Rooting agent and rooting method for promoting formation of pinus massoniana adventitious roots

Abstract

The present application relates to the field of biotechnology, in particular to a rooting agent and a rooting method for promoting formation of Pinus massoniana adventitious roots, and is the result of years of research by the applicant, and the inventor first discovered that lignin small molecules are related to formation of plant adventitious roots in breeding work, and conducted theoretical research on the influence of lignin small molecules on Pinus massoniana adventitious roots, and based on observation results of cell biology and metabolomics closely related to formation of adventitious roots, created a rooting agent composed of auxin-like substances, GA synthesis inhibitors and lignin synthesis enzyme inhibitors. The rooting agent has good rooting effect, significantly improves the rooting ability of Pinus massoniana cuttings, and has significant economic, ecological and social benefits. The rooting agent overcomes the defects that traditional rooting agents are mainly selected by experience, and the work has a large amount of blindness and contingency.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and in particular to a rooting agent and method for promoting the formation of adventitious roots in Pinus massoniana. [Background Technology]

[0002] Masson pine (Pinus massoniana) is a major afforestation tree species in southern my country, with high utilization value and broad application prospects, playing a pivotal role in my country's forestry economic development. High-quality varieties and robust seedlings are the prerequisite and guarantee for efficient cultivation of plantations. my country has conducted extensive research on the breeding of superior Masson pine varieties, and has successively bred a large number of excellent germplasm sources and families. However, Masson pine plantations established using seedlings as afforestation material exhibit significant differences in tree genetic differentiation, low stand productivity, and generally low stand quality. Using asexual rapid propagation technology to cultivate superior germplasm clonal lines is an effective shortcut to achieving scientific, efficient, and intensive management of Masson pine plantations.

[0003] In the asexual propagation of Masson pine, numerous Chinese scholars have conducted extensive research since the 1980s, confirming that Masson pine is a root-resistant species, making asexual reproduction difficult. The formation of adventitious roots in plants is related to endogenous hormone levels and cellular anatomical characteristics. While species with root primordia generally develop roots easily, Masson pine, being a species that induces root primordia, primarily relies on the differentiation of vascular cambium parenchyma cells into root primordia. These cells then further proliferate and elongate to form adventitious roots. Therefore, the formation of adventitious roots in Masson pine involves two steps: adventitious root formation and development. Hormones, as core regulatory factors in root formation, have shown that high levels of endogenous gibberellins (GAs) inhibit rooting in Masson pine. The endogenous IAA / GAs ratio shows a significant positive correlation with the rooting ability of Masson pine. Adding specific GAs biosynthesis inhibitors such as paclobutrazol (PBZ) or dichlorophenoxylate (DPC) to the culture medium can effectively improve the adventitious root formation rate of Masson pine. However, follow-up investigations revealed that in culture media with long-term addition of PBZ or DPC, although the rooting time and rooting rate of Pinus massoniana seedlings were significantly improved compared with the control, the effect was unstable. Overall, root growth was slow, the number of high-quality roots (length >2cm / 35 days) did not increase significantly, and root development was difficult.

[0004] Cuttings are a simple and inexpensive method of asexual propagation, and have become the most commonly used method in production. The physiological age effect of Masson pine cuttings is significant; the younger the cutting, the stronger its rooting ability. Studies have shown that the physiological activity of cuttings obtained from mature trees and young trees is completely different. The former has poorer activity, a lower endogenous IAA / GAs ratio, and is more prone to aging, resulting in extremely high lignification of branches. Therefore, this application, under the condition of controlling the genetic basis and physiological age of the cuttings, explores the effects of PBZ and DPC on the rooting effect of Masson pine cuttings from the perspectives of biological function, rooting anatomy, and lignin metabolism, based on the mechanism of adventitious root formation. The applicant found that PBZ and DPC have a phagocytic effect. By systematically optimizing the treatment time and concentration of PBZ, DPC, and related enzyme inhibitors, the root development of Masson pine was significantly accelerated, the number of roots increased substantially, and the root quality was significantly improved. Furthermore, the results showed good repeatability and stable rooting effects, providing strong scientific and technological support for the asexual propagation and utilization of superior Masson pine varieties. [Summary of the Invention]

[0005] In view of the above, it is necessary to conduct theoretical research on the effects of lignin small molecules on the adventitious roots of Masson pine, observe cell biology and metabolomics closely related to adventitious root formation, and summarize a new rooting agent suitable for the formation of adventitious roots of Masson pine. This rooting agent can significantly improve the rooting ability of Masson pine cuttings and effectively overcome the shortcomings of traditional rooting agent selection, which mainly relies on empirical methods and has a lot of blindness and randomness.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A rooting agent that promotes the formation of adventitious roots in Pinus massoniana, the rooting agent being composed of NAA, PBZ, DPC and AOA.

