Regeneration method of larix olgensis
By using mature zygotic embryos of Changbai larch as explants, a highly efficient and rapid regeneration system was established, which solved the problems of long breeding cycle and difficulty in rooting and transplanting of Changbai larch, and achieved the acquisition of highly efficient regenerated plants, laying the foundation for genetic improvement and industrial application.
Patent Information
- Application Number
- CN202512029568.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-01-30
AI Technical Summary
The regeneration system of Changbai larch has problems such as long breeding cycle, low efficiency, and difficulty in rooting and transplanting, which limits genetic improvement and industrial application.
Using mature zygotes of Changbai larch as explants, an efficient and rapid regeneration system was established through steps such as callus induction, adventitious bud induction, adventitious bud elongation, rooting culture, and transplantation. Tissue culture was carried out using specific culture media and light conditions.
It has achieved efficient and rapid regeneration of Changbai larch, improved rooting and transplanting rates, shortened the regeneration cycle to about 6 months, and provided technical support for genetic improvement and industrial application.
Smart Images
Figure CN121420892A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of tissue culture technology, and in particular to a method for the regeneration of Changbai larch. Background Technology
[0002] Larch is a member of the Pinaceae family ( Pinaceae Larch (genus) Larix Larch is a coniferous tree species, comprising 18 species, mainly distributed in temperate mountainous areas, cold-temperate plains, and alpine climate zones of the Northern Hemisphere. In my country, larch is mainly distributed in the high-altitude areas of the Greater and Lesser Khingan Mountains in Northeast China, North China, Northwest China, and Southwest China, forming large areas of pure or mixed forests. Larch is not only an essential tree species for short-cycle industrial timber forests in high-altitude areas, but also a major species for afforestation and forest regeneration in barren mountainous areas. Due to the increasing scarcity of global forest resources, the genetic improvement of larch species has attracted much attention. Larch species have high heterozygosity, large offspring variability, and long breeding cycles, and traditional breeding methods cannot meet the current needs of genetic improvement. Therefore, establishing its regeneration system is of great significance for variety improvement.
[0003] Plant somatic embryogenesis has significant advantages such as rapid growth, large number of explants, and high regeneration rate. Larch somatic embryogenesis mainly uses immature embryos as material, proceeding through stages including induction of embryogenic callus, subculture, somatic embryogenesis, germination, and plant regeneration. Currently, stable regeneration systems have been obtained through the somatic embryogenesis pathway for species such as European-Japanese hybrid larch, European larch, North China larch, Japanese larch, Dahurian larch, Changbai larch, and American western larch. However, the immature zygotic embryo stage in the annual cycle is relatively short, thus severely limiting the source of explants.
[0004] Compared to immature zygotic embryos, mature zygotic embryos have a wider range of explant sources, such as hypocotyls and needles, making the materials easier to obtain, but the practical application cycle is longer. Existing studies using mature zygotic embryos from hybrid larch, North China larch, and Xing'an larch as explants for adventitious bud induction and elongation to obtain complete regenerated plants have shown low efficiency. Studies using Changbai larch needles and mature zygotic embryos as explants have investigated direct adventitious bud induction, elongation, and adventitious root induction, constructing a direct adventitious bud generation system from larch needles, ultimately leading to successful obtaining rooted larch seedlings. However, this approach faces challenges such as severe explant browning, difficulty in callus differentiation, rooting difficulties, vitrification of regenerated plants, long cycle, and low efficiency. Currently, larch is widely recognized as one of the tree species most difficult to tissue culture and genetically transform, significantly limiting precise and efficient genetic improvement of intrinsic traits. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a regeneration method for Larix gmelinii. This invention establishes an efficient and rapid regeneration system for Larix gmelinii, using mature zygotic embryos of Larix gmelinii as starting explants to develop a novel regeneration culture system for Larix gmelinii. This solves the problem of the long breeding cycle of this species, creates technical means for the propagation of superior varieties, and lays the foundation for subsequent genetic transformation and targeted gene improvement research and application.
