A method for in-situ expansion of taxus cell and a method for in-vitro induction of root system of taxus cell

Abstract

The present application belongs to the field of plant tissue culture, and specifically discloses a taxus chinensis stem cell in-situ amplification method and a root in-vitro induction method. The present application realizes taxus chinensis stem cell in-situ amplification by orders of magnitude, and successfully establishes efficient in-vitro induction of taxus chinensis roots, thereby breaking through the technical bottleneck of tissue culture and rapid propagation of the species. The present application is expected to produce taxus chinensis roots in-vitro, and the roots are rich in high content of paclitaxel, thereby solving the problem of natural paclitaxel drug source.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture, specifically relating to a method for in situ expansion of Taxus chinensis stem cells and a method for in vitro induction of their roots. Background Technology

[0002] The yew, also known as the purple yew, belongs to the genus Taxus in the family Taxaceae. Taxus This plant is an ancient relict coniferous tree species, a national first-class protected plant, with a beautiful tree shape and bright fruits. It is an endangered species with good landscape effect and high ecological value.

[0003] The yew tree is rich in paclitaxel, a broad-spectrum anticancer active ingredient, and has extremely high medicinal value. In 1992, the U.S. Food and Drug Administration (FDA) officially approved paclitaxel as a clinical anticancer drug. However, the paclitaxel content in yew trees is extremely low; the highest content in the bark of the European shortleaf yew is only 0.069%, meaning that 3 to 12 century-old trees are needed to treat one ovarian cancer patient. The yew tree has become a "golden tree," a rare and endangered plant, and is under the key protection of 42 countries worldwide. In my country, the felling of wild yew trees is strictly prohibited.

[0004] Due to the low efficiency of seed propagation and the low survival rate of cuttings (Han Caixia, Xu Zhen, Qi Xiaoyang, Deng Guanghua. 2018. Research on the current status of propagation of Taxus species [J]. Modern Horticulture, (15): 35-37.), the propagation and industrial utilization of yew germplasm resources are severely limited. Plant tissue culture technology is the most widely used scientific research technology in the field of biotechnology today. It can carry out large-scale propagation of plant explant materials, enabling the propagation of thousands of high-quality seedlings of high-quality varieties in a short period of time. However, yew still lacks an effective in vitro rapid propagation technology system. The main difficulties are the large number of endophytic bacteria, the difficulty in preparing explant materials, the low propagation coefficient, and the difficulty in rooting. The inventors of this patent, Zhu Mulan et al., previously published the paper "In vitro culture of Taxane-rich Taxus chinensis" and the patent "A method for in vitro culture of Taxus chinensis with high taxane content" (patent number ZL202111576506.3). Using Taxus chinensis stem segments as starting material, adventitious buds can be induced synchronously at high frequency, with an induction rate of 87.5% and an average number of buds per explant of 34. This solves the problems of high endophytic bacteria, difficulty in explant material preparation, and low propagation coefficient in rapid propagation systems. In existing reports on Taxus chinensis rooting, the optimal rooting rate is 57%, with an average of 2.87 induced roots and a transplant survival rate of 69.45% (Liu Rui. 2024. Rapid in vitro propagation and plant regeneration technology of Taxus chinensis [D]. Northwest A&F University.), but the rooting step, which is the rate-limiting step in the rapid propagation technology system of Taxus chinensis, has not yet been effectively solved. Summary of the Invention

[0005] This invention uses regenerated yew seedlings as material and establishes a method for in situ expansion of yew stem cells and in vitro induction of roots through in vitro culture techniques such as high hormone enrichment culture of regenerated seedlings, construction of root development chassis, regulation of adventitious bud lignification, activation of root primordia, and quantitative induction of root system. This provides a theoretical basis and technical support for the large-scale and rapid propagation of the endangered yew species.

[0006] This invention provides a method for in situ expansion of Taxus chinensis stem cells, comprising the following steps:

[0007] Taxus chinensis seedlings were inoculated into MS medium containing hormones enriched with 0.5-2 mg / L BA and 0.5-2 mg / L ZT to expand Taxus chinensis stem cells in situ.

