Method for rapidly propagating desert shrub pitaya root system

Abstract

The invention relates to the technical field of plant culture, in particular to a desert shrub pitaya root system rapid propagation method which comprises the steps that S1, pitaya seeds are sequentially soaked and disinfected through ethyl alcohol and sodium hypochlorite and washed through sterile water, and then sterile seeds are obtained; s2, spreading the sterile seeds in a 1 / 2 MS solid culture medium, and culturing for 2-3 days at room temperature in a dark place to obtain sterile seedlings; s3, transferring the sterile seedlings into a 1 / 2 MS liquid culture medium, and continuously culturing by utilizing a shaking culture technology to obtain sterile pitaya seedlings; the method disclosed by the invention is simple and convenient to operate and short in culture period, and has important significance on rapid propagation, three-dimensional space observation and deep research of pitaya root systems.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, and in particular to a method for rapid propagation of the root system of desert shrub Xanthophyllum grandiflorum. Background Art

[0002] The super-xerophytic shrub Zygophyllum xanthoxylum is one of the dominant species and community-building species in the vegetation composition of the desert areas in northwest my country. It has strong drought resistance and barrenness tolerance, and plays an important role in preventing wind and fixing sand, protecting and improving the desert ecological environment. Studies on morphological structure and physiology have shown that the huge root system dominated by lateral roots increases the contact area between the roots and the soil, which is conducive to absorbing water and nutrients to supply the aboveground growth of the plant, thereby ensuring that the Zygophyllum xanthoxylum can survive in arid and barren environments, which is one of its effective strategies to resist drought stress. Therefore, in-depth research on the growth and development of the Zygophyllum xanthoxylum root system is of great significance for revealing its ecological value and promoting the protection of desert ecology, quicksand control and the development and utilization of sand-dwelling plant resources. In addition, a survey of the Zygophyllum xanthoxylum community in Minqin, Gansu, China found that the Zygophyllum xanthoxylum belongs to a declining population. Therefore, it is urgent to accelerate the in-depth research on the Zygophyllum xanthoxylum population in order to provide a scientific basis for the protection, rational utilization and sustainable management of the species and desert ecology.

[0003] As a small shrub, relevant studies have shown that under natural ecological conditions, the king of kings needs to go through a growth period of about 3 to 4 years before it can reach the mature stage in its life cycle. Its relatively slow growth cycle not only increases the complexity and time cost of scientific research, but also poses a challenge to in-depth research. Existing king of kings are mostly propagated by cuttings or sowing seedlings, which have the problems of seedling time being greatly affected by the season, low survival rate, and long seedling cycle. At present, although the in vitro culture of king of kings, such as tissue culture, has accelerated the seedling cycle of king of kings, its steps are cumbersome and complicated, and the cycle is relatively long; in addition, traditional culture substrates such as vermiculite or quartz sand limit the direct observation of the growth of the king of kings' roots, making it difficult to track its growth morphology and state in real time, and it is easy to damage the roots when collecting them, and it is not easy to obtain a complete plant root system, which limits the relevant research on the root system of king of kings. Using transparent substrates to cultivate plants is currently the main means of observing root growth. Existing commonly used methods such as transparent root chamber method, paper-based method, gel culture medium method and hydroponics method can allow roots to grow in a visually penetrating environment and meet the needs of non-destructive in situ observation, but they also have certain defects: due to the slow growth cycle of the desert shrub Xanthoxylum bungeana, such cultivation methods increase the time cost of research; and as a small shrub, Xanthoxylum bungeana has a very developed root structure, and the flat gel and paper-based culture space is limited, which restricts the normal development of the Xanthoxylum bungeana root system and is not conducive to its long-term observation and research.

[0004] In view of this, in order to more deeply reveal the characteristics and ecological value of the root system of the giant plant, the present invention aims to improve on the traditional hydroponic method and provide a new method suitable for the rapid reproduction and observation of the root system of the desert plant giant plant, so as to promote the growth of the root system of the giant plant and conduct real-time non-destructive observation and research, thereby providing strong support for a deep understanding of the ecological functions of the root system of the giant plant and its role in the desert ecosystem. Summary of the invention

[0005] The purpose of the present invention is to provide a method for rapid root propagation of desert shrub Xanthophyllum sophora japonica, so as to solve the problems existing in the existing cultivation methods.

[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0007] A method for rapid root propagation of desert shrub Xanthophyllum, comprising the following steps:

[0008] S1: The seeds of the king angelica are sequentially sterilized by soaking in alcohol and sodium hypochlorite and then rinsed with sterile water to obtain sterile seeds;

[0009] S2: Spread the sterile seeds on 1 / 2MS solid culture medium and culture them in the dark at room temperature for 2-3 days to obtain sterile seedlings;

[0010] S3: The sterile seedlings are transferred to 1 / 2MS liquid culture medium, and the culture is continued using shaking culture technology to obtain sterile Tyrannosaurus rex seedlings.

