Tea tree tissue culture-free regeneration method based on leaf cutting

Abstract

The invention discloses a tea tree tissue culture-free regeneration method based on leaf cutting, which comprises the following steps: firstly, selecting mature and healthy leaves of a tea tree variety, and reserving complete petioles; then soaking the petiole base part of the leaf with a first exogenous hormone solution, and then cutting the leaf into a substrate; illumination, temperature and air humidity conditions are set for culture, a second exogenous hormone solution is sprayed to the leaves, and the leaves are sequentially induced to grow calluses, adventitious roots and adventitious buds. By adopting a tissue culture-free mode, through specific hormone treatment and environment regulation and control, the tea tree leaves form calluses, adventitious roots and adventitious buds at the petiole bases, and finally complete plants are formed, so that the regeneration efficiency of tea tree leaf cuttage can be remarkably improved, meanwhile, rooting and germination are realized, the complete plants are formed, and the survival rate of the tea tree leaves is increased. The method has a wide application prospect and an important economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation and seed and seedling breeding, and particularly relates to a tea tree tissue culture-free regeneration method based on leaf cuttings. Background Art

[0002] Genetic transformation technology is a crucial tool for studying plant gene function and improving crop genetics. Traditional genetic transformation methods, such as Agrobacterium-mediated transformation and gene gun techniques, often rely on tedious tissue culture procedures, including explant preparation, callus induction, and screening of regenerated plants. These processes are not only time-consuming and labor-intensive (typically taking 3-6 months), but their success rate is also affected by numerous factors, including explant type, culture medium formulation, and hormone combination. Furthermore, many economically important woody and perennial herbaceous plants have long been difficult to genetically manipulate due to difficulties in regeneration, severely limiting the genetic improvement of these crops.

[0003] In recent years, the development of non-tissue culture genetic transformation systems has become an important research direction in plant biotechnology. These methods aim to bypass tissue culture steps and achieve direct gene delivery and stable transformation in intact plants or specific organs. These methods can be broadly divided into three categories: 1) germ cell-based transformation methods, such as the floral dip method; 2) viral vector-based delivery systems; and 3) transformation systems based on the regenerative capacity of specific organs.

[0004] However, each of the above technologies has its own advantages and disadvantages. The biggest advantage of the inflorescence dip method is that it is easy to operate, does not require tissue culture, and the transformation efficiency can reach 0.5%-3%. However, its scope of application is extremely limited. It is mainly suitable for Arabidopsis and its closely related species, and is not effective for other plants, especially monocotyledons. With the development of CRISPR-Cas9 gene editing technology, viral vectors are widely used to deliver gene editing tools. They have strong infection ability, simple operation, and can target multiple organs at the same time; however, most viral vectors cannot integrate exogenous genes into the host genome, resulting in the editing effect being mostly transient, and the viral genome capacity is limited, making it difficult to carry larger gene fragments; in addition, the host range of viral infection is usually narrow, and may cause plant pathological symptoms. In recent years, the development of non-tissue culture transformation systems using the natural regeneration ability of specific plant organs has become a research hotspot. Generally, Agrobacterium is used to infect specific explants (such as stem segments or roots) to induce transformed cells to directly regenerate complete plants, thereby bypassing the traditional tissue culture process. The operation process is extremely simplified: only the explant needs to be immersed in Agrobacterium suspension, cultured until hairy roots are formed, and then the positive root segments are placed on a moist substrate to obtain transgenic buds. However, it relies on the ability of roots to emerge, and the transformation cycle in woody plants is still relatively long (3-4 months), and the efficiency of bud regeneration needs to be improved.

