Issue 2 (202)/2025

ALSE and ACS Style
Elisovetcaia, D.; Ivanova, R.; Popovschi, E.; Brindza, J.  Germination of seeds and adaptation of beech seedlings of Plaiul Fagului-23 origin under the influence of bioregulators. Journal of Applied Life Sciences and Environment 2025, 58 (2), 301-321.
https://doi.org/10.46909/alse-582177

AMA Style
Elisovetcaia D, Ivanova R, Popovschi E, Brindza J. Germination of seeds and adaptation of beech seedlings of Plaiul Fagului-23 origin under the influence of bioregulators. Journal of Applied Life Sciences and Environment. 2025; 58 (2): 301-321.
https://doi.org/10.46909/alse-582177

Chicago/Turabian Style
Elisovetcaia, Dina, Raisa Ivanova, Ecaterina Popovschi, and Jan Brindza. 2025. “Germination of seeds and adaptation of beech seedlings of Plaiul Fagului-23 origin under the influence of bioregulators.” Journal of Applied Life Sciences and Environment 58, no. 2: 301-321.
https://doi.org/10.46909/alse-582177

Germination of seeds and adaptation of beech seedlings of Plaiul Fagului-23 origin under the influence of bioregulators

Dina ELISOVETCAIA¹*, Raisa IVANOVA¹, Ecaterina POPOVSCHI¹ and Jan BRINDZA²

¹Moldova State University, Institute of Genetics, Physiology and Plant Protection – Plant Physiology, 20, Pădurii Str. Chișinău MD-2002, Moldova (the Republic of); email: raisa.ivanova@sti.usm.md; ecaterina.popovschi@sti.usm.md

²Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, Nitra, Slovakia; email: jan.brindza@gmail.com

*Correspondence: elisovetcaia.dina@gmail.com 

Received: Sep. 23, 2024. Revised: Nov. 04, 2024. Accepted: Nov. 13, 2024. Published online: Aug. 04, 2025

ABSTRACT. The purpose of this research was to study the effect of bioregulators on the germination of beech seeds of Plaiul Fagului-23 origin and adaptation of seedlings under solarium and natural conditions. The highest mean daily germination (0.74) was observed for 0.001 and 0.0001% (w/v, dry residue) Juniperus sabina. During stratification under laboratory conditions, the highest seed germination (82.64%) occurred in 0.0001% J. sabina. The highest seed germination rate index was observed in 0.0001 and 0.0005% J. sabina (1.17 and 1.57, respectively). The average germination time of 0.01% alginite No. 2 and 0.0005% J. sabina was below that of the control by 1.05-1.11 days. The length of the roots of germinated beech seeds with alginite No. 2 and 0.001% J. sabina significantly exceeded that of the control (39.66 mm), reaching 56.04 and 52.05 mm, respectively. Under solarium conditions, the highest rate of seedling emergence was observed in variants alginite No. 6 (92.50%), 0.0005% J. sabina (91.11%), 0.001% J. sabina (90.00%) and alginite nano powder (82.35%), which exceeded the control by 1.6–1.8 times. The height of the plants in alginite No. 2 (13.50 cm), 0.0001% J. sabina (12.24 cm) and 0.001% J. sabina (10.08 cm) significantly exceeded the control. Under natural conditions, the germination of seeds treated with alginite nano powder (60.80%) and alginite No. 6 (47.47%) was significantly higher than that of the control (42.67%) and those treated with gibberellic acid (43.73%). The height of the plants treated with alginite nano powder (28.10 cm), 0.01% genistifolioside (29.70 cm) and 0.004% gibberellic acid (28.22 cm) significantly exceeded that of the control.

Keywords: Alginite; genistifolioside; Fagus sylvatica seeds; germination; Juniperus sabina.

 

INTRODUCTION

Fagus sylvatica is attracting increasing attention from scientists and foresters in many European countries, both from an economic and ecological point of view, especially due to the changes that beech forests have undergone in recent centuries. In Central Europe, beech forests form the most significant part of the ecosystem in the foothill and mountainous regions at an altitude of approximately 100–900 m above sea level (Kulla et al., 2023). Even though beech is the dominant species in many of its habitats, the area of natural beech forests in many European countries, including the Republic of Moldova, has significantly decreased (Postolache and Postolache, 2011; Schuldt et al., 2020; Scherrer et al., 2024). The decline was caused by a number of factors, including uncontrolled logging and ongoing climate change, which have led to an increase in the average daily temperature and a decrease in average annual precipitation (Roibu et al., 2023). Natural regeneration is inhibited in areas where parent plantations are not sufficiently protected due to high weed competition (Fuchs et al., 2024).

The depth of beech seed dormancy is determined by many factors, among which temperature, as an environmental pressure factor, has one of the highest priority values. The quality of seedlings depends on the high uniformity of seed germination under optimal conditions, which increases the chances of survival (Pawłowski et al., 2024; Varsamis et al., 2020). Breaking seed dormancy and reducing the germination time are essential for the success of plant establishment under environmental risk factors (Pausas et al., 2022; Zhang et al., 2022). To accelerate beech seed germination, a number of substances are currently used, such as ethephon, ethylene, and abscisic and gibberellic acids (Calvo et al., 2004a, b; Kolařova et al., 2010; Soltani, 2003; Staszak et al., 2019). One of the methods of increasing the seed germination of plant species, including beech, and often providing additional fungicidal action is the use of bioregulators of natural origin (Castro et al., 2020; Elisovetcaia et al., 2022; Njoku, 2023; Tanase et al., 2020; Volobueva et al., 2024).

Therefore, the aim of our work was to study the germination of beech seeds from the 2023 harvest originating from the “Plaiul Fagului” Nature Reserve (Republic of Moldova) under the influence of bioregulators and the adaptation of seedlings under solarium conditions and natural conditions.

