Rhodotorula mucilaginosa capable of stimulating seed germination and application of rhodotorula mucilaginosa in stimulation of seed germination and reduction of pollution rate in tissue culture
By isolating and identifying the contaminated bacteria JHJM during tissue culture of cucumber seeds, and using high-temperature treatment to eliminate endophytic bacterial contamination, the pollution problem in cucumber tissue culture was solved, the success rate of tissue culture was improved, and it was found that yeast gum red yeast has a promoting effect on the germination of cucumber seeds.
Patent Information
- Application Number
- CN202510264514.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-04-29
AI Technical Summary
In the cucumber tissue culture process, bacterial and fungal contamination often occurs, resulting in a decrease in the efficiency and success rate of the culture process. There are few specific studies on cucumber tissue culture contamination in existing studies.
By isolating and purifying the contaminated bacteria JHJM produced during tissue culture of cucumber seeds, it was classified and identified in combination with morphological and molecular biological characteristics, and it was determined that it was Rhodotorula mucilaginosa. Then, high-temperature treatment is used to eliminate endophytic contamination and verify its promoting effect on germination of cucumber seeds.
The bacteria, fermentation substances and fermentation supernatant of JHJM of jelly yeast JHJM have a promoting effect on the germination of cucumber seeds, reducing the contamination rate during tissue culture, improving the success rate of tissue culture, and providing a theoretical basis for cucumber tissue culture and cultivation.
Smart Images
Figure CN119899753A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to red yeast for stimulating seed germination and use of the red yeast for reducing contamination rate in stimulating seed germination and tissue culture. Background Art
[0002] As a large agricultural country, China has many Cucurbitaceae crops that play an important role in the national economy. These crops not only enrich our dining tables, but also become important cash crops, such as the well-known cucumbers, wax gourds and pumpkins. Among them, cucumber is an important vegetable widely cultivated in various parts of my country, and its nutritional value is quite rich. It is fresh, crisp and delicious, has anti-tumor, brain-strengthening and calming effects, is rich in various vitamins and mineral elements, and is deeply loved by everyone. Therefore, the market demand for cucumbers is huge. In order to prevent seasonal restrictions and achieve high yields of cucumbers, the area of cucumber facility cultivation has gradually increased.
[0003] Plant tissue culture, as an important form of cultivation technology, is an in vitro culture technology based on the theory of plant cell totipotency. It mainly involves the separation, culture and regeneration of plant organs, tissues, cells, anthers and microspores. Cucumber tissue culture includes explant culture method and reduced body culture method. Because it does not occupy land and has a fast reproduction rate, it is widely used in cucumber seedling production. However, in the process of tissue culture, pathogenic bacteria contamination often occurs. The main microbial contamination in tissue culture usually includes bacteria and fungi. Common bacterial contamination includes Bacillus sp., Staphylococcus sp., Pseudomonas sp., etc. In plant tissue culture, Bacillus is widely regarded as one of the most common contaminating bacteria. This bacterium has been reported as a common source of contamination in the tissue culture of many plants, such as Dendrobium officinale, Huaxuedan, and Giant Dragon Bamboo. Fungal contamination mainly includes Penicillium sp., Aspergillus niger, and Saccharomyces spp. The presence of these microorganisms will have an adverse effect on the culture process, reducing the culture efficiency and success rate. However, compared with the relevant reports on contamination problems in tissue culture of other plants, there are relatively few specific research reports on cucumber tissue culture contamination. With the improvement of people's living standards, people pay more and more attention to the quality of cucumbers. In recent years, the use of tissue rapid propagation in cucumber cultivation has gradually increased. In summary, removing contaminating bacteria in the tissue culture process and ensuring the rapid and efficient conduct of cucumber tissue culture have extremely important theoretical and practical significance in protecting germplasm resources, promoting the selection and breeding of new varieties, and improving production efficiency and quality. Summary of the invention
[0004] The present invention separates and purifies the contaminating bacteria JHJM generated during the cucumber seed tissue culture process, classifies and identifies them through morphological characteristics and molecular biological characteristics, and then inoculates the contaminating bacteria and their fermented products back into high-temperature treated healthy seeds to verify the function of the tissue culture contaminating bacteria, providing a theoretical basis for high-yield cucumber tissue culture.