[0005] Tea( Camellia sinensis (L.) O. Kuntze) is a perennial evergreen woody plant of the Theaceae family and the genus Camellia, usually a shrub or small tree. It is one of the most important beverages in the world and is widely used in food, health food and feed. Traditional tea seedling cultivation mainly relies on seed propagation. As a core tool for tea variety improvement and functional gene research, the construction of a genetic transformation system can not only accelerate the breeding of high-quality and highly resistant new varieties, but also provide technical support for analyzing important biological processes such as tea secondary metabolism and adversity response. However, due to the biological characteristics of the tea tree itself, such as a high degree of lignification and easy browning of explants, low genetic transformation efficiency and difficulty in plant regeneration have become the two major technical bottlenecks in the construction of this system, seriously restricting the progress of tea tree molecular breeding and the sustainable development of the tea industry. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies of the existing technology and provide a tea tree tissue culture-free regeneration method based on leaf cuttings. The method utilizes the characteristic that leaves have no pre-growth points, and through their own regeneration ability, combined with the action of exogenous hormones, regenerated plants are formed through callus at the base of the petiole, providing a basis for establishing a tea tree genetic transformation system that does not require tissue culture.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A tea plant tissue culture-free regeneration method based on leaf cuttings, comprising the following steps: Step (1), selecting mature and healthy leaves of tea varieties, and retaining intact petioles; Step (2), cutting the leaves into the matrix; optionally, before cutting, the base of the petiole of the leaves is dipped in a first exogenous hormone solution; Step (3), setting the light, temperature and air humidity conditions for cultivation, and inducing the leaves to grow callus tissue, adventitious roots and adventitious buds in sequence; wherein the light intensity during the callus growth induction stage is 2000-10000 Lux, and the light intensity during the adventitious root and adventitious bud growth induction stage is 5000-15000 Lux; in addition, optionally, a second exogenous hormone solution is sprayed on the leaves during the cultivation process.

[0008] Furthermore, the matrix is ​​pure vermiculite with a particle size of 1-4 mm, or a mixture of vermiculite and perlite in a volume ratio of 1:2-3.

[0009] Furthermore, the photoperiod of the illumination is 16 h of light and 8 h of darkness.

[0010] Furthermore, the temperature is 20-28°C.

[0011] Furthermore, the air humidity is 80%-100%.

[0012] Furthermore, the first exogenous hormone solution uses naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or indolebutyric acid (IBA); furthermore, the concentration of the first exogenous hormone solution is 25-100 mg / L.

[0013] Furthermore, the second exogenous hormone solution is a mixed solution of at least two of 6-benzylaminopurine (BAP), naphthaleneacetic acid (NAA), gibberellin (GA) and indolebutyric acid (IBA).

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a tea tree asexual propagation seedling raising method, which adopts leaf cuttings without tissue culture, directly utilizes the regeneration ability of tea leaves, realizes the differentiation of adventitious roots and adventitious buds, and finally forms complete regenerated plants. The method is simple to operate, low in cost, and suitable for large-scale application. It also realizes the application of leaf cuttings in the organ regeneration of tea plants, filling the gap in the relevant field. In addition, the method of the present invention can perform tissue culture-free regeneration throughout the year, has fewer restrictions, and can be regulated by exogenous hormones, further improving the rooting rate and budding rate, helping to improve the efficiency and quality of tea tree breeding and seedling raising, and is of great significance for the rapid propagation and promotion of excellent forest tree varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are diagrams of adventitious bud differentiation from leaf cuttings of different varieties of tea trees, among which A is Fuding Dabai, B is Huang Guanyin, C is Longjing No. 1, and D is Phoenix Narcissus.

[0016] Figure 2 These are the results of full-leaf and half-leaf cuttings at 30 days old, where A represents full leaf and B represents half leaf.

[0017] Figure 3 The figure shows a comparison between young and aged callus without adventitious root and bud differentiation, where A is young callus and B is aged callus.

[0018] Figure 4 These are diagrams showing the differentiation of adventitious buds from the callus tissue and adventitious roots of cuttings, where A is the "Phoenix Narcissus" tea variety and B is the "Longjing No. 1" tea variety.

[0019] Figure 5 These are toluidine blue stained sections (40x), where A is a sampling diagram of double bud differentiation, B is a longitudinal section of double bud differentiation, C is a diagram of callus bud organogenesis, D is a sampling diagram of single bud differentiation, E is a longitudinal section of single bud differentiation, F is a sampling diagram of adventitious root differentiation but not adventitious bud differentiation, and G is a transverse section of root differentiation.

