A method to improve the germination rate of Eucommia ulmoides seeds

Abstract

This invention discloses a method for improving the germination rate of Eucommia ulmoides seeds, comprising the following steps: selecting superior seeds; surface sterilizing the superior seeds, followed by mechanical damage treatment; and inoculating the treated seeds into a culture medium to cultivate them into complete sterile seedlings. The culture medium is either 1 / 2 MS medium + 20 g / L sucrose + 6.6 g / L agar or 1 / 2 MS medium + 0.5–2.0 mg / L activated carbon + 20 g / L sucrose + 6.6 g / L agar. This invention uses mechanical damage to treat Eucommia ulmoides seeds, causing less damage and breaking dormancy, effectively solving the problems of low germination rate and long germination time of existing Eucommia ulmoides seeds, achieving a germination rate of up to 95.7%, with seedlings emerging in just 14 days. This invention also improves seed coat aeration and water permeability, and enhances the absorption of nutrients from the culture medium.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, specifically to a method for improving the germination rate of Eucommia ulmoides seeds. Background Technology

[0002] Eucommia ulmoides Oliver, also known as eucommia bark, is a plant belonging to the genus Eucommia in the family Eucommiaceae. It thrives in sunny, warm, and humid environments, and is mostly distributed in southern Shaanxi, Sichuan, Yunnan, and Guizhou provinces of my country. Eucommia has high economic and medicinal value. The bark, sweet and warm in nature, can be used medicinally to treat liver and kidney deficiency, lower back and knee pain, weakness of muscles and bones, dizziness, and other ailments. It is a prized ingredient in ancient pharmacopoeias. Furthermore, extracts from the leaves of Eucommia can also be used medicinally.

[0003] Except for the xylem, the entire Eucommia ulmoides tree contains eucommia gum, which has excellent insulation properties and is resistant to acids, alkalis, and seawater corrosion. It can be used to produce high-strength submarine cables and high-strength adhesives, and is currently widely used in dental clinics. Its non-toxic, odorless, and wear-resistant properties are unmatched by any other material. The trunk of the Eucommia ulmoides tree is an excellent material for manufacturing boats, vehicles, high-end furniture, and handicrafts. With its straight trunk, abundant branches and leaves, and mostly round or conical crowns providing ample shade, the Eucommia ulmoides tree is highly resistant to pests and diseases, requiring no pesticides, making it an ideal tree species for urban landscaping.

[0004] With the increasing market demand for Eucommia ulmoides, its breeding work is receiving more and more attention. Currently, although there are reports on the use of plant tissue culture technology for Eucommia ulmoides propagation, these studies mainly focus on seedlings as explant sources, and suffer from problems such as serious pollution and low seed germination rates, severely limiting the germplasm preservation and large-scale propagation of superior Eucommia ulmoides varieties. Providing a rapid, sterile seedling breeding program lays the foundation for the establishment of Eucommia ulmoides regeneration systems and genetic transformation research.

[0005] Low germination rate and uneven emergence of Eucommia ulmoides seeds have been a long-standing and difficult problem to solve. The inability to provide a high germination rate and rapid propagation, hindering the testing of new cultivation techniques, is particularly detrimental to Eucommia ulmoides cultivation for research purposes. Therefore, a seed treatment method is needed to enable Eucommia ulmoides seeds to quickly achieve a high germination rate.

[0006] Chinese patent application CN112930749A discloses a method for breaking the dormancy of Eucommia ulmoides seeds. The method involves stratifying dried Eucommia ulmoides seeds with moist sand. By optimizing the stratification temperature, stratification time, sowing time, and sowing temperature, the method selects seeds that have been stratified in cold and moist conditions for 105 days and germinated at 10°C to achieve a maximum germination percentage of 97.7 ± 0.7%. However, the long germination time limits its application.

[0007] To meet the demand for sterile seedlings and the needs of scientific research, there is an urgent need to develop a method for efficient germination of Eucommia ulmoides seeds. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for treating Eucommia ulmoides seeds, which can enable Eucommia ulmoides seeds to quickly reach a high germination rate and shorten the seedling period.

