Micropropagation Technology Research Articles

A novel in vitro shoot excision technique for enhancing proliferation in banana cv. Chini Champa

Micropropagation technology has been commercially exploited for mass multiplication of banana. Several parameters such as nutrient media, explants, culture conditions etc have been standardized. However, a novel in vitro shoot excision technique designed to enhance the proliferation rate of banana cv. Chini Champa has been examined for the first time. We meticulously examined the impact of excision angles (45º and 90º) and excision site (Tip, middle and base) during shoot proliferation stage. Our findings unequivocally demonstrate that employing a 45º angle excision and tip excision yield the highest multiplication rates and biomass accumulation, surpassing other excision angles and methods. The substantial enhancement in shoot numbers, growth and biomass underscores the potential of this technique for improving banana propagation protocols, offering a valuable tool for sustainable banana production.

Improvement of the clonal micro-propagation technology elements for raspberries of various fruiting types

The results of research on clonal micropropagation of 17 raspberry varieties of the Altai and European selection are presented. Recently, researchers have been focusing on creating a universal nutrient medium that would be equally suitable for any variety of a particular plant species. This is especially important for mass clonal micropropagation in production nurseries. The regeneration potential of the raspberry varieties of different types of fruiting at each stage of reproduction has been assessed. At the stage of actual micropropagation, it was found that Driver and Kuniyuki medium in our modification was superior to the Murashige and Skoog medium in terms of the shoot length and averaged 9.0 mm for common type varieties and 12.3 mm for remontant varieties. This made it possible to eliminate the additional stage of elongation of the microshoots. Also, the appearance of microshoots improved on the new medium. It was found that for raspberry, regardless of its type, the optimal concentrations of the growth regulators lie in the following range: cytokinin 6-benzylaminopurine (BAP) 2.5–3.5 μM together with auxin ß-indolyl butyric acid (IBA) 0.5–0.7 μM. At the stage of rhizogenesis, the best results were shown by the Driver and Kuniyuki medium with 2-fold reduced content of all main components and with the addition of 2.0 μM IBA at the passage duration of 21 days. After that, all microcuttings with and without roots underwent the adaptation stage – growth completion in neutral substrates for 56 days under ex vitro conditions in a vegetation room. In most cases, there were no statistically significant differences in the length and number of leaves regardless of the presence or absence of roots at the initial stage of adaptation. By the end of this stage, 100% of the adapted raspberry plants of the usual type of fruiting and 98% of the remontant type were obtained.

INFLUENCE OF CULTIVATION CONDITIONS ON THE GROWTH AND ACCUMULATION OF BIOLOGICALLY ACTIVE SUBSTANCES OF HYSSOPUS OFFICINALIS L. UNDER IN VITRO CONDITIONS

Introduction. Since ancient times, aromatic plants have been widely used to treat diseases of various natures due to the content of valuable metab-olites and essential oils (EOs). In modern medicine, cosmetology and the food industry, the importance of aromatic plants is also not in doubt; the chemical composition, accumulation features, synthesis and properties of EO are being actively studied. With the development of clonal micropropaga-tion technology, it has become relevant to study the accumulation of biologically active substances in aseptic in vitro culture (microclones and cell cul-tures) under various propagation protocols. Material and methods. Hyssop (Hyssopus officinalis L.) is a common aromatic plant from the family Lamiaceae Martinov. The aerial parts and EO of the plant have a wide range of biological activities - anti-inflammatory, antioxidant, antimicrobial, etc., which determines the use of plant biomass and extracts in food and light industries, medicine, and pharmacology. In this work, the influence of the mineral and hormonal composition of nutrient me-dia on the growth of aseptic plants of two cultivars of Hyssopus officinalis – Lekar and Iney – was studied. Results and conclusion. A comparison was also made of the accumulation of the total flavonoids in an aseptic culture depending on the cultivation mode - the degree of aeration of the culture vessel and the addition of cytokinin or auxin components to the nutrient medium. It was found that the leaves of aseptic plants of Hyssopus officinalis accumulate more flavonoids (up to 3.6 mg/g fresh weight) than the stems (0.3–1.1 mg/g fresh weight for most experimental variants), in addition, the cv. Lekar was significantly richer in compounds of this group (2.7–3.6 mg/g fresh weight in leaves) compared to the cv. Iney (no more than 1.5 mg/g fresh weight in leaves).

