A tissue culture and rapid propagation method of agarwood tree

Abstract

The application discloses a tissue culture and rapid propagation method of Lignum Santali Albi, and belongs to the technical field of plant propagation. The method comprises the following steps: inoculating Lignum Santali Albi explants into a starting culture medium to induce adventitious buds, wherein the starting culture medium is WPM+0.05 mg / L NAA+0.25 mg / L TDZ; transferring the adventitious buds into a seedling strengthening culture medium to perform seedling strengthening and elongation culture, wherein the seedling strengthening culture medium is WPM+0.01 mg / L NAA+0.5 mg / L 6-BA+0.3 mg / L GA3; transferring the seedling-strengthened aseptic buds into a rooting culture medium to induce rooting and obtain regenerated plants, wherein the rooting culture medium is WPM+0.2 mg / L IBA. The application establishes a complete plant regeneration system for Lignum Santali Albi, has the advantages of high adventitious bud induction efficiency, good root quality and high transplanting survival rate, and provides technical support for germplasm resource preservation and industrialized rapid propagation of Lignum Santali Albi.

Description

Technical Field

[0001] This invention belongs to the field of plant propagation technology, specifically relating to a method for rapid propagation of Styrax styrax via tissue culture. Background Technology

[0002] Storax (Liquidambar orientalis Miller) is an important aromatic and medicinal tree species belonging to the genus *Liquidambar* in the family Altingiaceae. Its secreted natural resin, storax, has wide applications in the pharmaceutical and fragrance industries. Storax is rich in monoterpenes, sesquiterpenes, and triterpenoids, possessing various biological activities such as anti-inflammatory, antibacterial, and antioxidant properties. It is widely used in traditional Chinese medicine preparations, natural drug development, and daily chemical fragrance applications. Simultaneously, this tree species also has high ecological and landscape value, showing promising application prospects in functional forestry and landscaping.

[0003] However, the natural distribution of *Styrax storax* is narrow, mainly concentrated in southwestern Turkey. Due to long-term over-harvesting of its resin resources, the wild population has been declining continuously, and it is now an endangered species. To protect *Styrax storax* resources, the Turkish government has restricted or even banned the export of *Styrax storax* and related products, further exacerbating the supply-demand imbalance. Furthermore, due to resource constraints, traditional propagation methods for *Styrax storax* suffer from difficulties in artificial pollination, long cycles, and the inability to stably preserve superior germplasm, severely restricting its large-scale utilization. As a typical woody aromatic tree species, the direct explant regeneration system for *Styrax storax* is still imperfect, mainly facing problems such as severe explant browning, high contamination rates, and low adventitious bud induction efficiency, leading to unstable and poorly reproducible regeneration processes. At the same time, different explant types and hormone ratios show significant differences in regeneration responses, further increasing the difficulty of system construction.

[0004] Erdag et al. (Erdag B, Emek Y. In vitro Adventitious Shoot Regeneration of Liquidambar orientalis Miller. J Biol Sci, 2005, 5(6):805-808.) reported a method for adventitious bud regeneration using styrax leaves and buds as explants. However, their explants were taken from aseptic cultures of clustered buds induced from lateral buds of 25-30 year old trees. This method has the following limitations: (1) Aseptic bud clusters need to be established in advance, but endophytic fungi are severe in mature styrax trees, and establishing an aseptic system is itself a significant technical obstacle. This literature did not disclose any disinfection scheme; (2) The source of explants is limited to successfully established aseptic cultures, and it is impossible to start directly from field materials; (3) The extremely young aseptic leaf buds used have a low risk of browning, so this literature did not involve browning control and could not provide a browning solution for establishing a regeneration system from field materials.

[0005] The aforementioned bottlenecks directly limit the efficient regeneration from explants to complete plants, thus restricting the rapid propagation and genetic improvement of superior *Styrax storax* germplasm. Establishing a stable and efficient explant regeneration system is not only a key foundation for achieving large-scale asexual reproduction, but also an important prerequisite for conducting gene function analysis and molecular breeding research. It is of great significance for alleviating pressure on wild resources, protecting wild *Styrax storax* resources, and promoting the sustainable utilization of *Styrax storax* resources. Summary of the Invention

[0006] To address the problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for rapid propagation of Styrax styrax through tissue culture.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for rapid tissue culture propagation of Styrax styrax includes the following steps:

[0009] (1) Inoculate the explants of Styrax styrax into the initiation medium to induce the production of adventitious buds;

[0010] (2) The adventitious buds obtained in step (1) are transferred into the seedling culture medium for seedling elongation culture;

[0011] (3) Transfer the aseptic buds after the seedlings have grown strong in step (2) into the rooting medium to induce rooting and obtain regenerated plants.

