Methods for obtaining regenerated plants from hybrid Liquidambar formosana petioles via somatic embryogenesis

Abstract

This invention discloses a method for obtaining regenerated plants from the petioles of hybrid Liquidambar formosana via somatic embryogenesis. The method includes using mature vegetative organs of hybrid Liquidambar formosana as explants for callus induction culture; then, maturing the embryogenic callus; and finally, germinating the cotyledonary embryos obtained from the maturation culture. This method, using mature vegetative organs of hybrid Liquidambar formosana as explants for callus induction, achieves a high callus induction rate, high fresh weight of callus proliferation, a large number of mature somatic embryos, and a high somatic embryo germination rate, obtaining complete regenerated plants within 6 months. This method overcomes the bottleneck of difficulty in inducing somatic embryogenesis from mature organs; moreover, the establishment of a somatic embryogenesis system using this method enables rapid propagation of large-scale superior strains and provides technical support for subsequent genetic improvement of germplasm using genetic engineering techniques.

Description

Technical Field

[0001] This invention relates to a method for plant tissue culture, and more particularly to a method for obtaining regenerated plants by inducing somatic embryogenesis (or somatic embryogenesis) in Liquidambar species using petioles, belonging to the field of plant asexual reproduction technology. Background Technology

[0002] The genus *Liquidambar* (Liquidambar spp.) is a globally important forest resource and a key timber species in my country's "National Reserve Forest Construction Plan (2018-2035)". *Liquidambar formosana* Hance is distributed throughout most temperate and subtropical regions of China. The entire plant can be used medicinally. It grows rapidly, is highly adaptable, and is a pioneer species for barren mountains, as well as a popular urban landscaping tree. *Liquidambar styraciflua*, a common southern hardwood from the United States, is primarily used in the timber and paper industries and also has high ornamental value. The hybrid *Liquidambar styraciflua* × *Liquidambar formosana* exhibits superior growth rate, wood density, and biomass productivity compared to its parents, demonstrating significant hybrid vigor. It is suitable for cultivation in 20 provinces, municipalities, and autonomous regions of my country and is an important species for the future development and innovation of my country's forestry industry. Therefore, establishing an efficient asexual reproduction system to ensure the stable inheritance of superior traits has become a current research hotspot.

[0003] Asexual reproduction techniques mainly include cuttings, grafting, and tissue culture. Currently, the techniques for propagating Liquidambar formosana (Chinese sweetgum) through cuttings and grafting are unstable, with survival rates ranging from 17% to 98%. Furthermore, these two methods are seasonally limited and cannot utilize genetic engineering to achieve genetic improvement of the germplasm. Although some studies have successfully established in vitro organ regeneration systems using leaves and petioles of Liquidambar species, regenerating Liquidambar plants through a regeneration pathway different from somatic embryogenesis, this method has high labor costs, low propagation efficiency, and is difficult to implement for large-scale industrial production.

[0004] Currently, there are studies on the use of leaves and petioles as vegetative organs to establish in vitro organ regeneration systems for Liquidambar species. This involves inducing adventitious bud differentiation, elongation, and rooting through leaf culture to obtain regenerated plants. This method uses organ regeneration systems rather than somatic cell embryogenesis for plant propagation.

[0005] Somatic embryogenesis is an in vitro biological process in which somatic cells induce the formation of bipolar structures (somatic embryos) and regenerate into complete plants. It has advantages such as high reproductive efficiency and benefits for germplasm resource preservation. Furthermore, somatic embryos are ideal materials for genetic transformation and are crucial for the large-scale clonal propagation and molecular-assisted breeding of forest trees, making it a research hotspot in the field of asexual reproduction. Sommer and Brown (1980) successfully induced somatic embryogenesis using hypocotyl segments of *Liquidambar formosana* as explants. Subsequently, the Merkle team at the University of Georgia successfully established somatic embryogenesis systems for *Liquidambar formosana* and *Liquidambar formosana* using inflorescences of *Liquidambar formosana* and immature zygotes of hybrid *Liquidambar formosana*, respectively, and completed studies on embryogenic callus suspension culture and synchronized embryonic development (Merkle et al., 1998; Vendrame et al., 2001; Merkle et al., 2003). In China, Wang Xiaoqi successfully induced somatic embryogenesis using male flowers and inflorescence axes of *Liquidambar formosana* and immature zygotic embryos of hybrid *Liquidambar formosana* (Wang Xiaoqi, 2016). Building upon previous research, Qi Shuaizheng optimized the somatic embryogenesis process of hybrid *Liquidambar formosana*, establishing an efficient somatic embryogenesis system using immature zygotic embryos as explants (Qi Shuaizheng, 2022). However, somatic embryogenesis induced using immature zygotic embryos as explants is not only seasonally dependent, but the genetic value of the resulting regenerated plants remains unproven. Although this issue can be addressed through cryopreservation technology, extensive field trials are needed to determine their genetic quality.

