Impact of Plant Growth Regulators to Development of the Second Generation Energy Crop Miscanthus × giganteus Produced Two Years in Marginal Post-Military Soil

: The impact of the plant growth regulators (PGRs) Stimpo, Regoplant, and Charkor on the production of the second-generation energy crop Miscanthus × giganteus on marginal post-military soil was investigated during two vegetation seasons. The land, previously a tank training polygon, has not been in use since 1990 and has become marginal. Biological parameters (stem, shoot, and root lengths) and dry biomass values were evaluated in relation to the applied treatments. The multivariate general linear model (M-GLM) results showed a positive inﬂuence of Charkor on M. × giganteus development; the effect was markedly higher in the second year of vegetation. The impact of Stimpo and Regoplant was less noticeable; nevertheless, certain combinations of treatments showed satisfactory results. The M-GLM approach detected the inter-inﬂuence of the main factors of the production process, i.e., PGRs, soil, and year of growing. The results showed the predominant inﬂuence of year, PGRs and combined factor PGRs × year on the biological parameters; the other studied factors and their combinations were not as effective. Further research should focus on verifying the ﬁeld-scale results for the M. × giganteus plantation established in a post-military area and compare the lab and ﬁeld studies.


Introduction
Plant growth regulators (PGRs) are organic chemical compounds that modify or regulate physiological processes in plants when applied in small concentrations and appreciable measures. These substances include plant hormones (natural or synthetic) and non-nutrient chemicals that do not naturally occur in plants. PGRs are used to influence plant growth and development and better realize their genetic potential [1]. The type and working concentration of PGRs in the liquids applied depend on the plant species, the tissue or organ cultured, and the objective. These substances can influence plant physiology to change morphological (i.e., root biomass and architecture) and metabolic properties [2,3]. The commonly used PGRs are auxins, cytokinins, and gibberellins; some PGRs have as active ingredients biologically active substances synthesized by microorganisms, including fungi, such as micromycetes [3] or species of the genus Cylindrocarpon obtusiusculum Sacc. [4]. and field experiments, the enhancement of M. × giganteus productivity with beneficial soil microbes and Ascophyllum nodosum L. seaweed extract was observed on poor-quality, marginal land [25], which may be associated with increased access to limited soil nutrients due to PGR application.
The PGRs emistim, Agrostimulin, and Regoplant, which are based on aversectine and phytohormones produced by Cylindrocarpon obtusiusculum, were tested on M. × giganteus cultivation in fertile soil [27] and positively impacted the physiological parameters. They increased the value of the chlorophyll ratio, particularly in the early stages of vegetation growth, and enhanced morphometric indicators of plant height, the numbers of stems in the bush, and aboveground biomass yield. In addition, the application of PGRs reduced the dependence of survival rate on weather conditions, which was particularly evident for the late planting time.
A limited number of studies analyzed the impact of PGRs on Miscanthus spp. development in contaminated or marginal lands, including post-military areas. In one study, treatment of M. ×. giganteus rhizomes with the humic-based PGRs Gumifild Forte and Fulbital increased plant survival rates when a crop was cultivated in field conditions in previously oil-contaminated soil [35]. The application of PGRs stimulated the development of the root system, improved aboveground biomass, and increased the content of photosynthetic pigments in the leaves. Our previous study, in which Regoplant and Stimpo were tested during one vegetation year of M. × giganteus in soil slightly contaminated with TEs, indicated the effect of PGR utilization on morphological indicators and biomass parameters when the crop was produced in soil of moderate quality [36]. The influence was greatest when PGRs were combined to treat rhizomes before planting and used in plantation spraying during vegetation [36]. However, the research illustrated that the application of PGRs were not effective when the crop was cultivated in pure nutrients and organic matter soil.
