Method for increasing germination rate of ancient lotus seeds

Abstract

The invention provides a method for increasing the germination rate of ancient lotus seeds, and belongs to the technical field of ancient lotus seed reactivation, and the method comprises the following steps: selecting seeds, physically breaking seed coats of the selected seeds, soaking the seeds in water for germination after the seed coats are physically broken, controlling physical conditions, adding a nutrient solution, and regularly checking the germination state of the seeds. After the seeds reach the transplanting standard, transplanting the seeds into a matched matrix culture; the germination success rate can reach 82.7 + / -3.5% (n is equal to 300). All operations need to be carried out in a sterile operation table (the cleanliness is ISO 5 level), and microbial contamination is avoided.

Description

Technical Field

[0001] This invention relates to the field of ancient lotus seed revival technology, and more specifically, to a method for improving the germination rate of ancient lotus seeds. Background Technology

[0002] In 2017, during an archaeological excavation at the Ruyuan site in the southeastern corner of Changchun, ancient lotus seeds were discovered for the first time, with a total of 11 seeds unearthed. Last year, the Institute of Botany of the Chinese Academy of Sciences conducted cultivation experiments on 8 of them, and 6 of them successfully germinated. After growing leaves and developing rhizomes and overwintering in a greenhouse, they were transplanted to a lotus base in April this year and bloomed on Sunday (7th).

[0003] In 2022, Nanning Botanical Garden (Qingxiu Mountain) received three ancient lotus seeds and eight seeds of their descendants. These three ancient lotus seeds were unearthed in Pulandian District, Dalian City, Liaoning Province, and C14 radioisotope dating indicates they are approximately 1200 years old. In May 2023, the Guangxi Subtropical Garden Plant Research Center began attempting to "revive" these ancient lotus seeds. Recently, Nanning Botanical Garden (Qingxiu Mountain) exhibited two ancient lotus plants: one that it had independently "reviveed," and another a descendant of an ancient lotus "reviveed" by the Ancient Lotus Research Institute of Pulandian District, Dalian City. Both plants flowered for the first time in May 2024.

[0004] The lotus is one of the oldest dicotyledonous plants in the world, having existed on Earth in its current form for over 100 million years. The seed coat of a lotus seed, from the outside in, consists of the upper epidermis, palisade tissue, sclerenchyma tissue, spongy tissue, and lower epidermis. This structure prevents water and air from seeping in and out, maintaining the stability of the seed's internal environment and keeping it "dormant." Furthermore, ancient lotus seeds, buried in peat layers and subjected to long-term dry, low-temperature, and sealed conditions, have maintained their vitality for thousands of years. Therefore, lotus seeds unearthed from archaeological sites possess the potential to revive, germinate, and flower. Scientists are currently conducting repeated experiments to revive lotus seeds unearthed from ancient sites.

[0005] Therefore, there is an urgent need for a reliable method to promote the regrowth and flowering of lotus seeds that have been preserved from ancient times. Summary of the Invention

[0006] In view of this, the present invention proposes a method to promote the germination of ancient lotus seeds by selecting seeds, physically breaking the seed coat, controlling physical conditions, adding nutrient solution and substrate culture.

[0007] The technical solution of the present invention is implemented as follows: a method for improving the germination rate of ancient lotus seeds includes seed selection, physical breaking of the seed coat of the selected seeds, soaking the seeds in water for germination after physical breaking of the seed coat, controlling physical conditions, adding nutrient solution, regularly checking the germination status of the seeds, and transplanting the seeds into a prepared substrate culture medium when the seeds reach the transplanting standard.

[0008] Based on the above technical solutions, preferably, the seed selection includes two steps: appearance selection and buoyancy selection. Appearance selection involves selecting ancient lotus seeds that are plump, without obvious damage or pests. Buoyancy selection involves placing the ancient lotus seeds that have been selected in appearance into clean water and selecting those that sink to the bottom.

