Method for rapidly propagating waxberries by using waxberry leaves

Abstract

The invention belongs to the technical field of plant tissue culture, and relates to a method for rapid propagation of waxberry by using waxberry leaves, which comprises the following steps: S1, selection of waxberry leaves: selecting healthy and disease-free waxberry leaves which are cultured in a greenhouse and grow for 80-90 days, and cutting into small leaves; s2, disinfecting the waxberry leaves; s3, induced regeneration of waxberry seedlings: placing the disinfected explants on an induction culture medium for regeneration; s4, induced rooting of the waxberry seedlings: putting the induced waxberry seedlings on a 1 / 2MS + IBA (0.1-1mg / L) or NAA (0.1-1mg / L) rooting culture medium, and culturing to obtain waxberry regenerated seedlings. According to the method, the efficient waxberry seedling regeneration system is established by performing induced regeneration on the waxberry leaves, and the method has great economic value and is of great significance in realizing rapid propagation of waxberries and subsequently improving waxberry varieties through a transgenic technology.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology and relates to a method for rapid propagation of bayberry using bayberry leaves. Background Technology

[0002] The Chinese bayberry (Myrica rubra) is a plant belonging to the Myrica genus. In my country, it is mainly cultivated in the Jiangsu and Zhejiang regions. Its fruit is rich in vitamins, dietary fiber, minerals, anthocyanins, and other nutrients, and has a sweet and sour taste, making it a popular fruit among consumers. In addition, the Chinese bayberry has medicinal value, including relieving heat and thirst, invigorating the spleen and stomach, and stopping diarrhea. The Chinese bayberry is a perennial woody plant. Traditional propagation methods require one to two years from seed sowing or cuttings to seedling growth, and up to five or even decades to fruiting, consuming a significant amount of time. Furthermore, the seeds of the Chinese bayberry are covered with a thick seed coat, requiring shell breaking during seed production. Its germination rate is extremely low, the germination period is long, and it is easily affected by the environment, thus greatly increasing the cost and difficulty of cultivating the Chinese bayberry and making it difficult to meet market demand.

[0003] Plant regeneration technology is widely used in agricultural production. This technology utilizes the principle of totipotency of plant cells, meaning that, theoretically, isolated plant cells can differentiate into complete plants under certain conditions. Through plant regeneration, seedlings can be mass-produced via in vitro tissue culture in a short time, effectively shortening the growth cycle and enabling rapid asexual reproduction of superior varieties. The tissue culture process can also be used to detoxify seedlings, effectively eliminating the harmful effects of plant viruses and improving plant growth quality. Furthermore, establishing a regeneration system is essential for realizing transgenic technology. Transgenic technology can cultivate plants with superior traits such as insect resistance, disease resistance, stress tolerance, and high yield, significantly shortening the breeding cycle and effectively improving plant yield and quality.

[0004] The long growth cycle and difficulty in variety selection of waxberries necessitate the use of modern biological techniques for rapid propagation and improvement. However, due to the high anthocyanin content and difficulty in regeneration, as well as the tendency for browning and death, a widely applicable regeneration and transformation system for waxberries has yet to be established. While there are a few patent applications for rapid propagation of waxberries, such as CN118415071A (a method for waxberry tissue culture), CN103651144A (a method for rapid propagation of waxberries), and CN102119661A (a method for waxberry tissue culture), the materials used in these patent applications are all stem segments or branches. These materials have growth points containing plant stem cells, which inherently possess the ability to differentiate into plants, making rapid propagation relatively easy. However, using stem segments or branches as materials for rapid propagation also has limitations. These materials are available at specific stages of the waxberry growth cycle, and their acquisition is restricted by the plant's propagation time, making them difficult to obtain and limited in quantity, which is insufficient to meet the needs of large-scale industrial development. Summary of the Invention