[0008] Preferably, the hormone enrichment medium is MS medium + 0.9-1.1 mg / L BA + 0.9-1.1 mg / L ZT, more specifically MS medium + 1 mg / L BA + 1 mg / L ZT.

[0009] Specifically, the cultivation time is 42-63 days, with a transfer every 15-25 days, and the preferred cultivation time is 30-50 days; the yew seedlings are regenerated yew seedlings obtained through tissue culture, and single buds are taken for inoculation.

[0010] Specifically, MS medium was supplemented with 25-35 g / L sucrose and 4-5 g / L agar powder, the pH of the medium was adjusted to 5.5-6.2, the culture conditions were 24-26±2℃, the light intensity was 2000-5000 lx, and the light intensity was 14-18 h / d.

[0011] Preferably, the MS medium is supplemented with 28-32 g / L sucrose (e.g., 30 g / L), 4.2-4.8 g / L agar powder, and the pH of the medium is adjusted to 5.6-6.0. The culture conditions are 25±2℃, light intensity of 3000-4000 lx, and light intensity of 15-17 h / d.

[0012] This invention provides a method for in situ expansion of yew stem cells from roots in vitro, comprising the following steps:

[0013] S1. Obtain materials for in situ amplification of stem cells at the base of the yew stem according to the described in situ amplification method for yew stem cells.

[0014] S2. The stem base of the material obtained in step S1 was inoculated into MS medium containing 0.5-1.5 mg / L BA, 0.01-0.1 mg / L LTDZ, 0.1-1.0 mg / L KT, and 0.02-0.2 mg / L NAA for the first stage of root development and cultured for 12-18 days.

[0015] S3 involves inoculating the material after S2 culture into 2×MS medium supplemented with 0.3-1.0 mg / L BA, 0.1-0.7 mg / L KT, 0.1-1 g / L proline, and 0.01-0.1 g / L phenylalanine for root development chassis construction phase II culture, which lasts for 12-18 days.

[0016] 2MS medium is based on MS medium, with the content of macro-elements doubled, while other components remain unchanged;

[0017] S4 involves inoculating the material cultured in S3 with DCR + 0.05-0.7 mg / L BA + 0.01-0.07 mg / L NAA + 10-100 mg / L phloroglucinol + 0.02 mg / L methyl jasmonate to enhance the lignification of adventitious buds, and culturing for 20-30 days;

[0018] S5 involves inoculating the material after S4 culture into DCR + 0.05-0.5 mg / L BA + 0.01-0.05 mg / L NAA + 1-2 g / L activated carbon for root primordia activation, and culturing for 12-18 days.

[0019] Furthermore, it also includes the following steps:

[0020] S6 involves inoculating the yew material cultured from S5 into DCR + 1.0-3.0 mg / L IBA + 0.1-1.0 mg / L NAA for root induction, and cultured for 21-28 days.

[0021] Preferably,

[0022] S2. Take the stem base of the material obtained in step S1 and inoculate it into MS + 0.9-1.1 mg / L BA + 0.04-0.06 mg / LTDZ + 0.45-0.55 mg / L KT + 0.09-0.11 mg / L NAA (optimally, MS + 1 mg / L BA + 0.05 mg / LTDZ + 0.5 mg / L KT + 0.1 mg / L NAA) rooting tray construction phase I medium, and culture for 12-18 days, for example 14 days;

[0023] In S3, the material cultured via S2 was inoculated into 2×MS medium supplemented with 0.45-0.55 mg / L BA + 0.25-0.35 mg / L KT + 0.45-0.55 g / L Pro + 0.009-0.011 g / L Phe for Phase II rooting pedestal construction culture for 12-18 days, e.g., 14 days.

[0024] S4 involves inoculating the material cultured in S3 with DCR + 0.45-0.55 mg / L BA + 0.045-0.055 mg / L NAA + 45-55 mg / L PG + 0.015-0.25 mg / L MeJA (optimally, DCR + 0.5 mg / L BA + 0.05 mg / L NAA + 50 mg / L PG + 0.02 mg / L MeJA) to enhance the lignification of adventitious buds, and culturing for 21-28 days, for example, 25 days;

[0025] S5 involves inoculating the material after S4 culture into DCR + 0.28-0.32 mg / L BA + 0.028-0.032 mg / L NAA + 1.8-2.2 g / L AC for root primordia stimulation, and culturing for 12-18 days, e.g., 14 days;

[0026] S6 involves inoculating the yew material cultured from S5 with DCR + 1.8-2.2 mg / L IBA + 0.34-0.55 mg / L NAA for root induction, and then culturing for 21-28 days, for example, 25 days.