[0006] If a somatic embryogenesis system can be established using vegetative organs as explants, large-scale propagation can be achieved while maintaining the genetic characteristics of the mother plant, which will have potential economic value for forestry and horticulture. However, the ability of explants to acquire embryogenic potential decreases with increasing explant age. The further the explant development is from the zygotic embryo stage, the more its somatic embryogenesis ability is gradually suppressed. Once the apical meristem is formed, the potential for inducing somatic embryogenesis is limited. Therefore, only a few woody plants have achieved somatic embryogenesis induced by vegetative organs. For example, in Eucalyptus globulus Labill., Quercus robur L., and Cyphomandra betacea (Cav.) Sendt., the age problem of vegetative organ-induced somatic embryogenesis has been overcome by rejuvenating mature vegetative organ tissues.

[0007] Based on previous research, this invention first obtains young test-tube seedlings of hybrid Liquidambar formosana, and then uses the tender petioles of these seedlings as explants to induce somatic embryogenesis. The advantages of this invention are that the source of explants is not limited by season, and large-scale propagation can be achieved while maintaining the genetic characteristics of the mother plant. Currently, there are no research reports on somatic embryogenesis techniques using vegetative organs of hybrid Liquidambar formosana as explants, either domestically or internationally. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing hybrid Liquidambar formosana regeneration plant cultivation methods, which do not use mature vegetative organs as explants for somatic embryogenesis to obtain regenerated plants. Instead, they rely on somatic embryogenesis techniques using immature or mature zygote embryos. However, the somatic embryogenesis technique using immature zygotes is significantly limited by season and time, and the genetic value of the regenerated plants has not been confirmed, thus hindering large-scale propagation. This invention provides a method for propagating hybrid Liquidambar formosana regeneration plants. The method establishes a somatic embryogenesis system for hybrid Liquidambar formosana using vegetative organs as explants, overcoming the bottleneck of difficulty in inducing somatic embryogenesis with mature vegetative organs, and enabling large-scale asexual propagation of superior strains.

[0009] To achieve the objectives of this invention, one aspect of this invention provides a method for obtaining regenerated plants from the petioles of hybrid Liquidambar formosana through somatic embryogenesis, comprising: using mature vegetative organs of hybrid Liquidambar formosana as explants for callus induction culture; then performing mature culture of the embryogenic callus and germinating culture of the cotyledon embryos obtained from the mature culture, thereby obtaining the plant.

[0010] The mature vegetative organs of the hybrid Liquidambar formosana are leaves, petioles, or stem segments, preferably petioles.

[0011] In particular, select tender petioles from hybrid Liquidambar formosana seedlings that are 7-8 months old.

[0012] In particular, the tender petioles of 8-month-old hybrid Liquidambar formosana test-tube seedlings were selected.

[0013] The culture medium for inducing callus induction is: modified Blaydes' basal medium + 1.0–3.0 mg / L 2,4-D + 0.25–0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7; preferably: modified Blaydes' basal medium + 1.0 mg / L 2,4-D + 0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7.

[0014] In particular, the callus induction culture conditions are: 23-27℃, dark culture for 28-35 days.

[0015] The culture medium for the mature culture is: modified Blaydes' basal medium + 1 g / L activated carbon + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7.

[0016] In particular, the maturation conditions are: 23-27℃, dark culture for 28-35 days.

