Method for returning endangered mangrove plant sonneratia casegalis to wild

Abstract

This invention discloses a method for the reintroduction of the endangered mangrove plant *Avicennia marina* ovoidea into the wild, belonging to the field of rare and endangered plant reintroduction technology. The method includes steps such as seed collection and treatment, seed germination, seedling cultivation, wild acclimatization, and planting in the reintroduction site. Through scientifically proportioned seedbed substrate and temperature and salinity acclimatization, this invention significantly improves seedling survival rate and seedling quality. It solves the current problems of difficult seed germination, poor seedling quality, low survival rate, and poor natural regeneration of the mangrove plant *Avicennia marina* ovoidea.

Description

Technical Field

[0001] This invention belongs to the field of rare and endangered plant reintroduction technology, specifically relating to a method for the reintroduction of the endangered mangrove plant *Sonneratia ovata*. Background Technology

[0002] Sonnerntia ovata is a species of mangrove plant belonging to the genus Sonnerntia in the family Sonneraceae. In my country, it is only naturally distributed in the Qinglan Nature Reserve in Wenchang, Hainan.

[0003] *Avicennia ovata* seeds are light-demanding and exhibit narrow temperature adaptability; insufficient light and low temperatures under forest canopy conditions are detrimental to seed germination. *Avicennia ovata* also has poor salt tolerance; the high salinity of its habitat further inhibits seed germination. Sown seeds have a very low germination rate under natural conditions, and the understory habitat is unsuitable for *Avicennia ovata* seed germination; field surveys have revealed very few *Avicennia ovata* seedlings under mother trees. Zhong Cairong (Zhong Cairong, 2004, Seedling and Afforestation Techniques of *Avicennia ovata*) mentioned research on seed propagation and seedling cultivation techniques for the endangered mangrove plant *Avicennia ovata*. Seedling cultivation techniques encompass seed collection, seed treatment, seed germination, hardening-off, and field acclimatization. Due to the unique intertidal habitat of mangrove plants, they are subject to high light, high salinity, and physiological drought stress, making it difficult for endangered seedlings cultivated to survive in the wild. Existing breeding techniques mostly employ traditional seedling cultivation methods, which cannot fully address the survival rate issue of reintroducing Avicennia marina into the wild.

[0004] Propagating the endangered mangrove plant *Avicennia marina* ovata* to increase its population is the most direct and effective means of protecting my country's mangrove germplasm resources. Through exploration, we have developed a method for the field reintroduction of the endangered mangrove plant *Avicennia marina* ovata, providing a necessary means for the protection of its germplasm resources. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a method for the reintroduction of the endangered mangrove plant Aristolochia ovata into the wild. This method achieves the reintroduction of Aristolochia ovata into the wild by collecting and processing seeds, germinating seeds, cultivating strong seedlings, acclimatizing and cultivating seedlings, and planting them in the reintroduction site, thereby improving the survival rate of reintroduced Aristolochia ovata.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for the field reintroduction of the endangered mangrove plant *Avicennia marina* includes the following steps:

[0008] S1: Seed collection and treatment: Collect mature fruits of Avicennia marina ovata, soak them in seawater for 5-7 days, remove the rotten fruit peel, obtain the seeds, disinfect the seeds, soak them and then sow them.

[0009] S2: Seed germination: Spread the seeds treated in step S1 evenly on the seedling tray, cover with seedling soil of 1 to 2 times the volume of the seeds, spray fresh seawater on the soil surface to keep it moist, and spray with fungicide. Cultivate in a greenhouse.

[0010] S3: Seedling cultivation: Take seedlings that are 5cm to 6cm tall after S2 cultivation, transplant them into seedling bags containing nutrient soil and place them in a warm bed for temperature hardening;

[0011] The nutrient soil is composed of peat moss, sea mud, coconut coir and sawdust;

[0012] S4: Acclimation and cultivation: Transplant the seedlings with a height of more than 20cm obtained in step S3 to the acclimation seedbed for salinity acclimation.

[0013] S5: Returning to the original planting site: Transplant the seedlings with a height of more than 50cm obtained in step S4 to the original planting site. Apply cake fertilizer water one month after the transplanted ovoid-leafed sago palm survives.

[0014] Preferably, the S1 seed processing step is as follows:

[0015] Collect mature fruits of *Avicennia marina* ovalis and soak them in seawater for 5–7 days, changing the water daily until the fruit peel rots. Remove the rotten fruit peel by gently rubbing it while rinsing. After disinfecting with alcohol, soak the seeds in water at 42–45°C for 20 minutes, let them air dry, and then soak them in a 100 mg / L–200 mg / L gibberellin solution at 30°C–42°C for 2 hours before sowing.

