A tissue culture method using liquid medium

Abstract

The application discloses a tissue culture method using maple zygotic embryos as explants and relates to the technical field of plant tissue culture.The method comprises the following steps: S1, cutting the zygotic embryos of immature maple fruits and inoculating the zygotic embryos onto a callus induction culture medium to induce callus; S2, transferring the callus induced in S1 to a bud induction culture medium to induce adventitious buds; and S3, inoculating the adventitious buds formed in step S2 into a strong seedling rooting culture medium to induce rooting, directly forming regenerated seedlings and carrying out transplanting culture.Compared with the culture methods using other maple tissues as explants, the method is more convenient, has higher genotype universality and is more efficient in propagation.

Description

A tissue culture method using Liquidambar formosana zygote embryos as explants Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a tissue culture method using Liquidambar formosana zygote embryos as explants. Background Technology

[0002] Chinese sweetgum (Liquidambar formosana) is a multifunctional tree species unique to my country, integrating ecological value, ornamental aesthetics, and medicinal resources. Its upright and graceful shape, along with its autumn foliage that changes color according to environmental conditions, creates a unique seasonal landscape. However, the expression of sweetgum leaf color is influenced by environmental factors such as temperature, light intensity, and soil nutrients. Wild populations exhibit rich genetic diversity, making it difficult to guarantee the stability of key ornamental traits such as leaf color and tree shape. Traditional breeding methods are insufficient for targeted selection to produce varieties with uniform traits and outstanding ornamental value. Furthermore, sweetgum resin is hailed as a "holy medicine for promoting blood circulation" in traditional Chinese medicine, possessing detoxifying, tissue-regenerating, blood-stasis-dispersing, and pain-relieving effects. Modern research further reveals that its leaves and bark are rich in terpenoids and flavonoid derivatives, showing great potential in anti-inflammatory, antioxidant, and anti-tumor fields. Therefore, achieving efficient breeding and targeted improvement of Chinese sweetgum is of strategic significance for promoting its application in ecological restoration, landscape industry, and medicinal development.

[0003] Traditional asexual reproduction techniques, such as cuttings and grafting, can partially meet the demand for seedlings, but their limitations severely restrict industrialization. Cuttings are susceptible to rooting fluctuations due to substrate humidity and temperature, resulting in a survival rate of less than 70%, making large-scale propagation difficult. Grafting relies on specific rootstock matching and seasonal operations, with a short window of opportunity. Long-term asexual reproduction can also easily lead to genotypic degeneration, failing to overcome the limitations of the mother plant's genetic background, making it difficult to stably inherit desirable traits. While tissue culture technology provides a new approach for the propagation of Liquidambar formosana in China, the existing system still faces significant bottlenecks. For example, Zhuo Renying (2005) induced adventitious buds using zygotic embryos from seedlings through a two-step method, but the induction cycle was long (adventitious bud induction required 1-2 months) and the efficiency was low (less than 40%). Xu Lin (2008) used leaves from tissue-cultured seedlings propagated from superior tree stem segments as explants and achieved regeneration of the direct organogenesis pathway by optimizing the hormone ratio (TDZ and NAA), but this method had the disadvantages of high sterilization difficulty of superior tree stem segments, cumbersome operation, low seedling vigor coefficient (3.11), and limitation of mother tree genotype, and has not yet achieved a technological breakthrough. Yang Lanfang et al. (2016) used leaves from tissue-cultured seedlings propagated from superior tree stem segments as explants and obtained a patent (CN103975856B) through the embryoid regeneration pathway, but the stability of its system and the universality of its genotype still need to be verified.

[0004] Against this backdrop, constructing an efficient and stable tissue culture regeneration system has become a core task in overcoming technological barriers. This system needs to balance genotype universality, regeneration efficiency, and ease of operation. By optimizing explant pretreatment, hormone ratios in the culture medium, and the regulation of the culture environment, it can significantly improve the adventitious shoot induction rate (target >80%) and shorten the culture cycle, thereby reducing production costs. Crucially, an efficient regeneration system is the cornerstone of precise trait regulation in molecular breeding technology—only by establishing a stable and rapid plant regeneration platform can cutting-edge technologies such as gene editing and marker-assisted selection be efficiently integrated to directionally modify key genes in the anthocyanin synthesis pathway to stabilize leaf color phenotypes, or to screen for dominant genotypes with high medicinal component synthesis, thereby cultivating new varieties with consistent ornamental traits, strong stress resistance, and outstanding medicinal value. Furthermore, this technology can overcome the interference of environmental factors on in vitro culture, providing a new approach for the in vitro preservation and efficient propagation of rare wild germplasm, greatly improving the utilization efficiency of genetic resources and compensating for the shortcomings of traditional propagation techniques in diversity protection.

