The results are presented and discussed in two parts. In the first part the plant growth-regulating activity of synthesized compounds (PGR1, PGR2, and PGR3) are assessed. In the second part molecular interactions between microcapsule formulation constituents are analyzed and the essential physicochemical properties of microcapsule formulations without CS/(ALG/Ca) and with a PGR, CS/(ALG/(Ca+PGR)) discussed.
Part I. Assessment of plant growth-regulating activity of synthesized PGRs
Testing of plant growth-regulating activity. The results of testing plant growth-regulating activity are listed in Table 1.
Concentration %
|
0.01
|
0.001
|
0.0001
|
№ compound
|
root
|
stem
|
root
|
stem
|
root
|
stem
|
The length (weight) of plant parts to control (%)
|
Maize
|
PGR1
|
79/127
|
112/100
|
78/129
|
115/102
|
79/127
|
111/99
|
PGR2
|
121/164
|
129/131
|
100/148
|
109/117
|
97/140
|
114/108
|
PGR3
|
115/114
|
127/76
|
110/89
|
111/86
|
104/79
|
111/79
|
Humate
|
103/136
|
110/97
|
|
|
|
|
Barley
|
PGR1
|
105/109
|
94/116
|
110/123
|
96/119
|
95/115
|
90/116
|
PGR2
|
100/121
|
97/111
|
100/125
|
103/101
|
92/112
|
100/112
|
PGR3
|
81/104
|
89/72
|
86/107
|
86/91
|
87/96
|
88/90
|
Humate
|
103/102
|
100/103
|
|
|
|
|
Wheat
|
PGR1
|
102/121
|
104/119
|
125/132
|
107/122
|
119/126
|
104/118
|
PGR2
|
83/93
|
92/94
|
91/96
|
104/103
|
84/100
|
101/100
|
PGR3
|
94/116
|
110/122
|
105/118
|
107/118
|
119/137
|
109/123
|
Humate
|
111/134
|
106/118
|
|
|
|
|
Table 1. Growth-regulating activity testing of PGR1 (2,3-dehydroaspartic acid dimethyl ester), PGR2 (Z-isomer of the potassium salt of 2-amino-3-methoxycarbonylacrylic acid) and PGR3 (1-methyl-3-methylaminomaleinimide). The seeds grown or soaked in water was as a control. A 2.5% sodium humate solution was used as a standard.
The use of PGR1 in all tested concentrations accelerates plant growth, which is manifested in an increase in dry weight of seedlings, especially the mass of root systems. When PGR1 is used at a concentration of 0.001%, the plant root system and, to a lesser extent, the stem system for almost all studied cultures are at or above the standard level. This should positively affect the further growth of plants in adverse weather conditions, especially with a lack of moisture.
The PGR2 test on maize and barley seeds was found to affect the growth of roots and stems of seedlings following the standard in all aspects, especially at the level of 0.01%. At the same time, PGR2 did not have a positive effect and even slightly inhibited the growth of wheat seedlings. In contrast, PGR3 promoted the growth of wheat seedlings, especially at a concentration of 0.0001% and, to a lesser extent, maize seedlings at a concentration of 0.01%, inhibiting the development of barley seedlings in all concentrations.
Obtained data show a sharp dependence of the influence of the investigated compounds on the type of culture. The observed increase in the plant growth-regulating properties of the test compounds with a decrease in their concentration from 0.01% to 0.0001% solutions (inhibition at high concentrations - 0.01%), significantly exceeded the standard. Based on the obtained data, it can be considered that the most promising plant growth regulator is PGR1 showing high stimulating activity on all tested crops, especially on the development of their root system.
Testing of auxin- and gibberellin-like activity. Testing of auxin-like activity was performed by following the growth of segments of corn coleoptiles and mesocotyls in 24 hours of exposure and the growth of whole coleoptiles in 24, 48 and 72 hours of exposure to PGR solutions of different concentrations. During 24 hours of incubation in water, 1 cm of corn coleoptiles grew by 2.3-3.5 mm. The impact of indole-3-acetic acid (IAA) 5 mg dm-3 stimulated their growth by 2.5 times to 7.3-7.9 mm. Such an increase in the lengths of coleoptiles in auxin is normal and confirms the high sensitivity of this test object to auxin.
