Efficient in-vitro rapid propagation system for populus hopeiensis and construction method of efficient in-vitro rapid propagation system

Abstract

The invention discloses an efficient in-vitro rapid propagation system of populus hopeiensis and a construction method thereof, the construction method comprises the steps of explant starting culture, stem multiplication culture, rooting culture and tissue culture seedling transplanting, the explant is a semi-lignified twig of populus hopeiensis, and the explant is disinfected by ethanol and sodium hypochlorite; an MS culture medium is adopted in the explant starting culture, and 0.1-0.3 mg / L of 6-BA and 0.05-0.2 mg / L of NAA are added; the stem proliferation culture adopts an MS culture medium, and 0.2-0.4 mg / L of 6-BA and 0-0.2 mg / L of NAA are added; a 1 / 2 MS culture medium is adopted for rooting culture, IBA with the concentration being 0.2-0.4 mg / L is added, and mixed nutrient soil for tissue culture seedling transplanting is prepared from turfy soil, perlite and vermiculite with the ratio being 2: 1: 1. The bottleneck of the traditional breeding technology is broken through, the breeding period is shortened, and excellent characters are kept; efficient propagation of populus hopeiensis with different genotypes (including female plants and hybrid male plants) is realized; the method is suitable for large-scale production and lays a foundation for genetic transformation.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, in particular to a high-efficiency in vitro rapid propagation system of Hebei Populus and a construction method thereof. Background Art

[0002] Poplars from Hebei Province are a major native tree species in Northwest my country, possessing irreplaceable value in ecological restoration, urban greening, and timber forest development. However, the propagation and promotion of this species have long been hampered by multiple technical bottlenecks: First, its limited seedling propagation capacity. Existing Poplars from Hebei Province are mostly female, with no natural males found, making intraspecific hybridization virtually impossible to obtain high-quality clones. Furthermore, Poplars from Hebei Province exhibit significant heterozygosity between homologous chromosomes within their cells, leading to severe imbalance in homologous chromosome segregation during meiosis in megaspore mother cells of female plants and severe embryonic abortion, hindering research on hybridization of Poplars from Hebei Province. Second, traditional asexual propagation is inefficient. Commonly used techniques such as cuttings and grafting are subject to significant seasonal restrictions, resulting in rooting rates generally below 30%. This is particularly true for female plants and hybrid offspring, as endogenous hormone imbalances in the cuttings can lead to excessive callus proliferation and impaired root development. Furthermore, long-term asexual propagation can lead to species degradation and reduced seedling resistance, further increasing seedling production costs and making it impossible to meet the needs of large-scale seedling production. Third, the tissue culture technology system is imperfect. In recent years, tissue culture systems for poplars (such as Populus tomentosa and Populus xinjiangensis) have been extensively studied, but this has largely focused on single genotypes. Systematic research across different genotypes and sexes (female and male) is relatively scarce. Explant germination rates, proliferation coefficients, and rooting abilities vary significantly between genotypes. For example, under identical culture conditions, the germination rate of explants from natural female BT1 and hybrid male BT8 can differ by as much as 53.3%. Furthermore, existing culture medium formulations often suffer from inappropriate hormone ratios: high cytokinin concentrations can easily lead to vitrification of explants, and a single auxin concentration cannot adapt to diverse genotypes. This results in inconsistent tissue culture seedling quality and insufficient stability for large-scale production. Fourth, industrial applications face cost barriers. In traditional tissue culture, the propagation process typically involves dedifferentiating plant organs into callus tissue, which is then redifferentiated to produce adventitious buds. This process is complex, with long propagation cycles and high seedling costs. Consequently, existing technologies are unable to meet the modern forestry industry's demand for cutting-edge technologies such as rapid propagation of Hebei poplar varieties and gene editing.

[0003] In summary, the key to promoting the transformation of its ecological and economic value is to establish an efficient, stable, and multi-genotype in vitro rapid propagation system for Hebei Poplar, break through the genotype limitations of traditional propagation techniques, reduce seedling costs, and improve seedling consistency. This invention addresses these pain points by optimizing explant processing, hormone ratios, and culture conditions to establish a standardized technology system for the entire process, laying the foundation for the genetic improvement and industrial application of Hebei Poplar. Summary of the Invention

[0004] In response to the above problems, the present invention provides an efficient in vitro rapid propagation system for Hebei Poplar and a construction method thereof, which realizes the rapid propagation and large-scale production of Hebei Poplar varieties by optimizing the culture medium formula and culture conditions.

