The effect of the Mind Agro.Bio stimulator on the growth and regeneration of isolated sunflower cotyledons
The aim of our study is to investigate the effect of the drug Mind Agro.Bio on the growth and regeneration processes modeled in the culture of isolated cotyledons of dicotyledons.
RESEARCH METHODS
Isolated cotyledons of the Armavirsky 3497 sunflower variety were cultivated using a standard method approved by our researchers. The isolates were placed in sterile Petri dishes on filter paper soaked in 10 ml of Mind Agro.Bio solutions at concentrations of 1 mg/L and 10 mg/L, and placed in a dark incubator at a temperature of 26°±1°C. The dishes were ventilated once daily. Light exposure was 30 minutes.
Growth processes in the cotyledons were assessed based on changes in their fresh weight and area. Area was determined using a generally accepted modern method. Epidermal cell size and stomatal guard cell size were measured using an ocular micrometer on hand-cut sections embedded in glycerol-gelatin. Fat content was determined from the defatted residue.
RESULTS OF THE STUDY AND THEIR DISCUSSION
Analysis of the experiments revealed an S-shaped growth pattern of cotyledons based on fresh weight in all treatments. The cotyledon isolated from the rest of the embryo had a fresh weight of 43.77 mg. Over the next fifteen days, its fresh weight in the control increased by 4.1 times, while that of cotyledons cultivated in Mind Agro.Bio solutions with a concentration of 1 mg/L and 10 mg/L increased by 5.5 and 6.8 times, respectively. Thus, the most significant increase in cotyledon weight was observed with the 10 mg/L stimulant solution. The average cotyledon fresh weight in this treatment on the fifteenth day of cultivation was 71.4% higher than in the control.
As the graph shows, the water-based variant accumulates fresh weight at a slower rate than the variants containing the product. The most vigorous growth was observed in isolates grown in a Mind Agro.Bio solution at a concentration of 10 mg/L. In this case, fresh weight accumulation is expressed by the steepest curve.
The dry weight of cotyledons in the water and Mind Agro.Bio 1 mg/L treatments initially decreases, followed by a slight increase. In the control, the change in cotyledon dry weight during cultivation is graphically expressed by a curve with a maximum on the fourth day. In the 1 mg/L treatment, dry weight increases by the sixth day, and this increase is more significant than in the control.
In isolation plants grown in a 10 mg/L Mind Agro.Bio solution , dry matter accumulation is less pronounced than in the 1 mg/L solution. However, in the 10 mg/L solution, no drop in cotyledon dry weight is observed in the first days of cultivation. Dry weight begins to increase after just one day of cultivation, and it changes only slightly over the next six days. It then declines, as in the first two solutions. However, it should be noted that this decline is less intense, and by day 15, cotyledon dry weight is higher than in the other solutions.
The temporary increase in cotyledon dry matter may be due to the fact that photosynthesis continues even with such low light exposure, as greening of the isolants is observed. The degree of change in cotyledon dry matter depends on the ratio of its synthesis during light exposure under the experimental conditions to the costs of growth processes.
The earliest and most intense cotyledon greening occurs with the Mind Agro.Bio 10 mg/L treatment. This also delays cotyledon aging. While signs of yellowing of the cotyledons are observed as early as 15 days into cultivation in the control treatment, the cotyledons with the 10 mg/L treatment remain green even after a month of cultivation.
When converting dry matter to 1000 mg of wet weight, the following pattern is observed (Table 1). As the isolates were cultivated, this indicator dropped by 7.6 times over 15 days in the control, and by 7.5 and 8.0 times, respectively, in the 1 mg/L and 10 mg/L stimulant treatments. Moreover, the most intense weight loss was observed in the 1 mg/L treatment. By day 15, weight loss was approximately equal in all treatments.
Table 1. Dynamics of dry weight of cotyledons cultivated in water and Mind Agro.Bio solutions, calculated per 1000 mg of fresh weight
| Days of cultivation | Water | Mind Agro.Bio 1 mg/l | Mind Agro.Bio 10 mg/l |
|---|---|---|---|
| 0 | 789.7 | 789.7 | 789.7 |
| 1 | 616.8 | 599.8 | 601.0 |
| 2 | 503.9 | 439.4 | 440.5 |
| 4 | 301.3 | 389.2 | 298.9 |
| 6 | 209.5 | 226.3 | 210.1 |
| 10 | 151.6 | 118.8 | 151.6 |
| 15 | 103.3 | 106.7 | 96.9 |
Along with the decrease in dry matter content in the cotyledons, the percentage of water increases. In the experimental treatments ( Mind Agro.Bio 1 mg/L and 10 mg/L), this increase is more significant. It can be assumed that this increase in water content is caused by more intense cell elongation in the isolates of these treatments.
Dry weight loss correlates with the loss of fat, the main reserve nutrient in sunflower cotyledons. A decrease in fat content was observed in all variants during the cultivation of isolated sunflowers (Table 2).