[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for regenerating Changbai larch, comprising the following steps: 1) Callus induction was performed on mature zygotes of Changbai larch to obtain callus tissue; 2) Induce adventitious shoots from the callus tissue described in step 1) to obtain adventitious shoots; 3) Place the adventitious buds from step 2) in DCR medium for elongation culture, and then place them in elongation medium for further culture to obtain plants that have grown stems; 4) The plantlets that have grown stems as described in step 3) are subjected to rooting culture, and then placed in DCR medium to continue rooting culture to obtain regenerated larch plants. 5) Transplant the regenerated larch plants described in step 4).
[0007] Preferably, the culture medium used for callus induction in step 1) is: DCR medium + 0.5 mg / L 6-BA; The conditions for callus induction included: a time of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
[0008] Preferably, the culture medium used for adventitious bud induction in step 2) is: BM medium + 0.1 mg / L 2,4-D + 0.05 mg / L 6-BA + 0.05 mg / L KT; The conditions for inducing adventitious buds include: a duration of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
[0009] Preferably, the conditions for the elongation culture in step 3) include: an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16h light / 8h darkness; The elongation medium consisted of 1.64 g / L DCR basal salt (Phytotech-D146) + 0.2 g / L inositol (Biotopped-I6050D) + 30 g / L sucrose (Xilong-XL0009), without the addition of hormones; Preferably, the conditions for culturing in the elongation medium in step 3) include: an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h darkness.
[0010] Preferably, the culture medium used in step 4) for rooting culture is: DCR medium + 3 mg / L IBA + 1 mg / L NAA; The conditions for rooting culture included: a duration of 50 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
[0011] Preferably, the culture medium used for the continued rooting culture is DCR medium without added hormones; the conditions for the continued rooting culture include: a time of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16h light / 8h darkness.
[0012] Preferably, the substrate for transplanting in step 5) includes vermiculite and slag, wherein the volume ratio of vermiculite to slag is 1:2.
[0013] This method uses mature zygotic embryos of the superior larch variety Changbai Mountain as explants for tissue culture, successively undergoing five stages: callus induction, adventitious bud induction, adventitious bud elongation, adventitious root induction, and transplanting of rooted seedlings, ultimately obtaining complete regenerated larch seedlings. This method addresses the shortcomings of existing Changbai larch regeneration systems, focusing on overcoming the long-standing difficulties in rooting and transplanting. It establishes a complete, efficient, and rapid regeneration method using mature zygotic embryos of Changbai larch as explants, successfully improving the Changbai larch regeneration system and laying a solid foundation for variety improvement.
[0014] The invention lies in the fact that it uses the mature zygotic embryo of the superior variety of Changbai larch as an explant and has invented a complete culture method for cultivating it into rooted soil-grown seedlings. The entire culture process takes 6 months. In particular, it has invented a culture method that induces rooting of tissue culture seedlings by combining two culture media, which has achieved a breakthrough in tissue culture work.
