Tissue culture method using zygotic embryo of liquidambar formosana hance as explant

Abstract

The invention discloses a tissue culture method taking a liquidambar formosana hance zygotic embryo as an explant, and relates to the technical field of plant tissue culture. Comprising the following steps: S1, cutting zygotic embryos of immature fruits of liquidambar formosana hance, inoculating the zygotic embryos to a callus induction culture medium, and inducing calluses; s2, transferring the callus tissue induced in the step S1 to a bud induction culture medium, and performing adventitious bud induction; and S3, inoculating the adventitious buds formed in the step S2 into a seedling strengthening and rooting culture medium to induce rooting, directly forming regenerated seedlings, and transplanting and culturing. Compared with the conventional culture method using other liquidambar formosana hance tissues as explants, the method disclosed by the invention is simpler and more convenient to operate, higher in genotype universality and more efficient in propagation.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and more particularly to a tissue culture method using liquidambar formosana zygotic embryos as explants. Background Art

[0002] Liquidambarformosana is a multifunctional tree species unique to my country, integrating ecological value, ornamental aesthetics and medicinal resources. Its tree shape is upright and stretched, and the leaf color changes with environmental conditions in autumn, forming a unique seasonal landscape. However, the expression of Liquidambar leaf color is affected by environmental factors such as temperature, light intensity and soil nutrients. The wild population is rich in genetic diversity, and the stability of key ornamental traits such as leaf color and tree shape is difficult to guarantee. Traditional breeding methods are difficult to selectively breed varieties with uniform traits and outstanding ornamental value. In addition, Liquidambar resin (liquid gum) is known as the "holy medicine for promoting blood circulation" in traditional Chinese medicine. It has the effects of detoxification, muscle regeneration, blood stasis and pain relief. Modern research further reveals that its leaves and bark are rich in terpenoids and flavonoid derivatives, showing great potential in the fields of anti-inflammatory, antioxidant and anti-tumor. Therefore, achieving efficient breeding and targeted improvement of Liquidambar formosana is of strategic significance for promoting its application in ecological restoration, landscape industry and medicinal development.

[0003] Although traditional asexual propagation techniques such as cuttings and grafting can partially meet the demand for seedlings, their limitations seriously restrict the industrialization process. Cutting rooting is affected by substrate humidity and temperature fluctuations, and the survival rate is less than 70%, which is difficult to meet the needs of large-scale propagation; grafting technology relies on specific rootstock matching and seasonal operations, and has a short window period. Long-term asexual reproduction is also prone to genotype degeneration and cannot break through the limitations of the genetic background of the mother plant, resulting in the difficulty of stable inheritance of excellent traits. Although tissue culture technology has provided new ideas for the breeding of Chinese sweetgum, the existing system still has significant bottlenecks. For example, Zhuo Renying (2005) used zygotic embryos of seedlings to induce adventitious buds through a two-step method, but the induction period was long (adventitious bud induction required 1-2 months) and the efficiency was low (less than 40%); Xu Lin (2008) used leaves of tissue culture seedlings propagated by superior tree stem segments as explants, and achieved regeneration through direct organogenesis by optimizing the hormone ratio (TDZ and NAA), but this method has the disadvantages of high difficulty in sterilizing superior tree stem segments, cumbersome operation, low seedling coefficient (3.11) and limitation of mother tree genotype, and has not yet achieved a technological breakthrough; Yang Lanfang et al. (2016) used leaves of tissue culture seedlings propagated by superior tree stem segments as explants. Although they obtained a patent (CN103975856B) through the embryoid regeneration pathway, the stability of the system and the universality of genotypes still need to be verified.