[0016] Preferably, the greenhouse cultivation temperature for S2 seed germination is 30±2℃, with ventilation maintained and light exposure of 12h to 16h per day. Then, 0.25% to 1% water by weight is sprayed onto the soil surface regularly, and a fungicide is sprayed. The fungicide is prepared by mixing 30% hymexazol aqueous solution, 3% to 5% potassium permanganate, and distilled water in a mass ratio of 1.2:3.4:2500.

[0017] Preferably, the nutrient soil is prepared from peat moss, sea mud, coconut coir and sawdust in a volume ratio of 1-2:2-2.5:2-2.5:1.

[0018] Preferably, in step S3, the conditions for seedling temperature acclimatization are: a day-night temperature difference of 5-8℃, a daytime bed temperature of 20-25℃, and a nighttime temperature of 15-17℃.

[0019] Preferably, after the seedlings in the S4 culture reach a height of more than 20cm, the seedlings are transplanted to the acclimatization seedbed and watered continuously for 3-5 days, then watered with fresh seawater at a concentration of 0.25%-1% for 5-7 days, and then watered with seawater at a concentration of 2.0%-3.0% for 5-7 days.

[0020] Preferably, in step S4, the seedlings are domesticated by creating ridges in the nursery and opening water channels and ditches perpendicular to the ridges, applying organic fertilizer to the ridges, and then disinfecting the seedbed substrate with an 8% copper sulfate solution.

[0021] Preferably, step S5 is as follows: transplant the seedlings with a height of more than 50cm obtained in step S4 to the return site, with a planting spacing of 2m×2m. Apply cake fertilizer and water one month after the transplanted Avicennia marina has survived.

[0022] Preferably, the nutrient soil is prepared from peat moss, sea mud, coconut coir and sawdust in a volume ratio of 1.5:2:2.5:1.

[0023] Preferably, the return site is a coastal mudflat high tide zone with a seawater salinity of less than 2.0%.

[0024] The oilseed cake liquid is an organic fertilizer made from the residue left after pressing oil from plant seeds with high oil content (such as soybean cake, peanut cake, sesame cake, rapeseed cake, etc.) as the main raw material, which is fermented and decomposed to produce an organic liquid fertilizer that has a very good nourishing effect on plant growth.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention improves the germination rate and transplant survival rate of *Avicennia marina* seeds by adjusting seed germination factors, soaking the obtained seeds in diluted seawater to remove the pulp, disinfecting them, breaking dormancy with gibberellin and warm water to promote seed germination, and then rationally adjusting the seedling substrate and combining temperature acclimatization and salinity acclimatization methods. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0028] Example 1: Field reintroduction method of Avicennia ovata 'Ovate'

[0029] S1: Seed collection and treatment: Collect mature fruits of *Avicennia marina*, soak them in seawater for 5-7 days, changing the water once a day. After the fruit peel rots, gently rub the peel to remove the rotten material while rinsing with water. Disinfect the seeds with 70% alcohol and then soak them in 45℃ water for 20 minutes. After air drying, soak them in 100mg / L 30℃ gibberellin solution for 2 hours before sowing.

[0030] S2: Seed germination: Evenly scatter the seeds treated in step S1 onto seedling trays, cover with 1-2 times the volume of seedling soil, and cultivate in a greenhouse. The greenhouse cultivation temperature is 30±2℃, with ventilation maintained, 12 hours of light per day, and 20mL of fungicide sprayed per square meter of soil. Regularly spray the soil surface with 0.25% (w / w) fresh seawater to keep it moist. The fungicide is prepared by mixing 30% (w / w) hymexazol aqueous solution, 3% (w / w) potassium permanganate, and distilled water in a mass ratio of 1.2:3.4:2500.

[0031] S3: Seedling Cultivation: Take seedlings that are 5cm-6cm tall after cultivation and transplant them into seedling bags containing nutrient soil. Place them in a warm bed for temperature hardening. Set the day-night temperature difference to 5-8℃. The daytime bed temperature can be lowered to 20-25℃, and the nighttime temperature to 15-17℃. The seedling bags are 10cm diameter non-woven fabric seedling bags. The nutrient soil is prepared by mixing peat moss, sea mud, coconut coir, and sawdust in a volume ratio of 1:2:2:1. The particle size of the sawdust ranges from 3-5mm.