[0009] The present invention solves the above-mentioned technical problems through the following technical means:

[0010] A method for improving the germination rate of Eucommia ulmoides seeds includes the following steps:

[0011] S1. Selecting superior seeds;

[0012] S2. Disinfect the surface of the superior seeds and then treat them with mechanical damage.

[0013] S3. The seeds treated in S2 are inoculated into a culture medium and cultured to grow into complete sterile seedlings; wherein the culture medium is any one of 1 / 2MS + 20g / L sucrose + 6.6g / L agar or 1 / 2MS + 0.5-2.0mg / L activated carbon + 20g / L sucrose + 6.6g / L agar.

[0014] Preferably, in S1, the superior seeds are mature, plump, intact, and free from mold, current-year seeds.

[0015] Preferably, in S2, the surface disinfection includes the following steps: rinsing the superior seeds with running water; rinsing them with sterile water in a sterile operating table, then disinfecting them with 75% ethanol, then disinfecting them with a disinfectant solution, and finally rinsing them with sterile water.

[0016] Preferably, in S2, the surface disinfection includes the following steps: rinsing the superior seeds with running water for 10-30 minutes; rinsing them 3-6 times with sterile water in a sterile operating table, then disinfecting them with 75% ethanol for 20-60 seconds, then disinfecting them with disinfectant for 1-10 minutes, and then rinsing them 3-6 times with sterile water.

[0017] Preferably, the disinfectant is one of the following: a 10% NaClO solution, a 0.1% HgCl2 solution, or a 10% H2O2 solution.

[0018] Preferably, the disinfectant is a 10% NaClO solution, and the disinfection time is 10 minutes.

[0019] Preferably, in S2, the mechanical damage method includes seed coat disruption.

[0020] Preferably, the seed coat damage refers to cutting wounds at both ends of the seed.

[0021] Preferably, the seed coat damage refers to cutting 1-2 mm wounds at both ends of the seed.

[0022] Preferably, in S3, the culture medium is 1 / 2 MS + 2.0 mg / L activated carbon + 20 g / L sucrose + 6.6 g / L agar; it can achieve a seed germination rate of 95.7%, and the seedlings are growing well and uniformly.

[0023] Preferably, in S3, the culture is carried out in a constant temperature incubation chamber with a temperature set at 23±2℃, a light intensity of 2000~2500Lux, and a light cycle of 14h / 10h (light / dark).

[0024] Preferably, the culture time is 7 days.

[0025] The advantages of this invention are:

[0026] The method for improving the germination rate of Eucommia ulmoides seeds provided by this invention employs mechanical damage treatment, which causes minimal damage to the seeds and breaks seed dormancy. This effectively solves the problems of low germination rate and long germination time in existing Eucommia ulmoides seed treatments. It improves seed coat aeration and water permeability, and enhances the absorption of nutrients from the culture medium. The seed coat damage method breaks the binding of Eucommia ulmoides gum on the radicle or plumule, significantly increasing the seed germination rate. Specific advantages include:

[0027] (1) The disinfection procedure for Eucommia ulmoides seeds in the method of the present invention is simple. Soaking Eucommia ulmoides seeds in a 10% NaClO solution for 10 minutes is the optimal disinfection scheme. It has low toxicity to the seeds, and the contamination rate can be reduced to 0%, while the browning rate reaches 2%.

[0028] (2) The seed coat breaking method of the present invention (1-2 mm wounds are cut at both ends of the Eucommia ulmoides seed with a scalpel). It causes less damage to the Eucommia ulmoides seed and can break the binding of Eucommia ulmoides gum on the radicle or plumule, break the seed dormancy, and the seed will sprout on the second day, which greatly improves the seed germination rate.

[0029] (3) The preferred germination medium for Eucommia ulmoides seeds is 1 / 2MS + activated carbon (AC) 2.0 mg / L + 20 g / L sucrose + 6.6 g / L agar medium, which can achieve a seed germination rate of 95.7% and the seeds are growing well and growing relatively uniformly.