[0004] In this context, building an efficient and stable tissue culture regeneration system has become the core task of breaking through technical barriers. The system needs to take into account the universality of genotypes, regeneration efficiency and ease of operation. By optimizing explant pretreatment, culture medium hormone ratio and culture environment regulation, the adventitious bud induction rate can be significantly improved (target>80%) and the culture cycle can be shortened, thereby reducing production costs. More importantly, the efficient regeneration system is the cornerstone of molecular breeding technology to achieve precise regulation of traits. Only by establishing a stable and rapid plant regeneration platform can we efficiently integrate cutting-edge technologies such as gene editing and molecular marker-assisted selection, and modify key genes in the anthocyanin synthesis pathway to stabilize the leaf color phenotype, or screen for superior genotypes with high medicinal component synthesis, thereby cultivating new varieties with consistent ornamental traits, strong stress resistance and outstanding medicinal value. In addition, this technology can break through the interference of environmental factors on in vitro culture, provide a new way for the in vitro preservation and efficient propagation of wild rare germplasm, greatly improve the efficiency of genetic resource utilization, and make up for the shortcomings of traditional breeding technology in diversity protection.

[0005] The development of an efficient tissue culture regeneration system can not only solve the problem 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 sweetgum in ecological management, landscaping and biomedicine, etc., and has significant scientific research breakthrough value and industrial promotion prospects.

[0006] Therefore, providing a tissue culture method using Liquidambar formosana zygotic embryos as explants is an urgent problem 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 zygotic embryos as explants, and successfully obtains a batch of neat and healthy regenerated plants in a short period of time through steps such as callus and adventitious bud induction, adventitious bud rooting, seedling acclimatization and transplanting. Compared with other previous culture methods using Liquidambar formosana tissues as explants, the method is simpler to operate, has higher universality of genotypes, and is more efficient in propagation.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0010] S1. dissect the zygotic embryos of the immature fruits of Liquidambar formosana and inoculate them on callus induction medium to induce callus tissue;

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

[0012] S3. The adventitious buds formed in step S2 are inoculated into a rooting medium for seedling growth to induce rooting, directly forming regenerated seedlings for transplanting and cultivation;

[0013] The composition of the callus induction medium is as follows: 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 phytagel, 1 - 3 g / L casein hydrolysate, and the pH is 5.8 - 6.2;

[0014] The composition of the adventitious bud induction medium is as follows: WPM medium, 0.05 - 0.5 mg / L TDZ, 30 - 40 g / L sucrose, 2.6 - 3.5 g / L phytagel, and the pH is 5.8 - 6.2;

[0015] The composition of the strong seedling rooting medium is as follows: 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 phytagel, and the pH is 5.8 - 6.2.

[0016] Further, in the step S1, pick immature fruits, take out the seeds, sterilize them with 75% alcohol for 1 minute and then with 7.5% sodium hypochlorite solution for 28 minutes, dissect the zygotic embryos and inoculate them onto the callus induction medium.

[0017] Further, in the step S1, the culture conditions for callus induction are: temperature is 25°C, time is 10 - 12 d, and dark culture.

[0018] Further, in the step S2, the culture conditions for adventitious bud induction are: temperature is 25°C, time is 15 - 20 d, light intensity is 2500 lx, and light time is 12 h per day.

[0019] Further, in the step S3, the steps and culture conditions for strong seedling rooting are: light for 12 h per day, light intensity is 1500 lx, and the culture temperature during the whole rooting culture process is 25°C.

[0020] Further, the transplanting culture in the step S3 is to cultivate the obtained regenerated seedlings until the root length is 1 - 3 cm, then place them under natural light for acclimatization for 3 - 5 d, wash the medium on the roots of the regenerated seedlings, and transplant them into a moist sterilized substrate and keep it moist.

[0021] It can be seen from the above - mentioned technical solutions that, compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention innovatively uses immature zygotic embryos of Liquidambar formosana as explants, which have the advantages of convenient material collection, low sterilization difficulty, and high developmental potential. Zygotic embryos can be directly obtained from greenish-yellow cones without the need to breed elite tree mother plants or tissue culture seedlings, greatly shortening the time for obtaining materials. At the same time, this method shows high regeneration ability for different provenances (different provenances and families), and the genotype universality is significantly better than that of the existing technology, breaking through the bottleneck of genotype limitation in traditional methods and providing a stable platform for large-scale breeding and genetic improvement.