[0015] The beneficial effects of this invention are: Existing regeneration systems for Changbai larch are characterized by long cycles and low efficiency. This invention, through tissue culture technology, successfully induces rooted seedlings from mature zygotes of Changbai larch through a series of induction processes, leading to successful transplantation. This represents a significant improvement in the regeneration system for tissue-cultured seedlings of superior Changbai larch varieties. This technology offers several advantages. Firstly, it overcomes the limitations of difficult rooting and transplanting in Changbai larch tissue culture regeneration systems, achieving a highly efficient and rapid regeneration system with a cycle of only about 6 months, yielding complete regenerated plants. Furthermore, it improves callus induction, adventitious bud induction and elongation, rooting rate, and transplanting rate. Secondly, it provides technical support for subsequent genetic improvement and industrial application of Changbai larch trees, offering a feasible solution for the cultivation of valuable new larch varieties and representing a breakthrough in larch propagation technology. This invention solves the problems of slow cycles and low efficiency in Changbai larch regeneration systems, and has significant value and provides a reference method for future basic research, application promotion, and variety improvement. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0017] Figure 1 This describes the efficient regeneration process of Changbai larch plants. Detailed Implementation
[0018] This invention provides a method for regenerating Changbai larch, comprising the following steps: 1) Callus induction was performed on mature zygotes of Changbai larch to obtain callus tissue; 2) Induce adventitious shoots from the callus tissue described in step 1) to obtain adventitious shoots; 3) Place the adventitious buds from step 2) in DCR medium for elongation culture, and then place them in elongation medium for further culture to obtain plants that have grown stems; 4) The plantlets that have grown stems as described in step 3) are subjected to rooting culture, and then placed in DCR medium to continue rooting culture to obtain regenerated larch plants. 5) Transplant the regenerated larch plants described in step 4).
[0019] This invention involves inducing callus tissue from mature zygotes of *Larix decipiens* to obtain callus tissue. The preferred culture medium for callus induction is DCR medium + 0.5 mg / L 6-BA. The preferred conditions for callus induction include: a duration of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h darkness.
[0020] In this invention, the method for obtaining the mature zygotic embryo of the Changbai larch preferably includes: 1. Collection of Changbai larch seeds: In August, collect the cones of Changbai larch of superior varieties cultivated in the seed garden, dry them in the sun, and then take out the seeds for drying treatment. After drying, store the seeds in a refrigerator at -20℃. 2. Seed disinfection: On the first night, take out the Changbai larch seeds stored in the refrigerator, select about 200 mature and plump Changbai larch seeds, bring them to room temperature, and soak them in 100 mL of sterile water overnight. On the second day, pour out the sterile water, transfer the seeds to a new 100 mL of 75% anhydrous ethanol and shake for 3 minutes; pour out the 75% anhydrous ethanol, rinse the seeds once with sterile water, and then transfer the seeds to 100 mL of 5% sodium hypochlorite and shake for 8 minutes; pour out the 5% sodium hypochlorite, rinse the seeds four times with 100 mL of sterile water, 2 minutes each time; after rinsing, pour out the sterile water, transfer the seeds to semi-dry sterile filter paper to moisturize the material; 3. Zygote removal: Under sterile conditions, the seed coat and endosperm are removed with pointed forceps to extract the complete zygote, thus obtaining a mature zygote.
[0021] This invention induces adventitious shoots from the callus tissue to obtain adventitious shoots. In this invention, the preferred culture medium for adventitious shoot induction is: BM medium + 0.1 mg / L 2,4-D + 0.05 mg / L 6-BA + 0.05 mg / L KT. The preferred conditions for adventitious shoot induction in this invention include: a duration of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h darkness.
[0022] This invention involves placing adventitious buds in DCR medium for elongation culture, followed by cultivation in an elongation medium to obtain plants that have grown stems. In this invention, the preferred conditions for the elongation culture include: an ambient temperature of 25°C, humidity of 45%, light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h darkness. The preferred elongation medium is DCR basal salt (Phytotech-D146) 1.64 g / L + inositol (Biotopped-I6050D) 0.2 g / L + sucrose 30 g / L (Xilong-XL0009), without added hormones. In this invention, the preferred conditions for culturing in the elongation medium include: an ambient temperature of 25°C, humidity of 45%, light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h dark.
[0023] This invention involves rooting the plantlets that have sprouted stems, followed by further rooting culture in DCR medium to obtain regenerated larch plants. The preferred rooting culture medium is DCR medium + 3 mg / L IBA + 1 mg / L NAA. The preferred rooting culture conditions include: a duration of 50 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h dark. The preferred conditions for continued rooting culture include: a duration of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 h light / 8 h dark.
