Seed propagation method of endangered plant rhaphiolepis indica

Abstract

The application discloses a seed propagation method of endangered plant Xymaloschka apetala. The application realizes quick and sufficient swelling of the seed within 3-5 hours by performing minimally invasive treatment on the seed coat of the seed hilum of Xymaloschka apetala, and awakens the seed vitality. The seed coat after swelling is easy to peel off without damaging the cotyledon and seed embryo tissue. After peeling off the seed coat, the seed is completely immersed in sterile water for 3-5 days, and the water is changed every 12 hours. After the seed radicle is swollen, the seed is immediately sowed into a plug, and the sowing adopts a "shallow sowing half cotyledon exposed method", the radicle is partially buried in the substrate, and the cotyledon is exposed half to contact air and light. The method realizes that the seed dormancy breaking to the initial seed germination of Xymaloschka apetala only needs 15 days, and the seedling embryo germination only needs 20 days and the seedling emergence rate reaches 98%. The method greatly shortens the seed propagation time of Xymaloschka apetala, improves the seed germination efficiency, and saves the seedling seed cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of endangered plant conservation and seedling breeding, and particularly relates to a seed propagation method of endangered plant Ormosia henryi. BACKGROUND

[0002] Ormosia henryi is a plant of the genus Ormosia in the family Leguminosae, which is a good material for furniture and bridges, has medicinal value, and is a precious garden tree species, and is deeply loved by people. However, many rare Ormosia tree species, such as Ormosia henryi, are facing the danger of extinction, mainly due to the problem of difficult propagation. In order to quickly propagate good resources, plant rapid propagation technologies such as tissue culture, cutting, and micropropagation are increasingly used in the study of Ormosia tree species, but there are corresponding technical bottlenecks. The seeds of Ormosia henryi have a physical dormancy period, and the physical dormancy causes problems such as difficult seedling emergence and uneven seedling emergence. When using seed germination, breaking dormancy is a very critical step. The dormancy mechanism of Leguminosae seeds and how to break the seed dormancy period have always been a key problem in scientific research. Some studies have made preliminary exploration of the causes of physical dormancy of Ormosia seeds and the breaking of dormancy, and have commented on the causes of seed dormancy and the method of breaking dormancy, but there is a severe lack of efficient and rapid dormancy breaking methods and corresponding seedling cultivation techniques.

[0003] Ormosia henryi is a national second-class protected plant and a species endemic to China. It is naturally distributed in provinces such as Anhui, Fujian, Jiangxi, Hubei, Hunan, Guangdong, and Hainan in China's subtropical regions, and is mostly found in mountainous areas, riverbanks, and valley forests at altitudes of 600m to 1200m. Ormosia henryi has strict requirements for ecological growth environments, and its wild resources are scarce and unevenly distributed. Due to its narrow growth and distribution area and strict requirements for ecological environments, the wild resources of Ormosia henryi are becoming increasingly scarce. Although Ormosia henryi prefers warm conditions, it also has a certain degree of cold tolerance. It prefers moist soil and dislikes dry conditions. Ormosia henryi has great flexibility in terms of light intensity and duration, and can grow under full light or complete darkness, but the growth state of Ormosia henryi under complete darkness is poor, and it prefers scattered light under full light. Ormosia henryi is naturally rare and has a low seed setting rate, and the population from which seeds can be collected is limited. Therefore, vessel seedling is the preferred method for Ormosia henryi seedling cultivation.

[0004] The hard, dense seed coat of *Palmaria dentata* seeds hinders the supply of water and oxygen, resulting in poor water and air permeability and significantly inhibiting germination. Field seed germination experiments conducted using an artificially simulated soil seed bank revealed that, under natural conditions, the germination rate of *Palmaria dentata* seeds buried for one year was only 16%. Currently, physicochemical methods are commonly used to treat *Palmaria dentata* seeds to promote germination. One such method involves soaking the seeds in wood ash for one day, followed by soaking in warm water for three days, and finally stratifying them in moist sand for three to four days. Research on methods to break the dormancy of *Palmaria dentata* seeds has shown that 500 mg / L... -1 Soaking *Palmeria nivea* seeds in gibberellin for 12 hours followed by 45 days of wet storage in sand at 4℃ broke seed dormancy, increasing germination rate and germination potential by 36.6% and 32.7%, respectively, compared to the control. After wet storage in sand, the activities of acid phosphatase, amylase, and protease, as well as the contents of GA3 and ATP, increased with prolonged storage time, while the ABA content decreased. Furthermore, 2 hours of concentrated sulfuric acid etching effectively improved seed coat permeability, enhancing water absorption without damaging the seeds. Overall, chemical treatment and variable temperature methods improved seed coat permeability and increased water absorption in *Palmeria nivea*. Wet storage in sand played a crucial role in breaking seed dormancy by increasing the activity of related enzymes.

