Application of CLE small peptides in regulating peach adventitious root regeneration

Abstract

The application discloses application of CLE small peptides in regulation of peach adventitious root regeneration. Group-A CLE small peptides in the application can promote peach adventitious root regeneration, and specifically, PpCLE4 small peptides, PpCLE5 small peptides and PpCLE7. After the peach stem segments of the PpCLE4 small peptides, the PpCLE5 small peptides and the PpCLE7 are used as explants, the adventitious root regeneration efficiency is respectively increased to 69.70%, 43.33% and 38.79%, and the number of adventitious roots regenerated from a single explant is respectively increased to 2.40, 2.63 and 2.58, which is much better than that of a control group without adding CLE small peptides and a treatment group with other types of CLE small peptides. The Group-A CLE small peptides provided by the application have good research potential and wide application prospect, and provide a new method for promoting adventitious root regeneration of peach cutting rooting.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to the application of CLE peptides in regulating the regeneration of adventitious roots in peach. Background Technology

[0002] Peach( Prunus persica Peach (L.) is one of the most widely cultivated fruit trees in temperate and subtropical regions of the world, and is considered the eighth most important commercial fruit crop globally. Traditionally, peach trees are propagated primarily through seed rootstock. While this method is low-cost and easy to manage, it leads to genetic heterogeneity, potentially resulting in the loss of desirable traits from the parent plant (such as disease resistance and adaptability), and can negatively impact long-term orchard productivity due to outcrossing. Asexual propagation methods, such as stem cuttings (which rely on adventitious root formation), allow for the clonal propagation of superior rootstock genotypes, maintaining genetic uniformity and ensuring the stable transmission of rootstock traits. This is crucial for the large-scale production of grafted rootstocks with consistent traits.

[0003] The formation of adventitious roots is a crucial biological process in the asexual reproduction and rootstock cultivation of peach trees. It directly affects the rapid propagation of superior rootstocks and the cost and efficiency of orchard establishment, thus possessing extremely important agronomical and economic significance. However, peaches are among the fruit trees that are difficult to root through stem cuttings, and their low adventitious root formation ability is a major technical bottleneck restricting the asexual reproduction and promotion of superior rootstocks. Therefore, developing efficient adventitious root induction methods is of great importance for shortening the propagation cycle, reducing production costs, and meeting market demand.

[0004] CLE (CLAVATA3 / Embryo surrounding region) is one of the largest known small peptide families in plants. The CLE peptide family has distinctive structural features: an N-terminal signal peptide, a variable domain in the middle, and a conserved C-terminal domain. The CLE peptide family has been identified and analyzed in multiple species. Peach CLE peptides have been identified and named PpCLE1-PpCLE17, referencing those in Arabidopsis thaliana. CLE Genes are classified into four groups: Group-A, Group-B, Group-C, and Group-D. The molecular mechanisms and applications of CLE family peptides in regulating peach adventitious root formation have not yet been reported. Therefore, exploring the function and application potential of a class of CLE peptides in regulating peach adventitious root regeneration can provide important theoretical basis and technical support for optimizing and improving peach cutting rooting techniques in production practice. Summary of the Invention

[0005] Based on the shortcomings and deficiencies of existing technologies, this invention aims to provide the application of CLE peptides in regulating the regeneration of peach adventitious roots.

[0006] The first objective of this invention is to provide the application of peach CLE peptide in promoting the regeneration of adventitious roots in plants, wherein the peach CLE peptide is one or more of PpCLE4, PpCLE5, or PpCLE7; the amino acid sequence of PpCLE4 is shown in SEQ ID NO.1, the amino acid sequence of PpCLE5 is shown in SEQ ID NO.2, and the amino acid sequence of PpCLE7 is shown in SEQ ID NO.3.

[0007] A second objective of this invention is to provide the application of peach CLE peptide in the preparation of formulations that promote the regeneration of adventitious roots in plants, wherein the peach CLE peptide is one or more of PpCLE4, PpCLE5, or PpCLE7; the amino acid sequence of PpCLE4 is shown in SEQ ID NO.1, the amino acid sequence of PpCLE5 is shown in SEQ ID NO.2, and the amino acid sequence of PpCLE7 is shown in SEQ ID NO.3.