[0027] Specifically, MS medium was supplemented with 25-35 g / L sucrose and 4-5 g / L agar powder, the pH of the medium was adjusted to 5.5-6.2, the culture conditions were 24-26±2℃, the light intensity was 2000-5000 lx, and the light intensity was 14-18 h / d.

[0028] More specifically, MS medium is supplemented with 28-32 g / L sucrose (e.g., 30 g / L), 4.2-4.8 g / L agar powder, and the pH of the medium is adjusted to 5.6-6.0. The culture conditions are 25±2℃, light intensity of 3000-4000 lx, and light exposure of 15-17 h / d.

[0029] This invention enables in situ-scale expansion of yew stem cells and successfully establishes a highly efficient in vitro induction method for yew root systems, overcoming the technical bottleneck of rapid tissue culture propagation of this species. Natural paclitaxel is mainly found in the old roots and bark of mature trees, making the source extremely scarce. This invention holds promise for the in vitro production of yew root systems, enriching them with high levels of paclitaxel, thereby solving the problem of finding a natural source of paclitaxel. Attached Figure Description

[0030] Figure 1 The buds of the yew tree grow synchronously on a large scale.

[0031] Figure 2 High hormone enrichment culture (21 days).

[0032] Figure 3 High hormone enrichment culture (42 days).

[0033] Figure 4 Stem base section of seedlings expanded in situ using stem cells.

[0034] Figure 5 Stem base slices of material that has not undergone high hormone enrichment culture.

[0035] Figure 6 Phase I of the foundation construction.

[0036] Figure 7 Phase II of the foundation construction.

[0037] Figure 8 Adventitious buds after lignification degree regulation.

[0038] Figure 9 The root cause of stimulation.

[0039] Figure 10 Root system of Taxus chinensis in vitro. Detailed Implementation

[0040] To more clearly illustrate the purpose, technical solution, and advantages of this invention, a detailed description will be provided below in conjunction with the accompanying drawings and specific embodiments. The drawings illustrate only exemplary embodiments of the invention and are not intended to limit its implementation. This invention can be implemented in various forms, and its design concept and core technology are not limited to the embodiments shown in the drawings. These embodiments are provided to facilitate understanding of the principles, structure, and function of this invention by those skilled in the art, thereby enabling them to better master and apply its technical solutions. The terminology used in this specification is only for describing specific embodiments and does not constitute a limitation of the invention.

[0041] Example 1:

[0042] Step (1): In situ expansion of stem cells at the base of the stem of regenerated seedlings in vitro

[0043] 1) High hormone enrichment culture

[0044] High-frequency synchronous emergence of clustered shoots in Taxus chinensis was obtained through in vitro culture, specifically according to a method for in vitro culture of Taxus chinensis with high taxane content disclosed in Chinese Patent 2021115765063. The cultured clustered shoots are as follows: Figure 1 As shown.

[0045] Single buds were cut and inoculated into MS medium containing hormones enriched with 0.5-2 mg / L BA and 0.5-2 mg / L ZT, and cultured for 42-63 days, with subculturing every 21 days.

[0046] The experimental results are shown in Table 1. The results indicate that single buds of *Taxus chinensis* did not induce stem cell niches in MS medium without added hormones (materials with induced stem cell niches showed thicker stems and swollen stem bases). With increasing hormone concentration, stem cell niche induction first increased and then decreased. In media with excessively high hormone concentrations, some leaves of single buds turned yellow. The optimal medium for in situ stem cell expansion was MS + 1 mg / L BA + 1 mg / L ZT. After 21 days of culture, the number of leaves at the base of single buds increased (…). Figure 2 After 42 days of cultivation, the internodes of single-bud stems shortened, the stems thickened, and the number of basal leaves increased significantly. Figure 3 The highest stem cell niche induction rate was 93.3%.