 

MATERIALS AND METHODS

The experiments were carried out at the laboratory Natural Bioregulators of the Institute of Genetics, Physiology and Plant Protection, Moldova State University, and in the Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, under laboratory and solarium conditions during 2023–2024.

The study used Fagus sylvatica seeds from 3 populations collected in the fall of 2023 in the area of “Plaiul Fagului” Natural Reserve, Republic of Moldova (PF-23). The beech seeds were collected at an altitude of 200–310 m above sea level (Table 1).

The moisture content of beech seeds was determined both by the classical method (at a temperature of 103±2°C for 17 h) and using a RADWAG moisture analyser by gradual drying with a halogen lamp at a temperature of 160°C to a constant weight (if the change in weight does not exceed 1 mg in 10 sec) (ISTA, 2003; Elisovetcaia et al., 2023). For each determination, 3 g of seeds were used.

The weight of 1000 seeds was determined using an analytical balance with a readability of 0.0001 g (ISTA, 2019).

Seed viability was determined using two tests with 2.3.5-triphenyltetrazolium chloride (TTC) solution (Kerkez et al., 2018; França-Neto and Krzyzanowski, 2019) and hydrogen peroxide (HP) solution (Sharma et al., 2020). The root length of germinated seeds was measured in the HP test.

Preparation of alginite solutions

Alginite powder and nano powder were prepared using distilled water in calibrated flasks at concentrations of 0.01 and 0.00001%. Alginite powder and nano powder were weighed on an analytical balance and transferred quantitatively into a volumetric flask. Thereafter, the solution was thoroughly mixed and used immediately. Distilled water was used as a control.

Liquid products (liquid fractions of alginite preparations Nos. 2, 4 and 6) were prepared at the Institute of Agronomic Sciences, Department of Plant Production and Grassland Ecosystems, FAPZ SPU in Nitra (Brindza et al., 2021). Alginite liquid fraction No. 2 was purchased as 30 mL.L^-1ALGEX_r 2 (liquid). The sodium extract was obtained from crushed alginite (alginite from Pincina) after extraction (digestion) using an extraction solution containing a mixture of sodium pyrophosphate decahydrate and sodium hydroxide, followed by reduction (5:1). Alginite liquid fraction No. 4 was purchased as 30 mL.L^-1 ALGEX_r 4 (liquid). The potassium extract was obtained from crushed alginite (alginite from Pincina) after extraction (digestion) using an extraction solution containing a mixture of potassium hydrogen phosphate and potassium hydroxide, followed by reduction (5:1). Alginite liquid fraction No. 6 was purchased as 30 mL.L⁻¹ ALGEX_r 6 (liquid). The sodium-potassium extract was obtained from crushed alginite (alginite from Pincina) after extraction (digestion) using an extraction solution containing a mixture of sodium pyrophosphate decahydrate, potassium hydrogen phosphate and potassium hydroxide, followed by reduction (5:1).

For the studies, solutions were prepared from liquid fractions alginite No. 2, No. 4 and No. 6 in concentrations of 0.01 and 0.00001%.

Seed germination test

The seed germination test was carried out in accordance with the recommendations of the International Seed Testing Association (ISTA, 2006). Before germination using the stratification method, beech seeds were soaked in the aqueous solutions of Juniperus sabina plant extract (0.001, 0.0005 and 0.0001%) and various solutions of natural mineral alginite for 22–24 h at room temperature.

A total of 9 alginite solutions of different concentrations were used under laboratory and natural conditions, with 0.01% and 0.00001% aqueous solutions prepared from alginite powder, alginite nano powder and liquid fractions of alginite No. 2, No. 4 and No. 6. Distilled water was used as a control. Each variant consisted of 25 seeds with 4 replications (Table 2).

The following traits were determined using standard formulas: total germination percentage (TGP), mean daily germination (MDG), mean germination time (MDT) and germination rate index (GRI). At 112 days after stratification, the length of the seed roots was measured.

Beech seedling emergence studies

Seedling emergence studies were carried out under solarium conditions, similar to those presented by Elisovetcaia et al. (2022). Data are presented as the means and standard deviations of four biological replicates.

Seed germination under natural conditions

The seed germination under natural conditions was determined in accordance with the recommendations of the International Seed Testing Association (ISTA, 2006).

 

Table 1
Characteristics of Fagus sylvatica seed collection sites in “Plaiul Fagului” Natural Reserve, Republic of Moldova

Name population	Forest composition	Compartment	Altitude (above sea level), m	Age of beech trees as of 2023
PF-23 No. 2	2FS, 3CB, 2FE, 2TC, 1DS	29H	200-250	70
PF-23 No. 8	5FS, 2QP, 1TC, 2CB	48C	310	100
PF-23 No. 10	1FS, 2QP, 2QR, 2TC, 1FE, 2CB	44A	225-310	115

Note: FS – Fagus sylvatica L.; CB – Carpinus betulus L.; QR – Quercus robur L.; QP – Quercus petraea (Matt.) Liebl.; TC – Tilia cordata Mill.; DS – Diverse hardwood forest species; FE – Fraxinus excelsior L.

