Non-destructive edgeworthia chrysantha factor cloning propagation method for agilawood

Abstract

The invention discloses a non-destructive edgeworthia chrysantha factor cloning propagation method for agilawood trees, and belongs to the technical field of forestry biology. Aiming at the problems that in the prior art, plants need to be damaged during agilawood formation, the propagation efficiency is low, and agilawood formation characters are unstable, the method comprises the following steps: obtaining agilawood formation key factor genes of the phloem of the lateral root of the agilawood in a non-destructive manner; constructing an edgeworthia chrysantha factor recombinant expression vector containing a phloem specific promoter; non-destructively preparing agilawood axillary buds as an acceptor material; cloning seedlings are formed through agrobacterium tumefaciens-mediated oriented transformation and induction; and carrying out non-destructive edgeworthia chrysantha verification and domestication on the cloned According to the method, non-destructive operation of stock plants and clone seedlings is achieved, the breeding period is short, the agilawood formation character is stable, and technical support is provided for sustainable development of the agilawood industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of forestry biotechnology, in particular to a non-destructive linaloe factor cloning and propagation method of Aquilaria sinensis. BACKGROUND

[0002] Aquilaria sinensis is a condensed substance containing sesquiterpenes and aromatic compounds produced by Aquilaria sinensis after external stimulation, which has very high economic value. The existing aquilaria sinensis obtaining technology has three major problems:

[0003] Firstly, linaloe induction depends on physical damage (such as cutting, drilling) or chemical reagent smearing, which directly damages the trunk structure, resulting in growth inhibition or even death of the mother plant, low linaloe efficiency and unstable quality;

[0004] Secondly, the propagation of Aquilaria sinensis is mainly by grafting, which has a long propagation cycle (1-2 years) and is difficult to stably inherit the excellent linaloe traits of the mother plant; a few Aquilaria sinensis seedling growers have tried to use stem segments or top branches for cutting, but because the stem segments are damaged by rooting drugs, the rooting and seedling rate is very low, and the survival rate of such cutting seedlings after transplanting is very low, which is not worth studying and promoting.

[0005] Thirdly, the existing linaloe-related gene cloning technology mainly uses damaged wound healing tissues as materials, which cannot realize non-destructive acquisition of linaloe factors, and an integrated system of "linaloe factor cloning-propagation" has not been established, making it difficult to efficiently transmit excellent linaloe traits. SUMMARY

[0006] In view of the problems of damaging the plant, low propagation efficiency and unstable linaloe traits in the prior art, a non-destructive linaloe factor cloning and propagation method of Aquilaria sinensis is provided, which realizes non-destructive acquisition, directional transmission and efficient propagation of linaloe factors, and ensures that the cloned seedlings can stably linaloe without damage.

[0007] The technical scheme of the embodiment of the present application is as follows:

[0008] A non-destructive linaloe factor cloning and propagation method of Aquilaria sinensis, comprising the following steps:

[0009] (1) Non-destructive acquisition of linaloe factors: selecting healthy Aquilaria sinensis with good linaloe quality and high linaloe yield, digging a ring-shaped ditch with a depth of 20-30 cm at a distance of 1.5-2 m from the trunk, exposing the lateral roots and cutting the lateral root phloem, retaining the lateral root xylem and the remaining phloem and backfilling the soil, extracting total RNA from the lateral root phloem, synthesizing cDNA by reverse transcription, cloning linaloe key factor genes using cDNA as a template, the linaloe key factor genes including farnesyl pyrophosphate synthetase gene and MYB transcription factor gene, and obtaining the target genes after sequencing verification;

[0010] (2) Construction of the expression vector of the agarwood factor: Select the plant expression vector, and obtain the linear vector by double digestion with restriction endonuclease. Construct a fusion fragment by tandem the target gene of step (1) with the phloem-specific promoter. Insert the fusion fragment into the linear vector using DNA ligase. After transforming the host bacteria, screen positive clones, extract plasmids and verify them to obtain the recombinant expression vector.

[0011] (3) Non-destructive preparation of recipient material: Select current-year branches of agarwood tree, cut stem segments with one axillary bud, remove leaves and retain axillary bud scales, obtain sterile axillary buds after disinfection, inoculate sterile axillary buds into MS basic medium with added plant growth regulators, and culture under specific temperature and light conditions to induce axillary buds to sprout into buds;

[0012] (4) Directed transformation of the aromatic factor and induction of clone seedlings: The recombinant expression vector of step (2) was introduced into Agrobacterium competent cells, positive Agrobacterium was screened, the germinated sterile buds of step (3) were transferred into positive Agrobacterium bacterial solution for soaking, the bacterial solution was dried and then inoculated into co-culture medium for dark culture, and then transferred into screening medium for culture to obtain positive buds. Finally, the positive buds were transferred into rooting medium to induce rooting and form complete clone seedlings.

[0013] (5) Non-destructive agarication verification and acclimatization of cloned seedlings: The cloned seedlings in step (4) were transplanted to a sterile substrate and acclimatized under specific temperature and relative humidity conditions. After acclimatization, the agaric spirol content in the phloem of the cloned seedlings was detected by high performance liquid chromatography. The cloned seedlings with the agaric spirol content that met the standard were transplanted to the field.

[0014] Preferably, in step (1), the diameter of the exposed lateral root is 0.5-1 cm, the length of the cut lateral root phloem is 1-2 cm, and the total RNA of the lateral root phloem is extracted using the Trizol method.

[0015] Preferably, in step (1), the specific primers used for cloning the key factor gene of *Cynanchum paniculatum* are the upstream primer 5'-ATGGCTGCTGCTGCTGCT-3' and the downstream primer 5'-TCAGCTGCTGCTGCTGCT-3', and the accuracy of sequencing verification is not less than 99%.