[0032] S4: Acclimatization and Cultivation: After the seedlings reach a height of over 20cm, transplant them to an acclimatization seedbed. After transplanting, thoroughly water the acclimatization seedbed with fresh water for 3-5 consecutive days, then water it with 0.25% seawater for 5-7 days, and then water it with 2.0% seawater for 5-7 days. The seawater used is made with tap water as the solvent and sea salt as the solute.

[0033] The acclimatization seedbed is prepared by creating raised beds in the nursery and opening water channels and tidal ditches perpendicular to the raised beds, applying organic fertilizer to the raised beds, and then disinfecting the seedbed substrate with an 8% copper sulfate solution.

[0034] S5: Relocation and Planting: Transplant seedlings exceeding 50cm in height obtained in step S4 to the high tide zone of coastal mudflats with a seawater salinity of less than 2.0%. The planting spacing should be 2m x 2m. One month after the transplanted *Sonneratia ovata* has survived, apply 3-4 kg / m² of diluted organic fertilizer. 3 .

[0035] The oilseed cake fertilizer solution is made by mixing dried soybean cake powder with water (volume ratio 1:8), soaking it in a sealed container, and fermenting it at 25-28℃ for 40 days. Then, ferrous sulfate is added at 0.5% of the volume of the fermented oilseed cake fertilizer solution and mixed well.

[0036] Example 2: Field reintroduction method of Avicennia ovata 'Ovula'

[0037] S1: Seed collection and treatment: Collect mature fruits of *Avicennia marina*, soak them in seawater for 5-7 days, changing the water once a day. After the fruit peel rots, rinse the fruit while gently rubbing to remove the rotten fruit peel. Disinfect the seeds with 70% alcohol and then soak them in 42℃ water for 20 minutes. After air drying, soak them in 200mg / L 42℃ gibberellin solution for 2 hours before sowing.

[0038] S2: Seed germination: Evenly scatter the seeds treated in step S1 onto seedling trays, cover with 1-2 times the volume of seedling soil, and cultivate in a greenhouse. The greenhouse temperature is 30±2℃, with ventilation, 16 hours of light per day, and 20mL of fungicide per square meter of soil. Regularly spray 1% distilled water onto the soil surface to keep it moist. The fungicide is prepared by mixing 30% hymexazol aqueous solution, 5% potassium permanganate, and distilled water in a mass ratio of 1.2:3.4:2500.

[0039] S3: Seedling Cultivation: After cultivation, seedlings 5cm-6cm tall are transplanted into seedling bags containing nutrient soil and placed in a warm bed for temperature hardening. The day-night temperature difference is set at 5-8℃, with a daytime bed temperature of 20-25℃ and a nighttime temperature of 15-17℃. The seedling bags are 10cm diameter non-woven fabric seedling bags. The nutrient soil is prepared by mixing peat moss, sea mud, coconut coir, and sawdust in a volume ratio of 2:2.5:2.5:1. The sawdust particle size range is 3-5mm.

[0040] S4: Acclimatization and Cultivation: After the seedlings reach a height of over 20cm, transplant them to an acclimatization seedbed. After transplanting, thoroughly water the acclimatization seedbed with fresh water for 3-5 consecutive days, then water it with 1% (w / w) fresh seawater for 5-7 days, and then water it with 3.0% (w / w) seawater for 5-7 days. The fresh seawater uses tap water as the solvent and sea salt as the solute.

[0041] The acclimatization seedbed is prepared by creating raised beds in the nursery and opening water channels and tidal ditches perpendicular to the raised beds, applying organic fertilizer to the raised beds, and then disinfecting the seedbed substrate with an 8% copper sulfate solution.

[0042] S5: Relocation and Planting: Transplant seedlings exceeding 55cm in height obtained in step S4 to the high tide zone of coastal mudflats with a seawater salinity of less than 2.0%. The planting spacing should be 2m × 2m. One month after the transplanted *Sonneratia ovata* has survived, apply 3-4 kg / m² of diluted organic fertilizer. 3 .

[0043] The oilseed cake fertilizer solution is made by mixing dried soybean cake powder with water (volume ratio 1:8), soaking it in a sealed container, and fermenting it at 25-28℃ for 40 days. Then, ferrous sulfate is added at 0.5% of the volume of the fermented oilseed cake fertilizer solution and mixed well.