[0008] Furthermore, the concentrations of NAA, PBZ, DPC, and AOA are 50-300 mg / L, 50-150 mg / L, 50-200 mg / L, and 25-100 mg / L, respectively.

[0009] Furthermore, the concentrations of NAA, PBZ, DPC, and AOA are 50 mg / L, 150 mg / L, 200 mg / L, and 100 mg / L, respectively.

[0010] The present invention also includes a method for rooting Masson pine using the rooting agent, the method comprising the following steps:

[0011] (1) Prepare the rooting agent according to the concentration stated above and set it aside;

[0012] (2) Select superior individual plants, collect newly sprouted tender branches in the nursery for grafting, and cut off the top to promote sprouting after the grafting is successful. When the sprouted branches grow to 5-7cm in length, cut off the branches for short branch cuttings.

[0013] (3) Soak the base of the scion in different rooting agents for 4 hours, and then insert the scion directly into a mixture of peat moss, perlite and coconut coir of equal volume ratio;

[0014] (4) Cuttings are taken under artificially controlled environmental conditions. The temperature for cuttings is 28±1℃, the humidity is >90%, and the light intensity is 4000~5000lx.

[0015] The present invention has the following beneficial effects:

[0016] This invention is the result of the applicant's many years of research. The inventor first discovered in propagation work that lignin molecules are related to the formation of adventitious roots in plants. Theoretical research was conducted on the effects of lignin molecules on the adventitious roots of *Pinus massoniana*. Based on observations in cell biology and metabolomics closely related to adventitious root formation, a rooting agent composed of auxin-like substances, GA synthesis inhibitors, and lignin synthase inhibitors was created. It exhibits good root-promoting effects, significantly improving the rooting ability of *Pinus massoniana* cuttings, and has significant economic, ecological, and social benefits. It overcomes the shortcomings of traditional rooting agents, which rely mainly on empirical methods and are subject to considerable uncertainty and chance. [Attached Image Description]

[0017] Figure 1 This is a cross-sectional anatomical diagram of the stem 20 days after rooting of cuttings under different rooting agents; the arrows in the diagram indicate root primordia.

[0018] Figure 2 These are anatomical diagrams of the rootstocks of seedlings with different root system qualities;

[0019] Figure 3 This is a heatmap of differentially expressed lignin small molecules in rhizomes under different rooting agent treatments;

[0020] Figure 4 This is a metabolic pathway diagram of plant lignin;

[0021] Figure 5 This is a graph showing the changes in the lignin small molecule caffeic acid (pme0303) under different rooting agents;

[0022] Figure 6 This is a graph showing the changes in the lignin small molecule ferulic acid (pme0306) under different rooting agents. Figure 3-4In the diagram, R represents PBZ, D represents DPC, and C represents the control; 1 represents 0 days of rooting treatment; 2 represents 10 days of rooting treatment; 3 represents 20 days of rooting treatment; and 4 represents 35 days of rooting treatment.

[0023] Figure 7 These are the effect curves of different groups of rooting agents.

Detailed Implementation Methods

[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0025] Example 1:

[0026] This embodiment studies the root-promoting effect of PBZ / DPC, as detailed below:

[0027] 1. Experimental materials:

[0028] From a superior stand of mature Masson pine trees aged 15 years or older, four superior individual trees were selected using the five dominant trees method, numbered GLM-3, GLM-7, GLM-38, and GLM-72. Newly sprouted shoots were collected and grafted in the nursery. After successful grafting, the tops were pruned to encourage new growth. When the sprouted branches reached 5–7 cm in length, they were cut off for short-branch cuttings.

[0029] 2. Experimental methods:

[0030] Cuttings were propagated using the soaking method, where the base of the cuttings was first soaked in different rooting agents for 4 hours, and then the cuttings were directly inserted into a mixture of peat moss, perlite, and coconut coir in equal volumes. Three rooting agent treatments were used: control (200 mg / L NAA); PBZ (200 mg / L NAA + 100 mg / L PBZ); and DPC (200 mg / L NAA + 100 mg / L DPC). After propagation, humidity was maintained above 90% by misting, and light intensity was kept between 4000 and 5000 lx using shade netting. Two months after propagation, the rooting rate and the number of roots longer than 2 cm were recorded for each rooting agent treatment. Rooting time was defined as the time when more than 50% of the cuttings showed shoot emergence and root growth at the tip. Five months after cutting, the survival rate was calculated. The rooting rate was calculated as follows: (Number of rooted seedlings / Total number of cuttings) × 100%; the survival rate was calculated as follows: Survival rate (%) = (Number of surviving seedlings / Number of rooted seedlings) × 100%.