[0012] In some embodiments, the explant in step (1) is the petiole or leaf of the styrax tree.

[0013] In some embodiments, a step of disinfecting the explant is included before step (1), wherein the disinfectant is mercuric chloride or sodium hypochlorite.

[0014] In some embodiments, the starting medium in step (1) is: WPM basal medium + 0.05 mg / L NAA + 0.25 mg / L TDZ.

[0015] In some embodiments, the initiation medium further contains 30 g / L sucrose, 7 g / L agar, and has a pH of 5.8.

[0016] In some embodiments, the seedling culture medium in step (2) is: WPM basal medium + 0.01 mg / L NAA + 0.5 mg / L 6-BA + 0.3 mg / L GA3.

[0017] In some embodiments, the seedling culture medium further contains 30 g / L sucrose, 7 g / L agar, and has a pH of 5.8.

[0018] In some embodiments, after the seedling elongation culture in step (2), the height of the aseptic buds reaches 2.5-3.5 cm.

[0019] In some embodiments, the rooting medium in step (3) is: WPM basal medium + 0.2 mg / LIBA.

[0020] In some embodiments, the rooting medium further contains 30 g / L sucrose, 7 g / L agar, and has a pH of 5.8.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention addresses the challenges of disinfection for *Styrax storax*, a woody plant with abundant endophytic fungi. It employs a 0.1% mercuric chloride vacuum-assisted treatment for 3-5 minutes, effectively killing attached pathogens while minimizing damage to plant tissues from the disinfectant. Explant survival rates reach 90.0%, significantly superior to sodium hypochlorite treatment and prolonged mercuric chloride treatment. During the adventitious bud induction stage, the use of WPM basal medium combined with 0.05 mg / L NAA and 0.25 mg / L TDZ effectively induces callus formation and adventitious bud differentiation in petioles and leaves, resulting in vigorous growth.

[0023] This invention adds a seedling elongation stage before rooting culture, using a seedling-strengthening medium containing 0.3 mg / L GA3 to elongate the buds to 2.5-3.5 cm, resulting in robust stems and fully expanded leaves, significantly improving the quality of aseptic buds. During the rooting stage, WPM basal medium combined with 0.2 mg / L IBA is used. Root primordia begin to appear 20-25 days after inoculation, with a rooting rate of 93.0%. After 30-35 days, the average root length is 1.5 cm, with robust root systems, well-developed fibrous roots, and minimal basal callus tissue, forming healthy regenerated plants with a balanced root-to-shoot ratio. This invention successfully establishes a regeneration system from petioles and leaves of *Styrax styrax* to complete plants, providing technical support for its germplasm resource preservation and rapid industrial propagation. Attached Figure Description

[0024] Figure 1 Picture of a styrax sapling;

[0025] Figure 2 The results of sterilization culture of *Styrax styrax* explants; A is the petiole after 7 days of sterilization culture; B is the leaf after 7 days of sterilization culture; C is the leaf after 1 month of sterilization culture.

[0026] Figure 3 Diagrams showing the induction of adventitious buds on petioles and leaves of *Styrax styrax*; A. Induction of adventitious buds on petioles; B. Induction of adventitious buds on leaves;

[0027] Figure 4 Illustration of the cultivation of vigorous seedlings with adventitious buds of Styrax chinensis;

[0028] Figure 5 Image A shows the rooting culture of adventitious buds of Styrax chinensis; Image B shows the rooting culture of adventitious buds of Styrax chinensis; Image C shows a close-up of the root of an adventitious bud of Styrax chinensis. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0030] Example 1

[0031] 1. Materials and Methods

[0032] Plant material source: Seedlings of *Liquidambar orientalis* Miller, see Table 1 for details. Figure 1 .

