1. Introduction
The conservation of rare, endangered, and endemic plant species is one of the most critical tasks in nature conservation, to which great attention is paid worldwide. Despite all efforts, biodiversity continues to decline due to factors such as the destruction of natural habitats, overexploitation of natural resources, and water and soil pollution [1].
The genus Allium contains several rare species of scientific interest in terms of studying their biodiversity and the need for additional conservation measures [2,3]. One of them is Allium microdictyon Prokh (1930), a relic of broadleaved forests that has declined everywhere due to extreme habitat conditions and human factors [4].
Many wild species of Allium sp. are used for the prevention and treatment of cardiovascular disease. A. microdictyon has medicinal properties due to the presence of a wide range of secondary metabolites (thiosulfates, flavonoids, saponins, and vitamins). The volatile fractions, juice, and extracts exhibit high antibacterial and antifungal activity [5]. The above-ground parts of A. microdictyon contain significant amounts of flavonoids (3.3%), tannins (12.2%), pectin (6.2%), sugars (10.9%), and carotenoids (97%) [6]. This species is still poorly studied; relatively recently, flavonoid analysis was carried out for the first time, revealing the presence of about 14 compounds in its leaves, including the discovery of two new flavon glycosides: quercetin-3-O-neohesperidoside-7-O-glucuronide and kaempferol-3-O-neohesperidoside-7-O-glucuronide [7].
The leaves of A. microdictyon Prokh. (known as mountain garlic) are very popular among the population of Siberia and Altai and are used in the traditional cuisine of Korea, China, and Mongolia [8,9,10].
The high palatability of A. microdictyon causes its intensive consumption by the population, creating a good demand in the markets. This substantially reduces its range, affecting forests and meadows of the subalpine belt of Mongolia and Eastern Kazakhstan [11]. In Kazakhstan, A. microdictyon is found only on the ridges framing the Ridder depression: in spruce–fir and cedar forests of Ivanovsky Ridge, Kholzun, Lineysky, and Ubinsky. The climate of the Kazakhstani Altai is favorable for the growth and development of A. microdictyon plants. Due to high snow cover (up to 2.5 m), sub-snow growth is possible for A. microdictyon plants, and anthocyanin-generative shoots appear immediately after snow melting [12].
This species is biologically similar to other shady broad-leaved species such as Allium ursinum and Allium victorialis (called ramsons), but it differs in several morphological characteristics, reproductive biology, and peculiarities of reproduction as well as seasonal rhythm of development [13]. Despite its valuable nutritive qualities and medicinal properties, A. microdictyon is not used in the interspecific hybridization and selection of the traditional onion varieties of A. sepa due to incompatibility, resulting in meiosis disorders and offspring sterility [14].
In natural populations, A. microdictyon plants have low competitive ability; their vegetative reproduction potential is limited by competition with rhizomatous species of Poaceae sp. and dense turf Carex sp. Seed renewal of plants is significantly affected by the negative impact of anthropogenic factors. Immediately after the first shoots of A. microdictyon plants appear, the local population collects them en masse (called ramsons) for use as first greens. The mass collection of A. microdictyon reaches 25 tons of green mass annually, resulting in this species dropping out of forest cenoses. Large vegetative and generative shoots are usually harvested, and due to this, only 60% of the remaining plants are capable of secondary growth the following year. A minimum of 3–4 years is required for recovery and normal plant development [12]. The local population often pulls the entire plant together with the bulb, and the flowering shoots, which also have a high nutritional value, are used for food [10]. As a result, populations deprived of seed regeneration become old and die out.
Logging has a highly negative impact on the conservation of the species, disrupting the age balance in the populations of A. microdictyon. The prevalence of vegetative individuals of non-seed origin is noted at logging sites. Thus, it is evident that this species is threatened with extinction in Kazakhstan due to the damage caused by failure to conserve to the natural habitat through logging and uncontrolled harvesting by the population. Therefore, the search for conservation and breeding strategies is necessary.