[0031] (1) The rooting effect of Masson pine cuttings is shown in Table 1:

[0032] Table 1. Rooting effect of different rooting agents on pine cuttings.

[0033]

[0034] Note: Different lowercase letters in the table indicate significant differences between different genotypes treated with the same rooting agent, while different uppercase letters indicate significant differences between the average values ​​of each rooting agent treatment (P<0.05).

[0035] As shown in the table above, in the control treatment without added GA synthesis inhibitors, genotype significantly affected the rooting effect of cuttings, indicating that different genotypes resulted in different rooting times, rooting rates, and root numbers. Under PBZ and DPC treatments, genotype had no effect on rooting time, but the rooting times were significantly shorter than in the control treatment, indicating that adding GA synthesis inhibitors as rooting agents significantly accelerated the rooting progress of *Pinus massoniana* cuttings. However, regarding changes in rooting rate, root number, and survival rate, although the rooting rates under PBZ and DPC treatments were higher than the control, the number of roots did not change significantly, and the survival rate did not increase significantly, remaining generally low (63.9%–66.6%). Based on the input-output ratio requirements in production, the survival rate of cuttings generally needs to be above 80%, indicating that although GA synthesis inhibitors improved the ability of *Pinus massoniana* cuttings to develop adventitious roots, root development was not significantly improved, and root quality still needs to be improved. The differences between PBZ and DPC treatments show that different GA synthesis inhibitors have different root-promoting effects. Among them, PBZ has a stronger effect and promotes rapid rooting, but root development is difficult and the rooting rate is relatively low. DPZ, on the other hand, promotes rooting later than PBZ, but has better rooting stability and a higher rooting rate.

[0036] (2) Anatomical analysis of PBZ / DPC rooting

[0037] To analyze the differences in the rooting effects of rooting agents, the research group observed the anatomy of the rhizome. The cross-sectional anatomy of the stems of *Pinus massoniana* cuttings treated with different rooting agents for 20 days is shown in the following figures. Figure 1 As shown in the figure, blue represents parenchyma cells, purplish-red represents less active lignified and suberized cells, and red arrows indicate root primordia cells induced to form root primordia (small cells with distinct black dotted nuclei). The figure shows that different rooting agents result in different adventitious root development. Compared to the control (CK), the adventitious root primordia cells under DPC and PBZ treatments showed better development, but the lignification of root and stem cells was most pronounced under PBZ treatment.

[0038] Three rooted seedlings with significant differences in root quality were used as experimental materials for observation. The observation results are as follows: Figure 2 As shown, Figure 2The diagram shows the anatomical structure of the rhizomes of seedlings with different root system quality. The top row shows the anatomical structure of the rhizomes of seedlings with different root system quality; the bottom row shows the schematic diagram of different root system quality. In the diagram, ① represents good root quality, ② represents average root quality, and ③ represents poor root quality.

[0039] from Figure 2 It is evident that in the stem anatomy of group ③, where the root system quality was the worst, the most developed purplish-red, less active lignified and suberized tissues were present, while in group ①, where the root system quality was good, the purplish-red area was significantly less. Combining the root anatomy diagrams of the two groups, it can be seen that different rooting agents have different effects on the activity and lignification degree of root and stem cells.

[0040] (3) Small molecule metabolism analysis of PBZ / DPC root and stem lignin

[0041] Lignification is mainly regulated by lignin small molecule metabolism. Using rooted pine seedlings treated with different rooting agents as experimental materials, samples were taken on days 0, 10, 20, and 35 of rooting treatment for lignin small molecule determination. High-throughput LC-MS / MS technology was used to perform absolute quantitative detection of intermediate products of 14 lignin metabolic pathways. Specific results are as follows: Figures 3-6 As shown in the figure, the substances represented by each symbol are listed in Table 2. It can be seen from the figure that *Pinus massoniana* is a gymnosperm, and its lignin is mainly G-type lignin (Guaiacyl lignin). Analysis revealed that the levels of two lignin molecules closely related to G-type lignin metabolism, caffeic acid (pme0303) and ferulic acid (pme0306), showed no significant changes during the initial stage of adventitious root induction (0 days of rooting treatment) and the adventitious root development stage (10 days of rooting treatment). However, during the adventitious root development stage (20 days of rooting treatment) and the formation stage (35 days of rooting treatment), the levels of caffeic acid and ferulic acid significantly increased. Combining the physiological functions of two small lignin molecules (as cell wall components, forming the skeleton and leading to cell wall lignification; simultaneously, as signaling molecules, influencing protein phosphorylation through the MAPK pathway and regulating cell proliferation, differentiation, and apoptosis), this reflects that elevated levels of caffeic acid and ferulic acid lead to difficulties in adventitious root development. However, the timing and level of elevation vary depending on the rooting agent. This is the first discovery that small lignin molecules are related to adventitious root formation in plants. Specifically, at 20 days after rooting treatment, the levels of caffeic acid and ferulic acid were highest under PBZ(R3) treatment. This is consistent with the previously mentioned observations of the stem cross-section anatomy of cuttings 20 days after rooting treatment with different rooting agents (the purplish-red tissue was the most developed).