[0005] In order to clarify the classification status of cucumber seed tissue culture contaminants and their effects on cucumber seed germination, the present invention separates and purifies the contaminants (numbered JHJM) produced during cucumber seed tissue culture, classifies and identifies them by combining morphological characteristics with molecular biological characteristics, and then inoculates the contaminants and their cultures back into healthy seeds treated with high temperature (55-75°C for 8h) to verify the function of the tissue culture contaminants. The results show that the colonies are oval or round, orange-yellow, convex, moist and sticky, shiny, opaque, with smooth edges, easy to pick up, and the cell morphology is spherical or elliptical, 3-4μm, and its reproductive mode is budding reproduction, which is preliminarily identified as yeast; the ITS sequence is shown in SEQ ID No.1, and the ITS alignment and phylogenetic tree construction show that the contaminant JHJM is Rhodotorula mucilaginosa. The bacterial body, fermentation product and fermentation supernatant of Rhodotorula mucilaginosa all have a promoting effect on cucumber seed germination, which indicates that Rhodotorula mucilaginosa is an endophyte beneficial to cucumber seed germination. This provides a theoretical basis for cucumber tissue culture and cultivation.
[0006] The present invention takes the fermentation liquid, supernatant, OD 600 =0.5 and OD 600 =1 was used to culture cucumber seeds. After the culture dish was placed in a 28℃ constant temperature incubator in the dark and soaked for 48 hours, the germination rate and root length at 48 hours and 60 hours were observed. The study clearly showed that the bacteria, fermentation products and fermentation supernatant of Rhodotorula JHJM all promoted the germination of cucumber seeds. The research progress in the field of plant protection showed that it has potential application prospects and can be used as an environmentally friendly and effective biological control agent to improve the yield and quality of crops, reduce the use of chemical pesticides, and protect the health of agricultural ecosystems. This shows that Rhodotorula JHJM is a beneficial endophyte for cucumber seed germination. This provides a theoretical basis for promoting cucumber tissue culture.
[0007] Another object of the present invention is a method for improving the success rate of tissue culture, comprising reducing endophyte contamination by heat treatment, wherein the heat treatment comprises heat treatment at 55-75° C. for 8 hours; the endophyte comprises the above-mentioned Rhodotorula JHJM; the endophyte comprises the above-mentioned Rhodotorula JHJM.
[0008] The red yeast JHJM of the present invention was isolated by the inventor from cucumber tissue culture contamination bacteria in Qinhuangdao City, Hebei Province in April 2023. The colony was oval or round when cultured for 3 days, orange-yellow, convex, moist and sticky, shiny, opaque, with smooth edges, easy to be picked up, and the front and back colors were the same; when the colony grew to 7 days, the colony was orange-red, and the front and back colors were the same. The ITS sequence of the strain was determined, and it was determined to be red yeast (Rhodotorula mucilaginosa) through phylogenetic analysis and physiological and biochemical identification, and was named JHJM. The red yeast strain JHJM of the present invention has been proved by laboratory germination experiments that it can promote the germination of cucumber and peanut seeds, and has good application prospects.
[0009] Rhodotorula mucilaginosa, strain number JHJM, has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.32087 and the deposit date September 27, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 The morphology of JHJM bacteria, a: the contaminated state of cucumber seeds in tissue culture; b: the picture of JHJM stained with methylene blue under a microscope; c: the morphology of JHJM colonies at 3 days and 7 days of culture;
[0011] Figure 2 ITS electrophoresis and phylogenetic tree, in which a: electrophoresis of PCR products (M: markers; 1-3: strains JHJM); b: phylogenetic tree of strain JHJM based on ITS;
[0012] Figure 3 This is an efficacy diagram of strain JHJM, in which a: the contamination rate of cucumber seed tissue culture treated with high temperature (HC) and the contamination rate of cucumber seed tissue culture treated with no high temperature (UH); b: the curve of strain JHJM cultured in liquid at different temperatures for 8 hours. DETAILED DESCRIPTION
[0013] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0014] Example 1
[0015] 1. Materials
[0016] 1.1 Test samples
[0017] The cucumber inbred line 6457 bred by the cucumber innovation team of the College of Horticulture Science and Technology of Hebei University of Science and Technology, the contaminating bacteria produced during the cucumber seed tissue culture experiment (numbered JHJM), and the peanut seed Zhonghua 12 originated from the laboratory of this research group.
