A method for tissue culture and rapid propagation of bigflower purpleleaf willow

Abstract

The application discloses a method for tissue culture and rapid propagation of bigflower Lagerstroemia indica, and comprises the following steps: (1) selecting tender branches of bigflower Lagerstroemia indica; (2) disinfecting bigflower Lagerstroemia indica explants; (3) inducing and proliferating adventitious buds of bigflower Lagerstroemia indica; (4) rooting culture of bigflower Lagerstroemia indica; (5) acclimatization of bigflower Lagerstroemia indica tissue culture seedlings; and (6) transplanting bigflower Lagerstroemia indica. The method can realize rapid propagation of bigflower Lagerstroemia indica by optimizing the tissue culture and rapid propagation process of bigflower Lagerstroemia indica, and has high technical content, significant technical advantages and wide application prospect, solves problems, such as long cycle, low efficiency and genetic instability, in traditional propagation methods, and injects strong impetus into transformation and upgrading and high-quality development of the flower industry, and greatly meets the development demand of the tissue culture and rapid propagation industry of bigflower Lagerstroemia indica.

Description

Technical Field

[0001] This invention belongs to the field of plant propagation technology, specifically relating to a method for rapid propagation of Lagerstroemia indica via tissue culture. Background Technology

[0002] Lagerstroemia speciosa Pers., also known as the large-leaved crape myrtle, is a large tree belonging to the Lythraceae family and the Lagerstroemia genus. It is characterized by its large, brightly colored flowers, long flowering period, rapid growth, and lack of major pests and diseases, making it a major flowering tree species in tropical and subtropical regions. The tree can reach a height of 25 meters; the bark is gray and smooth; the columnar stalks are cylindrical, hairless or slightly pubescent. The leaves are leathery, oblong-elliptic or ovate-elliptic, rarely lanceolate, very large, with a blunt or short-pointed apex and a broadly cuneate to rounded base, hairless on both sides. The capsule is globose to obovate-oblong, brownish-gray, containing numerous seeds. Flowering occurs from May to July, and fruiting from October to November. Lagerstroemia speciosa prefers warm, humid environments, sunny locations but tolerates some shade, and thrives in calcareous soils. It is cultivated in Guangdong, Guangxi, and Fujian provinces. It is also distributed in Sri Lanka, India, Malaysia, Vietnam, and the Philippines. Crape myrtle is often cultivated in gardens for ornamental purposes; its wood is hard, rot-resistant, and bright red, and is often used for furniture, boats, bridges, utility poles, railway sleepers, and construction, as well as for aquatic use. Its economic value is said to be comparable to that of sapwood; its bark and leaves can be used as a laxative; its seeds have anesthetic properties; and its roots contain tannins and can be used as an astringent.

[0003] Select semi-lignified seedlings for transplanting to the field, applying sufficient base fertilizer in the planting hole before planting. Fertilize young trees monthly during their growth period, paying attention to bud removal and trunk support. After maturity, fertilize twice a year, applying a phosphorus-rich compound fertilizer during the flowering period to promote abundant flowering. Prune once after leaf fall and dormancy in winter. Remove residual flower branches, fruit branches, overly dense branches, and diseased or insect-infested branches, and apply base fertilizer (mainly organic fertilizer) around the base to promote growth and abundant flowering the following year. Pests and diseases are rare. Scale insects may damage branches; these should be controlled early by spraying pesticides such as dimethoate.

[0004] Select mother trees aged 5-20 years that are well-grown, robust, with straight, thick trunks, and free from pests and diseases. Seeds are ready for collection when the capsules turn from greenish-yellow to yellowish-red or light brown but have not yet split open. Seed collection is typically best done between October and November each year. Generally, the collected seeds can be classified and stored according to the flower color of the mother tree.

[0005] The "Technical Regulations for the Cultivation of Lagerstroemia indica" discloses that the seeds of Lagerstroemia indica are relatively small and can be sown by broadcasting. The seeds should be evenly scattered on the seedbed at a rate of 2-3 g / m². 2Before sowing, the seedbed should be thoroughly watered; seeds can be sown directly. After sowing, cover with fine sand or dried rice straw, just thick enough to cover the seeds. Sowing can be done in spring or autumn, but it is generally advisable to sow and raise seedlings in March or April. At this time, the seeds germinate in a shorter time, usually 15-20 days, but the germination rate is low.

[0006] Lagerstroemia speciosa can be propagated through layering. The best time for layering is generally from November to February of the following year. First, in a prepared nursery bed, dig trenches 20-30 cm deep with a row spacing of 40 cm. Add 2-3 cm of cow or horse manure as base fertilizer to the trenches. In conjunction with winter and spring pruning, select healthy plants of superior varieties with large, vibrant flowers and free from pests and diseases as mother plants. Take well-developed one- or two-year-old branches, requiring a diameter of 1-4 cm and no length limit. After soaking them in clean water for 2 hours, bury them obliquely along the trench, then cover with soil and tamp down the roots. Press the top down along the trench, cover with 10-15 cm of soil, and water thoroughly. Cover tightly with plastic film to increase warmth and moisture, and then cover with 2-3 layers of straw mats for frost protection. On sunny days, the straw mats can be removed during the day to increase warmth, and then covered again in the evening. With this treatment, buds will generally sprout and grow by mid-March, and tender shoots will appear above the ground in April. At this time, a small arched greenhouse can be rebuilt and covered with plastic film, removing the straw mat. If the temperature inside the greenhouse exceeds 30℃, ventilation should be provided to lower the temperature. Starting from mid-March, the film should be removed every 20 days, and soil should be mounded along the furrows, for a total of 3 to 4 times, so that the base of the seedlings becomes a 10cm high mound, promoting the production of numerous adventitious roots. At the end of May, the arched greenhouse should be removed, and the seedlings should be cut into sections according to the sprouting situation, with each sprout becoming an independent seedling. At this time, the top should be pinched to promote lateral branching, and farmyard manure, such as diluted cake fertilizer water or urea water, should be applied once. Soil should be mounded along the furrows again to promote growth. Afterward, apply fertilizer 3 to 4 more times. If flower buds appear, they should be removed promptly. Generally, the plant will become a full-crowned, lush plant in the same year. In autumn, on a rainy day, the seedlings should be dug up with soil, taking care not to damage the fine roots, and transplanted to a sunny place with sufficient water and fertilizer. After survival, water and fertilizer should be controlled to enhance its adaptability. Through comparison, under the same conditions, crape myrtle seedlings propagated by this method not only have well-developed root systems and a high survival rate after transplanting, but also show significant advantages over other propagated seedlings in terms of crown shape.