[0023] The present invention constructs an efficient regeneration process through callus induction → adventitious bud differentiation → elongation and rooting. Among them, callus induction only takes 10 - 14 days, and the callus has high quality and uniform state (it is an ideal receptor material for genetic transformation research); the induction efficiency of adventitious buds is significantly improved, and differentiation can occur in 7 days. On average, one zygotic embryo can produce 90 - 120 adventitious buds in 14 days (the highest number reported in existing reports is less than 10), and the overall cycle is shortened by more than 50%. The breakthrough of this technology solves the core problems of low efficiency and long cycle in the existing technology, laying a foundation for the industrial production of Liquidambar formosana seedlings.

[0024] The present invention innovatively designs an elongation and rooting medium to simultaneously achieve the functions of elongation, strengthening of seedlings, and rooting of adventitious buds, combining the traditional separate steps of "strengthening seedlings" and "rooting" into one, significantly simplifying the operation process. The root system develops robustly, and the transplanting survival rate reaches more than 95%, avoiding problems such as low proliferation coefficient (only 3.11) and poor transplanting adaptability in traditional methods. This integrated technology greatly reduces labor and time costs and provides an efficient solution for large-scale seedling cultivation.

[0025] The regeneration system established by the present invention provides efficient technical support for molecular biology research such as gene editing and genetic transformation of Liquidambar formosana. Through the high regeneration ability of zygotic embryos, a large number of uniform plants can be quickly obtained, breaking through the technical bottlenecks of low regeneration and transformation efficiency and unstable regeneration caused by genotype differences in Liquidambar formosana tissue culture. This technology not only significantly shortens the cultivation cycle of excellent varieties of Liquidambar formosana, but also provides core support for the innovation of germplasm resources and genetic improvement of Liquidambar formosana. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0027] Figure 1 The drawings are the tissue culture regeneration process of Example 1 of the present invention using zygotic embryos of Liquidambar formosana as explants;

[0028] Among them, A is the freshly picked greenish-yellow Liquidambar formosana cones;

[0029] B is the dissected zygotic embryo, used as the explant material;

[0030] C is the callus of the zygotic embryo in the medium of WPM + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 40 g / L sucrose + 3.0 g / L phytagel + 1 g / L casein hydrolysate, with a pH of 6.0;

[0031] D is the adventitious buds just induced when the callus is transferred to the medium of WPM + 0.1 mg / L TDZ + 40 g / L sucrose + 3.0 g / L phytagel, with a pH of 6.0;

[0032] E is the induction situation of adventitious buds after the callus is transferred to the medium of WPM + 0.1 mg / L TDZ + 40 g / L sucrose + 3.0 g / L phytagel, with a pH of 6.0 and cultured for 20 days;

[0033] F is the rooting situation when the adventitious buds are inoculated into the medium of 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 phytagel, with a pH of 6.0;

[0034] G is the regenerated plants after transplantation. Detailed implementation manners

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Sources of experimental materials

[0037] The WPM (lioyd&McCown WoodyPlant Medium) used in the following examples is the WPM medium prepared with the purchased WPM powder (product number L449), and the powder content of WPM in the medium is 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] The WPM powder and TDZ, 2,4-D, NAA, 6-BA, GA3 (sterile) hormones were all purchased from Phytotechnology Company. All hormones were in the form of pre-prepared liquid with a concentration of 1 mg / mL. Phytagel was purchased from Guangzhou Dingguo Biotechnology Co., Ltd., and sucrose was purchased from Shanghai Suishi Company. Casein Enzymatic Hydrolysate powder was purchased from coolaber company.

