A cultivation method for promoting seed germination of wild Hippophae rhamnoides

Abstract

This invention relates to the field of plant growth regulation technology, specifically disclosing a cultivation method for promoting the germination of wild sea buckthorn seeds. The main inventive concept lies in the synergistic application of amino oligosaccharides (AS) and sodium nitroprusside (SNP) in promoting the germination and hypocotyl elongation of wild sea buckthorn seeds. Using AS alone (optimal concentration 0.12 mmol / L) or SNP alone (optimal concentration 0.09 mmol / L) can significantly improve the germination rate (GP) and germination index (GI) of sea buckthorn seeds, while simultaneously promoting hypocotyl elongation. Combining AS (0.12 mmol / L) and SNP (0.08 mmol / L) in a 1:1 ratio produces a synergistic effect, promoting the germination of wild sea buckthorn seeds, increasing seed α-amylase activity, and promoting hypocotyl elongation. This invention provides a low-cost, high-efficiency technical solution for efficient sea buckthorn seedling cultivation, applicable to ecological restoration and economic forest cultivation.

Description

Technical Field

[0001] This invention relates to the field of plant growth regulation technology, and specifically discloses a cultivation method for promoting the germination of wild sea buckthorn seeds. Background Technology

[0002] Sea buckthorn (Hippophae rhamnoides), a plant with both ecological and economic value, is widely used in soil and water conservation, ecological restoration, and medicinal development. However, its low seed germination rate and slow seedling growth severely restrict large-scale seedling cultivation and artificial forest construction. Traditional methods often rely on single plant growth regulators (such as gibberellin and potassium nitrate) or physical treatments (warm water soaking), but the effects are limited, and high-concentration treatments can easily induce inhibitory effects, making it difficult to balance high efficiency and safety.

[0003] In recent years, amino oligosaccharides (AS) and sodium nitroprusside (SNP) have attracted attention in the field of plant growth regulation due to their functions of inducing stress resistance and releasing nitric oxide (NO) signals, respectively. Studies have shown that AS can enhance plant stress resistance by activating the expression of defense genes, while SNP promotes cell elongation through NO-mediated cell wall relaxation. However, the synergistic mechanism of AS and SNP and their application in sea buckthorn seed germination have not been reported in existing technologies, and single-component treatments are insufficient to overcome the bottlenecks in germination efficiency and growth quality.

[0004] Therefore, there is an urgent need to develop a technical solution based on the synergistic effect of multiple components to improve the germination potential of sea buckthorn seeds and seedling vigor in a low-cost and high-efficiency manner, so as to meet the needs of ecological restoration and industrial planting. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the synergistic effect of amino oligosaccharide (AS) and sodium nitroprusside (SNP). By optimizing the ratio and processing procedure, the germination rate, germination speed and hypocotyl elongation efficiency of wild sea buckthorn seeds can be significantly improved, solving the problems of low germination efficiency and significant inhibition effect at high concentrations in the prior art.

[0006] In one aspect, the present invention provides a cultivation method for promoting the germination of wild sea buckthorn seeds, comprising the following steps: sea buckthorn seeds are soaked in warm water and disinfected in sequence, then immersed in a mixed solution containing amino oligosaccharide (AS) and sodium nitroprusside (SNP), wherein the concentration of AS is 0.06-0.48 mmol / L, the concentration of SNP is 0.03-0.12 mmol / L, the molar ratio of AS to SNP is 1:0.8-1:1.2, the soaking time is 20-28 hours, and then germination culture is carried out at 25°C in the dark.

[0007] In some embodiments, the concentration of AS is 0.12 mmol / L, the concentration of SNP is 0.08 mmol / L, and the molar ratio of AS to SNP is 1:1.

[0008] In some embodiments, the soaking time of the mixed solution is 24 hours, and the germination culture conditions are a constant temperature of 25°C in the dark.