[0020] Figure 6This is a microscopic observation of the cross section of callus tissue, where Vb: vascular bundle, Co: cortex, Ep: epidermis, Ca: callus cell. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] As mentioned above, the present invention provides a tea plant tissue culture-free regeneration method based on leaf cuttings, comprising the following steps: Step (1), selecting mature and healthy leaves of tea varieties, and retaining intact petioles; Step (2), cutting the leaves into a matrix; wherein the matrix is ​​pure vermiculite with a particle size of 1-4 mm, or a mixture of vermiculite and perlite in a volume ratio of 1:2-3; optionally, before cutting, the base of the petiole of the leaves is dipped in a solution of naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or indolebutyric acid (IBA) at a concentration of 25-100 mg / L; Step (3), culturing at a temperature of 20-28 ° C and an air humidity of 80%-100%, inducing the leaves to grow callus tissue, adventitious roots and adventitious buds in sequence; wherein the light intensity during the callus growth induction stage is 2000-10000 Lux, and the light intensity during the adventitious root and adventitious bud growth induction stage is 5000-15000 Lux, and the photoperiod is 16 hours of light and 8 hours of darkness; in addition, optionally, a second exogenous hormone solution is sprayed on the leaves during the culturing process, and the second exogenous hormone solution is a mixed solution of at least two of 6-benzylaminopurine (BAP), naphthaleneacetic acid (NAA), gibberellin (GA) and indolebutyric acid (IBA).

[0023] The following describes the details in conjunction with specific embodiments.

[0024] Example 1: Effects of different seasons on tea leaf cuttings The leaves of the Huang Guanyin variety grown in the current year in different months were used for cuttings. The other cutting conditions were: temperature of 25 ℃, humidity of 90%, light intensity of 10,000 Lux, photoperiod of 16 h of light and 8 h of darkness, and pure vermiculite with a particle size of 1-4 mm was used as the cutting medium.

[0025] The results are shown in Table 1. The rooting rate of tea leaves cut in September-November in autumn is relatively higher than that in March-May (spring) and June-August (summer), and the cycle of adventitious bud differentiation of tea trees is shorter. This may be related to the growth rhythm and more sufficient nutrient accumulation of tea trees in autumn, which is more conducive to the physiological activities of tea leaf cuttings.

[0026] In addition, the survival rate of cuttings is related to the maturity of the leaves of the mother tree. On the strong mother tree of the same year, the buds with 1-2 leaves are too tender and will wilt due to water loss. The buds with 1-2 leaves and the buds with 6-8 leaves are more lignified, forming less callus tissue, slower and smaller in size, causing them to stop growing and unable to obtain enough nutrients for rooting and sprouting. However, the cuttings with 3-5 leaves in the middle of the bud grow well.

[0027] Table 1 Results of leaf cuttings of Huang Guanyin in different seasons month Number of cuttings Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) Germination time (weeks) March-May 400 297 74.3% 3 0.8% 24 June-August 400 310 77.5% 8 2.0% 24 September-November 400 321 80.3% 17 4.3% 16 Example 2: Effects of different varieties on tea leaf cuttings Four different tea tree varieties were selected: Phoenix Narcissus ( C. sinensis cv. Fenghuang Shuixian ) Fuding White Tea ( C. sinensis cv. Fuding Dabaicha )、Huang Guanyin( C. sinensis cv.Huangguanyin )、Longjing No. 1( C. sinensis cv. Longjing 1 ), all healthy, disease-free, mature leaves from the current year were used, with intact petioles. Other cutting conditions included a temperature of 25°C, a humidity of 90%, a light intensity of 10,000 Lux, a photoperiod of 16 hours of light and 8 hours of darkness, and pure vermiculite with a particle size of 1-4 mm as the cutting medium.

[0028] The results are shown in Table 2 and Figure 1 As shown, different tea tree varieties have the ability to generate plants through complete adventitious roots and adventitious buds through leaf cuttings. Huang Guanyin and Longjing No. 1 varieties have stronger adventitious root generation capabilities (83%, 84%). Longjing No. 1 has better adventitious root growth and a thicker root system during the cutting process, but is less likely to generate adventitious buds. The adventitious bud differentiation of Huang Guanyin is relatively better than that of other varieties; the root systems of Fuding Dabai and Phoenix Narcissus are relatively weak.