Miscanthus spp. is a perennial grass, and the first two production cycles are the most important for establishing a good stable feedstock [37]. In this regard, PGR influence must be monitored in at least two growing processes. In the previous study [36], it was concluded that the development of plants in pure soil was not sensitive to the application of PGRs; however, the conclusion was based on simple graphical analysis, while the influence of multi-factor interactions, i.e., soil-PGRs, soil-years of cultivation, PGRs-years of cultivation was not statistically evaluated. To overcome this gap, the current research was designed to examine the influence of PGRs on M. × giganteus cultivation during two vegetation seasons with a statistical evaluation of the primary process factors: soil, time, and PGR type.
Consequently, the goals of the current study were as follows: − To test the impact of three commonly used PGRs applied in the Miscanthus spp. production scheme, i.e., Regoplant, Stimpo, and Charkor, on dry biomass value during two vegetation seasons; − To analyze the impact and value of three main factors involved in the production cycle: soil, PGRs, and year of cultivation on plant development.

Soil Characteristics
The laboratory study was carried out in 2017-2018 with soil taken from the postmilitary site in Dolyna, Ivano-Frankivsk Region, Ukraine. This field was not under exploitation since 1991 and was previously used as a tank training polygon of the former Soviet Union army; currently, this land can be considered as marginal post-military area [38]. The physicochemical characteristics of the studied soil were presented and discussed in detail earlier [36]. The field consists of two plots; each plot has a size of 161 m 2 . The GPS coordinates are: Plot #1, 48 • [39] from one 5 m × 5 m testing square. The collected soil was first dried to constant weight and then passed through a sieve with a pore diameter of 2 mm [40] to remove the plant materials and stones, followed by thorough mixing. Soil collected for research from Plot #1 was named Soil I; soil collected from Plot #2 was named Soil II.
The agrochemical parameters of the research soils determined by standard methods are presented in Table 1. The physicochemical characteristics of the studied soil were presented and discussed in detail previously [36]. Before setting up the PGR experiment, the particle size distribution of the soils was determined following [47]. The results are presented in Table 2.

Design of the Pot Experiment and Characteristics of PGRs
The laboratory experiments were performed in greenhouse conditions in pots. First, 1 kg of sand and 1 kg of expanded clay for drainage were placed in the bottom of each pot, followed by 10 kg of the collected soil. The planting material was M. ×. giganteus J.M. Greef & Deuter ex Hodkinson & Renvoize (Angiospermae: Poaceae), and the rhizomes of the variety "Rankova Zorya" were three years old and had an average weight of 20 ± 2 g. The planting materials were obtained from the nursery of the Institute of Sugar Beets and Bioenergy Crops, Ukraine, Kyiv. Two rhizomes were planted in each pot to a depth of 10 cm. Then, the experiment plan in space was performed according to the Latin square [48]; each of the experiments was repeated in three replications. Three PGRs, Stimpo, Regoplant, and Charkor, were utilized to treat planting materials before the experiment and during vegetation.
Charkor belongs to toxicity class IV, the lowest under the standard [49]; LD50 related to mice and rats is 5300 mg kg −1 . The active substance is a complex of 2,6-dimethylpiridin-1-oxide with C 12 H 10 O 2 -naphtylacethic acid. The complex of the substances formed the active component of emistim C with the natural background phytohormones of aucsine, citokininon nature, saturated and unsaturated fatty acids, amino acids, carbohydrates, and biogenic ion microelements. Charkor does not have cumulative properties and is not accumulated in soil and produced crops. It is a slightly yellow, homogenous, transparent liquid, easily dissolved in water, acetone, ethyl alcohol, and other organic solvents.
Regoplant is a substance belonging to toxicity class IV [49]; LD50 related to mice and rats is 5800 mg kg −1 . The active substance is a complex of physiologically active compounds: metabolic products of the micromycetic fungi Cylindrocarpon obtusiusculum, a modified compound of PGR; emistim C, a complex of biogenic microelements; and aversectin C, a metabolic product of actymonycetic fungi Streptomyces avermytilis. This product does not have cumulative properties and is not accumulated in soil and produced crops. Regoplant is a homogeneous, light green, transparent liquid that can be indifferently dissolved in water, acetone, ethyl alcohol, and other organic solvents.