[0009] Based on the above technical solutions, preferably, the physical breaking of the seed coat is achieved by using a scalpel to make an incision along the length of the ancient lotus seed at a point 3-5 mm from one end of the seed hilum, with the incision depth being 35-45% of the seed coat thickness, exposing the radicle primordium but without damaging the embryo.

[0010] Based on the above technical solutions, preferably, the incision angle is maintained at 45±5° with the tangent direction of the seed coat, and the incision length is 8-12mm.

[0011] Based on the above technical solutions, preferably, the ancient lotus seeds after physical breaking of the seed coat are placed in a container filled with warm water at 28-30℃, and the water temperature in the container is maintained at 28-30℃, with the water being changed 1-2 times a day.

[0012] Based on the above technical solutions, preferably, the controlled physical conditions include controlling bacterial growth by adding 50 ppm sodium hypochlorite or a sterilizing solution every 8 hours.

[0013] Based on the above technical solutions, preferably, the controlled physical conditions also include light parameter control: in the early stage of germination, the light intensity is maintained at 1000-1500 lux; in the young leaf stage, the light intensity is maintained at 3000-5000 lux; and the daily light duration is 14-16 hours.

[0014] Based on the above technical solutions, preferably, the controlled physical conditions also include temperature parameter control, with the temperature maintained at 25±2℃ during the early germination and young leaf stages; the controlled physical conditions also include humidity control, with the humidity maintained at 80-85% RH during the early germination and young leaf stages to control excessive water loss from the seed coat.

[0015] Based on the above technical solutions, preferably, in the nutrient solution, Including 2.0 (mM), Including 2.5 (mM), Including 0.5 (mM), ·7 Including 1.0 (mM); Including 50 (μM), Including 12 (μM), Including 2 (μM), Including 1 (μM), Including 0.5 (μM), The concentration is 0.1 μM; the nutrient solution is changed every three days, and the EC value is between 1.2 and 1.5 ms / .

[0016] Based on the above technical solutions, preferably, when the radicle grows to 2-3 cm and the first young leaf appears, the seedlings are transplanted. The substrate culture includes pond mud, vermiculite and perlite, and the volume ratio of the pond mud, vermiculite and perlite is 6:2:2. The substrate culture also includes 5% biochar, and the particle size of the biochar is 2-3 mm.

[0017] The method for improving the germination rate of ancient lotus seeds proposed in this invention has the following advantages over existing technologies: The germination success rate can reach 82.7±3.5% (n=300). All operations must be performed in a sterile operating table (ISO Class 5 cleanliness) to avoid microbial contamination. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] A method for improving the germination rate of ancient lotus seeds includes seed selection, physical breaking of the seed coat after selection, immersion in water for germination after physical breaking, control of physical conditions, addition of nutrient solution, regular monitoring of seed germination status, and transplanting of seeds into a prepared substrate culture medium once the seeds meet the transplanting criteria. The germination success rate can reach 82.7±3.5% (n=300). All operations must be performed in a sterile operating table (ISO Class 5 cleanliness) to avoid microbial contamination.

[0020] The seed selection process includes two steps: visual selection and buoyancy selection. Visual selection involves choosing ancient lotus seeds that are plump, without obvious damage or pests. Buoyancy selection involves placing the visually selected ancient lotus seeds in clean water and selecting those that sink to the bottom. Visual selection eliminates lotus seeds with localized damage or insect infestation, while buoyancy selection removes lotus seeds with damaged or dead internal tissues, ensuring the viability of the cultivated lotus seeds and improving the success rate of cultivation.

[0021] The physical breaking of the seed coat involves making an incision along the length of the ancient lotus seed, 3-5 mm from the hilum end, using a scalpel. The incision depth is 35-45% of the seed coat thickness, exposing the radicle primordium without damaging the embryo. This physical breaking of the seed coat avoids the difficulty of seedling emergence caused by internal tissue damage or nutrient deficiency, thus improving the germination rate. Furthermore, strictly controlling the incision depth helps the seedling break the shell without damaging it, protecting the inside of the lotus seed from bacteria or impurities that could affect germination.

