997) Conjugated polyamines and reproductive development: Biochemical, molecular and physiological approaches. Physiol Plant 100:675–688. https://doi.org/10.1111/j.1399-3054.1997.tb03074.x
Martin C, Glover BJ (2007) Functional aspects of cell patterning in aerial epidermis. Curr. Opin. Plant Biol. 10:70–82
McCormack SA, Wang JY, Johnson LR (1994) Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells. Am J Physiol - Cell Physiol 267:. https://doi.org/10.1152/ajpcell.1994.267.3.c715
Muir CD (2018) Light and growth form interact to shape stomatal ratio among British angiosperms. New Phytol 218:242–252. https://doi.org/10.1111/nph.14956
Mulligan RM, Chory J, Ecker JR (1997) Signaling in plants. Proc Natl Acad Sci U S A 94:2793–2795. https://doi.org/10.1073/pnas.94.7.2793
Nadeau JA, Sack FD (2002) Control of stomatal distribution on the Arabidopsis leaf surface. Science (80- ) 296:1697–1700. https://doi.org/10.1126/science.1069596
Oriol-Audit C (1978) Polyamine‐Induced Actin Polymerization. Eur J Biochem 87:371–376. https://doi.org/10.1111/j.1432-1033.1978.tb12386.x
Pal Bais H, Ravishankar GA (2002) Role of polyamines in the ontogeny of plants and their biotechnological applications. Plant Cell. Tissue Organ Cult. 69:1–34
Panahirad S, Dadpour M, Peighambardoust SH, et al (2021) Applications of carboxymethyl cellulose- and pectin-based active edible coatings in preservation of fruits and vegetables: A review. Trends Food Sci. Technol. 110:663–673
Papanatsiou M, Amtmann A, Blatt MR (2016) Stomatal spacing safeguards stomatal dynamics by facilitating guard cell ion transport independent of the epidermal solute reservoir. Plant Physiol 172:254–263. https://doi.org/10.1104/pp.16.00850
Pascoli M, Lopes-Oliveira PJ, Fraceto LF, et al (2018) State of the art of polymeric nanoparticles as carrier systems with agricultural applications: a minireview. Energy, Ecol Environ 3:137–148. https://doi.org/10.1007/s40974-018-0090-2
Peighambardoust SH, Dadpour MR, Dokouhaki M (2010) Application of epifluorescence light microscopy (EFLM) to study the microstructure of wheat dough: a comparison with confocal scanning laser microscopy (CSLM) technique. J Cereal Sci 51:21–27. https://doi.org/10.1016/j.jcs.2009.09.002
Pereira AES, Silva PM, Oliveira JL, et al (2017) Chitosan nanoparticles as carrier systems for the plant growth hormone gibberellic acid. Colloids Surfaces B Biointerfaces 150:141–152. https://doi.org/10.1016/j.colsurfb.2016.11.027
Qi F, Zhang F (2020) Cell cycle regulation in the plant response to stress. Front Plant Sci 10:. https://doi.org/doi: 10.3389/fpls.2019.01765
Ramachandran D, Brijitta J, Raj NN, et al (2013) Synthesis and characterization of Zinc Oxide nanorods. In: Proceedings of the International Conference on “Advanced Nanomaterials and Emerging Engineering Technologies”, ICANMEET 2013. pp 566–568
Rohlf FJ (2015) The tps series of software. Hystrix 26:1–4. https://doi.org/10.4404/hystrix-26.1-11264
Romero-Aranda R, Soria T, Cuartero J (2001) Tomato plant-water uptake and plant-water relationships under saline growth conditions. Plant Sci 160:265–272. https://doi.org/10.1016/S0168-9452(00)00388-5
Rudall PJ, Hilton J, Bateman RM (2013) Several developmental and morphogenetic factors govern the evolution of stomatal patterning in land plants. New Phytol. 200:598–614
Sabzi A, Rahmani A, Edalati M, et al (2020) Targeted co-delivery of curcumin and doxorubicin by citric acid functionalized Poly (ε-caprolactone) based micelle in MDA-MB-231 cell. Colloids Surfaces B Biointerfaces 194:. https://doi.org/10.1016/j.colsurfb.2020.111225
Salama DM, Abd El-Aziz ME, Rizk FA, Abd Elwahed MSA (2021) Applications of nanotechnology on vegetable crops. Chemosphere 266
Samadi S, Saharkhiz MJ, Azizi M, et al (2020) Multi-walled carbon nanotubes stimulate growth, redox reactions and biosynthesis of antioxidant metabolites in Thymus daenensis celak. in vitro. Chemosphere 249:126069. https://doi.org/10.1016/j.chemosphere.2020.126069
Sánchez-Elordi E, de los Ríos LM, Vicente C, Legaz ME (2019) Polyamines levels increase in smut teliospores after contact with sugarcane glycoproteins as a plant defensive mechanism. J Plant Res 132:405–417. https://doi.org/10.1007/s10265-019-01098-7
Savarin P, Barbet A, Delga S, et al (2010) A central role for polyamines in microtubule assembly in cells. Biochem J 430:151–159. https://doi.org/10.1042/BJ20091811