[0009] In some embodiments, the synergistic effect of the AS and SNP enables sea buckthorn seeds to achieve a germination rate (GP) of over 90% and a hypocotyl elongation of over 45% on day 4.

[0010] In one aspect, the present invention provides a compound plant growth regulator of amino oligosaccharide (AS) and sodium nitroprusside (SNP), which contains AS at a concentration of 0.06-0.48 mmol / L and SNP at a concentration of 0.03-0.12 mmol / L, with a molar ratio of 1:0.8-1:1.2, and is suitable for promoting the germination of sea buckthorn seeds and the growth of seedlings.

[0011] In some embodiments, the concentration of AS is 0.12 mmol / L and the concentration of SNP is 0.08 mmol / L.

[0012] In one aspect, the present invention provides the application of compound plant growth regulators in sea buckthorn seedling cultivation or ecological restoration.

[0013] In one aspect, the present invention provides a method for treating wild sea buckthorn seeds, the method comprising: disinfecting the sea buckthorn seeds and then treating them using the above method, wherein the hypocotyl elongation growth rate reaches 45%-50% on the 4th day.

[0014] In some embodiments, the disinfection process includes soaking in a 3 g / L potassium permanganate solution for 15 minutes and rinsing with distilled water 2-3 times.

[0015] In one aspect, the present invention provides a sea buckthorn seedling cultivation system, including the above-mentioned compound regulator and an intelligent climate chamber for constant temperature cultivation at 25°C under dark conditions.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] For the first time, the synergistic effect of AS and SNP was revealed: by using a 1:1 ratio of AS (0.12 mmol / L) and SNP (0.08 mmol / L), the concentration limit of a single component was broken, and the germination rate and hypocotyl elongation rate were simultaneously improved.

[0018] High efficiency and safety go hand in hand: the combined treatment significantly reduces the concentration of SNP used, avoids oxidative damage caused by high concentrations, and reduces the amount of AS used, resulting in an overall cost reduction of 30%-40%.

[0019] Standardized and universal operation: The standardized process of 24-hour soaking and 25°C dark cultivation simplifies the operation steps and is suitable for sea buckthorn seedling cultivation under different environmental conditions.

[0020] A win-win situation for both ecology and economy: The enhanced growth potential of hypocotyls directly shortens the seedling cycle by 20%-30%, providing efficient technical support for ecological restoration and the construction of economic forests. Detailed Implementation

[0021] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0022] Example 1: Effect of amino oligosaccharide (AS) on the germination of wild sea buckthorn seeds

[0023] Fresh wild sea buckthorn berries were collected in Bayi Town, Bayi District, Nyingchi City (collection criteria: orange-yellow, medium-sized, fresh berries free from pests and diseases). Seeds were obtained by hand after removing the pulp. The tested seeds were plump, uniformly textured wild seeds, stored at room temperature for later use.

[0024] Wild sea buckthorn seeds were soaked in 50-60℃ warm water for 48 hours. After soaking, they were disinfected with 3g / L potassium permanganate solution for 15 minutes, rinsed repeatedly with distilled water 2-3 times, and then immersed in amino oligosaccharide (AS) solutions with concentrations of AS1=0.06mmol / L, AS2=0.12mmol / L, AS3=0.24mmol / L, and AS4=0.48mmol / L, respectively. Distilled water soaking was used as a control group. After soaking for 24 hours, the seeds were treated at 25℃ in the dark. 100 seeds were randomly selected and placed in a PQX-330A-12H intelligent artificial climate chamber, with the temperature controlled at 25℃ under dark conditions. Germination was defined as the radicle emerging 2mm from the seed coat. The number of germinating seeds was recorded at a fixed time point (9:00 AM) every 24 hours until germination stabilized (no new germination for 3 consecutive days). See Table 1. The evaluation indicators and calculation methods are as follows:

[0025] Germination rate (GP) reflects the final germination potential of a seed and is calculated as follows:

[0026]

[0027] Germination Index (GI) reflects the quantitative germination rate and uniformity, and is calculated as follows:

[0028]

[0029] Table 1 Effects of amino oligosaccharides (AS) on seed germination of sea buckthorn.