[0024] This invention involves transplanting regenerated larch plants. In this invention, the transplanting substrate preferably comprises vermiculite and loose soil, with a volume ratio of vermiculite to loose soil of 1:2.
[0025] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0026] Example 1 A rapid regeneration method using mature zygotic embryos of Changbai larch as explants is described in detail below: (1) Collection of Changbai larch seeds: In August, the cones of Changbai larch of superior varieties cultivated in the seed garden are picked, dried and the seeds are taken out for drying treatment. After drying, the seeds are stored in a -20℃ refrigerator.
[0027] (2) Seed disinfection: On the first night, take out the Changbai larch seeds stored in the refrigerator, select about 200 mature and plump Changbai larch seeds to bring them to room temperature, soak them in 100 mL of sterile water overnight, pour out the sterile water on the second day, transfer the seeds to a new 100 mL of 75% anhydrous ethanol and shake for 3 minutes; pour out the 75% anhydrous ethanol, wash the seeds once with sterile water, and then transfer the seeds to 100 mL of 5% sodium hypochlorite and shake for 8 minutes; pour out the 5% sodium hypochlorite, wash the seeds 4 times with 100 mL of sterile water, 2 minutes each time; after washing, pour out the sterile water and transfer the seeds to semi-dry sterile filter paper to keep the material moist.
[0028] (3) Zygote removal: Under aseptic conditions, the seed coat and endosperm are removed using pointed forceps to extract the complete zygote, thus obtaining a mature zygote. Figure 1 (A)
[0029] (4) Callus induction: The extracted zygotes were placed in DCR medium supplemented with 0.5 mg / L 6-BA and placed in an environment with a temperature of 25℃, humidity of 45%RH, light intensity of about 40 μmol / (m²·s), and a photoperiod of 16h light / 8h dark for callus induction. The callus induction rate was as high as 98%. Figure 1 (B) After one month in the callus induction medium, the zygote swelled into an irregular mass about 1 cm in diameter, with a dark red color and a relatively soft texture. Figure 1 (C)
[0030] (5) Adventitious bud induction: The induced callus tissue was transferred to BM medium supplemented with 0.1 mg / L 2,4D, 0.05 mg / L 6-BA, and 0.05 mg / L KT, and cultured for 1 month in an environment with a temperature of 25℃, humidity of 45%RH, light intensity of about 40 μmol / (m²·s), and a photoperiod of 16h light / 8h darkness to induce the germination of adventitious buds. The adventitious bud induction rate reached 60%. Figure 1 (D), adventitious buds emerge from the callus tissue as small needles ( Figure 1 (E), through continuous growth, gradually grows into clusters of adventitious buds around the callus tissue. Figure 1 (Middle F).
[0031] (6) Adventitious bud elongation: After the adventitious buds germinate, they can be transferred to DCR medium without hormones to obtain the fastest elongation efficiency. When clusters of adventitious buds are transferred to the elongation medium, the vigorous adventitious buds will preferentially elongate out of the stem. Figure 1 (G), then the plants that have grown stems are removed and placed individually on hormone-free DCR medium for culture. They are kept in an environment with a temperature of 25℃, humidity of 45%RH, light intensity of about 40μmol / (m²·s), and a photoperiod of 16h light / 8h dark. After 40 days of elongation culture, the next step of culture can be carried out. Figure 1 (H).
[0032] (7) Adventitious root induction: Rooting culture was carried out on the strains whose stems had grown to a height of 3-4 cm. Figure 1 (I), that is, cultured for 50 days in DCR medium supplemented with 3 mg / L IBA + 1 mg / L NAA at a temperature of 25℃, humidity of 45%RH, light intensity of approximately 40 μmol / (m²·s), and a photoperiod of 16h light / 8h dark. Figure 1 (J), and then all of them were transferred to DCR medium without any hormones, and cultured for 30 days in an environment with a temperature of 25℃, humidity of 45%RH, light intensity of about 40μmol / (m²·s), and a photoperiod of 16h light / 8h dark to obtain rooted regenerated larch plants (J). Figure 1(K), thus achieving a rooting culture rate of up to 20%.