 

Table 2
Stratification of seeds of Fagus sylvatica populations of the Plaiul Fagului-23, Republic of Moldova, under laboratory and natural conditions

Variants for natural conditions	Number seeds/ repetitions/ total	Variants for laboratory conditions	Number seeds/ repetitions/ total
	PF-23 No. 2		PF-23 No. 8	PF-23 No. 10
Control	250×3=750	Control	25×4=100	250×3=750
Gibberellic acid 0.001%	250×3=750	Alginite powder 0.01%	25×4=100	250×3=750
Genistifolioside 0.01%	250×3=750	Alginite nano powder 0.01%	25×4=100	250×3=750
Alginite powder 0.00001%	250×3=750	Alginite No 2 0.01%	25×4=100	250×3=750
Alginite nano powder 0.00001%	250×3=750	Alginite No 4 0.01%	25×4=100	250×3=750
Alginite No. 2 0.00001%	250×3=750	Alginite No 6 0.01%	25×4=100	250×3=750
Alginite No. 4 0.00001%	250×3=750	Juniperus sabina 0.001%	25×4=100	250×3=750
Alginite No. 6 0.00001%	250×3=750	Juniperus sabina 0.0001%	25×4=100	250×3=750
	–	Juniperus sabina 0.005%	25×4=100	–
Total	6000		900	6000
Total seeds in all variants	12900

 

Each variant consisted of 250 seeds with 3 replications. Before germination by stratification under natural conditions, the beech seeds were soaked in aqueous solutions (0.00001%) of natural mineral alginite (5 alginite solutions – aqueous solutions of alginite powder, alginite nano powder and liquid fractions No. 2, No. 4 and No. 6 of alginite) and 0.01% aqueous solution bioregulator genistifolioside from Linaria genistifolia (L.) Mill) for 22–24 h at room temperature (Mascenсo et al., 2015). Distilled water was used as a control, and a 0.001% solution of gibberellic acid was used as a standard (Table 2).

Chlorophyll index

The relative chlorophyll content of beech plants was quantified as the chlorophyll content index (CCI), measured with a CCM-200 Plus Chlorophyll Content Meter (Opti-Sciences, Inc., Tyngsboro, MA, USA).

The measurements were carried out for the first and second pairs of juvenile leaves of seedlings. For each leaf, 10 measurements were collected at different locations (points), avoiding the mid-vein (Van Wittenberghe, 2012).

Statistical analysis

The results were processed using Statgraphics Plus 5.0 software based on general statistical procedures, including one-way analysis of variance (ANOVA) and multiple range tests (Tukey’s honestly significant difference, HSD).

Pearson’s correlation analysis was conducted using the CORR procedure.

 

RESULTS

The viability of the beech seeds of Plaiul Fagului-23 (PF-23) populations reached 82.0% for PF-23 No. 2, 88.0% for PF-23 No. 8 and 86.1% for PF-23 No. 10 (as the average of two tests TTC and HP, Pearson correlation coefficient = 0.6832).

The average weight of 1000 seeds for three populations was 353.2, 298.9 and 327.1 g for PF-23 No. 2, No. 8 and No. 10, respectively. The moisture content of beech seeds before the start of the experiments, determined both by the classical method and by the RADWAG device, fluctuated from 9.38±0.24 to 10.49±0.29%.

 

Study of the effect of bioregulators alginite and Juniperus sabina on germination and root length of Fagus sylvatica seeds after stratification under laboratory conditions

Germination of the first beech seeds of the PF-23 populations (average values for two populations PF-23 No. 8 and PF-23 No. 10) was observed at 70 days after stratification (0.4–3.0% of the total number of seeds in the experiment). The mass germination of seeds of PF-23 origin was observed 98 days after stratification or 4 weeks after the date of germination of the first seeds (Figure 1).

The increase in seed germination with the variant 0.0005% Juniperus sabina was so strong that it numerically exceeded the control by 2.79 times. Another 4 variants – 0.0001% J. sabina, 0.01% alginite nano powder, 0.01% alginite No. 2 and 0.01% alginite No. 4 – significantly exceeded the seed germination of the control in seed germination by 98 days after stratification (by 1.28–1.49 times). At 98 days after stratification, seed germination in the variant with 0.0005% J. sabina significantly exceeded not only the control but all other treatment variants treated with bioregulators from J. sabina and alginite solutions. On the 112th day, the germination of PF-23 beech seeds in almost all variants levelled out and reached 76.0–82.64% (Figure 2). The lowest total germination (61.08%) was observed with 0.01% alginite No. 2.

 

 

 

The analysis of means plot showed that the germination in variants 0.01% Alginite nano powder, 0.001% J. sabina and 0.0001% J. sabina was significantly higher than that in the other variants, including the control (Figure 3).

 

 

For the PF-23 populations (average PF-23 No. 8 and PF-23 No. 10), the germination indices of beech seeds at 112 days were determined (Table 3). The highest mean daily germination – MDG (0.74) was observed for J. sabina variants at concentrations of 0.001 and 0.0001%. However, the highest germination rate index (GRI) was observed in the variant 0.0005% J. sabina (1.57), and the highest seed germination (at 112 days after stratification) was noted after treatment with 0.0001% J. sabina (82.64%, Figure 1). Variants 0.01% alginite nano powder, 0.01% alginite No. 2, 0.01% alginite No. 4 and 0.0001% J. sabina showed an mean germination time (MGT) below the control by 1.15–5.16 days. The lowest MGT was noted in the variant with 0.0005% J. sabina, with a value 11.06 days less than that of the control. The differences between the minimum (0.01% alginite No. 6) and maximum (0.01% alginite powder) germination among the variants treated with bioregulators was 21.56%. The lowest GRI index was noted in the variants treated with 0.01% alginite No. 2 and 0.01% alginite No. 6, which was lower than that in the control (1.03) and reached 0.94 and 0.99, respectively (Table 3).