[0011] Furthermore, the culture temperature in steps S2 and S3 is 24-26°C.

[0012] Furthermore, in step S1, the 75% alcohol immersion disinfection time is 5-10 minutes.

[0013] Furthermore, the mass concentration of the sodium hypochlorite solution in step S1 is 5%.

[0014] Furthermore, the sodium hypochlorite immersion disinfection in step S1 is performed twice, the first time for 10-15 minutes and the second time for 5-10 minutes.

[0015] Furthermore, in the 1 / 2MS solid culture medium described in step S2, the sucrose concentration is 10 g / L, and the agar concentration is 8 g / L; the pH value of the 1 / 2MS solid culture medium is 5.6-5.7.

[0016] Furthermore, the culturing method in step S2 is as follows: after sealing, the seeds are placed at room temperature and protected from light for 2-3 days for germination.

[0017] Furthermore, in step S3, the seedlings are cultured in a 1 / 2MS liquid culture medium, wherein the sucrose concentration in the 1 / 2MS liquid culture medium is 10 g / L and the pH value is 5.6-5.7.

[0018] Furthermore, the culture method in step S3 is as follows: the conical flask is placed on a decolorization shaker at 100 rpm for shaking culture, the light intensity is 600 μmol / m2·s, and the light-dark cycle is 16h / 8h.

[0019] Beneficial effects of the present invention:

[0020] The present invention uses 1 / 2MS liquid culture medium and combines it with shaking culture technology to make the seedlings grow in a shaking liquid environment. The core of this method is that the mechanical movement of the shaking table can enhance the fluidity of the liquid culture medium, thereby improving the transfer efficiency of oxygen and nutrients. This dynamic culture environment not only promotes cell metabolic activities and rapid growth of the root system, but also improves the overall growth conditions of the plant, greatly shortening the time required for the plant to grow from seed to seedling. Shaking culture technology can also effectively avoid uneven nutrition and water, thereby improving the survival rate of seedlings.

[0021] In step S1, the seeds are strictly sterilized by using 75% alcohol and 5% sodium hypochlorite solution. This double sterilization step effectively removes microbial contamination on the surface of the seeds, creates a sterile culture environment, and ensures that the seedlings in the subsequent culture steps will not be invaded by exogenous microorganisms. The establishment of a sterile environment is the key to ensuring the healthy growth of plants, which reduces the incidence of diseases and thus improves the breeding efficiency and seedling quality.

[0022] The 1 / 2MS medium used in the present invention has been optimized. The appropriate sucrose concentration (10 g / L) provides sufficient carbon source, and the appropriate pH value (5.6-5.7) ensures the effective absorption of nutrients. The temperature is set between 24-26°C, simulating the optimal temperature range for natural plant growth. At the same time, by adjusting the light intensity (600 μmol / m 2 ·s) and light-dark cycle (16h / 8h) to better simulate the day-night changes in the natural environment. These optimized environmental conditions promote plant photosynthesis and metabolism, creating optimal conditions for the robust growth of the root system.

[0023] The use of transparent liquid culture medium allows researchers to observe the growth dynamics and morphological changes of plant roots in real time. In situ observation without destroying the plant's growth environment allows researchers to collect dynamic data on root development and response to environmental changes. This non-destructive observation technology provides reliable data support for revealing the adaptation strategies and ecological functions of the root system of Tyrannosaurus rex, helps to deeply understand its role in desert ecosystems, and provides a basis for scientific decision-making on ecological protection and resource utilization.

[0024] The existing technology for cultivating shrubs such as the king of kings uses solid culture media such as soil, vermiculite and sand. When studying root development, it is impossible to avoid interference from impurities and bacteria in the medium. However, this technology uses sterile transparent liquid culture media to cultivate roots, avoiding external interference and pollution, and is conducive to real-time sampling for direct observation or physiological and biochemical analysis; it is also convenient to select lateral roots at different development stages and make plant sections for cell tissue staining, in situ hybridization and other experiments. This technology is helpful for in-depth research on the molecular mechanism of the king of kings' root development and adaptation to desert habitats.