Currently, there are two complementary strategies for plant biodiversity conservation: in situ conservation (conservation in natural habitats) and ex situ conservation (conservation in botanical gardens, seed banks, cryogenic storage, and in vitro collections). The species A. microdictyon is listed in the Red Book of the Republic of Kazakhstan [15]. However, the methods of in situ protection of rare, endangered, and endemic plant species cannot ensure their complete conservation in all habitats of this species due to significant costs. In this regard, recent years have seen the development of a promising area of research—plant conservation biotechnology, which contributes to a substantial expansion of opportunities for biodiversity conservation [16,17].
The use of biotechnological methods based on the cultivation of isolated cells, tissues, and plant organs on artificial nutrient media is one of the solutions for species whose natural regeneration is weakened or hindered as well as for the conservation of their gene pool as a whole [18]. These methods have several advantages over traditional methods: There is no need for large areas occupied by mother and propagated plants; in regular maintenance of plantings, plant diseases and, as a consequence, loss of material is excluded; and there is a possibility of restoring the number of protected plant species by creating artificial populations in the natural area, obtaining sterile rare, endemic plant species without removal from natural habitats, which does not disturb the natural phytocenosis [19,20,21].
The propagation of endemic plants using biotechnological tissue culture techniques is essential due to the limited populations of these species [22,23,24,25,26]. In vitro regeneration by direct and indirect organogenesis using different explants and growth regulator formulations of Allium plants has been described in detail in the literature [23,27]. There are reports of different methods of in vitro cultivation for other Allium species, including direct and indirect organogenesis and various types of explants. The most frequently used explants in Alliaceae representatives are bulb stalks, root tips, mature and immature embryos, inflorescences, and leaf fragments [22,28,29,30,31,32]. In vitro cultivation protocols using different explants, growth regulators, and combinations were developed for the endemic A. tuncelianum, which grows only in Turkey [33]. However, for species with a small number of individuals or species in which seed reproduction dominates over vegetative reproduction, it is preferable to use seeds as explants. Thus, for developing micropropagation technology for the rare geophyte A. neriniflorum (Herb.) Backer., seeds were used as starting material, from which microbulbs were obtained on modified nutrient media [34,35,36,37].
At the same time, it should be taken into account that the use of seeds of many wild species of Alliaceae as explants for introduction into in vitro culture is complicated by their deep dormancy, and for overcoming this, it is necessary to additionally use various methods—long stratification and exposure to phytohormones (gibberellins and cytokinins) [38]. Many researchers point to the presence of dormancy in the seeds of A. microdictyon as well as in its related species A. ursinum and A. victoralis, which is due to the action of physiological mechanisms of germination inhibition [12,39,40,41,42]. At the same time, there is evidence that under favorable conditions, A. microdictyon seeds can partially germinate without a dormancy period [12].
The advantage of using seeds and immature embryos as explants is that as ontogenetically young explants, they have a higher regenerative potential than mature tissues [43]. In addition, using seeds as explants for specimens from natural populations ensures that the species’ genetic diversity is preserved at a maximum level [44]. Our research focuses on using mature seeds of A. microdictyon as explants to develop an in vitro micropropagation and conservation system for this rare and endemic species.
2. Materials and Methods
2.1. Plant Material
Mature seeds of A. microdictyon collected from a natural population on the north-western slope of the Ivanovsky Ridge (coordinates 50°20′33″ N 83°44′04″ E) were used as initial material. The population is located at an altitude of 1023 m above sea level. It is the largest population in Kazakhstan, with an area of about 12,700 ha. Ecological habitat conditions correspond to optimal conditions for developing A. microdictyon; the population is complete, and individuals of all age groups are represented, of which juvenile plants are dominant.
The timing of seed collection was linked to the temperature regime before the onset of frost. Inflorescences were carefully cut with scissors to prevent the ripe seeds from falling to the ground, after which the seeds were cleaned. Some cut inflorescences that were not ripe were left to dry and ripen in the laboratory (Figure 1).
We also used seeds from the Altai Botanical Garden collection, stored for 1 and 2 years in air-dried conditions at 4–5 °C. Dr. Yuri Kotukhov identified A. microdictyon plants and seeds, and a voucher specimen (No. 18.08.2021/16) was deposited at the Altai Botanical Garden’s herbarium.
2.2. Establishment of In Vitro Culture
Seeds were sterilized using different antiseptics: ethanol (EtOH), mercuric chloride (HgCl2), potassium permanganate (KMnO4), sodium hypochlorite (NaClO), and hydrogen peroxide (H2O2). The concentrations and exposure times for each sterilizing agent were selected empirically. For each variant, 100 seeds from three replications were used. A total of four sterilization schemes were used (Table 1).