[0033] (8) Transplanting: After the rooted seedlings have grown strong roots, remove the regenerated plants from the tissue culture bottles. After removal, clean the residual culture medium from the roots and take care to protect the root tips. Transplant them into a mixture of vermiculite (V): soil (V) = 1:2. After transplanting, cover the plants to keep them moist, maintaining the humidity inside the cover at 100% RH. After one month, uncover the plants and continue to cultivate them in a greenhouse, i.e., at a temperature of 25℃, humidity of 65% RH, light intensity of about 300 μmol / (m²·s), and a photoperiod of 16h light / 8h darkness. Figure 1 (Middle L), the survival rate of transplanted seedlings is over 80%.
[0034] This invention establishes a rapid regeneration method for improving the efficiency and synchronization of somatic embryogenesis in Changbai larch, encompassing the entire process from mature zygotic embryos to rooted seedling transplantation. It achieves a highly efficient and rapid tissue culture regeneration system using mature zygotic embryos as explants, significantly improving callus induction, adventitious bud induction and elongation, rooting rate, and transplanting rate. Complete regenerated plants can be obtained in approximately 6 months. This invention overcomes the technical barriers to rooting and transplanting in larch regeneration systems, providing a reliable method for advancing genetic improvement research and industrial applications, and has significant value for both theoretical exploration and practical application in related fields.
[0035] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for regenerating Changbai larch, characterized in that, Includes the following steps: 1) Callus induction was performed on mature zygotes of Changbai larch to obtain callus tissue; 2) Induce adventitious shoots from the callus tissue described in step 1) to obtain adventitious shoots; 3) Place the adventitious buds from step 2) in DCR medium for elongation culture, and then place them in elongation medium for further culture to obtain plants that have grown stems; 4) The plantlets that have grown stems as described in step 3) are subjected to rooting culture, and then placed in DCR medium to continue rooting culture to obtain regenerated larch plants. 5) Transplant the regenerated larch plants described in step 4).
2. The regeneration method according to claim 1, characterized in that, Step 1) The culture medium used for callus induction is: DCR medium + 0.5 mg / L 6-BA; The conditions for callus induction included: a time of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
3. The regeneration method according to claim 1, characterized in that, Step 2) The culture medium used for adventitious bud induction is: BM medium + 0.1 mg / L 2,4-D + 0.05 mg / L 6-BA + 0.05 mg / L KT; The conditions for inducing adventitious buds include: a duration of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
4. The regeneration method according to claim 1, characterized in that, Step 3) The conditions for the elongation culture include: a time of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness; The elongation medium consisted of 1.64 g / L DCR basal salt + 0.2 g / L inositol + 30 g / L sucrose, without the addition of hormones.
5. The regeneration method according to claim 1, characterized in that, Step 3) The conditions for culturing in the elongation medium include: subculturing the elongation line once every 30 days, the ambient temperature is 25℃, the humidity is 45%, the light intensity is 40μmol / (m²·s), and the photoperiod is 16h light / 8h dark.
6. The regeneration method according to claim 1, characterized in that, Step 4) The rooting culture medium used is: DCR medium + 3 mg / L IBA + 1 mg / L NAA; The conditions for rooting culture included: a duration of 50 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
7. The regeneration method according to claim 1, characterized in that, Step 4) The culture medium used for the continued rooting culture is: basal DCR medium, without added hormones; The conditions for continued rooting culture include: a duration of 30 days, an ambient temperature of 25°C, a humidity of 45%, a light intensity of 40 μmol / (m²·s), and a photoperiod of 16 hours of light / 8 hours of darkness.
8. The regeneration method according to claim 1, characterized in that, Step 5) The transplanting substrate includes vermiculite and soil, and the volume ratio of vermiculite to soil is 1:2.