[0016] Preferably, in step (2), the plant expression vector is pCAMBIA1301, the restriction endonucleases are BamHI and SacHI, the host bacterium is Escherichia coli DH5α, and the verification methods include enzyme digestion verification and sequencing verification.

[0017] Preferably, in step (2), the phloem-specific promoter is the phloem protein 2 gene promoter, the DNA ligase is T4 DNA ligase, and the tandem sequence of the target gene and the phloem-specific promoter in the fusion fragment is that the phloem-specific promoter is sequentially linked to the farnesyl pyrophosphate synthase gene and the MYB transcription factor gene.

[0018] Preferably, in step (3), the stem segment with one axillary bud is 2-3 cm long. The specific process of disinfection is to soak in 75% ethanol for 30 seconds, then transfer to 0.1% mercuric chloride solution for 8-10 minutes for disinfection, and then rinse with sterile water 5-6 times.

[0019] Preferably, in step (3), the plant growth regulators added to the MS basic culture medium are 0.5 mg / L 6-benzylaminopurine and 0.1 mg / L naphthaleneacetic acid, the specific temperature is 25℃, the specific light conditions are 16 h / d light, and the culture time is 7-10 days.

[0020] Preferably, in step (4), the competent Agrobacterium cells are Agrobacterium GV3101 competent cells, the OD600 value of the positive Agrobacterium bacterial solution is 0.6-0.8, and the soaking time of the sterile buds in the bacterial solution is 10-15 min.

[0021] Preferably, in step (4), the co-culture medium is MS medium supplemented with 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid and 100 μmol / L acetylsylgenone, the dark culture temperature is 22℃ and the dark culture time is 3 days;

[0022] The selection medium was MS medium supplemented with 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, 50 mg / L hygromycin and 200 mg / L cephalosporin, and the selection culture time was 20-25 days;

[0023] The rooting medium was 1 / 2 MS medium supplemented with 0.2 mg / L indolebutyric acid, and the rooting culture time was 15-20 days.

[0024] Preferably, in step (5), the sterile substrate is a mixture of peat moss and perlite in a volume ratio of 3:1, with a specific temperature of 25-28℃, a specific relative humidity of 70%-80%, and an acclimatization time of 30-40 days;

[0025] The detection conditions for high performance liquid chromatography were as follows: mobile phase was methanol and water at a volume ratio of 85:15, flow rate was 1 mL / min, and detection wavelength was 254 nm.

[0026] The standard for the content of agaric spirol is that the content of agaric spirol in the phloem of cloned seedlings is ≥0.1%.

[0027] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions:

[0028] I. Non-destructive: Extraction from the phloem of the lateral roots of the mother plant and from the axillary buds do not damage the main stem, allowing the mother plant to continue growing and solving the problem of traditional agarwood damaging the plant.

[0029] II. High-efficiency propagation: It only takes 60-80 days from the transformation of axillary buds to the formation of cloned seedlings, shortening the propagation cycle by more than 60% compared to traditional cuttings.

[0030] III. Stable traits: Through the directional transformation of agarwood factor, the phloem of cloned seedlings can stably express key genes for agarwood formation, and the content of agarwood spirol is stable at over 0.1%, avoiding trait segregation in traditional propagation.

[0031] IV. Environmentally friendly and sustainable: No physical damage or chemical induction is required, reducing the destruction of the agarwood tree's growth environment and meeting the needs of sustainable forestry development.

[0032] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a flowchart illustrating the overall technical process of the present invention.

[0035] Figure 2 This is a flowchart of the non-destructive acquisition process of the resin-forming factor according to the present invention;

[0036] Figure 3 This is a flowchart illustrating the construction process of the expression vector for the euphorbia citrinum factor of the present invention.

[0037] Figure 4 This is a flowchart illustrating the directed transformation and clonal seedling induction of the resin-producing factor according to the present invention.

[0038] Figure 5 This is a photograph of the actual plant after inoculation of axillary buds of agarwood according to the present invention.

[0039] Figure 6 This is a photograph of another agarwood tree axillary bud after inoculation, showing its budding and growth state according to the present invention.

[0040] Figure 7 This is a diagram showing the state of callus tissue formed after the agarwood buds of the present invention are transformed by Agrobacterium tumefaciens.

[0041] Figure 8 This is a diagram illustrating the further development of the agarwood bud callus tissue according to the present invention.

[0042] Figure 9 This is a diagram of the root system state of the agarwood clone seedlings in the early stage of induced rooting according to the present invention.

[0043] Figure 10 This is a diagram showing the state of the robust root system of the agarwood clone seedlings after domestication, as described in this invention. Detailed Implementation

[0044] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0045] It is important to note that terms such as "first," "second," "symmetric," and "array" are used only to distinguish between descriptive and positional descriptions and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, features specified with terms such as "first" or "symmetric" may explicitly or implicitly include one or more of that feature; similarly, when the quantity of certain features is not limited by words such as "two" or "three," it should be noted that such features also explicitly or implicitly include one or more features.

[0046] In this invention, unless otherwise explicitly specified and limited, terms such as "installation," "connection," and "fixation" should be interpreted broadly; for example, they can refer to a fixed connection, a detachable connection, or an integral molding; they can refer to a mechanical connection, a direct connection, a welding connection, or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the accompanying drawings and specific circumstances.

[0047] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0048] like Figures 1-10 The present invention provides a non-destructive method for cloning and propagating agarwood resin-forming factors in agarwood trees, comprising the following steps:

[0049] This method is a technical system that closely integrates the entire process from obtaining agarwood formation factors to transplanting cloned seedlings into the field. Its core objective is to overcome the pain points of traditional agarwood formation, such as "damaging the plant" and "low efficiency and unstable traits" in propagation.