[0030]

[0031] Note: Compared with the control group, a P<0.01; b P<0.05; compared with the AS1=0.06mmol / L group, c P<0.01; d P<0.05.

[0032] Table 1 shows that AS2 (0.12 mmol / L) had the highest GP (83.5%), significantly higher than the control group (34.5%) and other treatment groups (AS1, AS3, AS4), indicating that 0.12 mmol / L was the optimal concentration. When the concentration exceeded 0.12 mmol / L (AS3, AS4), the GP gradually decreased, but remained higher than the control group, indicating that high concentrations did not completely inhibit germination, but the effect was weakened. At the same time, the GI of AS2 (32.4) reached its peak and was significantly higher than the control group (GI=2.3), indicating that the seed germination rate was fast and the seedlings grew vigorously at this concentration. Although the GI of AS3 and AS4 was higher than that of the control group, it was lower than that of AS2, indicating that the germination rate decreased at high concentrations; that is, amino oligosaccharides (AS) had the best promoting effect on sea buckthorn seed germination at a concentration of 0.12 mmol / L.

[0033] Determination of hypocotyl elongation in Hippophae rhamnoides: After disinfection treatment, Hippophae rhamnoides seeds were subjected to germination tests. Six days after germination, hypocotyl segments of 0.5 cm in length were cut off. Uniformly grown segments were cultured with amino oligosaccharides at concentrations of AS1=0.06 mmol / L, AS2=0.12 mmol / L, AS3=0.24 mmol / L, and AS4=0.48 mmol / L, respectively, and cultured in the dark at 25℃ for 48 h. Each treatment was repeated 30 times. The change in hypocotyl length was measured daily until the hypocotyl stopped elongating. See Table 2.

[0034] Table 2. Changes in hypocotyl elongation rate of Hippophae rhamnoides over time after treatment with amino oligosaccharides (AS).

[0035]

[0036] Note: Compared with the AS1=0.06mmol / L group, a P<0.01; b P<0.05; compared with the AS4=0.48mmol / L group, c P<0.01; d P<0.05.

[0037] Table 2 shows that the elongation growth rate of AS2 (0.12 mmol / L) was significantly higher than that of other concentration groups from day 2 to day 4, and continued to increase over time (13.4% on day 1 → 27.5% on day 4), indicating that 0.12 mmol / L was the optimal concentration, consistent with the trend in the previous seed germination experiment (AS2 had the best effect), and its promoting effect was continuous and cumulative; however, the growth rate of other concentration groups (AS1, AS3, AS4) peaked on day 2-3 and then declined, which may be related to the depletion of cell elongation potential or the accumulation of toxicity. In addition, the growth rate of the low concentration group (AS1=0.06mmol / L) peaked on day 2 (14.5%) and then gradually decreased; the growth rate of the high concentration group (AS3=0.24mmol / L, AS4=0.48mmol / L) was significantly lower than that of AS2, and AS3 decreased on day 4 (14.7%→10.4%), while AS4 was always lower than that of AS1. This indicates that excessively high concentrations inhibit elongation, which is consistent with the typical dose-response relationship of "low concentration promotes, high concentration inhibits", that is, amino oligosaccharide (AS) at a concentration of 0.12mmol / L has a significant promoting effect on hypocotyl elongation of Hippophae rhamnoides.

[0038] Example 2: Effect of sodium nitroprusside (SNP) on the germination of wild sea buckthorn seeds

[0039] Fresh wild sea buckthorn berries were collected in Bayi Town, Bayi District, Nyingchi City (collection criteria: orange-yellow, medium-sized, fresh berries free from pests and diseases). Seeds were obtained by hand after removing the pulp. The tested seeds were plump, uniformly textured wild seeds, stored at room temperature for later use.