[0005] The hard, dense seed coat of *Palmeria nivea* seeds, poor water and air permeability, and dormancy contribute to slow germination, long germination periods, and low germination rates, posing major challenges to artificial cultivation. Under most germination conditions, the germination rate of *Palmeria nivea* seeds is less than ideal, and germination is uneven. Although researchers have explored methods to break seed dormancy, shortening germination time and improving germination rates to some extent, the dormancy mechanism of *Palmeria nivea* seeds remains unclear, and the key factors restricting seed germination have not been definitively determined. Furthermore, *Palmeria nivea* seedlings grow very slowly, have poor drought and frost resistance, and are prone to death, significantly impacting the expansion of *Palmeria nivea* populations through seed propagation. Strengthening the protection of wild *Palmeria nivea* resources, focusing on breakthroughs in seed propagation techniques, protecting germplasm resources, enhancing resource cultivation efforts, and promoting artificial propagation are of great significance for the conservation of this endangered plant. Summary of the Invention

[0006] Breaking the dormancy of *Palmeria nivea* seeds quickly is a critical bottleneck in seed propagation. Breaking seed dormancy requires a certain amount of time, and given the extremely long dormancy period and slow growth rate of this endangered plant, shortening the dormancy-breaking time is crucial for maximizing seedling germination and development.

[0007] This invention solves the problem of shortening the dormancy period of Dalbergia odorifera seeds and promoting rapid seed germination. It achieves a rapid seedling cultivation technology that takes only 15 days from breaking dormancy to seed germination and only 20 days from seedling embryo germination, with a germination rate of 98%. This greatly shortens the seed propagation time of Dalbergia odorifera seeds, improves seed germination efficiency, and provides more growth time for promoting seedling growth and development in the later stages.

[0008] A method for seed propagation of the endangered plant *Palmeria nivea* according to the present invention includes the following steps:

[0009] S1. Collect naturally matured pods of the current year, collect plump and uniformly colored pods of the pods, dry them at room temperature within 5 days, and select seeds with a thousand-seed weight of not less than 200g.

[0010] S2. Use a blade to remove a 2mm*3mm area of ​​the seed coat from the hilum, being careful not to damage the cotyledons, hypocotyl, plumule, and radicle at the hilum; soak the seeds in sterile water for 3-5 hours in the dark at room temperature, and then completely peel off the seed coat, being careful not to damage the cotyledons, hypocotyl, plumule, and radicle when peeling off the seed coat.

[0011] S3. Under dark conditions at room temperature, soak the seeds with the seed coat removed in sterile water for 3-5 days, changing the water every 12 hours during this period;

[0012] S4. After the seed radicle swells and grows to 1-2 mm, sow the seeds into the cultivation substrate for germination culture. The sowing method is to bury half of the seed radicle and half of the cotyledons in the cultivation substrate, with half of the cotyledons exposed outside the cultivation substrate. The germination culture conditions are: temperature 25℃, relative humidity 70%, light intensity 5000 lx, and light duration 12 h / d. Keep the cultivation substrate completely moist and spray sterile water 2-3 times a day to keep the cotyledons moist. Stop spraying water after the cotyledons turn green.

[0013] Preferably, the cultivation substrate is peat moss.

[0014] Preferably, in step S2, the seeds are soaked in sterile water for 5 hours.

[0015] Preferably, in step S3, the seeds with the seed coat removed are soaked in sterile water for 3 days.