[0017] The germination culture medium is: modified Blaydes' basal medium + 0-0.5 mg / L 6-BA + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7, preferably modified Blaydes' basal medium + 0.5 mg / L 6-BA + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7.

[0018] In particular, the germination culture conditions are as follows: light intensity of 1500-3000 Lux, light duration of 10-16 h light / 8-14 h dark (preferably 16 h light / 8 h dark), temperature of 23-27 °C, and culture for 30-45 days.

[0019] Specifically, the modified Blaydes' basal medium consists of: 20 g / L KNO3, 20 g / L NH4NO3, 10 g / L Ca(NO3)2·4H2O, 6 g / L KH2PO4, 1.43 g / L MgSO4·7H2O, 1.3 g / L KCl, 8 g / L MnSO4·4H2O, 4 g / L ZnSO4·7H2O, 2 g / L H3BO3, 0.6 g / L KI, 0.025 g / L Na2MoO4·2H2O, 0.025 g / L CuSO4·5H2O, 0.025 g / L CoCl2·6H2O, 0.2 g / L Vitamin B1, 0.02 g / L Niacin, 0.02 g / L Vitamin B6, 10 g / L Inositol, and 3.73 g / L NH4NO3. Na2EDTA·2H2O, 2.78g / L FeSO4·7H2O.

[0020] Another aspect of the present invention provides a method for obtaining regenerated plants from hybrid Liquidambar formosana petioles via somatic embryogenesis, comprising the following steps:

[0021] 1) Under sterile conditions, the tender petioles of hybrid Liquidambar seedlings that have been cultured for 7-8 months are cut off and inoculated into callus induction medium for callus induction culture to obtain primary callus tissue.

[0022] 2) Under sterile conditions, the obtained primary callus tissue was detached from the explant with forceps and transferred into the callus proliferation culture medium for callus proliferation culture to obtain embryogenic callus tissue.

[0023] 3) Under sterile conditions, embryogenic callus tissue was transferred into somatic embryo maturation culture medium for somatic embryo maturation culture to obtain mature somatic cotyledon embryos.

[0024] 4) Under sterile conditions, mature somatic embryos are transferred to somatic embryo germination medium for germination culture to obtain regenerated plants.

[0025] The callus proliferation culture medium mentioned in step 2) is: modified Blaydes' basal medium + 0.5-1.0 mg / L 2,4-D + 0.25-0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7; or modified Blaydes' basal medium + 0.5-1.0 mg / L NAA + 0.25-0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7.

[0026] In particular, the preferred callus proliferation culture medium is: modified Blaydes' basal medium + 0.5-1.0 mg / L 2,4-D + 0.25-0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7.

[0027] In particular, the callus proliferation culture medium is further preferably: modified Blaydes' basal medium + 1.0 mg / L 2,4-D + 0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7.

[0028] In particular, the culture conditions for the callus proliferation culture are: dark culture at 23-27℃, subcultured every 25-30 days, for a total of 2-3 subcultures.

[0029] In particular, callus tissue culture yielded embryogenic callus tissue that was slightly yellow and had distinct granules.

[0030] The present invention provides a method for obtaining regenerated plants from hybrid Liquidambar formosana petioles via somatic embryogenesis. The method uses petioles from 8-month-old hybrid Liquidambar test-tube seedlings as explants, inoculating them into a callus induction medium to induce callus formation. These callus tissues are then inoculated into a proliferation medium to induce proliferating embryogenic callus. Subsequently, these callus tissues are inoculated into a somatic embryo maturation medium to induce mature somatic embryos. Finally, the mature cotyledonary embryos are transferred to a somatic embryo germination medium to germinate into complete regenerated plants. The specific steps include:

[0031] Eight months of sterile hybrid Liquidambar formosana test-tube seedlings were cultured. Under sterile conditions, tender petioles were cut from the test-tube seedlings and inoculated into callus induction medium. After inoculation, the seedlings were cultured in the dark at 23-27℃ for one month to obtain white, nearly spherical, and dense primary callus tissue.

[0032] Under sterile conditions and under a stereomicroscope, the induced primary callus was detached from the explant and transferred to the callus proliferation medium. It was then cultured in the dark at 23–27°C. Subculture was performed every 25–30 days. After 2–3 subcultures, a slightly yellow embryogenic callus with obvious granules was obtained.

