Method for inducing leaf stalk callus of adult tree of tung oil tree and regenerating plant

Abstract

The invention discloses a method for inducing petiole calluses of adult trees of field tung tree and regenerating plants, and belongs to the technical field of tung tree tissue culture.The method comprises the following steps that S1, petiole explants are obtained from the adult trees of field tung tree, surface disinfection is conducted through a disinfectant, and sterile explants are obtained; s2, inoculating the sterile explant into a first culture medium for culturing, and inducing to generate callus; s3, inoculating the calluses obtained in the step S2 into a second culture medium for culture, and inducing and differentiating adventitious buds; s4, inoculating the adventitious buds obtained in the step S3 into a third culture medium for culturing, and promoting the adventitious buds to grow robustly; s5, inoculating the seedlings obtained in the step S4 into a fourth culture medium for culture, and inducing the seedlings to root to obtain complete regenerated plants. The problem that an existing tung tree tissue culture technology depends on sterile seedlings is solved.

Description

Technical Field

[0001] This invention relates to the field of tung tree tissue culture technology, and more specifically, to a method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants. Background Technology

[0002] Tung oil tree (Vernicia fordii Hesml.) belongs to the genus Vernicia in the family Euphorbiaceae. It is a deciduous tree widely distributed in my country, with the largest cultivation areas in Guizhou, Hunan, Chongqing, and Hubei provinces. Tung oil is an important industrial oilseed tree species in my country. Due to its high unsaturated fatty acid content (84%-86%), it is a high-quality drying oil with excellent properties such as fast drying, good gloss, and strong adhesion, making it an environmentally friendly raw material with multiple uses. Furthermore, tung oil can be used to make canning coatings, which are natural, pollution-free, and highly sought after; tung oil-based power strips offer excellent performance while reducing costs; and the remaining tung oil cake and bran are high-quality fertilizers. As a traditional economic tree species, tung oil has significant social and economic benefits. Therefore, strengthening the protection and research of tung oil tree species, cultivating superior varieties, and improving its comprehensive benefits are inevitable trends.

[0003] However, with the development of science and technology, many new raw materials have emerged, hindering the development of tung oil trees. Meanwhile, current tung oil tree breeding methods still primarily rely on traditional methods. As a large tree, the tung oil tree grows slowly in its natural environment, and propagation through seed involves a long growth and breeding cycle, which is time-consuming and labor-intensive. Tissue culture technology is an important way to achieve rapid seedling production and shorten the growth cycle of tung oil trees, and it has broad commercial prospects.

[0004] Currently, although there are reports on tung oil tree tissue culture, existing tung oil tree tissue culture systems all use sterile seedlings as explants. Due to the complex field growing environment, sterilizing field-grown tung oil tree materials is extremely difficult, making it hard to obtain sterile seedlings, which severely limits the rapid propagation of existing superior tung oil tree varieties through tissue culture.

[0005] In view of this, the present invention provides a method for inducing callus tissue from the petioles of mature tung trees and regenerating plants. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing callus tissue from the petioles of mature tung trees in the field and regenerating plants, which solves the problem that existing tung tree tissue culture technology relies on sterile seedlings.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants includes the following steps: S1. Obtain petiole explants from mature tung trees in the field, and disinfect the surface with a disinfectant to obtain sterile explants; S2. The sterile explants are inoculated into a first culture medium for culture to induce callus formation; the first culture medium contains 6-BA and auxin. S3. The callus tissue obtained in step S2 is inoculated into the second culture medium for culture to induce the differentiation of adventitious shoots; the second culture medium contains 6-BA, auxin and GA3. S4. The adventitious buds obtained in step S3 are inoculated into a third culture medium for cultivation to promote their vigorous growth; the third culture medium contains 6-BA, IBA and GA3. S5. The seedlings obtained in step S4 are inoculated into the fourth culture medium for culture to induce rooting and obtain complete regenerated plants; the fourth culture medium contains IBA.

[0008] A further preferred embodiment is that the surface disinfection includes: first soaking the explant in 75% ethanol for 30 seconds, and then soaking the explant in 10% sodium hypochlorite solution for 5 minutes.

[0009] Further preferably, the first culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 3.0-6.0 mg / L, and the auxin is IAA with a concentration of 0.05-0.1 mg / L.

[0010] A further preferred embodiment is that, in the first culture medium, the concentration of 6-BA is 3.0 mg / L and the concentration of IAA is 0.05 mg / L.

[0011] Further preferably, the second culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 3.0-5.0 mg / L, the auxin is IAA with a concentration of 0.05-0.1 mg / L, and the concentration of GA3 is 0.1-0.5 mg / L.

