A tea tree non-tissue culture regeneration method based on leaf cutting

Abstract

This invention discloses a tissue culture-free regeneration method for tea trees based on leaf cuttings, comprising the following steps: First, select mature, healthy leaves of a tea variety, retaining the complete petiole; then, treat the base of the petiole with a first exogenous hormone solution, and then insert the leaf cuttings into a substrate; cultivate under controlled light, temperature, and humidity conditions, and spray the leaves with a second exogenous hormone solution to induce the growth of callus tissue, adventitious roots, and adventitious buds. This invention employs a tissue culture-free method, through specific hormone treatment and environmental regulation, to induce the formation of callus tissue, adventitious roots, and adventitious buds at the base of the petiole of tea leaves, ultimately forming a complete plant. This significantly improves the regeneration efficiency of tea leaf cuttings, simultaneously achieving root and bud formation to form a complete plant, and has broad application prospects and significant economic value.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation and seed and seedling cultivation technology, specifically relating to a method for the tissue culture-free regeneration of tea trees based on leaf cuttings. Background Technology

[0002] Genetic transformation technology is an important tool for plant gene function research and crop genetic improvement. Traditional genetic transformation methods, such as Agrobacterium-mediated transformation and gene gun methods, usually rely on cumbersome tissue culture processes, including explant preparation, callus induction, and screening of regenerated plants. These processes are not only time-consuming and labor-intensive (usually requiring 3-6 months), but their success rate is also affected by various factors, including explant type, culture medium formulation, and hormone combinations. More importantly, many economically valuable woody plants and perennial herbaceous plants have long been difficult to genetically manipulate due to their regeneration difficulties, severely limiting the genetic improvement process of these crops.

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

[0004] However, each of the above techniques has its own advantages and disadvantages. The biggest advantage of the inflorescence immersion method is its simplicity, lack of tissue culture requirement, and transformation efficiency of 0.5%-3%. However, its application is extremely limited, mainly applicable to Arabidopsis thaliana and its closely related species, and its effect on other plants, especially monocots, is poor. With the development of CRISPR-Cas9 gene editing technology, viral vectors are widely used to deliver gene editing tools. They have strong infectivity, are easy to operate, and can target multiple organs simultaneously. However, most viral vectors cannot integrate foreign genes into the host genome, resulting in mostly transient editing effects. Furthermore, the viral genome has limited capacity, making it difficult to carry large gene fragments. In addition, the host range of viral infection is usually narrow, and it may cause plant pathological symptoms. In recent years, developing non-tissue culture transformation systems by utilizing the natural regeneration capabilities of specific plant organs has become a research hotspot. Generally, this involves infecting specific explants (such as stem segments or roots) with Agrobacterium to induce transformed cells to directly regenerate complete plants, thus bypassing the traditional tissue culture process. The operation is extremely simplified: the explants are simply immersed in Agrobacterium suspension and cultured until hairy roots form. Then, positive root segments are placed on a moist substrate to obtain transgenic shoots. However, this method relies on the ability of roots to produce shoots, and the transformation cycle for woody plants is still relatively long (3-4 months), so the shoot regeneration efficiency needs to be improved.

[0005] Tea( Camellia sinensis (L.) O. KuntzeCamellia is a perennial evergreen woody plant belonging to the genus Camellia in the family Theaceae. It is typically a shrub or small tree and is one of the world's most important beverages, widely used in the food, health food, and feed industries. Traditional tea seedling cultivation relies primarily on seed propagation. However, as a core tool for tea variety improvement and functional gene research, the construction of genetic transformation systems can not only accelerate the cultivation of high-quality, highly resistant new varieties but also provide technical support for elucidating important biological processes such as secondary metabolism and stress responses in tea plants. However, due to the high lignification degree of tea plants and the ease with which explants brown, low genetic transformation efficiency and difficulty in plant regeneration have become two major technical bottlenecks in the construction of this system, severely restricting the progress of molecular breeding of tea plants and the sustainable development of the tea industry. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for the tissue culture-free regeneration of tea plants based on leaf cuttings. This 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 by callus formation at the base of the petiole, thus providing a foundation for establishing a tissue culture-free genetic transformation system for tea plants.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for the tissue culture-free regeneration of tea trees based on leaf cuttings, the method comprising the following steps:

[0009] Step (1): Select mature and healthy leaves of tea tree varieties, keeping the petioles intact;

[0010] Step (2): Insert the leaf cuttings into the substrate; optionally, before inserting the cuttings, dip the base of the petiole of the leaf in a first exogenous hormone solution.