[0007] The "Technical Regulations for the Propagation of Crape Myrtle by Cuttings" discloses a method for propagating Crape Myrtle by cuttings, including the following steps: Select healthy, disease-free tender branches or semi-lignified branches, cut them into 12-15cm sections, each section retaining 3-5 buds; cut the top of the cutting flat and the bottom at a 45° angle, remove the lower leaves, retaining 0.5-1 leaves at the top; before planting, soak the base of the cuttings in a 300mg / L solution of ABT1 rooting powder for 2-3 hours. Drill holes in the substrate, with the hole diameter roughly the same as the cutting diameter and the hole depth 1 / 3 to 1 / 2 of the cutting length. Insert the cuttings into the holes, press them firmly into the substrate, and water until the substrate is thoroughly moistened. Construct small arched sheds on the seedbeds, with an arch height of 50-60cm and a width slightly wider than the seedbed surface. Cover with white plastic film to retain moisture and prevent rain. The temperature inside the small arched sheds should ideally be 25-30℃, and the relative humidity should ideally be 80%-90%. Open the arched sheds for ventilation every morning and evening, and water as needed to keep the substrate moist. This method of propagating Lagerstroemia indica by cuttings can produce high-quality cuttings, but it suffers from slow propagation speed, which cannot meet the needs of large-scale, rapid industrialization.

[0008] "Cultivation Techniques and Applications of Lagerstroemia indica" reveals that using cuttings from current year's old branches for pot cultivation is an effective method for quickly developing bonsai. Because Lagerstroemia indica branches are flexible, potted plants can be trained and shaped appropriately. Generally, this is done when spring shoot growth stops and the branches become semi-lignified, using fine hemp rope to create bonsai in various postures.

[0009] The aforementioned public reports about Lagerstroemia indica primarily focus on traditional propagation methods, such as seed propagation, layering, cuttings, and general cultivation and management strategies. However, these traditional methods have gradually revealed their inherent limitations and shortcomings in practical application and industrial development, making it difficult to meet the rapidly growing market demand for Lagerstroemia indica, a plant variety that combines both ornamental and economic value.

[0010] Firstly, regarding seed propagation, although seeds are the cornerstone of natural reproduction, they suffer from significant variability. The germination rate of Lagerstroemia indica seeds is unstable, and the offspring exhibit severe phenotypic segregation, making it difficult to guarantee the stable inheritance of superior varietal characteristics, posing a challenge to large-scale, standardized production. Furthermore, the collection, storage, and germination processes of seeds require specific environmental and technical conditions, increasing operational difficulty and cost.

[0011] Secondly, while layering can effectively preserve the superior traits of the mother plant, its propagation efficiency is extremely low, it is time-consuming, and it causes significant damage to the mother plant, which is detrimental to the sustainable utilization and protection of resources. In large-scale production, the limitations of this method are particularly evident, making it difficult to meet the demands of rapid propagation.

[0012] While cutting propagation offers faster propagation speeds compared to the previous two methods, its survival rate is significantly affected by various factors such as season, cutting quality, substrate selection, and environmental control, resulting in poor stability and requiring highly skilled technicians. Especially when facing pests, diseases, or environmental changes, cuttings often exhibit weak resistance, impacting overall production efficiency.

[0013] In conclusion, existing propagation methods for Lagerstroemia indica, whether through seed propagation, layering, or cutting, all have varying degrees of shortcomings and challenges, making it difficult to fully meet the needs of the rapidly developing industry. Therefore, strengthening the innovation and optimization of Lagerstroemia indica propagation techniques and exploring more efficient, stable, and low-cost propagation methods has become crucial for promoting the sustainable and healthy development of the Lagerstroemia indica industry. Summary of the Invention

[0014] The purpose of this invention is to provide a method for rapid propagation of Lagerstroemia indica via tissue culture, thereby optimizing the process and enabling rapid propagation of Lagerstroemia indica to meet the needs of industrial development.

[0015] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0016] A method for rapid propagation of Lagerstroemia indica via tissue culture includes the following steps:

[0017] (1) Select tender branches of Lagerstroemia indica;

[0018] (2) Disinfect the explants of Lagerstroemia speciosa;

[0019] (3) Induction and proliferation culture of adventitious buds of Lagerstroemia indica;

[0020] (4) Rooting and cultivation of Lagerstroemia speciosa;

[0021] (5) Hardening off tissue culture seedlings of Lagerstroemia indica;

[0022] (6) Transplanting of Lagerstroemia speciosa.

[0023] Preferably, the crape myrtle shoots mentioned in step (1) are crape myrtle shoots that have just sprouted and are 10-15cm long.

[0024] Preferably, step (2) involves disinfecting the explants of Lagerstroemia indica as follows:

[0025] The explants were selected from tender shoots that had just sprouted 10-15cm. The tender shoots were cut from the mother plant with surgical scissors and the leaves were removed. The tender shoots were then soaked in an 800-fold carbendazim solution for 30-60 minutes. After soaking, they were rinsed with tap water for 1-2 hours.