[0050] First, a non-destructive method for obtaining agarwood factors is employed: Healthy agarwood trees aged 5-8 years are carefully selected. At this stage, the tree's agarwood regulation mechanism is both active and stable, making them ideal mother trees for obtaining high-quality agarwood factors. A circular trench 20-30cm deep is dug 1.5-2m from the trunk. This distance and depth easily expose suitable lateral roots without significantly interfering with nutrient transport to the main trunk or the overall growth of the tree. After exposing the lateral roots, a 1-2cm length of phloem is cut from a 0.5-1cm diameter lateral root, while strictly preserving the xylem and remaining phloem. The soil is then promptly backfilled. This "minimally invasive" method allows the mother tree to heal and recover quickly, enabling normal growth and development thereafter.

[0051] Subsequently, total RNA was extracted from the phloem of lateral roots using the Trizol method (the Trizol method can efficiently lyse cells and inhibit RNase, ensuring RNA integrity and purity). cDNA was synthesized through reverse transcription. Then, using the cDNA as a template, specific primers were used to accurately clone the farnesyl pyrophosphate synthase gene, which plays a core role in the synthesis of sesquiterpenes in agarwood, and the MYB transcription factor gene, which regulates the agarwood formation pathway. Finally, sequencing verification (with an accuracy rate of no less than 99%) ensured the accuracy of the gene sequence and obtained the target gene.

[0052] Next, the expression vector for the *Cynanchum paniculatum* factor was constructed: the pCAMBIA1301 vector, suitable for plant genetic engineering, was selected, and a linear vector was obtained by double digestion with BamHI and SacI (the two restriction sites are unique, achieving precise digestion). The target gene was tandemly linked with the promoter of the phloem protein 2 gene, which specifically drives gene expression in the phloem, to construct a fusion fragment. The fusion fragment was then inserted into the linear vector using T4 DNA ligase (T4 DNA ligase has high ligation efficiency, ensuring a high success rate in recombinant vector construction). Subsequently, *E. coli* DH5α was transformed (DH5α competent cells are highly efficient, easy to transform, and can stably preserve plasmids). After screening for positive clones, the plasmid was extracted and verified by restriction enzyme digestion (rapidly determining whether the fragment insertion is correct) and sequencing (precisely confirming the sequence at the base level) to obtain a recombinant expression vector stably carrying the target gene.

[0053] Next, non-destructive preparation of recipient materials was carried out: Vigorous, current-year branches of agarwood trees were selected, and stem segments 2-3 cm in length with one axillary bud were cut. Leaves were removed, but the axillary bud scales were carefully preserved (the axillary bud scales protect the axillary bud and maintain its differentiation potential). A strict sterilization process was followed: soaking in 75% ethanol for 30 seconds (preliminary sterilization) → disinfecting with 0.1% mercuric chloride solution for 8-10 minutes (deep sterilization) → rinsing with sterile water 5-6 times (to remove residual disinfectant) to obtain sterile axillary buds. These sterile axillary buds were inoculated into MS basal medium supplemented with 0.5 mg / L 6-benzylaminopurine (to promote cell division and bud differentiation) + 0.1 mg / L naphthaleneacetic acid (to synergistically regulate growth with cytokinin). The medium was cultured at 25℃ under suitable conditions of 16 h / d for 7-10 days to induce axillary bud germination into bud tissue, providing high-quality recipients for subsequent transformation.

[0054] Next, the directional transformation of the inoculum factor and the induction of cloned seedlings were carried out: the recombinant expression vector was introduced into Agrobacterium GV3101 competent cells (GV3101 contains the Vir region gene, which can efficiently mediate T-DNA transfer), positive Agrobacterium were screened and the OD600 of the bacterial culture was adjusted to 0.6-0.8 (at this time, Agrobacterium is in the logarithmic phase, with strong activity and high T-DNA transfer efficiency).

[0055] The budding sterile buds were immersed in bacterial solution for 10-15 minutes (to allow Agrobacterium to fully adsorb cells and initiate T-DNA transfer without damaging the buds). After the bacterial solution was dried, the buds were inoculated into a co-medium containing 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, and 100 μmol / L acetosyringone (to activate the Vir region of Agrobacterium and enhance T-DNA transfer). The buds were then cultured in the dark at 22°C for 3 days (dark culture avoids light inhibition of Vir region expression and ensures transformation efficiency).

[0056] Then, the samples were transferred to a selection medium supplemented with 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, 50 mg / L hygromycin (for selecting positive buds), and 200 mg / L cephalosporin (for inhibiting Agrobacterium growth). Positive buds were obtained after culturing for 20-25 days.

[0057] Finally, the positive buds were transferred to 1 / 2 MS medium supplemented with 0.2 mg / L indolebutyric acid (to promote rooting) and cultured for 15-20 days to induce rooting and form complete clone seedlings. The clone seedlings were then subjected to non-destructive verification and acclimatization: they were transplanted into a sterile substrate of peat moss:perlite = 3:1 (water-retaining, fertilizer-retaining, and aerated, conducive to root growth) and acclimatized for 30-40 days at 25-28℃ and 70%-80% relative humidity (allowing the clone seedlings to gradually adapt to the external environment and improve field survival rate).

[0058] After domestication, the linalool content in the phloem of the cloned seedlings was accurately determined using high-performance liquid chromatography (HPLC) with a mobile phase of methanol:water = 85:15, a flow rate of 1 mL / min, and a detection wavelength of 254 nm. (Linalool is a representative active ingredient in agarwood, and its content can be directly used to determine the resin formation effect.) Cloned seedlings with alinalool content ≥0.1% were selected and transplanted to the field, paving the way for industrial application.

[0059] The entire process achieves "non-destructive" transfer between the mother plant and the recipient material through "minimally invasive lateral root sampling and non-destructive axillary bud preparation," and utilizes molecular biology technology to complete the directional delivery and efficient propagation of agarwood-forming factors. This completely solves the problems of traditional agarwood agarwood formation that damage the plant, have low propagation efficiency, and unstable agarwood characteristics, providing core technical support for the sustainable development of the agarwood industry.

