A method for cultivating white nymphaea seeds

Abstract

The present application belongs to the field of white snow lotus cultivation, and particularly relates to a white snow lotus seed cultivation method. The method comprises the following steps: (1) white snow lotus seeds are treated with carbendazim, alcohol and sodium hypochlorite to obtain surface-sterilized seeds; (2) the seeds treated in step (1) are subjected to 24-hour low-temperature stimulation to break dormancy and obtain seeds to be soaked and germinated; (3) the seeds treated in step (2) are treated by different hull breaking methods, then soaked and placed in different temperature incubators to obtain the optimal hull breaking method and germination temperature of the seeds; (4) the seeds treated in step (3) are soaked in gibberellin (GA3) solutions with different concentrations for 12 hours, then transferred to distilled water for continuous soaking to obtain the optimal gibberellin (GA3) soaking concentration; and (5) the seedlings germinated by soaking in step (4) are transplanted into different substrates for seedling culture to obtain white snow lotus seedlings.

Description

Technical Field

[0001] This invention belongs to the field of water lily cultivation, specifically relating to a method for cultivating snow-white water lily seeds. Background Technology

[0002] Snow White Water Lily (Nymphaea candida J. Presl.) is a perennial aquatic herbaceous plant. Wild resources of Snow White Water Lily are only distributed in Xinjiang, my country, and it is listed in the "National Key Protected Wild Plants List (First Batch)" as a Class II protected rare and endangered wild plant. In 2013, it was listed in the "International Union for Conservation of Nature Red List of Threatened Species" (INCN) as Endangered (EN). Snow White Water Lily seeds require demanding environmental conditions to germinate; favorable external conditions are necessary to break dormancy and allow for successful germination. Current research on Snow White Water Lily mainly focuses on habitat protection, with limited research on seed germination conditions. Therefore, to protect the water lily population in Xinjiang, it is necessary to adopt human intervention methods for seedling cultivation and to expand the population of Snow White Water Lily plants. Summary of the Invention

[0003] To address the above problems, this invention provides a method for cultivating snow-white water lily seeds, comprising the following steps:

[0004] (1) Snow-white water lily seeds were treated with carbendazim, alcohol and sodium hypochlorite to obtain surface-sterilized seeds;

[0005] (2) The seeds treated in step (1) were subjected to low-temperature stimulation for 24 hours to break dormancy and obtain seeds to be soaked and germinated.

[0006] (3) After the seeds in step (2) are cracked, they are soaked and placed in an incubator;

[0007] (4) Soak the seeds treated in step (3) in gibberellin solution for 12 hours, then transfer them to distilled water for further soaking;

[0008] (5) Transplant the seedlings that have been soaked and germinated in step (4) into the substrate for seedling cultivation to obtain snow-white water lily seedlings.

[0009] Furthermore, 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%.

[0010] Furthermore, the low-temperature stimulation mentioned in step (2) involves placing the object at 4°C for 24 hours.

[0011] Furthermore, the method of breaking the shell in step (3) is to use tweezers to peel off the seed coat from the top of both ends of the softened snow-white water lily seed; the temperature in the incubator is 28°C.

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

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

[0014] Furthermore, the seedling cultivation conditions described in step (5) are: temperature 28℃, light 3000 lux, and pot culture.

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

[0016] The germination and cultivation method of snow-white water lily seeds provided by this invention has a low infection rate and a good germination rate during the germination stage, and the seeds grow well after transplanting. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Harvesting and observation of snow-white water lily seeds;

[0019] Figure 2 The whitening rate of snow-white water lily seeds under low-temperature stimulation and at room temperature;

[0020] Figure 3 The whitening rate of snow-white water lily seeds under different temperatures (A) and different shell-breaking treatments (B);

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

[0022] Figure 5 The growth of snow-white water lily seeds in agar and absorbent cotton germination media. A and C are agar, and B and D are absorbent cotton.