[0040] Example 1

[0041] (1) Inducing callus: Picking the greenish-yellow cones of Liquidambar formosana ( Figure 1 A), taking out the seeds and sterilizing them, then dissecting out the zygotic embryos ( Figure 1 B) and inoculating them onto the medium of WPM + 0.5 mg / L 6-BA + 1.0 mg / L 2,4-D + 40 g / L sucrose + 3.0 g / L Phytagel + 1 g / L Casein Enzymatic Hydrolysate with a pH of 6.0, and culturing them under the conditions of a temperature of 25°C and complete darkness. Callus induction was completed 10 days after inoculation ( Figure 1 C).

[0042] (2) Inducing adventitious buds: Transferring the white granular callus induced in step (1) to the medium of WPM + 0.1 mg / L TDZ + 40 g / L sucrose + 3.0 g / L Phytagel with a pH of 6.0, and culturing them under the conditions of a temperature of 25°C, a light intensity of 2500 lx, and a light duration of 12 h per day. Adventitious buds appeared around 7 days ( Figure 1 D), and a large number of adventitious buds formed around 20 days ( Figure 1 E).

[0043] (3) Rooting culture: Cutting the adventitious buds formed in step (2) and inoculating them onto the medium of 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 Phytagel with a pH of 6.0 for inducing root formation, and culturing them under the conditions of 12 h of light per day, a light intensity of 1500 lx, and a culture temperature of 25°C until rooting ( Figure 1 F).

[0044] (4) Hardening-off and transplanting: Loosening the caps of the culture bottles of the regenerated seedlings with roots growing to 2 cm long and having good rooting, placing them under natural light for hardening-off for 5 days, then carefully removing the regenerated seedlings from the culture bottles and carefully washing the medium on the roots with water; then transplanting the regenerated seedlings into the moist sterilized substrate ( Figure 1 G), and covering the culture pots with plastic wrap to maintain a relatively high humidity.

[0045] Example 2

[0046] (1) Inducing callus: Pick the greenish cones of Liquidambar formosana, take out the seeds, sterilize them, dissect the zygotic embryos and inoculate them onto the medium of WPM + 0.1 mg / L 6-BA + 0.1 mg / L 2,4-D + 30 g / L sucrose + 2.6 g / L phytagel + 2 g / L casein hydrolysate with a pH of 6.2, and culture them under the conditions of a temperature of 25°C and complete darkness.

[0047] (2) Inducing adventitious buds: Transfer the white granular callus cultured for 12 days in step (1) to the medium of WPM + 0.05 mg / L TDZ + 30 g / L sucrose + 2.6 g / L phytagel with a pH of 6.2, and culture them under the conditions of a temperature of 25°C, a light intensity of 2400 lx, and a light duration of 12 hours per day.

[0048] (3) Rooting culture: Cut the adventitious buds cultured for 18 days in step (2) and inoculate them onto the medium of 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 phytagel with a pH of 6.2 for inducing rooting, and culture them until rooting under the conditions of a light duration of 12 hours per day, a light intensity of 1500 lx, and a culture temperature of 25°C.

[0049] (4) Acclimatization and transplantation: Loosen the cap of the culture bottle of the regenerated seedlings with roots growing to 2 cm long and good rooting, place them under natural light for acclimatization for 5 days, then carefully remove the regenerated seedlings from the culture bottle and carefully wash the medium on the roots with water; then transplant the regenerated seedlings into the moist sterilized substrate, and cover the culture pot with plastic wrap to maintain a high humidity.

[0050] Example 3

[0051] (1) Inducing callus: Pick the greenish cones of Liquidambar formosana, take out the seeds, sterilize them, dissect the zygotic embryos and inoculate them onto the medium of WPM + 2.0 mg / L 6-BA + 0.5 mg / L 2,4-D + 35 g / L sucrose + 3.5 g / L phytagel + 3 g / L casein hydrolysate with a pH of 5.8, and culture them under the conditions of a temperature of 25°C and complete darkness.

[0052] (2) Inducing adventitious buds: Transfer the white granular callus cultured for 10 days in step (1) to the medium of WPM + 0.5 mg / L TDZ + 35 g / L sucrose + 3.6 g / L phytagel with a pH of 6.0, and culture them under the conditions of a temperature of 25°C, a light intensity of 2500 lx, and a light duration of 12 hours per day.

