Clonal Micropropagation of Promising Genotypes of Amygdalus communis L. for Population Restoration and Gene Pool Conservation
by
, , , , and
Timur Turdiyev
1,2,
Kumissay Duisenova
3,*,
Irina Kovalchuk
1,2,
Aigul Madenova
1,2,
Saule Baizhumanova
1,
Kamila Yemesheva
1,
Natalya Mikhailenko
1 and
Zakir Tuigunov
1,4 1
Institute of Plant Biology and Biotechnology, Almaty 050060, Kazakhstan
2
Kazakh Fruit and Vegetable Research Institute, Almaty 050060, Kazakhstan
3
Faculty of Natural Sciences and Geography, Abai Kazakh National Pedagogical University, Almaty 050010, Kazakhstan
4
Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty 050060, Kazakhstan
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 999; https://doi.org/10.3390/horticulturae11090999
Submission received: 3 July 2025
/
Revised: 3 August 2025
/
Accepted: 15 August 2025
/
Published: 22 August 2025
(This article belongs to the Special Issue Advances in Developmental Biology in Tree Fruit and Nut Crops—2nd Edition)
Abstract
The southern region of Kazakhstan represents the northernmost boundary of the natural habitat of five wild almond species, among which Amygdalus communis L. is of particular interest due to a range of favorable traits for use in breeding programs and cultivation in the region. The current distribution range of common almond growth was clarified using GPS to determine precise coordinates, and a schematic map was developed. Monitoring revealed a significant reduction in population size. In the surveyed areas, 54 trees were selected and described. Seed material was collected from 34 genotypes and characterized according to a descriptor. Genotypes A3, A8, and A15 were identified as having favorable trait combinations. To restore populations and preserve the gene pool of Amygdalus communis L., a method of clonal micropropagation was employed. The composition of the nutrient medium was optimized for establishment, multiplication, and rhizogenesis. It was determined that Murashige and Skoog (MS) medium without phytohormones is effective for in vitro establishment (70% regeneration rate). For multiplication, MS medium with 0.5 mg/L BAP (6-benzylaminopurine) was used (with a multiplication rate of 3.5 per explant). For rhizogenesis, MS medium with 0.5 mg/L BAP, 0.02 mg/L gibberellic acid (GA), and 0.1 mg/L IBA (indole-3-butyric acid) was used. A total of 340 clonal Amygdalus communis L. plants with closed root systems were grown for field collection. The research results can be applied for the restoration, propagation, and conservation of populations both in vitro and in situ, as well as for the inclusion of selected high-performing genotypes in breeding programs.
1. Introduction
Almond is a valuable fruit crop in the subtropical zone, characterized by early ripening, resistance to adverse climatic conditions, and high adaptability to various types of soil [1]. This crop was domesticated from wild forms through a long evolutionary process [2]. Due to its excellent taste, nutritional value, application in the confectionery industry, and low cultivation requirements, almond remains in high demand and plays a significant role in agricultural regions [3]. Almond belongs to the Rosaceae family and Prunoideae subfamily and includes around 50 species, 17 of which grow in the CIS countries. The most important among them is the common almond (Amygdalus communis L.), which is considered the ancestor of cultivated varieties [4,5,6,7]. The nuts of many wild-growing almond species are only slightly inferior in taste to those of cultivated varieties and serve as an additional source of income for the local population, contributing to improved socioeconomic conditions and playing a crucial role in strengthening regional economies [8].
Amygdalus communis L. is found in the South Caucasus, Central Asia, Afghanistan, Iran, and Asia Minor [4,9]. Within Central Asia, almonds grow in the foothill areas of Uzbekistan, Tajikistan, Turkmenistan, and Kyrgyzstan, with smaller patches in the southern regions of Kazakhstan [10]. Kazakhstan represents the northernmost natural range for nut-bearing crops such as walnut, pistachio, and almond [11], which may serve as a promising source of donors carrying genes for high adaptability to extreme environmental conditions when included in breeding programs.
According to data presented by Zverev N.E., natural populations of Amygdalus communis L. in Kazakhstan are limited to the riparian zones of the Ugam River [12]. The primary causes of habitat reduction are anthropogenic–tree cutting, livestock grazing, and forest fires. Therefore, studies aimed at clarifying the current distribution, preservation, restoration, and expansion of the natural populations of Amygdalus communis L. are highly relevant, as this species holds great potential for cultivation in Kazakhstan [13]. Natural regeneration of the species does not occur for various reasons, the most significant of which is human activity. Artificial propagation by seed does not ensure genetic identity with the original plant due to the high intraspecific polymorphism [14].
In addition to conventional propagation methods, biotechnological techniques are widely and successfully used in global practice [15,16]. These methods allow the production of genetically identical plants from small amounts of plant tissue year-round, regardless of the season, and significantly reduce the time required for propagation and obtaining large volumes of planting material for new varieties, valuable clones, and promising genotypes [15,17]. Biotechnological methods also make it possible to produce healthy, virus-free planting materials of agricultural crops [18], reliably preserve the gene pool of woody species, and aid in population recovery.
The research presented in this article focuses on clarifying the current natural range of Amygdalus communis L. populations, assessing the quality characteristics of selected genotypes, and optimizing microclonal propagation methods. These efforts aim to restore and conserve the declining populations of common almond, ensure a sustainable resource base, and expand the cultivation area under the agro-climatic conditions of Kazakhstan.
2. Materials and Methods
2.1. Plant Material
The objects of the study were wild genotypes of Amygdalus communis L.
Monitoring of the current natural range of wild populations of common almond, Amygdalus communis L., was carried out at places of natural growth in the Sairam-Ugam State National Nature Park, specifically in the Keles Forestry and Sastobe District of the Turkestan region. The geographic coordinates of growing areas were recorded using a GPS device (Garmin Etrex SE, Lenexa, KS, USA) and, during office data processing, were mapped onto electronic topographic maps at a scale of 1:700,000 using the specialized software ArcGIS 10.2.
