Influence of Humic and Fulvic Acid and pH on the Anatomical Structure of the Embryonic Root of Apple during Stratification

During the stratification of seeds, the embryo undergoes complex biochemical, physiological, structural, and other transformations that affect germination, further growth, and plant development.

The complex processes occurring in seeds are influenced by physiologically active substances. One such substance, as numerous studies have shown, is humic and fulvic acid. Water-soluble humic substances in anaerobic conditions promote seed germination. Even old seeds that would not germinate under normal conditions gained viability under the influence of humic substances. This prompted us to study the effect of humic acid on the anatomical structure of the apple seed embryo under different environmental pH conditions during stratification.

This issue has not been sufficiently studied to date.

Research Methodology The experiment used seeds of wild apple (Malus silvestris), obtained from the Novomoskovsk Forestry in the Dnipropetrovsk region, collected from mother trees in the early fourth age period. Fruits were selected from the western and southwestern sides of the upper parts of the crown, in a stage close to full biological ripeness, of uniform size with an average weight of 43–46 g. After extraction from the fruits, the seeds were divided into three fractions. For stratification and further studies, carefully selected seeds of the large fraction were used, distinguished by high uniformity in shape and color. The seeds had the following average characteristics: weight of 1000 seeds — 34.66 g; length — 8.6 mm; width — 4.9 mm; thickness — 2.3 mm. Seeds were stratified in washed and calcined sand in polyethylene bags. At the time of placing the seeds for stratification, a solution of potassium humate Agro.Bio of a specific pH and concentration was added to the bags. Humate was applied in the form of dialyzed potassium humate at concentrations of 0.001%, 0.01%, 0.1%, and 0.5% at pH levels of 4.94, 5.91, 6.98, and 8.04 in a Sørensen III buffer mixture. The control group consisted of all pH levels without humic acid. In a refrigeration unit, a temperature of +5°C was maintained for 70 days of stratification, then reduced to +1°C and maintained until the end of stratification.

On the 122nd day of stratification, to determine germination energy, seeds were sown on filter paper in Petri dishes, and for studying growth intensity — in vegetative vessels with soil culture. For anatomical studies on the 70th and 90th days of stratification, 10 apple seeds from each variant were selected, peeled to the cotyledons, and fixed in Navashin's mixture. Material fixation, washing, passage through a series of alcohols and an intermediate liquid, embedding in paraffin, cutting on a microtome, and mounting sections on slides were performed using standard methods. The average thickness of the obtained sections was 5–7 µm. Staining of the preparations was done with hematoxylin and an aqueous eosin solution using the Ganzin method, followed by embedding in polystyrene. In series of cross-sections made on the 70th and 90th days of stratification, measurements of the tissues of the embryonic root and cells by length and width at the root base were conducted at 300x magnification using Yurtsev’s method. Numerical values of anatomical changes represent the average of 50 measurements.

Research Results

When studying the effect of humic and fulvic acid on the anatomical structure of the embryonic root of apple, significant interest lies in identifying the degree of tissue differentiation, particularly of primary phloem and xylem, which ensure the transport of synthesized organic substances and mineral compounds. Additionally, it was important to investigate the effect of potassium humate Agro.Bio on cell sizes. There are only isolated reports in the literature regarding the degree of tissue differentiation under the influence of humic and fulvic acids. The effect of humic and fulvic acids on cell sizes has been demonstrated in studies by several authors. The anatomical structure of roots, stems, and leaves of tomatoes, sugar beets, and spring wheat shows that humic and fulvic acids have the greatest impact on the root system, followed by the leaf, and finally the stem. In these experiments, humic and fulvic acids increased the length of root cortex cells by 78–180%.

Water-soluble humic substances, when in direct contact with the root tissues of Sinapis alba, cause rapid cell division and also affect their elongation. Our studies on the influence of humic and fulvic acid at different environmental pH levels on the size of parenchymatous cells and differentiation of the central cylinder of the embryonic root of apple seeds revealed a general pattern of cell size increase under their influence (Table 1). Cell elongation occurs in the epiblem, exodermis, primary cortex parenchyma, endodermis, pericycle, and central cylinder of the embryonic root. The largest sizes of parenchymatous cells of the primary cortex and central cylinder were observed at humic acid concentrations of 0.1% and 0.01% and a pH of 6.98. Humic and fulvic acids also had a significant impact on the differentiation of central cylinder cells. Without humic acid, on the 90th day of stratification, only primary phloem is clearly visible in the cross-section of the embryonic root, while in the variant with 0.1% humic acid, as early as the 70th day, in addition to this tissue, the pericycle and vessels are outlined, and by the 90th day of stratification, they are clearly visible, especially at a pH of 6.98 (Fig. 1). Without humic acid, parenchymatous cells are smaller in size. Table 1. Effect of humic and fulvic acid on the size of parenchymatous cells and differentiation of the central cylinder