[0026] Place the explants in a clean bench and irradiate with ultraviolet light for 20-30 minutes, disinfect with 75% alcohol for 30-60 seconds, rinse with sterile water 3-5 times, then disinfect the explants with 0.1% mercuric chloride for 15-45 minutes, and rinse with sterile water 3-5 times.

[0027] After disinfection, the injured tissue at both ends of the explant was cut off with surgical scissors, and the explant was inoculated into MS medium. The explant was then placed in the dark for 10 days and cultured under light for 12 hours / day. The culture temperature was 25~28℃ and the light intensity was 2000~3000lx.

[0028] More preferably, the disinfection combination for the explants of Lagerstroemia speciosa is: 60 min of carbendazim disinfection + 20-30 min of ultraviolet irradiation + 45 s of alcohol disinfection + 15 min of 0.1% mercuric chloride disinfection + 11-15 cm of the lower part of the tender branch.

[0029] Preferably, the induction and proliferation culture of adventitious buds of Lagerstroemia indica in step (3) is as follows:

[0030] Thirty days after explant inoculation, adventitious buds were cut from uncontaminated plants using surgical scissors. The adventitious buds were then transferred to a proliferation medium and cultured in a sterile culture room for 25 days to obtain sterile seedlings. The temperature was 25-28℃, the light intensity was 2000-3000 lx, and the photoperiod was 12h / d.

[0031] Preferably, the proliferation medium comprises MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L + agar 6 g / L + white sugar 30 g / L.

[0032] Preferably, the rooting culture of Lagerstroemia indica in step (4) is as follows:

[0033] Sterile seedlings were transferred to the rooting medium of Lagerstroemia speciosa for 20 days to obtain complete plants. The rooting medium of Lagerstroemia speciosa was 1 / 2 MS + NAA 0.5 mg / L + activated carbon 0.1 g / L.

[0034] Preferably, the hardening-off of the crape myrtle tissue culture seedlings in step (5) is as follows:

[0035] After rooting, the tissue culture seedlings, still in their bottles (without opening the caps), are moved to a greenhouse with 60%~70% shading and a temperature of 25±2℃ for hardening off.

[0036] Preferably, the transplanting of Lagerstroemia indica in step (6) is as follows:

[0037] Before transplanting, spray the substrate thoroughly with a 0.1% potassium permanganate solution, leave it for 2-3 days, then spray thoroughly with tap water to rinse off the potassium permanganate solution. useRemove the crape myrtle tissue culture seedlings from the tissue culture bottle using tweezers, wash off the culture medium from the roots, soak them in an 800-fold carbendazim solution for 8-10 minutes, and immediately transplant them into the substrate. The transplanting depth should be such that the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to settle the roots, then spray with an 800-fold carbendazim solution. Cover the soil surface with a thin film to retain moisture, and then cover with a shade net for shading. After 15 days, remove the film and manage the seedlings as usual.

[0038] More preferably, the matrix comprises peat moss, perlite, and vermiculite in a mass ratio of 5:1:1.

[0039] Compared with the prior art, the present invention has the following technical advantages:

[0040] (1) In exploring the optimization path of rapid propagation technology of Lagerstroemia indica tissue culture, this invention systematically studied the combined effects of four key parameters: disinfection time with carbendazim (A), disinfection time with 75% alcohol (B), disinfection time with 0.1% mercuric chloride (C), and location (D). Through a carefully designed set of 9 experimental configurations, this invention optimized and screened the optimal disinfection strategy: namely, the 11-15cm section of the lower part of the tender branches, disinfected with carbendazim for 60min, followed by disinfection with 75% alcohol for 45s, and then disinfected with 0.1% mercuric chloride for 15min. This combination significantly reduced the contamination rate, achieving a low contamination level of only 33.3%, laying a solid foundation for the aseptic culture of Lagerstroemia indica explants.

[0041] (2) To improve the proliferation efficiency of Lagerstroemia speciosa, this invention explored the synergistic effect of changes in the concentrations of basal culture media (MS and WPM) and growth regulators (6-BA, NAA). By constructing an experimental matrix covering 24 unique culture media, this invention precisely identified the optimal proliferation formulation: MS basal culture medium supplemented with 0.5 mg / L 6-BA, 0.1 mg / L NAA, 6 g / L agar, and 30 g / L sucrose. This formulation not only promoted significant proliferation but also achieved a proliferation coefficient as high as 4.67, marking a major advancement in the in vitro culture technology of Lagerstroemia speciosa.

[0042] (3) Further focusing on the optimization of rooting in Lagerstroemia speciosa, this invention compared the rooting performance under different basal media (MS and 1 / 2MS at half concentration) and NAA concentration gradients. Through the careful planning of 12 media configurations, this invention precisely identified the optimal solution for promoting rooting in Lagerstroemia speciosa: using 1 / 2MS as the basal medium, combined with 0.5 mg / L NAA and 0.1 g / L activated carbon. This combination not only achieved a 100% rooting rate but also obtained a root effect index of 1.32, demonstrating its excellent efficacy in inducing rooting in Lagerstroemia speciosa and providing reliable technical support for large-scale propagation.

[0043] (4) Significantly Improved Economic Benefits: The three key technological optimizations of this invention—disinfection strategy, propagation formula, and rooting optimization scheme—together constitute a highly efficient system for the rapid propagation of Lagerstroemia indica. This system not only significantly shortens the propagation cycle and improves propagation efficiency, but also directly reduces production costs by lowering the contamination rate, increasing the propagation coefficient, and improving the rooting rate. Specifically, a low contamination rate reduces the loss of culture materials due to contamination, while a high propagation coefficient and rooting rate mean that more healthy plants can be obtained in the same amount of time, thereby greatly improving production efficiency and economic benefits. For the flower industry, this is undoubtedly a major technological breakthrough, providing the market with a more stable and abundant supply of high-quality seedlings and promoting the healthy development of the industry.