[0044] Example 3: Field reintroduction method of Avicennia ovata 'Ovate'

[0045] S1: Seed collection and treatment: Collect mature fruits of *Avicennia marina*, soak them in seawater for 5-7 days, changing the water once a day. After the fruit peel rots, gently rub the peel to remove the rotten material while rinsing with water. Disinfect the seeds with 70% alcohol and then soak them in 45℃ water for 20 minutes. After air drying, soak them in 100mg / L 30℃ gibberellin solution for 2 hours before sowing.

[0046] S2: Seed germination: Evenly scatter the seeds treated in step S1 onto seedling trays, cover with 1-2 times the volume of seedling soil, and cultivate in a greenhouse. The greenhouse cultivation temperature is 30±2℃, with ventilation maintained, 12 hours of light per day, and 20mL of fungicide sprayed per square meter of soil. Then, periodically spray 0.50% concentration of fresh seawater onto the soil surface to keep the soil moist. The fungicide is prepared by mixing 30% concentration of hymexazol (aqueous solution), 4% concentration of potassium permanganate, and distilled water in a mass ratio of 1.2:3.4:2500.

[0047] S3: Seedling Cultivation: After cultivation, take seedlings that are 5cm-6cm tall, transplant them into seedling bags containing nutrient soil, and place them in a warm bed for temperature hardening. Set the day-night temperature difference to 5-8℃, with a daytime bed temperature of 20-25℃ and a nighttime temperature of 15-17℃. The seedling bags are 10cm diameter non-woven fabric seedling bags. The nutrient soil is prepared by mixing peat moss, sea mud, coconut coir, and sawdust in a volume ratio of 1.5:2:2.5:1. The particle size of the sawdust ranges from 3-5mm.

[0048] S4: Acclimatization and Cultivation: After the seedlings reach a height of over 20cm, transplant them to an acclimatization seedbed. After transplanting, thoroughly water the acclimatization seedbed with fresh water for 3-5 consecutive days, then water it with 0.75% seawater for 5-7 days, and then water it with 3.0% seawater for 5-7 days. The seawater used is made with tap water as the solvent and sea salt as the solute.

[0049] The acclimatization seedbed is prepared by creating raised beds in the nursery and opening water channels and tidal ditches perpendicular to the raised beds, applying organic fertilizer to the raised beds, and then disinfecting the seedbed substrate with an 8% copper sulfate solution.

[0050] S5: Relocation and Planting: Transplant the seedlings obtained in step S4 that are over 60cm in height to the high tide zone of the coastal mudflats where the seawater salinity is less than 2.0%. The planting spacing is 2m × 2m. One month after the transplanted *Sonneratia ovata* has survived, apply 3-4 kg / m² of cake fertilizer solution. 3 .

[0051] The oilseed cake fertilizer solution is made by mixing dried soybean cake powder with water (volume ratio 1:8), soaking it in a sealed container, and fermenting it at 25-28℃ for 40 days. Then, ferrous sulfate is added at 0.5% of the volume of the fermented oilseed cake fertilizer solution and mixed well.

[0052] Comparative Example 1: Field Regression Method of Avicennia ovata 'Ovum'

[0053] The difference between this embodiment and embodiment 3 is that the nutrient soil in S3 is a mixture of peat moss, sea mud, coconut coir and sawdust in a volume ratio of 1:2:2:0.2.

[0054] Comparative Example 2: Field Regression Method of Avicennia ovata 'Ovulata'

[0055] The difference between this comparative example and Example 3 is that the seedlings were transplanted into the wild after being irrigated with fresh water without undergoing salinity acclimatization.

[0056] Comparative Example 3: Field Regression Method of Avicennia ovata 'Ovum'

[0057] The difference between this comparative example and Example 1 is that it did not undergo seedling temperature acclimation. The seedling culture temperature in step S4 was a bed temperature of 20-25°C.

[0058] Experimental determination 1

[0059] Each group consists of 30 plants. After 6 months of planting in the return site, growth data are collected, and the growth and development of the returned plants and their survival rate are recorded.

[0060] Table 1

[0061]

[0062] Experimental results show that, in Examples 1-3, adjusting seed germination factors, scientifically proportioning seedling substrates, temperature acclimatization, and domestication effectively improved the reintroduction planting effect of the endangered mangrove plant *Avicennia marina*. The transplanted seedlings from the reintroduced sites had high survival rates and were robust. In particular, Example 3 showed the best results, with seedlings cultivated using a nutrient soil mixture of peat moss, sea mud, coconut coir, and sawdust in a 1.5:2:2.5:1 ratio producing the highest quality seedlings.