 

Table 3
Germination indices of Fagus sylvatica seeds from Plaiul Fagului-23, Republic of Moldova, treated with alginite and Juniperus sabina during stratification (2024)

Variants	Germination indices
	MDG	MGT	GRI
Control	0.72	106.93	1.03
Juniperus sabina 0.001%	0.74	106.60	1.06
Juniperus sabina 0.0005%	0.68	95.87	1.57
Juniperus sabina 0.0001%	0.74	105.05	1.17
Alginite powder 0.01%	0.73	107.12	1.03
Alginite nano powder 0.01%	0.72	105.78	1.08
Alginite No. 2 0.01%	0.55	101.77	0.94
Alginite No. 4 0.01%	0.69	105.22	1.09
Alginite No. 6 0.01%	0.73	107.80	0.99

Note: MDG – mean daily germination; MDT – mean germination time; GRI – germination rate index

 

In the germinated seeds, at 112 days after stratification in the PF-23 populations, the root length was determined (Figure 4). The beech seeds responded differently to treatment with alginite solutions and J. sabina extract. The longest roots were observed in the variant with a seed treatment of 0.01% alginite No. 2 and 0.001% J. sabina extract. The smallest roots during seed germination were noted with 0.01% alginite powder and 0.01% alginite No. 4.

We carried out a statistical analysis of the data according to the following methods: analysis of means plots with 95% decision limits (Figure 4 a), box-and-whisker plot (Figure 4 b) and multiple range test with Tukey’s HSD (Table 4). The effect of alginite solutions and J. sabina extract on the root length of germinated beech seeds was very heterogeneous. The maximum root length was noted in the variants treated with 0.01% alginite No. 2 (56.04 mm), 0.0005% J. sabina (52.42 mm), 0.001% J. sabina (52.05 mm) and 0.0001% J. sabina (49.75 mm) (Figure 4 a, b; Table 4). According to the analysis of means plot (with 95% decision limits) and the box-and-whisker plot, the root length after treatment with 0.01% alginite No. 2, 0.0005% J. sabina and 0.001% J. sabina significantly exceeded that of the control and other variants (Figure 4 a, b).

According to the multiple range test (Tukey’s HSD), the root length of germinated seeds treated with 0.01% alginite No. 2, 0.0005% J. sabina and 0.001% J. sabina at all confidence levels (α = 0.05, 0.01 and 0.001) significantly exceeded only the length in the variants treated with 0.01% alginite powder (36.06 mm) and the control (39.66 mm). At the level of significance α = 0.05, the root length in these variants significantly exceeded the length in variant 0.01% alginite No. 4 (41.56 mm). The root length of germinated seeds in the variant treated with 0.0001% J. sabina significantly exceeded the variant treated with 0.01% alginite powder at all significance levels (α = 0.05, 0.01 and 0.001). At a significance level of α = 0.05, it also exceeded the control. Although the average root lengths in the 0.01% alginite nanopowder (43.74 mm), 0.01% alginite No. 4 (41.56 mm), and 0.01% alginite No. 6 (43.21 mm) treatments were higher than that of the control, the differences were not statistically significant at any level according to the Multiple Range Test (Table 4).

 

 

Table 4
The distribution of variants in groups for the length of beech seeds (Fagus sylvatica) from Plaiul Fagului-23, Republic of Moldova, under laboratory conditions after stratification and treatments with alginite and Juniperus sabina in 2024 (Multiple Range Test)

Variants / population

Length of roots (mm) and distribution of variants into groups based on the Method Tukey HSD for confidence levels

Coefficients of variation

L

α = 0.05 “+/- limits“ 9.21

α = 0.01 “+/- limits“ 10.73

α = 0.001 “+/- limits“ 11.87

1

2

3

4

1

2

3

4

5

1

2

3

4

Alginite powder 0.01%

36.06

×

×

×

18.94**

Control

39.66

×

×

×

×

×

19.24**

Alginite No 4 0.01%

41.56

×

×

×

×

×

×

×

×

21.31***

Alginite No 6 0.01%

43.21

×

×

×

×

×

×

×

×

×

×

17.71**

Alginite nano powder 0.01%

43.74

×

×

×

×

×

×

×

×

×

×

23.34***

Juniperus sabina 0.0001%

49.75

×

×

×

×

×

×

×

×

×

×

20.40***

Juniperus sabina 0.001%

52.05

×

×

×

×

×

×

×

22.26***

Juniperus sabina 0.0005%

52.42

×

×

×

×

×

×

18.89**

Alginite No 2 0.01%

56.04

×

×

×

17.21**

Note: “+/- limits” – confidence thresholds (critical difference values). For groups without significant differences (4–5 homogeneous groups), Multiple Range Tests (95% Tukey’s HSD) were used. In statistics:

• Coefficient of variation <10%: insignificant dispersion (*)
• 10–20%: average dispersion (**)
• 20–33%: significant dispersion (***)
• ≤33%: population is homogeneous; >33%: heterogeneous

 

According to the obtained coefficients of variation, the lowest degree of data dispersion occurred in variants treated with 0.01% alginite No. 2 (17.21%), 0.01% alginite No. 6 (17.71%), 0.0005% J. sabina (18.89%), 0.01% alginite powder (18.94%) and the control (19.24%); the coefficients of variation were less than 20% (average degree of data dispersion).

In the other variants, the coefficient of variation was 0.40–3.34% higher than the 20% level, which corresponded to a significant degree of data dispersion (Table 4).

Despite the lowest total seed germination of the 0.01% alginite No. 2 variant, the length of germinated roots of beech seeds was highest with the lowest coefficient of data dispersion compared to the control and other treatment variants treated with bioregulators of J. sabina and alginite solutions.