[0047] Table 1. Effects of high hormone enrichment culture on Taxus chinensis stem cell niche induction

[0048]

[0049] 2) Analyze the size of the stem cell niche by sectioning.

[0050] Paraffin sections of yew wood material cultured with high hormone enrichment were observed to compare stem cell niche sizes. Stem bases from both the high hormone enrichment cultured material (Example 1.6) and the uncultured material were fixed with FAA. Paraffin sections of the stem segments were prepared. The sections were first immersed in xylene I for 20 min, then in xylene II for 20 min, followed by transfer to anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then washed with water. The plant tissue sections were stained with safranin for 1-2 h, rinsed briefly with tap water to remove excess dye, and then destained in a gradient of 50%, 70%, and 80% ethanol. The sections were stained with Fast Green for 30-60 s, rinsed in three cycles of anhydrous ethanol, and dehydrated. The sections were then cleared by immersion in n-butanol and xylene for 5 min, slightly dried, and mounted with neutral resin. The sections were examined under a microscope, and images were acquired and analyzed. The results showed that the stem bases of seedlings cultured with high hormone enrichment (…) Figure 4The stem base of the material that was not cultured with high hormone enrichment ( Figure 5 The number of cells has increased by a significant margin, the cells have become smaller, and the tissue density is high, indicating that the number of stem cells has increased by a corresponding significant margin.

[0051] 3) The expression of genes such as WUS, STM, and CUC is correlated with the scale of stem cells.

[0052] Transcriptome analysis was performed on the stem bases of materials after high hormone enrichment culture and those without high hormone enrichment culture (CK) from Example No. 1.6. High-quality sequencing data were compared with the reference genome using HISAT2 software, and gene transcripts were counted and statistically analyzed using FeatureCounts software to perform differentially expressed gene analysis.

[0053] The results of RNA-seq differential expression analysis are shown in Table 2. The results indicate that the expression levels of stem cell characteristic maintenance genes such as WUS, STM, and CUC in the stem cell expansion culture materials were significantly upregulated; while the gene CLV3 only showed weak sequence similarity to KI387_003151 in the yew genome, and its expression level did not differ significantly among the materials, indicating that the cells after in situ expansion did not initiate differentiation. The pluripotent stem cells after in situ expansion possess the dual regeneration potential of adventitious shoots or adventitious roots.

[0054] Table 2. Differential expression results of genes related to Taxus chinensis stem cells

[0055]

[0056] Step (2) Foundation Construction

[0057] 1) Phase I construction of the root chassis

[0058] The material from the in situ expansion of stem base stem cells of Taxus chinensis (Example 1.6) was inoculated into MS medium containing 0.5-1.5 mg / L BA, 0.01-0.1 mg / L TDZ, 0.1-1.0 mg / L KT, and 0.02-0.2 mg / L NAA for Phase I root development and cultured for 14-21 days. The results showed that the MS medium containing 1 mg / L BA, 0.05 mg / L TDZ, 0.5 mg / L KT, and 0.1 mg / L NAA, cultured for 14 days, resulted in the best root development, with a 91.7% basal stem expansion, compact internodes, and robust buds. Figure 6 ).

[0059] Table 3. Effects of different plant hormone combinations on the formation of the root basal plate.

[0060]

[0061] 2) Phase II Construction of the Root Chassis

[0062] After the first-stage culture of rooting tray construction as in Example No. 2.4, the material was inoculated into 2MS medium supplemented with 0.3-1.0 mg / L BA, 0.1-0.7 mg / L KT, 0.1-1 g / L proline (Pro), and 0.01-0.1 g / L phenylalanine (Phe) for the second-stage culture of rooting tray construction, and cultured for 12-18 days. 2MS medium is based on MS medium, with the macro-element content doubled, while other components remain unchanged. The results (Table 4) showed that the rooting discs constructed by formulations in examples 2.9-2.12 were all larger than 10 mm. Formulation 2.11 induced the largest rooting disc at 15.6 mm, but its disc was relatively loose. A dense rooting disc is more conducive to subsequent root induction. Considering both the size and density of the rooting disc, the optimal culture medium was 2MS + 0.5 mg / L BA + 0.3 mg / L KT + 0.5 g / L Pro + 0.01 g / L Phe. After 14 days of culture, the rooting disc was 13.9 mm, indicating a dense disc. Figure 7 ), and induce a small number of adventitious buds.