[0030] (4) The seed coat destruction method of the present invention can treat ungerminated Eucommia ulmoides seeds, and the germination rate can still reach 91.5% after 7 days. Attached Figure Description

[0031] Figure 1This is a graph showing the effect of different disinfectants on the contamination rate of Eucommia ulmoides after disinfection in Examples 1-3 of this invention;

[0032] Figure 2 This is a graph showing the effect of different disinfectants on the browning rate of Eucommia ulmoides after disinfection in Examples 1-3 of this invention;

[0033] Figure 3 This is a graph showing the effect of different disinfectants on the germination rate of Eucommia ulmoides after disinfection in Examples 1-3 of this invention;

[0034] Figure 4 The figures show the germination rates of Eucommia ulmoides seeds under different mechanical damage treatments in Examples 4-7 of this invention.

[0035] Figure 5 This is a germination diagram of Eucommia ulmoides seeds cultured for 7 days after treatment with the seed coat destruction method in Example 7 of this invention;

[0036] Figure 6 The images show the germination of Eucommia ulmoides seeds after 14 days of cultivation in Example 7 of this invention, in the untreated group and the group treated with the seed coat destruction method. The left image shows the germination of the untreated group, and the right image shows the germination of the group treated with the seed coat destruction method.

[0037] Figure 7 The graph shows the effect of different wound sizes at both ends of Eucommia ulmoides seeds on their germination rate in Examples 7-9 of this invention.

[0038] Figure 8 This is a germination diagram of Eucommia ulmoides seeds with wounds at both ends greater than 2 mm in Example 9 of the present invention;

[0039] Figure 9 The germination rate of Eucommia ulmoides seeds after 14 days of cultivation in Examples 7 and 10-12 of this invention is shown in the figure.

[0040] Figure 10 The following are root system diagrams of seeds germinated on activated carbon culture media of different concentrations in Examples 7 and 10-12 of this invention;

[0041] Figure 11 This is a flowchart of the method for improving the germination rate of Eucommia ulmoides seeds in Embodiment 12 of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0044] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0045] In the following examples, the Eucommia ulmoides seeds used were provided by Henan Flora Agricultural Technology Co., Ltd., and the variety was Qin Zhong No. 1, which was a new variety of the current year.

[0046] Example 1

[0047] The specific steps for selecting a disinfectant solution for Eucommia ulmoides seed explants are as follows:

[0048] (1) First, remove the wing of the fruit of Eucommia ulmoides, wash it with water to remove debris and seeds, and select mature, plump, intact new seeds of the current year without mold.

[0049] (2) The selected superior Eucommia seeds were rinsed with running water for 15 minutes; then rinsed with sterile water 4 times in a sterile operating table, then disinfected with 75% ethanol for 40 seconds, then disinfected with 0.1% HgCl2 solution for 1 minute, 3 minutes, 5 minutes, 7 minutes and 10 minutes respectively, and then rinsed with sterile water 4 times. During the disinfection process, the seeds were shaken continuously.

[0050] (3) After draining, Eucommia ulmoides seeds were inoculated into 1 / 2 MS medium containing 20 g / L sucrose and 6.6 g / L agar, and cultured in a constant temperature incubator. The temperature in the incubator was set at 23 ± 2 ℃, the light intensity was 2000–2500 Lux, and the photoperiod was 14 / 10 h (light / dark). After 5 days of culture, the contamination rate, browning rate, and germination rate were statistically analyzed.

[0051] Example 2

[0052] The only difference from Example 1 is that the disinfectant used is a 10% H2O2 solution instead of the 0.1% HgCl2 solution used in Example 1.

[0053] Example 3

[0054] The only difference from Example 1 is that the disinfectant used is a 10% NaClO solution instead of the 0.1% HgCl2 solution used in Example 1.

[0055] Figure 1 , Figure 2 and Figure 3 The study statistically analyzed the contamination rate of Eucommia ulmoides seeds after disinfection with different disinfectants. Figure 1 ), browning rate ( Figure 2 ) and germination rate ( Figure 3Considering the influence of these three factors, the optimal disinfection method for Eucommia ulmoides seeds was determined to be soaking in a 10% NaClO solution for 10 minutes, which achieved a contamination rate of 0%, a browning rate of 2%, and a germination rate of 16.9%.

[0056] Example 4

[0057] (1) First, remove the wing of the fruit of Eucommia ulmoides, wash it with water to remove debris and seeds, and select mature, plump, intact new seeds of the current year without mold.