[0025] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0027] Figure 1 This is a physical picture of the Bawang shaking culture in Example 2;

[0028] Figure 2 This is a picture of the root system of Xanthophylla in Example 3 taken under a microscope;

[0029] Figure 3 This is the phenotypic analysis of X. xanthoxylum seedlings under different osmotic stress treatments in Example 3; Note: Figure A, phenotypic analysis under normal and different osmotic stress treatments, scale = 1 cm; Figures B, C and D, statistical analysis of taproot length, lateral root density and lateral root primordium density of each plant; different letters on the error bar represent significant differences (P<0.05; n>10);

[0030] Figure 4 This is a cross-sectional structure diagram of the root system of the king oak of Example 4;

[0031] Figure 5 This is a cross-sectional structural diagram of the root system of the king of kings cultivated in quartz sand. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] The method for rapid root propagation of the desert shrub Xanthophyllum grandiflorum described in this embodiment comprises the following steps:

[0035] S1: The seeds of the king angelica are sequentially sterilized by soaking in alcohol and sodium hypochlorite and then rinsed with sterile water to obtain sterile seeds;

[0036] S2: Spread the sterile seeds on 1 / 2MS solid culture medium and culture them in the dark at room temperature for 2-3 days to obtain sterile seedlings;

[0037] S3: The sterile seedlings are transferred to 1 / 2MS liquid culture medium, and the culture is continued using shaking culture technology to obtain sterile Tyrannosaurus rex seedlings.

[0038] In this embodiment, the culture temperature in steps S2 and S3 is 24-26°C.

[0039] In this embodiment, the seeds of the king mulberry are peeled from the capsule of the king mulberry, and the king mulberry seeds with clean seed coat are selected, and the king mulberry seeds are sequentially soaked and disinfected with alcohol and sodium hypochlorite and rinsed with sterile water to obtain sterile seeds. In the present invention, the purpose of selecting the king mulberry seeds with clean seed coat is to avoid contamination by miscellaneous bacteria.

[0040] In this embodiment, in step S1, the 75% alcohol immersion disinfection time is 5-10 minutes.

[0041] After the alcohol disinfection of the Bawang seeds in this embodiment, they are rinsed with sterile water, and the number of sterile water rinses is preferably 3-5 times; the purpose of the sterile water rinse is to remove the residual alcohol on the surface of the Bawang seeds.

[0042] In this embodiment, the mass concentration of the sodium hypochlorite solution in step S1 is 5%.

[0043] In this embodiment, the sodium hypochlorite immersion disinfection in step S1 is performed twice, the first time for 10-15 minutes, and the second time for 5-10 minutes. The mass concentration of the sodium hypochlorite solution is 5%; the purpose of the sodium hypochlorite disinfection is to deeply disinfect the seeds and ensure that the surface of the seeds is completely sterile.

[0044] The Bawang seeds described in this embodiment are soaked in a sodium hypochlorite solution for disinfection and then rinsed with sterile water; the number of sterile water rinses is preferably 8-10 times; the purpose of the sterile water rinse is to remove the residual sodium hypochlorite on the surface of the Bawang seeds.

[0045] After obtaining sterile Bawang seeds, the sterile Bawang seeds were spread on the surface of 1 / 2MS solid culture medium and cultured in dark at room temperature for 2-3 days to obtain sterile seedlings. The 1 / 2MS solid culture medium of the present invention is obtained by halving the concentration of components in the conventional MS culture medium, the sucrose concentration of the 1 / 2MS culture medium is 10g / L; the agar concentration of the 1 / 2MS solid culture medium is 8g / L; the pH value of the 1 / 2MS solid culture medium is 5.6-5.7.

[0046] In this embodiment, the aseptic bud culture is preferably carried out in a culture dish, the culture dish preferably has a diameter of 10-15 cm, and the seed inoculation density is 25-50 seeds per dish.

[0047] In this embodiment, the culture method in step S3 is as follows: placing the conical flask on a decolorization shaker at 100 rpm for shaking culture, the light intensity is 600 μmol / m2·s, and the light-dark cycle is 16h / 8h; the culture time of the shaking culture is preferably 5-10 days; the temperature of the shaking culture is preferably 24-26°C.

[0048] Example 2

[0049] The method for rapid root propagation of the desert shrub Xanthophyllum grandiflorum described in this embodiment comprises the following steps:

[0050] S1: Seed disinfection: Peel the seeds from the capsule of the king mulberry, select the king mulberry seeds with clean seed coat, disinfect them with 75% alcohol for 10 minutes in a clean bench, and rinse them with sterile water for 5 times; then use 5% sodium hypochlorite solution to deeply disinfect the seeds, the first time for 10 minutes, the second time for 10 minutes; finally rinse them with sterile water for 10 times.

[0051] S2: Aseptic seedling culture: Spread the sterilized seeds on the surface of 1 / 2MS solid culture medium, which also contains 10g / L sucrose, 8g / L agar, and pH 5.6. The diameter of the culture dish is 15cm, 50 seeds are inoculated in each dish, and cultured at 25℃ in the dark for 3 days.