[0018] 1.2 Test drugs and reagents
[0019] The drugs and reagents required for this study and their manufacturers are as follows:
[0020] Table 1 Test drugs and reagents
[0021]
[0022]
[0023] 1.3 Culture medium and primers
[0024] The culture medium and primers required for this study are as follows:
[0025] Table 2 Culture medium formula
[0026]
[0027] Table 3 Primers required for PCR in vitro amplification
[0028]
[0029] 2 Methods
[0030] 2.1 Statistics on contamination of cucumber seed tissue culture
[0031] Tissue culture test of healthy seeds without high temperature treatment: Soak cucumber seeds in 30℃ warm water for 4-6h, peel off the seed shell, rinse with tap water 2-3 times, disinfect with 75% alcohol surface for 30s, 2% sodium hypochlorite surface for 10min, rinse with sterile water 4-5 times, inoculate in 1 / 2MS solid culture medium, culture in the dark at 30℃ for 36-48h, and then culture in the light at 26℃ for 16h. Statistic the contamination situation, pick the tissue culture contaminated bacteria and inoculate them in PDA culture medium, culture at 28℃ for 3-7 days, obtain the purified strain JHJM, and purify it three times. Store at 4℃ for later use.
[0032] 2.2 Classification and identification of endogenous contaminating bacteria JHJM
[0033] (1) Morphological observation
[0034] The growth and morphological characteristics of JHJM were recorded, and the taxonomic status of JHJM was preliminarily established by observing the colony color, shape, texture and luster, and by observing the methylene blue-stained microscopy of JHJM.
[0035] (2) Molecular Biological Identification
[0036] The DNA of JHJM strain was extracted using the fungal genomic DNA extraction kit of Beijing Solebow Technology Co., Ltd. The primers used in the experiment were ITS1 and ITS4; the reaction system was 20 μL, including: 1 μL DNA template, 1 μL primer ITS1, 1 μL primer ITS4, 10 μL 2×SuperPfx MasterMix, and sterile water was added to 20 μL. The amplification steps were as follows: 94°C pre-denaturation for 90 s, 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 1 min, 30 cycles of reaction, 72°C, 5 min, and then the resulting PCR product was sent to Bioengineering (Shanghai) Co., Ltd. for sequencing analysis.
[0037] Sequences were assembled using DNAMAN and aligned using Blastn in NCBI, and a phylogenetic tree was constructed using the neighbor-joining method. The evolutionary status of JHJM in taxonomy was determined in combination with morphological characteristics.
[0038] 2.3 Optimization of high temperature treatment to eliminate endophytic contamination of cucumber seed tissue culture
[0039] Mix 30 cucumber seeds and 10 3 cfu / mL JHJM bacterial solution was fully mixed and treated at 55℃, 65℃ and 75℃ for 8h, and each treatment was repeated 3 times. The effect of high temperature on the germination of cucumber seeds was detected by seed germination index test, and the killing of endogenous contamination bacteria JHJM by high temperature was detected by plate colony counting method. The temperature used in the treatment group where the seed germination index did not decrease significantly and the endogenous contamination bacteria JHJM were basically removed was the optimal temperature for eliminating endogenous contamination bacteria in cucumber seed tissue culture. Cucumber seeds were treated with the optimal temperature for tissue culture, and a normal temperature treatment group was set as a control to further verify the contamination.
[0040] 2.4 Verification of the growth-promoting function of endophyte JHJM
[0041] The fermentation liquid of endophyte JHJM was prepared by shaking culture at 37℃ and 180rpm for 8h, 24h and 72h. The germination index of peanut seeds and cucumber seeds (treated at 65℃ for 8h) was tested with 2-fold dilution and 10-fold dilution, respectively, and the PDA liquid medium with corresponding dilution was used as control. Each treatment was repeated 3 times. The germination rate and root length were calculated by dark culture in a 28℃ constant temperature incubator for 48h and 60h.