[0033] Under sterile conditions, the obtained embryogenic callus was transferred into somatic embryo maturation medium and cultured in the dark at 23–27°C for one month to induce somatic embryo development.

[0034] Under sterile conditions, cotyledonary embryos with distinct cotyledons are taken and transferred to somatic embryo germination medium. The light intensity is 1500–3000 Lux, the light duration is 16 hours of light followed by 8 hours of darkness, and the temperature is 23–27℃. After 30–45 days of culture, they germinate and grow into complete regenerated plants.

[0035] Selecting appropriate types and developmental stages of explants is crucial for the successful induction of embryogenic callus and embryonic development. In many woody plants, somatic embryogenesis is induced using immature or mature zygotes as explants. The ability of explants to acquire embryogenicity declines with increasing explant age; that is, the further the explant's developmental stage is from the zygote stage, the weaker its somatic embryogenic capacity becomes. Once the explant forms apical meristem, its potential to acquire embryogenic tissue is significantly affected. The basal culture medium provides the necessary inorganic and organic matter, as well as iron salts, for plant growth during in vitro culture. The exogenous addition of plant growth regulators can promote the dedifferentiation of plant tissues, inducing tissues with strong meristematic capacity. These meristems continue to differentiate and develop, thus forming embryogenic tissues. Common plant growth regulators include auxins and cytokinins. Sucrose provides a carbon source for plant tissue growth, while hydrolyzed casein provides an organic nitrogen source. Plant gels provide fixation and support during plant tissue culture, and pH maintains normal osmotic pressure. These factors play important roles in plant tissue culture.

[0036] Most woody plants induce somatic embryogenesis using immature or mature zygotes as explants. These two types of tissues are closer to the zygote stage in their development, making them more prone to dedifferentiation and initiating regeneration. However, as the explant develops, more mature explants become increasingly difficult to restart regeneration.

[0037] However, the genetic stability and growth performance of regenerated plants obtained from somatic embryogenesis induced by immature or mature zygotes are uncertain, and they may exhibit genetic variations or poor growth. Confirming the genetic stability of regenerated plants requires subsequent field trials to observe phenotypic performance, which is time-consuming. Woody plants have longer growth cycles and require more time for verification than non-woody plants. Therefore, obtaining genetically stable regenerated plants is a lengthy and inefficient process.

[0038] Compared with the prior art, the present invention has the following advantages and benefits:

[0039] The present invention relates to a method for obtaining regenerated plants from the petioles of mature vegetative organs of hybrid Liquidambar formosana through somatic embryogenesis (i.e., somatic embryogenesis). This method belongs to the field of efficient propagation technology of plant tissue culture. The somatic embryo callus induction rate of the present invention is high, reaching more than 47%; the fresh weight of callus proliferation is high, reaching more than 3.07g; moreover, the number of mature somatic embryos is large, reaching 22 / g; and the somatic embryo germination rate is high, reaching more than 13%.

[0040] Using the method of this invention, petioles are used as explants for somatic embryogenesis culture, and complete regenerated plants can be obtained within 6 months. Not only is the reproduction time of regenerated plants short and the reproduction efficiency high, but it also allows for the large-scale reproduction of hybrid Liquidambar formosana plants, thereby improving the reproduction efficiency of regenerated plants.

[0041] The method of this invention for the propagation of regenerated plants is not restricted by time or season, and the heritability and genetic value of the regenerated plants are confirmed, eliminating the need for subsequent field trials to verify the growth phenotype of the regenerated plants.

[0042] This invention not only overcomes the shortcomings of existing regeneration technologies that use immature zygotes for somatic embryogenesis, such as seasonal constraints and the inability to verify the genetic value of regenerated plants, but also breaks through the bottleneck of inducing somatic embryogenesis in mature vegetative organs, enabling large-scale asexual reproduction of superior strains. At the same time, the establishment of a somatic embryogenesis system induced by petioles in hybrid Liquidambar formosana provides important technical support for subsequent molecular-assisted breeding.