[0022] The incision angle is maintained at 45±5° with the tangent direction of the seed coat, and the incision length is 8-12mm. The incision angle of 45±5° with the tangent direction of the seed coat is conducive to the lotus seed breaking through the seed shell after germination, thereby improving the germination rate. The incision length of 8-12mm can accommodate the size of the lotus seed breaking through the shell during germination, avoiding some young buds being blocked by the shell and unable to germinate well.

[0023] After the seed coat has been physically broken, the ancient lotus seeds are placed in a container filled with warm water at 28-30℃, and the water temperature is maintained at 28-30℃. The water is changed 1-2 times a day. Warm water can promote the activity of α-amylase in the seeds (optimal activity temperature 28-32℃), accelerate the physiological and biochemical reactions inside the seeds, and facilitate seed germination.

[0024] Comparative data of lotus seed temperature activity test The above lotus seed activity test shows that the relative enzyme activity of lotus seeds is highest when the water temperature is between 25-35 degrees Celsius. Therefore, lotus seeds are most likely to survive when the temperature is between 25-35 degrees Celsius, and the relative enzyme activity is highest at 30 degrees Celsius. In order to ensure the survival rate of lotus seeds, the water temperature should be controlled between 28-32 degrees Celsius.

[0025] Lotus seeds in warm water are prone to bacterial growth. To reduce bacteria in the petri dish, the controlled physical conditions include controlling bacterial growth by adding 50 ppm sodium hypochlorite or a sterilizing solution every 8 hours. This embodiment uses 50 ppm sodium hypochlorite as the disinfectant, but any general sterilizing solution can also be used.

[0026] The controlled physical conditions also include light parameter control. During the early germination stage, maintain light intensity at 1000-1500 lux; during the young leaf stage, maintain light intensity at 3000-5000 lux, with a daily light duration of 14-16 hours. Initially, 1000-1500 lux (equivalent to cloudy daytime light) is maintained. As seed germination progresses, the light intensity and duration are gradually increased to meet the seedling's photosynthetic needs. After the young leaf stage, the light intensity needs to be increased to 3000-5000 lux to promote photosynthetic pigment synthesis. The daily light duration is extended to 14-16 hours to simulate the extended summer light cycle.

[0027] Comparative experiment of light intensity with germination rate and hypocotyl length: The above four sets of comparative experiments show that the germination rate and hypocotyl length are highest when the light intensity is 1500 lux. The data do not change much when the light intensity is 1000-1500 lux, but the values ​​drop drastically when the light intensity is 1500-2000 lux. Therefore, we choose to maintain the light intensity at 1000-1500 lux during the early stage of germination.

[0028] The controlled physical conditions also include temperature control, maintaining the temperature at 25±2℃ during the early germination and young leaf stages; and humidity control, maintaining the humidity at 80-85% RH during the early germination and young leaf stages to prevent excessive seed coat dehydration. Experimental data indicate that this temperature range is close to the optimal temperature for seed germination, providing a suitable environment for internal material transformation and physiological activities, and promoting rapid seed germination. The optimal humidity is 80-85% RH to prevent excessive seed coat dehydration; experimental data shows that humidity <70% leads to radicle atrophy.

[0029] Germination monitoring standards 1. Radicle breakthrough period (days 5-8 of treatment): Radicle length ≥ 3mm is considered successful hatching; 2. Cotyledon expansion stage (days 12-15 of treatment): Start nutrient solution culture when the cotyledon expansion angle is ≥150°.

[0030] In the nutrient solution, in the nutrient solution, Including 2.0 (mM), Including 2.5 (mM), Including 0.5 (mM), ·7 Including 1.0 (mM); Including 50 (μM), Including 12 (μM), Including 2 (μM), Including 1 (μM), Including 0.5 (μM), The concentration is 0.1 μM; the nutrient solution is changed every three days, and the EC value is between 1.2 and 1.5 ms / .