[0005] Developing an efficient tissue culture regeneration system can not only solve the problems of cutting and grafting techniques being affected by environmental conditions, but also provide key technical support for molecular breeding, promote the innovative application of Chinese Liquidambar formosana in ecological governance, landscape beautification and biomedicine, and has significant scientific research breakthrough value and industrialization prospects.

[0006] Therefore, providing a tissue culture method using Liquidambar formosana zygote embryos as explants is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for efficient regeneration of Liquidambar formosana. The method uses Liquidambar formosana zygote as explants and successfully obtains a batch of uniform and healthy regenerated plants in a short period of time through steps such as callus and adventitious bud induction, adventitious bud rooting, hardening and transplanting. Compared with other previous culture methods using Liquidambar formosana tissue as explants, the method is simpler to operate, has higher genotypic universality, and is more efficient in propagation.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A tissue culture method using Liquidambar formosana zygote embryos as explants includes the following steps:

[0010] S1. The zygotic embryos of immature Liquidambar formosana fruits were dissected and inoculated onto callus induction medium to induce callus tissue.

[0011] S2. Transfer the callus induced in S1 to the bud induction medium to induce adventitious buds;

[0012] S3. Inoculate the adventitious buds formed in step S2 into the seedling rooting medium to induce rooting, directly forming regenerated seedlings for transplanting and cultivation;

[0013] The callus induction medium consists of: WPM medium, 0.10–2.0 mg / L 6-BA, 0.1–1.0 mg / L 2,4-D, 30–40 g / L sucrose, 2.6–3.5 g / L plant gel, 1–3 g / L hydrolyzed casein, and pH 5.8–6.2.

[0014] The adventitious bud induction medium consists of: WPM medium, 0.05–0.5 mg / L TDZ, 30–40 g / L sucrose, 2.6–3.5 g / L plant gel, and pH 5.8–6.2.

[0015] The seedling rooting culture medium consists of: WPM medium, 0.5–2.0 mg / L 6-BA, 0.01–0.5 mg / L NAA, 0.05–3.0 mg / L GA3, 20–30 g / L sucrose, 2.6–3.5 g / L plant gel, and a pH of 5.8–6.2.

[0016] Furthermore, in step S1, immature fruits are harvested, seeds are removed, sterilized with 75% alcohol for 1 minute, and then sterilized with 7.5% sodium hypochlorite solution for 28 minutes. The zygotic embryos are then dissected and inoculated into callus induction culture medium.

[0017] Furthermore, in step S1, the conditions for inducing callus culture are: temperature of 25°C, time of 10-12 days, and culture in the dark.

[0018] Furthermore, in step S2, the culture conditions for inducing adventitious buds are: temperature of 25℃, time of 15-20 days, light intensity of 2500 lx, and light duration of 12 hours per day.

[0019] Furthermore, in step S3, the steps and culture conditions for inducing strong seedlings to root are as follows: 12 hours of light per day, light intensity of 1500 lx, and a culture temperature of 25℃ throughout the rooting culture process.

[0020] Furthermore, in step S3, the transplanting culture involves cultivating the regenerated seedlings until the root length is 1-3 cm, then placing them under natural light for hardening off for 3-5 days. After washing off the culture medium from the roots of the regenerated seedlings, they are transplanted into a moist, sterilized substrate and kept moist.

[0021] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention innovatively uses immature zygotic embryos of Liquidambar formosana as explants, offering advantages such as convenient material acquisition, low sterilization difficulty, and high developmental potential. The zygotic embryos can be obtained directly from green cones, eliminating the need for propagation from superior mother trees or tissue culture seedlings, significantly shortening material acquisition time. Furthermore, this method demonstrates highly efficient regeneration capabilities across different germplasm sources (different germplasm sources and families), exhibiting significantly better genotypic universality than existing technologies. It overcomes the bottleneck of genotypic limitations in traditional methods, providing a stable platform for large-scale breeding and genetic improvement.