PGR1 inhibited the elongation of coleoptiles, showing a decrease in increments from 13 to 31% compared to the increment in water with increasing concentration from 0.1 to 100 mg dm-3.
PGR2 was tested not only on the growth of one-centimeter segments of coleoptiles, but also on the growth of mesocotyl segments of corn seedlings, as well as on the growth of whole coleoptiles. The growth rate of one-centimeter segments of mesocotyls in the control variant was two times weaker than that for segments of coleoptiles. Specimen for PGR2 did not reveal a significant increase in the germination of the studied objects. There was no influence on the growth of the corn coleoptile segment at concentrations from 0.1 to 100 mg dm-3, but at lower concentrations (from 0.001 to 10 mg dm-3) PGR2 exhibited some auxin-like stimulating effect on the growth of corn coleoptile segments. The increase in mesocotyls under the influence of all PGR2 concentrations either did not exceed the control increase, or it is slightly lower than the control.
At the same concentrations, PGR3 did not show an unambiguous effect on the growth of the test object, showing either a slight stimulation or a slight decrease in growth.
Testing of gibberellin-like activities of dehydroamino acid derivatives was performed using corn seedlings as test objects. Treatment of corn seedlings with gibberellic acid at a concentration of 10 mg dm-3 accelerates the growth of the first leaf of coleoptiles almost 1.5 times indicating a high sensitivity of test object to gibberellin. PGR1 used at a concentration of 0.1 mg dm-3 did not affect the leaf growth in 24 hours of exposure, while PGR2 and PGR3 at the same concentration increased the leaf growth by 16 and 7%, respectively.
Field trials. The synthesized compounds showed distinct plant growth regulation activity. The most stimulating activity on all laboratory tested cultures showed PGR and this compound was applicated in field experiment. PGR1 was used by treating seeds (semi-dry treatment, inlay), tubers, cuttings, grafts, the root system of seedlings and vegetative plants. Field trials revealed a significant increase in the productivity of winter wheat (Table S3) and barley (Table S4). In the absence of pre-sowing treatment, spraying vegetative plants at the beginning of flowering increased the productivity of barley by 0.3 t ha-1 (9% control) and oats by 0.4 t ha-1 (8% control).
In the latter case, the increase in productivity was facilitated by an increase in the productive grain of the broom (53% decrease in empty grain relative to control).
Seed treatment before sowing with 0.0001% aqueous solution of PGR1 protected plants during the growing season. The artificial infestation of winter wheat plants during the flowering season by the Fusaria population did not lead to reduced yields. In contrast, it remained above the values observed for uninfected control plants (Table S5).
Treatment of potato tubers before planting with aqueous solutions of PGR1 led to a significant increase in yield (Table S6). At the same time, the quality of tubers improved (Table S7), the incidence of bacteriosis and viral diseases, late disease, fusarium wilt and nesting decreased (Table S8).
Field experiments have shown that the first treatment with PGR1 - seeds before sowing, tubers before planting, cuttings when cutting and grafting, root systems of seedlings when sewing or planting - ensured early seed germination, good rooting, and increased plant resistance to adverse natural factors. Another treatment carried out in the budding phase and at the beginning of flowering revealed a decrease in ripening and an increase in harvest.
Based on the above data we can assume that PGR1 is a universally effective stimulator of plant development. Depending on the culture, time and method of processing, it increased germination and germination energy of seeds, accelerated the awakening and development of tubers, stimulated the formation of roots and blisters, improved photosynthesis by increasing the number of leaves and leaf surface of plants, increased crop yields, improved product quality, increased frost resistance and accelerated the regeneration of damaged plants, increases plant disease resistance and contributed to the safety of plant products during long-term storage. According to the principle of action, it is probably a promoter of endogenous phytohormone biosynthesis and/or an immunomodulator. In addition, PGR1 is environmentally friendly due to its low toxicity (LD50 5163.4 mg kg-1) and extremely small application rate (10-300 mg ) kg-1 (see details in Supplementary information)
Part II. Physicochemical properties of microcapsule formulations
Molecular interactions between constituents in microcapsule formulations. Spectra of microcapsules prepared without CS/(ALG/Ca) and with PGR CS/(ALG/(Ca+PGR)) are presented in Figure 1. All spectra exhibited bands of lower intensities in comparison with the CS/(ALG/Ca) spectrum. The bands corresponding to CS/(ALG/Ca) overlap those bands of microcapsule loaded with PGR. All spectra of CS/(ALG/(Ca+PGR)) exhibit changes of the strong and broad absorption band in the range 3000-3600 cm-1 (O–H and N-H stretchings) and asymmetric and symmetric stretching peaks of carboxylate (COO-) groups. The band intensity decreasing suggests a decrease in intermolecular hydrogen bonds which is connected with the presence of nitrogen atoms in encapsulated PGR.
