Two-phase method for producing potato microtubers using medium enriched with beta-aminobutyric acid (BABA) and proline

Abstract

FIELD: plants biotechnology.SUBSTANCE: two-phase method for obtaining potato microtubers is proposed, excluding the use of hormones. In the metabolic “priming” phase – 7–10 days, plants are cultivated on MS medium with the addition of BABA – 25–50 mg / l and proline – 100–200 mg / l. Then, without replanting, the medium is replaced with an induction medium (MS with sucrose 100–120 g / l, without growth regulators, with Fe-EDDHA). Cultivation is carried out in the dark at 19 °C. The invention allows for synchronization of tuber formation up to 98%, producing uniform tubers – 8–12 mm with an increased dry matter content of up to 23% and high shelf life.EFFECT: protocol is simplified due to the non-transplantation and stability of the Fe-EDDHA medium.7 cl, 1 tbl, 2 ex

Description

[0001] The invention relates to plant biotechnology, namely to methods of accelerated clonal micropropagation and production of potato seed material in vitro culture through induction of microtuber formation.

[0002] Various methods for inducing potato microtubers in vitro are known. The most common of these include:

[0003] – Use of growth regulators (hormones): Addition of cytokinins (6-BAP, kinetin) or jasmonic acid and its derivatives to the nutrient medium. Disadvantages: high cost of hormones, risk of somatic variations, residual hormone levels in the resulting microtubers, which can affect their physiology and subsequent germination.

[0004] – Use of high sucrose concentrations (osmotic stress): Use of media containing 60-100 g / L of sucrose. Disadvantages: poor synchronicity of tuber formation, formation of tubers of different sizes, frequent death of some explants due to severe stress, need for precise control of conditions.

[0005] – Combination methods: A combination of high sucrose levels with lower temperatures and / or a short photoperiod. Disadvantages: increased cycle length, difficulty in control, and genotype-dependent effectiveness.

[0006] A common drawback of many protocols is the stage of mechanical transplantation of explants from one medium to another, which increases the risk of microbiological contamination and trauma to the material.

[0007] The technical problem solved by the invention is to create a highly efficient, synchronous and technically simple method for obtaining aligned potato microtubers with increased shelf life, eliminating the use of exogenous growth regulators and minimizing mechanical impact on plants.

[0008] The technical result consists of achieving a record high synchronicity of tuber formation (up to 98%), obtaining aligned microtubers with a diameter of 8-12 mm with an increased dry matter content, increasing shelf life during storage, as well as simplifying the protocol by eliminating the transplantation stage and using a stable nutrient medium.

[0009] To solve this problem, a two-phase method for producing potato microtubers is proposed. The novelty of this method lies in the concept of metabolic "priming" (preparation) of the plants during the first phase.

[0010] Phase 1 (Priming): Cultivation of microshoots on a medium enriched with a combination of the non-protein amino acids BABA (a primer for systemic acquired resistance) and proline (an osmoprotectant and signaling molecule). This combination gently induces a state of stress readiness, activating defense and metabolic pathways that are subsequently redirected toward storage function without inhibiting biomass growth.

[0011] Phase 2 (Induction): Direct transfer to a medium with a very high sucrose concentration (100-120 g / L) in the complete absence of any exogenous growth regulators, vitamins (except for basic MS), and amino acids. Preliminary "priming" dramatically increases the efficiency of sucrose's osmotic signal, making it a pure and powerful tuberization inducer.

[0012] Key technological features:

[0013] – Iron source replacement: Using Fe-EDDHA instead of traditional Fe-EDTA ensures the chelate stability over a wide pH range, preventing precipitation in high sucrose concentration environment.

[0014] – Non-transplantation: Replacing the liquid phase of the medium in the same culture vessel (e.g. in temporary immersion systems or by gentle draining) minimizes trauma and contamination.

[0015] – Strict temperature regime: Cultivation at a constant temperature of 19°C is optimal for inducing tuberization under complete darkness.