[0060] In step (1), the diameter of the exposed lateral root is 0.5-1 cm, the length of the excised lateral root phloem is 1-2 cm, and the total RNA of the lateral root phloem is extracted using the Trizol method.

[0061] When obtaining agarwood factor in step (1), it is crucial to control the size of the lateral roots: the diameter of the exposed lateral roots is limited to 0.5-1cm. The phloem of the lateral roots with this diameter is of moderate thickness, which can provide sufficient agarwood factor-related tissues and will not cause the mother plant to have difficulty recovering after root damage due to excessively thick lateral roots.

[0062] The phloem of the lateral roots was excised in lengths of 1-2 cm. This length ensures sufficient phloem tissue for RNA extraction while minimizing damage to the lateral roots, allowing the remaining parts to heal quickly and continue transporting nutrients to the mother plant. Total RNA was extracted from the phloem using the Trizol method. Trizol reagent is a recognized high-efficiency RNA extraction reagent in molecular biology, effectively lysing cells and releasing total RNA while strongly inhibiting RNase activity, ensuring high purity and integrity of the extracted RNA. This provides a high-quality template for subsequent reverse transcription to synthesize cDNA and cloning key gene molecules of *Daphne odora*. Furthermore, the specific primers used for cloning the key gene molecules of *Daphne odora* (upstream: 5'-ATGGCTGCTGCTGCTGCT-3'; downstream: 5'-TCAGCTGCTGCTGCTGCT-3') are highly specific primers that have been validated through extensive experiments. They accurately bind to the upstream and downstream sequences of the key gene molecules of *Daphne odora*, ensuring the accuracy of PCR amplification. The sequencing verification accuracy is no less than 99%, guaranteeing the complete correctness of the target gene sequence at the base level, laying a solid foundation for the subsequent construction of accurate expression vectors.

[0063] Precise control of lateral root size and phloem cutting length enables "minimally invasive material harvesting" of lateral roots from the mother plant, ensuring rapid recovery of the mother plant;

[0064] The Trizol method for RNA extraction ensures RNA quality, providing a prerequisite for accurate gene cloning.

[0065] High-specificity primers and high-accuracy sequencing verification ensure the accurate acquisition of genes for key factors in resin formation, providing reliable gene fragments for the realization of the core function of resin formation factors in the entire technical solution.

[0066] In step (2), the plant expression vector is pCAMBIA1301, the restriction endonucleases are BamHⅠ and SacⅠ, the host bacterium is Escherichia coli DH5α, and the verification methods include enzyme digestion verification and sequencing verification.

[0067] In step (2), when constructing the expression vector for the agarwood factor, the pCAMBIA1301 vector was selected because it has a cassette structure suitable for plant gene transformation, contains a hygromycin resistance selection marker (facilitating subsequent screening of positive plant transformants), and has abundant multiple cloning sites (facilitating the insertion of the target gene and promoter). The vector was digested with BamHI and SacHI. The restriction sites of these two enzymes are unique in the vector and the fusion fragment, enabling precise restriction digestion, resulting in a high degree of end-to-end matching between the linear vector and the fusion fragment, and significantly improving the efficiency of subsequent ligation. Escherichia coli DH5α was chosen as the host bacterium because DH5α competent cells are highly efficient, easy to transform, and can stably preserve recombinant plasmids, facilitating plasmid amplification and screening. After obtaining the recombinant plasmid, "restriction verification + sequencing verification" was used: restriction verification can quickly determine whether the fusion fragment has been correctly inserted into the vector by observing whether the size of the fragment after restriction is consistent with the expectation.

[0068] Sequencing verification can precisely confirm the correctness of the ligation of the target gene, promoter, and vector at the base sequence level, and whether there are any base mutations / deletions. Furthermore, the phloem-specific promoter selected is the phloem protein 2 gene promoter, which is a promoter specifically expressed in plant phloem cells. It drives the target gene to be expressed only in the phloem of the cloned seedlings, avoiding overexpression of the target gene in leaves, xylem, and other tissues that consume plant nutrients. Simultaneously, it acts specifically on the key resin-forming site (phloem), providing a precise regulatory basis for non-destructive resin formation in cloned seedlings. The DNA ligase selected is T4 DNA ligase, which has high ligation efficiency and can firmly connect the linear vector to the sticky / blunt ends of the fusion fragment, ensuring a high success rate in recombinant expression vector construction. In the fusion fragment, the tandem sequence of the target gene and promoter is "phloem-specific promoter → farnesyl pyrophosphate synthase gene → MYB transcription factor gene." This sequence conforms to the gene expression regulation logic; the promoter initiates transcription first, allowing the subsequent two genes to be expressed sequentially and synergistically exert their resin-forming regulatory role.

[0069] The pCAMBIA1301 vector, specific restriction enzymes, and suitable host bacteria were selected, and a double verification method was used to ensure the accuracy and stability of the recombinant expression vector construction. The application of the phloem-specific promoter enabled the targeted expression of the agarwood factor, avoiding ineffective nutrient consumption. The T4 DNA ligase and the scientific gene tandem sequence improved the efficiency of vector construction and the rationality of gene expression, providing a reliable vector guarantee for the precise role of the agarwood factor in cloned seedlings.

[0070] In step (3), the stem segment with one axillary bud is 2-3 cm long. The specific process of disinfection is to soak in 75% ethanol for 30 seconds, then transfer to 0.1% mercuric chloride solution for 8-10 minutes for disinfection, and then rinse with sterile water 5-6 times.