Serna L (2009) Cell fate transitions during stomatal development. BioEssays 31:865–873
Shah P, Swiatlo E (2008) A multifaceted role for polyamines in bacterial pathogens. Mol. Microbiol. 68:4–16
Sharma N, Singh D, Rani R, et al (2019) Chitosan and its nanocarriers: Applications and opportunities. In: Tripathi DK, Ahmad P, Sharma S, et al. (eds) Nanomaterials in Plants, Algae and Microorganisms: Concepts and Controversies: Volume 2. Elsevier, pp 267–286
Shpak ED, McAbee JM, Pillitteri LJ, Torii KU (2005) Plant science: Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science (80- ) 309:290–293. https://doi.org/10.1126/science.1109710
Slocum RD, Kaur-Sawhney R, Galston AW (1984) The physiology and biochemistry of polyamines in plants. Arch Biochem Biophys 235:283–303. https://doi.org/10.1016/0003-9861(84)90201-7
Subbenaik SC (2016) Physical and chemical nature of nanoparticles. In: Plant Nanotechnology: Principles and Practices. Springer International Publishing, pp 15–27
Tiburcio AF, Alcázar R (2018) Potential applications of polyamines in agriculture and plant biotechnology. In: Methods in Molecular Biology. Humana Press Inc., pp 489–508
Torii KU (2007) Stomatal patterning and guard cell differentiation. In: Verma DPS, Hong Z (eds) Cell Division Control in Plants. Plant Cell Monographs, vol 9. Springer, Berlin, Heidelberg, pp 343–359
Torii KU (2015) Stomatal differentiation: The beginning and the end. Curr. Opin. Plant Biol. 28:16–22
Trewavas AJ, Malhó R (1997) Signal perception and transduction: The origin of the phenotype. Plant Cell 9:1181–1195. https://doi.org/10.1105/tpc.9.7.1181
Walden R, Cordeiro A, Tiburcio AF (1997) Polyamines: Small molecules triggering pathways in plant growth and development. Plant Physiol. 113:1009–1013
Webb AAR, Baker AJ (2002) Stomatal biology: New techniques, new challenges. New Phytol. 153:365–369
Weiger TM, Hermann A (2014) Cell proliferation, potassium channels, polyamines and their interactions: A mini review. Amino Acids 46:681–688
Yadav JS, Rajam MV (1997) Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: Efficient somatic embryogenesis with putrescine. J Exp Bot 48:1537–1545. https://doi.org/10.1093/jxb/48.8.1537
Yamashita T, Nishimura K, Saiki R, et al (2013) Role of polyamines at the G1/S boundary and G2/M phase of the cell cycle. Int J Biochem Cell Biol 45:1042–1050. https://doi.org/10.1016/j.biocel.2013.02.021
Yang R, Xiao CF, Guo YF, et al (2018) Inclusion complexes of GA3 and the plant growth regulation activities. Mater Sci Eng C 91:475–485. https://doi.org/10.1016/j.msec.2018.05.043
Yuan Y, Zhong M, Shu S, et al (2015) Effects of exogenous putrescine on leaf anatomy and carbohydrate metabolism in cucumber (Cucumis sativus L.) Uunder salt stress. J Plant Growth Regul 34:451–464. https://doi.org/10.1007/s00344-015-9480-2
Zelditch ML, Swiderski DL, Sheets HD (2012) Landmarks and Semilandmarks. Geom Morphometrics Biol 23–50. https://doi.org/10.1016/b978-0-12-386903-6.00002-2
Zhu C, Chen Z (2005) Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber in vitro. Plant Cell Tissue Organ Cult 81:45–53. https://doi.org/10.1007/s11240-004-2773-y

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Novel Methodology for Delivering Putrescine into the Apple Explants Using Hormone Anchored Nanotube

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Abstract

Figures

Introduction

Material And Methods

Preparation of nanoparticles

In-vitro Plant culture and treatment

Biochemical analysis and enzyme assay

Electron microscopy

Epi-fluorescence microscopy (EFLM)

Image processing

Estimating the density of stomata using image analysis procedure

Geometric morphometric characteristics of stomta

Results

Preparation and characterization of MWCNT-Put-Fl

Localization of MWCNT-Put-Fl in the plantlets

Effects of the MWCNT-Put on the growth and development of in-vitro plantlets

Effects of the MWCNT-Put on the biochemical characteristics of in-vitro plantlets

Effects of the MWCNT-Put on the stomatal density and form

Discussion

Synthetizing MWCNT-Put-Fl

Bio-imaging and localization of the MWCNT-Put-Fl

Effects of MWCNT-Put on the growth and development of plantlets

Effects of MWCNT-Put on the biochemical characteristics

Form and distribution of Stomata

Conclusion

Declarations

References

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