Method for surface sterilization of medicagosativa l. explants for clonal micropropagation

Abstract

FIELD: biotechnology.SUBSTANCE: method for sterilizing plant explants of alfalfa ( Medicago sativa L.) with their subsequent introduction into invitro culture. The plant material used is basal segments of shoots, selected from the zone located 10-15 cm below the apical meristem of the mother plants. The segments are divided into explants of 20±5 mm in length, containing isolated axillary buds, and then subjected to a two-stage sterilization under aseptic conditions. The preliminary stage is treatment with a 70% aqueous ethanol solution for 30 seconds, and then the main stage is immersion of the explants in an 8% solution of “Belizna” with an exposure of 6 minutes and rinsing 3 times with sterile distilled water for 3 minutes. The resulting sterile plant explants are used for cultivation on Murashigeand Skoog agar nutrient medium at a temperature of 24±2 °C, 16-hour photoperiod and illumination with fluorescent phytolamps with multispectral radiation intensity of 4000 lux.EFFECT: obtaining genetically stable and infection-free plant material of alfalfa invitro.1 cl, 1 tbl, 3 ex

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to the field of biotechnology for use in agriculture, in particular to methods for sterilizing plant explants used for clonal micropropagation of plants in vitro, and can be used in selection and for mass production of improved planting material.

[0003] The invention relates to the field of plant biotechnology for use in agriculture, in particular to methods for sterilizing plant explants to obtain genetically stable and infection-free planting material in vitro.

[0004] State of the art

[0005] Alfalfa (Medicago sativa L.) is a highly productive perennial legume crop, valuable for its fodder, honey, and soil-improving properties. Due to its ability to form symbioses with nodule bacteria, it accumulates nitrogen in the soil, which makes it an important element in crop rotation [Li A. et al. Effects of alfalfa crop rotation on soil nutrients and loss of soil and nutrients in semi-arid regions / / Sustainability. - 2023. - Vol. 15. - No. 20. - P. 15164]. The crop is distinguished by its exceptional fodder value - the protein content in the green mass reaches 18-22%, which is superior to many other forage grasses. Thanks to its powerful root system, which penetrates to a depth of 3-5 meters, alfalfa effectively utilizes soil moisture and nutrients from deeper soil layers, demonstrating high drought resistance.In crop rotation, alfalfa not only enriches the soil with nitrogen (up to 300 kg / ha during the growing season), but also improves its structure, promotes the accumulation of organic matter and suppresses the development of weeds [Testa G., Gresta F., Cosentino SL Dry matter and qualitative characteristics of alfalfa as affected by harvest times and soil water content / / European journal of agronomy. - 2011. - Vol. 34. - No. 3. - Pp. 144-152]. The honey productivity of the crop is 150-200 kg per hectare, while alfalfa honey has special taste qualities and healing properties [Fernandez AL et al. Alfalfa, wildlife, and the environment / / National Alfalfa and Forage Alliance. - 2019. - Pp. 1-31]. The long-term nature of development (3-8 years of productive use) and the ability to produce 2-4 cuts per season make alfalfa an economically profitable crop for agricultural production [Tucak M. et al.Assessment of alpha populations for forage productivity and seed yield potential under a multi-year field trial / / Agronomy. - 2023. - T. 13. - No. 2. - P. 349].

[0006] The strategic importance of alfalfa as a highly productive and multifunctional crop necessitates the continuous development of new, improved varieties. In the face of a rapidly changing climate and growing demands for agricultural sustainability, traditional breeding methods, which take 10-12 years, cannot keep pace with environmental changes. Against this backdrop, genomic selection, a cutting-edge field in modern crop production, offers revolutionary opportunities. Unlike traditional approaches that require years of field testing, genomic selection enables the prediction of valuable agricultural traits through genome analysis. This makes the breeding process more efficient.

[0007] The key to successful implementation of genomic selection is the availability of high-quality assembly and accurate genome annotation. However, in the case of alfalfa (Medicago sativa L.), this task is significantly complicated by the autotetraploid nature of the species and facultative cross-pollination, which causes an exceptionally high level of heterozygosity and genetic diversity even within a single cultivar [Havananda T., Brummer EC, Doyle JJ Complex patterns of autopolyploid evolution in alfalfa and allies (Medicago sativa; Leguminosae) / / American Journal of Botany. - 2011. - Vol. 98. - No. 10. - P. 1633-1646]. The most effective approach to solving this problem is clonal micropropagation in vitro. This method allows one to obtain a significant amount of genetically identical material from one initial genotype.