After sterilant treatment, seeds were washed three times with sterile distilled water and dried on sterile filter paper. Next, sterile A. microdictyon seeds were placed in Petri dishes on ½ Murashige and Skoog nutrient medium (½ MS) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with growth regulators—10 mg L−1 gibberellic acid (Sigma-Aldrich, St. Louis, MO, USA) and 0.1 mg L−1 indole-3-acetic acid (IAA) (Sigma-Aldrich, St. Louis, MO, USA). As a control, seed germination was carried out on 1/2 MS media without hormones. The media contained 30 g/L sucrose (PhytoTech Labs, Lenexa, KS, USA) and 5 g/L plant agar (Condalab, Madrid, Spain). The seed cups were placed under phytotron conditions (25 °C, 16 h light). The effectiveness of sterilization was evaluated after 30 days. The efficiency of the seed sterilization protocol was assessed only by the number (%) of sterile viable seedlings. Infected explants were defined as contaminated seeds and seedlings infected by latent infection for 30 days. Sterile seeds that did not germinate throughout the experiment and seedlings that stopped developing were classified as non-viable.
2.3. Protocol for In Vitro Micropropagation of A. microdictyon
Sterile seedlings of A. microdictyon (12–15 mm) were placed on Murashige and Skoog basic medium (MS) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 6-benzylaminopurine (BAP) (Sigma-Aldrich, St. Louis, MO, USA) (0.1 mg L−1, 0.5 mg L−1 and 1.0 mg L−1) and zeatin (ZEA) (Sigma-Aldrich, St. Louis, MO, USA) (0.1 mg L−1, 0.5 mg L−1, 1.0 mg L−1, 1.5 mg L−1 and 2.0 mg L−1). MS medium without growth stimulants was used as a control. Thirty explants were used for each variant. The cultivation period was six weeks. The morphometric parameters (number and length of adventitious shoots) of A. microdictyon regenerate plants were evaluated after the cultivation period.
Organogenesis was initiated on nutrient medium MS containing 0.1 mg L−1 BAP and 0.2 mg L−1 auxins (IAA, indole-3-butyric acid (IBA) (Sigma-Aldrich, St. Louis, MO, USA), 1-naphthaleneacetic acid (NAA) (Sigma-Aldrich, St. Louis, MO, USA), Paclobutrazol (PAC) (Duchefa Biochemie, Haarlem, The Netherlands). After 4 and 8 weeks of cultivation, morphometric measurements (number and length of leaves and roots) were performed.
All media contained 30 g/L sucrose (PhytoTech Labs, Lenexa, KS, USA) and 5 g/L plant agar (Condalab, Madrid, Spain).
2.4. Adaptation to Ex Vitro Conditions
To adapt A. microdictyon regenerants to non-sterile conditions, regenerants with a well-developed root system and 5–7 leaves were selected. A mixture of peat and perlite in a ratio of 1:1 was used as a substrate. Adaptation of regenerants was carried out at a temperature of +23 ± 2 °C and illumination of 61 μM m−2. During the dormancy period, plants were transferred to conditions of low, favorable temperatures up to +5 °C for a period of 16–20 weeks, after which the plants were again placed in a light room with a temperature of +23 ± 2 °C and illumination of 61 μM m−2. The degree of plant adaptation was assessed by the number of plants that formed assimilating leaves ex vitro.
2.5. Statistical Analyses
Data on the effects of growth regulators on regeneration and effect of cytokinins on the induction of adventitious organogenesis were analyzed using one-way analysis of variance (ANOVA) with the post hoc Tukey HSD test. R version 4.2.1, R Foundation for Statistical Computing, Vienna, Austria, was used for statistical analyses [45].
3. Results
3.1. Establishment of In Vitro Culture of A. microdictyon
The optimal sterilization method was selected for freshly harvested A. microdictyon seeds to exclude the influence of storage time on germination.
The results showed that the type of antiseptic used determined the level of surface contamination (Figure 2).