[0005] The purpose of this invention is to solve the aforementioned problems in the rapid propagation of *Myrica rubra* (Chinese bayberry) and to provide a method for rapid propagation of *Myrica rubra* using leaves. This method establishes a highly efficient and reliable regeneration system for *Myrica rubra* using leaves as explants. Since the cells on leaves are mature somatic cells, not stem cells with differentiation capacity, using leaves for rapid propagation of *Myrica rubra* represents true plant regeneration. Compared to plant materials such as branches and stem segments with growth points, leaves exist at every stage of plant growth and development, are easier to obtain, are more abundant, and are less limited by the plant's development time. Therefore, using *Myrica rubra* leaves as explant material for regeneration, compared to branches and stem segments, can achieve a significantly larger, more efficient, and more readily available rapid propagation of *Myrica rubra* seedlings, meeting the needs of large-scale industrial development.

[0006] The present invention adopts the following technical solution:

[0007] A method for rapid propagation of waxberry using waxberry leaves includes the following steps:

[0008] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 80 to 90 days and cut them into small pieces;

[0009] S2. Disinfection of bayberry leaves: Disinfect small pieces of leaves with ethanol solution and sodium hypochlorite solution in sequence, and then rinse with distilled water;

[0010] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon + IBA (0.1-2 mg / L) + BA (0.1-5 mg / L) or TDZ (0.01-5 mg / L). The pH was adjusted to 5.6 to 6.0. The culture dishes were placed in the light conditions of 25℃±3℃ (16 hours light / 8 hours dark).

[0011] S4. Induction of rooting of bayberry seedlings: The induced bayberry seedlings were placed on 1 / 2 MS + IBA (0.1-1 mg / L) or NAA (0.1-1 mg / L) rooting medium to obtain regenerated bayberry seedlings.

[0012] As described in the background section, existing rapid propagation techniques for waxberry based on tissue culture use materials with growth points, such as branches and stem segments, as explants for tissue culture. These materials contain stem cells with differentiation capacity, and during tissue culture, bud induction medium is mostly used to induce buds in the explants. However, this method is not suitable for waxberry leaves, which do not have growth points.

[0013] Myrica rubra leaves are abundant and readily available, making them a subject of ongoing research. However, progress has been slow. Apart from the lack of growth points on myrica rubra leaves, there is currently no literature indicating other reasons why tissue culture of myrica rubra leaves cannot be successfully performed.

[0014] The inventors also studied how to use bayberry leaves as explants for tissue culture to obtain bayberry plants, aiming to establish an efficient bayberry seedling regeneration system.

[0015] The present invention describes the key steps and techniques for rapid propagation of *Myrica rubra* plants through tissue culture using *Myrica rubra* leaves as explants. First, the selection of *Myrica rubra* leaves is crucial; relatively young leaves should be chosen, as they exhibit higher physiological activity. However, further experiments have shown that even the youngest leaves are not necessarily the best. The optimal results are achieved using leaves from *Myrica rubra* seedlings that are 80 to 90 days old. Leaves from this stage, when used as explants for tissue culture, result in a higher success rate in differentiating seedlings compared to leaves from seedlings less than 80 days old or older than 90 days.

[0016] Secondly, based on the selection of tissue culture materials, the above-mentioned scheme also optimized the induction medium. IBA (indolebutyric acid), BA (benzylaminopurine), and TDZ (thiafenuron) were added to the MS medium, with the corresponding amounts controlled. In particular, activated carbon was added, which played a crucial role in ensuring the successful differentiation of *Myrica rubra* leaves. Without activated carbon, browning occurred in the early and mid-stages of differentiation, leading to differentiation failure.

[0017] Preferably, in step S1, leaves from healthy bayberry seedlings that have grown for 85 to 90 days in a greenhouse are selected. Bayberry seedling leaves at this stage, as explant materials for tissue culture, exhibit the highest seedling survival rate under the same culture conditions.