Seeds were collected during the ripening period. Trees with positive (valuable) characteristics were recorded. The examination and description of the selected genotypes were carried out according to the descriptor “Descriptors list for Almond (Prunus amygdalus)”. Morphological traits (shape and size of the nut, ease of husk removal, and softness of the shell), phenological traits (periods of flowering and ripening), and agronomically valuable characteristics (yield and drought resistance) were assessed [19]. Nut dimensions (length, width, and thickness) were measured using a Vernier caliper (ShTsR-250, 0.1 mm, Moscow, Russia).
2.2. In Vitro Culture Initiation
Genotypes A3, A8, A15, A28, and A30 were used for in vitro culture initiation and microclonal propagation experiments. Two approaches were employed: (a) lignified cuttings were cut into 6–7 cm segments with 2–3 buds, sterilized, and placed on a nutrient medium, and (b) dormant lignified cuttings were germinated under laboratory conditions, and the resulting green shoots were divided into segments with 1–2 buds, sterilized, and transferred to nutrient media [16].
2.3. Sterilization of Plant Tissues
Three sterilization methods were used to obtain plant materials free from saprophytic microflora: (1) Treatment with a 1:1 aqueous solution of bleach (“Belizna”) for 10 min, followed by 0.1% mercury chloride (HgCl2) for 5 min, and three rinses with sterile distilled water; (2) Washing with soap solution for 10 min, then treatment with a 1:1 solution of “Belizna” for 10 min and 0.1% HgCl2 for 7 min, followed by three rinses; (3) rinsing under running water for 15 min, then treatment with 0.1% HgCl2 for 6 min, followed by three rinses with sterile distilled water [16].
2.4. Latent Infection Screening
To detect internal systemic infections, basal parts of shoots were placed on a diagnostic medium (VISS) containing 10.0 g/L sucrose, 8.0 g/L casein hydrolysate, 4.0 g/L yeast extract, 2.0 g/L KH2PO4, 15.0 g/L MgSO4·7H2O, and 6.0 g/L Gelrite. Cultivation was carried out for 1–3 weeks at 23–25 °C [20].
2.5. Microclonal Propagation
Murashige and Skoog (MS, Lenexa, KS, USA), Driver and Kuniyuki Woody Plant Medium (DKW, Lenexa, KS, USA), and Woody Plant Medium (WPM, Shawnee Mission, KS, USA) were used for in vitro initiation and propagation. All media were supplemented with vitamins: thiamine (B1, St. Louis, MO, USA)—0.1 mL, pyridoxine (B6, Mumbai, India)—0.5 mL, nicotinic acid (PP, St. Louis, MO, USA)—0.5 mL, 30 g/L sucrose (Darmstadt, Germany), and adjusted to pH 5.7. To stimulate shoot growth, proliferation, and multiplication, the optimal concentrations of phytohormones were tested: 6-Benzylaminopurine (BAP, Mumbai, Maharashtra, India): 0.1, 0.2, 0.5, and 1.0 mg/L; Gibberellic acid (GA, Moscow, Russia): 0.01 and 0.02 mg/L, and Indole-3-butyric acid (IBA, Lenexa, KS, USA): 0.01 and 0.1 mg/L in different concentrations. For in vitro cultivation, each culture tube (15 cm height, 2.5 cm diameter, PhytoTech LABS, N.C2035, Lenexa, KS, USA) was filled with 10 mL of nutrient medium. The pH was then adjusted to 5.7. The nutrient media were sterilized using an autoclave (UTKBS-150LV, MRC, Holon, Israel) at 121 °C under a pressure of 0.8–1.0 atmospheres for 25 min. Aseptic plants obtained in vitro were propagated on nutrient media by microcutting in culture jars (6.5 cm height, 9.5 cm diameter, Arkansas Glass A0008-140, Jonesboro, AR, USA) [21].
Each experiment included 15 plants with three replicates. Observations and records were made on a monthly basis. Results were assessed after the third subculture by recording the condition and number of newly formed shoots. The average multiplication rate per subculture for each genotype was calculated using the formula: MR = a/(b·c), where a is the number of new shoots, b is the number of shoots used for propagation, and c is the number of subcultures. Cultivation was carried out at a temperature (Tc) of +23–25 °C, with a light intensity (Eph) of 40 μmol·m−2·s−1 and a 16-h photoperiod (tsv).
2.6. Data Analysis
Statistical analysis was performed using GraphPad Prism 10.5.0 software. To evaluate the effects of different media and hormone combinations on each genotype, a one-way analysis of variance (ANOVA) was conducted. When statistically significant differences were detected (p < 0.05), Tukey’s HSD post-hoc test was applied for multiple comparisons between treatment groups. Statistically significant differences are indicated by different superscript letters in the tables. All quantitative data are presented as mean ± standard deviation (SD).
3. Results
3.1. Mapping the Distribution and Selection of Amygdalus communis L. Genotypes
As a result of field expeditions, wild almond populations in the southern region of Kazakhstan were surveyed, specifically in the Turkestan Region within the Sairam-Ugam State National Nature Park: (a) in the Keles Forestry Area and (b) in the Sastobe District. The coordinates of selected superior (elite) almond genotypes were recorded using a GPS device (Garmin Etrex SE). In the Keles Forestry Area, 25 superior forms were selected, and in the Sastobe District, 29 samples were selected (Table 1).
The coordinates of the selected superior genotypes were processed and plotted on electronic topographic maps at a scale of 1:700,000 (Figure 1).