[0044] (5) A Double Leap in Scientific Research and Application Value: The success of this invention is not only reflected in the significant technological advancements, but also in the double leap in scientific research and application value. From a scientific research perspective, this study deeply analyzes the complex relationships between various factors during disinfection, proliferation, and rooting processes, providing a new perspective and experimental basis for theoretical research in the field of plant tissue culture. Simultaneously, by constructing a precise experimental design and data analysis model, it provides a reference method and approach for tissue culture technology research on other plant species. From an application perspective, the rapid propagation technology of this invention is not only suitable for the large-scale production of Lagerstroemia speciosa, but also has broad potential for promotion, applicable to the rapid propagation of various ornamental plants and economic crops, injecting new vitality into the development of modern agriculture. Furthermore, this technology is expected to play an important role in ecological restoration, urban greening, and other fields, promoting the progress of green ecological construction. Attached Figure Description

[0045] Figure 1 Diagram showing the bud induction of Lagerstroemia speciosa;

[0046] Figure 2 Image of sterile seedlings propagated from Lagerstroemia indica;

[0047] Figure 3 A complete picture of a Lagerstroemia speciosa plant after it has taken root;

[0048] Figure 4 This is a picture of transplanted seedlings of Lagerstroemia speciosa. Detailed Implementation

[0049] To facilitate a better understanding of the present invention, the following embodiments are provided. These embodiments are within the scope of protection of the present invention, but do not limit the scope of protection of the present invention.

[0050] 1.1 Test Materials

[0051] For the Lagerstroemia speciosa, newly sprouted shoots of 10-15cm were selected as experimental materials.

[0052] 1.2 Experimental Design and Methods

[0053] 1.2.1 Explant disinfection

[0054] Disinfection time with carbendazim (A), disinfection time with 75% alcohol (B), disinfection time with 0.1% mercuric chloride (C), and site of infection (D) were used as test factors. Each factor had three levels, and the results were calculated according to L9(3). 4 The orthogonal experimental design table (Table 1) was used to conduct the experiment and study the optimal combination of various factor levels for disinfection of Lagerstroemia speciosa explants.

[0055]

[0056] The explants are selected from newly sprouted shoots, 10-15cm long, with the optimal time being 13:00-14:00. Using surgical scissors, cut 10-15cm shoots from the mother plant, then divide them into three sections: upper 1-5cm, middle 6-10cm, and lower 11-15cm (containing 1-2 dormant buds). Remove the leaves and soak the shoots in an 800-fold dilution of carbendazim solution for 0 min, 30 min, and 60 min, respectively. After soaking, rinse with tap water for 1-2 hours.

[0057] The explants were placed in a clean bench and irradiated with ultraviolet light for 30 minutes, then disinfected with 75% alcohol for 30, 45, and 60 seconds, rinsed with sterile water 3-5 times, and then disinfected with 0.1% mercuric chloride for 15, 30, and 45 minutes, and rinsed with sterile water 3-5 times.

[0058] After disinfection, the injured tissue of the explants was removed using surgical scissors, and the explants were inoculated onto MS medium. Each treatment consisted of 30 bottles, with one explant per bottle, and three replicates were performed, for a total of 90 bottles. The explants were cultured in the dark for 10 days, followed by light culture (12 h / d) at a temperature of 25–28℃ and a light intensity of 2000–3000 lx. Contamination and induced shoot development were assessed after 30 days.

[0059] 1.2.2 Induction and Proliferation Culture of Adventitious Buds of Lagerstroemia indica (6-BA+NAA)

[0060] Twenty-four different culture media were designed for the induction and proliferation of adventitious buds of Lagerstroemia speciosa (see Table 2 for details). When preparing the culture media, 6 g / L of agar and 30 g / L of white sugar were added.

[0061] Thirty days after explant inoculation, adventitious buds were surgically removed from uncontaminated plants and transferred to the corresponding culture medium. Three plants were transferred per bottle, with 30 bottles per treatment, and the process was repeated three times. The plants were then cultured in a sterile environment at 25–28°C, with a light intensity of 2000–3000 lx and a photoperiod of 12 h / d. Plant growth and proliferation were observed after 25 days.

[0062]

[0063] 1.2.3 Rooting Culture of Lagerstroemia indica

[0064] The rooting medium design for Lagerstroemia indica (Table 3) was used. To promote better and stronger growth, activated carbon (0.1 g / L) was added, as activated carbon has a certain adsorption effect on phenolic substances, thus promoting root growth. After 25 days of propagation culture, sterile seedlings were transferred to the rooting medium, with 3 seedlings per bottle, 30 bottles per treatment, and 3 replicates. Rooting status was assessed after 20 days.

[0065]

[0066] 1.2.4 Transplanting of Lagerstroemia indica tissue culture seedlings

[0067] 1.2.4.1 Seedling hardening

[0068] After 20 days of rooting culture, the rooted tissue culture seedlings were transferred (without opening the cap) to a greenhouse with 60%~70% shading and a temperature of 25±2℃.

[0069] 1.2.4.2 Transplanting

[0070] Before transplanting, mix peat moss, perlite, and vermiculite in a 5:1:1 ratio to obtain a substrate. Spray the substrate thoroughly with a 0.1% potassium permanganate solution and let it sit for 2-3 days. Then, spray thoroughly with tap water to rinse off the potassium permanganate solution. Carefully remove the crape myrtle tissue culture seedlings from the culture bottle using tweezers. Wash the culture medium off the roots and soak them in an 800x carbendazim solution for 8-10 minutes. Immediately transplant them into the substrate, ensuring the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to settle the roots, then spray with an 800x carbendazim solution. Cover the soil surface with a thin film to retain moisture and then cover with a shade net for shading. After 15 days, remove the film and manage the seedlings as usual.