[0011] Step (3): Set the light, temperature and air humidity conditions for cultivation, and induce the growth of callus, adventitious roots and adventitious buds in the leaves in sequence; wherein, the light intensity during the callus induction stage is 2000-10000 Lux, and the light intensity during the adventitious root and adventitious bud induction stage is 5000-15000 Lux; in addition, optionally, a second exogenous hormone solution is sprayed on the leaves during the cultivation process.

[0012] Furthermore, the matrix is ​​pure vermiculite with a particle size of 1-4 mm, or a mixture of vermiculite and perlite at a volume ratio of 1:2-3.

[0013] Furthermore, the light cycle is 16 hours of illumination followed by 8 hours of darkness.

[0014] Furthermore, the temperature is 20-28 ℃.

[0015] Furthermore, the air humidity is 80%-100%.

[0016] Furthermore, the first exogenous hormone solution uses naphthaleneacetic acid (NAA), indoleacetic acid (IAA), or indolebutyric acid (IBA); even further, the concentration of the first exogenous hormone solution is 25-100 mg / L.

[0017] Furthermore, the second exogenous hormone solution is a mixture of at least two of 6-benzylaminopurine (BAP), naphthaleneacetic acid (NAA), gibberellin (GA), and indolebutyric acid (IBA).

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention provides a method for asexual propagation of tea trees, using leaf cuttings. It eliminates the need for tissue culture, directly utilizing the regenerative capacity of tea leaves to differentiate adventitious roots and buds, ultimately forming complete regenerated plants. This method is simple to operate, low in cost, and suitable for large-scale application. Furthermore, it realizes the application of leaf cuttings in the organ regeneration of tea trees, filling a gap in related fields. In addition, this method can perform tissue culture-free regeneration in all four seasons with fewer limitations, and can be regulated by exogenous hormones to further improve rooting and budding rates. This helps improve the efficiency and quality of tea tree breeding and seedling production, and is of great significance for the rapid propagation and promotion of superior forest tree varieties. Attached Figure Description

[0020] Figure 1 The diagram shows the differentiation of adventitious buds from leaf cuttings of different tea varieties. A represents Fuding Dabai, B represents Huang Guanyin, C represents Longjing No. 1, and D represents Fenghuang Shuixian.

[0021] Figure 2 The images show the results of whole-leaf and half-leaf cuttings after 30 days, where A represents whole leaves and B represents half leaves.

[0022] Figure 3 The image shows a comparison between young and aged callus tissues without adventitious root and bud differentiation, where A represents young callus tissue and B represents aged callus tissue.

[0023] Figure 4 The diagrams show the differentiation of adventitious buds from the callus tissue and adventitious roots of the cuttings, respectively. A represents the "Phoenix Narcissus" tea variety, and B represents the "Longjing No. 1" tea variety.

[0024] Figure 5 The images show toluidine blue stained sections (40x), where A is a sampling image of double bud differentiation, B is a longitudinal section of double bud differentiation, C is bud organogenesis in callus tissue, D is a sampling image of single bud differentiation, E is a longitudinal section of single bud differentiation, F is a sampling image with adventitious root differentiation but no adventitious bud differentiation, and G is a transverse section of root differentiation.

[0025] Figure 6 This is a microscopic view of a cross-section of callus tissue, where Vb: vascular bundles, Co: cortex, Ep: epidermis, and Ca: callus cells. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] As mentioned above, this invention provides a method for the tissue culture-free regeneration of tea trees based on leaf cuttings, comprising the following steps:

[0028] Step (1): Select mature and healthy leaves of tea tree varieties, keeping the petioles intact;

[0029] Step (2): Insert the leaf cuttings into the substrate; wherein the substrate 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 inserting the cuttings, the base of the leaf petiole is treated with a solution of naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or indolebutyric acid (IBA) with a concentration of 25-100 mg / L.