Common almond (Amygdalus communis L.) also exhibits considerable fruit diversity (in shape, taste, shell softness, and ease of husk removal). Seed collection and description were carried out during the ripening season in natural habitats. Trees with superior (valuable) characteristics were recorded (Table 2).
Seed material was collected from various almond genotypes. Among them, 22 genotypes had sweet-tasting kernels, and 12 had bitter ones. Nut shape distribution: 11 genotypes had round nuts, 12 had ovate nuts, 7 had heart-shaped nuts, and 4 had elongated nuts. Based on nut size, 10 genotypes had small nuts, 18 had medium-sized nuts, and six had large nuts. Ease of husk removal: low in seven genotypes, intermediate in five, and high in 22. Shell hardness: hard in 12 genotypes, intermediate in five, papery in five, and soft in 12.
Among the selected genotypes, three were identified: A3, A8, and A15. Nuts of genotype A3 were uniform in size and small (28.1 × 16.5 mm), with a round shape and moderately pointed apex. Shell hardness was intermediate (between hard and soft). Husk removal is easy. The average nut weight was 3.4 g. The kernel is well-formed and elongated oval in shape, with a golden-brown skin. The taste is sweet and has a distinct buttery flavor. The average kernel weight was 0.8 g. No double kernels were detected. This genotype demonstrates stable and high productivity, up to 6.1 kg of dry nuts from a 15-year-old tree under natural conditions. Key advantages of the genotype include a compact crown, consistently high yield, and high-quality kernel taste (Figure 2).
Nuts of genotype A8 were uniform and medium-sized (32.7 × 19.8 mm), heart-shaped with a pointed apex and a hard shell (Figure 3). Husk removal is easy. The average fruit weight was 3.1 g. The kernel is elongated, oval, well-formed, and meets international quality standards. The kernel skin is smooth and light-brown. Taste is sweet. The average kernel weight was 0.6 g. No double kernels were detected. This genotype shows a high and consistent yield of up to 4.8 kg per 20-year-old tree. Key advantages of this genotype are its high and regular productivity and excellent kernel quality.
Nuts of genotype A15 were large (28.7 × 17.8 mm), light brown, and ovate with a small tip (Figure 4). The shell was smooth, thin, and soft. Husk removal was easy. The average nut weight was 3.6 g. The kernel is elongated, oval, well-formed, and sweet in taste. The average kernel weight was 1.0 g. The proportion of double kernels was about 10%. This genotype is characterized by a high yield of up to 5.2 kg of dry nuts from an 8-year-old tree. Key advantages of this genotype are early ripening, high yield, and low percentage of double kernels (Table 3 and Figure 5).
In total, 34 genotypes of common almond were selected. Of particular interest are genotypes A3, A8, and A15, which demonstrated stable performance across multiple traits, including taste quality and agronomic values.
3.2. Microclonal Propagation
The most effective method for obtaining aseptic plants for in vitro culture introduction was the use of actively growing green shoots with 2–3 buds, sprouted under laboratory conditions after winter dormancy from lignified one-year-old cuttings. The optimal sterilization method against saprophytic microflora was a sequential treatment of explants: first with an aqueous solution of bleach (“Belizna”) at a 1:1 ratio for 10 min, followed by a 0.1% mercury chloride (HgCl2) solution for 5 min, and then rinsed three times with sterile distilled water. Using this method, the rate of aseptic plants under in vitro conditions was 60–68%. As shown in Figure 6, the plants exhibited active growth and development with bright green leaves. The introduction of lignified cuttings into in vitro culture was less effective, and the number of sterile plants obtained did not exceed 20%. Their growth and development were weak; the leaves were light green, many were vitrified, necrosis was observed at the shoot tips, and callus formation occurred at the shoot base.
In some cases, symptoms of bacterial infection were observed in individual shoots at various cultivation stages. These included darkening of the shoot bases and leaves, as well as clouding of the culture medium, which ultimately led to plant death. Testing on a provocative medium [20] confirmed bacterial contamination (Figure 6). It is likely that the surface sterilization procedure effectively eliminated fungal microflora but was insufficient for the complete elimination of bacterial infections. All infected samples were removed from the in vitro cultures.
To introduce explants into in vitro culture, cultivation was carried out on Murashige and Skoog (MS) nutrient medium with various modifications—both with and without the addition of phytohormones. The results showed that when 0.01 mg/L gibberellic acid (GA) was used in combination with 6-benzylaminopurine (BAP) at concentrations of 0.1, 0.2, and 0.3 mg/L, the regeneration activity was low; the number of newly formed shoots was minimal, and the multiplication coefficient did not exceed 1.2. The condition of the plants was assessed as unsatisfactory, and yellowing of stems and leaves, followed by leaf drop, was observed. More than 60% of the shoots were vitrified. In contrast, on hormone-free medium, 70% of the plants exhibited active growth, forming vigorous and bright green shoots and leaves.
For micropropagation, shoots were cultured on MS, WPM, and DKW media supplemented with the phytohormones 6-benzylaminopurine (BAP), gibberellic acid (GA), and indole-3-butyric acid (IBA) at various concentrations (Figure 6). All variants contained sucrose at 30 g/L and were adjusted to pH 5.7. Each genotype was tested in triplicate, with 45 explants per treatment (Table 4).