[0012] Further preferably, in the second culture medium, the concentration of 6-BA is 3.0 mg / L, the concentration of IAA is 0.05 mg / L, and the concentration of GA3 is 0.1 mg / L.

[0013] Further preferably, the third culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 0.5-3.0 mg / L, the concentration of IBA is 0.025-0.1 mg / L, and the concentration of GA3 is 0.1-0.5 mg / L.

[0014] More preferably, in the third culture medium, the concentration of 6-BA is 0.5 mg / L, the concentration of IBA is 0.025 mg / L, and the concentration of GA3 is 0.1 mg / L.

[0015] Further preferably, the fourth culture medium is based on 1 / 2 MS medium, wherein the concentration of IBA is 0.1-0.5 mg / L.

[0016] A further preferred embodiment is that the concentration of IBA in the fourth culture medium is 0.5 mg / L.

[0017] In summary, the present invention has the following beneficial effects: (1) Unlike existing technologies that start with sterile laboratory seedlings, this invention primarily addresses the fundamental challenge of obtaining explants from complex open-air environments—efficient disinfection. Mature tung tree materials from field-grown plants carry a large number of unknown and persistent microbial communities, and conventional disinfection methods easily lead to high contamination or browning rates, resulting in experimental failure. This invention successfully overcomes this challenge through an optimized combination of surfactant pretreatment, short-term ethanol treatment, and appropriate concentration of sodium hypochlorite disinfection. (2) This invention directly uses the petioles of superior mature tung trees in the field as explants to obtain regenerated plants through asexual reproduction. It can completely maintain the genetic traits of the mother plant and effectively overcome the problem of trait segregation caused by tissue culture using sterile seedlings. It provides a guarantee for the accurate cloning and preservation of superior genotypes. (3) The present invention successfully cultivated tung tree tissue culture seedlings using the petioles of mature field tung tree leaves. The present invention has the characteristics of good induction of adventitious buds and vigorous growth of tissue culture seedlings. (4) Compared with existing tung oil tissue culture techniques that rely on sterile materials, this invention achieves significant optimization in the adventitious bud differentiation stage. Existing techniques often face problems such as unstable differentiation rates and the need for multiple rounds of subculture to obtain sufficient material, resulting in cumbersome processes and high costs. This invention, through an optimized differentiation and seedling strengthening culture medium formula, can directly and efficiently differentiate a large number of robust adventitious buds from callus tissue in one go, with a high differentiation rate and high seedling quality, without the need for independent subculture steps. This breakthrough simplifies the operation process, shortens the seedling cycle, and significantly reduces production costs, providing a reliable and efficient technical solution for the large-scale, factory-scale production of tung oil tissue culture seedlings. Attached Figure Description

[0018] Figure 1 This is a photograph of the mother tree from which the explants of the tung tree in Datian were obtained in Embodiment 1 of the present invention; Figure 2 This is a photograph of the part of the tung tree from the field used in Embodiment 1 of the present invention; Figure 3This is a photograph of the disinfection process of the tung tree explants in Example 1 of the present invention; Figure 4 This is a photograph of the early stage of callus induction in the petiole of *Vernicia fordii* in the B7 treatment group of Example 1 of this invention; Figure 5 These are photos of callus induction in the petiole of *Vernicia fordii* in the B7 treatment group 15-30 days after the invention. Figure 6 This is a photograph of the early stage of adventitious bud differentiation of the petiole callus tissue of the C7 treatment group in Example 1 of the present invention; Figure 7 This is a photograph of the differentiation of adventitious buds from the callus tissue of the field-grown tung oil petiole in the C7 treatment group of Example 1 of the present invention; Figure 8 This is a photograph of the early stage of vigorous seedlings with adventitious buds on the petioles of tung oil trees in the D1 treatment group of Example 1 of the present invention. Figure 9 These are photos of vigorous seedlings with adventitious buds on petioles of *Vernicia fordii* in the D1 treatment group of Example 1 of this invention, taken 15-20 days later. Figure 10 This is a photograph of the early stage of adventitious bud rooting on the petiole of the field tung oil tree in the E4 treatment group of Example 1 of the present invention; Figure 11 These are photos of adventitious buds on the petioles of *Tungus tung* var. *mairei* in the E4 treatment group of Example 1 of this invention, taken 15-30 days after rooting. Figure 12 This is a photograph of the sterile tung tree seedlings cultivated during the experiment in Example 1 of the present invention after hardening and transplanting. Detailed Implementation

[0019] Unless otherwise defined, all terms used herein should be interpreted in accordance with their meaning as commonly understood by those skilled in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention.

