Method for cultivating snow white water lily seeds

Abstract

The invention belongs to the field of snow-white water lily cultivation, and particularly relates to a snow-white water lily seed cultivation method. The method comprises the following steps: (1) treating snow white water lily seeds with carbendazim, alcohol and sodium hypochlorite to obtain seeds with sterilized surfaces; (2) performing low-temperature stimulation on the seeds treated in the step (1) for 24 hours to break dormancy so as to obtain seeds to be soaked and germinated; (3) treating the seeds treated in the step (2) in different shell breaking modes, and soaking and placing the seeds in incubators with different temperatures to obtain the most suitable shell breaking mode and germination temperature for seed germination; (4) soaking the seeds treated in the step (3) into gibberellin (GA3) solutions with different concentrations for 12 hours, and then transferring the seeds into distilled water for continuous soaking to obtain the optimal gibberellin (GA3) soaking concentration; and (5) transplanting the seedlings soaked and germinated in the step (4) into different matrixes, and culturing the seedlings to obtain the snow white water lily seedlings.

Description

Technical Field

[0001] The present invention belongs to the field of water lily cultivation, and particularly relates to a cultivation method for seeds of Nymphaea candida Background Art

[0002] Nymphaea candida J.Presl is a perennial aquatic herb. The wild resources of Nymphaea candida are only distributed in Xinjiang region of China. It is included in the "List of Wild Plants under State Key Protection (the First Batch)", and the protection level is Class II key protected wild rare plants. In 2013, it was included in the "Red List of Threatened Species of the International Union for Conservation of Nature (INCN)", and the level is Endangered (EN). The environmental conditions required for the germination of Nymphaea candida seeds are harsh, and good external conditions are necessary to break dormancy and enable them to germinate smoothly. At present, the research on Nymphaea candida mainly focuses on the protection of its habitat, and there is less research on the germination conditions of Nymphaea candida seeds. Therefore, in order to protect the water lily population in Xinjiang region, it is necessary to take artificial intervention to raise seedlings of Nymphaea candida and expand the number of its population plants. Summary of the Invention

[0003] To solve the above problems, the present invention provides a cultivation method for seeds of Nymphaea candida, comprising the following steps:

[0004] (1) After the seeds of Nymphaea candida are treated with carbendazim, alcohol and sodium hypochlorite, seeds with surface sterilization are obtained;

[0005] (2) The seeds treated in step (1) are subjected to low-temperature stimulation for 24 hours to break dormancy, and seeds to be soaked and germinated are obtained;

[0006] (3) The seeds treated in step (2) are subjected to shell-breaking treatment and then soaked and placed in an incubator;

[0007] (4) The seeds treated in step (3) are soaked in gibberellin solution for 12 hours and then transferred to distilled water for continuous soaking;

[0008] (5) The seedlings germinated by soaking in step (4) are transplanted into a substrate for seedling cultivation, and seedlings of Nymphaea candida are obtained.

[0009] Further, in step (1), the concentration of carbendazim is 10 g / L, the alcohol concentration is 75%, and the concentration of the sodium hypochlorite solution is 10% or 3%.

[0010] Further, in step (2), the low-temperature stimulation is to place at 4°C for 24 hours.

[0011] Further, in step (3), the shell-breaking method is to use forceps to peel off the seed coats at the top of both ends of the softened seeds of Nymphaea candida; the temperature in the incubator is 28°C.

[0012] Further, the concentration of the gibberellin solution in step (4) is 15 mg / L.

[0013] Further, the substrate in step (5) is agar and MS medium.

[0014] Further, the seedling culture conditions in step (5) are: temperature 28 °C, light 3000 lux, and culture in a sunken pot.