[0071] 1.3 Data Statistics

[0072] After inoculation, observe the contamination status daily for 30 consecutive days, count the contamination number, and calculate the contamination rate. After 30 days of transplanting, observe the plant proliferation and calculate the proliferation coefficient. Once the crape myrtle plants are growing vigorously, transfer them to rooting medium, observe the rooting status daily, record and photograph the rooting status, and calculate the rooting rate.

[0073] Contamination rate (%) = Number of contaminated items / Number of inoculated items × 100

[0074] Proliferation coefficient = Number of proliferators / Number of inoculated individuals

[0075] Rooting rate (%) = Number of roots / Number of inoculated plants × 100

[0076] Root system effectiveness index = (average root length × average number of roots × rooting rate) / number of plants surveyed

[0077] 1.4 Results and Analysis

[0078] 1.4.1 Effects of different treatments on sterilization of Lagerstroemia indica stem segments

[0079] As shown in Table 4, treatment 8 had the lowest contamination rate (33.3%), followed by treatment 9, while treatments 1 and 2 had the highest contamination rates. Therefore, the best combination for disinfecting the explants of Lagerstroemia indica in Table 4 is: carbendazim disinfection for 60 min + 75% alcohol disinfection for 45 s + 0.1% mercuric chloride disinfection for 15 min + 11-15 cm section of the lower part of the tender branches.

[0080]

[0081] 1.4.2 Effects of different treatments on the subgeneration and propagation of Lagerstroemia indica

[0082] The concentrations of cytokinin 6-BA and auxin NAA significantly affected the subculture proliferation of Lagerstroemia indica. Table 5 shows that when the culture medium was MS, the highest proliferation coefficient (4.67) was observed with increased cytokinin 6-BA concentration (0.5 mg / L) and auxin NAA concentration (0.1 mg / L), characterized by excellent growth, robust plants, and dark green leaves (treatment 6). Treatment 7 was the second highest, with a proliferation coefficient of 2.73, also showing good growth, robust plants, and green leaves. The lowest proliferation coefficients were observed in treatments 1, 11, and 12 (0), indicating generally weak growth, small plants, and pale green leaves. The proliferation coefficient decreased with increasing NAA concentration, indicating that high NAA concentrations had an inhibitory effect on the subculture proliferation of Lagerstroemia indica. When the culture medium was WPM, the highest proliferation coefficient was observed in treatment 18 (2.20), followed by treatment 16, with treatments 13 and 24 showing the worst results. Based on the above analysis, the optimal culture medium for the subculture and proliferation of Lagerstroemia indica in Table 5 is treatment 6 (MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L).

[0083]

[0084] 1.4.3 Effects of different treatments on the rooting of Lagerstroemia indica

[0085] The effects of different treatments on the rooting of Lagerstroemia indica (Table 6) were analyzed 20 days after inoculation using rooting media treated from 1 to 12. The results showed significant differences in rooting rates among the treatments. Treatment 7 had the lowest rooting rate (100%) compared to the other groups. The highest average number of roots was in treatment 10, followed by treatment 9, and the lowest was treatment 6. The longest average root length was in treatment 6, followed by treatment 7, and the shortest was treatment 12. The highest root system effect index was in treatment 10, followed by treatment 9, and the lowest was treatment 12. In summary, the effect of different NAA concentrations on rooting of Lagerstroemia indica was observed. With the same basic culture medium, the concentration of NAA increased with increasing concentration, reaching a promoting effect at 0.5 mg / L, resulting in the highest average number of roots and the highest root system effect index. However, when NAA exceeded 0.5 mg / L, it inhibited rooting. Therefore, treatment 10 (1 / 2 MS + 0.5 mg / L NAA) showed the best rooting effect for Lagerstroemia indica.

[0086]

[0087] Note: Root system effectiveness index = (average root length × average number of roots × rooting rate) / number of plants surveyed

[0088] 1.5 Conclusion

[0089] (1) The rapid propagation technology of Lagerstroemia indica tissue culture was studied using the disinfection time of carbendazim (A), the disinfection time of 75% alcohol (B), the disinfection time of 0.1% mercuric chloride (C) and the location (D) as reference factors. Nine combinations were set up for research. The results showed that the best combination for disinfecting explants of Lagerstroemia indica was 60 min of carbendazim disinfection + 45 s of 75% alcohol disinfection + 15 min of 0.1% mercuric chloride disinfection + 11-15 cm of the lower part of the tender branch.

[0090] (2) By using different basal culture media (MS and WPM) and different mass concentrations of 6-BA and NAA, 24 kinds of culture media were set up for experimental screening. It can be concluded that the culture medium with better proliferation of Lagerstroemia speciosa is MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L + agar 6 g / L + white sugar 30 g / L.

[0091] (3) By using different basal media (MS+1 / 2MS) and different concentrations of NAA, 12 kinds of media were set up to culture the rooting of Lagerstroemia speciosa. The results showed that the best medium for the rooting of Lagerstroemia speciosa was 1 / 2MS+NAA0.5 mg / L+activated carbon0.1 g / L.

[0092] Using the optimized process parameters explored above, the following embodiments were obtained.

[0093] Example 1

[0094] A method for rapid propagation of Lagerstroemia indica via tissue culture includes the following steps:

[0095] (1) Select tender branches of large-flowered crape myrtle that have just sprouted 15cm;

[0096] (2) Disinfect the explants of Lagerstroemia speciosa as follows:

[0097] The explants were selected from newly sprouted 15cm shoots at 13:00 in the afternoon. Using surgical scissors, 15cm shoots were cut from the mother plant, selecting the 11-15cm section (1-2 dormant buds) of the lower part of the shoot and removing the leaves. The shoots were then soaked in an 800-fold dilution of carbendazim solution for 60 minutes. After soaking, they were rinsed with tap water for 1 hour.