[0031] Exception handling.

[0032] If you find that the seeds are moldy, rotten, or otherwise abnormal, take the following steps: 1. Remove the infected seeds immediately. 2. Soak in 1% hydrogen peroxide for 3 minutes. 3. Transfer to a culture medium containing 0.1% azoxystrobin. Principle: Hydrogen peroxide destroys the fungal cell membrane, while pyraclostrobin inhibits mitochondrial respiration, thereby killing the bacteria.

[0033] True leaf differentiation period (days 18-22 of treatment): Transplant when the length of the first true leaf is ≥15mm.

[0034] When the radicle grows to 2-3 cm and the first young leaf appears, the seedlings are transplanted. The substrate culture includes pond mud, vermiculite and perlite, with a volume ratio of 6:2:2. The substrate culture also includes 5% biochar with a particle size of 2-3 mm.

[0035] Water layer management Start with a water depth of 5cm (water temperature 25±1℃), and increase the depth by 2cm each week until a stable depth of 15cm is reached. As the lotus seeds grow and the young shoots grow taller, gradually increase the water depth.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the germination rate of ancient lotus seeds, comprising seed selection, characterized in that: After selection, the seeds undergo physical cracking of the seed coat. After cracking, the seeds are immersed in water for germination. Physical conditions are controlled, nutrient solution is added, and the germination status is checked regularly. Once the seeds meet the transplanting standards, they are transplanted into a prepared substrate culture medium.

2. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: The selection process includes two steps: appearance selection and buoyancy selection. Appearance selection involves choosing ancient lotus seeds that are plump, without obvious damage or pests. Buoyancy selection involves placing the selected ancient lotus seeds in clean water and choosing those that sink to the bottom.

3. The method for improving the germination rate of ancient lotus seeds as described in claim 2, characterized in that: The seed coat is physically broken by making an incision along the length of the ancient lotus seed at a point 3-5 mm from one end of the hilum with a scalpel. The incision depth is 35-45% of the seed coat thickness, exposing the radicle primordium but without damaging the embryo.

4. The method for improving the germination rate of ancient lotus seeds as described in claim 3, characterized in that: The incision angle is maintained at 45±5° with the tangent direction of the seed coat, and the incision length is 8-12mm.

5. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: After the seed coat has been physically broken, the ancient lotus seeds are placed in a container filled with warm water at 28-30℃, and the water temperature is maintained at 28-30℃. The water is changed 1-2 times a day.

6. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: The controlled physical conditions include controlling bacterial growth by adding 50 ppm sodium hypochlorite or a sterilizing solution every 8 hours.

7. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: The controlled physical conditions also include light parameter control: during the early germination stage, maintain light at 1000-1500 lux; during the young leaf stage, maintain light at 3000-5000 lux; and maintain daily light duration of 14-16 hours.

8. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: The controlled physical conditions also include temperature control, with the temperature maintained at 25±2℃ during the early germination and young leaf stages; the controlled physical conditions also include humidity control, with the humidity maintained at 80-85% RH during the early germination and young leaf stages to control excessive water loss from the seed coat.

9. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: In the nutrient solution Including 2.0 (mM), Including 2.5 (mM), Including 0.5 (mM), ·7 Including 1.0 (mM); Including 50 (μM), Including 12 (μM), Including 2 (μM), Including 1 (μM), Including 0.5 (μM), The concentration is 0.1 μM; the nutrient solution is changed every three days, and the EC value is between 1.2 and 1.5 ms / .

10. The method for improving the germination rate of ancient lotus seeds as described in claim 1, characterized in that: When the radicle grows to 2-3 cm and the first young leaf appears, the seedlings are transplanted. The substrate culture includes pond mud, vermiculite and perlite, with a volume ratio of 6:2:

2. The substrate culture also includes 5% biochar with a particle size of 2-3 mm.