[0016] This invention is based on minimally invasive treatment of the seed coat of *Dalbergia odorifera* seeds, breaking down mechanical barriers in the seed coat and eliminating problems such as difficulty in water and oxygen absorption caused by poor seed coat permeability. The minimally invasive removal of the seed coat at the hilum is effective for two reasons: First, the seed coat at the hilum encloses the embryo tissue; enabling the embryo to expel water and oxygen in the shortest possible time is crucial for breaking seed dormancy. Removing the seed coat from other parts obviously cannot achieve the same rapid imbibition effect in the same time. Second, *Dalbergia odorifera* seed coats contain substances that inhibit germination. The longer the soaking time, the higher the permeability of these substances, which, to some extent, enter the cotyledons and embryo tissue during imbibition, also inhibiting subsequent germination. Furthermore, the dry seed coat of *Dalbergia odorifera* seeds is very tightly adhered to the cotyledons, making it almost impossible to remove the dried seeds without damage, resulting in a certain rate of cotyledon damage. Therefore, this invention uses minimally invasive treatment at the hilum to break down the mechanical barriers to germination in the first step, achieving a very good imbibition effect in a short time. In the seed tray planting process, this invention employs a self-developed "shallow sowing with partially exposed cotyledons" method. The purpose is to keep the embryo of the seed in darkness after the seed coat is removed, allowing it to fully absorb moisture while being buried in the seed tray substrate, promoting radicle elongation and development. The cotyledons, partially exposed to sunlight, effectively inhibit mold growth and promote cotyledon activation and rapid chlorophyll formation. Once the cotyledons begin synthesizing chlorophyll, they enter the dormancy-breaking germination phase. After the seed coat is removed, half of the cotyledons containing the embryo tissue are kept in darkness, promoting root elongation. The other half, exposed to air, allows for full contact with oxygen and light-induced chlorophyll production, maximizing seed vigor. This entire process achieves rapid dormancy breaking and rapid germination of the *Dalbergia odorifera* seeds, resulting in unprecedentedly rapid germination rates and germination rates.

[0017] The seeds of the rare and endangered plant Dalbergia odorifera are already precious. This invention achieves efficient dormancy breaking and germination, providing important technical support for the protection and conservation of Dalbergia odorifera. Attached Figure Description

[0018] Figure 1 These are images of minimally invasive treatment of the hilum of a Dalbergia odorifera seed; Figure A shows the appearance of the seed coat at the hilum before treatment; Figure B shows the appearance after minimally invasive removal of the seed coat at the hilum.

[0019] Figure 2 These are images of the appearance of rosewood seeds before and after imbibition; Figure A shows the appearance of the seeds before imbibition; Figure B shows the appearance of the seeds after imbibition and removal of the seed coat.

[0020] Figure 3The T1 treatment involves planting *Palmeria nivea* seeds after peeling and shallow sowing with half cotyledons exposed. Figure A shows the seeds after peeling and planting using the shallow sowing method with half cotyledons exposed. Figure B shows the cotyledons turning completely green 7 days after planting. Figure C shows seeds starting to germinate 12 days after planting.

[0021] Figure 4 This is a comparison of the effects of different seed treatments using the shallow sowing method with half-exposed cotyledons after 25 days of growth and development. Among them, Figure A shows the effect of planting the seed with micro-treatment of the seed hilum and seed coat in the T1 treatment, with uniform emergence; Figure B shows the germination effect of the untreated seeds in the T2 treatment, with no signs of germination; Figure C shows the effect of the T5 treatment with only micro-injury of the seed coat followed by imbibition treatment without removing the seed coat, with widespread mold growth. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Example 1

[0024] This invention achieves rapid dormancy breaking and rapid germination of Pterocarpus santalinus seeds by combining the following technologies:

[0025] First, the seed coat at the hilum needs to be removed with minimal invasiveness. Second, it needs to be allowed to fully absorb water and swell. Then, the seed coat is peeled off, and the seeds are soaked to swell and awaken the radicle. Finally, the seeds are cultured under moderate light conditions using the "shallow sowing with half-exposed cotyledons method" to stimulate chlorophyll synthesis in the cotyledons and promote seed germination.

[0026] 1. Materials and Methods

[0027] 1.1 Screening of Dalbergia odorifera seeds

[0028] The experimental seed material was obtained from approximately 75-year-old *Dalbergia odorifera* trees in Wuhua County, Meizhou City, Guangdong Province. The seeds were collected on November 15, 2021, from naturally matured seed pods with dark brown pericarps. After shelling, plump, round, and uniformly colored *Dalbergia odorifera* seeds were selected and placed in a drying box for 2 days. The seeds were then thoroughly washed for 3-5 minutes with detergent (1 mL of Libai dishwashing liquid diluted in 500 mL of tap water) to remove impurities and attached substances. After rinsing with running water, the seeds were repeatedly washed 3-5 times with sterile water and then blotted dry with sterile filter paper. Seeds with a thousand-seed weight of at least 200 g were selected for subsequent experiments.

[0029] 1.2 Design of Seed Treatment and Sowing Methods

[0030] Configure the following settings:

[0031] (1) T1: The seed coat with a surface area of ​​2mm*3mm at the hilum of the selected rosewood seeds was minimally invasively removed.Figure 1 B), avoid damaging the cotyledons, hypocotyl, plumule, and radicle at the seed hilum. Soak the seeds in sterile water for 5 hours at room temperature in the dark, then completely remove the seed coat. Figure 2 Do not damage the cotyledons, hypocotyl, plumule, or radicle. Under dark conditions at room temperature, soak the seeds (with the seed coat removed) in sterile water for 3 days (after soaking for 3 days, the radicle will swell and elongate to 1-2 mm), changing the water every 12 hours. Then, use the "shallow sowing method with partially exposed cotyledons" (…). Figure 3 A) Sow seeds in seed trays. The “shallow sowing with half cotyledon exposed method” involves burying the part of the seed on the radicle side and half of the cotyledon (the half of each cotyledon closest to the radicle side) in the cultivation substrate, with half of the cotyledon exposed outside the cultivation substrate.