[0005] According to one object of the present invention, the present invention provides a method for constructing an efficient in vitro rapid propagation system for Hebei Poplar, comprising the steps of explant initiation culture, stem segment proliferation culture, rooting culture, and tissue culture seedling transplantation, wherein the explant is a semi-lignified young branch of Hebei Poplar, and the explant is disinfected with ethanol and sodium hypochlorite; the explant initiation culture adopts MS medium supplemented with 0.1-0.3 mg / L 6-BA and 0.05-0.2 mg / L NAA; the stem segment proliferation culture adopts MS medium supplemented with 0.2-0.4 mg / L 6-BA and 0-0.2 mg / L NAA; the rooting culture adopts 1 / 2 MS medium supplemented with 0.2-0.4 mg / L NAA, and the mixed nutrient soil composition for transplanting the tissue culture seedling is: peat soil: perlite: vermiculite = 2:1:1.

[0006] Furthermore, the optimal culture medium for initiating culture of the explants is: MS + 0.1 mg / L 6-BA + 0.05 mg / L NAA.

[0007] Furthermore, the optimal culture medium for the stem segment proliferation culture is: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA.

[0008] Furthermore, the optimal culture medium for the rooting culture is: 1 / 2 MS + 0.2 mg / L IBA or 1 / 2 MS + 0.4 mg / L IBA.

[0009] According to another object of the present invention, the present invention provides an efficient in vitro rapid propagation system for Hebei Poplar constructed by the above method, including a priming culture medium, a proliferation culture medium, a rooting culture medium and supporting culture conditions; the priming culture medium is used to induce Hebei Poplar explants to sprout, with an average germination rate ≥83.3%; the proliferation culture medium is used for adventitious bud proliferation, with an average proliferation coefficient ≥5.3; the rooting culture medium is used to induce adventitious roots, with an average rooting rate ≥100.0% and an average number of adventitious roots ≥7.3.

[0010] Furthermore, the rapid propagation system is suitable for in vitro rapid propagation of female Populus Hebeiensis plants (such as BT1 and BT3) and hybrid male plants (such as BT8).

[0011] The beneficial effects of the present invention are: The present invention breaks through the bottleneck of traditional breeding technology, shortens the breeding cycle, and maintains excellent traits; realizes the efficient reproduction of Hebei poplars with different genotypes (including female plants and hybrid male plants); reduces the cost of seedling cultivation, is suitable for large-scale production, and lays the foundation for genetic transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the rapid in vitro propagation growth of Populus Hebeiensis in Example 1 of the present invention; a. explant initiation culture; b. stem segment proliferation culture; c. sterile seedling rooting culture.

[0013] Figure 2 This is a histogram showing the effects of different treatment combinations on the germination rate of Hebei Poplar explants in initiation culture according to Example 2 of the present invention.

[0014] Figure 3 This is a histogram showing the effects of different treatment combinations on the proliferation coefficient of Hebei Populus stem segments in value-added culture according to Example 3 of the present invention.

[0015] Figure 4 This is a histogram showing the effects of different IBA concentrations on the rooting rate of Hebei Populus in rooting culture in Example 4 of the present invention.

[0016] Figure 5 This is a histogram showing the effects of different IBA concentrations on the number of adventitious roots of Hebei Populus in rooting culture according to Example 4 of the present invention.

[0017] Figure 6 This is a diagram showing the growth status of the Hebei Poplar tissue culture seedlings after transplantation according to Example 4 of the present invention. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] Example 1 A method for constructing an efficient in vitro rapid propagation system of Hebei Populus, comprising the following steps: Explant initiation culture: Use MS medium containing 6-BA and NAA to screen out the medium combination with high germination rate; Stem segment proliferation culture: By adjusting the concentration ratio of 6-BA and NAA, the adventitious bud proliferation coefficient can be increased; Rooting culture: Use IBA to induce adventitious root formation and optimize the rooting medium; Transplanting of tissue culture seedlings: Improve the survival rate of tissue culture seedlings by hardening the seedlings and transplanting them in mixed nutrient soil.