A spectrum of the CS/(ALG/(Ca+PGR1)) shows a peak at approximately 1750 cm-1 due to the C=O stretching of the free carboxylic acid, alginic acid arising from hydrolysis of sodium alginate in the reaction vessel. This indicates that not all carboxylic acids will form a carboxylate with amine and some remain as free acids with a lower effect on the amino group 16. New bands with a maximum at 1327 cm-1 and 1217 cm-1 corresponding to C-N stretching (aliphatic amines) appear in CS/(ALG/(Ca+PGR2)) spectrum. Also, new bands in the spectrum of CS/(ALG/(Ca+PGR3)) appear in the range of 1800 to 1600 cm-1; peaks at 1783 cm-1 and one wide peak with two peaks at 1730 and 1698 cm-1. These bands and peaks represent C=O stretching and secondary amine N-H band. Also, the appearance of a small peak at 1253 cm-1 represents the stretching of the C-N tertiary amines.
Fig. 1. Characteristic FTIR spectra of dry microcapsule formulations; CS/(ALG/Ca) (green line); CS/(ALG/(Ca+PGR1)) (black line), CS/(ALG/(Ca+PGR2)) (red line) and CS/(ALG/(Ca+PGR3)) (blue line).
In all FTIR spectra peaks of chitosan and alginate, functional groups are somewhat shifted due to electrostatic interactions. Characteristic peaks of loaded and unloaded microcapsules are peaks at approximately 1420 cm-1 (C=O stretching band) and approximately 1610 cm-1 (carboxyl group) 17. All shifts indicate complex electrostatic interactions and changes in hydrogen bonds 18. Information about molecular interactions between microcapsule components and synthesized PGR revealed functional groups of all constituents interact with each other including mainly electrostatic interactions and hydrogen bonds.
Physicochemical characterization of microcapsule formulations. The morphology, size and shape analysis of the wet and dry microcapsules were carried out by an optical microscope (Figure 2) immediately after the preparation and scanning electron microscopy (SEM) after drying to constant mass (approximately four weeks on air at room temperature) (Figure 3). All wet microcapsules were almost spherical, but after drying to constant mass, their sphericity was lost. The analysis of wet microcapsules showed that PGR encapsulation did not have significant effect on their size which ranged from 1706.07 to 2120.64 µm, respectively (CS/(ALG/Ca) = 2053±114 µm; CS/(ALG/(Ca+PGR1)) = 2120.64±173.30 µm; CS/(ALG/Ca+PGR2)) = 1706.07±175.51 µm; CS/(ALG/Ca+PGR3)) = 2104.60±188.23 µm).
The colors of prepared formulations are white (PGR1), light yellow (PGR2) and yellow (PGR3) as a consequence of entrapped PGR (Figs. 2a,c,e,g). The surface of wet microcapsules was smooth, dense and round. The smooth surface is a consequence of chitosan binding on sodium alginate chains 19. Upon the drying to constant mass, microcapsules lost their spherical shape and uniformity in size (Figs. 2b,d,f,h). The surface of all dry microcapsule formulations were not smooth anymore, the wrinkles appeared and the surface became very rough. This could be explained by the partial collapse of the polymer network during dehydration 20. Also, all prepared microcapsule formulations contained ~ 98% water. With the removal of water and with humidity loss, the size of all prepared microcapsules dramatically decreased by more than double which is associated with biopolymer strain-relaxation processes 21. The size of dry microcapsules ranged from 745 to 810 µm (CS/(ALG/Ca) = 810±94 µm, CS/(ALG/(Ca+PGR1)) = 806±123 µm, CS/(ALG/(Ca+PGR2)) = 745±121 µm, CS/(ALG/(Ca+PGR3)) = 797 ± 88 µm). Furthermore, we have observed more intense color change in dried microcapsules compared to the wet microcapsules due to the higher concentration of PGR, respectively to the mass.