The highest number of viable seedlings, 92.7%, was obtained by sterilization with 0.01% HgCl2, while the development of pathogenic microflora was observed in only 3.1% of explants (Figure 3A). When 20% NaClO and 3% H2O2 were used, 11.5% and 10.5% of non-viable seedlings and non-germinated seeds were observed, respectively. The worst sterilization option was the use of 2% KMnO4, which had little effect on pathogenic microflora—up to 84.3% of explants were infected.
The use of H2O2 at a low concentration of 3% was ineffective (scheme 1); as it was not possible to achieve a sterilizing effect on the microflora, up to 58.1% of seeds in this variant were contaminated with pathogens.
The results of the studies showed that germination also depended on the storage period of seeds. Thus, the lowest percentage of germination was observed during a 2-year storage period at 1.3%, and the percentage of germination of seeds after one year of storage was 5.7%. The highest germination rate was in freshly harvested seeds was 92.7%, and on the 7th day, the number of germinated seeds was 25 pieces. Seed germination increased during cultivation; the maximum value was observed at 30 days (Table 2).
The results show that freshly collected seeds of A. microdictyon have a maximum germination percentage of 92.7%. In seeds stored for one year, germination decreased to 5.7%. Seeds stored for two years germinated sporadically, and only 1.3% formed typically developed seedlings. On hormone-free medium, single seedlings were obtained only from fresh seeds. Further cultivation with regular passaging on fresh media for the next six months did not allow obtaining germinated seeds.
3.2. In Vitro Micropropagation of A. microdictyon
The effect of different hormone combinations on adventitious shoot formation was studied (Table 3). Proliferation was manifested in the appearance of new adventitious shoots in seedlings depending on the hormonal profile of the medium.
The average number of shoots per explant on different media variants differed significantly compared to the control medium MS, which had the lowest values of the number of adventitious shoots and their length. However, the length of the adventitious shoots did not show statistically significant differences (p > 0.05).
Evaluation of the ability to form adventitious shoots in A. microdictyon showed that media MSPR9 and MSPR10 were optimal regarding the number and length of adventitious shoots. The number of adventitious shoots per explant on media MSPR9 and MSPR10 was 18.4 and 18.2, respectively, and the length of adventitious shoots was 9.4 and 6.8 cm, respectively. The minimum values in the experiment were obtained on media MSPR1 and MSPR2, containing low concentrations of BAP and ZEA hormones. On MSPR9 and MSPR10, numerous conglomerates consisting of thin leaves firmly fused at the rhizome-like base were observed (Figure 3B). The conglomerates, divided into segments or clones of one leaf on a rhizome-like base, were then passed onto media for organogenesis (Figure 3C).
After 4 weeks, insignificant development of cloned A. microdictyon plants was observed on the control variant of MS (without phytohormones). On nutrient media supplemented with growth regulators, an increase in the number of leaves from 1.2 to 2.5 leaves per plant was observed (Table 4). Increasing the cultivation period up to 8 weeks increased this indicator from 1.2 to 7.2 leaves per explant. Leaf length growth in regenerates after 4 weeks of in vitro cultivation ranged from 1.0 to 1.3 cm. The maximum values in the experiment were obtained when cultured on medium MSB-IBA, containing 0.1 mg L−1 BAP and auxin hormone 0.2 mg L−1 IBA. This combination of hormones was the most favorable for leaf and root system development. In addition, in this variant, all regenerates showed the formation of microbulbs covered with filmy scales and the presence of intensely anthocyanin-colored cover scales on shoots, which was observed in juvenile plants of A. microdictyon in natural populations (Figure 3D). No such changes in the development of regenerants were observed in other variants.
3.3. Ex Vitro Adaptation of A. microdictyon
Plants with a well-formed root system were transferred into vessels with soil and placed in phytotron conditions with a light intensity of 61 μM m−2. The adaptation period lasted 2 months; during this time, the regenerants gradually died off of all leaves, which is typical for A. microdictyon plants from natural populations during the autumn dormancy period of bulbs. However, root formation continued in the root system during this period, which was visible in the cultivation vessels’ drainage holes. After leaf die-off, all plants were transferred to a dark room at a temperature of +5 °C for 16–20 weeks and then again to phytotron conditions under the same lighting conditions. After low-temperature adaptation, the appearance of true leaves was observed, which was evidence of successful rooting of regenerants under ex vitro conditions (Figure 3E). The rooting rate of the regenerated plants was 60–70%. As a result of the research, 88 plants of 127 planted in the soil rooted. The dormancy period was overcome, and 74 plants survived (Figure 3F). The remaining regenerates showed weak growth of assimilating leaves and slow development; some plants never emerged from dormancy.