Table 2. The influence of Mind Agro.Bio on the dynamics of lipids in sunflower cotyledons during their cultivation (in % of dry weight)
| Days | Water | Mind 1 mg/L | % to control | Mind 10 mg/L | % to control |
|---|---|---|---|---|---|
| 1 | 60.03±1.10 | 59.85±0.92 | 99.7 | 60.18±0.96 | 102.52 |
| 2 | 58.45±0.42 | 56.50±1.91 | 96.66 | 58.52±0.46 | 100.13 |
| 4 | 56.63±0.07 | 53.01±0.86 | 93.6 | 54.44±1.41 | 96.32 |
| 5 | 55.25±0.12 | 39.30±1.39 | 71.11 | 57.75±1.40 | 91.85 |
| 7 | 52.75±0.96 | 35.95±1.08 | 68.15 | 50.38±0.72 | 95.57 |
| 10 | 50.60±1.80 | 33.73±1.15 | 66.65 | 47.23±0.84 | 93.24 |
| 12 | 40.33±1.05 | 30.77±0.98 | 76.29 | 37.35±1.42 | 92.61 |
| 15 | 37.33±1.48 | 28.93±1.14 | 74.82 | 35.95±1.43 | 96.30 |
As can be seen from the table, the most intensive reduction in fat is observed in the variant with a stimulant at a concentration of 1 mg/l, and the least significant consumption is in the control.
Cotyledon growth is measured by changes in their area. In the experiments with water and 1 mg/L of the preparation, the area increases up to 15 days of cultivation, while in the experiment with 10 mg/L of Mind Agro.Bio , it increases up to 20 days (Table 3).
An increase in area was observed in all treatments by 4.2, 4.8, and 10.1 times, respectively. The preparation, especially at a concentration of 10 mg/L, contributed to a more significant increase in the isolated cotyledon compared to the control. On the 20th day of cultivation, the cotyledon area in this treatment was 209.2% greater than in the control.
Table 3. Change in the area of isolated sunflower cotyledons under the influence of Mind Agro.Bio, cm²
| Days | Water | Mind 1 mg/L | % to control | Mind 10 mg/L | % to control |
|---|---|---|---|---|---|
| 0 | 0.38±0.02 | 0.38±0.02 | 100 | 0.38±0.02 | 100 |
| 1 | 0.39±0.03 | 0.43±0.01 | 110.28 | 0.52±0.02 | 133.33 |
| 2 | 0.48±0.02 | 0.73±0.08 | 150.20 | 0.56±0.03 | 116.66 |
| 3 | 0.77±0.11 | 1.11±0.10 | 144.14 | 1.25±0.07 | 162.32 |
| 4 | 0.88±0.08 | 1.38±0.12 | 156.81 | 1.65±0.05 | 198.87 |
| 6 | 0.98±0.12 | 1.57±0.04 | 160.20 | 2.12±0.17 | 216.30 |
| 10 | 1.49±0.09 | 2.06±0.09 | 134.90 | 2.82±0.15 | 189.21 |
| 15 | 1.58±0.25 | 2.57±0.18 | 162.68 | 3.85±0.20 | 243.73 |
| 20 | 1.52±0.16 | 2.58±0.47 | 170.50 | 4.70±0.50 | 309.20 |
It is characteristic that the increase in area is more rapid than the accumulation of fresh weight. Thus, compared to the control, fresh weight increased by 39.5% at a concentration of 1 mg/L and by 80.3% at a concentration of 10 mg/L on the 15th day of cultivation. Meanwhile, area increased by 62.7% and 143.7% during this period. This is due to the fact that cotyledons grown on the stimulant are thinner in the dorsoventral direction. The fresh and dry weight per unit area of cotyledons on the 15th day will be highest in the water-treated variant and lowest in the 10 mg/L variant.
Given such a significant increase in area in isolated plants, especially under the influence of our stimulant, studying cell size is of considerable interest. Cotyledon epidermal cells increase in size as they are cultured (Table 4).
Table 4. Sizes of epidermal cells in isolated sunflower cotyledons (in divisions of a 40x10 ocular micrometer)
| Day | Water (width) | Water (length) | Mind 1 mg/L (width) | Mind 1 mg/L (length) | Mind 10 mg/l (width) | Mind 10 mg/L (length) |
|---|---|---|---|---|---|---|
| 1 | 5.50±0.17 | 7.10±0.11 | 6.56±0.24 | 7.56±0.12 | 5.91±0.20 | 7.63±0.14 |
| 20 | 10.80±0.25 | 14.28±0.40 | 11.37±0.11 | 15.01±0.16 | 14.36±0.40 | 21.11±0.75 |
The most significant increase in cell size was observed in the Mind Agro.Bio variant at a concentration of 10 mg/L. In this experiment, cell width increased 2.4-fold and length 2.7-fold on day 20 of cultivation, compared to day 1. In the control, cell width increased 2.0-fold and length 1.8-fold.