[0018] Preferably, in step S3, the amount of activated carbon added to the induction medium is 8-12 mg / L. Under the conditions of adding IBA (0.1-2 mg / L), BA (0.1-5 mg / L), or TDZ (0.01-5 mg / L) to the induction medium, the amount of activated carbon added between 8-12 mg / L promotes differentiation. Furthermore, it was found that adding activated carbon at this concentration not only improves the success rate of differentiation but also shortens the differentiation time, allowing leaves to differentiate earlier and shortening the induction period.

[0019] Preferably, in step S2, the disinfection process is as follows: the small leaf pieces are soaked in 75% ethanol for 1-2 minutes for disinfection; then the explants are disinfected again with a 5-10% sodium hypochlorite solution; the explants are rinsed with sterile distilled water 4-6 times; and the surface moisture is absorbed with filter paper.

[0020] Preferably, after disinfecting the small leaf pieces in step S2, the small leaf pieces are soaked in ascorbic acid solution.

[0021] Preferably, the mass concentration of the ascorbic acid solution is 0.5%-1.5%.

[0022] Preferably, the small piece in step S1 is roughly square with a side length of 0.5-1cm.

[0023] Preferably, in step S4, the induced bayberry seedlings are placed on a rooting medium and cultured for 45 to 60 days before undergoing hardening-off treatment.

[0024] Preferably, the seedling hardening treatment is performed as follows: open the cap of the tissue culture bottle and spray it with clean water every day. After 3 to 5 days, treat the bayberry seedlings at 30-35℃ for 1-2 hours every day for 3-5 consecutive days. Then, clean the culture medium from the roots of the bayberry seedlings and transplant them into the soil.

[0025] By implementing the above technical solution, the present invention achieves the following specific beneficial effects:

[0026] This invention establishes an efficient waxberry seedling regeneration system by inducing leaf regeneration. Using tissue culture technology, a large number of asexually propagated waxberry seedlings were successfully obtained with a regeneration efficiency of over 85%. This system can be used for the rapid propagation of waxberry tissue culture seedlings, providing an important prerequisite for the subsequent exploration of waxberry transgenic technology. It has great economic value and is of great significance for achieving rapid propagation of waxberry and for the subsequent improvement of waxberry varieties through transgenic technology. Attached Figure Description

[0027] Figure 1 In the method of Example 1, young bayberry leaf explants were placed in an induction culture medium.

[0028] Figure 2 This describes the differentiation of young bayberry leaf explants into seedlings after 35 days of cultivation on an induction medium, as described in Example 1.

[0029] Figure 3 The image shows the rooting status of bayberry seedlings after 30 days of cultivation on rooting medium in Example 1. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Example 1

[0033] This embodiment provides a method for rapid propagation of waxberry using waxberry leaves, including the following steps:

[0034] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 80 days and cut them into small pieces;

[0035] S2. Disinfection of bayberry leaves: Soak the small leaf pieces in 75% ethanol for 2 minutes for disinfection; then disinfect the explants again with 5% sodium hypochlorite solution, rinse the explants 4 times with sterile distilled water, and use sterile filter paper to absorb the surface moisture.

[0036] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon (8 mg / L) + IBA (0.5 mg / L) + BA (3 mg / L). The pH was adjusted to 5.8. The culture dishes were placed in the light conditions of 25℃ (16 hours light / 8 hours dark).

[0037] S4. Induction of rooting of bayberry seedlings: The induced bayberry seedlings were placed on 1 / 2 MS + IBA (0.5 mg / L) rooting medium to obtain regenerated bayberry seedlings.

[0038] Example 2

[0039] This embodiment provides a method for rapid propagation of waxberry using waxberry leaves, including the following steps:

[0040] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 85 days and cut them into small pieces;

[0041] S2. Disinfection of bayberry leaves: Soak the small leaf pieces in 75% ethanol for 2 minutes for disinfection; then disinfect the explants again with 5% sodium hypochlorite solution, rinse the explants 4 times with sterile distilled water, and use sterile filter paper to absorb the surface moisture.