[0029] Table 2 Results of leaf cuttings of different tea varieties variety Number of cuttings Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) Fuding Dabai 300 211 70.3% 4 1.3% Huang Guanyin 300 249 83.0% 13 4.3% Longjing No. 1 300 252 84.0% 1 0.3% Phoenix Narcissus 300 215 71.6% 8 2.6% Example 3: Results of regeneration from whole leaf and half leaf cuttings Considering that, unlike plant branch cuttings, leaf nutrient accumulation in the early stages of cuttings comes from both the parent plant and the leaves' own photosynthetic accumulation, we investigated the effects of leaf nutrient accumulation and photosynthetic respiration on leaf cuttings. We used both full-leaf and half-leaf cuttings of the Huang Guanyin variety. Other cutting conditions included a temperature of 25°C, a humidity of 90%, a light intensity of 10,000 Lux, and a photoperiod of 16 hours of light and 8 hours of darkness. The cutting medium was pure vermiculite with a particle size of 1-4 mm.

[0030] The results are shown in Table 3 and Figure 2As shown, tea cuttings from full-leaf cuttings grow rapidly, accumulate sufficient nutrients, and can rapidly develop within 30 days, with callus tissue expanding and a small number of rooting. Cuttings from half-leaf cuttings grow slowly, accumulate insufficient nutrients themselves, and show significant differences in callus development. They are smaller in size, and a small number of them stop growing. Furthermore, adventitious rooting is much lower in half-leaf cuttings than in full-leaf cuttings.

[0031] Table 3 Whole leaf and half leaf cutting treatments Cutting method Number of cuttings Number of wounds healed after 30 days Healing rate (%) Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) Whole leaf 256 224 87.5% 148 57.8% 0 0 Half Leaf 256 188 73.4% 85 33.2% 1 0.3% Example 4: Regeneration results of tea leaves treated with different light intensities The growth of tea leaf cuttings under different light intensities was tested. The other cutting conditions were: the yellow Guanyin variety of the current year was selected, the temperature was 25 ℃, the humidity was 90%, the photoperiod was 16 h of light and 8 h of darkness, and the cutting medium was pure vermiculite with a particle size of 1-4 mm.

[0032] The results are shown in Table 4. Under 20,000 Lux, the light intensity is too strong, which leads to the loss of leaf photosynthesis and respiration, and the leaves are prone to death and poor growth, which is not conducive to the formation of callus tissue and adventitious roots. Compared with 5,000 Lux, the growth conditions at 10,000 Lux and 15,000 Lux are relatively better at the adventitious rooting stage, which is more conducive to plant rooting development and adventitious bud differentiation.

[0033] Table 4 Treatments with different light intensities Light intensity Number of cuttings Number of wounds healed after 30 days Healing rate (%) Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) 5000Lux 224 192 85.7% 65 29.0% 0 0 10000Lux 224 201 89.7% 124 55.3% 2 0.8% 15000Lux 224 194 86.6% 108 48.2% 1 0.4% 20000Lux 224 64 28.5% 25 11.1% 0 0 Example 5: Regeneration results of tea leaves treated with different substrates The rooting and development stage of tea leaf cuttings is a critical period for success and is influenced by a combination of factors. Among the abiotic factors affecting tea leaf cuttings, the substrate is also crucial. The substrate's air permeability, water retention, and fertility directly impact the growth environment of tea leaves. A well-permeable substrate ensures sufficient oxygen to the roots, promoting respiration and providing energy for bud formation. A substrate with adequate water retention maintains a balanced moisture level around the leaves, preventing them from losing water and drying out. A nutrient-rich substrate provides essential nutrients for bud growth. Callus tissue will form in 2-3 weeks after cutting, and rooting will begin around 4 weeks later. The cutting substrates used in the experiment consisted of pure vermiculite of varying particle sizes and varying ratios of vermiculite to perlite (the vermiculite particle size in each vermiculite to perlite ratio was uniformly 2-4 mm). In order to explore the possible relationship between different stress response conditions of plants and the differentiation of regenerative organs of tea trees, different cutting media were treated; the other cutting conditions were: the current year Huang Guanyin variety was selected, the temperature was 25 ℃, the humidity was 90%, the light intensity was 10000 Lux, and the photoperiod was 16 h of light and 8 h of darkness.