The change in the size of the guard cells of the stomata does not differ significantly between the variants (Table 5).
Table 5. Sizes of stomatal cells of isolated cotyledons (in divisions of an ocular micrometer 40x10)
| Day | Water (length) | Water (width) | Mind 1 mg/L (length) | Mind 1 mg/L (width) | Mind 10 mg/L (length) | Mind 10 mg/l (width) |
|---|---|---|---|---|---|---|
| 20 | 8.42±0.08 | 7.19±0.11 | 8.91±0.21 | 7.39±0.09 | 9.25±0.14 | 8.20±0.07 |
A study of the number of stomata per field of view at 40x10 magnification showed that their number did not differ significantly between the control and the 1 mg/L stimulant treatment. A decrease in the number of stomata per field of view was observed in the 10 mg/L treatment. This is likely due to significant elongation of the epidermal cells (Table 6).
Table 6. Number of stomata in the field of view of the microscope (10x40)
| Day | Water | Mind 1 mg/L | Mind 10 mg/L |
|---|---|---|---|
| 20 | 14.55±0.12 | 12.10±0.95 | 7.98±1.12 |
It is characteristic that in cotyledons cultivated in a 10 mg/l solution of the preparation, the stomatal slit is open wider than in other variants, which is consistent with modern physiological data.
The effect of Mind Agro.Bio on root formation in isolated plants was as follows. During the first days of cultivation, adventitious roots formed more vigorously in the control. Thus, by the sixth day in the experiment with 1 mg/L of the product, only 51.3% of the rooted cotyledons in the control regenerated. By the tenth day, the percentage of regenerating cotyledons leveled off. Characteristically, this leveling off occurs after the period of the steepest increase in cotyledon growth has passed. Apparently, the more intense cotyledon growth in the 1 mg/L treatment inhibits root formation. However, by the 20th day, the number of adventitious roots in the 1 mg/L treatment is higher than in the control.
In isolates cultivated in a solution at a concentration of 10 mg/l, root formation was not observed at all (Table 7).
Table 7. Effect of Mind Agro.Bio on root regeneration in isolated cotyledons, %
| Day | Water | Mind 1 mg/L | Mind 10 mg/L |
|---|---|---|---|
| 6 | 18.07±0.20 | 9.37±0.43 | — |
| 10 | 43.84±1.15 | 41.56±1.36 | — |
| 20 | 82.33±4.31 | 91.33±3.18 | — |
In an experiment where, along with intensive cotyledon growth, root formation is also observed, a more vigorous loss of fats occurs.
Data on the effect of the stimulator on root growth are presented in Table 8.
Table 8. The effect of Mind Agro.Bio on the length and number of lateral roots in isolates
| Option | Length of the main root, mm (by stage of development: I, II, III, IV) | Average root length per cotyledon, mm | Number of lateral roots per 1 cotyledon |
|---|---|---|---|
| Water | 66.66±0.62; 49.30±0.15; 41.19±0.15; 41.31±0.65; 22.15±0.55 | 36.23 | 65.91±1.25 |
| Mind 1 mg/L | 23.46±0.41; 14.92±0.26; 16.57±0.38 | 32.66 | 82.80±1.73 |
The most significant differences in length are observed in the roots that developed first in the isolated plants. When roots developed later (after 10 days), the lengths of the different treatments are virtually identical. The average root length is one cotyledon longer in the control.
Thus, the stimulator inhibits the growth of the first roots to form in isolated plants. Modern research confirms the suppression of high concentrations of phytohormones in the primary root growth. Regarding the number of lateral roots, the Mind Agro.Bio formulation has shown some stimulation of their formation.
CONCLUSIONS
- Mind Agro.Bio at concentrations of 1 mg/L and 10 mg/L promotes more intensive growth of isolated sunflower cotyledons. This is indicated by an increase in the fresh weight and area of the isolated cotyledons.
- The most significant effect on cotyledon growth is achieved with a stimulator at a concentration of 10 mg/L. It increases not only the rate of growth in the isolated seeds but also their lifespan.
- Mind Agro.Bio at a concentration of 10 mg/l suppresses root formation, and at a concentration of 1 mg/l slightly reduces its intensity during the period of the most rapid growth of cotyledons and increases the total percentage of adventitious roots on the 20th day of cultivation.
- The stimulator at a concentration of 1 mg/l inhibits the growth of roots that are the first to form in isolated plants.
- The increase in cotyledon area correlates with the increase in the size of epidermal cells.
Agro.Bio Expert Note: Agro.Bio specialists continue to develop the area described in the article using advanced biotechnology. Today, the Mind line of products is successfully used in Ukraine's agricultural sector for targeted growth regulation, increased germination energy, and maximum plant genetic potential.