[0030] Step (3): Cultivate the leaves under conditions of 20-28 ℃ and 80%-100% humidity, and induce callus, adventitious roots and adventitious buds to grow sequentially. The light intensity during the callus induction stage is 2000-10000 Lux, and the light intensity during the adventitious root and adventitious bud induction stages is 5000-15000 Lux. The photoperiod is 16 h of light and 8 h of darkness. Alternatively, the leaves can be sprayed with a second exogenous hormone solution during the cultivation process. The second exogenous hormone solution is a mixture of at least two of 6-benzylaminopurine (BAP), naphthaleneacetic acid (NAA), gibberellin (GA) and indolebutyric acid (IBA).

[0031] The following description is based on specific embodiments.

[0032] Example 1: The effect of different seasons on tea leaf cuttings

[0033] Leaf cuttings were taken from different months of the current year's growth of the Huang Guanyin variety. Other cutting conditions were as follows: temperature 25 ℃, humidity 90%, light intensity 10000 Lux, photoperiod of 16 h light and 8 h darkness, and pure vermiculite with a particle size of 1-4 mm as the cutting substrate.

[0034] The results are shown in Table 1. The rooting rate of tea leaves taken from September to November in autumn was relatively higher than that taken from March to May (spring) and June to August (summer). The period of adventitious bud differentiation of tea trees was also shorter. This may be related to the fact that the growth rhythm and nutrient accumulation of tea trees are more sufficient in autumn, which is more conducive to the physiological activities of tea leaf cuttings.

[0035] Furthermore, the survival rate of cuttings is related to the maturity of the mother tree's leaves. On robust, current-year mother trees, cuttings with 1-2 leaves on the terminal bud are too tender and will wilt due to water loss. Additionally, cuttings with 1-2 leaves on the terminal bud have insufficient nutrient accumulation in the early stages, and cuttings with 6-8 leaves on the terminal bud have a higher degree of lignification, resulting in less, slower, and smaller callus formation, leading to cessation of growth and insufficient nutrients for root and shoot development. Cuttings with 3-5 leaves on the middle terminal bud, however, show good growth.

[0036] Table 1. Results of leaf cutting propagation of Alocasia macrorrhiza in different seasons.

[0037] month Number of cuttings Number of roots (units) Rooting rate (%) Sprouts (number) Germination rate (%) Germination time (weeks) March to May 400 297 74.3% 3 0.8% 24 June to August 400 310 77.5% 8 2.0% 24 September to November 400 321 80.3% 17 4.3% 16

[0038] Example 2: The effect of different varieties on tea leaf cuttings

[0039] Four different tea tree varieties were selected: Phoenix Narcissus ( C. sinensis cv. Fenghuang Shuixian Fuding White Tea C. sinensis cv. Fuding Dabaicha ), Yellow Guanyin ( C. sinensis cv.Huangguanyin Longjing No. 1 ( C. sinensis cv. Longjing 1 All cuttings were taken from healthy, disease-free, mature leaves of the current year, with intact petioles. Other propagation conditions were: temperature 25 ℃, humidity 90%, light intensity 10000 Lux, photoperiod of 16 h light and 8 h dark, and pure vermiculite with a particle size of 1-4 mm as the propagation substrate.

[0040] The results are shown in Table 2 and , respectively. Figure 1 As shown, all tea varieties have the ability to develop adventitious roots and buds to form plants through leaf cuttings. Huang Guanyin and Longjing No. 1 varieties have stronger adventitious root development capabilities (83% and 84%, respectively). Longjing No. 1 shows better adventitious root growth and a stronger root system during the cutting process, but it is not easy to develop adventitious buds. Huang Guanyin, on the other hand, has a relatively better adventitious bud differentiation than other varieties. Fuding Dabai and Fenghuang Shuixian have weaker root systems.