[0020] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0021] Currently, research on tung oil tissue culture mainly focuses on using the hypocotyl, cotyledon, leaf blade, or petiole of sterile seedlings as explants. While these methods can obtain regenerated plants, they have the following fundamental limitations: I. Limited source of explants: It relies on sterile seedlings cultivated in the laboratory and cannot directly utilize the superior mature tung tree germplasm resources screened in the wild.

[0022] Second, it cannot maintain the superior traits of the mother plant: seedlings exhibit genetic segregation, and plants obtained through their regeneration cannot be guaranteed to fully maintain the characteristics of the superior mother tree, making them unsuitable for direct and rapid propagation of superior clones.

[0023] Third, difficulties in utilizing field materials: directly obtaining mature tree materials from the field environment for tissue culture faces major technical bottlenecks such as complex bacterial contamination of explants and extreme difficulty in disinfection, resulting in a very high contamination rate and leading to experimental failure.

[0024] Existing technologies involving the regeneration of tung oil tree petioles all use sterile seedlings as explants, failing to address the core challenge of successfully regenerating explants directly from mature trees in heavily polluted fields. Therefore, this invention provides a method for inducing callus tissue from petioles of mature tung oil trees in the field and regenerating plants. It aims to use young petioles from mature tung oil trees in the field as explants, optimize sterilization methods to provide materials for callus induction, and study the effects of different combinations of plant growth regulators on callus induction, differentiation, seedling vigor, and rooting. This successfully overcomes key technical challenges related to sterilization, induction, differentiation, and seedling vigor. A stable in vitro culture and regeneration system for petioles of mature tung oil trees in the field has been established, providing important technical support for the preservation of tung oil germplasm resources and the rapid propagation of superior varieties.

[0025] A method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants includes the following steps: S1. Acquisition and disinfection of explants Petiole explants were obtained from mature tung oil trees in Datian, and the surfaces were disinfected with a disinfectant to obtain sterile explants.

[0026] Preferably, surface disinfection includes treatment with a 70-75% (v / v) ethanol solution and a 8-10% (w / w) sodium hypochlorite solution in sequence.

[0027] Preferably, before surface disinfection, the procedure further includes a pretreatment step for the explant: soaking it in a solution containing a surfactant and rinsing it with running water; after surface disinfection, washing away any disinfectant residue with sterile water.

[0028] The specific process is as follows: Select young petioles from healthy mature tung trees in the field, wash them with clean water for 5-10 minutes, then soak them in soap or laundry detergent solution for 2-4 minutes, and rinse them with clean water for later use; in a sterile operating table, first soak the explants in 75% ethanol for 30-60 seconds, then soak them in 10% sodium hypochlorite solution for 5-9 minutes, and then wash them 5-6 times with sterile water sterilized at 121°C to ensure that there is no disinfectant residue, and then sterile explants can be obtained.

[0029] Furthermore, the rinsing time with clean water is 5 minutes; the soaking time with soap or laundry detergent solution is 2-4 minutes; the soaking time with ethanol is 30 seconds; and the soaking time with sodium hypochlorite solution is 5 minutes.

[0030] S2, Inducing callus tissue Sterile explants were inoculated into a first culture medium and cultured to induce callus formation; the first culture medium contained 6-benzylaminopurine (6-BA) and auxin.

[0031] Preferably, the first culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 3.0-6.0 mg / L and the auxin is indoleacetic acid (IAA) with a concentration of 0.05-0.1 mg / L.

[0032] The specific process is as follows: First, the parts of the sterile explant that were in contact with the disinfectant at both ends are removed. Then, the explant is inoculated into 1 / 2 MS medium containing 3.0-6.0 mg / L 6-BA and 0.05-0.1 mg / L IAA to induce callus tissue. The explant is cultured under light for 15-30 days at a temperature of 26±2℃, a light intensity of 2100-2200 lx, and a light-to-dark ratio of 14-16 h / d. After adventitious bud differentiation, seedling strengthening culture, and rooting culture, complete plants are obtained and finally, the plants are hardened off and transplanted.

[0033] Furthermore, the first culture medium was 1 / 2 MS + 3.0 mg / L 6-BA + 0.05 mg / L IAA.

[0034] S3, Inducing Adventitious Buds The callus tissue obtained in step S2 was inoculated into the second culture medium for culture to induce the differentiation of adventitious shoots; the second culture medium contained 6-BA, auxin and gibberellin (GA3).

[0035] Preferably, the second culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 3.0-5.0 mg / L, the auxin is IAA with a concentration of 0.05-0.1 mg / L, and the concentration of GA3 is 0.1-0.5 mg / L.