[0033] Table 1. Explant Details

[0034]

[0035] 1.2 Main Instruments and Reagents

[0036] (1) Instruments: ultra-clean workbench, autoclave, incubator / room, etc.

[0037] (2) Reagents: basal culture medium (such as MS, WPM, B5, etc.), plant growth regulators (6-BA, TDZ, NAA, IBA, etc.), disinfectants (mercuric chloride HgCl2, sodium hypochlorite NaClO), sucrose, agar, etc.

[0038] 1.3 Experimental Design

[0039] (1) Optimization of disinfection scheme: Compare the effects of mercuric chloride (HgCl2) or sodium hypochlorite (NaClO) treatment at different times, and screen the best sterilization combination.

[0040] (2) Callus / bud induction: An orthogonal design was used to test the effect of different concentration gradients of cytokinins (such as 6-BA, TDZ) and auxins (such as NAA, IBA) on adventitious bud induction.

[0041] (3) Proliferation and rooting: Screen the optimal proliferation factors and rooting hormone concentrations to determine the optimal subculture cycle.

[0042] 1.4 Cultivation Conditions

[0043] Temperature 25 ± 2 ℃, light intensity 40 μmol·m -2 ·s -1 The photoperiod is 16 hours light / 8 hours dark, and the relative humidity is about 60%.

[0044] 2. Steps and Results

[0045] 2.1 Explant sterilization and initiation

[0046] Since *Styrax styrax* is a woody plant, the petioles or leaves used as explants carry a large number of endophytic bacteria. The key to initiating culture is to effectively remove contaminants while minimizing the damage of disinfectants to plant tissues. Select healthy, non-lignified leaves and their corresponding petioles from 1-2 year old plants or sterile seedlings, with the 2nd-4th fully unfolded but not yet mature leaves and tender petioles approximately 0.5-1.5 cm in length being the best. After collection, rinse the explants under tap water for 20-30 minutes, followed by a shaking wash with 0.1% Tween-20 to remove surface contaminants. Then, cut the leaves into 1-2 cm square pieces and the petioles into 1-2 cm long segments, and then disinfect them. In this embodiment, 75% alcohol was used for disinfection for 30 seconds, followed by treatment with two disinfectants, mercuric chloride (HgCl2) and sodium hypochlorite (NaClO), with different concentrations and time combinations (Table 2). The treated explants were then placed in tLif2A starter medium (WPM + 0.05 mg / L NAA + 0.25 mg / LTDZ + 30 g / L sucrose + 7 g / L agar, pH=5.8) for culture. The screening results are shown in Table 2.

[0047] Table 2. Effects of different disinfection treatments on the sterilization effect of *Styrax styrax* explants.

[0048]

[0049] Results and Analysis: The experimental results showed that sodium hypochlorite (NaClO) treatment had a poor disinfection effect on *Styrax styrax* explants, with a contamination rate still as high as 35% after 4 minutes of treatment. While extending the treatment time reduced the contamination rate, the browning mortality rate also increased. In contrast, mercuric chloride (HgCl2) showed significantly better sterilization effects than sodium hypochlorite. Treatment with 0.1% mercuric chloride under vacuum (-0.08 MPa) for 3-5 minutes (treatment 3) yielded the best results. This treatment effectively killed the attached pathogens while minimizing damage to the explants, ultimately achieving an ideal explant survival rate of 90.0%. Figure 2 (A and 2B). When the treatment time was extended to 8 minutes, the toxic effects of mercuric chloride led to a sharp increase in mortality, while the survival rate decreased significantly.

[0050] Liquidambar species are rich in resins and polyphenols, and severe browning of explants after cutting is a primary obstacle to establishing tissue culture systems. Erdag et al. (2005) used extremely young leaf buds from established sterile cultures, where browning was not a significant problem, but their literature did not report browning rates or anti-browning measures. This invention uses field-collected seedling leaves / petioles as starting materials, facing a higher risk of browning. Experimental results show that vacuum-assisted treatment with 0.1% mercuric chloride for 3-5 min (treatment 3) not only achieved an extremely low contamination rate of 10%, but also a browning rate of only 14% and a survival rate of 90.0%, achieving an ideal balance between sterilization and tissue damage.