[0042] Table 2. Corresponding numbers of 14 lignin small molecules

[0043]

[0044] Example 2:

[0045] Based on the research results of Example 1, this embodiment optimizes the rooting agent scheme based on the regulation of lignin small molecule activity.

[0046] Based on the above experimental observations, four factors were selected as rooting agent components: highly active auxin-like substance NAA (naphthaleneacetic acid), GA synthesis inhibitors PBZ (paclobutrazol) and DPC (mepiride), and aminooxyacetic acid (AOA), a specific inhibitor of phenylalanine ammonia-lyase (PAL), a key upstream enzyme in the synthesis of lignin small molecules caffeic acid and ferulic acid. The rooting agent scheme was optimized.

[0047] Each component has 4 levels:

[0048] Table 3. Component levels of rooting agents

[0049]

[0050] Sixteen rooting agent schemes were designed using orthogonal design. Each rooting agent scheme had 10 replicates, with 70 seedlings per replicate. Cuttings were taken under artificially controlled environmental conditions: temperature 28±1℃, humidity >90%, and light intensity 4000–5000 lx. The cutting experiment was repeated four times consecutively, and the average value was then analyzed.

[0051] Table 4. Analysis of the rooting effects of different rooting agent regimens

[0052]

[0053]

[0054] Note: The table shows significant differences in rooting indicators among different rooting agent schemes with lowercase letters (P<0.05).

[0055] The results in the table show that different rooting agent regimens resulted in different rooting effects. Among them, under regimen 4 (50mg / L NAA + 150mg / L PBZ + 200mg / L DPC + 100mg / L AOA), the rooting time of Masson pine cuttings was short, and the rooting rate, number of roots, and survival rate were the highest.

[0056] The effect curves of the above rooting agent components are shown in the figure. Figure 7 As shown in the figure, the effect curves of each rooting agent component reveal that different rooting agents produce different rooting indicators and have different effective concentrations. This application, for the first time, comprehensively evaluates the adventitious root formation process of Masson pine from the perspectives of rooting time, rooting rate, number of roots, and survival rate, addressing the problems of difficult development, poor root quality, and low survival rate after adventitious root formation, by considering both the occurrence and development stages of adventitious root formation.

[0057] In summary, the inventors of this application have for the first time discovered the relationship between lignin small molecules and adventitious root formation in plants. Based on theoretical research and observations from cell biology and metabolomics closely related to adventitious root formation, this application has developed a rooting agent composed of auxin-like substances, GA synthesis inhibitors, and lignin synthase inhibitors. This rooting agent exhibits excellent root-promoting effects, significantly improving the rooting ability of Masson pine cuttings, and possesses significant economic, ecological, and social benefits. It overcomes the shortcomings of traditional rooting agents, which rely heavily on empirical methods and involve significant blind spots and chance.

[0058] The examples described above are merely illustrative of several embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A rooting agent that promotes the formation of adventitious roots in Masson pine, characterized in that, The rooting agent is composed of NAA, PBZ, DPC and AOA; the concentrations of NAA, PBZ, DPC and AOA are 50 mg / L, 150 mg / L, 200 mg / L and 100 mg / L, respectively.

2. A method for rooting Masson pine using the rooting agent as described in claim 1, characterized in that, The method includes the following steps: (1) Prepare the rooting agent according to the concentration stated above and set aside; (2) Select superior individual plants, collect newly sprouted tender branches in the nursery for grafting, and cut off the top to promote sprouting after the grafting is successful. When the sprouted branches grow to 5-7 cm in length, cut off the branches for short branch cuttings. (3) Soak the base of the scion in the rooting agent for 4 hours, and then insert the scion directly into a mixture of peat moss, perlite and coconut coir of equal volume ratio; (4) Cuttings are taken under artificially controlled environmental conditions. The temperature for cuttings is 28±1℃, the humidity is >90%, and the light intensity is 4000~5000 lx.

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