[0036] The specific process is as follows: cut the well-induced petiole callus tissue with good texture into 2cm pieces. 2 Small pieces were inoculated into a second medium consisting of 1 / 2 MS + 3.0-5.0 mg / L 6-BA + 0.05-0.1 mg / L IAA + 0.1-0.5 mg / L GA3 to induce adventitious shoot differentiation. The culture was carried out under light conditions for about 45-60 days, at a temperature of 26±2℃, a light intensity of 2100-2200 lx, and a light-to-dark ratio of 14-16 h / d.

[0037] Furthermore, the second culture medium was 1 / 2 MS + 3.0 mg / L 6-BA + 0.05 mg / L IIAAA + 0.1 mg / L GA3.

[0038] S4. Seedling cultivation The adventitious buds obtained in step S3 were inoculated into the third culture medium for cultivation to promote their vigorous growth; the third culture medium contained 6-BA, indolebutyric acid (IBA) and GA3.

[0039] Preferably, the third culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 0.5-3.0 mg / L, the concentration of IBA is 0.025-0.1 mg / L, and the concentration of GA3 is 0.1-0.5 mg / L.

[0040] The specific process is as follows: Healthy adventitious shoots obtained from callus differentiation culture are cut off and inoculated into the third medium of 1 / 2 MS + 0.5-3.0 mg / L 6-BA + 0.025-0.1 mg / L LIBA + 0.1-0.5 mg / L GA3 for adventitious shoot seedling culture; cultured under light conditions for about 15-20 days, with a culture temperature of 26±2℃, a light intensity of 2100-2200 lx, and a light duration of 14-16 h / d.

[0041] Furthermore, the third culture medium consisted of 1 / 2 MS + 0.5 mg / L 6-BA + 0.025 mg / L IBA + 0.1 mg / L GA3.

[0042] S5, Induced Rooting The robust seedlings obtained in step S4 were inoculated into the fourth culture medium for cultivation to induce rooting and obtain complete regenerated plants; the fourth culture medium contained IBA.

[0043] Preferably, the fourth culture medium is based on 1 / 2 MS medium, wherein the concentration of IBA is 0.1-0.5 mg / L.

[0044] The specific process is as follows: 2-4cm seedlings obtained from strong seedling culture are inoculated into 1 / 2MS medium + 0.1-0.5 mg / L IBA for rooting culture; cultured under light conditions for about 15-30 days, with a culture temperature of 26±2℃, a light intensity of 2100-2200lx, and a light duration of 14-16h / d.

[0045] Furthermore, the fourth culture medium was 1 / 2 MS + 0.5 mg / LIBA.

[0046] In steps S1-S5, the culture media were supplemented with 30 g / L sucrose, 7 g / L agar, and the pH was adjusted to 5.4-5.8.

[0047] This invention addresses the shortcomings of current tung oil tree tissue culture techniques, which primarily focus on inducing seedlings from aseptic seed embryos, leaves, and petioles via callus induction or direct induction, and then obtaining regenerated plants through tissue culture. This invention selects superior trees from the field as explants, providing abundant and readily available materials. Compared with tissue culture methods of the same genus, this method is simple to operate, produces high adventitious bud differentiation rates and robust young buds, eliminating the need for adventitious bud subculture and significantly shortening the growth and seedling cycle, thus increasing the possibilities for future industrialized seedling production and transgenic technology. This invention effectively overcomes the challenge of sterilizing field materials, efficiently and stably inducing regenerated plants from mature leaf petioles, providing a pathway for the rapid propagation of superior tung oil trees. Furthermore, it offers possibilities for future genetic engineering methods to improve tung oil tree resistance, accelerate molecular breeding processes, enhance oil quality, and establish a genetic system for tung oil trees.

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Example 1: A method for inducing callus tissue from the petioles of mature tung trees in the field and regenerating plants, comprising the following steps: S1. Acquisition and disinfection of explants This step aims to solve the core technical bottleneck of complex bacterial contamination and difficult disinfection of mature tung oil tree materials from the field.

[0050] S11. Material Selection and Pretreatment: In spring, select young petioles of the current year from healthy, mature tung trees grown in the field (refer to...). Figure 1 , 2 Rinse with clean water for 5 minutes, then soak in a 1% (w / v) laundry detergent solution for 2 minutes. Rinse the collected petioles under running water for 5 minutes to remove most of the dust and microorganisms adhering to the surface. Then, immerse in a 1% (w / v) laundry detergent solution for 2 minutes, gently agitating to further remove surface wax, lint, and organic dirt using surfactants. After pretreatment, rinse thoroughly with running water and set aside.

[0051] S12. Surface Disinfection Scheme Screening: To determine the optimal disinfection parameters, an orthogonal experiment was designed. Pretreated petioles were randomly divided into 9 groups (A1-A9) and treated using a two-step disinfection method of "ethanol-sodium hypochlorite" in a clean bench (refer to...). Figure 3 ), and the specific parameters and results are shown in Table 1.