[0023] Figure 6 The germination process of snow-white water lily seeds;

[0024] Figure 7 Some water lily seed culture plants were successfully grown. Detailed Implementation

[0025] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] This invention provides a method for collecting, germinating, and cultivating snow-white water lily seeds, comprising the following steps:

[0031] (1) Snow-white water lily seeds were treated with carbendazim, alcohol and sodium hypochlorite to obtain surface-sterilized seeds;

[0032] (2) The seeds treated in step (1) were subjected to low-temperature stimulation for 24 hours to break dormancy and obtain seeds to be soaked and germinated.

[0033] (3) After the seeds in step (2) are cracked, they are soaked and placed in an incubator;

[0034] (4) Soak the seeds treated in step (3) in gibberellin solution for 12 hours, then transfer them to distilled water for further soaking;

[0035] (5) Transplant the seedlings that have been soaked and germinated in step (4) into the substrate for seedling cultivation to obtain snow-white water lily seedlings.

[0036] This invention involves surface sterilization of snow-white water lily seeds before soaking and germination: the snow-white water lily seeds are treated with carbendazim, alcohol and sodium hypochlorite to obtain surface-sterilized seeds.

[0037] In this invention, the surface sterilization treatment is preferably performed as follows: the seeds are soaked and shaken in a 10 g / L carbendazim solution for 20 min, dispensed into 2 mL centrifuge tubes, sprayed with 75% ethanol, transferred to a clean bench, soaked in 75% alcohol for 1 min, rinsed with distilled water, and then soaked in a 10% sodium hypochlorite solution for 10 min. After that, the seed coat of the snow-white water lily seeds is peeled off at one end and placed in a 75% alcohol solution for 20 min, rinsed with distilled water, and then transferred to a 3% sodium hypochlorite solution for 5 min. Finally, the seeds are rinsed with clean water.

[0038] The seeds treated in step (1) were subjected to 24 hours of low-temperature stimulation to break dormancy, resulting in seeds ready for soaking and germination.

[0039] In this invention, the preferred method for low-temperature stimulation is immersion in water at 4°C for 24 hours.

[0040] After the seeds were treated in step (2), they were soaked and placed in incubators at different temperatures after being treated with different shell-breaking methods to obtain the optimal shell-breaking method and germination temperature for seed germination.

[0041] In this invention, the preferred method of breaking the seed coat and the preferred culture temperature are: peeling off the top two ends of the softened snow-white water lily seed with tweezers and then cultured at 28°C.

[0042] After soaking the seeds treated in step (3) in GA3 solutions of different concentrations for 12 hours, the seeds were transferred to distilled water for further soaking to obtain the optimal GA3 soaking concentration.

[0043] In this invention, the preferred GA3 soaking concentration is 15 mg / L, and the soaking time is 12 hours.

[0044] After soaking and germinating in step (4), the seedlings are transplanted into different substrates for seedling cultivation to obtain snow-white water lily seedlings. In this invention, the preferred culture medium is agar + MS medium, and the preferred culture conditions are a temperature of 28°C, a light intensity of 3000 lux, and submerged pot culture.

[0045] The pot cultivation method is as follows: Fill a bottomless flowerpot with aquatic plant soil, filling it to 2-3 cm below the rim. Evenly sow the pre-germinated seeds into the aquatic plant soil, cover with 1 cm of soil, press firmly, and then fill with shallow water, ensuring the water level is 3-4 cm above the soil surface. Place the flowerpot in a light-controlled incubator, setting the temperature to 28℃ and the light intensity to 70% of natural light. Observe the growth daily.