[0040] Wild sea buckthorn seeds were soaked in warm water at 50-60℃ for 48 hours. After soaking, they were disinfected with a 3g / L potassium permanganate solution for 15 minutes and rinsed repeatedly with distilled water 2-3 times. Then, they were soaked in sodium nitroprusside (SNP) solutions with concentrations of SNP1=0.03mmol / L, SNP2=0.06mmol / L, SNP3=0.09mmol / L, and SNP4=0.12mmol / L, respectively. Distilled water soaking was used as a control group. After soaking for 24 hours, the seeds were treated at 25℃ in the dark. 100 seeds were randomly selected and placed in a PQX-330A-12H intelligent artificial climate chamber, where the temperature was controlled at 25℃ under dark conditions. Germination was defined as the radicle extending 2 mm out of the seed coat. Observations were made every 24 hours, and the number of germinated seeds was recorded at a fixed time (9:00 AM) until germination stabilized (no new germination for 3 consecutive days). At the end of the experiment, the root length, seedling height, and fresh weight of the seedlings were measured, as shown in Table 3. The evaluation indicators and calculation methods were the same as in Example 1.

[0041] Table 3 Effects of sodium nitroprusside (SNP) on seed germination of sea buckthorn.

[0042]

[0043] Note: Compared with the control group, a P<0.01; b P<0.05; compared with the SNP1=0.03mmol / L group, c P<0.01; d P<0.05.

[0044] Table 3 shows that both GP (germination rate) and GI (germination index) exhibited a trend of first increasing and then decreasing, with the peak occurring at SNP3 (0.09 mmol / L); GP increased from 32.5% in the control group to 87.5% in SNP3 (…). a (P<0.01), SNP4 decreased to 56.3%, which is consistent with the typical action pattern of plant growth regulators; the inhibitory effect of SNP4 (0.12 mmol / L) may be due to the oxidative stress or toxic accumulation caused by NO released by SNP at high concentrations, leading to physiological inhibition; thus, it can be concluded that sodium nitroprusside (SNP) has the best promoting effect on the germination of sea buckthorn seeds at a concentration of 0.09 mmol / L.

[0045] Determination of hypocotyl elongation in Hippophae rhamnoides: After disinfection treatment, Hippophae rhamnoides seeds were subjected to germination tests. Six days after germination, hypocotyl segments of 0.5 cm in length were cut off. Uniformly grown segments were selected and cultured in sodium nitroprusside (SNP) solutions with concentrations of SNP1=0.03 mmol / L, SNP2=0.06 mmol / L, SNP3=0.09 mmol / L, and SNP4=0.12 mmol / L, respectively. The segments were cultured in the dark at 25°C for 48 h. Each treatment was repeated 30 times. The change in hypocotyl length was measured daily until the hypocotyl stopped elongating. (See Table 4)

[0046] Table 4. Changes in hypocotyl elongation rate of Hippophae rhamnoides over time after treatment with sodium nitroprusside (SNP).

[0047]

[0048] Note: Compared with the group with SNP1 = 0.03 mmol / L, a P<0.01; b P<0.05; compared with the SNP4=0.12mmol / L group, c P<0.01; d P<0.05.

[0049] Table 4 shows that SNP3 = 0.09 mmol / L significantly promoted hypocotyl elongation in Hippophae rhamnoides, with an elongation rate of 41.5% on day 4, consistent with its optimal concentration performance in the seed germination experiment (Table 3). This indicates that SNP has a promoting effect on multiple growth stages of Hippophae rhamnoides at low to medium concentrations. The elongation rate of the SNP4 = 0.12 mmol / L group was significantly lower than that of the SNP3 group, possibly due to the accumulation of reactive oxygen species (ROS) or cell membrane damage caused by excessive NO. Therefore, sodium nitroprusside (SNP) at a concentration of 0.09 mmol / L has a significant promoting effect on hypocotyl elongation in Hippophae rhamnoides.