[0098] The explants were placed in a clean bench and irradiated with ultraviolet light for 22 minutes, then disinfected with 75% alcohol for 45 seconds, rinsed with sterile water 4 times, and then disinfected with 0.1% mercuric chloride for 15 minutes, and rinsed with sterile water 4 times.

[0099] After disinfection, the injured tissue at both ends of the explant was cut off with surgical scissors, and the explant was inoculated into MS medium. The explant was placed in the dark for 10 days and then cultured under light (12h / d) to obtain induced buds of Lagerstroemia speciosa. The culture temperature was 25~28℃ and the light intensity was 2000~3000lx.

[0100] (3) Induction and proliferation culture of adventitious buds of Lagerstroemia indica

[0101] When preparing the proliferation medium for Lagerstroemia speciosa seedlings (MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L), add 6 g / L agar and 30 g / L white sugar.

[0102] Thirty days after explant inoculation, adventitious buds were cut from uncontaminated plants using surgical scissors. The adventitious buds were then transferred to a proliferation medium and cultured in a sterile culture room for 25 days to obtain sterile seedlings of Lagerstroemia indica. The temperature was 25-28℃, the light intensity was 2000-3000 lx, and the light duration was 12h / d.

[0103] (4) Rooting culture of Lagerstroemia indica

[0104] Sterile seedlings were transferred to the rooting medium of Lagerstroemia speciosa for 20 days to obtain complete plants. The rooting medium for Lagerstroemia speciosa was 1 / 2 MS + NAA 0.5 mg / L. In order to make the Lagerstroemia speciosa grow better and stronger, activated carbon 0.1 g / L was added to the rooting medium. Activated carbon has a certain adsorption effect on phenolic substances and promotes root growth.

[0105] (5) Hardening off tissue culture seedlings of Lagerstroemia indica

[0106] After rooting, the tissue culture seedlings, still in their bottles (without opening the caps), were moved to a greenhouse with 62% shading and a temperature of 25±2℃ for hardening off.

[0107] (6) Transplanting

[0108] Before transplanting, thoroughly spray the substrate (comprising peat moss, perlite, and vermiculite in a 5:1:1 mass ratio) with a 0.1% potassium permanganate solution. After 2 days, thoroughly spray with tap water to rinse off the potassium permanganate solution. Carefully remove the crape myrtle seedlings from the tissue culture bottle using tweezers. Wash the culture medium off the roots of the seedlings and soak them in an 800-fold carbendazim solution for 8 minutes. Immediately transplant them into the substrate, ensuring the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to settle the roots, then spray with an 800-fold carbendazim solution. Cover the soil surface with a thin film to retain moisture and then cover with a shade net for shading. After 15 days, transplanted crape myrtle seedlings are obtained. Remove the film and manage the seedlings as usual.

[0109] Example 2

[0110] A method for rapid propagation of Lagerstroemia indica via tissue culture includes the following steps:

[0111] (1) Select tender branches of large-flowered crape myrtle that have just sprouted 15cm;

[0112] (2) Disinfect the explants of Lagerstroemia speciosa as follows:

[0113] The explants were selected from newly sprouted 15cm crape myrtle shoots, and the sampling time was 13:30 in the afternoon. Using surgical scissors, 15cm shoots were cut from the mother plant, selecting the 11-15cm section of the lower part of the shoot (1-2 dormant buds), removing the leaves, and then soaking the shoots in an 800-fold diluted carbendazim solution for 60 minutes. After soaking, the shoots were rinsed with tap water for 1 hour.

[0114] The explants were placed in a clean bench and irradiated with ultraviolet light for 20 minutes, then disinfected with 75% alcohol for 45 seconds, rinsed three times with sterile water, and then disinfected with 0.1% mercuric chloride for 15 minutes, and rinsed three times with sterile water.

[0115] After disinfection, the injured tissue at both ends of the explant was cut off with surgical scissors, and the explant was inoculated into MS medium. The explant was placed in the dark for 10 days and then cultured under light (12h / d) to obtain induced buds of Lagerstroemia speciosa. The culture temperature was 25~28℃ and the light intensity was 2200~3000lx.

[0116] (3) Induction and proliferation culture of adventitious buds of Lagerstroemia indica

[0117] When preparing the proliferation medium for Lagerstroemia speciosa seedlings (MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L), add 6 g / L agar and 30 g / L white sugar.

[0118] Thirty days after explant inoculation, adventitious buds were cut from uncontaminated plants using surgical scissors. The adventitious buds were then transferred to a proliferation medium and cultured in a sterile culture room for 25 days to obtain sterile seedlings of Lagerstroemia indica. The temperature was 25-28℃, the light intensity was 2000-3000 lx, and the light duration was 12h / d.

[0119] (4) Rooting culture of Lagerstroemia indica

[0120] Sterile seedlings were transferred to rooting medium for 20 days to obtain complete crape myrtle plants. The rooting medium for crape myrtle was 1 / 2 MS + 0.5 mg / L NAA. To promote better and stronger growth of crape myrtle, 0.1 g / L of activated carbon was added to the rooting medium. Activated carbon has a certain adsorption effect on phenolic substances, which promotes root growth.

[0121] (5) Hardening off tissue culture seedlings of Lagerstroemia indica

[0122] After rooting, the tissue culture seedlings, still in their bottles (without opening the caps), were moved to a greenhouse with 62% shading and a temperature of 25±2℃ for hardening off.

[0123] (6) Transplanting

[0124] Before transplanting, thoroughly spray the substrate (comprising peat moss, perlite, and vermiculite in a 5:1:1 mass ratio) with a 0.1% potassium permanganate solution. After 2 days, thoroughly spray with tap water to rinse off the potassium permanganate solution. Carefully remove the Lagerstroemia indica tissue culture seedlings from the tissue culture bottle using tweezers. Wash the culture medium off the roots of the seedlings and soak them in an 800-fold carbendazim solution for 9 minutes. Immediately transplant them into the substrate, ensuring the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to settle the roots, then spray with an 800-fold carbendazim solution. Cover the soil surface with a thin film to retain moisture and then cover with a shade net for shading. After 15 days, transplanted Lagerstroemia indica seedlings are obtained. Remove the film and manage the seedlings as usual.