[0053] (3) Rooting culture: The adventitious buds cultured for 15 days in step (2) were cut and inoculated onto a medium of 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 phytagel with a pH of 6.0 for induced rooting, and cultured until rooting under the conditions of 12 h of light per day, a light intensity of 1500 lx, and a culture temperature of 25 °C.

[0054] (4) Acclimatization and transplantation: The cap of the culture bottle of the regenerated seedlings with roots growing to 2 cm long and good rooting was loosened, and after acclimatizing the seedlings under natural light for 5 days, the regenerated seedlings were carefully removed from the culture bottle and the medium on the roots was carefully washed off with water; then the regenerated seedlings were transplanted into a moist and sterilized substrate, and a plastic wrap was covered on the culture pot where they were located to maintain a high humidity.

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

[0056] Comparative Example 1

[0057] The selected explant was the hypocotyl of a sterile seedling. After being cut into 1-cm segments, it was inoculated into the induction medium in a horizontal position, and the remaining steps were the same as in Example 1.

[0058] Comparative Example 2

[0059] The selected explant was the cotyledon of a sterile seedling. After being cut into small pieces of about 0.3 × 0.3 cm, it was inoculated into the induction medium with the adaxial surface facing up, and the remaining steps were the same as in Example 1.

[0060] Comparative Example 3

[0061] The selected explant was the leaf of a sterile seedling. After cutting 3 times perpendicular to the main vein (without cutting off the segments), it was inoculated into the induction medium with the adaxial surface facing up, and the remaining steps were the same as in Example 1.

[0062] Table 1 Statistical results of the efficiency of tissue culture regeneration of Liquidambar formosana zygotic embryos

[0063]

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

[0065] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present 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 present invention. Thus, the present invention is not intended 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 the zygotic embryo of Liquidambar formosana as an explant, characterized in that, It includes the following steps: S1. Dissect the zygotic embryos of immature Liquidambar formosana fruits and inoculate them onto the callus induction medium to induce callus; S2. Transfer the callus induced in S1 to the bud induction medium to induce adventitious buds; S3. Inoculate the adventitious buds formed in step S2 into the strong seedling rooting medium to induce root formation, directly form regenerated seedlings, and carry out transplanting culture; The composition of the callus induction medium is: 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 phytagel, 1 - 3 g / L casein hydrolysate, and the pH is 5.8 - 6.2; The composition of the adventitious bud induction medium is: WPM medium, 0.05 - 0.5 mg / L TDZ, 30 - 40 g / L sucrose, 2.6 - 3.5 g / L phytagel, and the pH is 5.8 - 6.2; The composition of the strong seedling rooting medium is: 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 phytagel, and the pH is 5.8 - 6.

2.

2. The method according to claim 1, wherein In step S1, pick the immature fruits, take out the seeds, sterilize them with 75% alcohol for 1 minute and then with 7.5% sodium hypochlorite solution for 28 minutes, and dissect the zygotic embryos and inoculate them into the callus induction medium.

3. The method according to claim 1, wherein In step S1, the culture conditions for inducing callus are: temperature is 25°C, time is 10 - 12 d, and dark culture.

4. The method according to claim 1, characterized in that, In step S2, the culture conditions for adventitious bud induction are: temperature is 25°C, time is 15 - 20 d, light intensity is 2500 lx, and light time is 12 h per day.

5. The method according to claim 1, characterized in that In step S3, the steps and culture conditions for inducing strong seedling rooting are: light for 12 h per day, light intensity is 1500 lx, and the culture temperature during the whole rooting culture process is 25°C.

6. The method according to claim 1, characterized in that The transplanting culture in step S3 is to cultivate the obtained regenerated seedlings until the root length is 1 - 3 cm, then place them under natural light for acclimatization for 3 - 5 d, wash the medium on the roots of the regenerated seedlings, and transplant them into a moist sterilized substrate and keep it moist.

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