[0008] A third objective of this invention is to provide an agent for promoting the regeneration of adventitious roots in plants, which contains one or more of peach CLE peptides PpCLE4, PpCLE5, or PpCLE7; wherein the amino acid sequence of PpCLE4 is shown in SEQ ID NO.1, the amino acid sequence of PpCLE5 is shown in SEQ ID NO.2, and the amino acid sequence of PpCLE7 is shown in SEQ ID NO.3.

[0009] A fourth objective of this invention is to provide a method for promoting the regeneration of adventitious roots in plants by inoculating plant material into an adventitious root regeneration culture medium containing the aforementioned preparation.

[0010] Preferably, the adventitious root regeneration medium is based on MS medium and contains sucrose, agar and IBA.

[0011] Preferably, the concentration of peach CLE peptide in the adventitious root regeneration medium is 100 μM.

[0012] Preferably, the adventitious root regeneration medium contains 25 g / L sucrose, 8 g / L agar, and 2.0 mg / L IBA.

[0013] Preferably, the plant material is a tissue culture seedling segment.

[0014] Preferably, the culture is carried out at a temperature of 25°C, with 16 hours of light / 8 hours of darkness per day and a light intensity of 1500 lux for 1-2 weeks.

[0015] Preferably, the plant is a peach.

[0016] The beneficial effects of this invention are: This invention explores the application of CLE peptides from peaches in regulating adventitious root regeneration. The invention discovers that peptides in Group-A significantly promote adventitious root regeneration in peaches, filling a gap in the application of CLE in peaches. By exogenously adding one or more of the peptides PpCLE4, PpCLE5, and PpCLE7, the number of regenerated adventitious roots in peaches can be significantly increased, providing a novel method for adventitious root growth in peach cuttings during practical production, and demonstrating promising prospects for practical development and application. Attached Figure Description

[0017] Figure 1 This refers to the rooting status of stem explants in the Control group and those treated with PpCLE4, PpCLE5, PpCLE7, PpCLE10, PpCLE12, PpCLE13 and PpCLE17 peptides for 2 weeks in Example 1. Detailed Implementation

[0018] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0019] Example 1 (1) Artificial synthesis of peach CLE peptide Based on the transcriptome of adventitious root regeneration, CLE sequences specifically expressed in stem segments and roots were screened. Seven small peptides from peach were screened and artificially synthesized. Their peptide names, amino acid sequences, and groupings are shown below.

[0020] PpCLE4 small peptide: KRLVPSGPNPLHN (SEQ ID NO.1), Group-A; PpCLE5 small peptide: KRRVPTGPNPLHH (SEQ ID NO.2), Group-A; PpCLE7 small peptide: KRIVHTGPNPLHN (SEQ ID NO.3), Group-A; PpCLE10 small peptide: KRRVPNGPDPIHN (SEQ ID NO.4), Group-C; PpCLE12 small peptide: KRVSPGGPDPRHH (SEQ ID NO.5), Group-B; PpCLE13 small peptide: KRKVRKGSDPIHN (SEQ ID NO.6), Group-C; PpCLE17 small peptide: AHEVPSGPNPISN (SEQ ID NO. 7), Group-D.

[0021] The artificially synthesized small peptides were dissolved in ddH2O to prepare a 10 mM stock solution, which was then stored at -80°C.

[0022] (2) Obtaining stem segment explants Take mature 'Zaoyoutao' seeds, soak them in 75% alcohol for 3-5 minutes, rinse them with sterile water, and then soak them in a self-prepared disinfectant solution (sterile water: 84 disinfectant = 2:1, v / v) for 15 minutes. Rinse them with sterile water and set aside. Remove the seed coat from the disinfected seeds and culture them in a sowing medium (1 / 2 MS + 25 g / L sucrose + 8 g / L agar + 0.05 mg / L IBA) at 25℃, with a light intensity of 1500 lux and approximately 16 hours of light / 8 hours of darkness per day. After about two months, sterile tissue culture seedlings will be obtained. Remove the leaves and terminal buds from the obtained sterile seedlings and cut them into 1 cm stem segments for explants.

[0023] (3) Group processing Group 1 (Control group): The stem explants obtained in (2) were transferred to MS medium containing 25 g / L sucrose, 8 g / L agar, and 2.0 mg / L IBA for adventitious root regeneration. The culture was carried out for 2 weeks at a temperature of 25°C, approximately 16 h of light / 8 h of darkness per day, and a light intensity of 1500 lux.