[0042] 3. Results Analysis
[0043] 3.1 Classification and identification of contaminating bacteria
[0044] 3.1.1 Morphological characteristics of contaminating bacteria
[0045] The strain JHJM was cultured on PDA solid medium for 3 days. The colonies were oval or round, orange-yellow, convex, moist and sticky, shiny, opaque, with neat edges, smooth surface, easy to pick up, and the front and back colors were the same (see Figure 1 c); After 7 days of culture, the colony was orange-red, with the same color on the front and back (c3, c4), and no conidia were produced. Microscopic observation showed that the cell morphology was spherical or elliptical, with a diameter of 3.5±0.5×5±0.5μm, and budding. Based on morphological identification, the contaminating bacteria were preliminarily identified as yeast (see Figure 1 b) 。
[0046] 3.1.2 Molecular identification of contaminating bacteria
[0047] The ITS1 / ITS4 primers were used to amplify the genome of strain JHJM by PCR to obtain the ITS fragment ( Figure 2 a), about 600 bp. After sequencing, the sequence information was submitted to Genebank and the gene accession number was PQ865371. The blastn comparison showed that the similarity with R. mucilaginosa AAN6 (gene accession number LC473094.1) was more than 99%. Sequences with high similarity were selected to construct a phylogenetic tree based on the neighbor-joining method (such as Figure 2 The results showed that the obtained sequence was clustered on the same branch as R. mucilaginosa CBS 316. Combining molecular biology and morphological characteristics, the strain JHJM was identified as R. mucilaginosa.
[0048] 3.2 Optimization of high temperature treatment to eliminate endophytes in cucumber seed tissue culture
[0049] The germination rate and seed germination index of cucumber seeds treated at 65℃ for 8h were 96.67±5.77% and 108.13±24.47%, respectively, which were significantly higher than those treated at 55℃ and 75℃. At the same time, the growth curves of strain JHJM cultured in liquid at different temperatures for 8h showed that the optimal growth temperature was 35℃ and the lethal temperature was around 60℃. Figure 3 The results of tissue culture of cucumber seeds treated at 65℃ for 8 hours showed that the JHJM contamination rate of the treated group (HC) was 1%, while that of the control group at normal temperature (UH) was as high as 62%. Figure 3 This indicates that the tissue culture contamination bacteria JHJM is actually an endophyte of cucumber seeds. Treating cucumber seeds at 65℃ for 8h for tissue culture can not only eliminate the contamination of endophyte JHJM, but also activate seed germination, which is an excellent method for eliminating endophytic contamination bacteria in cucumber seed tissue culture by high temperature treatment.
[0050] Table 4 Effect of high temperature treatment on cucumber seed germination
[0051]
[0052] 3.3 Verification of the growth-promoting function of endophyte JHJM
[0053] Cucumber seeds were treated with the fermentation liquid of endophyte JHJM (treated at 65℃ for 8h). The germination rates of seeds treated with 10-fold diluted liquid were 66.7% and 93.3% at 24h and 72h, respectively, which were significantly higher than those of the conventional control and blank control cultured for the same time, indicating that endophyte JHJM also has a growth-promoting effect on cucumber seeds.
[0054] When peanut seeds were treated with the fermentation liquid of endophyte JHJM, the germination rates of its 2-fold dilution were 70%, 90% and 90% at 24h, 48h and 72h, respectively, which were significantly higher than those of the conventional control and blank control cultured for the same time, indicating that the endophyte JHJM also has a growth-promoting effect on peanut seeds.
[0055] Table 5 Effect of strain JHJM fermentation liquid on cucumber seed germination
[0056]
[0057]
[0058] Table 6 Effect of strain JHJM fermentation liquid on peanut seed germination
[0059]
[0060] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A Rhodotorula mucilaginosa JHJM for stimulating seed germination, characterized in that: The biological preservation number of the Rhodotorula glutinosus JHJM is CGMCC NO.32087.
2. A primer pair for detecting the ITS sequence of Rhodotorula glutinosus JHJM according to claim 1, characterized in that: It comprises an upstream primer with a nucleotide sequence as shown in SEQ ID No.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID No.
3.
3. An agent for stimulating seed germination prepared by using the red yeast JHJM according to claim 1.
4. The method for preparing the reagent according to claim 3, characterized in that: The method comprises culturing the red yeast JHJM according to claim 1, and the obtained product is the reagent.
5. The preparation method according to claim 4, characterized in that: The product comprises at least one of the following: fermentation broth, supernatant of fermentation broth and bacterial cells.
6. The preparation method according to claim 4, characterized in that: The culture medium used in the culture includes PDA medium.
7. Use of the agent according to claim 3 in promoting seed germination.
8. A method for promoting seed germination, characterized in that: The method comprises culturing plant seeds in the agent of claim 3.
9. The method according to claim 8, characterized in that: Including soaking seeds in the dark at 28°C.
10. A method for improving the success rate of tissue culture, characterized in that: The method comprises reducing endophyte contamination by heat treatment, wherein the heat treatment comprises heat treatment at 55 to 75° C. for 8 hours; The endophyte comprises the Rhodotorula glutinosus JHJM according to claim 1.