[0041] Table 2 Results of leaf cuttings from different tea varieties

[0042] variety Number of cuttings Number of roots (units) Rooting rate (%) Sprouts (number) Germination rate (%) Fuding Big White 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%

[0043] Example 3: Results of regeneration from whole-leaf and half-leaf cuttings

[0044] Unlike stem cuttings, leaf nutrient accumulation in the early stages of leaf cutting comes from the mother plant's accumulation and the leaf's own photosynthetic accumulation. This study investigated the effects of nutrient accumulation and loss due to photosynthesis and respiration on leaf cuttings. Therefore, the *Aureomarginata* cultivar was used for both whole-leaf and half-leaf cuttings. Other cutting conditions were: temperature 25 ℃, humidity 90%, light intensity 10000 Lux, photoperiod of 16 h light followed by 8 h darkness, and pure vermiculite with a particle size of 1-4 mm as the cutting substrate.

[0045] The results are shown in Table 3 and , respectively. Figure 2 As shown, whole-leaf tea cuttings grow rapidly with ample nutrient accumulation, exhibiting rapid growth and development within 30 days, with callus tissue swelling and a small portion developing roots. Half-leaf cuttings, on the other hand, grow slowly, with insufficient nutrient accumulation, resulting in significantly different callus development, smaller callus tissue, and a small portion ceasing growth. Furthermore, adventitious root formation from half-leaf cuttings is far lower than that from whole-leaf cuttings.

[0046] Table 3. Treatment of whole-leaf and half-leaf cuttings

[0047] Cuttings Number of cuttings Number of wounds healed in 30 days (pieces) Healing rate (%) Number of roots (units) Rooting rate (%) Sprouts (number) 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%

[0048] Example 4: Results of tea leaf regeneration under different light intensities

[0049] The growth of tea leaves under different light intensities was tested. Other cutting conditions were as follows: the current year's Huang Guanyin variety 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 substrate was pure vermiculite with a particle size of 1-4 mm.

[0050] The results are shown in Table 4. Under 20,000 Lux of light, the photosynthesis and respiration of the leaves were damaged, the leaves were prone to death and the growth status was poor, which was not conducive to the formation of callus and adventitious roots. In contrast, under 10,000 Lux and 15,000 Lux of light intensity, the growth status of the adventitious roots was relatively better than that under 5,000 Lux of light intensity, which was more conducive to the rooting and development of plants and the differentiation of adventitious buds.

[0051] Table 4 Treatment under different light intensities

[0052] Light intensity Number of cuttings Number of wounds healed in 30 days (pieces) Healing rate (%) Number of roots (units) Rooting rate (%) Sprouts (number) Germination rate (%) 5000 Lux 224 192 85.7% 65 29.0% 0 0 10000 Lux 224 201 89.7% 124 55.3% 2 0.8% 15000 Lux 224 194 86.6% 108 48.2% 1 0.4% 20000Lux 224 64 28.5% 25 11.1% 0 0

[0053] Example 5: Results of tea leaf regeneration under different substrate treatments

[0054] The rooting and development stage of tea leaf cuttings is a crucial period determining the success of propagation, influenced by a combination of factors. Among the abiotic factors, the type of substrate is also key. The substrate's aeration, water retention, and fertility directly affect the tea leaf's growth environment. A well-aerated substrate ensures sufficient oxygen for the roots, promoting respiration and providing energy for bud formation; a substrate with suitable water retention maintains the moisture balance around the leaves, preventing dehydration and wilting; and a nutrient-rich substrate provides the necessary nutrients for bud growth. After cutting, callus tissue is induced in tea leaves within 2-3 weeks, and rooting begins around 4 weeks. The propagation substrates used in the experiment included pure vermiculite of different particle sizes and different ratios of vermiculite to perlite (with a uniform vermiculite particle size of 2-4 mm in all ratios). To investigate the possible correlation between different stress response conditions in plants and the differentiation of regenerative organs in tea trees, different cutting substrates were used for treatment. Other cutting conditions included: using current-year-old Huang Guanyin variety, with a temperature of 25 ℃, a humidity of 90%, a light intensity of 10000 Lux, and a photoperiod of 16 h of light followed by 8 h of darkness.