 

Study of the effect of bioregulators alginite and Juniperus sabina on the growth of Fagus sylvatica plants under solarium conditions

Germination was determined during stratification of Fagus sylvatica seeds from Plaiul Fagului-23 (No. 8 and No. 10), Republic of Moldova, sown under solarium conditions. Sprouting of germinated seeds after sowing began in the soil after 5 days. Seeds of Plaiul Fagului-23 (No. 8) origin had extremely low germination under solarium conditions. Even the seeds that germinated often did not shed the pericarp and did not open the cotyledons. A high seedling mortality was noted – more than 80% of the population. Observations showed that among the seeds that germinated during stratification in the Plaiul Fagului-23 No. 10 population, the proportion of seeds with infected cotyledons ranged from 35 to 45%. In the Plaiul Fagului-23 (No. 8) population from the Republic of Moldova, the highest percentage of seedling germination was noted in the variants 0.001% J. sabina (90.00%), 0.0005% J. sabina (91.11%), 0.0001% J. sabina (65.45%), 0.01% alginite nano powder (82.35%), 0.01% alginite No. 2 (61.90%) and 0.01% alginite No. 6 (92.50%). At the same time, 1.3–1.8-fold fewer seedlings emerged in the control (50.77%) than in the above-mentioned variants (Figure 5).

According to the analysis of means plot with 95% decision limits (Figure 7 a), the germination of beech seedlings was significantly higher in variants treated with 0.001 and 0.0005% J. sabina and 0.01% alginite nano powder and alginite No. 6 than in the control (Figure 5 a). According to the box-and-whisker plot (Figure 5 b), the germination of beech seedlings was significantly higher in variants treated with 0.001, 0.0005 and 0.0001% J. sabina and 0.01% alginite nano powder, alginite No. 2 and alginite No. 6 than in the control (Figure 5 a, b).

Statistical analysis of the data using the multiple range test (95.0% Tukey’s HSD) showed that the germination of beech seedlings was significantly lower in the 0.01% alginite No. 4 (36.73%) variant than in the control (50.77%). The germination of beech seedlings treated with alginite powder (48.65%) was similar to the control. All other variants of seed treatment with extracts from J. sabina and alginite solution showed seedling germination values that were significantly higher than that in the control. Moreover, the highest rate of germinated plants (90.00–92.50%) was noted in three variants: 0.001 and 0.0005% J. sabina and 0.01% alginite No. 6 (Table 5; Figure 5 a, b).

 

 

Table 5
Distribution of variants in groups for the germination of beech seedlings from Plaiul Fagului-23, Republic of Moldova, under solarium conditions and treatments with alginite and Juniperus sabina in 2024 (Multiple Range Test)

Variants	Distribution of variants into groups based on the Method Tukey HSD for confidence level α = 0.05, “+/- limits“ 4.28464						Coefficients of variation
	Germination of beech seedlings (%)	1	2	3	4	5
Alginite No. 4 0.01%	36.73	×					6.07*
Alginite powder 0.01%	48.65		×				4.48*
Control	50.77		×				2.99*
Alginite No. 2 0.01%	61.90			×			2.76*
Juniperus sabina 0.0001%	65.45			×			2.09*
Alginite nano powder 0.01%	82.35				×		2.20*
Juniperus sabina 0.001%	90.00					×	1.81*
Juniperus sabina 0.0005%	91.11					×	1.81*
Alginite No. 6 0.01%	92.50					×	2.07*

Note: “+/- limits” – confidence thresholds (critical difference values)

 

After 1 month of growth (from the first seedlings appeared), the height of plants of Plaiul Fagului-23 origin ranged from 2–3 to 5–7 cm. The heights of 3-month-old seedlings were 3–17 cm and reached 9.46 cm, on average (Table 6).

According to the statistical analysis using the multiple range test (95.0, 99.0 and 99.9% Tukey’s HSD), the shortest plants (7.33 cm on average) were found in the control at all three levels of significance (α = 0.05, 0.01 and 0.001). The tallest plants were found in the 0.0001% J. sabina and 0.01% alginite No. 2 treatments; their height averaged 12.24 and 13.50 cm, respectively, and was significantly higher than other variants, including the control, at all three significance levels (Table 6). The height of plants in the variant treated with 0.001% J. sabina was also significantly higher than in the control at all significance levels and reached an average of 10.08 cm. The coefficient of variation ranged from average to significant, and for the population as a whole, it was significant at 31.27% (Table 6).

Study of the effect of bioregulators on the growth of Fagus sylvatica plants under natural conditions

The germination of seeds or the emergence of beech seedlings under natural conditions means the appearance and blossoming of cotyledons. The overall emergence of seedlings and their length were determined 1 and 3 months after appearance. The seeds, after preliminary treatment with bioregulator solutions (Table 2), were sown under natural conditions in the last third of November 2023.

The first seedlings appeared in the last third of March 2024. The appearance of the first pairs of true leaves was observed in 10% of the seedlings in the first ten-day period of April 2024. The appearance of the second pair of true leaves was recorded in the last ten-day period of April 2024. The mass appearance of the first pairs of true leaves occurred in the last ten-day period of April 2024. An examination of seedlings with 1 or 2 pairs of true leaves, carried out 2 months after the first germination, showed that 85% of seedlings had one pair of leaves and 40% of seedlings had two pairs of leaves (Figure 6). The highest proportion of plants with 2 pairs of true leaves was noted in the variant treated with 0.001–42.0% gibberellic acid as well as 0.00001–30.0% alginite powder. The lowest proportion (18.0%) was noted in variants treated with 0.01–18.0% alginite No. 2. The remaining variants treated with 0.00001% solutions of alginite and 0.01% genistifolioside were similar to the control (Figure 6).