[0020] like Figure 1 As shown in FIG. 1 , this is a diagram showing the rapid in vitro propagation growth of Populus Hebei in Example 1; wherein, a is the explant initiation culture; b is the stem segment proliferation culture; and c is the sterile seedling rooting culture.

[0021] Example 2 A method for constructing an efficient in vitro rapid propagation system of Hebei Populus, comprising the following steps: Initiation of culture of Populus Hebei explants: Materials: Newly sprouted semi-lignified shoots of one-year-old seedlings of genotypes BT1 (natural female plant), BT3 (natural female plant), and BT8 (male hybrid of Hebei Populus × Xinjiang Populus); Methods: Newly sprouted semi-lignified shoots of one-year-old seedlings were collected, rinsed with tap water for 30 min, disinfected with 75% ethanol for 30 s, washed three times with sterile water, disinfected with 1% sodium hypochlorite for 20 min, and washed five times with sterile water. The shoots were then cut into 2 cm single-bud stem segments and inoculated into MS medium supplemented with different concentrations of 6-BA (0, 0.1, and 0.3 mg / L) and NAA (0, 0.05, and 0.2 mg / L) for initiation culture. A total of 9 treatment combinations were used, and each treatment was replicated three times (see Table 1).

[0022] Table 1. Hebei Poplar explant initiation culture medium Culture conditions: temperature 25°C, light intensity 2000 lx, photoperiod 14 h light / 10 h dark. After 30 days of culture, the germination rate of Hebei poplar explants was calculated. Data analysis: Excel and IBM SPSS Statistics 20 statistical analysis software were used for data analysis. Significance of differences was analyzed using the general linear model (GLM), and multiple comparisons were tested using the least significant difference (LSD) method. Percentage data were square-root arcsine transformed before analysis of significance and multiple comparisons. Table 2 Multiple comparisons of germination rates at different hormone concentrations Note: Data represent the mean ± SD of three replicates; different lowercase letters indicate significant differences at the 0.05 level based on LSD, the same below.

[0023] Results: As Figure 2As shown in the results, BT1 had the highest germination rate (83.3±5.8%) in treatment 5 (0.1 mg / L 6-BA + 0.05 mg / L NAA); BT3 had the lowest germination rate (13.3±5.8%) in treatment 7 (0.3 mg / L 6-BA). The results of variance analysis and multiple comparisons (Table 2) showed that the optimal starting medium was: MS + 0.1 mg / L 6-BA + 0.05 mg / L NAA.

[0024] Example 3 A method for constructing an efficient in vitro rapid propagation system of Hebei Populus, comprising the following steps: Proliferation culture of Hebei Populus stem segments: Materials: Sterile seedling stem segments (2 cm single buds) obtained in Example 2; Methods: 2 cm single bud stem segments were cut and inoculated into MS medium supplemented with different concentrations of 6-BA (0.2, 0.3 and 0.4 mg / L) and NAA (0, 0.05, 0.1 and 0.2 mg / L) for proliferation culture. A total of 12 treatment combinations were used, and each treatment was repeated 3 times (see Table 3).

[0025] Table 3 Proliferation culture medium for Hebei Populus stem segments Culture conditions: Same as in Example 2, and the proliferation coefficient was calculated after 30 days of culture; Data processing: same as Example 2; Table 4 Multiple comparisons of proliferation coefficients at different hormone concentrations Results: As Figure 3 As shown in the results, BT8 had the highest proliferation coefficient (5.3±0.5) in treatment 8 (0.3 mg / L 6-BA + 0.1 mg / L NAA); BT3 had the lowest proliferation coefficient (0.9±0.3) in treatment 3 (0.4 mg / L 6-BA). The results of variance analysis and multiple comparisons (Table 4) showed that the optimal proliferation medium was: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA.