Stimpo is a substance belonging to toxicity class IV [49]; LD50 related to mice and rats is 5800 mg kg −1 . It is formed of a complex of physiologically active compounds: metabolic products of the micromycetic fungi Cylindrocarpon obtusiusculum, a modified compound of PGR; emistim C, a complex of biogenic microelements; and aversectin C, a metabolic product of the actymonycetic fungi Streptomyces avermytilis. The substance does not have cumulative properties and is not accumulated in soil and produced crops during application. Stimpo is a homogeneous, colorless, transparent liquid easily dissolved in water, acetone, ethyl alcohol, and other organic solvents.
All three selected PGRs were produced by the Inter-institutional Scientific and Technological Centre "Agrobiotech" (Agrobiotech) under the jurisdiction of the National Academy of Science and Ministry of Education, Ukraine (ISTC NAS&MOE), (https:// www.agrobiotech.com.ua, accessed on 24 December 2021). The substances are produced on an industrial scale, however, the producer does not provide detailed formulations [50]. Publicly available characteristics of selected PGRs are presented in Table 3. Charkor, Regoplant and Stimpo are widely utilized in Ukraine and surrounding countries for improving the development and biomass production of different agricultural plants, including first-generation energy crops: barley, wheat, sugar beet, fruits, and vegetables [6][7][8]50]. Recently, Stimpo and Regoplant obtained Organic Standard certification and can be used in organic agriculture [54]. The type of M. × giganteus treatment with selected PGRs was chosen based on their function type [6,9,[51][52][53]55]. The composition of Charkor includes chemically synthesized analogues of phytohormones, i.e., 2,6-Dimethylpyridine-N-oxide and 1-naphthylacetic acid; these substances are plant root initiators/ stimulators. Charkor was successfully utilized when green and woody cutting seedlings of fruit and ornamental trees, shrubs, flowers, and medicinal plants were treated. Hence, Charkor's standard application method involves the soaking of vegetative plant parts in a water solution of this substance before planting; spraying biomass during vegetation is not utilized in the case of Charkor. The recommended concentration (Table 3) of Charkor in the water solution used to treat vegetative parts before planting is between 0.1% and 4%; the recommended exposition time is between 2 and 20 h. Following these recommendations, M. × giganteus rhizomes were soaked in a Charkor water solution with concentrations of 0.1% and 0.4% and an exposure time of 12 h. In the control experiment, rhizomes were soaked in distilled water without PGR at the same exposition time. Regoplant and Stimpo do not have chemically synthesized analogues of plant hormones and, unlike Charkor, they are not characterized with a strong root initiation effect. Based on testing with wheat and barley (which belong to the same family, Poaceae, as M. × giganteus), these PGRs are recommended [6,55] for utilization in a combined treatment, i.e., treatment of planting material before planting followed by spraying of the vegetation: it is recommended to perform the the first spray when three to four pairs of leaves have appeared, and the second when six pairs have appeared. For Regoplant, the proposed treatment dose is between 1.22-2.44% with an exposition of 2-24 h. In the current study, we used a 2.44% water solution of Regoplant to treat rhizomes before planting and 1.22% and 2. 44% water solutions for spraying, with an exposure time of 12 h. In the control experiment, rhizomes were soaked in distilled water without PGR at the same exposition time or sprayed with distilled water. For Stimpo, the proposed treatment dose for the initial planting material is between 0.25-0.50% with an exposition of 2-24 h. In the current study, we used a 0.25% water solution of Stimpo to treat rhizomes before planting and 0.25% and 0.50% water solutions for spraying with an exposure time of 12 h. In the control experiment, rhizomes were soaked in distilled water without PGR at the same exposition time or sprayed with distilled water.
The study was started on 11 May 2017 and completed on 10 October 2018. Plants were watered with tap water two to three times a week during both vegetation seasons. After harvest on 13 October 2017, the pots containing the roots of the plants were stored in the dark over winter. On 30 April 2018, the experiment re-started when the first green shoots appeared, and the pots were returned to the light in the greenhouse.

