Tissue culture method for reducing polyploidy bamboo reed somatic cell variation rate

Abstract

The invention discloses a tissue culture method for reducing the somatic variation rate of bamboo reed by adding chitosan oligosaccharide. According to the method, 50-200 mg / L of chitosan oligosaccharide with the specific molecular weight of (3000-5000 Da) is added into a callus induction culture medium, and 1.5 mg / L of 2, 4-D and 0.2 mg / L of 6-BA are combined, so that the variation rate of somatic cells is reduced from 23.7% to 9.8%. The SSR molecular marker and flow cytometry prove that the genetic stability is remarkably improved, and the biomass of the regenerated plant is increased by 15-20%. The method is suitable for large-scale cloning propagation of energy plants.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, in particular to a tissue culture method for regulating genetic stability during somatic embryogenesis of Arundo donax L. by adding chitosan oligosaccharides with a specific molecular weight. Background Art

[0002] As a high-biomass energy crop, Arundo donax suffers from a high somatic cell mutation rate of 20-30% during tissue culture, severely impacting clonal consistency. Existing technologies, such as CN1132340C, employ low-temperature pretreatment to reduce variation, but this is complex and energy-intensive. CN112166968A utilizes antioxidant compositions, but this presents challenges such as high cost and inconsistent efficacy. While chitosan oligosaccharides (COS) have been used in crop stress tolerance (CN108522646A), there are no reports of their use in regulating the genetic stability of somatic cells in energy plants. Summary of the Invention

[0003] Technical problem: Solve the technical bottleneck of high somatic cell clone mutation rate and genetic inconsistency of propagation materials in Arundo donax tissue culture.

[0004] Core technology: It was discovered that chitosan oligosaccharides with a molecular weight of 3000-5000Da can significantly reduce the mutation rate by regulating the expression of DNA methyltransferase in the concentration range of 50-200 mg / L. Beneficial effects

[0005] 1. The mutation rate dropped from 23.7% to 9.8% (a 58.6% decrease) 2. The embryonic callus induction rate increased by 8.3% 3. The biomass of regenerated plants increased by 15-20% DETAILED DESCRIPTION

[0006] Example 1: Explant treatment Take the stem segment of Phragmites australis → treat with 70% ethanol for 30 seconds → sterilize with 0.1% HgCl2 for 8 minutes → rinse with sterile water 5 times Example 2: Induction culture (key steps) Culture medium formula: - MS basal medium - 2,4-D 1.5 mg / L - 6-BA 0.2mg / L - Chitosan oligosaccharide (4000±500Da) 100mg / L - Sucrose 30g / L - Agar 6.5g / L Adjust pH to 5.8 and sterilize by autoclave at 121℃ for 20min Example 3: Mutation rate detection 1. Flow cytometry: Take callus tissue and use CyStain PI staining kit to detect DNA content. The coefficient of variation (CV value) is less than 5% and is considered qualified. 2. SSR analysis: PCR amplification was performed using 10 pairs of primers (e.g., ADO03, ADO17, etc.). Band consistency of ≥90% was considered genetically stable. Experimental data table Group Embryogenic callus induction rate (%) Mutation rate of regenerated plants (%) DNMT1 expression level (relative value) control group 76.3±3.2 23.7±2.1 1.00±0.12 50mg / l cos 83.5±2.8 14.2±1.5 0.82±0.09 100mg / l cos 87.2±3.1 9.8±0.8 0.61±0.07 200mg / l cos 79.6±4 12.5±1.2 0.75±0.10 (p<0.05, p<0.01 vs control group, n=5).

Claims

1. A tissue culture method for reducing the somatic cell mutation rate of Arundo donax, characterized by: During the somatic embryo induction stage, chitosan oligosaccharide with a molecular weight of 3000-5000Da is added to the culture medium at a concentration of 50-200 mg / L.

2. The method according to claim 1, wherein the molecular weight of the chitosan oligosaccharide is 4000±500Da and the optimal concentration is 80-120mg / L.

3. The method according to claim 1, wherein the culture medium comprises MS basal salts, 1.0-2.0 mg / L 2,4-D, and 0.1-0.5 mg / L 6-BA.

4. The method according to claim 3, wherein the concentration of 2,4-D is 1.5 mg / L and the concentration of 6-BA is 0.2 mg / L.

5. A culture medium composition for reducing somatic cell variation, comprising: MS basal medium, 1.5 mg / L 2,4-D, 0.2 mg / L 6-BA, 100 mg / L chitosan oligosaccharide (4000 Da), and 30 g / L sucrose.

6. Use of the method according to any one of claims 1 to 4 in tissue culture of Miscanthus and Panicum virgatum energy plants.