[0042] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon (10 mg / L) + IBA (1.0 mg / L) + TDZ (3 mg / L), and the pH was adjusted to 5.8. The culture dishes were placed in the light conditions of 25℃ (16 hours light / 8 hours dark).

[0043] S4. Induction of rooting of bayberry seedlings: The induced bayberry seedlings were placed on 1 / 2 MS + IBA (1.0 mg / L) rooting medium to obtain regenerated bayberry seedlings.

[0044] Example 3

[0045] This embodiment provides a method for rapid propagation of waxberry using waxberry leaves, including the following steps:

[0046] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 90 days and cut them into small pieces;

[0047] S2. Disinfection of bayberry leaves: Soak the small leaf pieces in 75% ethanol for 2 minutes for disinfection; then disinfect the explants again with 8% sodium hypochlorite solution, rinse the explants 6 times with sterile distilled water, and use sterile filter paper to absorb the surface moisture.

[0048] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon (10 mg / L) + IBA (0.1 mg / L) + BA (2 mg / L), and the pH was adjusted to 5.8. The culture dishes were placed in the light conditions of 25℃ (16 hours light / 8 hours dark).

[0049] S4. Induction of rooting of bayberry seedlings: The induced bayberry seedlings were placed on 1 / 2 MS + IBA (0.5 mg / L) rooting medium to obtain regenerated bayberry seedlings.

[0050] Example 4

[0051] This embodiment provides a method for rapid propagation of waxberry using waxberry leaves, including the following steps:

[0052] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 90 days and cut them into small pieces;

[0053] S2. Disinfection of bayberry leaves: Soak the small leaf pieces in 75% ethanol for 2 minutes for disinfection; then disinfect the explants again with 8% sodium hypochlorite solution, rinse the explants 6 times with sterile distilled water, and use filter paper to absorb the surface moisture; then soak in 1.0% filtered sterile ascorbic acid solution for 20 minutes, and use sterile filter paper to absorb the surface moisture.

[0054] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon (10 mg / L) + IBA (0.1 mg / L) + BA (2 mg / L), and the pH was adjusted to 5.8. The culture dishes were placed in the light conditions of 25℃ (16 hours light / 8 hours dark).

[0055] S4. Induction of rooting of bayberry seedlings: The induced bayberry seedlings were placed on 1 / 2 MS + IBA (0.5 mg / L) rooting medium to obtain regenerated bayberry seedlings.

[0056] Example 5

[0057] This embodiment provides a method for rapid propagation of waxberry using waxberry leaves, including the following steps:

[0058] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 90 days and cut them into small pieces;

[0059] S2. Disinfection of bayberry leaves: Soak the small leaf pieces in 75% ethanol for 2 minutes for disinfection; then disinfect the explants again with 8% sodium hypochlorite solution, rinse the explants 6 times with sterile distilled water, and use filter paper to absorb the surface moisture; then soak in 1.0% filtered sterile ascorbic acid solution for 20 minutes, and use sterile filter paper to absorb the surface moisture.

[0060] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon (10 mg / L) + IBA (0.1 mg / L) + BA (2 mg / L), and the pH was adjusted to 5.8. The culture dishes were placed in the light conditions of 25℃ (16 hours light / 8 hours dark).

[0061] S4. Induction of rooting of bayberry seedlings: After 60 days of culture on 1 / 2 MS + IBA (0.5 mg / L) rooting medium, the cap of the tissue culture bottle was opened and water was sprayed every day. After 3 days, the bayberry seedlings were treated at 30℃ for 1 hour every day for 5 consecutive days. The culture medium was cleaned from the roots of the bayberry seedlings and they were transplanted into the soil to obtain regenerated bayberry seedlings.

[0062] Comparative Example 1

[0063] This comparative example provides a method for rapid propagation of bayberry using bayberry leaves. The difference from Example 1 is that in step S1, healthy, disease-free bayberry leaves grown in a greenhouse for 30 days are selected and cut into small pieces.