According to the data presented in Figure 7, the number of developed shoots varied depending on the BAP concentration. Statistical analysis revealed significant differences between the tested treatments. The highest number of shoots and most vigorous growth were observed when the MS medium was supplemented with 0.5 mg/L BAP, 0.02 mg/L GA, and 0.1 mg/L IBA. In this treatment, the average multiplication coefficient over three subcultures for genotypes A3, A8, A15, A28, and A30 was 3.5. The average shoot length after 4–6 weeks ranged between 5.0 and 6.3 cm. These results confirm the critical role of BAP in inducing in vitro shoot formation in almond. At lower BAP concentrations (0.1 and 0.2 mg/L), the average number of shoots per explant decreased, and the multiplication coefficient dropped to 2.0. On WPM medium with the same levels of growth regulators (0.5 mg/L BAP and 0.1 mg/L IBA), the plants exhibited a suppressed state: the multiplication coefficient was 1.0–1.5, and the average shoot height was about 1.2 cm. Leaf yellowing, reddening, vitrification, and callus formation were observed. On DKW medium with the same regulator concentrations, there was a sharp decline in culture viability; the multiplication coefficient did not exceed 1.0. Callus formation was noted, and the green coloration of leaves and stems was retained for no more than one week, after which yellowing occurred rapidly. On MS media containing BAP 1.0 mg/L (with or without GA 0.01 mg/L and IBA 0.1 mg/L), strong vitrification of leaves and stems occurred, and overall plant growth and development were extremely low.
The MS medium with 0.5 mg/L BAP, 0.02 mg/L GA, and 0.1 mg/L IBA was also effective in inducing root system development. Root formation began two weeks after transfer, and after 4–5 weeks, the rooting rate reached 80% (Figure 8).
Plantlets with developed root systems were transplanted into containers filled with substrates composed of various ratios of peat, chernozem (black soil), and sand, and were cultivated under greenhouse conditions. The highest survival rate (65–76.5%) over 2–4 weeks was observed in the substrate mixture with a peat: chernozem: sand ratio of 50:40:10. In other substrate compositions, survival did not exceed 35–40% (Figure 7).
Based on these experiments, a protocol for the large-scale propagation of 34 selected superior genotypes of common almond was optimized, with the aim of establishing a field collection. A total of 340 clonal plantlets with closed root systems were obtained in containers.
4. Discussion
Comprehensive studies, including the analysis of current habitats, selection of genotypes with desirable traits, and development of a biotechnological system for mass propagation and seedling production of superior almond trees (Amygdalus communis L.) in containers, are of great significance for Kazakhstan.
In 2001, the renowned pomologist D.A. Dzhangaliev identified and described the preliminary habitats of five wild almond species in Kazakhstan: Amygdalus ledebouriana Schlecht., A. petunnikowii Litv., A. spinosissima Bunge, A. nana L., and A. communis L. [22]. Among these, A. communis L. has gained considerable interest due to its economically valuable traits, making it a promising candidate for breeding programs and regional cultivation. This has led to the development of effective propagation techniques, particularly given the limited availability of source materials and the risk of losing local gene pools. According to N.E. Zverev [12], natural populations of A. communis L. in Kazakhstan are now restricted to the floodplains of the Ugam River. Our recent monitoring in Sayram-Ugam National Nature Park in southern Kazakhstan revealed a significant decline in almond populations compared to previous reports. In response, we initiated conservation and restoration efforts, which required propagation of selected genotypes for reintroduction into their natural habitat, as well as preservation via both in vitro and in situ collections of A. communis L. Clonal micropropagation is a method that allows for the rapid multiplication of genetically identical plants from a small amount of starting tissue. However, to achieve high efficiency, specific protocols tailored to the species in question must be developed, including the selection of sterilizing agents and optimization of nutrient media composition [23]. The composition of the nutrient medium is critical for the in vitro establishment and microclonal propagation of nut-bearing species. The most commonly used media for in vitro culture include Murashige and Skoog (MS), Harvais, Gamborg, and Eveleg B5, Nitsch, and White [24]. There are relatively few studies on almond micropropagation. Filipova et al. developed a modified Quoirin & Lepoivre medium for in vitro almond cultivation [25]. Other studies have reported that MS medium supplemented with 6-benzylaminopurine (BAP) is optimal for in vitro culture initiation and micropropagation of Prunus ledebouriana L. and cultivars such as ‘Nonpareil’ and ‘Texas’ [26,27,28,29,30,31]. Research on artificial propagation of other nut crops has been more extensive. For instance, walnut (Juglans regia) is propagated using Driver and Kuniyuki Woody Medium (DKW), Murashige and Skoog (MS), Woody Plant Medium (WPM), and Glucose Dulbecco’s (GD), with BAP or thidiazuron as cytokinins, and indole-3-butyric acid (IBA) as the auxin [32]. The average number of new shoots per explant ranged from two to five [33,34]. In hazelnut in vitro cultures, multiplication coefficient and shoot elongation were positively influenced by DKW, WPM, and MS nutrient media [35,36,37]. Cytokinin 6-benzylaminopurine (6-BAP) in Murashige and Skoog (MS) nutrient medium significantly promoted embryo cell germination in Amygdalus communis L. [38].
Our findings are consistent with earlier reports but differ in terms of hormone composition, combination, and concentration at each stage of propagation. For in vitro culture initiation, MS medium without phytohormones was effective (regeneration rate: 70%). For multiplication, MS medium supplemented with 0.5 mg/L BAP yielded a multiplication rate of 3.5 per explant. For rooting, MS medium with 0.5 mg/L BAP, 0.02 mg/L GA, and 0.1 mg/L IAA was found to be optimal. Acclimatization to non-sterile conditions in a peat: chernozem: sand substrate resulted in survival rates of 65–76.5%. Currently, there is a lack of literature data on plant acclimatization under closed-field non-sterile conditions.
5. Conclusions
This study aimed to clarify the current distribution range of Amygdalus communis L. populations and identify genotypes with favorable traits for breeding programs. Monitoring results of this species in the Sayram-Ugam State National Nature Park in southern Kazakhstan revealed a significant decline in population size compared to previous research. Using GPS, precise coordinates of almond growth locations were recorded and mapped. Three genotypes exhibiting favorable qualitative traits were identified. To restore populations and conserve the almond gene pool, the method of clonal micropropagation was employed. The composition of the nutrient media was optimized for cultivation, propagation, and rhizogenesis. A total of 340 clonal Amygdalus communis L. plants were grown in open soil.