[0023] This invention establishes a highly efficient regeneration process through callus induction, adventitious bud differentiation, and root elongation. Callus induction takes only 10-14 days, and the callus quality is high and uniform (making it an ideal recipient material for genetic transformation research). Adventitious bud induction efficiency is significantly improved, with differentiation occurring in 7 days, and an average of 90-120 adventitious buds produced from a single zygotic embryo within 14 days (previous reports indicate a maximum of less than 10 buds), shortening the overall cycle by over 50%. This technological breakthrough solves the core problems of low efficiency and long cycles in existing technologies, laying the foundation for the industrial production of Liquidambar formosana seedlings.

[0024] This invention innovatively designs an elongation and rooting culture medium that simultaneously achieves the functions of adventitious bud elongation, seedling strengthening, and rooting, combining the traditionally separate "seedling strengthening" and "rooting" steps into one, significantly simplifying the operation process. The root system develops robustly, with a transplant survival rate exceeding 95%, avoiding problems such as low proliferation coefficient (only 3.11) and poor transplant adaptability in traditional methods. This integrated technology significantly reduces labor and time costs, providing an efficient solution for large-scale seedling cultivation.

[0025] The regeneration system developed in this invention provides efficient technical support for molecular biology research such as gene editing and genetic transformation in Liquidambar formosana. Through the high regeneration capacity of the zygotic embryo, a large number of uniform and homogeneous plants can be rapidly obtained, overcoming the technical bottleneck of low regeneration and transformation efficiency and unstable regeneration caused by genotypic differences in Liquidambar formosana tissue culture. This technology not only significantly shortens the breeding cycle of superior Liquidambar formosana varieties but also provides core support for the innovation and genetic improvement of Liquidambar formosana germplasm resources. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 illustrates the tissue culture regeneration process of Liquidambar formosana zygote embryos as explants in Example 1 of the present invention.

[0028] Among them, A represents the greenish-yellow cones of Liquidambar formosana that have been picked;

[0029] B is a dissected zygote used as explant material;

[0030] C represents callus tissue from a zygote in a medium containing WPM + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 40 g / L sucrose + 3.0 g / L plant gel + 1 g / L hydrolyzed casein at pH 6.0.

[0031] D represents adventitious shoots newly induced from callus tissue transferred to a medium containing WPM + 0.1 mg / L TDZ + 40 g / L sucrose + 3.0 g / L plant gel at pH 6.0.

[0032] E represents the induction of adventitious shoots after callus tissue was transferred to WPM medium containing 0.1 mg / L TDZ, 40 g / L sucrose, 3.0 g / L plant gel, and pH 6.0 for 20 days.

[0033] F represents the rooting status of adventitious buds inoculated into a medium containing WPM + 0.5 mg / L 6-BA + 0.03 mg / L NAA + 0.4 mg / L GA3 + 25 g / L sucrose + 3.2 g / L plant gel at pH 6.0.

[0034] G represents the regenerated plant from the transplant. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Source of experimental materials

[0037] The WPM (Lioyd & McCown WoodyPlant Medium) used in the following examples is a WPM culture medium prepared using purchased WPM powder (product number L449), with a WPM powder content of 2.41 g / L.

[0038] TDZ refers to thiabendazole; NAA refers to naphthaleneacetic acid; 2,4-D refers to 2,4-dichlorophenoxyacetic acid; 6-BA refers to 6-benzylaminopurine; GA3 refers to gibberellin.

[0039] WPM powder and TDZ, 2,4-D, NAA, 6-BA, and GA3 (sterile) hormones were all purchased from Phyto Technology, and all hormones were pre-prepared 1 mg / mL solutions. Plant gel was purchased from Guangzhou Dingguo Biotechnology Co., Ltd., and sucrose from Shanghai Suisheng Co., Ltd. Hydrolyzed casein powder (Casein Enzymatic Hydrolysate) was purchased from Coolaber.

[0040] Example 1

[0041] (1) Induction of callus: The green cones of Liquidambar formosana were harvested (Fig. 1A), the seeds were removed and sterilized, and the zygotic embryos were dissected (Fig. 1B) and inoculated onto a medium containing WPM + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 40 g / L sucrose + 3.0 g / L plant gel + 1 g / L hydrolyzed casein at pH 6.0. The medium was cultured at 25°C in complete darkness. Callus induction was completed 10 days after inoculation (Fig. 1C).