4. Discussion
The anthropogenic impact on the biosphere has become global, and the scale and pace of the effects continue to grow from year to year. Endemic species in nature are often subjected to anthropogenic threats in small numbers or minimal areas. Habitat destruction under the influence of anthropogenic factors is the leading cause of endemic species extinction [46]. Human activity due to agro-industrial development, industrialization, or urbanization leads to fragmentation of the distribution areas of most endemics, fragmentation of populations, and their complete extinction [47,48]. In this regard, the conservation of biodiversity of endemic plant species using biotechnology methods related to conservation and propagation in vitro culture, allowing the reproduction of valuable genotypes in natural populations, is seen as a global priority worldwide [49,50].
In vitro techniques have been quite effective in conserving for many endemic Allium species [22,23,29,30,32]. However, we found no publications on the conservation and reproduction for A. microdictyon under in vitro conditions, which indicates the relevance of the conducted research in connection with the threat of its extinction under the influence of anthropogenic factors.
The biological peculiarities of A. microdictyon make in vitro cultivation difficult and time-consuming. Under natural conditions, the species is propagated mainly by seeds; this method of propagation is of exceptional importance for the conservation and regeneration of natural populations [12]. Despite this, seed germination under natural conditions is very low; seeds need long (up to 20 months) stratification and lose germination within 1–2 years. Therefore, in vitro conservation and propagation technologies are the only feasible methods for preserving the germplasm of such species, as they cannot be maintained in seed banks [51]. Exposure to exogenous growth regulators and optimization of cultivation conditions allows for overcoming the dormancy period of A. microdictyon seeds used as explants. The main problem when using seeds from wild plants is significant contamination by pathogenic microflora, often present in latent form. Obtaining the maximum number of viable and sterile seedlings is the main objective when introducing in vitro culture.
Surface sterilization using antiseptics such as EtOH, KMnO4, and H2O2 is sufficient to remove contamination induced by exogenous factors [50]. Numerous studies show that the most effective and safe use of NaClO involves its ability to penetrate the pores of seed coats, thereby inhibiting the development of latent forms of infection. However, bacterial pathogens are more sensitive to NaClO exposure, while endophytic fungi (e.g., Ascomycota) show increased resistance, forcing researchers to use either higher concentrations or longer exposure times. This can adversely affect plant explants, causing irreversible tissue necrosis and reducing viability [52]. The 0.1% solutions of HgCl2 that are used in similar studies can cause denaturation of proteins and inactivation of pathogenic enzymes [53]. Our studies used a 10-fold reduced concentration of 0.01% HgCl2, which was compelling enough to produce aseptic A. microdictyon seedlings. We did not observe the occurrence of latent infection on seeds and seedlings during the cultivation period on both the germination medium and for the subsequent micropropagation steps. The absence of necrotic seedlings and low infection rate of explants using this sterilization method may be due to the combinatorial effect of using 70% EtOH and 0.01% HgCl2 simultaneously; this is in agreement with the findings of Y. Si et al. [54].
The age of Allium sp. seeds has a significant effect on their germination and viability. The loss of viability in seed aging may be associated with metabolic disorders of enzyme proteins, decreased electrical conductivity, and loss of membrane integrity. Old seeds of Allium sp. show decreased or complete absence of germination or reduced viability as well as abnormal seedling development [55]. In the process of storage, A. microdictyon seeds acquire hard seediness, and germination becomes difficult; after two years of storage, most seeds go into deep morphophysiological rest, which causes low germination of seeds [12]. Our studies show that freshly harvested seeds of A. microdictyon are capable of germination immediately after maturation and, like the closely related species A. victoralis, do not experience morphological, physical, or physiological dormancy [56]. Seed germination under in vitro conditions after one year of storage decreased by 16 times, and after two years, this indicator decreased by 70 times. To overcome the dormancy period in wild Allium species, along with scarification and stratification, the most commonly used phytohormones of diterpene nature are gibberellins. Gibberellins trigger the gibberellin-dependent transcription factor GAMYB, leading to the expression of α-amylase genes in the aleurone layer [57,58]. We suggest that in our experiments, the germination of single A. microdictyon seeds after 2 years of storage was possible due to a combination of gibberellic acid and IAA. The ability of low concentrations of IAA in combination with gibberellic acid enhances the ability of aleurone to synthesize a-amylase. It activates calmodulin, ultimately stimulating embryo growth without stratification [59].