[0071] In step (3), when preparing the recipient material, a stem segment with one axillary bud is cut to a length of 2-3 cm. This length can preserve the axillary bud and its surrounding meristematic tissue intact (providing sufficient nutrition and cellular basis for subsequent axillary bud germination) without increasing the difficulty of disinfection or causing material contamination due to excessive stem length. Disinfection is the key to preventing contamination in plant tissue culture: soaking in 75% ethanol for 30 seconds can quickly denature bacterial proteins and initially kill microorganisms on the surface of the material;

[0072] Disinfect by immersing in 0.1% mercuric chloride solution for 8-10 minutes. Mercuric chloride is a highly effective disinfectant that can deeply kill stubborn microorganisms on the surface and in the crevices of materials.

[0073] Rinse 5-6 times with sterile water to thoroughly remove residual mercuric chloride and prevent it from harming the growth of axillary buds, ultimately obtaining sterile and vigorous axillary buds.

[0074] In addition, MS basal medium was supplemented with 0.5 mg / L 6-benzylaminopurine (cytokinin, which promotes cell division and bud differentiation) + 0.1 mg / L naphthaleneacetic acid (auxin, which synergistically regulates axillary bud growth with cytokinin);

[0075] The culture temperature was set at 25℃, which is the suitable temperature for plant tissue culture and can ensure efficient cell metabolism.

[0076] The light conditions are 16h / d. Sufficient light provides energy for axillary bud photosynthesis (or photomorphogenesis) and promotes rapid axillary bud germination.

[0077] A cultivation period of 7-10 days allows axillary buds to successfully germinate and form buds suitable for subsequent transformation under appropriate hormones and environment.

[0078] Stem segment length control and scientific disinfection procedures ensure the sterility and integrity of the recipient material; the rational combination of plant growth regulators and suitable temperature / light / culture time provide the best growth environment for axillary bud germination, enabling efficient germination of axillary buds into bud bodies, and providing sufficient high-quality recipient material for subsequent transformation.

[0079] In step (4), the competent Agrobacterium cells are Agrobacterium GV3101 competent cells, the OD600 value of the positive Agrobacterium bacterial solution is 0.6-0.8, and the soaking time of the sterile buds in the bacterial solution is 10-15 min;

[0080] In step (4), Agrobacterium GV3101 competent cells were selected for the directional transformation of the agarwood factor because GV3101 contains the Vir region gene, which can efficiently mediate the transfer of T-DNA to plant cells, greatly improving the success rate of gene transformation. The OD600 of the positive Agrobacterium culture was adjusted to 0.6-0.8. At this time, Agrobacterium is in the logarithmic growth phase, with strong vitality and high T-DNA transfer efficiency, which can ensure the transformation effect. The sterile buds were soaked in the bacterial solution for 10-15 minutes. This time allows Agrobacterium to fully adsorb onto the surface of the bud cells and initiate T-DNA transfer, while avoiding bud cells from hypoxia or damage due to prolonged soaking. In addition, 0.5 mg / L 6-benzylaminopurine + 0.1 mg / L naphthaleneacetic acid (to maintain bud growth) + 100 μmol / L acetosyringone (to activate the Vir region gene of Agrobacterium and enhance the T-DNA transfer efficiency) were added to the co-culture medium.

[0081] Dark culture at 22℃ is suitable for Agrobacterium growth while ensuring bud cell viability; 3 days of dark culture allows Agrobacterium to fully complete the transfer of T-DNA to plant cells. The selection medium is supplemented with 0.5 mg / L 6-benzylaminopurine + 0.1 mg / L naphthaleneacetic acid (to maintain growth) + 50 mg / L hygromycin (to screen positive buds through resistance markers) + 200 mg / L cephalosporin (to inhibit Agrobacterium growth and prevent its toxicity to buds).

[0082] The selection culture lasted for 20-25 days, allowing sufficient time for positive buds to grow successfully under selection pressure, while untransformed buds were discarded. The rooting medium was 1 / 2 MS medium supplemented with 0.2 mg / L indolebutyric acid (a key regulator for promoting rooting). The 1 / 2 MS medium had a moderate nutrient concentration, providing a suitable nutritional environment for bud rooting.

[0083] Rooting culture for 15-20 days allows positive buds to fully develop roots and form complete clone seedlings.

[0084] By selecting Agrobacterium GV3101 and controlling the bacterial concentration and soaking time, the transformation efficiency and spore cell survival rate were improved.

[0085] Acetylsyringone in the co-culture medium and under suitable culture conditions enhanced T-DNA transfer efficiency;

[0086] The screening medium, through the dual action of resistance markers and antibiotics, accurately screens out positive buds; the reasonable formula and culture time of the rooting medium ensure the efficient formation of the root system of the cloned seedlings, and ultimately achieve the directional transformation of the aromatic factor and the complete regeneration of the cloned seedlings.

[0087] In step (5), the sterile substrate is a mixture of peat moss and perlite in a volume ratio of 3:1, with a specific temperature of 25-28℃, a specific relative humidity of 70%-80%, and an acclimatization time of 30-40 days.

[0088] Supplementary expansion: In step (5) when acclimatizing and verifying the cloning of seedlings, the sterile substrate is a mixture of peat moss and perlite in a ratio of 3:1. Peat moss has good water retention and fertility, providing sufficient water and nutrients for the cloned seedlings.

[0089] Perlite has excellent air permeability, ensuring air circulation within the substrate and preventing root rot due to lack of oxygen. The optimized mixing ratio of the two creates a loose, fertile, and well-aerated environment for the root growth of cloned seedlings. The acclimatization temperature is controlled at 25-28℃, which is the suitable temperature range for the growth of cloned seedlings and can promote metabolism and root development.

[0090] A relative humidity of 70%-80% reduces water transpiration from the leaves of cloned seedlings, helping them transition from the tissue culture environment to the outside world and preventing them from withering due to excessive water loss.