Wet microcapsules
|
Dry microcapsules
|
(a) CS/(ALG/Ca)
|
(b) CS/(ALG/Ca)
|
(c) CS/(ALG/(Ca+PGR1))
|
(d) CS/(ALG/(Ca+PGR1))
|
(e) CS/(ALG/(Ca+PGR2))
|
(f) CS/(ALG/(Ca+PGR2))
|
(g) CS/(ALG/(Ca+PGR3))
|
(h) CS/(ALG/(Ca+PGR2))
|
Fig. 2. Optical microscope microphotographs of wet (left) and dry (right) microcapsules as denoted. Microcapsule were prepared without (a, b) and with PGR1 (c, d), PGR2 (e, f) and PGR3 (g, h). Bars are indicated.
SEM microphotographs of dried microcapsules are shown in Figure 3. The size of dry microcapsules observed by SEM follows values obtained by OM.
(a) CS/(ALG+Ca)
|
(b) CS/(ALG/(Ca+PGR1))
|
(c) CS/(ALG/(Ca+PGR2))
|
(d) CS/(ALG/(Ca+PGR3))
|
Fig. 3. SEM microphotographs of dry microcapsule formulations (as denoted). Microcapsules were prepared without (a) and with PGR1 (b), PGR2 (c) and PGR3 (d). Aggregated nubs are marked by black lines (c,d). Bars are indicated.
After drying the spheric form of wet microcapsules was lost. Striped and fibrous surfaces representing the chitosan layer can be seen on CS/(ALG/Ca) surface (Fig. 3a). In comparison with the microspheres, the microcapsules' surface porosity was reduced 21. Loading of CS/(ALG/Ca) with PGR resulted in the appearance of locally aggregated PGR (nubs) (aggregated nubs marked by black lines, Figs. 3b,c,d). Energy dispersive X-ray (EDX) analysis showed that chemical compositions on these small nubs are equal to the chemical composition of PGR loaded into microcapsules. The surface of a microcapsule showed fibrous network propagation along the gel membrane surface 22. It can be seen that some microcapsule surface parts exhibit the partial collapse of the polymer network as a result of drying.
Loading efficiency and loading capacity determination were performed to obtain information on the yield and the content of PGR in chitosan/alginate microcapsule. It is known that the entrapped amount of active agents in chitosan/alginate microcapsule depends on the type and concentration of both biopolymers, gelling cation and active agents, as well as the method of preparation 22,23. Results listed in Table 2 show significant differences in both, EE and LC between samples. The difference in the EE reflects the extent of electrostatic interactions and hydrogen bonds involved in interactions of PGRs with both, calcium chloride and sodium alginate during preparation. The most water-soluble PGR - PGR2 exhibited the highest EE and LC values indicating a greater extent of molecular interaction in solution.
In addition to molecular interactions, the encapsulation capacity depends on the structure of the PGR, too. The smallest LC was determined for PGR3 which is a cyclic compound with a primary and tertiary amino group and two keto functional groups differing significantly in the structures from aliphatic primary amine (see details in Supplementary information).
PGR
|
EE/%
|
LC/mg g-1
|
Sw/%
|
PGR1
|
53.31±0.01
|
7.79±0.08
|
27.76±2.40
|
PGR2
|
89.94±1.43
|
15.60±0.09
|
1.76±1.04
|
PGR3
|
75.73+1.14
|
0.24±0.00
|
1.05±0.18
|
Table 2. Encapsulation efficiency (EE), loading capacity (LC) and swelling degree (Sw) of CS/(ALG/(Ca+PGR)) microcapsule formulations.