[0042] (2) Induction of adventitious shoots: The white granular callus induced in step (1) was transferred to WPM + 0.1 mg / L LTDZ + 40 g / L sucrose + 3.0 g / L plant gel medium with pH 6.0. It was cultured at 25℃, light intensity of 2500 lx and light time of 12 h per day. Adventitious shoots appeared after about 7 days (Fig. 1D), and a large number of adventitious shoots formed after about 20 days (Fig. 1E).

[0043] (3) Rooting culture: The adventitious buds formed in step (2) were divided and inoculated into a medium containing WPM + 0.5 mg / L 6-BA + 0.03 mg / L NAA + 0.4 mg / L GA3 + 25 g / L sucrose + 3.2 g / L plant gel at pH 6.0 to induce rooting. The medium was cultured under the conditions of 12 h light per day, light intensity of 1500 lx, and culture temperature of 25 ℃ until rooting (Figure 1F).

[0044] (4) Hardening and transplanting: Loosen the cap of the culture bottle of the regenerated seedling with roots that are 2cm long and well rooted, and place it under natural light for 5 days to harden the seedling. Then carefully remove the regenerated seedling from the culture bottle and carefully wash off the culture medium on the roots with clean water. Then transplant the regenerated seedling into a moist sterilized substrate (Fig. 1G) and cover the culture pot with plastic wrap to maintain high humidity.

[0045] Example 2

[0046] (1) Induction of callus: The green cones of Liquidambar formosana were harvested, the seeds were removed and sterilized, the zygotes were dissected and inoculated onto a medium containing WPM + 0.1 mg / L 6-BA + 0.1 mg / L 2,4-D + 30 g / L sucrose + 2.6 g / L plant gel + 2 g / L hydrolyzed casein, pH 6.2, and cultured at 25°C in complete darkness.

[0047] (2) Induction of adventitious shoots: The white granular callus tissue cultured for 12 days in step (1) was transferred to WPM + 0.05 mg / L TDZ + 30 g / L sucrose + 2.6 g / L plant gel medium with pH 6.2 and cultured at 25℃, light intensity of 2400 lx and light duration of 12 h per day.

[0048] (3) Rooting culture: After the adventitious buds cultured for 18 days in step (2) were divided, they were inoculated into a medium containing WPM + 1.0 mg / L 6-BA + 0.01 mg / L NAA + 0.05 mg / L GA3 + 20 g / L sucrose + 2.6 g / L plant gel at pH 6.2 to induce rooting. The medium was cultured under the conditions of 12 h light per day, light intensity of 1500 lx, and culture temperature of 25 ℃ until rooting occurred.

[0049] (4) Hardening off and transplanting: Loosen the cap of the culture bottle for the regenerated seedling with roots that have grown to 2cm in length and have good roots, and place it under natural light for 5 days to harden off. Then carefully remove the regenerated seedling from the culture bottle and carefully wash off the culture medium on the roots with clean water. Then transplant the regenerated seedling into a moist, sterilized substrate and cover the culture pot with plastic wrap to maintain high humidity.

[0050] Example 3

[0051] (1) Induction of callus: The green cones of Liquidambar formosana were harvested, the seeds were removed and sterilized, the zygotes were dissected and inoculated onto a medium containing WPM + 2.0 mg / L 6-BA + 0.5 mg / L 2,4-D + 35 g / L sucrose + 3.5 g / L plant gel + 3 g / L hydrolyzed casein, pH 5.8, and cultured at 25℃ in complete darkness.

[0052] (2) Induction of adventitious shoots: The white granular callus tissue cultured for 10 days in step (1) was transferred to WPM + 0.5 mg / L TTDZ + 35 g / L sucrose + 3.6 g / L plant gel medium with pH 6.0 and cultured at 25℃, light intensity of 2500 lx and light duration of 12 h per day.

[0053] (3) Rooting culture: After the adventitious buds cultured for 15 days in step (2) were divided, they were inoculated into a medium containing WPM + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 3.0 mg / L GA3 + 30 g / L sucrose + 3.2 g / L plant gel at pH 6.0 to induce rooting. The medium was cultured under the conditions of 12 h light per day, light intensity of 1500 lx, and culture temperature of 25 ℃ until rooting occurred.