[0008] In vitro clonal micropropagation is a powerful biotechnological tool that not only enables the mass production of genetically uniform planting material but also guarantees the preservation of the target traits of the selected genotype. This technology ensures genetic stability while reproducing a full range of economically valuable characteristics, including:

[0009] 1. high productivity and yield of green mass;

[0010] 2. resistance to pathogens and diseases;

[0011] 3. tolerance to abiotic stressors (drought, salinity, low temperatures);

[0012] 4. Stable feed quality with optimal biochemical composition.

[0013] Modern global practice in plant breeding and cultivation demonstrates the ever-increasing importance and application of clonal micropropagation as a method for replicating unique genotypes. This is especially relevant for perennial cross-pollinated crops, where traditional breeding and seed production methods fail to effectively preserve and propagate valuable genetic lines. The expanding adoption of biotechnological approaches in agricultural production confirms the growing demand for in vitro methods for preserving and replicating elite plant genetic material.

[0014] The main method of propagating alfalfa is by seed, and for breeding purposes, dividing the bush by dissection of the root collar is used [Bhattarai S. et al. Evaluating the effectiveness of clonal plant selection of alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) in mixtures: Mean performance and stability in a multi-environment trial / / Plant Breeding. - 2024. - Vol. 143. - No. 5. - Pp. 713-724]. However, these methods have both advantages and disadvantages. For example, when propagating by seeds, valuable genotypes are lost, since alfalfa is a cross-pollinating plant. When using the classical method of dividing the bush, the planting material may become susceptible to various fungal, bacterial and viral infections. In this regard, modern biotechnology offers a fundamentally different approach - clonal micropropagation in vitro, which allows us to overcome the limitations of both traditional methods.This technology is based on the introduction of pathogen-free explants into a sterile culture, followed by their propagation under controlled conditions. A key step in introducing them into culture is effective sterilization of the explants—a process based on the integrated use of physicochemical methods for disinfecting plant material. In particular, protocols for the use of axillary and apical meristems, stem segments, and leaf cuttings have been described for various plant species [Loyola-Vargas VM, Ochoa-Alejo N. An introduction to plant tissue culture: advances and perspectives / / Plant cell culture protocols. - 2018. - Pp. 3-13]. However, existing sterilization methods often result in a high percentage of explant mortality due to the phytotoxicity of sterilizing agents or are insufficiently effective in suppressing endogenous microflora.

[0015] The most common sterilizing agent is sodium hypochlorite (NaOCl), used in concentrations ranging from 0.5% to 5.0% available chlorine with exposure times ranging from 5 to 20 minutes [Sahu PK et al. Surface sterilization for isolation of endophytes: Ensuring what (not) to grow / / Journal of Basic Microbiology. - 2022. - Vol. 62. - No. 6. - Pp. 647-668]. However, its effectiveness is often insufficient for field samples with a high microbial load, resulting in latent contamination that manifests itself in the late stages of cultivation. Furthermore, even with successful sterilization, high concentrations and prolonged exposure can cause phytotoxicity, burning of meristematic tissues and inhibition of subsequent rhizogenesis.

[0016] Another widely used agent is hydrogen peroxide (H2O2), in concentrations of 5-15%. Although it is less toxic to plants compared to some other sterilizing agents, its main disadvantage is low efficiency against fungal contaminants and spores, which limits its use as a single agent [Köse M. Ş. H., Doğan M., Sadi G. Surface sterilization of Staurogyne repens (Nees) Kuntze with hydrogen peroxide / / Bulletin of Biotechnology. - 2020. - Vol. 1. - No. 2. - P. 39-42].

[0017] For difficult-to-sterilize species and heavily contaminated material, solutions of mercury salts (e.g., corrosive sublimate, HgCl2) are sometimes used; they exhibit high antimicrobial activity. However, their use is associated with extremely high phytotoxicity, leading to massive necrosis of explants, and also poses a serious environmental and toxicological hazard to personnel. In addition, residual amounts of mercury can have a mutagenic and inhibitory effect on regeneration processes [Gu M. et al. Efficient in vitro sterilization and propagation from stem segment explants of Cnidoscolus aconitifolius (Mill.) IM Johnst, a multipurpose woody plant / / Plants. - 2022. - Vol. 11. - No. 15. - P. 1937].