[0046] Example 1

[0047] After the flower sinks in water, the petals and sepals on the bulb need to be removed. The bulb surface is green with spots, and the seed sac is obvious and round in shape. Figure 1 B). Place the bulbs, with petals and sepals removed, into a nylon stocking. Ensure the bulbs are completely encased in the stocking so that any detached seeds can be collected in the following steps. Figure 1 C). After a week of gestation, the bulb detaches from the stem. Gently knead the bulb inside the nylon stocking to release the seeds. Figure 1 D). After collecting the seeds, invert the stocking and gently tap it to allow the seeds to fall into a clean container. Then, carefully inspect the seeds, removing any remaining seed coat or other impurities. Soak the collected seeds in water and store them at 4°C.

[0048] Seeds soaked in warm water at 28℃ for 3 days were placed in 4℃ (medium-low temperature) and room temperature environments for 24 hours, respectively. They were then placed back into 250mL Erlenmeyer flasks, each with 200mL of distilled water added, and placed in a 28℃ incubator for germination. The water was changed daily to maintain cleanliness, and development was recorded daily. Three treatment methods were implemented: seeds without shelling, seeds that were only polished without shelling, and seeds with shells removed from both ends and tops. Twenty seeds from each treatment were placed in 250mL Erlenmeyer flasks, each with 200mL of distilled water added, and placed in incubators at 28℃ and 37℃ respectively for germination. The water was changed daily to maintain cleanliness, and development was recorded daily.

[0049] The white lily seeds, after being cultured at 4℃ for 24 hours in a low-temperature environment and at room temperature, respectively, showed white germination rates of 55.00% and 31.67% on the seventh day in a 28℃ constant-temperature incubator. Figure 2 The white water lily seeds cultured in constant temperature incubators at 28℃ and 37℃ showed a whitening rate of 30.00% and 46.67% respectively on the seventh day. However, the whitening water lily seeds cultured in the 37℃ environment showed signs of rotting at the whitening point.

[0050] The whitening rates of snow-white water lily seeds after being cultured in a 28℃ constant temperature incubator using three treatment methods—unpeeled seeds, seeds polished but not peeled, and seeds peeled from both ends—were 8.33%, 26.67%, and 72.50% respectively on day seven. Figure 3 ).

[0051] Using white water lily seeds as material, the seeds were removed from a 4℃ refrigerator after two days of experimentation, rinsed with running water for 2 hours, and then soaked in sterile water at room temperature to restore their activity. After either pretreatment with carbendazim or without, the seeds were aliquoted into 2mL centrifuge tubes, sprayed with 75% ethanol, and transferred to a clean bench for surface sterilization using different treatment methods (Table 1). The seeds were then transferred to sterilized filter paper and air-dried. After drying, they were inoculated into petri dishes to observe contamination. Five seeds were inoculated for each treatment and cultured at 28±1℃, light intensity of 2500–4000 lux, and a photoperiod of 16:8. After 20 days, the contamination rate was calculated, and the treatment method with the lowest contamination rate was selected.

[0052] Table 1 Different surface sterilization treatments for seeds

[0053]

[0054] For the first four days, the contamination rate was 0% for all treatments. On day 5, treatment 3 showed a 20% contamination rate, while treatments 1 through 4 were 0%. On day 6, the contamination rate for treatment 3 rose to approximately 40%, treatment 1 to 20%, and treatments 2 and 4 remained at 0%. By day 7, the contamination rate for treatment 3 increased to 60%, treatment 1 also rose to approximately 40%, treatment 2 to approximately 20%, and treatment 4 remained at 0%. On day 8, the contamination rates for treatments 1 and 3 reached approximately 80%, while treatment 2 rose to approximately 40%, and treatment 4 to 10%. Treatment 4 maintained a low contamination rate throughout all incubation days, demonstrating the best contamination prevention effect.

[0055] Seeds were subjected to concentrations of 0 (CK), 5, 10, 15, and 20 mg / mL, respectively. -1 After soaking in GA3 for 12 hours, the seeds were peeled at both ends, sterilized, and then inoculated into MS medium. They were then cultured in the dark at room temperature for 3–7 days, and the seed germination rate was calculated at 15 mg / mL. -1 The germination rate was highest after soaking in GA3 for 12 hours.