ВВЕДЕНИЕ В КУЛЬТУРУ IN VITRO И МИКРОКЛОНАЛЬНОЕ РАЗМНОЖЕНИЕ БОЛЬШЕГОЛОВНИКА СЕРПУХОВИДНОГО STEMMACANTHA SERRATULOIDES (GEORGI) M. DITTRICH

Stemmacantha serratuloides (Georgi) M. Dittrich is a perennial herbaceous medicinal plant, a source of a number of phytoecdysteroids with adaptogenic, anabolic and tonic effects. Due to the rarity of this plant species and the strong dependence of its seed productivity on weather conditions, the relevance of research aimed at its biotechnological cultivation and reproduction is high. The aim of this study was to introduce S. serratuloides plants from the Cis-Ural population of the Republic of Bashkortostan into in vitro culture and to develop an effective protocol for micropropagation of this species. It was shown that an effective method of preparing seeds for in vitro culture is their successive sterilization with 96% ethanol (1 min) and 20% bleach (20 min), as well as me- chanical scarification, without a preliminary stratification step. When using 1 mg/l of 2,4-dichlorophenoxyacetic acid and 1.5 mg/l of 6-benzylaminopurine on Murashige-Skoog medium, no regeneration of shoots on explants of cotyledons and hypocotyls occurred. Shoot regeneration was induced from root explants using Murashige-Skoog medium supplemented with 6-benzylaminopurine and indoleacetic acid at concentrations of 1 mg/l and 1.5 mg/l, respectively. Rooting of shoots was carried out on MS medium containing 0.25 mg/L IAA. This micropropagation technology can be used for rapid propagation of plants for various biotechnological purposes, for example, for further transformation by Agrobacterium rhizogenes and creating of hairy root cultures.

ДИНАМИКА РАЗМНОЖЕНИЯ ПОДВОЕВ ВИНОГРАДА В КУЛЬТУРЕ IN VITRO

In connection with the extension of areas under vineyards, it becomes necessary to use rootstocks, resistant to certain soil and climatic conditions (high content of active lime and salts in the soil, drought). Growth of the assortment of rootstocks available for propagation is one of the most important strategies of domestic nursery farming development, along with the production of improved planting material and better performance of its quality indicators. Today it is important to optimize clonal micro-propagation technology for promising grape rootstocks in order to increase net reproduction while maintaining the genetic identity of planting material. The purpose of the work was to study the dynamics of morphogenesis of grape rootstocks in the system in vitro to optimize the technology of clonal micro-propagation, taking into account the genetic specificity of particular genotypes. The study materials were the in vitro plants of grape rootstocks: Fercal clone 242, Ruggeri 140, and Gravesac (clones 11 and 12). The methods accepted in biotechnology and developed in the Breeding Department of the Institute Magarach were both used in the process of research. Cultivation was carried out on PG medium supplemented with NAA (α-naphthylacetic acid) 0.05 mg/l. The results showed that the dynamics of morphogenesis of rootstocks Ferсal clone 242, Gravesac clone 11 and Gravesac clone 12, analyzed on the basis of net propagation, did not differ significantly by varieties, with the exception of the second passage. The study of morphogenetic potential of Ruggeri 140 rootstock bud made it possible to reveal the dependence of net propagation on the line, it was especially noticeable in the first passage. Separate lines showed high net propagation in the second passage. The average indicator value for all lines was 2.59. In general for all rootstocks, similar results were obtained in successive passages, indicating a tendency for net propagation to be decreased. In order to increase the efficiency of technology, it is necessary to optimize the conditions of cultivation for industrial propagation, taking into account the genetic characteristics of every rootstock.