[0015] The present invention has the following beneficial effects;

[0016] The method for germination and cultivation of Nymphaea candida seeds provided by the present invention has a low contamination rate, a good germination rate during the germination stage, and good growth after transplantation. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 Harvest and observation of Nymphaea candida seeds;

[0019] Figure 2 Percentage of seeds showing radicle emergence of Nymphaea candida under low-temperature stimulation and normal temperature;

[0020] Figure 3 Percentage of seeds showing radicle emergence of Nymphaea candida under different temperatures (A) and different shell-breaking treatments (B);

[0021] Figure 4 Contamination rate of different sterilization methods;

[0022] Figure 5 Growth of Nymphaea candida seeds in agar and absorbent cotton germination substrates, A and C are agar, B and D are absorbent cotton;

[0023] Figure 6 Germination process of Nymphaea candida seeds;

[0024] Figure 7 Partially successful plants of Nymphaea seeds in culture. Detailed Embodiments

[0025] The various exemplary embodiments of the present invention will be described in detail. Unless otherwise specified, the methods in the examples are all conventional methods, and the reagents, unless otherwise specified, are all conventional commercially available reagents or reagents prepared by conventional methods. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] The present invention provides a method for collecting, germinating, and cultivating Nymphaea candida seeds, comprising the following steps:

[0031] (1) After treating Nymphaea candida seeds with carbendazim, alcohol, and sodium hypochlorite, surface-sterilized seeds are obtained;

[0032] (2) The seeds treated in step (1) are subjected to 24-hour low-temperature stimulation to break dormancy, and seeds ready for soaking and germination are obtained;

[0033] (3) The seeds treated in step (2) are subjected to shell-breaking treatment and then soaked and placed in an incubator;

[0034] (4) Immerse the seeds processed in step (3) in gibberellin solution for 12 hours and then transfer them to distilled water for continued immersion.

[0035] (5) Transplant the germinated seedlings obtained in step (4) into the substrate and conduct seedling cultivation to obtain Nymphaea candida seedlings.

[0036] In the present invention, surface sterilization treatment is carried out on Nymphaea candida seeds before soaking and germination acceleration: after Nymphaea candida seeds are treated with carbendazim, alcohol and sodium hypochlorite, surface-sterilized seeds are obtained.

[0037] In the present invention, the preferred surface sterilization treatment is as follows: soak the seeds in a 10 g / L carbendazim solution for 20 min with shaking, dispense them into 2 mL centrifuge tubes, spray 75% ethanol and then transfer them to a laminar flow bench. Soak them in 75% alcohol for 1 min, rinse with distilled water and then transfer them to a 10% sodium hypochlorite solution for 10 min. Then, peel the seed coat at one end of the Nymphaea candida seeds and place them in 75% alcohol for 20 min. Rinse with distilled water and transfer them to a 3% sodium hypochlorite solution for 5 min, and wash the seeds with clear water.

[0038] Subject the seeds processed in step (1) to 24-hour low-temperature stimulation to break dormancy and obtain seeds to be soaked and germinated.

[0039] In the present invention, the preferred low-temperature stimulation is: soak in water at 4°C for 24 hours.

[0040] Subject the seeds processed in step (2) to different shell-breaking methods and then soak and place them in incubators at different temperatures to obtain the optimal shell-breaking method and germination temperature for seed germination.

[0041] In the present invention, the preferred shell-breaking method and culture temperature are: use forceps to peel the seed coats at the top of both ends of the softened Nymphaea candida seeds and culture at 28°C.

[0042] Immerse the seeds processed in step (3) in GA3 solutions with different concentrations for 12 hours and then transfer them to distilled water for continued immersion to obtain the optimal GA3 soaking concentration.

[0043] In the present invention, the preferred GA3 soaking concentration is: 15 mg / L, soak for 12 hours.

[0044] Transplant the germinated seedlings obtained in step (4) into different substrates and conduct seedling cultivation to obtain Nymphaea candida seedlings. In the present invention, the preferred culture substrate is: agar + MS medium, and the preferred culture conditions are temperature 28°C, light 3000 lux, and submerged culture.

[0045] The sunken basin cultivation is as follows: Fill a flower pot without bottom holes with aquatic plant soil and fill the soil to 2-3 cm below the rim of the pot. Evenly sow the germinated seeds into the aquatic plant soil, cover the soil with 1 cm of soil, compact it, and then inject shallow water so that the water surface is 3-4 cm higher than the soil surface. Place the flower pot in a light incubator, set the temperature to 28 °C, and the light intensity to 70% of natural light. Observe the growth situation every day.