The findings of this study can be applied to the restoration, propagation, and conservation of Amygdalus communis L. populations both in vitro and in situ, as well as for the inclusion of identified promising genotypes in breeding programs. Future work is planned to restore populations by propagating both promising and other local Amygdalus communis L. genotypes under controlled conditions and reintroducing them into their natural habitats, along with other native-almond species.
Author Contributions
Conceptualization, T.T.; methodology, T.T. and S.B.; software, A.M.; investigation, S.B., K.D., and Z.T.; resources, T.T., K.Y., and N.M.; validation, K.Y.; formal analysis, K.D.; data curation, T.T.; writing—original draft preparation, K.D.; writing—review and editing, K.D. and K.Y.; visualization, K.D.; supervision, T.T. and I.K. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Ministry of Science and Higher Education of the Republic of Kazakhstan, grant number BR21882024 “Study of pistachio and almond biodiversity, development of methods to preserve their gene pool, selection and cloning of promising genotypes for breeding and horticulture”.
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Acknowledgments
We express our gratitude to the staff of the Sairam-Ugam State National Nature Park.
Conflicts of Interest
The authors declare no conflicts of interest.
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Figure 1.
Current distribution range of common almond (Amygdalus communis L.).
Figure 2.
Tree, nuts, kernel, and germinal axis of common almond Amygdalus communis L.: (a) A3 tree in natural conditions; (b) nuts; (c) kernel; (d) germinal axis.
Figure 2.
Tree, nuts, kernel, and germinal axis of common almond Amygdalus communis L.: (a) A3 tree in natural conditions; (b) nuts; (c) kernel; (d) germinal axis.
Figure 3.
Tree, nuts, kernel, and germinal axis of common almond Amygdalus communis L.: (a) A8 tree in natural conditions; (b) nuts; (c) kernel; (d) germinal axis.
Figure 3.
Tree, nuts, kernel, and germinal axis of common almond Amygdalus communis L.: (a) A8 tree in natural conditions; (b) nuts; (c) kernel; (d) germinal axis.
Figure 4.
Tree, nuts, kernel, and germinal axis of common almond Amygdalus communis L.: (a) A15 tree in natural conditions; (b) nuts; (c) kernel; (d) germinal axis.
Figure 4.
Tree, nuts, kernel, and germinal axis of common almond Amygdalus communis L.: (a) A15 tree in natural conditions; (b) nuts; (c) kernel; (d) germinal axis.
Figure 5.
Dimensions of nuts, kernels, and embryonic axes of selected common almond (Amygdalus communis L.) genotypes. Bar plots show mean (± SD) measurements for three seed components nuts, kernels, and embryonic axes in three almond genotypes (A3, A8, A15). Measurements were taken for (A) length, (B) width, and (C) thickness. Statistical comparisons were performed using one-way ANOVA, followed by Tukey’s HSD test. Brackets indicate pairwise comparisons within each component across genotypes. “ns” denotes p ≥ 0.05; denotes **** p < 0.0001.
Figure 5.
Dimensions of nuts, kernels, and embryonic axes of selected common almond (Amygdalus communis L.) genotypes. Bar plots show mean (± SD) measurements for three seed components nuts, kernels, and embryonic axes in three almond genotypes (A3, A8, A15). Measurements were taken for (A) length, (B) width, and (C) thickness. Statistical comparisons were performed using one-way ANOVA, followed by Tukey’s HSD test. Brackets indicate pairwise comparisons within each component across genotypes. “ns” denotes p ≥ 0.05; denotes **** p < 0.0001.
Figure 6.
Introduction of Amygdalus communis L. into in vitro culture and verification of latent infection of explants on VISS medium: (a) germination of cuttings under laboratory conditions; (b) aseptic plants; (c) no infection; and (d) bacterial infection.
Figure 6.
Introduction of Amygdalus communis L. into in vitro culture and verification of latent infection of explants on VISS medium: (a) germination of cuttings under laboratory conditions; (b) aseptic plants; (c) no infection; and (d) bacterial infection.
Figure 7.
Average multiplication coefficient (mean ± SD) for each culture medium (n = 5). All pairwise comparisons between media were statistically significant at p < 0.0001 (one-way ANOVA with Tukey’s HSD), denoted by “****”. These results emphasize the importance of medium selection for growth stimulation.
Figure 7.
Average multiplication coefficient (mean ± SD) for each culture medium (n = 5). All pairwise comparisons between media were statistically significant at p < 0.0001 (one-way ANOVA with Tukey’s HSD), denoted by “****”. These results emphasize the importance of medium selection for growth stimulation.
Figure 8.
Root system formation in vitro and subsequent growth and development of Amygdalus communis L. in substrate-filled containers. (a) Plantlets in culture tubes (height 15 cm, diameter 2.5 cm; PhytoTech LABS No. C2035); (b) Plantlets in culture jars (height 6.5 cm, diameter 9.5 cm; Arkansas Glass Jars A0008-140); and (c) Plantlets in containers (polyethylene container, height 9 cm, diameter 9 cm, volume 560 mL).
Figure 8.
Root system formation in vitro and subsequent growth and development of Amygdalus communis L. in substrate-filled containers. (a) Plantlets in culture tubes (height 15 cm, diameter 2.5 cm; PhytoTech LABS No. C2035); (b) Plantlets in culture jars (height 6.5 cm, diameter 9.5 cm; Arkansas Glass Jars A0008-140); and (c) Plantlets in containers (polyethylene container, height 9 cm, diameter 9 cm, volume 560 mL).
Table 1.