[0058] (2) The selected superior Eucommia seeds were rinsed with running water for 15 minutes; then rinsed with sterile water 4 times in a sterile operating table, then disinfected with 75% ethanol for 40 seconds, then disinfected with 10% NaClO solution for 10 minutes, and then rinsed with sterile water 4 times, shaking continuously during the disinfection process.

[0059] (3) After the Eucommia ulmoides seeds were drained, one part was left untreated and became the CK group, while the other part was subjected to mechanical damage treatment, namely: the sterilized Eucommia ulmoides seeds were placed in a sterile tissue culture bottle, 200mL of sterile water was added, and the seeds were sonicated for 5 minutes with an ultrasonic power of 700W.

[0060] (4) The treated seeds were inoculated in 1 / 2 MS medium containing 20 g / L sucrose and 6.6 g / L agar and placed in a constant temperature incubator. The temperature in the incubator was set at 23 ± 2 °C, the light intensity was 2000–2500 Lux, and the photoperiod was 14 / 10 h (light / dark). The germination rate was counted after 7 days of culture.

[0061] Example 5

[0062] The only difference from Example 4 is that the ultrasound was performed for 10 minutes.

[0063] Example 6

[0064] The only difference from Example 4 is that the ultrasound was performed for 15 minutes.

[0065] Example 7

[0066] The only difference from Example 4 is that the mechanical damage method is seed coat damage (1-2 mm wounds are cut at both ends of the Eucommia seed with a scalpel).

[0067] Figure 4 The figure shows the germination rate of Eucommia ulmoides seeds after different mechanical damage treatments: ultrasound for 5 min, ultrasound for 10 min, ultrasound for 15 min, and seed coat damage. Ultrasound treatment improved the germination rate compared to the control (CK), but the seed coat damage treatment achieved a germination rate of 88.6% (e.g., ...). Figure 5The percentage was significantly higher than the 19.55% in the control group. After 14 days of cultivation, the aseptic seedlings of *Eucommia ulmoides* showed healthy growth and consistent growth cycles. Figure 6 The middle left image is CK. Figure 6 The image in the middle right shows the seed coat damage treatment. The seed coat damage treatment causes less damage to Eucommia ulmoides seeds and can break the binding of Eucommia ulmoides gum on the radicle or plumule, break seed dormancy, and significantly improve seed germination rate.

[0068] Example 8

[0069] The only difference from Example 7 is that the wounds at both ends of the Eucommia ulmoides seeds are less than 0.5 mm.

[0070] Example 9

[0071] The only difference from Example 7 is that the wounds at both ends of the Eucommia ulmoides seeds are greater than 2 mm.

[0072] Figure 7 The effect of different wound sizes at both ends of Eucommia ulmoides seeds on their germination rate was statistically analyzed. The results showed that when the wound size was less than 0.5 mm, the germination rate was significantly higher than that of the control group (CK) in Example 4, but still less than 60%. However, when the wound size at both ends of the Eucommia ulmoides seeds was greater than 2 mm, the germination rate was significantly lower. Figure 8 As shown, although the seeds can germinate normally, their radicles are damaged and cannot be implanted into the culture medium for normal growth. Therefore, we selected 1-2 mm wounds made at both ends of the Eucommia ulmoides seeds with a scalpel as the optimal damage method.

[0073] Example 10

[0074] The only difference from Example 7 is that the treated seeds were inoculated in 1 / 2 MS medium containing 0.5 mg / L activated carbon (AC), 20 g / L sucrose, and 6.6 g / L agar.

[0075] Example 11

[0076] The only difference from Example 7 is that the treated seeds were inoculated in 1 / 2 MS medium containing 1.0 mg / L activated carbon (AC), 20 g / L sucrose, and 6.6 g / L agar.

[0077] Example 12

[0078] The only difference from Example 7 is that the treated seeds were inoculated in 1 / 2 MS medium containing 2.0 mg / L activated carbon (AC), 20 g / L sucrose, and 6.6 g / L agar, as shown in the flowchart below. Figure 11 As shown.