[0046] Example 1

[0047] After the flowers sink into the water, the petals and sepals on the bulb need to be removed. The surface of the bulb is green, with spots, and the seed capsule is obvious, and the shape is round ( Figure 1 B). Put the bulb without petals and sepals into a silk stocking. Ensure that the bulb is completely wrapped by the silk stocking so as to collect the shed seeds in the next step ( Figure 1 C). After a week of gestation, the bulb falls off the stem. Gently knead the bulb in the silk stocking by hand to make the seeds fall off the bulb ( Figure 1 D). After the seeds are collected, invert the silk stocking and gently pat it to make the seeds fall into a clean container. Then, carefully check the seeds, remove any remaining seed coats or other impurities, and soak the collected seeds in water and store them at 4 °C.

[0048] The seeds soaked in warm water in a 28 °C constant temperature incubator for 3 days are respectively placed in a 4 °C low temperature and a normal temperature environment for 24 h, and then are respectively placed in a 250 mL conical flask, 200 mL of distilled water is added, and they are placed in a 28 °C constant temperature incubator for germination. Change the water every day to keep the water quality clean, and count the development situation every day. Set 3 treatment methods: the seeds are not shelled, only polished without shelling, and shelled at both ends and the top. Respectively place 20 seeds with different shell-breaking treatments in a 250 mL conical flask, add 200 mL of distilled water, and place them in a 28 °C and a 37 °C constant temperature incubator for germination. Change the water every day to keep the water quality clean, and count the development situation every day.

[0049] The rates of showing white of Nymphaea candida seeds cultured in a 28 °C constant temperature incubator after being placed in a 4 °C low temperature and a normal temperature environment for 24 h are 55.00% and 31.67% respectively on the seventh day ( Figure 2 ). The rates of showing white of Nymphaea candida seeds cultured in a 28 °C and a 37 °C constant temperature incubator are 30.00% and 46.67% respectively on the seventh day, but the germinated seeds showing white in the 37 °C environment showed the phenomenon of rot at the white showing part.

[0050] The rates of showing white of Nymphaea candida seeds cultured in a 28 °C constant temperature incubator with 3 treatment methods of not shelling, only polishing without shelling, and shelling at both ends and the top are 8.33%, 26.67% and 72.50% respectively on the seventh day ( Figure 3 ).

[0051] Using the seeds of Nymphaea candida as materials, the seeds were taken out of the 4°C refrigerator for two days, rinsed with running water for 2 h, and then soaked in normal-temperature sterile water to restore their activity. After the seeds were pretreated with carbendazim or not, they were dispensed into 2-mL centrifuge tubes, sprayed with 75% ethanol, and then transferred to a laminar flow hood. Different treatment methods (Table 1) were used for surface sterilization, and then transferred to sterilized filter paper to dry. After drying, they were inoculated into petri dishes to observe the contamination situation. Five seeds were inoculated for each treatment, and they were cultured in an environment with a temperature of 28 ± 1°C, a light intensity of 2500 - 4000 lux, and a photoperiod of 16:8. After 20 days, the contamination rate was counted, and the treatment method with the lowest contamination rate was screened out.

[0052] Table 1 Different surface sterilization treatment methods for seeds

[0053]

[0054] In the first 4 days, the contamination rate of all treatment methods was 0%. On the 5th day, the contamination rate of treatment 3 showed 20%, while that of treatments 1 to 4 was 0%. On the 6th day, the contamination rate of treatment 3 increased to about 40%, the contamination rate of treatment 1 was 20%, and the contamination rates of treatments 2 and 4 remained 0%. By the 7th day, the contamination rate of treatment 3 increased to 60%, the contamination rate of treatment 1 also increased to about 40%, the contamination rate of treatment 2 was about 20%, while the contamination rate of treatment 4 remained 0%. On the 8th day, the contamination rates of treatments 1 and 3 reached about 80% respectively, the contamination rate of treatment 2 increased to about 40%, and the contamination rate of treatment 4 was 10%. Treatment 4 maintained a low contamination rate during all culture days, showing the best anti-contamination effect.

[0055] The seeds were soaked in GA3 at 0 (CK), 5, 10, 15, 20 mg·mL -1 for 12 h respectively, then the two ends were shelled, sterilized, and inoculated onto MS medium, and cultured in the dark at normal temperature for 3 - 7 d. The germination rate of the seeds was counted. The germination rate was the highest after soaking in 15 mg·mL -1 GA3 for 12 h.