[0043] The method of this invention uses mature vegetative organs, such as petioles, as explants. Petioles are easier to obtain and are available in large quantities, and the time, season, and region of collection are not limited. Compared with reproductive organs (such as embryos and seeds), the processing of vegetative organs is relatively simple, which is more conducive to large-scale asexual reproduction and molecular-assisted breeding in the later stages. Attached Figure Description

[0044] Figure 1 The image shows a hybrid Liquidambar formosana leaf petiole used as an explant.

[0045] Figure 2 This is a diagram of primary callus tissue induced in the petiole 18 days ago;

[0046] Figure 3 This is a diagram of primary callus tissue induced in the petiole 30 days ago, where the part shown in the box is the swollen petiole;

[0047] Figure 4 This is a diagram of embryogenic callus obtained after primary callus has been subcultured.

[0048] Figure 5 This is a diagram of a somatic embryo induced from embryonic callus.

[0049] Figure 6 This is a diagram showing the germination of a somatic embryo to form a complete regenerated plant. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0051] This invention discloses a method for obtaining regenerated plants from the petioles of hybrid Liquidambar formosana through somatic embryogenesis. The method uses the petioles of 8-month-old test-tube seedlings of hybrid Liquidambar formosana as explants, inoculates them into a callus induction medium to induce callus, then transfers them into a proliferation medium to induce proliferating embryogenic callus, then inoculates them into a somatic embryo maturation medium to induce mature somatic embryos, and finally transfers the mature cotyledonary embryos into a somatic embryo germination medium to germinate and form complete regenerated plants.

[0052] The specific method includes the following steps:

[0053] (1) Induction and culture of callus

[0054] Under aseptic conditions, tender petioles of 7-8 month old hybrid Liquidambar formosana plantlets were harvested and inoculated into callus induction medium. The plants were then cultured in the dark at 23-27°C for 28-35 days to induce callus formation and obtain primary callus. The callus induction medium consisted of a modified Blaydes' basal medium supplemented with different concentrations of auxins: 2,4-D (2,4-dichlorophenoxyacetic acid) 1.0-3.0 mg / L and cytokinins: 6-BA (6-benzylaminopurine) 0.25-0.50 mg / L, along with 1.0 g / L hydrolyzed casein, 40 g / L sucrose, and 2.6-2.7 g / L plant gel. The pH was adjusted to 5.6-5.7. The medium was then sterilized at 121°C for 15 minutes. The callus induction medium consisted of modified Blaydes' basal medium + 1.0–3.0 mg / L 2,4-D + 0.25–0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7.

[0055] (2) Proliferation culture of callus

[0056] Under sterile conditions and under a stereomicroscope, the primary callus obtained in step (1) was detached from the explant with forceps and transferred to the callus proliferation culture medium for callus proliferation culture to obtain a large amount of embryogenic callus; that is, it was cultured in the dark at 23-27℃, and subcultured every 25-30 days for 2-3 times to obtain slightly yellow embryogenic callus with obvious granules.

[0057] The callus proliferation medium is: modified Blaydes' basal medium supplemented with 0.5–1.0 mg / L 2,4-D + 0.25–0.50 mg / L 6-BA, or modified Blaydes' basal medium supplemented with 0.5–1.0 mg / L NAA + 0.25–0.50 mg / L 6-BA, along with 1.0 g / L hydrolyzed casein, 40 g / L sucrose and 2.6–2.7 g / L plant gel, and the pH is adjusted to 5.6–5.7.

[0058] The callus proliferation medium was: modified Blaydes' basal medium + 0.5–1.0 mg / L 2,4-D + 0.25–0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7; or

[0059] The callus proliferation medium was: modified Blaydes' basal medium + 0.5–1.0 mg / L NAA + 0.25–0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7.

[0060] (3) Maturation culture of somatic embryos

[0061] Under sterile conditions, the embryonic callus obtained in step (2) is transferred into somatic embryo maturation culture medium for somatic embryo maturation culture; that is, it is cultured in the dark at 23-27℃ for 28-35 days to induce somatic embryo maturation and development, and embryos at different developmental stages are obtained, including: globular embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos.

[0062] The culture medium for somatic embryo maturation was: modified Blaydes' basal medium + 1 g / L activated carbon + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7.