Acacia mangium × A. auriculiformis micropropagation in a non-sterile environment

ABSTRACT Autoclaving eliminates microbial contamination during micropropagation, but the process is complex, time-consuming and expensive. Chemical sterilisation also effectively disinfects culture media and is relatively simple and cost-effective. Prior studies have focused on the effects of chemical sterilisation on bud induction, but the effects of sterilant on proliferation and rooting are unknown. We investigated the effect of sterilant on Acacia mangium × A. auriculiformis bud induction, rooting and subculture rooting. The optimal bud induction medium comprised 1/8 Murashige and Skoog medium + 7 g l−1 agar + 0.2 g l−1 chlorothalonil + 0.5 mg l−1 6-benzylaminopurine. The maximum bud induction rate (99.54%) with zero contamination was achieved using the third to fifth stem segments collected in October and treated with 0.8 g l−1 carbendazim for 3 min. The maximum rooting rate (97.62%) was attained using a rooting medium consisting of 7 g l−1 agar + 0.2 g l−1 chlorothalonil + 1.5 mg l−1 indolebutyric acid + 0.5 mg l−1 naphthaleneacetic acid. Proliferation ratio and subculture duration were positively correlated. The maximum proliferation rate (3.58%) was realised in the fourth subculture rooting. Chlorothalonil can effectively replace autoclaving of A. mangium × A. auriculiformis bud induction and rooting media. The present study provides insights for improving the rapid propagation method of Acacia sp. and a new direction for the development of micropropagation technology.

Protocol for the Acclimatization of Coconut Vitro-Plants Under Ex Vitro Conditions.

The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.

Features of reproduction in vitro of Silene cretacea and Silene hellmannii

This article provides information about improving of the clonal micropropagation technology and in vitro reproduction peculiarities of Silene cretacea and S. hellmannii. As a result of the research, optimization of cultivation conditions at different stages of clonal micropropagation of S. cretacea and S. hellmannii cultures was carried out. The optimal mode of sterilization seeds of species S. cretacea and S. hellmannii has been selected - a 5 % solution of Lysofornin® 3000 at an exposure of 7 minutes. The maximum germination of S. hellmannii seeds was 75 % observed on day 11 of the experiment, and for S. cretacea 50 % on day 14. At the same time, the germination percentage of S. hellmannii is 30 % higher than that of S. cretacea. The highest reproduction coefficient for S. cretacea and S. hellmannii was observed when using Cytodef at a concentration of 0.5 mg/l. It was 23.2 ± 0.7 and 26.2 ± 0.4, respectively. As a result of the conducted research, optimal conditions for the long-term preservation of regenerating plants have been selected. Plants are stored in vitro in a climatic chamber SANYO MRL-351H on a nutrient medium of У MS supplemented with 6-BAP 0.4 mg/l, sucrose (20 mg/L), t (3-7 °C) and illumination (500-1200 lk).

Agricultural-based challenges, genetic enhancement, and obstacles to an industrially important medicinal plant, ashwagandha (Withania somnifera (L.) Dunal): A review

In the pharmaceutical enterprise, traditional/herbal remedies and commercial drug sources Withania somnifera and Withania coagulans received excellent scores. Numerous studies have been conducted on the phytochemistry and pharmacology of Withania somnifera (L.) Dunal. However, the toxicity of plant components extracted using different solvent systems is yet to be ascertained. It is important to find out and isolate active ingredients. Advanced analytical and separation chemistry studies will reveal data on the toxicity, isolation of novel compounds, chemotypic diversity, and ethnobotany of the Withania somnifera (L.) Dunal. Modern tools of speed breeding can be applied, and micropropagation technology can be developed and used to ensure the supply of superior germplasm/improved varieties with genetic fidelity for farming. Genomics-assisted breeding, transgenic technologies, and gene-, genome-, and epigenome editing approaches may be considered new potential tools for metabolic engineering beneficial compounds from Withania somnifera (L.) Dunal.

Liquid Culture System: An Efficient Approach for Sustainable Micropropagation

Micropropagation of important forestry, horticultural and medicinal plants have made revolutionary changes in terms of research and commercialization. However, there are a variety of factors which influence the scaling-up and commercialization aspects, which decide whether mass propagation will be effective and lucrative. Low rates of shoot multiplication, increased costs of media components, loss of cultures due to contamination, and difficulties with hardening and acclimation are the key obstacles to scaling up micropropagation technology. These restrictions have forced a large number of in vitro technologies developed for a range of plant species to be used only under research laboratories settings. To apply tissue culture technology to large-scale propagation, it is required to develop techniques that are relatively simple to adopt, have high multiplication rate with high levels of reproducibility, and exhibit higher survival of plantlets when transferred to ex vitro conditions. Efficient techniques include utilization of liquid culture systems and replacement of agar with other gelling agents. These techniques allow development of micropropagules that not only function better in post-vitro soil conditions and are comparatively less expensive, but will also help develop a workable micropropagation technique that can be applied to the mass production of desirable plant species. The current review describes liquid culture system as an efficient approach to produce large number of plants at low production cost.