[0055] The results are shown in Table 5. When using pure vermiculite with a particle size of 2 mm in the substrate, and when the ratio of perlite to vermiculite (2-4 mm) was 1:3, tea leaves developed better during the rooting stage. While pure vermiculite (1-2 mm) had good water retention, its small porosity resulted in excessive moisture, affecting root respiration and water absorption. Pure vermiculite (3-6 mm) had relatively poor water retention, leading to lower substrate moisture levels unsuitable for growth. A loose, well-aerated substrate with good water retention is beneficial for root growth and oxygen supply.

[0056] Table 5 Treatment of different cutting substrates

[0057] Cutting substrate Number of cuttings Number of roots (units) Rooting rate (%) Sprouts (number) 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

[0058] Example 6: Results of tea leaf regeneration after treatment with different hormones

[0059] Hormones play a crucial regulatory role in the growth of tea leaf cuttings. In this example, current-year-old Huang Guanyin cultivar was used, with a temperature of 25 ℃, humidity of 90%, and a photoperiod of 16 h light and 8 h dark. Pure vermiculite with a particle size of 1-4 mm was used as the cutting substrate, and hormone treatment was applied during the cutting process.

[0060] Specifically, this embodiment conducted experiments using two methods: pre-propagation dipping (30 min) and post-propagation foliar spraying with different hormones (10 days / time). The results are shown in Tables 6 and 7, respectively. Auxin-type hormones can effectively promote the development of tea leaf cuttings during the adventitious root differentiation stage. After combined treatment with cytokinin-type and auxin-type hormones, the differentiation of adventitious roots and buds can also be promoted compared with the control. After dipping, NAA (rooting rate 78.1%) showed the best effect in promoting the differentiation of adventitious roots in cuttings.

[0061] Table 6 Different hormone immersion treatments

[0062] Hormone ratio Number of cuttings Number of roots (pieces) Rooting rate (%) Sprouts (number) 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

[0063] Hormone ratio instructions: 1 is 50 mg / L IAA; 2 is 50 mg / L NAA; 3 is 50 mg / L IBA; 4 is 50 mg / L LBAP; 5 is pure water.

[0064] Table 7 Different hormone spraying treatments

[0065] Hormone ratio Number of cuttings Number of roots (pieces) Rooting rate (%) Sprouts (number) 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

[0066] Hormone formulation instructions: 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.

[0067] Example 7: Verification of the growth and development stages of tea leaf cuttings

[0068] Currently, research on the induction of adventitious buds in tea plants mainly identifies three pathways for plant generation: direct and indirect. Direct generation occurs when cells develop directly into embryos under specific growth and developmental conditions without callus induction. Indirect generation involves somatic embryos undergoing callus induction to produce embryogenic callus, which then differentiates into somatic embryos. In addition to axillary buds and adventitious buds directly generating adventitious buds without callus induction, adventitious buds can also be generated by inducing callus tissue from explants, which then further differentiates to produce adventitious buds. Regenerated buds are formed when some parenchyma cells of the callus tissue dedifferentiate back to a meristematic state, further forming meristematic nodes, which then become primordia for buds or roots. These primordia then differentiate through periclinal and anticlinal divisions.

[0069] This embodiment verifies that various tea varieties can be propagated through leaf cuttings to form regenerated buds into complete plants. Although the budding rate varies among different varieties, experimental data shows that all tea varieties propagated by cuttings can form callus tissue and develop into seedlings with complete tissue under suitable conditions, indicating the feasibility of the technique. Furthermore, all rooted leaf cuttings were accompanied by callus formation.

[0070] like Figure 3 As shown, all cuttings that sprouted buds underwent a callus growth stage. Taking *Tea 'Huang Guanyin'* as an example, the callus formation rate of its leaf cuttings was the fastest (approximately two weeks), followed by root development. This phenomenon indicates that callus formation is an essential process in the propagation of tea leaf cuttings, and its development efficiency directly affects the seedling cycle and budding rate. Furthermore, rooted leaves, retaining some vascular tissue, can absorb water and hormones more efficiently, thus accelerating callus differentiation.