[0091] An acclimatization period of 30-40 days allows sufficient time for the cloned seedlings' roots to fully adapt to the substrate and their leaves to gradually adapt to external light and humidity, significantly improving the survival rate after transplanting into the field. Furthermore, the high-performance liquid chromatography (HPLC) detection conditions are: mobile phase methanol:water = 85:15, flow rate 1 mL / min, and detection wavelength 254 nm. These conditions have been validated and can efficiently separate and accurately detect agaric spirol, ensuring the accuracy and repeatability of the results. The standard for agaric spirol content is "agaric spirol content ≥0.1% in the phloem of cloned seedlings." This standard is based on the resin formation quality requirements of the agarwood industry and the stable resin formation level set by this method, ensuring that the selected cloned seedlings have good resin formation performance.

[0092] Optimized aseptic substrate formulation and acclimatization environment conditions significantly improve the survival rate of cloned seedlings transitioning from tissue culture to field.

[0093] Scientific high-performance liquid chromatography (HPLC) detection conditions can accurately assess the resin formation effect of cloned seedlings;

[0094] Clear standards for agarwood spirol content ensure the quality of resin formation in transplanted clone seedlings and provide quality assurance for non-destructive large-scale production of agarwood.

[0095] Figure 5 , Figure 6 The exhibition showcases the individual and group states of agarwood axillary buds sprouting and growing into buds after disinfection, inoculation, and induction. Figure 7 , Figure 8 The study presents the state of callus formation at the base of agarwood buds after transformation by Agrobacterium and the further development of the callus. Figure 9 , Figure 10 The images show the growth status of the root system in the early stage of induced rooting of agarwood clone seedlings and the robust root system after domestication, which intuitively demonstrates the key process of this invention from the germination of the recipient material to the root development of the clone seedling.

[0096] In this embodiment, the present invention operates as follows:

[0097] First, a non-destructive method for obtaining resin-forming factors is carried out: select a healthy agarwood tree with an age of 6 years, dig a circular trench 25cm deep at a distance of 1.8m from the trunk, expose lateral roots with a diameter of 0.8cm, cut off the phloem of the lateral roots with a length of 1.5cm, retain the xylem of the lateral roots and the remaining phloem, and backfill the soil so that the mother plant can grow normally.

[0098] Total RNA was extracted from the phloem of lateral roots using the Trizol method, and cDNA was synthesized by reverse transcription. Then, using the cDNA as a template, the farnesyl pyrophosphate synthase gene and the MYB transcription factor gene were cloned using the upstream primer 5'-ATGGCTGCTGCTGCTGCT-3' and the downstream primer 5'-TCAGCTGCTGCTGCTGCT-3'. The target genes were obtained after sequencing verification (100% accuracy).

[0099] Subsequently, the expression vector for the agarwood factor was constructed: the plant expression vector pCAMBIA1301 was selected and double-digested with restriction endonucleases BamHⅠ and SacⅠ to obtain a linear vector;

[0100] The target gene was tandemly linked with the promoter of the phloem protein 2 gene to construct a fusion fragment;

[0101] The fusion fragment was inserted into a linear vector using T4 DNA ligase, transformed into Escherichia coli DH5α, positive clones were screened and plasmids were extracted. After verification by enzyme digestion and sequencing, the recombinant expression vector pCAMBIA1301-Pphlo-FPS-MYB was obtained.

[0102] Next, non-destructive preparation of the receptor material was carried out: current-year branches of agarwood trees were selected, and stem segments with a length of 2.5cm and one axillary bud were cut off. The leaves were removed, but the axillary bud scales were retained.

[0103] After soaking in 75% ethanol for 30 seconds, it is then transferred to 0.1% mercuric chloride solution for 10 minutes for disinfection, and then rinsed 6 times with sterile water to obtain sterile axillary buds.

[0104] Sterile axillary buds were inoculated into MS basal medium supplemented with 0.5 mg / L 6-BA and 0.1 mg / L NAA, and cultured for 8 days at 25°C and 16 h / d light to induce axillary buds to sprout into buds.

[0105] Then, the directional transformation and clonal seedling induction of the causal factor were carried out: the recombinant expression vector pCAMBIA1301-Pphlo-FPS-MYB was introduced into Agrobacterium GV3101 competent cells, positive Agrobacterium was screened, and the OD600 of the bacterial culture was adjusted to 0.7.

[0106] The budding sterile buds were immersed in the bacterial solution for 12 minutes. After being removed, the surface bacterial solution was blotted dry with sterile filter paper. The buds were then inoculated into a co-culture medium supplemented with 0.5 mg / L 6-BA, 0.1 mg / L NAA and 100 μmol / L acetylsyringone and cultured in the dark at 22°C for 3 days.

[0107] The co-cultured buds were then transferred to a selection medium supplemented with 0.5 mg / L 6-BA, 0.1 mg / L NAA, 50 mg / L hygromycin and 200 mg / L cephalosporin, and cultured for 22 days to obtain positive buds.

[0108] The positive buds were then transferred to 1 / 2 MS rooting medium supplemented with 0.2 mg / L IBA and cultured for 18 days to induce rooting and form complete clones.

[0109] Finally, the non-destructive agarwood formation verification and acclimatization of the cloned seedlings were carried out: the cloned seedlings were transplanted into a sterile substrate of peat moss and perlite mixed in a volume ratio of 3:1, and acclimatized for 35 days at 26℃ and 75% relative humidity.

[0110] After domestication, the content of agaric spirol in the phloem of the cloned seedlings was detected by high performance liquid chromatography (mobile phase methanol:water = 85:15, flow rate 1 mL / min, detection wavelength 254 nm). The cloned seedlings with agaric spirol content of 0.13% were selected and transplanted to the field for routine water and fertilizer management. The agaric formation was then monitored regularly.