When hydrophilic microcapsules are dispersed in the water they start to swell. This process starts with the penetration of the solution into the microcapsule through the surface and with polymer stress relaxation (transition of glassy structure to a rubbery state) 24. One of the main factors which determine alginate swelling is a concentration of calcium ions which has got a major effect on the kinetics of gelation and the characteristics of the gel formed 25. During gelation, calcium cations cooperatively interact with a block of L-guluronic acid groups forming ionic crosslinks between different polymer chains. The penetration of water into the denser network with high cross-linking density is difficult, that is, swelling is limited by crosslinks and the Sw can be used as a measure of the extent of crosslinking 26.
A peculiar property of the chitosan/alginate microcapsules in the dry form is their ability, after contact with an aqueous fluid, to rehydrate, take up the fluid, and undergo a swelling process, mainly associated with the hydration of the hydrophilic groups of chitosan. When the fluid is water, it penetrates the microcapsules, fills the pores on the surface causing swelling without erosion/disintegration 27. Chitosan/alginate microcapsules would interact with the water molecules through their available –COO− and –NH3+ groups 28. As can be seen in Table 2 all microcapsules show a relatively high degree of swelling which can be explained by the hydrophilic nature of the chitosan-alginate complex on the microcapsule surface 29. The addition of PGR alters the number of alginate strands held together in the three-dimensional network and thus changes its strength and microcapsule structure. The highest swelling degree value obtained for PGR1 could be explained by less resistance against expansion and penetration of water molecules into its structure due to less crosslinking. Figs. 3a to 3c revealed presence of an PGR on the microcapsule surface. It seems that the surface of microcapsules loaded with PGR1 contains the largest number of amino groups that would react with water contributing to a high swelling degree.
Mechanisms and kinetics of PGRs release from microcapsule formulations. The release of active agents from hydrophilic microcapsules involves several different interconnected processes from the penetration of the surrounding solution into microcapsules, swelling, diffusion of the active agent through the gel membrane, dissolution of the active agent in the medium to the erosion of the swelled gel matrix. Kinetics and mechanism of active agent release primarily depend on the characteristics of the core material and active agent. In the case of a hydrophilic biopolymer matrix, active components are uniformly distributed/dissolved in the gel matrix and the release occurs by diffusion, swelling and matrix erosion 24. The possible use of biopolymer microcapsule formulations loaded with PGR as a new potential fertilizer involves tunning and optimization of many characteristic parameters (preparation process, microcapsules chemical composition, geometry and size, the conditions during release, etc.) among which are the most important type and concentrations of biopolymer, gelling cation and active agents 30. All the above parameters and their combination have an important influence on PGRs release from microcapsule formulations.
Microcapsule formulations are reservoirs of microscopic size surrounded by a wall that can control the release from the reservoir. When they are dispersed in water, it penetrates them, fills the pores among the polymer chains causing swelling. The release profiles of PGRs from microcapsule formulations with time are presented in Fig. 4. All release profiles exhibit rapid initial PGR release followed by a slower release obeying the power-law equation. To explain and understand the kinetics and type of mechanism involved in the release, Korsmeyer-Peppa's model was applied 31. According to this model different controlling mechanisms may be distinguished by a simple empirical equation:
where k is a release constant characteristic for a particular system considering structural and geometrical aspects, n is the release exponent representing the release mechanism, and t is the release time.
Fig. 4. The fraction of released PGRs (f(PGR1, PGR2, or PGR3) with time from microcapsule formulations CS/(ALG/(Ca+PGR)): I – the first time interval, II – the second time interval.
According to Korsmeyer–Peppas empirical model, the release exponent n can be characterized by three different mechanisms (Fickian diffusion, anomalous (non-Fickian diffusion), or Type II transport). Values of n < 0.43 indicates the release is controlled by classical Fickian diffusion, n > 0.85 is controlled by Type II transport, involving polymer swelling and relaxation of the polymeric matrix, whereas values of n between 0.43 and 0.85 show the anomalous transport kinetics determined by a combination of the two diffusion mechanisms and Type II transport.
The values of the release constants, exponents and correlation coefficients of PGRs are listed in Table 3. Lower n values than 0.50 indicate that the release is controlled by a classical Fickian diffusion. The diffusion is much greater than the process of polymeric chain relaxation. The kinetics of this phenomenon are characterized by diffusivity. Fickian diffusion is characterized by the high velocity of solvent diffusion to the interior of the matrix and the low velocity of polymeric relaxation. This behavior induces the formation of a gradient of solvent penetration.