[0051] Observation of initiation status: Under optimal sterilization conditions, callus sprouting began on the petioles on day 10 and the leaves on day 15 after explant inoculation. Obvious bud sprouting was observed within 1-1.5 months, and overall growth was vigorous. Figure 2 C) indicates that the sterilization procedure successfully established a clean and viable sterile starting culture.

[0052] 2.2 Adventitious bud induction

[0053] Based on the successful acquisition of sterile materials, this example further investigates the effects of different basal culture media and plant growth regulator ratios on the induction of adventitious buds in *Styrax styrax*. The induction effects of combinations of cytokinin (6-BA, TDZ) and auxin (NAA) were compared, with polyvinylpyrrolidone (PVP) added to some treatments to prevent browning (Tables 3-1 and 3-2).

[0054] Table 3-1 Experimental formulation of adventitious bud induction medium for Styrax styrax

[0055]

[0056] Table 3-2 Induction effect of adventitious shoot induction medium

[0057]

[0058] Definition of effective buds: buds ≥ 0.5 cm in length and with normal morphology (no obvious vitrification, malformation, or yellowing). Percentage of effective buds (%) = number of effective buds ÷ total number of indeterminate buds × 100%.

[0059] Results and Analysis: The experimental results showed that both culture medium formulations could effectively induce callus formation in *Styrax styrax* explants, but there were significant differences in the induction of shoot development, with tLif2A showing the best induction effect. Figure 3 ).

[0060] Erdag (2005) reported obtaining 19.97 shoots per explant on WPM + 0.54 μM NAA + 11.1 μM BAP medium, but the shoots were "weak shoots of 0.5-1 cm" and required special shoot elongation culture before they could be used for rooting. This invention uses TDZ instead of BAP as the main cytokinin. On WPM + 0.05 mg / L NAA + 0.25 mg / L TDZ medium, the adventitious shoot induction effect differs from that reported by Erdag (2005) (on their respective explants): the adventitious shoots obtained in this invention are more robust (average shoot length 1.2 cm), with an effective shoot ratio of 85%, allowing direct entry into the seedling cultivation stage, reducing culture steps and improving overall efficiency.

[0061] 2.3 Successive generations of robust seedlings and elongation

[0062] This embodiment adds a seedling strengthening and elongation culture step before rooting culture. The induced clustered shoots are divided and transferred to a specially prepared seedling strengthening medium (seedling strengthening: WPM + 30 g / L sucrose + 7 g / L agar + 0.01 mg / L NAA + 0.5 mg / L 6-BA + 0.3 mg / L GA3, pH=5.8). Once the shoots reach 2.5-3.5 cm in length, with thick stems and fully expanded leaves, they are then transferred to a rooting medium (…). Figure 4 ).

[0063] Before seedling strengthening culture, the adventitious buds had an average length of 1.2 cm and an effective bud ratio of 85%. After 20 days of seedling strengthening culture, the buds elongated to 2.5-3.5 cm, the stems thickened significantly, and the leaves unfolded. In contrast, the adventitious buds obtained by Erdag (2005) were "weak buds of 0.5-1 cm," requiring bud elongation culture (WPM + 3.3 μM BAP) to elongate the buds from 0.5-1 cm to 2-3 cm before they could be cut for rooting. The seedling strengthening process in this application achieves both bud elongation and thickening and rejuvenation effects, resulting in superior bud quality.

[0064] 2.4 Inducing Rooting

[0065] Obtaining a robust and complete root system is crucial for the successful transplanting of tissue culture seedlings. In this example, aseptic buds with vigorous growth, reaching a height of approximately 2.5-3.5 cm after strong seedling elongation culture, were excised and transferred to different rooting media for induction, and the optimal rooting formula was screened.

[0066] The experiment compared the effects of different basal medium strengths (MS, 1 / 2MS, WPM) and different types and concentrations of auxins (IBA, NAA) on the rooting of aseptic shoots of *Styrax styrax*. The results showed that tLifRT2 had the best rooting effect (Table 4).

[0067] Table 4. Formulas for different culture media for the rooting of aseptic seedlings of Styrax styrax

[0068]

[0069] Results and Analysis:

[0070] Simultaneously, aseptic buds of *Styrax styrax* were placed in different rooting media, with 10 buds in each group. Experimental data showed that the rooting effect was most ideal in the medium treated with tLifRT2.