Induction of adventive organogenesis of A. microdictyon was largely dependent on the amount and ratio of cytokinin activity hormones in the medium. Our results confirm the significant effect of cytokinin hormones on the induction of adventitious shoot formation of A. microdictyon, established earlier for various Allium species, where successful results of microcloning using BAP in combinations with ZEA, NAA, KIN, and IAA were also obtained [60,61,62]. Efficient proliferation of adventitious shoots was observed in wild garlic (Allium ochotense Prokh.) in the presence of 1.0 mg/l of zeatin and 0.1 mg/l NAA [63].
Further development of A. microdictyon regenerates and micro bulbs formed under the influence of BAP and auxin hormones (IAA, NAA, IBA, and PAC), promoting the development of root system and leaf apparatus. To form microbulbs in vitro, it is necessary to reduce the content of cytokinin hormones and increase the concentration of auxins [64]. Our studies have shown that IBA is preferred for microbulbs formation in A. microdictyon compared to other auxins. The results obtained agree with other studies showing the excellent effect of IBA on microbulb formation using different types of explants, including seeds in Alliaceae species [65,66]. Also, a positive effect of using IBA in similar studies is the increase in adaptive potential of plants in soil when transferred to ex vitro conditions due to better rooting ability [67]. Medium with 2.0 mg/L IBA supplementation was found to be the best medium for rooting elite wild garlic Allium ochotense Prokh. [63].
The formation of microbulbs covered with anthocyanin-colored film scales, similar to our results, was observed in regenerants of the related species A. victoralis when cultivated on media with IBA [68].
Adaptation to ex vitro conditions is the most challenging stage of in vitro propagation. Our studies confirm that plants with well-developed bark systems have the highest survival rate at the ex-vitro adaptation stage.
The adaptation period to ex vitro conditions in A. microdictyon plants proceeded similarly to other representatives of the family Alliaceae, whose microbulbs under ex vitro conditions went into dormancy, the duration of which was determined by species affiliation [23,27]. According to Kotukhov Y. et al. [12], during the dormancy period in bulbs, the most critical organ-forming processes occur, preparing plants for further vegetation and forming renewal buds; during this period, bulbs do not lose roots. Microbulbs that had entered the dormancy period were placed in low light and low, favorable temperatures (+5 °C). After 16–20 weeks of cultivation, the emergence of shoots was observed, which was evidence of successful rooting of regenerants under ex vitro conditions.
This is the first report on developing a complete cycle of in vitro propagation of the rare endemic species A. microdictyon. The need for new knowledge on the peculiarities of in vitro cultivation of this species will ensure the conservation of natural biodiversity and rational utilization. The developed technology of in vitro microclonal propagation presented in this study opens new perspectives for preventing the extinction of an endemic species. It will serve as a basis for developing a strategy for renewing natural populations.
5. Conclusions
In this study, an efficient protocol for in vitro propagation of the rare endemic species A. microdictyon from mature seeds collected from natural populations was developed. We optimized the sterilization conditions for obtaining viable seedlings, the micropropagation stages, and the acclimatization of A. microdictyon regenerants for ex vitro conditions. Cultivation of A. microdictyon seedlings on media containing 0.5 mg L−1 BAP and 1.5–2 mg L−1 ZEA is an effective method for inducing adventitious shoot formation. The use of 0.2 mg L−1 IBA in combination with 0.1 mg L−1 BAP was more effective in comparison with other auxins (NAA, IAA, and PAC) for the growth and development of A. microdictyon regenerants as well as their adaptation to ex vitro conditions. This protocol is the effective method for the propagation and conservation of biodiversity of the endangered species A. microdictyon.