[0052] Table 1. Effects of different disinfectant treatment times on the disinfection effect of tung oil leaf petioles in the field. Note: The contamination rate is the proportion of explants that develop colonies within 7 days after inoculation; the browning rate is the proportion of explants that die from severe browning after 15 days of culture.

[0053] Table 1 shows that a short ethanol treatment time (15 seconds, group A1) was insufficient for effective sterilization, resulting in a contamination rate as high as 70%. While prolonged treatment with ethanol and / or sodium hypochlorite controlled contamination (contamination rates were all 0% in groups A2-A9), the browning rate of explants significantly increased, indicating tissue damage caused by excessive sterilization. Group A2 (75% ethanol treatment for 30 seconds, 10% sodium hypochlorite treatment for 5 minutes) achieved the best balance, thoroughly controlling contamination (0%) while maintaining a relatively low browning rate (16.67%), providing a large number of viable sterile explants for subsequent culture.

[0054] Based on the above screening results, the preferred disinfection method of the present invention is determined to be as follows: In a clean bench, first soak the explants in 75% ethanol for 30 seconds, then soak the explants in 10% sodium hypochlorite solution for 5 minutes, and then wash them 5-6 times with sterile water sterilized at 121°C to completely remove residual disinfectant, thus obtaining sterile explants for subsequent culture.

[0055] S2, Inducing callus tissue This step aims to screen and establish a culture medium formulation that can efficiently induce high-quality callus from sterile petioles from field sources.

[0056] Reference Figure 4 , 5 First, the ends of the sterile explants obtained in step S1 (A2 protocol) that had been in contact with the disinfectant were removed. The explants were then randomly grouped and inoculated into different plant growth regulator formulations in the first culture medium (basal medium was 1 / 2 MS, supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.6). A total of 12 treatment groups (B1-B12) were established to investigate the effects of different concentrations of cytokinin 6-BA and auxin IAA on callus induction efficiency and quality. Specific formulations and results are shown in Table 2.

[0057] The culture conditions for each group were as follows: temperature (26±2)℃, light intensity 2100-2200 lx, and 16 hours of light per day. After 30 days of culture, the callus induction rate (i.e., the percentage of explants that formed visible callus out of the total number of inoculated explants) was calculated, and the growth status of the callus (such as density) was observed and recorded.

[0058] Table 2. Effects of different plant growth regulators on callus induction in tung oil leaf petioles As shown in Table 2, the concentration and ratio of 6-BA and IAA have a decisive influence on the induction rate and quality of callus tissue.

[0059] Low concentration combinations (B1-B3): slow induction start-up, low induction rate (10%-33.33%), not suitable for efficient regeneration systems.

[0060] Medium concentration combination (B4-B6): The induction rate gradually increased (46.67%-70.00%), and the quality of callus tissue improved, but it was not optimal.

[0061] High concentration combinations (B7-B12): High induction rates (83.33%-100%) were achieved when 6-BA concentration was ≥3.0 mg / L. However, induction rate is not the only indicator. Observations revealed that while achieving high induction rates, IAA concentration significantly affected callus quality. Low IAA concentration (0.05 mg / L, B7 scheme): At a 6-BA concentration of 3.0 mg / L, the optimal balance between induction rate (83.33%) and callus quality was achieved. The induced callus tissue was abundant and dense, which is an ideal prerequisite for subsequent successful adventitious shoot differentiation.

[0062] Increasing IAA concentration (B8-B12 scheme): Although the induction rate reached or approached 100%, the callus tissue generally exhibited excessive growth and loose texture. This loose callus tissue has weak differentiation ability and is difficult to form high-quality adventitious shoots, which is not conducive to subsequent regeneration processes.

[0063] Considering both induction rate and callus quality, the culture medium formulation for the B7 treatment group was the optimal choice. This formulation ensured high induction efficiency while yielding high-quality callus most suitable for subsequent differentiation. Therefore, the preferred callus induction medium (i.e., the first medium) of this invention is determined to be: 1 / 2 MS basal medium + 3.0 mg / L 6-BA + 0.05 mg / L IIA + 30 g / L sucrose + 7 g / L agar, pH 5.6.

[0064] S3, Inducing Adventitious Buds This step aims to establish a key culture medium formula for the efficient differentiation of adventitious shoots from the high-quality callus tissue described above, which is the core step in obtaining a large number of regenerated seedlings.