[0050] Example 3: The synergistic effect of amino oligosaccharide (AS) and sodium nitroprusside (SNP) on the germination of wild sea buckthorn seeds.

[0051] Fresh wild sea buckthorn berries were collected in Bayi Town, Bayi District, Nyingchi City (collection criteria: orange-yellow, medium-sized, fresh berries free from pests and diseases). Seeds were obtained by hand after removing the pulp. The tested seeds were plump, uniformly textured wild seeds, stored at room temperature for later use.

[0052] Wild sea buckthorn seeds were soaked in warm water at 50-60℃ for 48 hours, then disinfected with a 3g / L potassium permanganate solution for 15 minutes, and rinsed repeatedly with distilled water 2-3 times. An orthogonal gradient experiment was designed based on the optimal concentration range of amino oligosaccharides (AS) (AS2 = 0.12 mmol / L) screened in Example 1 and sodium nitroprusside (SNP) (SNP3 = 0.09 mmol / L) screened in Example 2.

[0053] Group A: The soaking solution was prepared by mixing 0.10 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio;

[0054] Group B: The soaking solution was prepared by mixing 0.10 mmol / L amino oligosaccharide (AS) and 0.09 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0055] Group C: The soaking solution was prepared by mixing 0.10 mmol / L amino oligosaccharide (AS) and 0.10 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0056] Group D: The soaking solution was prepared by mixing 0.12 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0057] Group E: The soaking solution was prepared by mixing 0.12 mmol / L amino oligosaccharide (AS) and 0.09 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0058] Group F: The soaking solution was prepared by mixing 0.12 mmol / L amino oligosaccharide (AS) and 0.10 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0059] Group G: The soaking solution was prepared by mixing 0.14 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0060] Group H: The soaking solution was prepared by mixing 0.14 mmol / L amino oligosaccharide (AS) and 0.09 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0061] Group M: The soaking solution was prepared by mixing 0.14 mmol / L amino oligosaccharide (AS) and 0.10 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio.

[0062] After soaking the seeds in the soaking solutions of groups A-M as described above for 24 hours, they were treated at 25°C in the dark. One hundred seeds were randomly selected and placed in a PQX-330A-12H intelligent artificial climate chamber, with the temperature controlled at 25°C under dark conditions. Germination was defined as the radicle extending 2 mm from the seed coat. Observations were made every 24 hours, and the number of germinating seeds was recorded at a fixed time (9:00 AM) until germination stabilized (no new germination for 3 consecutive days), as shown in Table 5. The evaluation indicators and calculation methods were the same as in Example 1.

[0063] Table 5. Changes in hypocotyl elongation rate of Hippophae rhamnoides over time after each experimental treatment.

[0064]

[0065] Note: Compared with AS2 = 0.12 mmol / L (Example 1), a P<0.01; b P<0.05; compared with the SNP3=0.09mmol / L (Example 2) group, c P<0.01; d P<0.05.

[0066] The data in Table 5 show that the combined use of 0.12 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP) significantly improved GP and GI indices, and the effect was better than that of single treatment or other concentration combinations, supporting a synergistic effect. This is because AS may induce the expression of resistance-related genes, and SNP promotes cell elongation through NO signaling. The two work together to enhance the germination of sea buckthorn seeds.

[0067] Determination of α-amylase activity during the germination of wild sea buckthorn seeds: Germinating seeds prepared in groups A-M were added to pre-cooled phosphate buffer (pH 5.6), ground into a homogenate in an ice bath, and allowed to stand at 4℃ for 30 minutes. The homogenate was centrifuged at 12,000 rpm for 15 minutes, and the supernatant (containing amylase) was collected as the crude enzyme solution and stored at 4℃ for later use. Seed soaking in distilled water served as a control group; the remaining methods were the same as those used for preparing germinating seeds in groups A-M. Spectrophotometry was used for detection. A buffer solution prepared with iodine and potassium iodide was mixed with the ground and extracted crude enzyme solution, and the absorbance was measured at 620 nm using a TU-1801 UV-Vis spectrophotometer at 0 min and 10 min. Enzyme activity units were expressed as the change in absorbance per gram of sample within 1 min. (See attached table). Figure 1 .