[0034] The results, as shown in Table 5, show that tea leaves developed better during the rooting stage when pure vermiculite with a particle size of 2 mm and a perlite to vermiculite ratio of 1:3 (2-4 mm) were used in the substrate. Although pure vermiculite (1-2 mm) has good water retention, its small pores and high humidity impede root respiration and water absorption. Pure vermiculite (3-6 mm) has relatively poor water retention, resulting in a low humidity level that is unsuitable for growth. A loose, breathable substrate with good water retention is beneficial for root growth and oxygen supply.

[0035] Table 5 Treatments of different cutting media Cutting medium Number of cuttings Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) Pure vermiculite (1-2 mm) 360 216 60.0% 0 0 Pure vermiculite (2-4 mm) 360 290 80.8% 3 0.8% Pure vermiculite (3-6 mm) 360 205 56.9% 0 0 Perlite:Vermiculite (1:2) 360 223 61.9% 2 0.5% Perlite:Vermiculite (1:3) 360 273 75.8% 0 0 Example 6: Results of tea leaf regeneration treated with different hormones Hormones play a key regulatory role in the growth of tea leaf cuttings. This example uses a current-year Huang Guanyin tea plant, maintains a temperature of 25°C, a humidity of 90%, and a photoperiod of 16 hours of light and 8 hours of darkness. The cutting medium is pure vermiculite with a particle size of 1-4 mm. Hormone treatment is used during the cutting process.

[0036] Specifically, this embodiment conducted experiments in two ways: dipping before cutting (30 minutes) and foliar spraying of different hormones after cutting (10 days / time). The results are shown in Tables 6 and 7, respectively. Auxin hormones can effectively promote the development of tea leaf cuttings in the adventitious root differentiation stage. After combined treatment with cytokinins and auxin hormones, the differentiation of adventitious roots and adventitious buds can be promoted compared with the control. After dipping, NAA (rooting rate 78.1%) has the best effect on promoting the differentiation of adventitious roots of cuttings.

[0037] Table 6 Different hormone dipping treatments Hormone ratio Number of cuttings Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) 1 160 110 68.8% 0 0 2 160 125 78.1% 1 0.6% 3 160 103 64.4% 0 0 4 160 92 57.5% 0 0 Ck 80 42 52.5% 0 0 Hormone ratio description: 1 is 50 mg / L IAA; 2 is 50 mg / L NAA; 3 is 50 mg / L IBA; 4 is 50 mg / LBAP; 5 is pure water.

[0038] Table 7 Treatments with different hormones sprayed Hormone ratio Number of cuttings Number of roots Rooting rate (%) Sprouts (pieces) Germination rate (%) 1 160 96 60.0% 2 1.3% 2 160 93 58.1% 3 1.9% 3 160 88 55.0% 3 1.9% 4 160 97 60.6% 1 0.6% Ck 80 33 41.2% 0 0 Hormone ratio description: 1 is 2 mg / L BAP+0.1 mg / L NAA+3 mg / L GA; 2 is 2 mg / L BAP+0.8 mg / L NAA+1 mg / L IBA; 3 is 4 mg / L BAP+1 mg / L IBA; 4 is 2 mg / L BAP+1 mg / L GA; 5 is pure water.

[0039] Example 7: Verification of the growth and development stages of tea leaf cuttings Currently, research on inducing adventitious buds in tea plants focuses on three main pathways for generating plants: direct and indirect. The direct pathway involves cells developing directly into embryos under certain growth and development conditions without callus induction. The indirect pathway involves somatic embryos undergoing callus induction to produce embryonic calli, where the embryonic cells continue to differentiate and develop into somatic embryos. Furthermore, in addition to direct bud formation from axillary and adventitious buds without callus induction, plant organs can also regenerate into adventitious buds by inducing callus tissue from explants, which then differentiates into adventitious buds. Regenerated buds are formed when some parenchyma cells in the callus dedifferentiate back into meristematic cells, further forming meristematic nodes and ultimately forming the primordium of a bud or root. These cells then undergo tissue differentiation through periclinal and clinodial divisions.

[0040] This example demonstrates that all tea varieties can be regenerated from leaf cuttings into complete plants. While budding rates vary among varieties, experimental data show that all tea varieties can form callus and develop into complete plants under suitable conditions, demonstrating the feasibility of the technique. Rooting leaf cuttings also produce callus.