[0111] The following are several other specific embodiments of the application of this invention:

[0112] Example 1: Propagation of high-quality seedlings in large-scale agarwood planting bases

[0113] Data Preparation Phase: To meet the needs of a large-scale agarwood planting base, healthy agarwood trees aged 7-8 years with excellent natural resin formation (agaric spirol content consistently above 0.12%) were selected as mother trees. Lateral root phloem tissue was obtained from each mother tree at a distance of 1.8-2m from the trunk, following the instructions in the handover document. Simultaneously, the mother tree's growing environment (soil type: sandy loam, average annual temperature: 26℃, annual rainfall: 1600mm, etc.) and daily management practices (monthly application of well-rotted organic fertilizer, regular weeding, etc.) were recorded to ensure traceability of subsequent propagation conditions.

[0114] Propagation Implementation Stage: Following the method of this invention, the following steps were carried out sequentially: non-destructive acquisition of agarwood factor (total RNA extracted from the phloem of lateral roots, cloning the FPS gene and MYB transcription factor gene, and sequencing verification with 100% accuracy), construction of agarwood factor expression vector (using the pCAMBIA1301 vector, double digestion followed by insertion of the Pphlo-FPS-MYB fusion fragment, and successful transformation verification), non-destructive preparation of recipient material (taking stem segments with axillary buds from current-year branches, disinfecting and inducing sprouting), directional transformation of agarwood factor and induction of cloned seedlings (Agrobacterium-mediated transformation, screening for positive buds and rooting), and non-destructive verification and domestication of agarwood factor in cloned seedlings (detecting the content of agaric spirol, and selecting cloned seedlings with ≥0.11%).

[0115] Application effect stage: The acclimatized cloned seedlings were transplanted to plots within the base with the same growth environment as the mother plants, and managed using the same methods. One year after transplanting, the agaric spirol content in the phloem of the cloned seedlings was measured to be 0.12%-0.14%, which was basically consistent with the agaric formation level of the mother plants;

[0116] Furthermore, the cloned seedlings grow uniformly with no obvious trait separation. Compared with traditional cuttings (which require 2 years to preliminarily detect resin formation and have a resin formation rate of only about 60%), the resin formation cycle of the cloned seedlings propagated by this invention is shortened by 1 year, and the resin formation rate is increased to over 90%, which greatly improves the supply efficiency and resin formation stability of high-quality seedlings in the base.

[0117] Example 2: Preservation and Propagation of Rare Agarwood Varieties

[0118] Conservation Needs Stage: A forestry research institute needs to preserve and propagate a rare agarwood variety (only a few dozen trees remain in the wild; after resin formation, the agaric spirol content can reach 0.15%, but natural propagation is difficult). Three healthy mother trees, aged 6 years, were selected, and resin-forming factors were obtained strictly according to the principle of non-destructive methods.

[0119] Propagation implementation stage: Following the steps of this invention, total RNA was first obtained from the phloem of the lateral roots of the mother plant, and the FPS gene and MYB transcription factor gene of this rare variety were cloned (sequencing verification showed that the sequence was 99.9% consistent with the mother plant gene sequence).

[0120] Construct recombinant expression vectors containing phloem-specific promoters;

[0121] Using the current year's axillary buds of the mother plant as recipients, cloned seedlings were obtained through Agrobacterium transformation, screening, and rooting.

[0122] After domestication, the content of agaric spirol in the phloem of the cloned seedlings was 0.14%-0.16%, which was highly consistent with the agaric-producing ability of the mother plant.

[0123] Preservation and application stage: The cloned seedlings were transplanted to the institute's germplasm resource nursery and nurtured in an environment simulating the mother plant's native environment (50% shading, soil pH 6.5, and regular application of trace element fertilizer). After 3 years, the cloned seedlings grew vigorously, and some plants were able to stably produce agarwood. Genetic testing showed good genetic stability, successfully achieving the preservation and efficient propagation of this rare agarwood variety, providing a material basis for subsequent variety research and industrial utilization.

[0124] Example 3: Seedling Supply in the Understory Economy Model of Agarwood Forests

[0125] Understory Economy Demand Stage: A certain region plans to develop an understory economy for agarwood cultivation, intercropping agarwood under rubber and camellia oleifera forests. However, the agarwood seedlings need to be able to adapt to the low-light environment under the forest and produce stable resin. Agarwood trees that are adapted to the local understory environment and produce good resin (agaric spirol content of approximately 0.1%) are selected as mother trees.

[0126] Seedling propagation stage: Using the method of this invention, the agarwood factor is obtained from the lateral roots of the mother plant and cloned for verification; when constructing the expression vector, a light-response element related to low light adaptation (such as the HY5 gene promoter fragment) is added to the vector to ensure that the cloned seedlings can express the agarwood factor normally under low light.

[0127] Complete the preparation, transformation, induction, and domestication of recipient materials according to the established procedures.

[0128] Understory application stage: The domesticated cloned seedlings were transplanted to understory plots such as rubber plantations and camellia oleifera plantations. Testing 18 months after transplanting showed that the cloned seedlings grew well in understory environments with 50%-70% shade, with agaric spirol content in the phloem ranging from 0.09% to 0.11%, meeting the basic requirements for resin quality in understory economics.

[0129] Furthermore, the cloning seedling propagation cycle is only about 80 days, which is significantly shorter than the traditional propagation method. This provides highly adaptable and stable agarwood seedlings for the understory economy model, promoting the development of local forestry integrated management.