[0016] Example 1 (Basic, demonstration of effectiveness)

[0017] Object: potato microshoots (Solanum tuberosum L.) KRASA MESHCHERY, grown on standard MS medium.

[0018] Control groups (CG):

[0019] – KG-1: Standard hormonal method (MS medium + 5 mg / L 6-BAP + 80 g / L sucrose).

[0020] – KG-2: Osmotic method (MS medium + 110 g / l sucrose, without hormones, without priming). Experimental group (OG, proposed method):

[0021] – Phase 1 (10 days): MS medium containing: BABA – 40 mg / l, L-proline – 150 mg / l, sucrose – 30 g / l, Fe-EDDHA – 10 mg / l, agar – 6 g / l, pH 5.7.

[0022] – Phase 2 (21 days): without replanting, the medium was replaced. MS medium containing: sucrose – 110 g / L, Fe-EDDHA – 10 mg / L, agar – 7 g / L, pH 5.8. Growth regulators, additional vitamins and amino acids were absent. Conditions for all groups: Constant darkness, temperature 19°C.

[0023] Results (on day 21 of phase 2)

[0024] Indicator Synchronicity, % Average tuber diameter, mm Tuber yield >8 mm, pcs / vessel Dry matter, % KG-1 (Hormonal) 75 ± 5 7 ± 3 3.2 ± 0.8 18.5 KG-2 (Only sucrose) 60 ± 7 6 ± 4 2.1 ± 1.1 17.0 OG (Proposed method) 98 ± 2 10 ± 1 7.8 ± 0.4 23.2

[0025] Example 2 (Effect of BABA concentration within the stated range)

[0026] The protocol is similar to Example 1, but in Phase 1, BABA concentrations of 25, 40, and 50 mg / L were used with a constant proline concentration of 150 mg / L. Result: Concentrations of 40 and 50 mg / L showed statistically similar high results (synchronicity 96-98%). The concentration of 25 mg / L was slightly less effective (synchronicity 85%). The concentration of 40 mg / L was considered optimal based on economic and biological criteria.

[0027] Example 3 (Assessment of shelf life)

[0028] The microtubers obtained in Example 1 were stored for 90 days in the dark at 4°C. Result: The weight loss of OG microtubers was 3.1%, while that of KG-1 was 7.8%, and that of KG-2 was 12.5%. OG microtubers showed no signs of wrinkling or premature germination.

Claims

1. A method for producing potato microtubers in vitro culture, comprising a two-phase cultivation of microshoots on nutrient media, characterized in that in the first phase lasting 7-10 days, a Murashige-Skoog medium is used, containing beta-aminobutyric acid (BABA) at a concentration of 25-50 mg / l and proline at a concentration of 100-200 mg / l, and in the second phase, following the first without mechanical transplantation of explants, the medium is replaced with an induction medium containing an increased concentration of sucrose of 100-120 g / l and not containing exogenous growth regulators, while Fe-EDDHA at a concentration of 8-12 mg / l is used as a source of iron in both phases, and cultivation in both phases is carried out under conditions of constant darkness and a constant temperature of 19±1°C.

2. The method according to claim 1, characterized in that the concentration of beta-aminobutyric acid (BABA) in the first phase is 40 mg / l.

3. The method according to claim 1, characterized in that the concentration of proline in the first phase is 150 mg / l.

4. The method according to claim 1, characterized in that the concentration of sucrose in the second phase is 110 g / l.

5. The method according to paragraph 1, characterized in that the duration of the first phase is 10 days.

6. The method according to claim 1, characterized in that the induction medium of the second phase does not contain additional vitamins and amino acids other than those included in the basic composition of the Murashige-Skoog medium.

7. The method according to claim 1, characterized in that the replacement of the medium during the transition to the second phase is carried out by removing the liquid phase of the culture medium and adding new medium to the same culture container.