[0079] like Figure 9 and Figure 10 The diagram shows the germination of Eucommia ulmoides seeds and their root systems in Examples 7, 10, 11, and 12 after 14 days.1 / 2MS represents Example 7. 1 / 2MS+AC 0.5mg / L 1 / 2MS+AC 1.0mg / L 1 / 2MS+AC 2.0mg / L represent Examples 10-12 respectively; Figure 9 Studies have shown that the germination rate of Eucommia ulmoides seeds increases to 95.7% after the addition of activated carbon (AC), and root length and lateral roots increase with increasing AC concentration. Adding an appropriate amount of activated carbon to plant growth media can significantly improve the composition of the medium, create a dark environment for root growth, increase aeration, and adsorb substances in the medium that inhibit plant growth, such as phenols and quinones, thus promoting better tissue growth. Furthermore, a healthy root system also provides a better selection of rootstocks for grafting.

[0080] Example 13

[0081] (1) First, remove the wing of the fruit of Eucommia ulmoides, wash it with water to remove debris and seeds, and select mature, plump, intact new seeds of the current year without mold.

[0082] (2) The selected superior Eucommia seeds were rinsed with running water for 15 minutes; then rinsed with sterile water 4 times in a sterile operating table, then disinfected with 75% ethanol for 40 seconds, then disinfected with 10% NaClO solution for 10 minutes, and then rinsed with sterile water 4 times, shaking continuously during the disinfection process.

[0083] (3) After draining, Eucommia ulmoides seeds were inoculated into 1 / 2 MS medium containing 2.0 mg / L activated carbon (AC), 20 g / L sucrose, and 6.6 g / L agar and cultured in a constant temperature incubator. The temperature of the incubator was set at 23 ± 2 °C, the light intensity was 2000–2500 Lux, and the photoperiod was 14 / 10 h (light / dark). After 7 days of culture, the ungerminated seeds were treated with seed coat disruption as described in Example 7. It was found that these ungerminated seeds germinated again, with a germination rate of 91.5%. It is speculated that the disruption broke the binding of Eucommia ulmoides gum in the seed coat to the radicle or plumule, breaking the seed dormancy and thus enabling the seeds to germinate again.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for increasing the germination rate of Eucommia ulmoides Oliver seeds, characterized by: The method comprises the following steps: S1, selecting seeds to obtain fine seeds; S2, surface sterilizing the fine seeds, using a 10% NaClO solution as the sterilizing solution, and then treating the seeds by mechanical damage; the mechanical damage method comprises seed coat breakage; the seed coat breakage refers to cutting 1-2 mm wounds at both ends of the seeds; S3, inoculating the seeds treated in S2 into a culture medium to grow into complete sterile seedlings; the culture medium is a culture medium of 1 / 2MS+0.5-2.0 mg / L activated carbon+20 g / L sucrose+6.6 g / L agar.

2. The method for improving the seed germination rate of Eucommia ulmoides according to claim 1, characterized in that: In S1, the fine seeds are full and intact year-old seeds without mildew.

3. The method for improving the seed germination rate of Eucommia ulmoides according to claim 1, characterized in that: In S2, the surface sterilization comprises the following steps: washing the fine seeds with running water; washing the fine seeds with sterile water in a sterile operation table, then sterilizing the fine seeds with 75% ethanol, then sterilizing the fine seeds with a sterilizing solution, and then washing the fine seeds with sterile water.

4. The method for improving the seed germination rate of Eucommia ulmoides according to claim 1, characterized in that: In S2, the surface sterilization comprises the following steps: washing the fine seeds with running water for 10-30 min; washing the fine seeds with sterile water in a sterile operation table for 3-6 times, then sterilizing the fine seeds with 75% ethanol for 20-60 s, then sterilizing the fine seeds with a sterilizing solution for 1-10 min, and then washing the fine seeds with sterile water for 3-6 times.

5. The method for improving the seed germination rate of Eucommia ulmoides according to claim 1, characterized in that: In S3, the culture medium is a culture medium of 1 / 2MS+2.0 mg / L activated carbon+20 g / L sucrose+6.6 g / L agar.

6. The method for improving the seed germination rate of Eucommia ulmoides according to any one of claims 1-5, characterized in that: In S3, the culture is carried out in a constant-temperature culture room, the temperature is set to 23±2 ℃, the light intensity is 2000-2500 Lux, and the light cycle is 14 h / 10 h (light / dark).

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