[0032] (2) T2: No seed coat micro-invasive treatment is performed on the selected rosewood seeds. Figure 1 A) Under room temperature and darkness, soak the seeds in sterile water for 5 hours, then continue soaking in sterile water for 3 days, changing the water every 12 hours. Sow the seeds in seed trays using the "shallow sowing with half-exposed cotyledons" method described above. Specifically, bury half of the seed's hilum and cotyledon position in the cultivation substrate, leaving the other half of the cotyledon position exposed outside the cultivation substrate.

[0033] (3) T3: For the selected *Palmaria* seeds, a 2mm*3mm area of ​​the seed coat at the hilum is minimally invasively removed, taking care not to damage the cotyledons, hypocotyl, plumule, or radicle at the hilum. Under dark conditions at room temperature, the seeds are soaked in sterile water for 5 hours, then the seed coat is completely removed, again taking care not to damage the cotyledons, hypocotyl, plumule, or radicle. Under dark conditions at room temperature, the seeds with the seed coat removed are soaked in sterile water for 3 days, changing the water every 12 hours. Following conventional seed sowing methods, the entire seed is buried in the seed tray substrate.

[0034] (4) T4: No seed coat micro-invasive treatment was performed on the selected rosewood seeds. The seeds were soaked in sterile water for 5 hours under dark conditions at room temperature, and then soaked in sterile water for another 3 days, with the water changed every 12 hours. The entire seed was buried in the plug culture substrate according to the conventional seed sowing method.

[0035] (5) T5: For the selected *Palmaria* seeds, a 2mm*3mm area of ​​the seed coat at the hilum is minimally invasively removed, taking care not to damage the cotyledons, hypocotyl, plumule, or radicle at the hilum. Under room temperature and darkness, soak the seeds in sterile water for 5 hours, then continue soaking for 3 days without removing the seed coat, changing the water every 12 hours. Sow the seeds in seed trays using the "shallow sowing with half-exposed cotyledons method" described above. Specifically, bury half of the seed at the hilum and half at the cotyledon position in the cultivation substrate, leaving the other half exposed outside the substrate.

[0036] (6) T6: For the selected *Palmaria* seeds, a 2mm*3mm area of ​​the seed coat at the hilum is minimally invasively removed, taking care not to damage the cotyledons, hypocotyl, plumule, or radicle at the hilum. Under room temperature and darkness, the seeds are soaked in sterile water for 5 hours without removing the seed coat, and then soaked in sterile water for another 3 days, changing the water every 12 hours. Following standard seed sowing methods, the entire seed is buried in the seed tray substrate.

[0037] 1.3 Selection and treatment of substrate for ploughing trays

[0038] Seeds treated with T1-T6 were sown in plug trays filled with peat moss substrate. Each plug tray had a size of approximately 5*5*8 cm (length*width*depth). During germination, the trays were kept at a high water level (approximately 60% of the tray depth) to maintain a highly moist substrate within the holes. Each treatment had 20 seeds, with 3 biological replicates.

[0039] During the seedling tray planting stage, the seedlings were uniformly placed under the conditions of 25℃ temperature, 70% relative humidity, 5000lx light intensity, and 12h / d light duration for cultivation.

[0040] 2 Results and Analysis

[0041] (1) Analysis of imbibition effect of different treatments (T1-T6) on Pterocarya spp. seeds

[0042] After treating *Palmaria dentata* seeds with treatments T1-T6 for 5 hours under each treatment condition, the seed coat imbibition effect was compared. The results are shown in Table 1. The comparison revealed that the minimally invasive treatment at the seed hilum rapidly achieved imbibition in the seed coat and even the cotyledons. Seeds T1, T3, T5, and T6 achieved excellent imbibition effects after 5 hours of treatment. Seeds T2 and T4, which did not undergo any minimally invasive treatment, showed no change in imbibition. This indicates that the minimally invasive treatment can first break down the mechanical barriers to *Palmaria dentata* seed germination and promote the imbibition of the seed coat and cotyledons, achieving the first step in rapidly breaking seed dormancy.