[0024] Group 2 (PpCLE4 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE4 peptide and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0025] The third group (PpCLE5 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE5 peptide and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0026] Group 4 (PpCLE7 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE7 peptide and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0027] Group 5 (PpCLE10 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE10 peptides and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0028] Group 6 (PpCLE12 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE12 peptide and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0029] Group 7 (PpCLE13 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE13 peptide and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0030] Group 8 (PpCLE17 group): The stem explants obtained in (2) were transferred to an adventitious root regeneration optimized medium consisting of MS + 25 g / L sucrose + 8 g / L agar + 2.0 mg / L IBA + 100 μM PpCLE17 peptide and cultured for 2 weeks at a temperature of 25℃, approximately 16 h light / 8 h dark per day, and a light intensity of 1500 lux.

[0031] (4) After two weeks of cultivation, the adventitious root regeneration efficiency and the number of adventitious roots regenerated in a single explant were counted.

[0032] Table 1. Statistics on adventitious root regeneration efficiency and the number of adventitious roots regenerated from a single explant. Note: Results in the table are presented as mean ± SE standard deviation. Different letters represent significant differences (Student's-test). Each experimental group has three replicates. Adventitious root regeneration efficiency = number of rooted stem segment explants / total number of stem segment explants; number of roots per explant = total number of roots / number of rooted stem segment explants.

[0033] Rooting status of stem segment explants after 2 weeks of culture in different treatment groups is as follows: Figure 1 As shown in Table 1, the adventitious root regeneration efficiency was 29.29% in the control group, while it significantly increased to 69.70%, 43.33%, and 38.79% in the PpCLE4, PpCLE5, and PpCLE7 groups, respectively. The number of regenerated adventitious roots per explant in the control group was 1.58, while it increased to 2.40, 2.63, and 2.58 in the PpCLE4, PpCLE5, and PpCLE7 groups, respectively. PpCLE4, PpCLE5, and PpCLE7 belong to Group-A, indicating that the small peptides in Group-A can promote peach adventitious root regeneration. Among them, the small peptide PpCLE17, belonging to Group-D, significantly inhibited peach adventitious root regeneration, while PpCLE10, PpCLE12, and PpCLE13, belonging to Group-B or Group-C, had no significant effect on promoting peach adventitious root regeneration.

Claims

1. The application of peach CLE peptide in promoting adventitious root regeneration in plants, characterized in that, The peach CLE peptide is one or more of PpCLE4, PpCLE5, or PpCLE7; the amino acid sequence of PpCLE4 is shown in SEQ ID NO.1, the amino acid sequence of PpCLE5 is shown in SEQ ID NO.2, and the amino acid sequence of PpCLE7 is shown in SEQ ID NO.

3.

2. The application of peach CLE peptide in the preparation of formulations that promote adventitious root regeneration in plants, characterized in that, The peach CLE peptide is one or more of PpCLE4, PpCLE5, or PpCLE7; the amino acid sequence of PpCLE4 is shown in SEQ ID NO.1, the amino acid sequence of PpCLE5 is shown in SEQ ID NO.2, and the amino acid sequence of PpCLE7 is shown in SEQ ID NO.

3.

3. A preparation for promoting the regeneration of adventitious roots in plants, characterized in that, It contains one or more of the following small peptides: PpCLE4, PpCLE5, or PpCLE7; the amino acid sequence of PpCLE4 is shown in SEQ ID NO.1, the amino acid sequence of PpCLE5 is shown in SEQ ID NO.2, and the amino acid sequence of PpCLE7 is shown in SEQ ID NO.

3.

4. A method for promoting adventitious root regeneration in plants, characterized in that, Plant material is inoculated into an adventitious root regeneration medium containing the preparation of claim 3 and cultured therein.

5. The method according to claim 4, characterized in that, The adventitious root regeneration medium is based on MS medium and contains sucrose, agar, and IBA.

6. The method according to claim 5, characterized in that, The concentration of peach CLE peptide in the adventitious root regeneration medium is 100 μM.

7. The method according to claim 6, characterized in that, The adventitious root regeneration medium contains 25 g / L sucrose, 8 g / L agar, and 2.0 mg / L IBA.

8. The method according to claim 4, characterized in that, The plant material mentioned is a segment of tissue culture seedlings.

9. The method according to claim 4, characterized in that, The culture was carried out at a temperature of 25°C, with 16 hours of light / 8 hours of darkness per day and a light intensity of 1500 lux for 1-2 weeks.

10. The method according to claim 4, characterized in that, The plant in question is a peach.

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