[0041] like Figure 3 As shown, all the cuttings that sprouted passed through the callus growth stage. For example, Huang Guanyin tea, the callus formed fastest in its leaf cuttings (approximately two weeks), followed by rooting. This phenomenon indicates that callus formation is a necessary process for seedling formation from tea leaf cuttings, and its development efficiency directly affects the seedling cycle and budding rate. Furthermore, rooting leaves, because they retain some conductive tissue, can more efficiently absorb water and hormones, thereby accelerating callus differentiation.

[0042] The regenerated buds produced have two states: buds growing from nodules and buds growing on the roots of the plants. Through experimental observation and growth tracking, two bud-generating patterns of tea leaf cuttings were discovered: Path 1: Buds from nodule-like callus (stemoid manifestation) In this pathway, the callus tissue formed at the base of the tea leaf cutting gradually expands and takes on a tumor-like structure (approximately 2-3 mm in diameter), on which buds differentiate. These buds have the anatomical characteristics of a stem and eventually develop into independent plants.

[0043] Method 2: Direct sprouting from the roots In the tea variety Longjing No. 1 ( Figure 4 In B), after the callus and rooting stages, the bud points of the cuttings germinate directly from the roots of the root system without going through the callus tissue stem-like stage.

[0044] At different stages of tea leaf cuttings, different callus morphologies and nodule-like structures were observed to form at the callus site. Bud formation from nodule-like calli relies on the expression of cellular totipotency, while direct bud formation on the roots likely utilizes pre-existing lateral bud primordia in the root system. The proportion of these two pathways may be regulated by both the genetic characteristics of the variety and the external environment. Furthermore, this experiment observed that in tea leaf cuttings, callus tissue is not only the physical basis for bud differentiation but also a regulatory center for hormonal signaling, playing a central role in the differentiation of adventitious roots and buds.

[0045] In addition, the adventitious buds of the Huang Guanyin variety were differentiated by toluidine blue staining and section observation. Figure 5 Slice observation Figure 5 The structure of callus tissue and adventitious roots and buds is complex, and the cross section of callus tissue is also composed of epidermis, cortex and vascular bundles ( Figure 5 G in ), with complete stem-like structure and a large number of callus cells differentiated (see Figure 6 ), a large number of callus tissues proliferated and grew, arranged tightly and compactly, and divided vigorously. Microscopically, the callus cells in the dense part were stained darker, and the cytoplasm was dense ( Figure 5 B). In the longitudinal section of the double bud differentiation of Huang Guanyin, new buds are differentiated in the callus tissue ( Figure 5 C in ).

[0046] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A tea plant tissue culture-free regeneration method based on leaf cuttings, characterized in that: The method comprises the following steps: Step (1), selecting mature and healthy leaves of tea varieties, and retaining intact petioles; Step (2), inserting the leaves into the substrate; Step (3): setting the light, temperature and air humidity conditions for cultivation, and sequentially inducing the growth of callus tissue, adventitious roots and adventitious buds from the leaves; wherein the light intensity during the callus growth induction stage is 2000-10000 Lux, and the light intensity during the adventitious root and adventitious bud growth induction stage is 5000-15000 Lux.

2. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 1, characterized in that: The matrix is ​​pure vermiculite with a particle size of 1-4 mm, or a mixture of vermiculite and perlite in a volume ratio of 1:2-3.

3. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 1, characterized in that: The photoperiod of the illumination was 16 h of light and 8 h of darkness.

4. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 1, characterized in that: The temperature is 20-28°C.

5. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 1, characterized in that: The air humidity is 80%-100%.

6. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 1, characterized in that: In step (2), the base of the petiole of the leaf is dipped in the first exogenous hormone solution before cutting.

7. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 6, characterized in that: The first exogenous hormone solution adopts naphthylacetic acid, indoleacetic acid or indolebutyric acid.

8. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 6 or 7, characterized in that: The concentration of the first exogenous hormone solution is 25-100 mg / L.

9. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 1, characterized in that: During the cultivation process of step (3), the second exogenous hormone solution is sprayed on the leaves.

10. The tea plant tissue culture-free regeneration method based on leaf cuttings according to claim 9, characterized in that: The second exogenous hormone solution is a mixed solution of at least two of 6-benzylaminopurine, naphthaleneacetic acid, gibberellin and indolebutyric acid.

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