[0063] (4) Germination culture

[0064] Under aseptic conditions, the cotyledon embryos cultured in step (3) until they have obvious cotyledons are transferred to somatic embryo germination medium for germination culture, wherein: the light intensity is controlled at 1500-3000 Lux, the light duration is 10-16h light / 8-14h dark (preferably 16h light / 8h dark), the temperature is 23-27℃, and the culture is carried out for 30-45 days, during which the somatic embryos germinate and grow into complete regenerated plants.

[0065] The somatic embryo germination medium was: modified Blaydes' basal medium + 0-0.5 mg / L 6-BA + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7.

[0066] Example 1: Induction and Culture of Callus

[0067] Under sterile conditions, the tender petioles of hybrid Liquidambar formosana plantlets cultured for 8 months were cut off (e.g. Figure 1 The explants were inoculated into culture dishes containing callus induction medium and cultured in the dark at 23–27°C for 30 days (usually 28–35 days). Ten explants were inoculated into each culture dish, with six treatments and three replicates per treatment. The hormone concentrations for each treatment are shown in Table 1.

[0068] Eighteen days after explant petioles were inoculated into callus induction medium, white, ice-crystal-like primary callus tissue was observed (e.g. Figure 2Continue culturing for 30 days, then observe and record the primary callus tissue obtained by inducing explant petioles, and observe the state of the primary callus tissue (e.g., ...). Figure 3 Primary callus tissue, which is white, nearly spherical in shape, and dense in texture, was observed on the surface of the petiole; the induction rate of primary callus tissue was statistically analyzed and shown in Table 1.

[0069] Callus induction medium: Modified Blaydes' basal medium + 1.0–3.0 mg / L 2,4-D + 0.25–0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7. Sterilize at 121°C for 15 minutes.

[0070] The basic culture medium used in this invention is a modified Blaydes' basic culture medium, the formulation of which is shown in Table 2.

[0071] Table 1. Effects of different hormone concentrations in callus induction culture medium on petiole-induced callus.

[0072]

[0073] The experimental results in Table 1 show that during the primary callus induction stage, the addition of 1.0 mg / L 2,4-D and 0.5 mg / L 6-BA resulted in the optimal callus induction rate from petiole explants, reaching 96.67%. The callus induction was also at its best, with the highest quantity and white, nearly spherical, and densely packed primary callus observed on the petiole surface. Excessively high concentrations of 2,4-D not only failed to promote callus induction but also inhibited it. This resulted in a lower induction rate, poor callus condition, reduced quantity, fewer spherical primary callus, and more white filamentous material observed on the explant surface. Therefore, the modified Blaydes' basal medium with the addition of 1.0 mg / L 2,4-D + 0.5 mg / L 6-BA showed the best effect on primary callus induction.

[0074] The callus induction medium was: modified Blaydes' basal medium + 1.0 mg / L 2,4-D + 0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7. The medium was sterilized at 121°C for 15 minutes.

[0075] Table 2. Improved Blaydes' basal culture medium formulation

[0076]

[0077] Example 2: Callus proliferation culture

[0078] Under sterile conditions and under a stereomicroscope, the primary callus obtained in Example 1 was detached from the explant using forceps and transferred to a culture dish containing callus proliferation medium. 1g of primary callus was inoculated into each dish. The callus was cultured in the dark at 23–27°C (i.e., dark culture). Subculture was performed every 27 days (usually 25–30 days) during the proliferation culture, for a total of 3 subcultures (usually 2–3 times). Proliferated embryogenic callus was obtained. The proliferated callus was slightly yellow and had distinct granules. Figure 4 .

[0079] Four treatments were administered, with hormone concentrations for each treatment as shown in Table 3. Each treatment was repeated three times.

[0080] Thirty days after the primary callus was introduced, the proliferation status of the callus was observed and recorded, and the fresh weight of the proliferated callus was calculated. The results are shown in Table 3.