[0063] Table 4. Effects of different combinations of plant hormones and additives on the density of the root basal plate.

[0064]

[0065] Step (3) Induction of isolated root systems

[0066] 1) Increased lignification of adventitious buds

[0067] The yew material cultured using the rooting tray constructed in Example No. 2.9 was inoculated with DCR + (0.05, 0.1, 0.3, 0.5, 0.7 mg / L) BA + (0.01, 0.03, 0.05, 0.07 mg / L) NAA + (10, 50, 100 mg / L) phloroglucinol (PG) + 0.02 mg / L methyl jasmonate (MeJA) to regulate the lignification degree of adventitious buds, and cultured for 20-30 days. As the concentrations of 6-BA and NAA increased, the lignification degree of adventitious buds first increased and then decreased. The optimal combination was 0.5 mg / L BA and 0.05 mg / L NAA. At hormone concentrations of 0.7 mg / L BA and 0.07 mg / L NAA, new adventitious buds were induced, which was not conducive to improving lignification. Increasing PG concentration improved the lignification degree of the buds, but at excessively high concentrations (100 mg / L), the buds turned yellow. Therefore, the preferred culture medium formulation was DCR + 0.5 mg / L BA + 0.05 mg / L NAA + 50 mg / L PG + 0.02 mg / L MeJA. After 25 days of culture, the adventitious buds lignified and grew vigorously. Figure 8 ).

[0068] 2) Root primordium activation

[0069] Materials cultured to enhance the lignification of adventitious buds were inoculated into DCR + (0.05, 0.3, 0.5 mg / L) BA + (0.01, 0.03, 0.05 mg / L) NAA + (1, 2 g / L) activated carbon (AC) for root primordia activation, and cultured for 12-18 days. The results showed that different combinations of hormone concentrations were beneficial for root primordia activation, but higher hormone concentrations (e.g., 0.5 mg / L BA, 0.05 mg / L NAA) stimulated shoot growth (above-ground parts), leading to upward nutrient transport and a lower root primordia activation rate; lower hormone concentrations (e.g., 0.05 mg / L BA, 0.01 mg / L NAA) resulted in a lower root primordia activation rate; 2 g / L AC showed better root primordia activation than 1 g / L AC. Therefore, the preferred culture medium formulation is DCR + 0.3 mg / L BA + 0.03 mg / L NAA + 2 g / L AC, with optimal root primordium activation after 14 days of culture. Figure 9 ).

[0070] 3) Root system quantitative induction

[0071] Root induction was performed on yew trees after exogenous hormone unloading culture in a medium containing DCR + 1.0-3.0 mg / L IBA + 0.1-1.0 mg / L NAA for 21-28 days. The results (Table 5) showed that root induction was significantly improved after regulating the degree of adventitious bud lignification and unloading exogenous hormones. IBA was the main active plant hormone, and the optimal rooting effect was achieved when combined with different concentrations of NAA. The optimal culture medium was DCR + 2 mg / L IBA + 0.5 mg / L NAA, with an adventitious root induction rate of 82.2% and an adventitious root count of 33.7 roots / tree after 25 days of culture. Figure 10 With a well-developed root system, the tissue culture seedlings achieved a 100% survival rate upon transplanting.

[0072] Table 5. Effects of different hormone combinations on root induction in Taxus chinensis

[0073] .