[0052] S3: Sterile shaking seedling culture: The above sterile seedlings were transferred to 1 / 2MS liquid culture medium, which also contained 10g / L sucrose and had a pH value of 5.6. The medium was placed at 25°C, 600μmol / m2·s light conditions, and shaken on a decolorizing shaker at 100rpm for 5 days to obtain sterile shaking seedlings of Zanthoxylum bungeana ( Figure 1 ).

[0053] Example 3

[0054] Based on the results of Example 2, this example further analyzes and studies the effect of osmotic stress on the growth and development of the root system of Zanthoxylum bungeanum.

[0055] Experimental materials: Sterile seedlings of Bawang obtained in Example 2.

[0056] Experimental design and process: Three osmotic stress levels were set at 0, -0.270MPa, and -0.5MPa. Phenotypic photos were taken 48 hours after treatment, and the root length, lateral roots, and the number of lateral root primordia were counted.

[0057] Experimental results: The results showed that after 48 hours of osmotic stress at -0.270MPa and -0.5MPa, there was no significant change in the growth phenotype of Zygophyllum ( Figure 3 Further statistics show that ( Figure 2 ), compared with the control, after -0.27MPa treatment, the root length of the king tree decreased by 6.69%, the lateral root density increased by 32.93%, and the lateral root primordium density increased by 81.59%; after -0.5MPa treatment, the root length of the king tree decreased by 1.46%, the lateral root density decreased by 18.53%, and the lateral root primordium density decreased by 11.55% ( Figure 3 ). Under -0.270MPa osmotic stress, the density of lateral roots and lateral root primordium of X. wang were higher than those of the control, while -0.5MPa osmotic stress inhibited the development of lateral roots and lateral root primordium of X. wang.

[0058] Example 4

[0059] Based on the results of Example 2, this example further observed the cross-sectional structure of the root system of Zanthoxylum bungeanum.

[0060] Experimental materials: Sterile seedlings of Bawang obtained in Example 2.

[0061] Experimental design and process: Using the Zanthoxylum bungeana obtained in Example 1 and quartz sand culture as materials, take about 1 cm of roots and place them in pre-cooled PFA fixative at 4°C overnight; after dehydration with ethanol and xylene, embed them in paraffin; make paraffin sections and dewax them with xylene and ethanol.

[0062] Experimental results: After dewaxing, the sterile shaking culture ( Figure 4 ) and the cross-section structure of the root system of Zygophyllum grandiflorum cultivated in quartz sand ( Figure 5 ). The results showed that the X. wang seedlings cultured using the method of the present invention had a complete root cross-section structure, while the X. wang root epidermis cultured in traditional quartz sand was easily damaged, which was not conducive to further research on the X. wang root system.

[0063] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for rapid root propagation of desert shrub Xanthophyllum, characterized in that: The following steps are involved: S1: The seeds of the king angelica are sequentially sterilized by soaking in alcohol and sodium hypochlorite and then rinsed with sterile water to obtain sterile seeds; S2: Spread the sterile seeds on 1 / 2MS solid culture medium and culture them in the dark at room temperature for 2-3 days to obtain sterile seedlings; S3: The sterile seedlings obtained in step S2 are transferred to 1 / 2MS liquid culture medium, and the culture is continued using shaking culture technology to obtain sterile Tyrannosaurus rex seedlings.

2. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: The culture temperature in steps S2 and S3 is 24-26°C.

3. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: In step S1, the 75% alcohol immersion disinfection time is 5-10 minutes.

4. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: The mass concentration of the sodium hypochlorite solution in step S1 is 5%.

5. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: In step S1, the sodium hypochlorite immersion disinfection is performed twice, the first time for 10-15 minutes and the second time for 5-10 minutes.

6. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: In step S2, in the 1 / 2MS solid culture medium, the sucrose concentration is 10 g / L, and the agar concentration is 8 g / L; the pH value of the 1 / 2MS solid culture medium is 5.6-5.

7.

7. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: The culturing method in step S2 includes: sealing and standing for germination at room temperature in the dark for 2-3 days.

8. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: In step S3, the seedlings are cultured in a 1 / 2MS liquid culture medium, wherein the sucrose concentration in the 1 / 2MS liquid culture medium is 10 g / L and the pH value is 5.6-5.

7.

9. The method for rapid root propagation of desert shrub Xanthophyllum as claimed in claim 1, characterized in that: The culturing method in step S3 includes: placing the conical flask on a decolorizing shaker at 100 rpm for shaking culture, with a light intensity of 600 μmol / m2·s and a light-dark cycle of 16h / 8h.

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