[0056] Table 2 Different GA3 treatment methods for seeds

[0057]

[0058] A total of 3 treatments were set in the experiment (Table 3). The pH value of MS agar medium was adjusted to 5.8 with NaOH or HCl. For the absorbent cotton medium, absorbent cotton was added to 1 / 4 of the culture bottle, and the medium was watered with MS nutrient solution until it was moist. The bottle cap was covered and sterilized at 121°C for 20 min. It was cultured in an environment with a temperature of 26 ± 1°C, a light intensity of 2500 - 4000 lux, and a photoperiod of 16:8, and the germination potential and germination rate were counted.

[0059] Table 3 Culture Medium

[0060]

[0061] The seed coat of the seeds of Nymphaea candida has cracked in the agar culture medium, and the radicle has begun to emerge. As a solid culture medium, agar can provide stable support and sufficient moisture for the seeds, while maintaining a sterile environment in the culture medium. The germination process of the seeds in agar is relatively uniform, and due to the transparency of agar, it is convenient to observe the internal growth of the seeds ( Figure 5 A), the radicle of the seeds of Nymphaea candida has significantly elongated and begun to form roots ( Figure 5 C). The seeds of Nymphaea candida also showed signs of germination in the absorbent cotton culture medium. Absorbent cotton has good water absorption and air permeability, and can provide appropriate moisture and air exchange for the seeds. The germination of the seeds in absorbent cotton may be physically restricted by the cotton fibers, but generally it can still proceed normally ( Figure 5 B). The radicle of the seeds of Nymphaea candida also elongated in the absorbent cotton culture medium, but due to the physical properties of absorbent cotton, the growth direction of the radicle may not be as straight as in agar. Nevertheless, absorbent cotton can still provide sufficient moisture and air to support the germination of the seeds ( Figure 5 D).

[0062] Fill the bottomless flower pot with aquatic plant soil, and fill the soil to 2 - 3 cm below the rim of the pot. Evenly sow the germinated seeds into the aquatic plant soil, cover the seeds with 1 cm of soil, compact it, and then inject shallow water so that the water surface is 3 - 4 cm above the soil. Place the flower pot in a light incubator, set the temperature to 28 °C, and the light intensity to 70% of natural light. Observe the growth situation every day.

[0063] The seeds of Nymphaea candida show white tips at 5 - 7 days, germinate at 8 - 10 days, grow cotyledons at 11 - 14 days, and the cotyledon leaves unfold two days after transplanting into the aquatic plant soil ( Figure 6 ).

[0064] These plants show healthy leaves, strong roots and good growth trends. Through this series of steps, the tissue-cultured seedlings can not only be successfully transplanted, but also thrive in the aquatic plant soil and finally become mature plants.

[0065] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for cultivating snow-white water lily seeds, characterized in that: The following steps are involved: (1) Snow white water lily seeds are treated with carbendazim, alcohol and sodium hypochlorite to obtain surface sterilized seeds; (2) subjecting the seeds treated in step (1) to low temperature stimulation for 24 hours to break dormancy and obtain seeds to be soaked and germinated; (3) breaking the shells of the seeds treated in step (2), soaking them and placing them in an incubator; (4) soaking the seeds treated in step (3) in a gibberellin solution for 12 hours and then transferring them to distilled water for further soaking; (5) transplanting the seedlings after germination in step (4) into a substrate for seedling cultivation to obtain snow-white water lily seedlings.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of carbendazim is 10 g / L, the concentration of alcohol is 75%, and the concentration of sodium hypochlorite solution is 10% or 3%.

3. The preparation method according to claim 1, characterized in that: The low temperature stimulation in step (2) is placed at 4°C for 24 hours.

4. The preparation method according to claim 1, characterized in that: The shell breaking method in step (3) is to use tweezers to peel off the seed coat at the top of both ends of the softened snow-white water lily seeds; the temperature in the incubator is 28°C.

5. The preparation method according to claim 1, characterized in that: The concentration of the gibberellin solution in step (4) is 15 mg / L.

6. The preparation method according to claim 1, characterized in that: The matrix in step (5) is agar and MS culture medium.

7. The preparation method according to claim 1, characterized in that: The seedling culture conditions in step (5) are: temperature 28°C, light 3000 lux, and culture in a sinking pot.

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