Claims

1. A method for in vitro induction of red yew stem cell in situ expansion root system, characterized in that, Comprise the following steps: S1 inoculate the Taxus bud seedling to the hormone-rich culture medium of MS + 0.5-2 mg / L BA+ 0.5-2 mg / L ZT to amplify the Taxus stem cell in situ to obtain the material of Taxus stem cell in situ amplification; the Taxus bud seedling is the Taxus regenerated bud seedling obtained by tissue culture, and single bud is inoculated; S2 inoculate the stem base of the material obtained in S1 to the rooting medium construction phase I medium of MS + 0.5-1.5 mg / L BA+ 0.01-0.1 mg / L TDZ+ 0.1-1.0 mg / L KT + 0.02-0.2 mg / L NAA, and culture for 12-18 d; S3 inoculate the material after S2 culture to the medium of 2×MS+ 0.3-1.0 mg / L BA+0.1-0.7 mg / L KT+ 0.1-1g / L proline+0.01-0.1 g / L phenylalanine for rooting medium construction phase II culture, and culture for 12-18 d; 2MS medium is that the content of macroelement is doubled on the basis of MS medium, and other components remain unchanged; S4 inoculate the material after S3 culture to DCR + 0.05-0.7 mg / L BA+ 0.01-0.07 mg / L NAA +10-100 mg / L phloroglucinol+ 0.02 mg / L methyl jasmonate for improving the degree of adventitious bud lignification, and culture for 20-30 d; S5 inoculate the material after S4 culture to DCR + 0.05-0.5 mg / L BA+ 0.01-0.05 mg / L NAA +1-2 g / L activated carbon for root primordium excitation, and culture for 12-18 d; S6 inoculate the Taxus material after S5 culture to DCR + 1.0-3.0 mg / L IBA+ 0.1-1.0 mg / L NAA for root system induction, and culture for 21-28 d; The MS medium is added with 25-35 g / L sucrose and 4-5 g / L agar powder, and the pH value of the culture medium is adjusted to 5.5-6.

2.

2. The root system ex vivo induction method according to claim 1, wherein, S2 inoculate the stem base of the material obtained in S1 to the rooting medium construction phase I medium of MS + 1 mg / L BA+ 0.05 mg / L TDZ + 0.5 mg / L KT + 0.1 mg / L NAA, and culture for 12-18 d; S3 inoculate the material after S2 culture to the medium of 2×MS+ 0.5 mg / L BA + 0.3 mg / L KT + 0.5 g / L ‌proline + 0.01 g / L phenylalanine for rooting medium construction phase II culture, and culture for 12-18 d.

3. The root ex vitro induction method of claim 2, wherein The time of rooting medium construction phase I culture and rooting medium construction phase II culture is 14 d.

4. The root system ex vivo induction method according to claim 2, wherein, S4: the material after S3 is inoculated in DCR + 0.5 mg / L BA + 0.05 mg / L NAA + 50 mg / L phloroglucinol + 0.02 mg / L methyl jasmonate for improving the degree of adventitious bud lignification, and cultured for 21-28 d; S5: the material after S4 is inoculated in DCR + 0.3 mg / L BA + 0.03 mg / L NAA + 2 g / L activated carbon for root primordium excitation, and cultured for 12-18 d; S6: the taxus material after S5 is inoculated in DCR + 2 mg / L IBA + 0.5 mg / L NAA for root system induction, and cultured for 21-28 d.

5. The root system ex vivo induction method according to claim 4, wherein, the culture time for improving the degree of adventitious bud lignification is 25 d; the culture time for root primordium excitation is 14 d; the culture time for root system induction is 25 d.

6. The root system ex vivo induction method according to any one of claims 1-5, wherein, the culture condition is 24-26±2℃, light intensity 2000-5000lx, and light for 14-18 h / d.

7. The root ex-vivo induction method of claim 6, wherein the MS culture medium is added with 28-32 g / L sucrose and 4.2-4.8 g / L agar powder, and the pH value of the culture medium is adjusted to 5.6-6.0; the culture condition is 25±2℃, light intensity 3000-4000lx, and light for 15-17 h / d.

8. The root ex-vivo induction method of claim 7, wherein, the added sucrose is 30 g / L.

9. The root ex vitro induction method according to any one of claims 1 to 5, wherein the hormone-rich culture medium in S1 is MS culture medium + 1 mg / L BA + 1 mg / L ZT.

10. The root ex vitro induction method according to any one of claims 1 to 5, wherein the culture time in S1 is 42-63 d, and the material is transferred every 15-25 d.

11. The root ex-vivo induction method of claim 10, wherein, the culture time in S1 is 30-50 d.

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