[0125] Example 3

[0126] A method for rapid propagation of Lagerstroemia indica via tissue culture includes the following steps:

[0127] (1) Select tender branches of large-flowered crape myrtle that have just sprouted 15cm;

[0128] (2) Disinfect the explants of Lagerstroemia speciosa as follows:

[0129] The explants were selected from newly sprouted 15cm shoots at 13:00 in the afternoon. Using surgical scissors, 15cm shoots were cut from the mother plant, selecting the 11-15cm section (1-2 dormant buds) of the lower part of the shoot and removing the leaves. The shoots were then soaked in an 800-fold dilution of carbendazim solution for 60 minutes. After soaking, they were rinsed with tap water for 1.5 hours.

[0130] The explants were placed in a clean bench and irradiated with ultraviolet light for 25 minutes, then disinfected with 75% alcohol for 45 seconds, rinsed with sterile water 5 times, and then disinfected with 0.1% mercuric chloride for 15 minutes, and rinsed with sterile water 5 times.

[0131] After disinfection, the injured tissue at both ends of the explant was cut off with surgical scissors, and the explant was inoculated into MS medium. The explant was placed in the dark for 10 days and then cultured under light (12h / d) to obtain induced buds of Lagerstroemia speciosa. The culture temperature was 25~27℃ and the light intensity was 2000~2800lx.

[0132] (3) Induction and proliferation culture of adventitious buds of Lagerstroemia indica

[0133] When preparing the proliferation medium for Lagerstroemia speciosa seedlings (MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L), add 6 g / L agar and 30 g / L white sugar.

[0134] Thirty days after explant inoculation, adventitious buds were cut from uncontaminated plants using surgical scissors. The adventitious buds were then transferred to a proliferation medium and cultured in a sterile culture room for 25 days to obtain sterile seedlings of Lagerstroemia indica. The temperature was 25-28℃, the light intensity was 2000-3000 lx, and the light duration was 12h / d.

[0135] (4) Rooting culture of Lagerstroemia indica

[0136] Sterile seedlings were transferred to rooting medium for 20 days to obtain complete crape myrtle plants. The rooting medium for crape myrtle was 1 / 2 MS + 0.5 mg / L NAA. To promote better and stronger growth of crape myrtle, 0.1 g / L of activated carbon was added to the rooting medium. Activated carbon has a certain adsorption effect on phenolic substances, which promotes root growth.

[0137] (5) Hardening off tissue culture seedlings of Lagerstroemia indica

[0138] After rooting, the tissue culture seedlings, still in their bottles (without opening the caps), were moved to a greenhouse with 62% shading and a temperature of 25±2℃ for hardening off.

[0139] (6) Transplanting

[0140] Before transplanting, thoroughly spray the substrate (comprising peat moss, perlite, and vermiculite in a 5:1:1 mass ratio) with a 0.1% potassium permanganate solution. After 2 days, thoroughly spray with tap water to rinse off the potassium permanganate solution. Carefully remove the Lagerstroemia indica tissue culture seedlings from the tissue culture bottle using tweezers. Wash the culture medium off the roots of the seedlings and soak them in an 800-fold carbendazim solution for 10 minutes. Immediately transplant them into the substrate, ensuring the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to settle the roots, then spray with an 800-fold carbendazim solution. Cover the soil surface with a thin film to retain moisture and then cover with a shade net for shading. After 15 days, transplanted Lagerstroemia indica seedlings are obtained. Remove the film and manage the seedlings as usual.

[0141] Example 4

[0142] A method for rapid propagation of Lagerstroemia indica via tissue culture includes the following steps:

[0143] (1) Select tender branches of large-flowered crape myrtle that have just sprouted 15cm;

[0144] (2) Disinfect the explants of Lagerstroemia speciosa as follows:

[0145] The explants were selected from newly sprouted 15cm shoots at 2:00 PM. Using surgical scissors, 15cm shoots were cut from the mother plant, selecting the 11-15cm section (1-2 dormant buds) of the lower part of the shoot and removing the leaves. The shoots were then soaked in an 800-fold dilution of carbendazim solution for 60 minutes. After soaking, they were rinsed with tap water for 2 hours.

[0146] The explants were placed in a clean bench and irradiated with ultraviolet light for 30 minutes, then disinfected with 75% alcohol for 45 seconds, rinsed with sterile water 4 times, and then disinfected with 0.1% mercuric chloride for 15 minutes and rinsed with sterile water 3 times.

[0147] After disinfection, the injured tissue at both ends of the explant was removed using surgical scissors. The explants were then inoculated onto MS medium and cultured in the dark for 10 days, followed by light culture (12 h / d) to obtain induced buds of Lagerstroemia speciosa (see...). Figure 1 The cultivation temperature is 26~28℃, and the light intensity is 2100~2900lx.

[0148] (3) Induction and proliferation culture of adventitious buds of Lagerstroemia indica

[0149] When preparing the proliferation medium for Lagerstroemia speciosa seedlings (MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L), add 6 g / L agar and 30 g / L white sugar.

[0150] Thirty days after explant inoculation, adventitious buds were surgically removed from uncontaminated plants and transferred to proliferation medium. These buds were then cultured in a sterile culture room for 25 days to obtain sterile propagated seedlings of Lagerstroemia indica (see...). Figure 2 Temperature 25~28℃, light intensity 2000~3000lx, light duration 12h / d.