[0081] The callus proliferation medium was prepared by adding 0.5–1.0 mg / L 2,4-D and 0.25–0.50 mg / L 6-BA to a modified Blaydes' basal medium, or adding 0.5–1.0 mg / L NAA and 0.25–0.50 mg / L 6-BA to a modified Blaydes' basal medium, along with 1.0 g / L hydrolyzed casein, 40 g / L sucrose, and 2.6–2.7 g / L plant gel. The pH was adjusted to 5.6–5.7. The medium was then sterilized at 121°C for 15 minutes.

[0082] Table 3. Effects of different hormone concentrations on callus proliferation

[0083]

[0084]

[0085] The statistical results in Table 3 show that during the callus proliferation stage, the addition of 1.0 mg / L 2,4-D and 0.5 mg / L 6-BA resulted in the fastest callus proliferation rate and a more pronounced granular structure. The combination of 2,4-D and 6-BA significantly improved callus proliferation compared to the combination of NAA and 6-BA. Therefore, the modified Blaydes' basal medium supplemented with 1.0 mg / L 2,4-D + 0.5 mg / L 6-BA exhibits the best callus proliferation effect.

[0086] The callus proliferation medium was: modified Blaydes' basal medium + 1.0 mg / L 2,4-D + 0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7. It was sterilized at 121°C for 15 minutes.

[0087] Example 3: Maturation Culture of Somatic Cell Embryos

[0088] Under aseptic conditions, the embryogenic callus tissue obtained from the proliferation culture in Example 2 was transferred into a culture dish containing somatic embryo maturation medium. It was then cultured in the dark at 23–27°C for 30 days (usually 28–35 days) to obtain mature somatic embryos, including: globular embryos, heart-shaped embryos, torpedo embryos, and cotyledonary embryos. Figure 5 ).

[0089] 1g of callus tissue was inoculated into each culture dish, with 3 replicates. After 30 days, somatic embryos at different developmental stages were observed, including globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonary embryos, with a mature somatic embryo count of 22 / g.

[0090] The somatic embryo maturation medium was: modified Blaydes' basal medium + 1 g / L activated carbon + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7. It was sterilized at 121°C for 15 minutes.

[0091] Example 4: Germination and Seedling Formation of Somatic Cell Embryos

[0092] Under sterile conditions, the cotyledonary embryos with distinct cotyledons obtained in Example 3 were transferred to tissue culture flasks containing somatic embryo germination medium with different hormone concentrations for germination culture to obtain regenerated plants. The light intensity was 2000 Lux (usually 1500-3000 Lux), the light duration was 16 hours of light and 8 hours of darkness (usually 10-16 hours of light / 8-14 hours of darkness), and the temperature was 25°C (usually 23-27°C).

[0093] Ten mature cotyledonary embryos were inoculated into each tissue culture bottle, with three treatments and three replicates per treatment. After 30–45 days of culture, the somatic embryos germinated and grew into complete regenerated plants. Figure 6 The number of cotyledon embryos that germinated and grew into regenerated plants was counted, and the germination rate of somatic embryos was also counted, as shown in Table 4.

[0094] The somatic embryo germination medium was: modified Blaydes' basal medium + 0–0.5 mg / L 6-BA + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7. The medium was sterilized at 121°C for 15 minutes.

[0095] Table 4. Effects of different hormone concentrations on somatic embryo germination

[0096]

[0097] The statistical results in Table 4 show that the addition of 0.5 mg / L 6-BA resulted in the highest somatic embryo germination rate during the somatic embryo germination stage. 60% of the plants were able to develop into seedlings with true leaves.

[0098] The somatic embryo germination medium was: modified Blaydes' basal medium + 0.5 mg / L 6-BA + 40 g / L sucrose + 2.6–2.7 g / L plant gel, pH 5.6–5.7. The medium was sterilized at 121°C for 15 minutes.

[0099] Example 5: Culture of regenerated plants using leaves as explants

[0100] 1. Callus induction culture

[0101] Except for cutting off the tender leaves of the 8-month-old hybrid Liquidambar formosana test-tube seedlings, cutting them into uniform small leaves, and then laying them flat in a petri dish containing callus induction medium with the upper surface of the leaves in contact with the medium, the rest is the same as in Example 1.

[0102] After 30 days of dark culture, white, nearly spherical, and dense primary callus tissue was obtained.