Microcapsule formulations
|
k/h
|
n
|
R2
|
CS/(ALG/(Ca+PGR1))
|
10.36
|
0.104
|
0.991
|
CS/(ALG/(Ca+PGR2))
|
38.17
|
0.134
|
0.991
|
CS/(ALG/(Ca+PGR3))
|
4.84
|
0.441
|
0.992
|
Table 3. Variation of the release constant (k/h), exponent (n) and correlation coefficient (R2) of PGR released from CS/(ALG/(Ca+PGR)) microcapsule formulations.
It can be seen in Table 3 that the release rate of PGR2 is the fastest and of PGR3 is the slowest. This can be attributed to ring structure of PGR3 molecule which slows down diffusion through the biopolymer matrix.
In the case of microcapsule formulations loaded with PGR1 two separate time regions in the release, the profile can be recognized. In the first time interval, the release profile is characterized by rapid initial release followed by slower release obeying power low, and in the second time interval, the release profile is characterized by linear release with time. The second part of the release curve can be easily described as zero-order kinetics, described with the equation 32:
where fi represents the fraction of active agent dissolved during the time t and K0 is the zero-order rate constant and t is the time.
For zero-order kinetics, the release of PGR1 is only a function of time and the process takes place at a constant rate independent of PGR1 concentration. This phenomenon can be explained by the high degree of swelling (Table 2), which can prove to be a good property that will allow release for a long time. When chitosan coating is permeable to active agent and water, after swallowing, the core becomes hydrated and PGR1 dissolves until it reaches its saturation concentration or solubility.
Concluding remarks. Novel plant growth regulators based on the derivatives of dehydroamino acids (2,3-dehydroaspartic acid dimethyl ester (PGR1), Z-isomer potassium salt of 2-amino-3-methoxycarbonyl acrylic acid (PGR2) or 1-methyl-3-ethylamino imine of maleic acid (PGR3)) were synthesized. Laboratory testing and in field experiments on several plant species from Ukraine reveal their high growth-regulating activities. PGR1 showing the most stimulating activity on all laboratory tested cultures were used in field experiments (treating seeds (semi-dry treatment, inlay), tubers, cuttings, grafts, the root system of seedlings and vegetative plants). The first treatment seeds before sowing, tubers before planting, cuttings at cutting and grafting, root systems of seedlings at quilting or planting provided early germination of seeds, good rooting, and increased resistance of plants to adverse natural factors. The second treatment carried out in the budding phase and at the beginning of flowering reduced the ripening time and increased the yield.
Calcium alginate microcapsules coated with chitosan layer loaded with synthesized enamines are designed as new microcapsule formulations suitable for application in agriculture. Information about molecular interactions between biopolymers and plant growth regulators by FTIR spectroscopy revealed functional groups of all constituents interact with each other. A complex electrostatic interaction and hydrogen bonding between all constituents in microcapsule formulations as well as PGR molecular properties caused changes in microcapsule structure, and consequently, in physicochemical characteristics. The addition of PGRs alters the number of alginate strands held together in the three-dimensional gel network and thus the extent of crosslinks between polymer chains. The highest swelling rate value obtained on the microcapsule formulation loaded with PGR1 can be attributed to the less crosslinked polymer chains and the partial localization of PGR1 on the microcapsule surface.
All release profiles exhibit PGR rapid initial release followed by a slower release obeying the power-law equation. The peculiar property of microcapsule loaded with PGR1 is that after initial swelling and release, their core becomes hydrated and dissolves PGR1 until it reaches its saturation concentration or solubility resulting in linear release dependence with time. Differences in microcapsule structure due to PGRs encapsulation and surface properties do not affect the controlling release mechanism identified as Fickian diffusion. A better understanding of the structure/property relationship in microcapsule formulations and mechanisms controlling plant growth regulator release enhances the ability to control the release behavior and may aid in developing microparticles with specifically tailored properties
With all these results, it seems that the new formulations of microcapsules with PGRs have great potential for application in the agricultural production of various crops. The advantages of using microcapsule formulations are in protecting the encapsulated PGR from degradation, reducing the amount required, and achieving controlled release to regulate growth for the entire crop cycle.