Locations and coordinates of collected common almond (Amygdalus communis L.) samples.
| Sample | Latitude | Longitude | Elevation | Collection Site |
|---|---|---|---|---|
| A 1 | 41°44′53.16″ | 069°42′13.26″ | 1352 | Keles Forestry of the Ugam Branch, Sairam-Ugam State National Nature Park, 3 km from the village of Turbat |
| A 2 | 41°44′53.52″ | 069°42′13.14″ | 1355 | |
| A 3 | 41°44′53.04″ | 069°42′12.90″ | 1340 | |
| A 4 | 41°44′52.98″ | 069°42′12.96″ | 1344 | |
| A 5 | 41°44′52.80″ | 069°42′12.78″ | 1346 | |
| A 6 | 41°44′52.50″ | 069°42′12.84″ | 1352 | |
| A 7 | 41°44′53.10″ | 069°42′12.36″ | 1341 | |
| A 8 | 41°44′52.62″ | 069°42′12.84″ | 1339 | |
| A 9 | 41°44′52.26″ | 069°42′13.50″ | 1349 | |
| A 10 | 41°44′54.00″ | 069°42′11.40″ | 1347 | |
| A 11 | 41°44′54.24″ | 069°42′11.16″ | 1348 | |
| A 12 | 41°44′55.05″ | 069°42′09.25″ | 1343 | |
| A 13 | 41°44′55.28″ | 069°42′09.05″ | 1345 | |
| A 14 | 41°44′55.71″ | 069°42′07.54″ | 1344 | |
| A 15 | 41°44′57.21″ | 069°42′03.76″ | 1342 | |
| A 16 | 41°44′57.20″ | 069°42′03.66″ | 1342 | |
| A 17 | 41°44′57.08″ | 069°42′03.50″ | 1342 | |
| A 18 | 41°44′57.24″ | 069°42′03.10″ | 1340 | |
| A 19 | 41°44′57.28″ | 069°42′03.14″ | 1341 | |
| A 20 | 41°44′57.20″ | 069°42′03.14″ | 1337 | |
| A 21 | 41°44′57.86″ | 069°42′02.76″ | 1337 | |
| A 22 | 41°44′57.72″ | 069°42′03.02″ | 1338 | |
| A 23 | 41°44′57.59″ | 069°42′03.29″ | 1336 | |
| A 24 | 41°44′55.45″ | 069°42′01.49″ | 1336 | |
| A 25 | 41°44′55.83″ | 069°42′02.29″ | 1338 | |
| A 26 | 42°32′02.81″ | 070°01′19.46″ | 603 | Village of Sastobe, Turkistan Region, 45 km from the city of Shymkent |
| A 27 | 42°32′03.15″ | 070°01′18.39″ | 602 | |
| A 28 | 42°32′03.04″ | 070°01′17.56″ | 600 | |
| A 29 | 42°32′02.12″ | 070°01′17.35″ | 599 | |
| A 30 | 42°32′01.69″ | 070°01′17.01″ | 597 | |
| A 31 | 42°32′01.54″ | 070°01′17.00″ | 597 | |
| A 32 | 42°32′01.48″ | 070°01′16.66″ | 598 | |
| A 33 | 42°32′04.71″ | 070°01′18.38″ | 600 | |
| A 34 | 42°32′04.73″ | 070°01′18.46″ | 601 | |
| A 35 | 42°32′04.88″ | 070°01′18.57″ | 601 | |
| A 36 | 42°32′05.12″ | 070°01′18.66″ | 601 | |
| A 37 | 42°32′02.30″ | 070°01′17.50″ | 608 | |
| A 38 | 42°32′03.17″ | 070°01′18.23″ | 606 | |
| A 39 | 42°32′03.11″ | 070°01′18.69″ | 606 | |
| A 40 | 42°32′05.43″ | 070°01′18.65″ | 603 | |
| A 41 | 42°32′05.09″ | 070°01′19.03″ | 604 | |
| A 42 | 42°32′04.90″ | 070°01′20.75″ | 606 | |
| A 43 | 42°32′03.66″ | 070°01′19.67″ | 608 | |
| A 44 | 42°32′02.91″ | 070°01′19.45″ | 607 | |
| A 45 | 42°32′02.50″ | 070°01′18.69″ | 606 | |
| A 46 | 42°32′01.45″ | 070°01′17.09″ | 599 | |
| A 47 | 42°32′01.14″ | 070°01′16.26″ | 596 | |
| A 48 | 42°32′03.92″ | 070°01′18.80″ | 615 | |
| A 49 | 42°32′03.41″ | 070°01′19.00″ | 614 | |
| A 50 | 42°32′02.95″ | 070°01′19.43″ | 613 | |
| A 51 | 42°32′04.58″ | 070°01′18.93″ | 609 | |
| A 52 | 42°32′04.83″ | 070°01′19.29″ | 608 | |
| A 53 | 42°32′05.14″ | 070°01′21.03″ | 609 | |
| A 54 | 42°32′05.43″ | 070°01′21.10″ | 608 |
Table 2.