[0065] Reference Figure 6 , 7 Select healthy callus tissue with dense texture induced by the preferred formula (B7 scheme) in step S2, and cut it into 2cm pieces. 2Small, uniformly sized pieces were randomly grouped and inoculated into secondary culture media (basal medium was 1 / 2 MS, supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.6) with different ratios of plant growth regulators. A total of 12 treatment groups (C1-C12) were established to systematically investigate the effects of different concentrations of cytokinin (6-BA), auxin (IAA), and gibberellin (GA3) on adventitious shoot differentiation efficiency. Specific formulations are shown in Table 3.

[0066] The culture conditions for each group were as follows: temperature (26±2)℃, light intensity 2100-2200 lx, and 16 hours of light per day. After 45-60 days of culture, the adventitious bud differentiation rate (i.e., the percentage of callus tissue blocks with visible adventitious buds out of the total number of inoculated callus tissue blocks) was calculated, and the number and growth status of adventitious buds were observed and recorded.

[0067] Table 3. Effects of different plant growth regulators on callus differentiation of tung oil leaf petioles As shown in Table 3, the ratio of plant growth regulators has a very significant effect on adventitious bud differentiation, and there is a clear concentration window effect.

[0068] Low 6-BA concentration (1.5-2.0 mg / L, C1-C6): The overall differentiation rate is low (≤46.67%), indicating that the cytokinin concentration is insufficient and it is difficult to effectively initiate the redifferentiation process of callus tissue.

[0069] At medium 6-BA concentrations (3.0 mg / L, C7-C9), the differentiation rate fluctuated dramatically, demonstrating the key regulatory role of IAA concentration. When the IAA concentration was maintained at a low level (0.05 mg / L, C7 formulation), combined with 3.0 mg / L 6-BA and 0.1 mg / L GA3, the highest and most significant adventitious shoot differentiation rate (70.00%) was achieved, and the adventitious shoots grew vigorously. This reflects the advantage of choosing a low IAA formulation (B7) during the callus induction stage to obtain the persistence of dense callus. Once the IAA concentration was increased (C8, C9), the differentiation rate dropped sharply (23.33%, 13.33%), indicating that the higher auxin environment inhibited the occurrence of adventitious shoots.

[0070] High 6-BA concentration (5.0 mg / L, C10-C12): All treatments showed extremely low or even zero differentiation rates, indicating that excessively high cytokinin concentrations had a toxic effect on cell differentiation and completely inhibited the formation of bud primordia.

[0071] Comparing all treatment groups, the C7 treatment group (6-BA 3.0 mg / L, IAA 0.05 mg / L, GA3 0.1 mg / L) showed significantly better adventitious bud differentiation rate than all other combinations, and the differentiated adventitious buds were of high quality. This formulation successfully achieved efficient conversion of field-derived petiole callus tissue into a large number of adventitious buds. Therefore, the preferred adventitious bud differentiation medium (i.e., the second medium) of this invention is determined to be: 1 / 2 MS basal medium + 3.0 mg / L 6-BA + 0.05 mg / L IAA + 0.1 mg / L GA3 + 30 g / L sucrose + 7 g / L agar, pH 5.6.

[0072] S4. Seedling cultivation This step aims to optimize the culture medium formulation to promote further elongation and thickening of the adventitious buds obtained in step S3, forming robust and complete seedlings suitable for rooting.

[0073] Reference Figure 8 , 9 Healthy adventitious shoots differentiated using the optimized formula (C7 scheme) in step S3 were selected and randomly grouped. They were then inoculated into different media (basal medium: 1 / 2 MS, supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.6) containing different ratios of plant growth regulators. A total of 16 treatment groups (D1-D16) were established to systematically investigate the effects of different concentrations of cytokinin (6-BA), auxin (IBA), and gibberellin (GA3) on the seedling vigor of adventitious shoots. Specific formulations and evaluation results are shown in Table 4.

[0074] The cultivation conditions for each group were as follows: temperature (26±2)℃, light intensity 2100-2200 lx, and 16 hours of light per day. The effects were evaluated after 15-20 days of cultivation. The growth status of the seedlings was also recorded in detail.

[0075] Table 4. Effects of different plant growth regulators on vigorous growth of adventitious buds in tung oil trees. The goal of the seedling strengthening stage is to promote the axial elongation of existing buds (due to the effect of GA3) and stem thickening (due to moderate cell division). At the same time, it is crucial to strictly control the excessive production of callus tissue at the base, otherwise it will consume nutrients and lead to weak seedlings. Table 4 shows that the concentrations of IBA and 6-BA are key to controlling callus production.

[0076] As shown in Table 4, group D1 (0.5 mg / L 6-BA, 0.025 mg / L IBA, 0.1 mg / L GA3) achieved an overwhelmingly optimal seedling vigor rate (86.67%). This formulation, using low concentrations of 6-BA and extremely low concentrations of IBA, combined with a low concentration of GA3, perfectly achieved the goals of high seedling vigor and low callus formation. The seedlings exhibited ideal overall traits.