D.T. and O.R. performed the experiments, prepared figures and tables, authored or reviewed paper drafts, and approved the final draft; A.D. collected A. microdictyon samples and performed morphological descriptions of plants; A.T. analyzed the data, prepared figures and tables, authored or reviewed paper drafts, and approved the final draft; O.K. conceived and designed the experiments, analyzed the data, prepared figures and tables, authored or reviewed paper drafts, and approved the final draft. All authors have read and agreed to the published version of the manuscript.
The original contributions presented in the study are included in the article material, further inquiries can be directed to the corresponding author.
The authors would like to acknowledge Premina N.V., Researcher of the RSI “West Altai State Nature Reserve”, for providing photographs of plants A. microdictyon in their natural habitats.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. Plants of A. microdictyon in natural habitats on the Ivanovsky Ridge: (A) shoot regrowth after snowmelt; (B) formation of buds; (C) blossom; (D) seed ripening (photo by Premina N.).
Enlarge this image.Figure 2. Viability and contamination rates of A. microdictyon seeds depending on the method of sterilization: Scheme 1 (EtOH + H2O2), scheme 2 (EtOH + KMnO4), scheme 3 (EtOH + NaClO), and scheme 4 (EtOH + HgCl2).
Enlarge this image.Figure 3. Micropropagation of a rare and endemic species A. microdictyon. (A) Germination of fresh seeds under in vitro conditions. (B) Formation of multiple conglomerates. (C) Clones on a rhizome-like base. (D) Formation of microbulbs in regenerants. (E) Appearance of true leaves after dormancy. (F) Regenerant plants adapted to ex vitro conditions. The black arrow shows the formation of microbulbs covered with filmy scales on MCB-IBA medium; the white arrow shows anthocyanin-colored cover scales on shoots.
A. microdictyon seed sterilization schemes.
| Scheme No. | Sterilizing Agent (%) and Exposure Time | ||||
|---|---|---|---|---|---|
| EtOH | HgCl2 | H2O2 | KMnO4 | NaClO | |
| 1 | 70% 30″ | 3% 20′ | |||
| 2 | 70% 30″ | 2% 20′ | |||
| 3 | 70% 30″ | 20% 20′ | |||
| 4 | 70% 30″ | 0.01% 20′ | |||
Germination of A. microdictyon seeds under in vitro conditions.
| Seeds | Seed Quantity, pcs. | Number of Germinated Seeds, pcs | Germination, % | |||
|---|---|---|---|---|---|---|
| 7th Day | 14th Day | 21th Day | 30th Day | |||
| ½ MC +10 mg L−1 GA3+ 0.1 mg L−1 IAA | ||||||
| Two years of storage | 300 | 0 | 0 | 1 | 4 | 1.3 |
| One year of storage | 300 | 0 | 0 | 11 | 17 | 5.7 |
| Freshly harvested | 300 | 25 | 81 | 175 | 278 | 92.7 |
| ½ MS without hormones | ||||||
| Two years of storage | 96 | 0 | 0 | 0 | 0 | 0 |
| One year of storage | 92 | 0 | 0 | 0 | 0 | 0 |
| Freshly harvested | 95 | 0 | 0 | 3 | 17 | 17.9 |
Effect of cytokinins on the induction of adventitious organogenesis.