[0018] Alfalfa protocols often describe combined sterilization methods, such as pretreatment with 70% ethanol followed by sodium hypochlorite. However, existing descriptions do not describe the parameters of a regime that ensures high sterilization efficiency for clonal micropropagation of difficult-to-sterilize field samples. Optimal parameters that simultaneously ensure maximum sterility (minimal contamination) and maximum explant viability (minimal necrosis) for basal segments of alfalfa shoots have not been established. Uncertainty in parameters leads to high contamination rates or, conversely, to material loss.

[0019] Field samples characterized by a high degree of contamination with microorganisms are particularly challenging for micropropagation [Babu GA et al. Improved sterilization techniques for successfulin vitromicropropagation / / Commercial scale tissue culture for horticulture and plantation crops. - Singapore: Springer Nature Singapore, 2022. - Pp. 1-21]. Due to these difficulties, an optimized protocol was developed to minimize contamination when working with field samples, including the selection of the most regeneratively competent tissues and their subsequent processing [Borodayeva Zh. A., Chernyavskikh V.I., Dumacheva E.V. Study of the features of introducing individual selections of Medicago varia Mart into in vitro culture for accelerated propagation of breeding samples / / Fruit and berry growing of Russia. - 2019. - Vol. 59. - Pp. Basal segments of shoots, selected 10-15 cm below the apical meristem of maternal plants, were used as the starting material.The segments were divided into single-node explants 20±5 mm long with an axillary bud, which were subjected to multi-stage sterilization.

[0020] Thus, the presented data confirm the need to develop specialized sterilization protocols for field samples of alfalfa, combining the selection of morphogenically competent tissues with optimized chemical sterilization regimens.

[0021] Disclosure of the essence of the invention

[0022] The technical result of the invention is a method for sterilizing vegetative explants of alfalfa, ensuring a high percentage of material survival with a minimum level of contamination.

[0023] In its first embodiment, the invention involves preparing explants. For in vitro culture, basal segments of alfalfa shoots, selected 10-15 cm below the apical meristem of the mother plants, were used. The segments were divided into single-node explants 20±5 mm long with an axillary bud. After selection, the plant material was pre-washed for 30 minutes in running water with a 0.1% detergent solution.

[0024] In the second embodiment, the invention features the sterilization of explants performed under aseptic conditions. The first step of this embodiment involves treating the plant material with a 70% aqueous ethanol solution for 30 seconds. The next step of this embodiment* is sterilizing the explants by immersing them in an 8% bleach solution for 6 minutes. The final step of this embodiment involves washing the explants with sterile distilled water, with periodic stirring, to remove the sterilizing agent.

[0025] In its further embodiment, the invention is a confirmation of the preservation of the viability and morphology of the explants.

[0026] In another embodiment, the invention consists of introducing sterile explants into in vitro culture and confirming the absence of visible signs of microbial contamination after 10-14 days of cultivation.

[0027] Brief description of figures and tables

[0028] Table 1. Influence of the method and time of sterilization on obtaining an aseptic culture of Medicago sativa L.

[0029] Implementation of the invention

[0030] The purpose of the invention was to develop an optimal method for sterilizing the vegetative part of alfalfa (Medicago sativa L.).

[0031] The invention is aimed at developing a method for sterilizing vegetative explants of alfalfa, ensuring the preservation of a high percentage of material survival with a minimum level of contamination, with their subsequent cultivation on an agarized Murashige-Skoog nutrient medium.

[0032] Experimental studies demonstrated the high regenerative capacity of basal segments of alfalfa shoots, collected 10-15 cm below the apical meristem. For in vitro culture, these segments were mechanically fragmented into single-node explants 20±5 mm long, containing isolated axillary buds.

[0033] These segments are characterized by:

[0034] 1. Increased ability to rhizogenesis;

[0035] 2. Developed meristematic activity;

[0036] 3. Reduced sensitivity to aggressive chemical influences;

[0037] 4. Less surface microflora compared to the apical parts.

[0038] To select a sterilization regimen for the initial explants, a comparative analysis of various sterilizing agents, their concentrations, and exposure times was conducted. To compare the effectiveness of the various sterilization regimens, 20 explants were used for each treatment. The criteria for sterilization regimen effectiveness were the percentage of explant survival after sterilization and the absence of visible signs of microbial contamination after 10-14 days of cultivation.

[0039] In the process of implementing the invention, the conditions for sterilization of explants were selected, and sterilizing agents and processing modes were also studied:

[0040] 1. Pre-treatment: All explants were washed with running water for 30 minutes with the addition of 0.1% detergent solution before sterilization.