[0151] (4) Rooting culture of Lagerstroemia indica

[0152] Sterile seedlings were transferred to rooting medium for 20 days to obtain complete crape myrtle plants (see...). Figure 3 The rooting medium for Lagerstroemia indica was 1 / 2 MS + NAA 0.5 mg / L. To promote better and stronger growth of Lagerstroemia indica, activated carbon 0.1 g / L was added to the rooting medium. Activated carbon has a certain adsorption effect on phenolic substances, which promotes root growth.

[0153] (5) Hardening off tissue culture seedlings of Lagerstroemia indica

[0154] After rooting, the tissue culture seedlings, still in their bottles (without opening the caps), were moved to a greenhouse with 62% shading and a temperature of 25±2℃ for hardening off.

[0155] (6) Transplanting

[0156] Before transplanting, thoroughly spray the substrate (comprising peat moss, perlite, and vermiculite in a 5:1:1 mass ratio) with a 0.1% potassium permanganate solution. After 2 days, thoroughly spray with tap water to rinse off the potassium permanganate solution. Carefully remove the crape myrtle seedlings from the tissue culture bottle using tweezers. Wash the culture medium off the roots of the seedlings and soak them in an 800-fold carbendazim solution for 9 minutes. Immediately transplant them into the substrate, ensuring the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to settle the roots, then spray with an 800-fold carbendazim solution. Cover the soil surface with a thin film to retain moisture and then cover with a shade net for shading. After 15 days, transplanted crape myrtle seedlings are obtained (see...). Figure 4 Then uncover the film and manage the seedlings as usual.

[0157] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapid propagation of Lagerstroemia indica via tissue culture, characterized in that, Includes the following steps: (1) Select tender branches of Lagerstroemia indica; (2) Disinfect the explants of Lagerstroemia speciosa; (3) Induction and proliferation culture of adventitious buds of Lagerstroemia indica; (4) Rooting and cultivation of Lagerstroemia speciosa; (5) Hardening off tissue culture seedlings of Lagerstroemia indica; (6) Transplanting of Lagerstroemia speciosa; The crape myrtle shoots mentioned in step (1) are crape myrtle shoots that have just sprouted and are 10-15cm long. Step (2) Disinfect the explants of Lagerstroemia speciosa as follows: The explants were selected from tender shoots that had just sprouted 10-15cm. The tender shoots were cut from the mother plant with surgical scissors and the leaves were removed. The tender shoots were then soaked in an 800-fold carbendazim solution for 30-60 minutes. After soaking, they were rinsed with tap water for 1-2 hours. Place the explants in a clean bench and irradiate with ultraviolet light for 20-30 minutes, disinfect with 75% alcohol for 30-60 seconds, rinse with sterile water 3-5 times, then disinfect the explants with 0.1% mercuric chloride for 15-45 minutes, and rinse with sterile water 3-5 times. After disinfection, the injured tissue at both ends of the explant was cut off with surgical scissors, and the explant was inoculated into MS medium. The explant was placed in the dark for 10 days and then cultured under light for 12 hours / day. The culture temperature was 25~28℃ and the light intensity was 2000~3000lx. The induction and proliferation culture of adventitious buds of Lagerstroemia indica in step (3) is as follows: Thirty days after explant inoculation, adventitious buds were cut from uncontaminated plants using surgical scissors. The adventitious buds were then transferred to a proliferation medium and cultured in a sterile culture room for 25 days to obtain sterile seedlings. The temperature was 25-28℃, the light intensity was 2000-3000 lx, and the photoperiod was 12h / d. The proliferation medium was MS + 6-BA 0.5 mg / L + NNA 0.1 mg / L + agar 6 g / L + sucrose 30 g / L; The rooting culture of Lagerstroemia indica described in step (4) is as follows: Sterile seedlings were transferred to the rooting medium of Lagerstroemia speciosa for 20 days to obtain complete plants. The rooting medium of Lagerstroemia speciosa was 1 / 2 MS + NAA 0.5 mg / L + activated carbon 0.1 g / L.

2. The method for rapid propagation of Lagerstroemia indica via tissue culture according to claim 1, characterized in that, The disinfection combination for explants of Lagerstroemia speciosa is: 60 min of carbendazim disinfection + 20-30 min of ultraviolet irradiation + 45 s of alcohol disinfection + 15 min of 0.1% mercuric chloride disinfection + 11-15 cm of the lower part of the tender branch.

3. The method for rapid propagation of Lagerstroemia indica via tissue culture according to claim 1, characterized in that, The hardening-off of the crape myrtle tissue culture seedlings in step (5) is as follows: After rooting, the tissue culture seedlings, still in their bottles with the caps on, are moved to a greenhouse with 60%~70% shading and a temperature of 25±2℃ for hardening off.

4. The method for rapid propagation of Lagerstroemia indica via tissue culture according to claim 1, characterized in that, The transplanting of the large-flowered crape myrtle in step (6) is as follows: Before transplanting, spray the substrate thoroughly with a 0.1% potassium permanganate solution, leave it for 2-3 days, then spray thoroughly with tap water to rinse off the potassium permanganate solution. use Use tweezers to remove the crape myrtle tissue culture seedlings from the tissue culture bottle, wash off the culture medium on the roots of the seedlings, soak them in an 800-fold carbendazim solution for 8-10 minutes, and immediately transplant them into the substrate. The transplanting depth should be such that the soil just covers the roots. Compact the substrate to stabilize the seedlings. After transplanting, water thoroughly to help the roots establish, then spray with an 800-fold dilution of carbendazim solution. Cover the soil surface with a thin film to retain moisture, and then cover with a shade net for shading. After 15 days, remove the film and manage the seedlings as usual.

5. The method for rapid propagation of Lagerstroemia indica via tissue culture according to claim 4, characterized in that, The matrix consists of peat moss, perlite, and vermiculite in a mass ratio of 5:1:1.

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