| Medium | The Concentration of Growth Regulators, | Number of Adventitious Shoots per Explant, pcs. | Length of Adventitious Shoots, cm | |
|---|---|---|---|---|
| BAP | ZEA | |||
| MS | 0 | 0 | 1.0 ± 0 l | 1.5 ± 0.6 |
| MSPR1 | 0.1 | 0.1 | 2.0 ± 0.1 k | 1.2 ± 0.5 |
| MSPR2 | 0.1 | 0.5 | 2.5 ± 0.1 j | 2.6 ± 0.7 |
| MSPR3 | 0.1 | 1.0 | 12.2 ± 0.4 e | 2.5 ± 0.5 |
| MSPR4 | 0.1 | 1.5 | 13.7 ± 0.1 d | 2.2 ± 0.7 |
| MSPR5 | 0.1 | 2.0 | 10.3 ± 0.1 g | 3.6 ± 0.7 |
| MSPR6 | 0.5 | 0.1 | 11.5 ± 0.1 f | 1.0 ± 0.0 |
| MSPR7 | 0.5 | 0.5 | 14.8 ± 0.2 c | 4.5 ± 0.9 |
| MSPR8 | 0.5 | 1.0 | 16.1 ± 0.1 b | 3.4 ± 0.8 |
| MSPR9 | 0.5 | 1.5 | 18.4 ± 0.1 a | 9.4 ± 2.3 |
| MSPR10 | 0.5 | 2.0 | 18.2 ± 0.0 a | 6.8 ± 1.0 |
| MSPR11 | 1.0 | 0.1 | 13.7 ± 0.0 d | 2.5 ± 0.5 |
| MSPR12 | 1.0 | 0.5 | 11.3 ± 0.1 f | 1.6 ± 0.7 |
| MSPR13 | 1.0 | 1.0 | 7.7 ± 0.1 h | 5.6 ± 0.8 |
| MSPR14 | 1.0 | 1.5 | 2.6 ± 0.1 j | 9.5 ± 1.9 |
| MSPR15 | 1.0 | 2.0 | 3.8 ± 0.1 i | 4.8 ± 0.7 |
| F ratio | 75.9 | 1.0 | ||
| Sig. level | 0.000 *** | n.s. | ||
Note: *** p ≤ 0.001 denotes values significantly different from control among tested by Tukey test. Different superscript letters indicate statistically significant differences exist between the groups. The values are presented as means ± SD. n.s. means not significant.
Influence of growth regulators on the dynamics of growth and development of A. microdictyon.
| Medium | Hormones | Number of Leaves, pcs. | Leaf Length, cm | Number of Roots, pcs. | Root Length, cm | ||||
|---|---|---|---|---|---|---|---|---|---|
| 4 Weeks | 8 Weeks | 4 Weeks | 8 Weeks | 4 Weeks | 8 Weeks | 4 Weeks | 8 Weeks | ||
| MS | 1.1 ± 0.1 c | 1.5 ± 0.1 cd | 1.1 ± 0.2 | 1.1 ± 0.3 b. | 1.2 ± 0.2 bc | 1.3 ± 0.1 d | 0.7 ± 0.2 b | 0.7 ± 0.2 c | |
| MSB-IAA | 0.1 BAP | 1.2 ± 0.2 c | 1.2 ± 0.2 d | 1.3 ± 0.2 | 2.1 ± 0.2 a | 1.0 ± 0.1 c | 3.2 ± 0.1 b | 0.9 ± 0.1 b | 1.4 ± 0.1 b |
| MSB-IBA | 0.1 BAP | 2.5 ± 0.3 a | 7.2 ± 0.2 a | 1.0 ± 0.2 | 2.3 ± 0.1 a | 2.2 ± 0.2 a | 4.3 ± 0.2 a | 1.9 ± 0.2 a | 4.6 ± 0.2 a |
| MSB-NAA | 0.1BAP | 1.6 ± 0.3 bc | 1.9 ± 0.2 bc | 1.1 ± 0.2 | 2.0 ± 0.2 a | 1.5 ± 0.4 b | 2.4 ± 0.3 c | 0.7 ± 0.3 b | 1.3 ± 0.2 b |
| MSB-PAC | 0.1 BAP | 1.7 ± 0.2 b | 2.2 ± 0.1 b | 1.2 ± 0.3 | 1.9 ± 0.1 a | 2.0 ± 0.1 a | 2.6 ± 0.2 c | 0.8 ± 0.1 b | 1.1 ± 0.1 bc |
| F ratio | 13.3 | 331.9 | 1.1 | 17.9 | 19.1 | 56.4 | 48.4 | 92.1 | |
| Sig. level | 0.000 *** | 0.000 *** | n.s. | 0.000 *** | 0.000 *** | 0.000 *** | 0.000 *** | 0.000 *** | |
Note: *** p ≤ 0.001 denotes values significantly different from control among tested by Tukey test. Different superscript letters indicate statistically significant differences exist between the groups. The values are presented as means ± SD. n.s. means not significant.
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Details
; Danilova, Alevtina 2 ; Turzhanova, Ainur 1
; Khapilina, Oxana 1
1 National Center for Biotechnology, Korgalzhin hwy 13/5, Astana 010000, Kazakhstan;
2 Altai Botanical Garden, Yermakova Str 1, Ridder 070000, Kazakhstan;