Розмноження та укорінення клонових підщеп сливової групи в умовах культури in vitro

Stone crops of the plum group are one of the important economic crops in horticulture of Ukraine. Cultivation of plum, myrabolan, peach and apricot fruit trees is a high-tech process that demands use of planting material based on clonal rootstocks. The rootstock should be characterized by good compatibility with various varieties, ensure uniformity in the garden, appropriate vigor strength, early fruit bearing, high resistance to biotic and abiotic factors etc. Traditional propagation of Prunus spp. is time-consuming and limited by a short growing season and harsh winter conditions. Rootstocks Wavit and Myrabolan 29C have prospects for use in horticulture in Ukraine, so our objective was to optimize their micropropagation technology. The optimal composition of the nutrient medium for micropropagation of two clonal rootstocks of the plum group was established, the influence of different cytokinin concentrations on proliferation of these rootstocks was investigated. The highest coefficients of shoot formation for rootstocks Myrabolan 29C and Wavit were obtained on media MS with 1 mg/l BAP and QL with 1.5 mg/l BAP, respectively. The effect of different concentrations of auxin on the rhizogenesis process was also studied. The optimized medium for rooting of Myrabolan 29C was ½ MS in combination with 1.0 mg/l IBA, which ensured rooting of 90 % of microshoots with well-developed roots, it is also possible to use MS medium with 0.75 mg/l NAA, which provides 100 % of rooted microshoots, but the development of the root system is weaker. The best auxin for Wavit was naphthylacetic acid, but its effect needs to be further investigated. Key words: rootstock, stone crops, propagation, rooting, cytokinin, auxin, medium.

Screening of different Fe(III) and Fe(II) chelate complexes in clonal micropropagation technology of highbush blueberry (Vaccinium corymbosumL.)

The element iron plays a key role in plant physiology. For plant nutrition, chelated forms of iron are preferred. Therefore, in clonal micropropagation media, FeSO4 x 7H2O is usually introduced together with Na2EDTA. At the same time, other effective iron chelates with both carboxyl and phosphorus chelates are known. So far, there are few scientific studies on the effect of iron chelates complexes on blueberry plants underin vitroconditions. Therefore, studies were conducted with different iron chelate complexes in 4 concentrations on highbush blueberry (Vaccinium corymbosumL.) cv. Brigitta Blue in clonal micropropagation technology. The best results were obtained using Fe(III)-DTPA in increased (×1.5) and doubled (×2.0) concentrations, where the multiplication factor was 4.59-4.65.

Features of in vitro cultivation of rare species of the family Iridaceae Juss.

This article provides information about improving of the clonal micropropagation technology and in vitro reproduction peculiarities of some species of family Iridaceae Juss. In vitro collection of Volgograd regional botanical garden (VRBG) contains 6 species of genus Iris. As a result of the research, optimization of cultivation conditions at different stages of clonal micropropagation of species genus Iris L. cultures was carried out.
 The optimal mode of sterilization seeds of species of the genus Iris has been selected - a 5% solution of Lysofornin® 3000 at an exposure of 10 minutes. The features of the development of Iris notha and Iris pseudonotha seedlings on nutrient media with a high content of cytokinins and auxins have been studied. The greatest regenerative activity in Iris pseudonotha and Iris notha was observed on the nutrient media У MS 2 iP 1.0 mg/l + 0.5 mg/l IBA and MS + 13 mg/l BAP + 7.0 mg/l IAA, the reproduction coefficient was 7.5 and 4.7, respectively. The optimal nutrient medium for Iris pumila for long-term cultivation in vitro is the Knudson nutrient medium; regenerate plants have a slow uniform growth, while the number of abnormal and non-viable plants is minimal, which allows Iris pumila to be preserved in the collection in vitro for a long time.