 

Table 6
Distribution of variants in groups based on the plant height of Fagus sylvatica from Plaiul Fagului-23, Republic of Moldova, under solarium conditions and treatments with alginite and Juniperus sabina in 2024 (Multiple Range Test)

Variants / population	Plant height (cm) and distribution of variants into groups based on the Method Tukey HSD for confidence level										Coefficients of variation
	Height (cm)	α = 0.05 “+/- limits“ 1.74			α = 0.01 “+/- limits“ 0.02			α = 0.001 “+/- limits“ 2.13
		1	2	3	1	2	3	1	2	3
Control	7.33	×			×			×			25.21***
Alginite powder 0.01%	8.00	×			×			×	×		30.17***
Alginite No. 6 0.01%	8.07	×			×	×		×	×		23.96***
Juniperus sabina 0.0005%	8.47	×	×		×	×		×	×		22.44***
Alginite nano powder 0.01%	8.63	×	×		×	×		×	×		17.61**
Alginite No 4 0.01%	8.83	×	×		×	×		×	×		23.67***
Juniperus sabina 0.001%	10.08		×			×			×		15.88**
Juniperus sabina 0.0001%	12.24			×			×			×	22.89***
Alginite No 2 0.01%	13.50			×			×			×	19.28**
Average	9.46										31.27***

Note: “+/- limits” – confidence thresholds (critical difference values)

 

 

The germination of Fagus sylvatica seeds of Plaiul Fagului-23 (No. 2) origin varied from 35.73 to 60.80%, depending on the variant of pre-sowing seed treatment with bioregulators, after stratification under natural conditions. The beech seeds treated with 0.00001% alginite nano powder and 0.00001% alginite No. 6 showed the highest germination rate (60.80 and 47.47%, respectively), which was higher than the control at all confidence levels according to statistical analysis carried out using the multiple range test (95.0, 99.0 and 99.9% Tukey’s HSD) (Table 7). The variants treated with 0.001% gibberellic acid, 0.01% genistifolioside and 0.00001% alginite No. 4 showed germination rates similar to the control at all confidence levels (Table 7). In total, 2656 of 6000 sown seeds germinated, an average of 44.27% of all sown seeds.

The tallest beech plants were noted in the variants treated with aqueous extracts of 0.00001% alginite No. 6 and 0.001% gibberellic acid under natural conditions one month after emergence (16.03 and 16.05 cm, respectively). The average plant height one month after seedling emergence was 15.27 cm.

The height of plants determined 3 months after emergence varied from 23.40 to 29.70 cm, depending on the variant. According to statistical analysis carried out using the multiple range test (95.0, 99.0 and 99.9% Tukey’s HSD), the tallest plants were noted in variants treated with 0.01% genistifolioside, 0.001% gibberellic acid and 0.00001% alginite nano powder; their heights were 29.70, 28.22 and 28.10 cm, respectively, exceeding the height of the control plants at all confidence levels (Table 8).

The height of plants in variants treated with 0.00001% alginite No. 6 (27.85 cm) and 0.00001% alginite powder (26.48 cm) also exceeded the control (23.40 cm) at all significance levels, whereas the height in variant 0.00001% alginite No. 4 (25.80 cm) exceeded the control plants only at a confidence level of 0.05.

 

Table 7
Distribution of variants in groups for the germination of beech seedlings from Plaiul Fagului-23 (Republic of Moldova) under natural conditions and treated with bioregulators (2024) (Multiple Range Test)

Variants

Germination (%)

Distribution of variants into groups based on the Method Tukey HSD for confidence levels

Coefficients of variation (%)

α = 0.05 “+/- limits “ 1.34768

α = 0.01 “+/- limits “ 1.67329

α = 0.001 “+/- limits “ 1.90616

1

2

3

4

5

6

1

2

3

4

5

1

2

3

4

5

Alginite No 2 0.00001%

35.33

×

×

×

1.73*

Alginite powder 0.00001%

39.47

×

×

×

0.59*

Alginite No 4 0.00001%

42.13

×

×

×

0.55*

Genistifolioside 0.01%

42.13

×

×

×

1.09*

Control

42.93

×

×

×

×

0.54*

Gibberellic acid 0.001%

43.73

×

×

×

1.39*

Alginite No 6 0.00001%

47.47

×

×

×

0.49*

Alginite nano powder 0.00001%

60.80

×

×

×

1.32*

Note: “+/- limits” – confidence thresholds (critical difference values).

 

Table 8
Distribution of variants in groups for the height of beech seedlings from Plaiul Fagului-23 (Republic of Moldova) under natural conditions and treated with bioregulators (2024) (Multiple Range Test)

Variants

Height (cm)

Distribution of variants into groups based on the Method Tukey HSD for confidence levels

Coefficients of variation (%)

α = 0.05+/- limits 2.29102

α = 0.01+/- limits 2.67021

α = 0.001+/- limits 2.85375

1

2

3

4

5

1

2

3

4

1

2

3

4

Control

23.40

×

×

×

21.35***

Alginite No 2 0.00001%

24.23

×

×

×

×

×

×

15.52**

Alginite No 4 0.00001%

25.80

×

×

×

×

×

×

×

×

15.24**

Alginite powder 0.00001%

26.48

×

×

×

×

×

×

×

16.99**

Alginite No 6 0.00001%

27.85

×

×

×

×

×

×

×

13.25**

Alginite nano powder 0.00001%

28.10

×

×

×

×

×

×

10.08**

Gibberellic acid 0.001%

28.22

×

×

×

×

×

×

17.94**

Genistifolioside 0.01%

29.70

×

×

×

12.53**

Note: “+/- limits” – confidence thresholds (critical difference values).

 

The plant height determined 3 months after seedling appearance in variant 0.00001% alginite No. 2 (24.23 cm) was similar to the control at all confidence levels (Table 8).