Concentration of humic and fulvic acid, %	Primary cortex parenchyma cells, µm	Central cylinder parenchyma cells, µm	Differentiation in the central cylinder
	70th day Length	90th day Width	70th day Length	90th day Width	70th day	90th day
pH 4.94
Control (without humic acid)	20	20	23	23	None	None
0.001	23	23	27	24	None	None
0.01	30	27	33	20	None	None
0.1	35	26	37	25	None	Emerging
0.5	36	30	27	27	Cell deformation	Cell deformation
pH 5.91
Control (without humic acid)	20	19	23	23	None	None
0.001	23	23	27	24	None	None
0.01	30	27	33	20	None	None
0.1	35	26	37	25	None	Emerging
0.5	36	30	27	27	Cell deformation	Cell deformation
pH 6.98
Control (without humic acid)	20	19	26	25	None	None
0.001	26	25	33	30	None	Emerging
0.01	30	27	36	30	None	Present
0.1	39	30	40	32	Emerging	Present
0.5	40	24	30	27	Cell deformation	Cell deformation
pH 8.04
Control (without humic acid)	19	19	25	24	None	None
0.001	25	24	30	29	None	Emerging
0.01	27	24	33	24	None	Emerging
0.1	33	27	35	29	Emerging	Present
0.5	36	24	27	24	Cell deformation	Cell deformation

Acidification and alkalinization of the environment, as well as a decrease in the concentration of humic and fulvic acid, have a less favorable effect on cell development. A concentration of 0.5% humic acid is clearly too high and leads to pathological cell changes: in many cases, cell deformation, structural changes, or excessive elongation were observed. The degree of differentiation and development of primary xylem and phloem are of significant importance for increasing seed germination energy and seedling growth. Experimental data indicate a significant increase in the areas of conductive tissues of the embryonic root of apple and their differentiation under the influence of humic and fulvic acid (Table 2). The energy of primary phloem and xylem formation varies depending on stratification conditions. Thus, on the 70th day, the area of primary phloem and xylem relative to the total section area is: without humic acid — 3.7–6.3% (at different pH levels), and with humic acid — 5.4–13.6%; on the 90th day — 8.4–14% and 12–23.8%, respectively. The highest degree of differentiation of primary phloem and xylem was observed at humic acid concentrations of 0.1–0.01% and a pH of 6.98. Comparison of anatomical data, germination energy, and, as further studies showed, subsequent seedling growth rates led us to conclude that the degree of differentiation of conductive tissues is, alongside biochemical changes, an important indicator of seed readiness for germination. Table 2. Anatomical changes in the embryonic root of wild apple under the influence of potassium humate Agro.Bio

Concentration of humic acid, %	Tissue area, thousand µm²	Percentage of total section area, %
	Primary cortex	Phloem and xylem	70th day	90th day
pH 4.94
Control (without humic acid)	560.3	522.5	3.8	9.4
0.001	578.6	497.4	5.0	12.8
0.01	560.4	565.6	6.4	12.0
0.1	569.7	462.4	7.5	18.9
0.5	593.5	536.3	7.5	6.4
pH 5.91
Control (without humic acid)	530.4	469.4	6.3	14.0
0.001	584.0	504.9	7.4	14.3
0.01	514.3	462.4	10.6	18.9
0.1	568.3	425.5	9.3	22.4
0.5	---	---	Cell deformation	---
pH 6.98
Control (without humic acid)	578.9	496.2	6.1	12.3
0.001	568.3	512.4	7.7	14.9
0.01	588.2	431.7	7.8	23.1
0.1	532.4	431.7	13.6	23.8
0.5	534.1	526.9	---	---
Note. At all pH values, the section radius was 450±5.3 µm, total area — 635.8±15 thousand µm², experiment accuracy — 0.47.

Conclusions Humic and fulvic acids significantly affect the anatomical structure of the embryonic root of wild apple seeds during stratification. They accelerate the degree of differentiation of conductive tissues and increase cell sizes. The optimal effect is observed with 0.1% and 0.01% solutions of potassium humate Agro.Bio at a pH of 6.98. The degree of differentiation of conductive tissues is an important indicator of seed readiness for germination, influencing germination energy and subsequent seedling growth.