Description of seeds from selected common almond (Amygdalus communis L.) genotypes.
| №. of Sample | Kernel Taste | Nut Shape | Nut Size | Ease of Husk Removal | Shell Hardness | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sweet | Bitter | Round | Ovate | Elongated | Sweet | Bitter | Round | Ovate | Elongated | Sweet | Bitter | Round | Ovate | Elongated | Sweet | |
| A 1 | + | + | + | + | + | |||||||||||
| A 2 | + | + | + | + | + | |||||||||||
| A 3 | + | + | + | + | + | |||||||||||
| A 4 | + | + | + | + | + | |||||||||||
| A 5 | + | + | + | + | + | |||||||||||
| A 6 | + | + | + | + | + | |||||||||||
| A 7 | + | + | + | + | + | |||||||||||
| A 8 | + | + | + | + | + | |||||||||||
| A 9 | + | + | + | + | + | |||||||||||
| A 10 | + | + | + | + | + | |||||||||||
| A 11 | + | + | + | + | + | |||||||||||
| A 12 | + | + | + | + | + | |||||||||||
| A 13 | + | + | + | + | + | |||||||||||
| A 14 | + | + | + | + | + | |||||||||||
| A 15 | + | + | + | + | + | |||||||||||
| A 16 | + | + | + | + | + | |||||||||||
| A 17 | + | + | + | + | + | |||||||||||
| A 18 | + | + | + | + | + | |||||||||||
| A 19 | + | + | + | + | + | |||||||||||
| A 20 | + | + | + | + | + | |||||||||||
| A 21 | + | + | + | + | + | |||||||||||
| A 22 | + | + | + | + | + | |||||||||||
| A 23 | + | + | + | + | + | |||||||||||
| A 24 | + | + | + | + | + | |||||||||||
| A 25 | + | + | + | + | + | |||||||||||
| A 26 | + | + | + | + | + | |||||||||||
| A 27 | + | + | + | + | + | |||||||||||
| A 28 | + | + | + | + | + | |||||||||||
| A 29 | + | + | + | + | + | |||||||||||
| A 30 | + | + | + | + | + | |||||||||||
| A 31 | + | + | + | + | + | |||||||||||
| A 32 | + | + | + | + | + | |||||||||||
| A 33 | + | + | + | + | + | |||||||||||
| A 34 | + | + | + | + | + | |||||||||||
Table 3.
Actual dimensions of nuts, kernels, and embryonic axes of selected common almond (Amygdalus communis L.) genotypes.
Table 3.
Actual dimensions of nuts, kernels, and embryonic axes of selected common almond (Amygdalus communis L.) genotypes.
| Sample | Nuts | Kernel | Embryonic Axis | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Length | Width | Thickness | Length | Width | Thickness | Length | Width | Thickness | |
| A 3 | 3.02 ± 0.13 a | 2.04 ± 0.12 a | 1.3 ± 0.1 a | 2.8 ± 0.24 abc | 2.12 ± 0.18 a | 1.2 ± 0.07 ac | 1.8 ± 0.11 abc | 1.2 ± 0.07 ac | 0.7 ± 0.1 a |
| A 8 | 3.5 ± 0.24 b | 2.35 ± 0.19 b | 1.6 ± 0.13 bc | 2.7 ± 0.18 abc | 1.8 ± 0.11 bc | 1.4 ± 0.17 bc | 1.7 ± 0.11 abc | 1.6 ± 0.07 b | 0.9 ± 0.04 bc |
| A 15 | 3.9 ± 0.18 c | 2.6 ± 0.2 c | 1.7 ± 0.17 bc | 2.8 ± 0.11 abc | 1.8 ± 0.14 bc | 1.3 ± 0.08 abc | 1.7 ± 0.12 abc | 1.1 ± 0.05 ac | 0.9 ± 0.1 bc |
Footnote: Means in the same column not sharing a superscript letter differ significantly at p < 0.05 (one-way ANOVA, Tukey’s HSD test).
Table 4.
Effect of nutrient medium composition on microclonal propagation of Amygdalus communis L.
| Nutrient Medium | Callus Formation, pcs * | Average Number of Shoots (pcs ± SD) | Average Multiplication Coefficient | Average Shoot Length (cm ± SD) | Leaf Color ** |
|---|---|---|---|---|---|
| Genotype A3 | |||||
| MS, BAP 0.1 mg/L, GA 0.01 mg/L | 5 | 77 ± 1.58 a | 1.71 | 2.6 ± 0.5 abcd | 1 |
| MS, BAP 0.2 mg/L, IBA 0.01 mg/L | 8 | 94 ± 1.58 b | 2.08 | 2.4 ± 0.6 abcd | 4 |
| MS, BAP 1.0 mg/L, GA 0.01 mg/L, IBA 0.1 mg/L | 13 | 68 ± 1.00 c | 1.51 | 1.9 ± 0.5 abcdfg | 3 |
| MS, BAP 1.0 mg/L, IBA 0.1 mg/L | 11 | 55 ± 1.58 d | 1.22 | 1.7 ± 0.4 abcdfg | 4 |
| MS, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 9 | 158 ± 1.58 e | 3.51 | 5.7 ± 0.3 e | 5 |
| WPM, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 14 | 36 ± 1.00 f | 0.8 | 1.1 ± 0.3 cdfg | 2 |
| DKW, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 17 | 45 ± 0.00 g | 1 | 0.9 ± 0.2 cdfg | 3 |
| Genotype A8 | |||||
| MS, BAP 0.1 mg/L, GA 0.01 mg/L | 10 | 76 ± 1.00 a | 1.68 | 2.7 ± 0.6 abcd | 3 |
| MS, BAP 0.2 mg/L, IBA 0.01 mg/L | 9 | 96 ± 1.00 b | 2.13 | 2.5 ± 0.4 abcd | 3 |
| MS, BAP 1.0 mg/L, GA 0.01 mg/L, IBA 0.1 mg/L | 16 | 69 ± 1.00 c | 1.5 | 2.1 ± 0.5 abcdfg | 3 |
| MS, BAP 1.0 mg/L, IBA 0.1 mg/L | 14 | 54 ± 1.00 d | 1.2 | 1.9 ± 0.5 abcdfg | 4 |