[0077] Negative effects of changes in hormone concentrations: Increasing IBA concentration (D2-D4, D6-D8, etc.): Even with the 6-BA concentration remaining constant (0.5 mg / L), the seedling vigor rate decreased sharply with increasing IBA concentration, and "excessive callus formation" became a common problem. This indicates that low concentrations of IBA are sufficient to support seedling development before rooting, while excessively high concentrations strongly induce callus formation.

[0078] Increasing 6-BA concentration (D5-D16): In any IBA / GA3 combination, increasing the 6-BA concentration from 0.5 mg / L to 1.0 mg / L or higher significantly reduced the seedling vigor rate, and the problem of excessive callus formation became more prominent. This indicates that the cytokinin level required during the seedling vigor stage is much lower than that during the differentiation stage, and even a slightly higher level will disrupt the growth balance.

[0079] A comprehensive comparison of the various treatment groups revealed that the culture medium formulation of the D1 treatment group demonstrated exceptional performance in promoting robust adventitious bud growth, exhibiting a significantly higher seedling vigor rate and the best seedling quality compared to all other combinations. Therefore, the preferred seedling vigor medium (i.e., the third medium) of this invention is determined to be: 1 / 2 MS basal medium + 0.5 mg / L 6-BA + 0.025 mg / L IBA + 0.1 mg / L GA3 + 30 g / L sucrose + 7 g / L agar, pH 5.6.

[0080] S5, Induced Rooting This step is the final stage of the regeneration system, designed to establish an efficient and high-quality rooting system for robust tissue culture seedlings, thereby obtaining complete regenerated plants that can be transplanted.

[0081] Reference Figure 10 , 11 Robust seedlings cultured using the optimized formula (D1 scheme) in step S4 were selected and randomly divided into groups. They were then inoculated into a fourth culture medium (basal medium was 1 / 2 MS, supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.6) containing different types and concentrations of auxin. A total of nine treatment groups (E1-E9) were established to systematically compare the effects of single auxin IBA and NAA on the rooting of *Vernicia fordii* tissue culture seedlings. Specific formulations and rooting results are shown in Table 5.

[0082] The culture conditions for each group were as follows: temperature (26±2)℃, light intensity 2100-2200 lx, and 16 hours of light per day. After 15-30 days of culture, the rooting rate (i.e., the percentage of seedlings that produced adventitious roots out of the total number of inoculated seedlings) was calculated, and the average root length and average number of roots were measured to comprehensively evaluate the rooting quality.

[0083] Table 5. Effects of different plant growth regulators on adventitious bud rooting in tung oil trees. Table 5 shows that the NAA-treated groups (E6-E9) generally exhibited short, thick, and clustered roots with poor morphology; while the IBA-induced roots (such as E4) were long, robust, and had abundant lateral roots, which was more conducive to transplant survival. This indicates that IBA is a better choice for inducing high-quality rooting in tung oil tissue culture seedlings.

[0084] Comparing rooting efficiency and root quality, the culture medium formula of the E4 treatment group was the optimal choice for inducing rooting in tung oil tree tissue culture seedlings. This formula ensured a high rooting rate while obtaining an adventitious root system with excellent length, quantity, and morphology. Therefore, the preferred rooting medium (i.e., the fourth medium) of this invention is determined to be: 1 / 2 MS basal medium + 0.5 mg / L IBA + 30 g / L sucrose + 7 g / L agar, pH 5.6.

[0085] After rooting culture, the obtained complete plants can be hardened off and transplanted (refer to...). Figure 12 ).

[0086] Example 2: A method for inducing callus from petioles of mature tung trees and regenerating plants. To more objectively evaluate the technical advantages of this invention, especially the unique effect of the optimized culture medium formula for the special explant of petioles of mature tung trees, this example selects 9 existing patents (numbered A1-A9) closely related to this technical field and compares them with this invention (numbered A10) for key technical aspects, as follows:

[0087] (1) The existing patent numbers are as follows: A1:201310271826.7 A method for regenerating plants from tung oil tree leaves; A2:201410326807.4 Methods for inducing callus tissue from tung oil tree hypocotyls and generating highly efficient regenerated plants; A3:201410327567.X A method for efficient direct regeneration of tung oil tree petioles into plants; A4:201610089435.7 A method for directly inducing plant regeneration using the hypocotyl of the tung tree as an explant; A5:201710077542.2 Method for rooting tung oil tissue culture seedlings in bottles; A6:201710079785.X A tissue culture method for rapid propagation of tung oil trees; A7:201910934260.9 A method for physically promoting the regeneration of tung oil tree stem segments with buds; A8:202110161400.0 A method for disinfecting and rapidly propagating stem segments of the ancient Chinese tung tree; A9:202410092026.7 A method for establishing a genetic transformation system for tung oil callus.