Micropropagation of the endangered species Malus niedzwetzkyana for conservation biodiversity in Kazakhstan

Biodiversity conservation requires advanced and effective ex situ plant propagation techniques. The present study was conducted to optimize the micropropagation of a rare and endangered species of Malus niedzwetzkyana. The objective was the ex situ conservation and reintroduction, via micropropagation, of this species. Further, we aimed to obtain healthy planting material on a large-scale for commercial implementation. To study shoot multiplication, we examined the effects of three cytokinins (6-benzylaminopurine, kinetin, and thidiazuron) supplemented to Quoirin–Lepoivre (QL) culture medium. To examine the rooting of the shoots, we tested the effects of the macronutrient concentration of the QL medium (at 1×, 0.5×, and 0.25× concentration), and of its sucrose content (0%, 10%, 20%, and 30%). The optimized micropropagation technology provided a high propagation rate (28.77 new shoots per explant) on QL medium supplemented with 0.5 mg L−1 6-benzylaminopurine and 0.01 mg L−1 indole-3-butyric acid (IBA). All shoots developed roots on 0.5× QL media with the addition of 10 mg L−1 sucrose and 1.5 mg L−1 IBA, producing a mean of 11.8 roots per explant. Using nine pairs of simple sequence repeat primers, no somaclonal variation was detected among the plantlets produced using the 13 treatments. Thus, the proposed technology makes it possible to preserve and reproduce the valuable genetic material of the M. niedzwetzkyana in the laboratory and natural conditions. This study demonstrates how biotechnology can be effectively used as an ex situ conservation strategy for this endangered species.

Establishment of a micropropagation supporting technology for the Fraxinus mandshurica × Fraxinus sogdiana

The interspecific hybridization can take advantage of heterosis and combine the double parental traits most extensively, and is an effective strategy for improving species and stress tolerance breeding for cross-pollinated trees. Species of Fraxinus L. are important for landscaping, timber, and afforestation tree species. To solve problems of poor cold adaption associated with the introduction of Fraxinus sogdiana in Maoershan (Heilongjiang province, Northeast China), to improve pest resistance of Fraxinus mandshurica, and to take advantages of the characteristics of F. mandshurica and F. sogdiana, the interspecific hybridization of F. mandshurica × F. sogdiana was conducted. Tissue culture is the key technology for the promotion of excellent clones and is a powerful tool for germplasm conservation of endangered or valuable plant species. Basing on the embryo culture of hybridization seeds, a complete and efficient micropropagation technology of the Fraxinus mandshurica × Fraxinus sogdiana for its production was established by the uniform design, regression analysis, and verification tests for optimal conditions. In this micropropagation technology, induction rates of callus, adventitious shoot, and adventitious root were 83.5%, 65.9%, and 80.0%, respectively; the number of adventitious shoots was 12.5 produced per callus and the survival rate of the seedlings grew in the greenhouse could be 91.3% after acclimatization. These findings enriched the germplasm resources of Fraxinus species, supplied a micropropagation supporting technology for production of F1 hybrids with ideal traits and accelerated the genetic crossbreeding process of F. mandshurica.

Composition and structural characterization of pectin in micropropagated and conventional plants of Premma puberula Pamp.

The metabolites produced by plants can be enhanced by plant tissue culture. In Premma puberula Pamp., the pectin content in leaves is 30 %–40 %, and it is widely used in the food industry and medicine. However, inefficient propagation has seriously restricted the utilization of pectin resources. Therefore, we established an efficient micropropagation technology for P. puberula through comparative analysis in mature leaves of regenerated and conventionally propagated plants. The results showed that the pectin composition of their leaves was similar in terms of galacturonic acid, monosaccharide composition, degree of esterification, functional groups, nuclear magnetic resonance spectrum and morphological characteristics. Furthermore, micropropagated plants had better hardness, gumminess and chewiness characteristics than conventionally propagated plants and were similar in emulsion stability, adhesiveness, springiness, cohesiveness and viscoelasticity. Therefore, micropropagation technology will provide an important guarantee for the industrial production of pectin from P. puberula. The technical essentials include callus induction, embryoid formation, and root induction, followed by acclimatization and transplanting.

Obtaining micro cuttings of potatoes by clonal micropropagation

At present, high-quality healthy planting material of potato tubers cannot be obtained without the use of clonal micropropagation technology, with the help of which, through micro-cuttings, it is possible to recover from viral, viroid, bacterial and fungal infections and mass propagation of economically valuable varieties. The wrong choice of explants and ineffective sterilization are the most common causes of suppression of the growth of microplants, bacterial infections, necrosis of shoot tips, habituation and growth of tissues, lack of rooting at the stage of rhizogenesis and successful acclimatization at the stage of adaptation to non-sterile conditions, etc.