[0054] (4) Hardening off and transplanting: Loosen the cap of the culture bottle for the regenerated seedling with roots that have grown to 2cm in length and have good roots, and place it under natural light for 5 days to harden off. Then carefully remove the regenerated seedling from the culture bottle and carefully wash off the culture medium on the roots with clean water. Then transplant the regenerated seedling into a moist, sterilized substrate and cover the culture pot with plastic wrap to maintain high humidity.

[0055] In Examples 1-3, the entire process from the culture of Liquidambar formosana zygote explants to the formation of complete regenerated seedlings can be completed within 2 months.

[0056] Comparative Example 1

[0057] The selected explants were hypocotyls of sterile seedlings, cut into 1cm segments, and inoculated into the induction medium in a horizontal manner. The remaining steps were the same as in Example 1.

[0058] Comparative Example 2

[0059] The explants selected were cotyledons of sterile seedlings, cut into small pieces of about 0.3×0.3cm, and inoculated into the induction medium with the adaxial surface facing up. The remaining steps were the same as in Example 1.

[0060] Comparative Example 3

[0061] The selected explants were leaves from sterile seedlings. After making three cuts perpendicular to the main vein (without cutting the leaf segment), the leaves were inoculated into the induction medium with the adaxial surface facing upwards. The remaining steps were the same as in Example 1.

[0062] Table 1. Efficiency statistics of tissue culture regeneration examples of Liquidambar formosana embryos.

[0063]

[0064] As can be seen from Table 1, the adventitious bud induction rate of the Liquidambar formosana zygote explants obtained in Examples 1-3 of the present invention reached over 80%, and the multiplication coefficient was between 90 and 120.

[0065] 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 tissue culture method using Liquidambar formosana zygote embryos as explants, characterized in that, The procedure includes the following steps: S1. Extracting the zygotic embryo from an immature Liquidambar formosana fruit and inoculating it onto a callus induction medium to induce callus tissue; S2. Transferring the callus induced in S1 to a shoot induction medium to induce adventitious shoots; S3. Inoculating the adventitious shoots formed in step S2 into a seedling rooting medium to induce rooting, directly forming regenerated seedlings for transplanting and cultivation; the callus induction medium consists of: WPM medium, 0.10–2.0 mg / L 6-BA, 0.1–1.0 mg / L 2,4-D, 30–40 g / L sucrose, 2.6–3.5 g / L plant gel, 1–3 g / L hydrolyzed casein, and a pH of 5.8–6.2; the adventitious shoot induction medium consists of: WPM medium, 0.05–0.5 mg / L... The seedling rooting medium consists of: TDZ, 30–40 g / L sucrose, 2.6–3.5 g / L plant gel, and a pH of 5.8–6.2; the seedling rooting medium consists of: WPM medium, 0.5–2.0 mg / L 6-BA, 0.01–0.5 mg / L NAA, 0.05–3.0 mg / L GA3, 20–30 g / L sucrose, 2.6–3.5 g / L plant gel, and a pH of 5.8–6.

2.

2. The method according to claim 1, characterized in that, In step S1, immature fruits are harvested, seeds are removed, sterilized with 75% alcohol for 1 minute, and then sterilized with 7.5% sodium hypochlorite solution for 28 minutes. The zygote is then dissected and inoculated into callus induction medium.

3. The method according to claim 1, characterized in that, In step S1, the conditions for inducing callus culture are: temperature 25℃, time 10-12 days, and culture in the dark.

4. The method according to claim 1, characterized in that, In step S2, the culture conditions for inducing adventitious shoots are: temperature 25℃, time 15-20 days, light intensity 2500 lx, and light duration 12 hours per day.

5. The method according to claim 1, characterized in that, In step S3, the steps and conditions for inducing strong seedlings to root are as follows: 12 hours of light per day, light intensity of 1500 lx, and a culture temperature of 25℃ throughout the rooting culture process.

6. The method according to claim 1, characterized in that, The transplanting culture in step S3 involves cultivating the regenerated seedlings until the root length is 1-3 cm, then placing them under natural light for hardening off for 3-5 days. After washing off the culture medium on the roots of the regenerated seedlings, they are transplanted into a moist, sterilized substrate and kept moist.

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