[0088] (2) Comparative experimental design: The dense callus tissue derived from the petioles of mature field trees (induced by the preferred first culture medium of the present invention) obtained in step S2 of Example 1 of the present invention was divided into two groups: Experimental group: Continue to use the series of culture media preferred in Example 1 of the present invention for subsequent culture (i.e., use the second culture medium preferred in the present invention for adventitious bud differentiation, use the third culture medium preferred in the present invention for seedling strengthening, and use the fourth culture medium preferred in the present invention for rooting).

[0089] Control group: The callus differentiation medium, seedling strengthening / proliferation medium and rooting medium disclosed in patents A1-A9 for other parts of tung tree (such as sterile seedling leaves, hypocotyls, petioles or stem segments) were used to replace the corresponding medium of the present invention, and the same batch of callus tissue was subsequently cultured.

[0090] All cultures were conducted under the same environmental conditions (temperature, light, etc.) to eliminate environmental interference.

[0091] (3) Comparison results and analysis: Comparative culture results showed that the control group (using the A1-A9 patented culture medium) was significantly inferior to the experimental group (using the A10 patented culture medium of this invention) in key indicators such as adventitious bud differentiation rate, seedling vigor, and rooting quality. This indicates that culture medium formulations optimized for specific parts of the tung tree (such as hypocotyls and leaves of sterile seedlings) cannot be directly applied to and effectively solve the technical problem of inducing regeneration of plants from the petioles of mature trees in the field. The responses of different organs and physiological states (mature trees and seedlings) of the tung tree to plant growth regulators differ fundamentally. This invention is simple to operate, has low cultivation costs, and provides a foundation for the development of industrialized seedling production and transgenic technology.

[0092] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for inducing callus tissue from the petioles of mature tung trees in the field and regenerating plants, characterized in that: Includes the following steps: S1. Obtain petiole explants from mature tung trees in the field, and disinfect the surface with a disinfectant to obtain sterile explants; S2. The sterile explants are inoculated into a first culture medium for culture to induce callus formation; the first culture medium contains 6-BA and auxin. S3. The callus tissue obtained in step S2 is inoculated into the second culture medium for culture to induce the differentiation of adventitious shoots; the second culture medium contains 6-BA, auxin and GA3. S4. The adventitious buds obtained in step S3 are inoculated into a third culture medium for cultivation to promote their vigorous growth; the third culture medium contains 6-BA, IBA and GA3. S5. The seedlings obtained in step S4 are inoculated into the fourth culture medium for culture to induce rooting and obtain complete regenerated plants; the fourth culture medium contains IBA.

2. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 1, characterized in that: The surface disinfection includes: first soaking the explants in 75% ethanol for 30 seconds, and then soaking the explants in 10% sodium hypochlorite solution for 5 minutes.

3. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 1, characterized in that: The first culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 3.0-6.0 mg / L and the auxin is IAA with a concentration of 0.05-0.1 mg / L.

4. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 3, characterized in that: In the first culture medium, the concentration of 6-BA is 3.0 mg / L and the concentration of IAA is 0.05 mg / L.

5. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 1, characterized in that: The second culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 3.0-5.0 mg / L, the auxin is IAA with a concentration of 0.05-0.1 mg / L, and the concentration of GA3 is 0.1-0.5 mg / L.

6. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 5, characterized in that: In the second culture medium, the concentration of 6-BA is 3.0 mg / L, the concentration of IAA is 0.05 mg / L, and the concentration of GA3 is 0.1 mg / L.

7. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 1, characterized in that: The third culture medium is based on 1 / 2 MS medium, wherein the concentration of 6-BA is 0.5-3.0 mg / L, the concentration of IBA is 0.025-0.1 mg / L, and the concentration of GA3 is 0.1-0.5 mg / L.

8. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 7, characterized in that: In the third culture medium, the concentration of 6-BA is 0.5 mg / L, the concentration of IBA is 0.025 mg / L, and the concentration of GA3 is 0.1 mg / L.

9. The method for inducing callus tissue from the petioles of mature field-grown tung trees and regenerating plants according to claim 1, characterized in that: The fourth culture medium is based on 1 / 2 MS medium, wherein the concentration of IBA is 0.1-0.5 mg / L.

10. The method for inducing callus tissue from the petioles of mature tung trees and regenerating plants according to claim 9, characterized in that: In the fourth culture medium, the concentration of IBA is 0.5 mg / L.

Images