Clonal micropropagation of decorative cereal <i>Molinia caerulea</i> (L.) Moench

The research is devoted to the development of clonal micro-propagation technology of the decorative cereal Molinia caerulea (L.) Moench. The concentration of 2.0 mg /l of cytokinin 6-benzylaminopurine (6-BAP) contributed to obtaining the maximum number of microshoots: 6.3 and 7.9 pcs. on Anderson's and Murashige-Skoog's (MS) media, respectively, which exceeded the control (by 4.7 and 6.3, respectively, with LSD05 = 2.3). The length shoots were observed on hormone-free media, this indicator significantly decreased with an increase in 6-BAP content. On Anderson and MS media with 1.0 mg/l 6-BAP, the shoot length averaged 21.5 and 26.4 mm, respectively, which made it possible to transplant them for rooting, bypassing planting on a medium for elongation. The inclusion of the Siliplant micro-fertilizer in the MS medium at doses of 1.0 and 2.0 ml/l contributed to a significant increase in shoot size, by 16.7 and 10.7 mm (LSD05 = 8.9), respectively, in comparison with the control (MS). It is recommended to use Anderson's medium and 0.5 mg/l of indole-3-acetic acid as a medium for rhizogenesis: after two weeks of cultivation, the regenerants had a standard appearance with developed roots suitable for planting for adaptation. At the adaptation stage, watering the substrate with the biofungicide «Trichoderma veride» according to the instructions and a cereals single spraying with the micro-fertilizer «Siliplant» at a dose of 1.5 ml/l contributed to their 100 % survival rate.

In vitro culture and rapid propagation of Ilex asprella

Ilex asprella is usually used as the main material for a variety of traditional Chinese medicines and herbal tea. With the destructive excavation of the roots of I. asprella, its wild resources have been drastically reduced and is not available in quantity to meet the current market demand. In order to meet the needs of Chinese medicine market and environmental protection, it is necessary to develop a suitable and rapid micro-propagation technology. In this study, we have established an effective in vitro tissue culture protocol for mass production of I. asprella seedlings. Stems were used as explants and 0.1% mercuric chloride solution treating the explants for 7 min was the best disinfecting method. The optimum medium for induction and proliferation of adventitious buds was developed with modified MS medium with 2.0 mg/L 6-benzylaminopurine (6-BA), 0.1 mg/L 1-naphthaleneacetic acid (NAA) and 0.5 mg/L adenine sulfate (AD). The modified ½ MS medium with 0.5 mg/L NAA, 0.5 mg/L indole-3-butyric acid (IBA) and 0.5 mg/L activated carbon was suitable for rooting. The method established in this study provides the basis for large-scale propagation I. asprella seedlings to meet the market demand of traditional Chinese medicine.

Vitrification during development of clonal micropropagation technology for rare plant species of Salvia scabiosifolia Lam.

Aim. Anatomical structure comparison of normal and vitrified Salvia scabiosifolia Lam plants in vitro; nutrient medium growth regulator selection for vitrification minimization. Methods. In vitro culture methods were used. Plants were grown on MS nutrient medium, enriched by peroxyacetic and indoleacetic acids and BAP with different combinations and concentrations. Anatomical leaf slices 10 µm thick were dyed with aceto-orcein and safranin. Results. Anatomical differences between thew structures of normal and vitrified S. scabiosifolia leaves were detected. Minimal vitrification was achieved by using nutrient mediums with 0.2 mg/l IAA + 1 mg/l BAP; 0.2 mg/l IAA + 2.5 mg/l BAP; 0.2 mg/l IAA + 0.1 mg/l NAA + 2 mg/l BAP. Conclusions. Anatomical structural features of vitrified leaves indicate abnormal transpiration, division and growth speed of epidermis and mesophyll cells. Vitrification can be lowered by variation of growth regulation content in the nutrient medium. Major role in causing vitrification in S. scabiosifolia is played by auxins, but combining them with cytokinins lowers vitrification significantly.
 Keywords: Salvia scabiosifolia Lam., in vitro, plant anatomy, vitrification, auxins, BAP.