Measuring of Biological Parameters and Dry Biomass Value
Biological parameters: stem length, stem number, and shoots were monitored over vegetation. At the end of each vegetation when the leaves turned yellow the final measurements of stem length, number of stems, number of shoots, and root length were made.
The cut aboveground biomass was dried on an open surface till the contact weight. Further, in order to determine the dry biomass value (DW), a certain amount of biomass was transferred to the metal box and dried in a thermostat at 100-105 • C to the constant weight. Initially, biomass was dried for eight hours, then, it was weighed and put in the oven again; the procedure was repeated until the difference between the two weights was less than 0.0001 g.

Statistical Analysis
The results were evaluated using multivariate general linear models (M-GLMs) using the Statistics v. 12.0 PL software package from TIBCO Software, Inc. (Palo Alto, CA, USA) [56]. Two multivariate GLMs were performed, and the following factors were considered: the Effect (PGR type and control (nine levels)), Soil (Soil I and Soil II), and Year (two levels). Year × Soil, Year × Effect, Soil × Effect, Year × Soil × Effect (interaction) were used as categorical predictors to explain M. × giganteus trait variations, i.e., number of stems, number of shoots, length of roots, and length of stems. The differences between individual treatments were tested using a planned comparison.

Results and Discussion
The lignocellulose biomass, which contains carbohydrate polymers (cellulose and hemicellulose) and an aromatic polymer (lignin), is identified as a prospective alternative for biofuel production [19][20][21]. The conversion of lignocellulose biomass to heat, bio-oil, syngas, ethanol, methane, and hydrogen is valuable in climate change mitigation [57]. The biomass of second-generation industrial crops, including Miscanthus spp., can be processed to produce energy through the thermochemical routes of gasification and pyrolysis [58]. The effectiveness of the processes is determined by the operational conditions of feed properties, oxidizer, temperature, heating rate, and residence time [21]. Efforts are focused on utilizing different agricultural factors to ensure the proper development of plants and deriving an economically valuable amount of biomass [12,59]. These goals can be achieved with the application of soil amendments, such as sludge [23,26,60], carbon contented amendments [61], fertilizers [62], and by influencing the initial planting materials with PGRs [27,32,36] or microbial isolates [63]. The harvest value of Miscanthus spp. biomass is determined by plant development during vegetation [64] and plant stress [16], which can be evaluated by measuring the common biological indicators: plant health, number of stems, number of shoots, and dry biomass at harvest.
According to the analysis (Tables 1 and 2), Soil I is slightly neutral, while Soil II is acidic. Both soils are weak in phosphorus and rich enough in potassium. Sulfur content is low in both soils, and both soils have high organic matter values. Based on the standard evaluation [65], the researched soils included enough nutrients; however, Soil I is less acceptable for agricultural purposes due to its higher swelling and shrinking characteristics, which implies a higher content of clay (Table 2) [65,66] and, following Jones et al. [67], led to worst plant develop in this soil. Based on soil properties (Tables 1 and 2), the differences in the influence of PGR treatment on the M. × giganteus production cycle may be expected.
The influence of PGR treatments was more pronounced in the second year of vegetation than in the first year (Figure 1). For the second vegetation year, the differences in impact on the bioparameters depending on soil type are more prominent with the plants grown in Soil II compared to those grown in Soil I. This may be due to the slightly better quality of Soil II in terms of pH value and swelling and shrinking characteristics (Tables 1 and 2). The impact of the studied factors, i.e.: PGRs, soil type (Soil I and Soil II), and time (year of vegetation), on M. × giganteus bioparameters are presented in Tables 4 and S1, Figures S1-S4 (Supplementary Materials). These studied factors explained 66-94% of the variation of the traits and were statistically significantly influenced to the number of stems (Table 4). The stronger influence of PGR treatment in the second year of M. × giganteus vegetation (Figures S1-S4) may explain the reduction in PGR impact when the plant grew in the pure quality soil [36] because results were obtained only for one vegetation of M. × giganteus.