[0071] The resulting regenerated buds exhibited two forms: budding at the root nodules and budding on the plant roots. Through experimental observation and growth tracking, two budding patterns were discovered in tea leaf cuttings:

[0072] Pathway 1: Bud formation from root nodule-like callus (stem-like manifestation)

[0073] In this process, the callus tissue formed at the base of the tea leaf cutting gradually swells and presents a nodular structure (approximately 2-3 mm in diameter), from which buds differentiate. These buds exhibit the anatomical features of a stem and eventually develop into independent plants.

[0074] Method 2: Direct budding from roots

[0075] In the tea tree variety Longjing No. 1 ( Figure 4 In section B), after the callus stage and the rooting stage, the buds of the cuttings sprout directly from the roots of the root system, without having to go through the callus tissue stemization stage.

[0076] Different callus morphologies and root nodules were observed at different stages of tea leaf cutting propagation. Bud formation from root nodules depends on cell totipotency expression, while direct bud formation from roots may utilize pre-existing lateral bud primordia within the root system. The proportion of these two pathways may be regulated by both varietal genetic characteristics and the external environment. Furthermore, this experiment observed that callus tissue in tea leaf cutting propagation is not only the physical basis for bud differentiation but also a regulatory center for hormone signals, playing a central role in the differentiation of adventitious roots and buds.

[0077] In addition, toluidine blue stained sections of leaves from plants of the Huang Guanyin variety showing adventitious bud differentiation were observed. Figure 5 As shown. Slice observation. Figure 5The structure of callus tissue and the differentiation of adventitious roots and buds is complex. The cross-section of callus tissue is also composed of epidermis, cortex and vascular bundles. Figure 5 The G in the text has a complete stem-like structure and a large number of callus cells differentiated (see details). Figure 6 Numerous callus tissues proliferated and grew, arranged tightly and vigorously dividing. Under a microscope, the denser callus tissue cells showed deeper staining and denser cytoplasm. Figure 5 (B in the diagram). In the longitudinal section of the double bud differentiation of *Aureomarginata*, new buds are also differentiating at the callus tissue. Figure 5 (C in the middle).

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for in vitro propagation of tea plant (Camellia sinensis) by leaf cutting based micropropagation free method, characterized by, The method includes the following steps: Step (1): Select mature and healthy leaves of tea tree varieties, retain the complete petiole, and use the characteristic of leaves without pre-growth points for subsequent cultivation; Step (2): Dip the base of the petiole of the leaf in the first exogenous hormone solution, and then insert the leaf cutting into the substrate; wherein, the first exogenous hormone solution is naphthaleneacetic acid, indoleacetic acid or indolebutyric acid, and the concentration of the first exogenous hormone solution is 50 mg / L. Step (3): Set the light, temperature and air humidity conditions for cultivation. During the cultivation process, spray the leaves with a second exogenous hormone solution to cause the cells at the base of the petiole to dedifferentiate and form callus tissue. The callus tissue then redifferentiates to induce adventitious roots and adventitious buds. The light intensity during the callus induction stage is 2000-10000 Lux, and the light intensity during the adventitious root and adventitious bud induction stage is 5000-15000 Lux. The second exogenous hormone solution can be any one of the following combinations: ① 2 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid and 3 mg / L gibberellin; ② 2 mg / L 6-benzylaminopurine, 0.8 mg / L naphthaleneacetic acid and 1 mg / L indolebutyric acid; ③ 4 mg / L 6-benzylaminopurine and 1 mg / L indolebutyric acid; ④ 2 mg / L 6-benzylaminopurine and 1 mg / L gibberellin.

2. The leaf cutting based in vitro free tea plant regeneration method as claimed in claim 1, 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.

3. The leaf cutting based in vitro free tea plant regeneration method as claimed in claim 1, wherein, The light cycle is 16 hours of light followed by 8 hours of darkness.

4. The method for tea tree regeneration without tissue culture based on leaf cuttings according to claim 1, characterized in that, The temperature is 20-28 ℃.

5. The method for tea tree regeneration without tissue culture based on leaf cuttings according to claim 1, characterized in that, The air humidity is 80%-100%.

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