[0043] Table 1 Comparison of imbibition effects of different seed coat treatments

[0044] Seed pre-treatment regime Effect after 5h T1 Complete seed coat swelling T2 No change T3 Complete seed coat swelling T4 No change T5 Complete seed coat swelling T6 Complete seed coat swelling

[0045] (2) Analysis of germination effects of different planting treatments (T1-T6) on Pterocarya stenoptera seeds

[0046] Seeds treated with T1-T6 were planted in plug trays according to the respective treatment conditions, and seed germination was tracked and investigated during the period. The investigation revealed (Table 2) that on day 7, the cotyledons of the T1 treatment showed greening upon light stimulation. Figure 3B), the appearance and germination of seeds in other treatments remained unchanged. On day 12, the cotyledons of treatment T1 continued to turn dark green due to light exposure. Figure 3 C), and 5.12% of the cotyledons were slightly open, and 2.98% of the seeds began to sprout; treatments T2 and T4 showed no changes; treatments T3, T5, and T6 showed signs of mold growth on the seed surface. At day 25 ( Figure 4 In the T1 treatment, the cotyledons developed well under the influence of light, with 98.12% of the cotyledons slightly open and 90.56% of the seeds showing buds, achieving excellent germination results. The T2 and T4 treatments showed no changes. The T3, T5, and T6 treatments showed large-scale mold growth on the seed surface, and the vast majority of the seeds lost their germination vitality and entered a state of decay. Only 3.23% of the seeds in the T3 treatment remained viable and showed signs of radicle elongation.

[0047] Table 2 Comparison of germination effects of different seed treatments

[0048]

[0049]

[0050] (3) Screening of the optimal and most efficient treatment method for quickly breaking the dormancy of rosewood seeds

[0051] Table 2 analysis revealed the optimal combination of different seed treatments and germination methods for *Palmaria rubra*. Treatment T1 achieved the highest seed germination rate and germination efficiency, with the shortest dormancy breaking time. Compared to treatments T2-T6, treatment T1 clearly demonstrated higher efficiency. Treatment T1 achieved a germination rate of 98%, significantly superior to other currently reported techniques for *Palmaria rubra* seed propagation.

[0052] Example 2

[0053] Seeds treated according to Example 1 (T1) and sown using the same method as in Example 1 were placed in seed trays containing peat moss substrate. Each seed tray had a size of approximately 5*5*8 cm (length*width*depth). During germination, the trays were kept at a high water level (approximately 60% of the tray depth) to maintain a highly moist substrate within the cells. Forty seeds were used for each treatment, with three biological replicates.

[0054] During the tray planting stage, different temperatures and light intensities were set (Table 3), with a relative humidity of 70% and a light duration of 12 h / d. Under these cultivation conditions, the seed germination was investigated 12 days after sowing, and the results are shown in Table 3. The results indicate that 25℃ is the optimal temperature and 5000 lx is the optimal light intensity.

[0055] Table 3. Results of the survey 12 days after sowing under different temperatures and light intensities during the plug planting stage.

[0056]

[0057]

Claims

1. A method for seed propagation of the endangered plant *Dalbergia odorifera*, characterized in that, Includes the following steps: S1. Collect naturally matured pods of the current year, collect plump and uniformly colored pods of the pods, dry them at room temperature within 5 days, and select seeds with a thousand-seed weight of not less than 200g. S2. Use a blade to remove a 2mm*3mm area of ​​the seed coat from the hilum, being careful not to damage the cotyledons, hypocotyl, plumule, and radicle at the hilum; soak the seeds in sterile water for 3-5 hours in the dark at room temperature, and then completely peel off the seed coat, being careful not to damage the cotyledons, hypocotyl, plumule, and radicle when peeling off the seed coat. S3. Under dark conditions at room temperature, soak the seeds with the seed coat removed in sterile water for 3-5 days, changing the water every 12 hours during this period; S4. After the seed radicle swells and grows to 1-2 mm, sow the seeds into the cultivation substrate for germination culture. The sowing method is to bury half of the seed radicle and half of the cotyledons in the cultivation substrate, with half of the cotyledons exposed outside the cultivation substrate. The germination culture conditions are: temperature 25℃, relative humidity 70%, light intensity 5000 lx, and light duration 12 h / d. Keep the cultivation substrate completely moist and spray sterile water 2-3 times a day to keep the cotyledons moist. Stop spraying water after the cotyledons turn green.

2. The method according to claim 1, characterized in that, The cultivation substrate is peat moss.

3. The method according to claim 1, characterized in that, In step S2, the seeds are soaked in sterile water for 5 hours.

4. The method according to claim 1, characterized in that, In step S3, the seeds with the seed coat removed are soaked in sterile water for 3 days.

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