The treatment with various PGRs impacted M. × giganteus biological parameters differently. For example, while Ch1, Ch2, and S2 treatments increased the number of stems (Figure 1a), R2, R3, and Ch2 treatments impacted mainly the number of shoots ( Figure S2a), and R1, R2, R3, and Ch2 treatments were the most essential in case of root length ( Figure S3a).
The biomass is mainly formed by stems when M. × giganteus is harvested [59]. Therefore, the enhancement of biomass is among the targeted goals when the crop is grown on different marginal lands, including post-military areas [60]. This goal may be achieved by applying varied agricultural practices, including treatment by PGRs [36]. The results (Table 5) showed that the dry biomass value is primarily dependent on the length of stems (Figure 1) and the variety of PGR applied (Figure 2) (p < 0.001). Table 5. Evaluation of the effect of the research factors, PGRs, soil type, and year of vegetation, on dry biomass yield following M-GLM procedures (Radj 2 = 0.74, F = 8.9, p < 0.001).  The stronger influence of PGR treatment in the second year of M. × giganteus vegetation ( Figures S1-S4) may explain the reduction in PGR impact when the plant grew in the pure quality soil [36] because results were obtained only for one vegetation of M. × giganteus.
The treatment with various PGRs impacted M. × giganteus biological parameters differently. For example, while Ch1, Ch2, and S2 treatments increased the number of stems (Figure 1a), R2, R3, and Ch2 treatments impacted mainly the number of shoots ( Figure S2a), and R1, R2, R3, and Ch2 treatments were the most essential in case of root length ( Figure S3a).
The biomass is mainly formed by stems when M. × giganteus is harvested [59]. Therefore, the enhancement of biomass is among the targeted goals when the crop is grown on different marginal lands, including post-military areas [60]. This goal may be achieved by applying varied agricultural practices, including treatment by PGRs [36]. The results ( Table 5) showed that the dry biomass value is primarily dependent on the length of stems ( Figure 1) and the variety of PGR applied (Figure 2) (p < 0.001). Table 5. Evaluation of the effect of the research factors, PGRs, soil type, and year of vegetation, on dry biomass yield following M-GLM procedures (R adj 2 = 0.74, F = 8.9, p < 0.001).

F-Ratio p-Level
Year  In this regard, the gradation of factors influenced stem length and the determination of the extent to which these results depended on PGR treatment is essential for enhancing M. × giganteus biomass. The results shown in Figure 1 illustrate that in the first year of vegetation in Soil I the impact of PGRs had the following order: Ch2 > Ch1 > R1 > S2; for another treatment, the effect was neglected and close to the treatment with water (control). In the second year of vegetation, the order of PGR impact was changed for Soil I: S2 > Ch2 > R2 ≥ R3; S1 > Ch1, S3; for other treatments, the effect was neglected and close to the control. These results showed that, while for the first year of vegetation the influence of Charkor on increased root growth was the main factor related to enhancing biomass values (Figure 2), for the second year, the treatment of aboveground biomass competed with the treatment of rhizomes and resulted in longer stem length under this treatment. For the first year of vegetation in Soil II, the impact of PGRs had the following order: Ch2, Ch1 > R1, S2; for other treatments, the effect was neglected and close to the control treatment. In the case of Soil II, the effect of treatment by Charkor, which influences root In this regard, the gradation of factors influenced stem length and the determination of the extent to which these results depended on PGR treatment is essential for enhancing M. × giganteus biomass. The results shown in Figure 1 illustrate that in the first year of vegetation in Soil I the impact of PGRs had the following order: Ch2 > Ch1 > R1 > S2; for another treatment, the effect was neglected and close to the treatment with water (control). In the second year of vegetation, the order of PGR impact was changed for Soil I: S2 > Ch2 > R2 ≥ R3; S1 > Ch1, S3; for other treatments, the effect was neglected and close to the control. These results showed that, while for the first year of vegetation the influence of Charkor on increased root growth was the main factor related to enhancing biomass values (Figure 2), for the second year, the treatment of aboveground biomass competed with the treatment of rhizomes and resulted in longer stem length under this treatment. For the first year of vegetation in Soil II, the impact of PGRs had the following order: Ch2, Ch1 > R1, S2; for other treatments, the effect was neglected and close to the control treatment. In the case of Soil II, the effect of treatment by Charkor, which influences root system development [9], was the main factor for the first year of growing, similar to Soil I. For the second year, the influence of PGRs had the following order: Ch2 ≥ Ch1 > S2, S1; for other treatments, the influence of PGRs was the same as for the control. These results highlighted that for the second year the influence of PGRs in the better quality Soil II was altered compared to the influence in the poorer quality soil of Soil I. For both years, the prevailing impact of Charkor in the better quality Soil II revealed that this particular PGR could be recommended for increasing stem length (and biomass value) when the crop is produced in good quality soil. When the soil quality is worse (Soil I), combined treatment with the other PGRs studied, Stimpo and Regoplant, gives almost the same effect as Charkor for increasing stem length (biomass value).