[0056] Table 2 Different GA3 treatments for seeds

[0057]

[0058] Three treatments were set up in the experiment (Table 3). The pH of MS agar medium was adjusted to 5.8 with NaOH or HCl. For the defatted cotton medium, defatted cotton was added to 1 / 4 of the culture bottle, and MS nutrient solution was poured to moisten the medium. The bottle was then capped and sterilized at 121℃ for 20 min. The culture was carried out in an environment with a temperature of 26±1℃, a light intensity of 2500~4000 lux, and a photoperiod of 16:8. Germination potential and germination rate were recorded.

[0059] Table 3 Culture substrate

[0060]

[0061] The seeds of the snow-white water lily have cracked their outer shells and the radicle has begun to emerge in the agar medium. Agar, as a solid culture medium, provides stable support and sufficient moisture for the seeds while maintaining a sterile environment. Seed germination in agar is relatively uniform, and due to the transparency of agar, the internal growth of the seeds can be easily observed. Figure 5 A) The radicle of the snow-white water lily seed has clearly elongated and has begun to form a root system. Figure 5 C). Seeds of the snow-white water lily also showed signs of germination in a culture medium of absorbent cotton. Absorbent cotton has good water absorption and air permeability, providing suitable moisture and air exchange for the seeds. Seed germination in absorbent cotton may be physically limited by the cotton fibers, but it can still proceed normally overall. Figure 5 B). The radicles of the white water lily seeds also elongated in the absorbent cotton medium, but perhaps due to the physical properties of the absorbent cotton, the growth direction of the radicles was not as straight as in agar. Nevertheless, the absorbent cotton still provided sufficient moisture and air to support seed germination. Figure 5 D).

[0062] Fill bottomless flowerpots with aquatic plant soil, filling it to 2-3 cm below the rim. Sow the pre-germinated seeds evenly into the aquatic plant soil, cover with 1 cm of soil, press firmly, and then add shallow water, ensuring the water level is 3-4 cm above the soil surface. Place the flowerpot in a light-controlled incubator at 28℃ with 70% of natural light. Observe the growth daily.

[0063] Snow-white water lily seeds show white tips in 5–7 days, germinate in 8–10 days, and develop cotyledons in 11–14 days. Two days after transplanting into aquatic plant soil, the cotyledon leaves will unfold. Figure 6 ).

[0064] These plants exhibit healthy leaves, strong root systems, and good growth. Through this series of steps, tissue-cultured seedlings can not only be successfully transplanted, but also thrive in aquatic plant mud, eventually becoming mature plants.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for cultivating seeds of Nymphaea alba, characterized in that, The method comprises the following steps: (1) the seed of Nymphaea alba is treated with carbendazim, alcohol and sodium hypochlorite to obtain surface-sterilized seed; (2) the seed treated in step (1) is subjected to 24-hour low-temperature stimulation to break dormancy and obtain seed to be soaked and germinated; (3) the seed treated in step (2) is subjected to hull breaking treatment and then soaked and placed in an incubator; (4) the seed treated in step (3) is soaked in a gibberellin solution for 12 hours and then transferred to distilled water for continuous soaking; (5) the seedling germinated in step (4) is transplanted into a substrate for seedling culture to obtain Nymphaea alba seedling; 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%; In step (4), the concentration of gibberellin solution is 15 mg / L; In step (5), the substrate is agar and MS medium; In step (5), the seedling culture conditions are as follows: temperature 28℃, illumination 3000 lux, and sink culture; In step (2), the low-temperature stimulation is placing in 4℃ for 24 hours.

2. The method of claim 1, wherein, In step (3), the hull breaking method is using tweezers to peel off the seed coat at the top of both ends of the softened Nymphaea alba seed; and the temperature in the incubator is 28℃.

Images