[0064] Comparative Example 2

[0065] This comparative example provides a method for rapid propagation of bayberry using bayberry leaves. The difference from Example 1 is that in step S1, healthy, disease-free bayberry leaves grown in a greenhouse for 45 days are selected and cut into small pieces.

[0066] Comparative Example 3

[0067] This comparative example provides a method for rapid propagation of bayberry using bayberry leaves. The difference from Example 1 is that in step S1, healthy, disease-free bayberry leaves grown in a greenhouse for 60 days are selected and cut into small pieces.

[0068] Comparative Example 4

[0069] This comparative example provides a method for rapid propagation of bayberry using bayberry leaves. The difference from Example 1 is that activated carbon is not added to the induction medium. The formula of the induction medium is: MS + IBA (0.1 mg / L) + BA (2 mg / L), and the pH is adjusted to 5.8.

[0070] The regeneration efficiency of explants and the rooting of seedlings in the methods of Examples 1-5 and Comparative Examples 1-4 were statistically analyzed, and the results are shown in Table 1.

[0071] The differentiation success rate is calculated as (number of explants in differentiated seedlings / total number of explants)%.

[0072] Table 1. Regeneration efficiency of explants and rooting status of seedlings in each example and comparative example.

[0073]

[0074]

[0075] As shown in Table 1, in Examples 1 to 5 of the present invention, using leaves from specific stages of *Myrica rubra* as explants for tissue culture and employing specific induction culture media, the final differentiation success rate of the explants can reach over 70%, and even over 90%. Furthermore, a single explant differentiates into a large number of seedlings, reaching 5-7 seedlings. This efficiently achieves rapid propagation of *Myrica rubra*.

[0076] Compared with Example 3, Example 1 added a step of soaking the leaves in ascorbic acid solution after disinfection. The results showed that the differentiation success rate was improved, indicating that this added step is beneficial to improving the differentiation success rate of bayberry leaves.

[0077] The comparison between Comparative Examples 1 to 3 and Example 1 shows that the timing of selecting bayberry leaves has a significant impact on the results and is directly related to the differentiation success rate.

[0078] The comparison between Comparative Example 4 and Example 1 shows that the addition of activated carbon to the induction medium also directly affects the differentiation success rate.

[0079] During the experiment, the inventors also discovered that adding activated carbon to the induction culture medium could help shorten the differentiation cycle. Therefore, the inventors conducted related experiments.

[0080] A method for rapid propagation of waxberry using waxberry leaves includes the following steps:

[0081] S1. Selection of bayberry leaves: Select healthy, disease-free bayberry leaves that have been grown in a greenhouse for 90 days and cut them into small pieces;

[0082] S2. Disinfection of bayberry leaves: Soak the small leaf pieces in 75% ethanol for 2 minutes for disinfection; then disinfect the explants again with 8% sodium hypochlorite solution, rinse the explants 6 times with sterile distilled water, and use sterile filter paper to absorb the surface moisture.

[0083] S3. Induction and regeneration of bayberry seedlings: The sterilized explants were placed on the induction medium for regeneration. The formula of the induction medium was: MS + activated carbon (0, 3, 5, 8, 10, 13, 15 mg / L) + IBA (0.1 mg / L) + BA (2 mg / L), and the pH was adjusted to 5.8. The culture dishes were placed in the light conditions of 25℃ (16 hours light / 8 hours dark).

[0084] S4. Induction of rooting of bayberry seedlings: The induced bayberry seedlings were placed on 1 / 2 MS + IBA (0.5 mg / L) rooting medium to obtain regenerated bayberry seedlings.

[0085] Using induction culture media with varying amounts of activated carbon, 30 leaves from the same batch of *Myrica rubra* plants were cultured for differentiation. The differentiation success rate and time were statistically analyzed using the method described above. Differentiation time was defined as the time when the first green differentiated tissue appeared. The results are shown in Table 2.