The chlorophyll index of the first pair of true leaves of beech seedlings determined 1 month after emergence under natural conditions ranged from 17.88±4.13 to 21.54±3.98 g/dm³. Statistical analysis of the data using the analysis of means plot with 95% decision limits showed that the chlorophyll index in two variants, 0.00001% alginite No. 2 and 0.00001% alginite nano powder, was 21.54±3.98 and 21.48±3.07 g/dm³ respectively, and exceeded the values in both the control (19.29±4.83 g/dm³) and the variant treated with gibberellin (17.98±2.54 g/dm³). The chlorophyll index in variants treated with 0.00001% alginite No. 4 (19.60±3.08 g/dm³), 0.00001% alginite No. 6 (20.72±2.92 g/dm³) and 0.00001% alginite powder (19.33±1.53 g/dm³) was at the control level and significantly exceeded the values in the gibberellic acid variant (Figure 7).

 

DISCUSSION

Beech seeds from the three studied populations, collected in 2023 from the Plaiul Fagului Nature Reserve at an altitude of 200–310 m above sea level from trees aged 70–115 years, were characterised by a fairly high weight of 298.9–353.2 g / 1000 seeds, which amounted to an average of 326.4 g (1000 seeds).

The seeds of Serbian populations collected in 2004 in Avala, Fruska Gora, Ger and Boranja were also characterised by a high weight (241.14–336.54 g / 1000 seeds, and an average of 299.38 g) (Šijačić-Nikolić et al., 2007). The weight of 1000 seeds, was significantly lower in the other beech populations described in scientific publications. For example, seeds collected in Poland from trees aged 105–130 years (7 locations) varied from 185.7 to 217.2 g (Kaliniewicz et al., 2018). Seeds of 6 populations collected in 2007 in Bosnia and Herzegovina weighed from 143.2 (Bosansko Grahovo) to 274.8 g (Banja Luka) per 1000 seeds (Mekić et al., 2010). Seeds collected in 2001 and 2003 from two regions in the Czech Republic (Stredomoravske Karpaty and Luzicka piskovcova vrchovina, at an altitude of 400–550 and 600–700 m above sea level) after storage in sealed plastic bags at –7°C for 2–4 years at the Tree Seed Center in Tyniste with a moisture content of 8.6–9.0% ranged from 241.4 to 277.4 g (Kolařova et al., 2010).

The average seed viability of Plaiul Fagului-23 populations from the Republic of Moldova (PF-23) reached 85.37%. Thus, given the high level of seed viability and weight, and therefore the size, the PF-23 populations had a high germination potential. However, the germination of seeds collected in Plaiul Fagului in autumn 2023 was observed only 70 days after stratification under laboratory conditions. The germination of seeds collected in Plaiul Fagului in 2020 was observed 27 days after stratification (Elisovetcaia et al., 2023). These circumstances may indicate a state of deep dormancy in beech seeds of the PF-23 populations.

It was previously established (Elisovetcaia et al., 2022) that the bioregulators capsicoside and genistifolioside at a concentration of 0.01% had a positive effect on the germination of beech seeds originating from Slovakia (Nitra-19) and the Republic of Moldova (Cioresti-21) during stratification under laboratory conditions. Bioregulators genistifolioside and capsicoside increase the rate of seed germination and seedling adaptation in beech from Slovakia. The rate of emergence of beech seedlings of Cioresti-21 (Republic of Moldova) origin under solarium conditions in the variants treated with genistifolioside and capsicoside was 1.5–2.0 times higher than that in the control. The total number of adapted beech seedlings of Cioresti-21 origin was 82.79±4.32 and 81.41±3.75% in variants treated with genistifolioside and capsicoside, respectively, and significantly higher than that in the control (72.63±4.75%) and in the standard of 0.01% gibberellic acid (80.23±4.25%) (Elisovetcaia et al., 2022).

The use of aqueous solutions of an alcoholic extract of J. sabina as a growth regulator to increase beech seed germination and seedling adaptation showed the potential of the extract as a stimulator of germination, increasing the adaptive properties of beech plants of PF-23 origin.

Thus, the germination of PF-23 beech seeds in the variants treated with 0.0001 and 0.0005% J. sabina extracts was so strong that 98 day after stratification under laboratory conditions, it numerically exceeded the control by 1.49 and 2.79 times, respectively. The root length of germinated seeds in these variants also significantly (according to the multiple range test) exceeded the control.

The variants treated with 0.001 and 0.0005% J. sabina had a high proportion of germinated seedlings under solarium conditions (90.00 and 91.11%, respectively), which was 1.77 times higher than that in the control.

 

 

Our data on the application of J. sabina extract to stimulate seed germination are in good agreement with our previously obtained results on the use of bioregulators genistifolioside and capsicoside (Moldstim) for the germination of beech seeds of Nitra-19 from Slovakia, Cioresti-21 from the Republic of Moldova and Codrii-20 from the Republic of Moldova (Elisovetcaia et al., 2022). For example, the best results of an increase in daily germination (up to 18.5%) were obtained under the influences of capsicoside and genistifolioside at a concentration of 0.001%. Under the influence of the preparations capsicoside and genistifolioside at a concentration of 0.001%, the germination time of total beech seeds of Nitra-19 origin was reduced by 20–22 days. Foliar treatment of beech seedlings with preparations under solarium conditions led to an increase in the leaf blade by 1.7–2.9 cm in length and 0.3–1.3 cm in width compared with the control. The relative chlorophyll index determined in the phase of three pairs of true leaves on seedlings treated with capsicoside and genistifolioside significantly exceeded that of the control by 9.2–24.1 g/m². For the beech population of Cioresti-21 origin, at 114 day after stratification, the largest number of germinated seeds was noted in the variants treated with genistifolioside (82.8%) and capsicoside (80.5%), which was significantly higher than in the control (73.8%) and the standard (76.8%). Genistifolioside (0.001%) also increased the germination of seeds of Codrii-20 origin by 11.2% compared to the control, while significantly reducing the proportion of seed infection during germination by 1.84–8.5% compared to the control and treatment with 0.01% gibberellic acid.