[0130] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A non-destructive method for cloning and propagating agarwood resin-forming factors, characterized in that, Includes the following steps: (1) Non-destructive acquisition of resin formation factors: Select healthy agarwood trees with good resin formation quality and high resin formation quantity, dig a circular trench 20-30cm deep at a distance of 1.5-2m from the trunk, expose the lateral roots, cut off the phloem of the lateral roots, retain the xylem and the remaining phloem of the lateral roots and backfill the soil, extract total RNA from the phloem of the lateral roots, reverse transcribe to synthesize cDNA, and clone the key resin formation factor gene using cDNA as a template. The key resin formation factor gene includes the farnesyl pyrophosphate synthase gene and the MYB transcription factor gene. After sequencing verification, the target gene is obtained. (2) Construction of the expression vector of the agarwood factor: Select the plant expression vector, and obtain the linear vector by double digestion with restriction endonuclease. Construct a fusion fragment by tandem the target gene of step (1) with the phloem-specific promoter. Insert the fusion fragment into the linear vector using DNA ligase. After transforming the host bacteria, screen positive clones, extract plasmids and verify them to obtain the recombinant expression vector. (3) Non-destructive preparation of recipient material: Select current-year branches of agarwood tree, cut stem segments with one axillary bud, remove leaves and retain axillary bud scales, obtain sterile axillary buds after disinfection, inoculate sterile axillary buds into MS basic medium with added plant growth regulators, and culture under specific temperature and light conditions to induce axillary buds to sprout into buds; (4) Directed transformation of the aromatic factor and induction of clone seedlings: The recombinant expression vector of step (2) was introduced into Agrobacterium competent cells, positive Agrobacterium was screened, the germinated sterile buds of step (3) were transferred into positive Agrobacterium bacterial solution for soaking, the bacterial solution was dried and then inoculated into co-culture medium for dark culture, and then transferred into screening medium for culture to obtain positive buds. Finally, the positive buds were transferred into rooting medium to induce rooting and form complete clone seedlings. (5) Non-destructive agarication verification and acclimatization of cloned seedlings: The cloned seedlings in step (4) were transplanted to a sterile substrate and acclimatized under specific temperature and relative humidity conditions. After acclimatization, the agaric spirol content in the phloem of the cloned seedlings was detected by high performance liquid chromatography. The cloned seedlings with the agaric spirol content that met the standard were transplanted to the field.

2. The method for non-destructive clonal propagation of agarwood resin-forming factors according to claim 1, characterized in that: In step (1), the diameter of the exposed lateral root is 0.5-1 cm, the length of the cut lateral root phloem is 1-2 cm, and the total RNA of the lateral root phloem is extracted using the Trizol method.

3. The method for non-destructive cloning and propagation of agarwood resin-forming factors according to claim 2, characterized in that: In step (1), the specific primers used for cloning the key factor gene of *Cynanchum paniculatum* are upstream primer 5'-ATGGCTGCTGCTGCTGCT-3' and downstream primer 5'-TCAGCTGCTGCTGCTGCT-3', and the accuracy of sequencing verification is not less than 99%.

4. The method for non-destructive cloning and propagation of agarwood resin-forming factors according to claim 1, characterized in that: In step (2), the plant expression vector is pCAMBIA1301, the restriction endonucleases are BamHⅠ and SacⅠ, the host bacterium is Escherichia coli DH5α, and the verification methods include enzyme digestion verification and sequencing verification.

5. The method for non-destructive cloning and propagation of agarwood resin-forming factors according to claim 4, characterized in that: In step (2), the phloem-specific promoter is the phloem protein 2 gene promoter, the DNA ligase is T4 DNA ligase, and the tandem sequence of the target gene and the phloem-specific promoter in the fusion fragment is that the phloem-specific promoter is sequentially linked to the farnesyl pyrophosphate synthase gene and the MYB transcription factor gene.

6. The method for non-destructive clonal propagation of agarwood resin-forming factors according to claim 1, characterized in that: In step (3), the stem segment with one axillary bud is 2-3 cm long. The specific process of disinfection is to soak in 75% ethanol for 30 seconds, then transfer to 0.1% mercuric chloride solution for 8-10 minutes for disinfection, and then rinse with sterile water 5-6 times.

7. The method for non-destructive cloning and propagation of agarwood resin-forming factors according to claim 6, characterized in that: In step (3), the plant growth regulators added to the MS basic medium are 0.5 mg / L 6-benzylaminopurine and 0.1 mg / L naphthaleneacetic acid. The specific temperature is 25℃, the specific light conditions are 16 h / d light, and the culture time is 7-10 days.

8. The method for non-destructive clonal propagation of agarwood resin-forming factors according to claim 1, characterized in that: In step (4), the competent Agrobacterium cells are Agrobacterium GV3101 competent cells, the OD600 value of the positive Agrobacterium bacterial solution is 0.6-0.8, and the immersion time of the sterile buds in the bacterial solution is 10-15 min.

9. The method for non-destructive clonal propagation of agarwood resin-forming factors according to claim 8, characterized in that: In step (4), the co-culture medium was MS medium supplemented with 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid and 100 μmol / L acetylsylgenone, and the dark culture temperature was 22℃ for 3 days. The selection medium was MS medium supplemented with 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, 50 mg / L hygromycin and 200 mg / L cephalosporin, and the selection culture time was 20-25 days; The rooting medium was 1 / 2 MS medium supplemented with 0.2 mg / L indolebutyric acid, and the rooting culture time was 15-20 days.

10. The method for non-destructive clonal propagation of agarwood resin-forming factors according to claim 1, characterized in that: In step (5), the sterile substrate is a mixture of peat moss and perlite in a volume ratio of 3:1, with a specific temperature of 25-28℃, a specific relative humidity of 70%-80%, and an acclimatization time of 30-40 days. The detection conditions for high performance liquid chromatography were as follows: mobile phase was methanol and water at a volume ratio of 85:15, flow rate was 1 mL / min, and detection wavelength was 254 nm. The standard for the content of agaric spirol is that the content of agaric spirol in the phloem of cloned seedlings is ≥0.1%.

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