[0041] 2. Sterilization modes: different modes were compared, such as 10-minute exposure of explants to a 10% hydrogen peroxide solution, 15-minute exposure to a 15% hydrogen peroxide solution, 1-minute, 2-minute, or 6-minute exposure to a 0.1% mercuric chloride solution, 8-minute exposure to a 6% Bleach solution (Sanfor, article 1966, sodium hypochlorite solution), and 6-minute exposure to an 8% Bleach solution.

[0042] 3. Sterilization procedure: explants were pre-treated with 70% ethanol for 30 seconds, then treated with the indicated sterilizing solutions. The final step was rinsing the explants three times with sterile distilled water. The process of treatment with the sterilizing solution and rinsing with water was carried out with periodic agitation.

[0043] 4. Evaluation of the effectiveness of the influence of time and methods of sterilization on obtaining aseptic culture (Table 1).

[0044] A comparative analysis of the effectiveness of various sterilizing agents was carried out:

[0045] When using corrosive sublimate (0.1%) the following was observed:

[0046] 1. Massive tissue damage (necrosis in 80-100% of explants);

[0047] 2. Low level of latent contamination (0-20%);

[0048] 3. Pronounced stress effect on plant tissues.

[0049] When using hydrogen peroxide (10-15%):

[0050] 1. Insufficient effectiveness against fungal contaminants;

[0051] 2. The contamination level reached 45-65%;

[0052] 3. Tissue necrosis in 25-50% of explants.

[0053] The results of the comparative analysis presented in Table 1 showed the advantage of using an 8% solution of “Belizna” for sterilization of plant explants, which ensured the maximum yield of viable explants (85%) with a minimum level of contamination (15%), as well as complete preservation of morphogenic competence (95%) and genetic stability of regenerants (100%).

[0054] The technical and economic advantages of the developed method are the use of an available reagent, reduction in the duration of the sterilization stage, simplification of the technology due to the exclusion of hormonal treatments and increased reproducibility of the results.

[0055] The invention will now be illustrated by examples, which are intended to provide a better understanding of the essence of the claimed invention, but should not be considered as limiting this invention.

[0056] Example 1. Collection and preparation of plant explants.

[0057] The most suitable segments for sterilization and subsequent micropropagation are single-node segments, which are selected from the lower and middle parts of the shoot, 10-15 cm below the apical bud. The optimal segment length is 20±5 mm. The segment should contain the axillary bud and adjacent internodal areas. The shoots are cut with a sterile scalpel or blade. The explants are then washed under running water for 30 minutes with a 0.1% detergent solution.

[0058] Example 2. Sterilization of plant explants.

[0059] Prepared explants (Example 1) are treated with 70% ethanol for 30 seconds and soaked in an 8% bleach solution for 6 minutes, stirring occasionally. The explants are then rinsed three times with sterile distilled water, stirring occasionally.

[0060] Example 3. Introduction of explants into in vitro culture.

[0061] Sterile plant explants obtained in Example 2 are cultured on Murashige-Skoog agar medium for 10-14 days at 24±2°C and a 16-hour photoperiod. The photoperiod is provided by fluorescent phytolamps with multispectral radiation of 4000 lux.

[0062] Thus, the advantages of the claimed method of sterilizing plant explants are the use of an accessible reagent, a reduction in the duration of the sterilization stage, simplification of the sample preparation technology by eliminating hormonal treatments and an increase in the reproducibility of the results.

[0063] It was found that the optimal sterilizing agent is sodium hypochlorite in the form of the household product “Whiteness” at a concentration corresponding to 8% (8% “Whiteness” solution), with an exposure time of 6 minutes.

[0064] Benefits of using "Whiteness":

[0065] 1. Minimum level of tissue necrosis (no more than 20%);

[0066] 2. Low contamination rate (15-30%);

[0067] 3. Absence of pronounced stress impact on explants;

[0068] 4. Maintaining genotype stability during micropropagation;

[0069] 5. Rapid adaptation and onset of regeneration in vitro.

Claims

A method for sterilizing explants of Medicago sativa L. for introduction into in vitro culture, including the preparation and sterilization of plant material, characterized in that, when preparing the explants, alfalfa shoots with axillary buds are used, selected 10-15 cm below the apical meristem of the mother plant, washed in running water with the addition of a 0.1% detergent solution for 30 minutes, and the sterilization of the explants occurs in two successive stages, the first of which is treatment with a 70% aqueous ethanol solution for 30 seconds, and the second is keeping the explants for 6 minutes in an 8% solution of "Belizna" with periodic stirring, followed by three washes with sterile distilled water for 3 minutes each.

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