| MS, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 10 | 159 ± 1.00 e | 3.53 | 5.6 ± 0.4 e | 4 |
| WPM, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 12 | 35 ± 1.00 f | 0.77 | 1.2 ± 0.3 cdfg | 2 |
| DKW, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 15 | 45 ± 0.00 g | 1 | 1.0 ± 0.3 cdfg | 1 |
| Genotype A15 | |||||
| MS, BAP 0.1 mg/L, GA 0.01 mg/L | 7 | 75 ± 1.58 a | 1.66 | 2.8 ± 0.4 abcd | 2 |
| MS, BAP 0.2 mg/L, IBA 0.01 mg/L | 5 | 95 ± 1.00 b | 2.11 | 2.5 ± 0.6 abcd | 4 |
| MS, BAP 1.0 mg/L, GA 0.01 mg/L, IBA 0.1 mg/L | 12 | 70 ± 1.00 c | 1.55 | 2.0 ± 0.5 abcdfg | 3 |
| MS, BAP 1.0 mg/L, IBA 0.1 mg/L | 10 | 56 ± 1.00 d | 1.24 | 1.8 ± 0.4 abcdfg | 4 |
| MS, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 13 | 160 ± 1.00 e | 3.55 | 5.9 ± 0.4 e | 5 |
| WPM, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 17 | 38 ± 1.41 f | 0.84 | 1.2 ± 0.3 cdfg | 3 |
| DKW, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 21 | 45 ± 0.00 g | 1 | 1.1 ± 0.3 cdfg | 2 |
| Genotype A28 | |||||
| MS, BAP 0.1 mg/L, GA 0.01 mg/L | 6 | 79 ± 2.00 a | 1.75 | 2.8 ± 0.6 abcd | 2 |
| MS, BAP 0.2 mg/L, IBA 0.01 mg/L | 7 | 93 ± 1.58 b | 2.06 | 2.6 ± 0.5 abcd | 2 |
| MS, BAP 1.0 mg/L, GA 0.01 mg/L, IBA 0.1 mg/L | 10 | 67 ± 1.00 c | 1.48 | 2.0 ± 0.6 abcdfg | 4 |
| MS, BAP 1.0 mg/L, IBA 0.1 mg/L | 12 | 53 ± 1.00 d | 1.17 | 1.9 ± 0.5 abcdfg | 4 |
| MS, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 10 | 157 ± 1.00 e | 3.48 | 5.6 ± 0.4 e | 5 |
| WPM, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 18 | 37 ± 1.00 f | 0.82 | 1.2 ± 0.3 cdfg | 2 |
| DKW, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 8 | 45 ± 0.00 g | 1 | 0.9 ± 0.3 cdfg | 1 |
| Genotype A30 | |||||
| MS, BAP 0.1 mg/L, GA 0.01 mg/L | 5 | 78 ± 1.58 a | 1.73 | 2.7 ± 0.5 abcd | 2 |
| MS, BAP 0.2 mg/L, IBA 0.01 mg/L | 8 | 92 ± 1.58 b | 2.04 | 2.5 ± 0.4 abcd | 2 |
| MS, BAP 1.0 mg/L, GA 0.01 mg/L, IBA 0.1 mg/L | 11 | 66 ± 1.00 c | 1.46 | 2.1 ± 0.5 abcdfg | 3 |
| MS, BAP 1.0 mg/L, IBA 0.1 mg/L | 13 | 52 ± 1.00 d | 1.15 | 1.8 ± 0.4 abcdfg | 4 |
| MS, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 9 | 161 ± 1.58 e | 3.57 | 5.8 ± 0.3 e | 5 |
| WPM, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 12 | 34 ± 1.58 f | 0.75 | 1.3 ± 0.3 cdfg | 2 |
| DKW, BAP 0.5 mg/L, GA 0.02 mg/L, IBA 0.1 mg/L | 7 | 45 ± 0.00 g | 1 | 1.0 ± 0.3 cdfg | 1 |
* Callus formation—number of shoots in which callus formation was observed during the experiment. ** Leaf color—assessed on a 5-point scale, where: 1—necrosis, 2—yellow, 3—yellow with green veins, 4—light green, 5—bright green. Means in the same column not sharing a superscript letter differ significantly at p < 0.05 (one-way ANOVA, Tukey’s HSD test).
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Turdiyev, T.; Duisenova, K.; Kovalchuk, I.; Madenova, A.; Baizhumanova, S.; Yemesheva, K.; Mikhailenko, N.; Tuigunov, Z. Clonal Micropropagation of Promising Genotypes of Amygdalus communis L. for Population Restoration and Gene Pool Conservation. Horticulturae 2025, 11, 999. https://doi.org/10.3390/horticulturae11090999
AMA Style
Turdiyev T, Duisenova K, Kovalchuk I, Madenova A, Baizhumanova S, Yemesheva K, Mikhailenko N, Tuigunov Z. Clonal Micropropagation of Promising Genotypes of Amygdalus communis L. for Population Restoration and Gene Pool Conservation. Horticulturae. 2025; 11(9):999. https://doi.org/10.3390/horticulturae11090999
Chicago/Turabian StyleTurdiyev, Timur, Kumissay Duisenova, Irina Kovalchuk, Aigul Madenova, Saule Baizhumanova, Kamila Yemesheva, Natalya Mikhailenko, and Zakir Tuigunov. 2025. "Clonal Micropropagation of Promising Genotypes of Amygdalus communis L. for Population Restoration and Gene Pool Conservation" Horticulturae 11, no. 9: 999. https://doi.org/10.3390/horticulturae11090999
APA StyleTurdiyev, T., Duisenova, K., Kovalchuk, I., Madenova, A., Baizhumanova, S., Yemesheva, K., Mikhailenko, N., & Tuigunov, Z. (2025). Clonal Micropropagation of Promising Genotypes of Amygdalus communis L. for Population Restoration and Gene Pool Conservation. Horticulturae, 11(9), 999. https://doi.org/10.3390/horticulturae11090999
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