PGR treatment impacts on dry biomass yield showed (Figure 2) that the most potent influence was observed for Ch2; the order of impact was the following: Ch2 followed by S2, R2, R2, Ch1; for another treatment, the effect was neglected and almost the same as for control. This observation is correlated with the impact of PGR treatment on length and is in line with the results of [9], which showed that Charkor is an effective substance for enhancing M. × giganteus development due to stimulation of the root system, which is among the main factors in plant development [64].
The stimulating effects of two factors (Year and PGR) were essential for the second year of vegetation and noted for almost all PGR treatments besides Ch1, S1, and S3. Comparison of Ch1 and Ch2 showed that Ch2 had a greater stimulating influence on the effects of the two factors.

Conclusions
The impact of three studied factors, PGRs, soil type, and year of vegetation, were evaluated for two vegetation seasons during the growth of the second-generation energy crop M. × giganteus in marginal post-military soils supported by the treatment of rhizomes with PGRs. The M-GLM statistical approach showed that these factors influenced plant bioparameters (stems, shoot and root lengths) and dry biomass yield. As a result, increased plant height and a larger amount of M. × giganteus biomass were ultimately achieved. However, stimulation of other aspects of individual plant growth can increase competition between individuals, which may not increase the proportional growth of biomass and its energy output.
The results of two years of production of M. × giganteus stimulated by various PGR treatments emphasized that treatment by Charkor in higher concentrations positively influenced biological parameters and dry biomass value, and the effect was much more vigorous for the second vegetation year. Therefore, this PGR can be recommended for practical application on M. × giganteus plantations to enhance plant development and biomass production. However, other studied PGR treatments were not as effective in terms of biomass production despite certain combinations of treatments showing satisfactory results.
To the best of our knowledge, the results reported here represent the first systematic study comparing the influence of the common PGRs Charkor, Regoplant, and Stimpo on the production of M. × giganteus over two years in lab conditions. The results obtained permitted the conclusion that when a crop is grown at a field scale on post-military marginal soil, only Charkor may be utilized; in particular, the effect of its positive influence has to be evident with years of growing. Two other researched PGRs (Stimpo and Regoplant), despite earlier reported positive influence on the development of common crops (corn and barley) [7][8][9][10]55], appeared not to be so effective in the case of the development of M. × giganteus and cannot be recommended for broad utilization in field conditions when economics is among the main decision-making factors. Future research should focus on verifying the field-scale recommendations for M. × giganteus plantations established on post-military land and on comparing the results of lab and field-scale experiments.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/app12020881/s1. Table S1. Descriptive statistics of morphometric traits of Miscanthus; Figure S1. The effect of PGRs on number of steams (a) depending of soil's type (b). Squares and circles show least square means. Vertical bars denote 0.95 confidence intervals; Figure S2. The effect of PGRs on shoot number (a) depending of soil's type (b). Squares and circles show least square means. Vertical bars denote 0.95 confidence intervals; Figure S3. The effect of PGRs on root length (a) depending of soil's type (b). Squares and circles show least square means. Vertical bars denote 0.95 confidence intervals; Figure S4. The effect of PGRs on stem's length (a) depending of soil's type (b). Squares and circles show least square means. Vertical bars denote 0.95 confidence intervals.