[0086] Table 2 Differentiation time under different activated carbon addition amounts

[0087]

[0088] As shown in Table 2, without activated carbon, the differentiation success rate was very low, only 6.7%, and the differentiation time was also very long. When the activated carbon concentration was below 8 mg / L, the improvement in differentiation success rate compared to not adding activated carbon was not significant, and the differentiation time was not much different from that without activated carbon. When the activated carbon concentration was above 8 mg / L, especially below 12 mg / L, the differentiation success rate was significantly improved, and the differentiation time was also significantly shortened.

Claims

1. A method for rapid propagation of Myrica rubra using leaf blades of Myrica rubra, characterized by, The method comprises the following steps: S1. Selection of leaves of Myrica rubra: selecting healthy and disease-free leaves of Myrica rubra seedlings grown for 80 to 90 days in a greenhouse and cutting the leaves into small pieces; S2. Disinfection of leaves of Myrica rubra: disinfecting the small pieces of leaves with an ethanol solution and a sodium hypochlorite solution in sequence and then rinsing with distilled water; S3. Induction of regeneration of seedlings of Myrica rubra: placing the disinfected explants on an induction medium for regeneration, wherein the induction medium is prepared by adding activated carbon, IBA and BA or activated carbon, IBA and TDZ to MS medium and adjusting the pH to 5.6 to 6.0, and culturing the culture dish under illumination conditions of 25℃±3℃, 16 hours of illumination and 8 hours of darkness; S4. Induction of rooting of seedlings of Myrica rubra: placing the induced seedlings of Myrica rubra on 1 / 2MS+IBA or NAA rooting medium to obtain regenerated seedlings of Myrica rubra.

2. The method for rapid propagation of waxberry using waxberry leaves according to claim 1, characterized in that, In step S1, the leaves of healthy seedlings of Myrica rubra grown for 85 to 90 days in a greenhouse are selected.

3. The method for rapid propagation of waxberry using waxberry leaves according to claim 1, characterized in that, In step S3, the amount of IBA added is 0.1-2mg / L, the amount of BA added is 0.1-5mg / L, and the amount of TDZ added is 0.01-5mg / L.

4. The method for rapid propagation of waxberry using waxberry leaves according to claim 3, characterized in that, In step S3, the amount of activated carbon added in the induction medium is 8-12mg / L.

5. The method for rapid propagation of waxberry as claimed in claim 1, wherein the waxberry leaves are used. In step S2, the disinfection process is as follows: the small pieces of leaves are soaked in 75% ethanol for disinfection for 1-2 minutes; then the explants are disinfected again with a 5-10% sodium hypochlorite solution, and the explants are rinsed with sterilized distilled water for 4-6 times and the surface water is absorbed with filter paper.

6. The method for rapid propagation of waxberry as claimed in claim 1, wherein the waxberry leaves are used. After the small pieces of leaves are disinfected in step S2, the small pieces of leaves are soaked in an ascorbic acid solution.

7. The method for rapid propagation of waxberry using waxberry leaves according to claim 6, characterized in that, The mass concentration of the ascorbic acid solution is 0.5%-1.5%.

8. The method for rapid propagation of waxberry using waxberry leaves according to claim 1, characterized in that, The small pieces in step S1 are roughly square-shaped with a side length of 0.5-1cm.

9. The method for rapid propagation of waxberry using waxberry leaves according to claim 1, characterized in that, In step S4, the induced seedlings of Myrica rubra are cultured on the rooting medium for 45 to 60 days and then subjected to hardening treatment.

10. The method for rapid propagation of waxberry using waxberry leaves according to claim 9, characterized in that, The hardening treatment is performed by opening the lid of the tissue culture bottle and spraying clean water every day, and after 3 to 5 days, the seedlings of Myrica rubra are subjected to 30-35℃ treatment for 1-2 hours every day for 3-5 days continuously, the rooting medium of the seedlings of Myrica rubra is cleaned and the seedlings are transplanted into soil.

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