Various preparative forms of alginite have been previously used to improve the germination of tomato, corn, Poa pratensis, medicinal plants, and some other crop seeds (Horčinová Sedláčková et al., 2021). Alginite is widely used as a mineral supplement to improve rooting (Brindza et al., 2021). In this study, the aqueous solutions of alginite (0.00001–0.001%) also showed stimulating properties, reducing germination time and increasing overall germination, depending on the formulation and concentration. For example, the seed germination 98 days after stratification under laboratory conditions in variants treated with 0.01% alginite nano powder, 0.01% alginite No. 2 and 0.01% alginite No. 4 exceeded the control by 1.28-1.37 times. However, the root length of germinated seeds was significantly higher than that of the control (1.55 times) in the variant treated with 0.01% alginite No. 2 0.01%. Under solarium conditions, among the alginite treatment variants, the most effective were 0.01% alginite nano powder and 0.01% alginite No. 6; seedling germination was 1.62–1.82 times higher than in the control. However, the tallest seedlings under solarium conditions were noted in the 0.01% alginite No. 2 treatment (1.84 times higher than in the control). Under natural conditions, the highest germination was also observed in the alginite nano powder and alginite No. 6 variants, while the concentration of the preparations was 1000 times lower and amounted to 0.00001%. The height of beech seedlings under natural conditions in the alginite No. 6 seed treatment variant was one of the highest and similar to the 0.01% genistifolioside and 0.001% gibberellic acid variants.

Overall, our studies showed that the proportion of plants obtained from seeds under natural conditions was higher than those under laboratory stratification and solarium conditions. Our data are supported by the findings of other studies showing slow seed germination under natural conditions with treatments increasing the proportion of seed germination and seedling adaptation, with a balance between the required signalling events for germination and oxidative damage caused by abiotic and biotic factors during imbibition (Sharma et al., 2020).

The PF-23 population showed generally higher values for the chlorophyll index (from 17.88±4.13 to 21.54±3.98 g/dm³), which was determined in the phase of three pairs of true leaves, compared to the population obtained in 2020 from the seeds of Nitra-19 from the Slovak Republic. The chlorophyll index for the Slovak populations in the variants treated with 0.001% capsicoside and genistifolioside reached 144.9±3.5 and 145.6±3.7 g/m², respectively, which significantly exceeded the control (135.7±3.2 g/m²) and the variant with gibberellic acid (121.5±2.9 g/m²) (Elisovetcaia et al., 2022). At the same time, when treated with 0.00001% alginite No. 2 and 0.00001% alginite nano powder in our study, the chlorophyll index reached 21.54±3.98 and 21.48±3.07 g/dm³ respectively, which was 1.48 times higher than the values in the Nitra-19 population.

Vannini et al. (2022) examined whether the seed germination and growth of Fagus sylvatica L. and Quercus cerris L. species were affected by the addition of biochar (BC) obtained from deciduous broadleaf trees. They showed that BC amendments did not affect either germination time or germination percentage for either investigated plant species (p > 0.05). Plants of both species grown with BC showed a higher chlorophyll content when compared to the controls (p < 0.05), with no differences detected between the two amendments (p > 0.05). In contrast, no significant influence of BC was recorded for the functionality of photosystem II (FV/FM; p > 0.05).

Research on bioregulators of J. sabina and alginite should be continued to identify general trends in relation to beech seeds of different origins as well as to select concentrations and preparations that have maximum efficiency. Seed treatment before stratification should also be combined with foliar treatment during the growing season.

 

CONCLUSIONS

The application of bioregulators, such as Juniperus sabina extract and aqueous solutions from alginite, for seed treatment before stratification under controlled laboratory conditions and before sowing under natural conditions clearly stimulated germination compared to the control, confirming our assumptions.

Under laboratory conditions, the germination rate and total germination of beech seeds improved, and the root length increased compared to the control. The adaptive properties of seedlings were enhanced.

Under natural conditions, some alginite preparations improved seed germination and significantly increased the chlorophyll index not only in comparison to the control but also in comparison to treatment with gibberellic acid (0.001%).

All alginite preparations significantly stimulated plant growth compared to the control and was similar to the gibberellic acid treatment variant under natural conditions. The biopreparation genistifolioside, previously proven to be a stimulator of beech seed germination and seedling adaptation under solarium and natural conditions, demonstrated the highest seedling growth rate under natural conditions.

 

Author contributions: Conceptualization: DE, RI, JB; Methodology: DE, RI; Analysis: DE, RI, JB; Investigation: DE, EP; Resources: DE, RI, JB; Data curation: DE, RI; Writing: DE; Supervision: JB. All authors declare that they have read and approved the publication of the manuscript in this present form.

Funding: Research was carried out within the bilateral scholarship program “Changes in the mor-pho-physiological features of cultivated plants under the influence of growth bioregulators of mineral and or-ganic origin”, Elisovetcaia Dina, PhD, for 2024-2025 financed by Ministry of Education, Science, Research and Sport of the Slovak Republic (https://oblak.iedu.sk/index.php/s/ooFeFygFn3BoRWp?dir=undefined&openfile=1313967) and within the Subprogramme 011101 “Genetic and biotechnological approaches to agroecosystem management under cli-mate change conditions”, financed by the Ministry of Education and Research (MEC) of the Republic of Moldova.

Acknowledgments: The authors thank the management of the “Plaiul Fagului” Natural Reserve for the orga-nized collection of beech seeds in the autumn 2023. The authors express their gratitude to Mascenco N.E. for the provided bioregulator genistifolioside. The authors also thank Nikolae Platovschi for assistance in deter-mining the chlorophyll indices.

Conflicts of interest: There are no conflicts of interest regarding this article. 

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Academic Editor: Dr. Iuliana MOTRESCU

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