[0063] Experimental determination 2: Selection of the return location

[0064] Seedlings with a height exceeding 60cm cultivated in Example 3 were selected as field transplantation sites in the high tide, mid tide, and low tide zones of coastal mudflats with seawater salinity less than 2.0%. 100 seedlings were planted at each transplantation site. Growth was assessed after 6 months, and the results are shown in Table 2.

[0065] Table 2

[0066] Orgasm 100 100％ 126.7 Mid-tidal zone 87 87％ 108.1 Low tide zone 73 73％ 105.2

[0067] As shown in Table 2, the survival rates of the high tide zone, mid-tide zone, and low tide zone were 100%, 87%, and 70%, respectively. The high tide zone had the highest survival rate, followed by the mid-tide zone, and the low tide zone had the lowest survival rate. Seedlings planted in the high tide zone grew faster, were taller, and were more robust.

[0068] 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 returning a critically endangered mangrove plant, Sonneratia caseolaris, to the wild, characterized by, Includes the following steps: S1: Seed collection and treatment: Collect mature fruits of *Avicennia marina*, soak them in seawater for 5-7 days, changing the water once a day until the pericarp rots. Remove the rotten pericarp by gently rubbing it while rinsing it with water. After disinfecting with alcohol, soak the seeds in water at 42-45℃ for 20 minutes, air dry them naturally, and then soak them in a 100mg / L-200mg / L gibberellin solution at 30℃-42℃ for 2 hours before sowing. S2: Seed germination: Evenly scatter the seeds treated in step S1 on the seedling tray, cover with soil, spray with fungicide, spray fresh seawater on the soil surface to keep it moist, and cultivate in a greenhouse. S3: Seedling cultivation: Take seedlings that are 5cm-6cm tall after S2 cultivation and transplant them into seedling bags containing nutrient soil and place them in a warm bed for temperature hardening. The nutrient soil is prepared by mixing peat moss, sea mud, coconut coir and sawdust in a volume ratio of 1-2:2-2.5:2-2.5:

1. The temperature hardening conditions for seedlings are: a day-night temperature difference of 5-8℃, a daytime bed temperature of 20-25℃ and a nighttime temperature of 15-17℃. The nutrient soil is composed of peat moss, sea mud, coconut coir and sawdust; S4: Acclimatization and cultivation: After the seedlings reach a height of over 20cm, transplant them to an acclimatization seedbed and water them continuously for 3-5 days, then water them with 0.25%-1% fresh seawater for 5-7 days, and then water them with 2.0%-3.0% seawater for 5-7 days. S5: Relocation to the original planting site: Transplant the seedlings with a height of more than 50cm obtained in step S4 to the original planting site. Apply cake fertilizer water one month after the transplanted ovary-leaved sago palm survives. The original planting site is the high tide zone of the coastal mudflats with a seawater salinity of less than 2.0%.

2. The method for returning the endangered mangrove plant of C. ovata to the wild according to claim 1, wherein, Step S2 is as follows: The seeds treated in step S1 are evenly scattered on the seedling tray, covered with seedling soil of 1 to 2 times the volume of the seeds, sprayed with fungicide, and 0.25% to 1% water (by mass concentration) of fresh seawater is sprayed on the soil surface regularly for greenhouse cultivation. The greenhouse cultivation temperature is 30±2℃, ventilation is maintained, and the light is 12h to 16h per day. The fungicide is prepared by mixing 30% hymexazol aqueous solution, 3% to 5% potassium permanganate, and distilled water in a mass ratio of 1.2:3.4:2500.

3. The method for returning the endangered mangrove plant of C. ovata to the wild according to claim 1, wherein: In step S4, seedling acclimatization involves creating raised beds in the nursery and opening water channels and ditches perpendicular to the raised beds. Organic fertilizer is then applied to the raised beds, and the seedbed substrate is disinfected with an 8% copper sulfate solution.

4. The method of claim 1, wherein the endangered mangrove plant is Gracilaria lichenoides. 5 Step S5 is as follows: Transplant the seedlings with a height of more than 50cm obtained in step S4 to the return site, with a planting spacing of 2m×2m. Apply cake fertilizer water one month after the transplanted ovoid-leafed mangrove has survived.

5. The method for the field reintroduction of the endangered mangrove plant *Avicennia marina* as described in claim 1, characterized in that, The nutrient soil is prepared from peat moss, sea mud, coconut coir and sawdust in a volume ratio of 1.5:2:2.5:1.

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