[0026] Example 4 A method for constructing an efficient in vitro rapid propagation system of Hebei Populus, comprising the following steps: Rooting culture and transplanting of Hebei Populus Materials: Adventitious shoots grown to 2 cm in Example 3; Methods: Rooting culture: 2 cm adventitious buds were cut and inoculated into blank control medium and 1 / 2 MS medium supplemented with 0.2, 0.4, 0.6, 0.8, and 1.0 mg / L IBA for rooting. Six treatment combinations were used, with three replicates for each treatment. Transplanting of tissue-cultured seedlings: Rooted Populus Hebeiense seedlings cultured on rooting medium for 30 days were selected and, after 7 days of hardening, transplanted into plastic pots containing a nutrient soil mixture (peat soil: perlite: vermiculite = 2:1:1) and transferred to the greenhouse for cultivation.

[0027] Culture conditions: Same as in Example 2. After 30 days of culture, the rooting rate and number of adventitious roots were counted. Data processing: same as Example 2.

[0028] Table 5 Multiple comparisons of rooting rate and adventitious root number at different IBA concentrations Results: As Figure 4 and Figure 5 As shown in the results, BT1 had the highest rooting rate (100%) in 0.2 mg / L IBA, with an average number of adventitious roots of 7.3; BT8 had the highest rooting rate (90%) in 0.4 mg / L IBA, with an average number of adventitious roots of 4.5. Analysis of variance and multiple comparisons showed (Table 5) that the optimal rooting medium was 1 / 2 MS + 0.2 mg / L IBA or 1 / 2 MS + 0.4 mg / L IBA. The survival rate of tissue culture seedlings after transplantation reached over 95.56% after 7 days of hardening.

[0029] like Figure 6 The following is a graph showing the growth of the Hebei Poplar tissue culture seedlings after transplantation in Example 4; wherein, a is the growth of the Hebei Poplar tissue culture seedlings 7 days after transplantation; b is the growth of the Hebei Poplar tissue culture seedlings 90 days after transplantation. This invention addresses the traditional propagation difficulties of Hebei Poplar and the shortcomings of existing tissue culture technology. Through multi-dimensional technological innovation, it realizes the construction of an efficient in vitro rapid propagation system. Its core innovations are as follows: 1. Breaking through genotype limitations and covering multiple types of Hebei poplar For the first time, a systematic in vitro rapid propagation study was conducted on natural female plants (BT1, BT3) and hybrid male plants (BT8) of Hebei Poplar, revealing significant differences in explant germination rate, proliferation coefficient and rooting ability among different genotypes, and optimizing the culture medium formula in a targeted manner.

[0030] Existing technologies mostly focus on single-genotype or single-sex Poplar species. The method of the present invention realizes the universal reproduction of male and female plants and hybrid offspring, especially solving the bottleneck of genetic improvement caused by the lack of natural male plants, laying the foundation for the large-scale utilization of Hebei Poplar seed resources.

[0031] 2. Optimize hormone ratios and establish a precise culture medium system 1. Start-up cultivation stage: low-concentration hormone synergistic effect A two-factor completely randomized trial screened out a starting culture medium combination of 0.1 mg / L 6-BA + 0.05 mg / L NAA. Compared with traditional single hormones or high-concentration combinations (such as 0.3 mg / L 6-BA), the germination rate increased by 50%-60% (for example, the BT1 germination rate increased from 23.3% to 83.3%).

[0032] Breaking through the conventional understanding that "high concentrations of cytokinins promote germination", the synergistic effect of low concentrations of 6-BA and NAA is utilized to significantly improve the activity of explants while avoiding the vitrification phenomenon, significantly shortening the period during which explants start germination.

[0033] 2. Proliferation culture stage: dynamic regulation of hormone ratios 0.3 mg / L 6-BA + 0.1 mg / L NAA was determined to be the optimal proliferation medium, with a proliferation coefficient of 5.3 (BT8), an increase of 212% compared with the blank control (1.7), and no bud deformity or vitrification caused by high concentrations of 6-BA occurred.

[0034] By balancing the concentrations of cytokinin and auxin (6-BA:NAA=3:1), it promotes the massive proliferation of adventitious buds while also promoting their effective growth, achieving simultaneous optimization of "bud proliferation-bud elongation", breaking through the contradiction between proliferation and growth in existing technologies, and providing high-quality seedlings for subsequent rooting.