[0071] The highest rooting rate was achieved: root primordia began to appear 20-25 days after inoculation (Tables 5-1 and 5-2), with a rooting rate of 93%.

[0072] The root system exhibited optimal quality: at 30 days, the average root length reached 1.5 cm, the root system was robust with well-developed fibrous roots, minimal callus tissue at the base, and a tight connection between the root system and the stem. Figure 5 ).

[0073] Harmonious plant growth: While rooting, the above-ground parts continue to grow, the leaves unfold and turn dark green, forming a robust regenerated plant with a harmonious root-to-shoot ratio. Figure 5 ).

[0074] Table 5-1 Rooting status of aseptic seedlings of *Styrax styrax* under different culture medium formulations

[0075]

[0076] Table 5-2 Rooting effect of tLifRT2 medium

[0077]

[0078] Erdag (2005) clearly stated that the rooting rate of styrax was only 32% at 3.7 μM IBA, and the IBA concentration needed to be increased to 9.8 μM to achieve a rooting rate of 83.3%. The applicant unexpectedly discovered that using only 0.2 mg / L IBA (≈0.99 μM, approximately 1 / 10 of the concentration used in the prior art) achieved a rooting rate of 93%, with robust roots and minimal basal callus tissue.

[0079] In summary, this embodiment successfully established a regeneration system from petiole and leaf blade to complete plant, and clarified the optimal disinfection scheme, the optimal culture medium formula and culture conditions for each stage.

[0080] The final optimized formula for the styrax tree plant regeneration system is as follows:

[0081] Start-up medium: tLif2A: WPM + 30 g / L sucrose + 7 g / L agar + 0.05 mg / L NAA + 0.25 mg / L TDZ, pH=5.8;

[0082] Seedling growth medium: WPM + 30 g / L sucrose + 7 g / L agar + 0.01 mg / L NAA + 0.5 mg / L 6-BA + 0.3 mg / L GA3, pH=5.8;

[0083] Rooting medium: tLifRT2: WPM + 30 g / L sucrose + 7 g / L agar + 0.2 mg / L IBA, pH=5.8.

[0084] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of Styrax styrax via tissue culture, characterized in that, Includes the following steps: (1) Inoculate the explants of Styrax styrax into the initiation medium to induce the production of adventitious buds; (2) The adventitious buds obtained in step (1) are transferred into the seedling culture medium for seedling elongation culture; (3) Transfer the aseptic buds after the seedling elongation culture in step (2) into the rooting medium to induce rooting and obtain regenerated plants.

2. The method according to claim 1, characterized in that, The explants mentioned in step (1) are styrax leaves cut into 1-2 cm square pieces, or petioles cut into 1-2 cm pieces.

3. The method according to claim 2, characterized in that, The leaves and petioles of Styrax styrax mentioned in step (1) are taken from the 2nd to 4th fully unfolded but not yet aged leaves at the top and the tender petioles about 0.5 to 1.5 cm in length.

4. The method according to claim 1, characterized in that, Before step (1), there is also a step of disinfecting the explant, specifically: disinfecting with 75% alcohol for 30 seconds, and then disinfecting with 0.1% mercuric chloride under vacuum conditions of -0.08 MPa.

5. The method according to claim 1, characterized in that, The starting medium mentioned in step (1) is: WPM basal medium + 0.05 mg / L NAA + 0.25 mg / L TDZ.

6. The method according to claim 5, characterized in that, The initiation medium also contains 30 g / L sucrose, 7 g / L agar, and has a pH of 5.

8.

7. The method according to claim 1, characterized in that, The seedling culture medium mentioned in step (2) is: WPM basal medium + 0.01 mg / L NAA + 0.5 mg / L 6-BA + 0.3 mg / L GA3.

8. The method according to claim 7, characterized in that, The seedling culture medium also contains 30 g / L sucrose, 7 g / L agar, and has a pH of 5.

8.

9. The method according to claim 1, characterized in that, The rooting medium mentioned in step (3) is: WPM basal medium + 0.2 mg / L IBA.

10. The method according to claim 9, characterized in that, The rooting medium also contains 30 g / L sucrose, 7 g / L agar, and has a pH of 5.8.

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