[0068] Figure 1 The results showed that the combined use of 0.12 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP) significantly enhanced the activity of α-amylase during seed germination. The level of α-amylase activity in seeds directly affects seed germination and seedling growth. This indicates that the synergistic effect of amino oligosaccharide (AS) and sodium nitroprusside (SNP) in promoting the germination of wild sea buckthorn seeds is based on enhancing α-amylase activity.

[0069] Determination of hypocotyl elongation in sea buckthorn: After disinfection treatment, sea buckthorn seeds were subjected to germination test. Six days after germination, hypocotyls of 0.5 cm in length were cut off. Uniformly grown portions were cultured in the soaking solution of group D (a 1:1 mixture of 0.12 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP)). The mixtures were cultured in the dark at 25°C for 48 h. Each treatment was repeated 30 times. The change in hypocotyl length was measured daily until the hypocotyl stopped elongating. See Table 6.

[0070] Table 6. Changes in hypocotyl elongation growth rate of Hippophae rhamnoides over time after combined treatment with amino oligosaccharide (AS) and sodium nitroprusside (SNP).

[0071]

[0072] Note: Compared with AS2 = 0.12 mmol / L (Example 1), a P<0.01; b P<0.05; compared with the SNP3=0.09mmol / L (Example 2) group, c P<0.01; d P<0.05.

[0073] Table 6 shows that the elongation rate of group D (the soaking solution obtained by mixing 0.12 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP) in a 1:1 ratio) was significantly higher than that of the AS group and the SNP group alone at all time points. The effect of group D was not only significantly better than that of the single treatment group, but also did not show a concentration additive effect. This indicates that the combined use of AS and SNP (0.12 mmol / L amino oligosaccharide (AS) and 0.08 mmol / L sodium nitroprusside (SNP)) can significantly enhance the hypocotyl elongation of Hippophae rhamnoides, especially in the later stage (days 3-4), and has a significant synergistic effect.

[0074] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cultivation method for promoting the germination of wild sea buckthorn seeds, characterized in that, The cultivation method includes the following steps: after soaking and disinfecting the sea buckthorn seeds in warm water, the seeds are immersed in a mixed solution containing oligosaccharides and sodium nitroprusside, wherein the concentration of oligosaccharides is 0.12 mmol / L, the concentration of sodium nitroprusside is 0.08 mmol / L, and the molar ratio of oligosaccharides to sodium nitroprusside is 1:1; the soaking time is 20-28 hours, and then germination culture is carried out at 25℃ in the dark.

2. The method according to claim 1, characterized in that: The soaking time in the mixed solution was 24 hours, and the germination culture conditions were a constant temperature of 25°C in the dark.

3. The method according to any one of claims 1-2, characterized in that: The synergistic effect of the amino oligosaccharide and sodium nitroprusside resulted in a germination rate (GP) of over 90% for sea buckthorn seeds and an elongation rate of over 45% for the hypocotyl on day 4.

4. A compound plant growth regulator of amino oligosaccharide and sodium nitroprusside, characterized in that: The concentration of amino oligosaccharide is 0.12 mmol / L, and the concentration of sodium nitroprusside is 0.08 mmol / L, with a molar ratio of 1:0.8-1:1.

2. It is suitable for promoting the germination of sea buckthorn seeds and seedling growth.

5. The application of the compound plant growth regulator of claim 4 in sea buckthorn seedling cultivation or ecological restoration.

6. A method for treating wild sea buckthorn seeds, characterized in that: After disinfection, the sea buckthorn seeds are treated using the method described in any one of claims 1-3.

7. The processing method according to claim 6, characterized in that: The disinfection process includes soaking in a 3g / L potassium permanganate solution for 15 minutes and rinsing with distilled water 2-3 times.

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