[0035] 3. Rooting stage: dual-concentration IBA for suitable genotypes It was found that the differentiated rooting schemes of 0.2 mg / L IBA (applicable to female plants BT1 and BT3) and 0.4 mg / L IBA (applicable to hybrid male plants BT8) had a rooting rate of up to 100% and an average number of adventitious roots of 7.3, which were significantly improved compared with the hormone-free control (rooting rate 56.7%-83.3%, number of roots 2.6-5.4). In addition, the sterile seedlings grew very vigorously during the rooting culture stage, with a significant increase in plant height, fully expanded leaves with dark green color, a well-developed root system, a large number of lateral roots and even distribution, and uniform growth of the seedlings after transplanting, with good seedling quality.

[0036] It breaks through the limitations of traditional breeding technology on different genotypes, and solves the problem of large differences in rooting ability under different genetic backgrounds through precise matching of genotype-hormone concentration. In particular, it improves the rooting efficiency of hybrid offspring (such as BT8), reduces seedling cultivation costs and improves seedling consistency, laying the foundation for the genetic improvement and industrial application of Hebei poplar.

[0037] 3. Integrate efficient breeding processes to reduce industrialization costs This technique employs a direct induction of adventitious bud regeneration from stem axillary buds, eliminating the cumbersome traditional process of first dedifferentiating the isolated organ to form callus tissue and then inducing adventitious bud differentiation. This technique significantly shortens the proliferation cycle while increasing the adventitious bud multiplication coefficient, reducing seedling production costs for the efficient production of improved Hebei poplar varieties.

[0038] Existing forest tissue culture technology often leads to high costs due to losses in the propagation process. The present invention significantly shortens the propagation cycle of tissue culture seedlings by optimizing the propagation process, while finely adjusting the hormone ratio to significantly reduce the vitrification phenomenon of tissue culture seedlings, providing technical connection for subsequent low-cost and rapid propagation, and providing standardized receptor materials for Hebei poplar genetic transformation (such as gene editing and stress resistance gene introduction). The overall seedling cost is reduced by 30%-50% compared with traditional methods.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an efficient in vitro rapid propagation system for Populus Hebei, characterized in that: The method comprises the steps of explant initiation culture, stem segment proliferation culture, rooting culture and tissue culture seedling transplanting. The explant is a semi-lignified young branch of Hebei poplar, and the explant is disinfected with ethanol and sodium hypochlorite. The explant initiation culture adopts MS medium supplemented with 0.1-0.3 mg / L 6-BA and 0.05-0.2 mg / L NAA; the stem segment proliferation culture adopts MS medium supplemented with 0.2-0.4 mg / L 6-BA and 0-0.2 mg / L NAA; the rooting culture adopts 1 / 2 MS medium supplemented with 0.2-0.4 mg / L IBA, and the mixed nutrient soil composition for transplanting the tissue culture seedling is: peat soil: perlite: vermiculite = 2:1:

1.

2. The method for constructing an efficient in vitro rapid propagation system of Populus Hebei according to claim 1, characterized in that: The optimal culture medium for initiating explant culture is: MS + 0.1 mg / L 6-BA + 0.05 mg / L NAA.

3. The method for constructing an efficient in vitro rapid propagation system of Populus Hebei according to claim 1, characterized in that: The optimal culture medium for the stem segment proliferation culture is: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA.

4. The method for constructing an efficient in vitro rapid propagation system of Populus Hebei according to claim 1, characterized in that: The optimal culture medium for the rooting culture is: 1 / 2 MS + 0.2 mg / L IBA or 1 / 2 MS + 0.4 mg / L IBA.

5. The efficient in vitro rapid propagation system of Populus Hebeiensis constructed according to any one of the methods of claims 1-4, characterized in that: It includes a priming culture medium, a proliferation culture medium, a rooting culture medium and supporting culture conditions; the priming culture medium is used to induce Hebei poplar explants to sprout, with an average germination rate of ≥83.3%; the proliferation culture medium is used to proliferate adventitious buds, with an average proliferation coefficient of ≥5.3; the rooting culture medium is used to induce adventitious roots, with an average rooting rate of ≥100.0% and an average number of adventitious roots of ≥7.

3.

6. The efficient in vitro rapid propagation system of Populus Hebei according to claim 5, characterized in that: The rapid propagation system is suitable for in vitro rapid propagation of female Populus Hebeiensis plants (such as BT1 and BT3) and hybrid male plants (such as BT8).

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