[0103] 2. Callus proliferation culture

[0104] Except for separating the primary callus obtained from leaf induction culture from the explant leaves and transferring it into the callus proliferation medium, the rest is the same as in Example 2.

[0105] After three subcultures, callus tissue with a slightly yellow color and obvious granules was obtained.

[0106] 3. Mature cultivation

[0107] The obtained callus tissue was transferred into somatic embryo maturation medium and cultured under the same conditions as in Example 3 to induce the development of somatic embryos; ultimately, no somatic embryos were induced.

[0108] Example 6: Regeneration of plants using stem segments as explants

[0109] 1. Callus induction culture

[0110] Except for cutting off the tender stem segments of the 8-month-old hybrid Liquidambar formosana test-tube seedlings, cutting them into uniform small segments with scissors, and then inoculating them into a culture dish containing callus induction medium, the rest is the same as in Example 1.

[0111] After 30 days of dark culture, primary callus tissue was obtained that was white, had high water content, was dense, and was difficult to separate from the explant with tweezers.

[0112] The primary callus obtained from stem segment induction culture was non-embryonic callus. Embryonic callus should be granular and easily separable from the explant. Furthermore, stem segments require a high number of in vitro seedlings as explants; therefore, stem segments were not used in subsequent experiments.

[0113] Therefore, the callus tissue obtained by induction culture of leaves and stem segments is non-embryonic callus tissue, and regenerated plants cannot be obtained through somatic embryogenesis using leaves and stem segments as explants.

[0114] Non-woody plants have relatively low cell differentiation levels, making somatic embryogenesis induction relatively easy. Woody plants, on the other hand, have higher cell differentiation levels, especially in non-embryonic organs such as leaves, petioles, and stem segments, which are in a very complex differentiation stage. Induction of somatic embryogenesis is more difficult, and the induction of somatic embryogenesis is greatly influenced by genotype in different woody plants.

[0115] Although leaves, petioles, and stem segments are all mature vegetative organs, their appearance, cellular structure, and internal gene expression differ. Through repeated and rigorous experiments, the inventors selected petioles as explants for somatic embryogenesis, creatively obtaining regenerated hybrid Liquidambar formosana plants.

[0116] The above embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A method for obtaining regenerated plants from hybrid Liquidambar formosana petioles via somatic embryogenesis, characterized in that, The steps are performed in the following order: 1) Under sterile conditions, tender petioles of hybrid Liquidambar seedlings cultured for 7-8 months were cut and inoculated into callus induction medium for callus induction culture to obtain primary callus. The callus induction medium was: modified Blaydes' basal medium + 1.0-2.0 mg / L 2,4-D + 0.25-0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7; 2) Under sterile conditions, the obtained primary callus tissue was detached from the explant using forceps and transferred to callus proliferation culture medium for callus proliferation culture to obtain embryogenic callus tissue. The callus proliferation culture medium was: modified Blaydes' basal medium + 0.5-1.0 mg / L 2,4-D + 0.25-0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7; or modified Blaydes' basal medium + 0.5-1.0 mg / L NAA + 0.25-0.50 mg / L 6-BA + 1.0 g / L hydrolyzed casein + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.7; 3) Under sterile conditions, embryogenic callus tissue was transferred into somatic embryo maturation medium for somatic embryo maturation culture to obtain mature somatic cotyledon embryos. The somatic embryo maturation medium was: modified Blaydes' basal medium + 1 g / L activated carbon + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.

7. 4) Under sterile conditions, mature somatic embryos are transferred to somatic embryo germination medium for germination culture to obtain regenerated plants. The somatic embryo germination medium is: modified Blaydes' basal medium + 0.5 mg / L 6-BA + 40 g / L sucrose + 2.6-2.7 g / L plant gel, pH 5.6-5.

7. The modified Blaydes' basal culture medium formula is as follows:

2. The method as described in claim 1, characterized in that, The culture conditions for callus proliferation culture described in step 2) are: dark culture at 23-27℃, subcultured every 25-30 days, for a total of 2-3 subcultures.

3. The method as described in claim 1 or 2, characterized in that, The culture conditions for callus induction culture in step 1) are: 23-27℃, dark culture for 28-35 days.

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