Method for in-vitro regeneration of plants from leaf and fruit dual-purpose tea tree seed embryos
By optimizing the culture medium formula of tea tree seed embryos and the method of generating regenerated plants, the problem of low regeneration rate of tea tree explants with leaf and fruit is solved, and the rapid proliferation and efficient rooting of tea tree embryos are achieved, which significantly shortens the breeding cycle and improves the reproduction efficiency.
Patent Information
- Application Number
- CN202411749672.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-04-15
AI Technical Summary
In the prior art, the regeneration rate of the explants of the dual-purpose tea tree with leaf fruit is low, resulting in a long breeding cycle and difficulty in obtaining the target trait varieties.
By optimizing the medium formula of 6-benzyl aminopurine, indole butyric acid, zein and 6-furylaminopurine, a method including ex vivo embryo disinfection, proliferation, rooting, seedling cultivation and transplantation was developed to promote the regeneration of tea tree seed embryos.
It has achieved rapid proliferation and efficient rooting of tea tree embryos, significantly shortened the traditional breeding cycle, improved the reproduction efficiency, and screened tea tree varieties with excellent traits such as high yield, disease resistance, and insect resistance in a short time.
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Figure CN119817465A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of packaging, and in particular relates to a method for in vitro regeneration of tea tree seed embryos for leaf and fruit purposes. Background Art
[0002] Tea trees that can be used to make tea from leaves or to extract oil from fruits have a high fruit-bearing rate. However, the growth cycle of this type of tea tree is long, and it usually takes more than 20 years to breed a new variety. At present, the main breeding methods include single-plant breeding, population separation, hybrid breeding, and physical and chemical mutagenesis, all of which require a lot of pollination and screening. In order to shorten the breeding cycle and increase the probability of obtaining varieties with target traits, the application of directional breeding and modern biotechnology is particularly important.
[0003] Through the study of tissue culture technology, the pace of germplasm innovation can be accelerated. In this process, the selection of explants, the optimization of light culture conditions, and the preparation of induction and proliferation culture medium are the key steps to cultivate tea tree regeneration plants. However, the disinfection process of explants is complicated, which can easily lead to seed inactivation, slow growth in the later stage, and generally low callus, proliferation rate and seedling rate. Summary of the invention
[0004] The invention provides a method for in vitro regeneration of tea tree seed embryos for leaf and fruit use, aiming to solve the technical problem of low regeneration rate of tea tree explants for leaf and fruit use in the prior art.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:
[0006] In a first aspect, the present invention provides a method for in vitro regeneration of tea plant seed embryos having both leaf and fruit functions, comprising:
[0007] S1. In vitro embryo disinfection: peel off the tea fruit peel, take out the tea seeds, and soak them in ethanol for 25-35 minutes, take out the tea seeds and ignite them until the ethanol on the surface of the tea seeds is completely burned, take out the seed kernels from the disinfected tea seeds, and take out the embryos and inoculate them on the induction medium to produce seedlings;
[0008] S2, in vitro embryo proliferation: removing the root system of the seedlings, selecting seedlings with tender stems larger than 0.5 cm, cutting them into stem segments of 0.4-1.2 cm, transferring them to proliferation medium, and culturing them under light to obtain proliferated seedlings;
[0009] The proliferation culture medium comprises 0.9-1.1 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L 6-furfurylaminopurine, 0.4-0.6 mg / L indolebutyric acid, 1.0-1.5 mg / L zeatin and WPM culture medium;
[0010] S3, in vitro embryonic rooting: transfer the proliferated seedlings with 2-4 leaves and 2.5-3.5 cm in size to rooting medium and culture for 28-32 days to obtain test tube seedlings;
[0011] The rooting medium comprises 0.5-0.7 mg / L indolebutyric acid, 0.8-1.5 mg / L 6-benzylaminopurine and half-concentration MS medium;
[0012] S4. Training and transplanting: Place the test tube seedlings in a climate box with a humidity of 55-65% for 2-4 days, transplant them into nutrient soil, and after culturing them under greenhouse conditions for 12-16 days, transplant them to outdoor cultivation to obtain leaf-fruit dual-purpose tea tree regeneration plants.
[0013] In a preferred embodiment, in step S2, the proliferation culture medium includes 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L 6-furfurylaminopurine, 0.5 mg / L indolebutyric acid, 1.2 mg / L zeatin and WPM culture medium.
[0014] Based on the above scheme, the present invention has obtained an optimal proliferation culture medium formula through experiments: 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L 6-furfurylaminopurine, 0.5 mg / L indolebutyric acid, 1.2 mg / L zeatin and WPM culture medium. This formula shows the best proliferation effect, with a proliferation rate of 3.2, healthy growth of seedlings, dark green leaves, and clusters of buds.
[0015] In a preferred embodiment, in step S2, the proliferation culture medium includes 0.92 mg / L 6-benzylaminopurine, 1.0 mg / L 6-furfurylaminopurine, 0.4 mg / L indolebutyric acid, 1.5 mg / L zeatin and WPM culture medium.
[0016] In a preferred embodiment, in step S2, the proliferation culture medium comprises 1.1 mg / L 6-benzylaminopurine, 0.8 mg / L 6-furfurylaminopurine, 0.6 mg / L indolebutyric acid, 1.0 mg / L zeatin and WPM culture medium.
[0017] Based on the above scheme, the present invention uses a relatively low concentration of 6-benzylaminopurine (0.9-1.1 mg / L) in combination with indolebutyric acid to improve the proliferation rate and make the leaf color healthier. Further experiments show that after the combination of zeatin and 6-furfurylaminopurine, the proliferation rate is significantly improved, and the tea seedlings grow healthily and form clusters of buds. Adding an appropriate amount of the cytokinin zeatin to the proliferation medium is significantly better than using 6-BA alone. This combination not only promotes the healthy growth of tea seedlings, the leaf color is dark green, but also effectively improves the proliferation rate, showing good growth potential.
[0018] In a preferred embodiment, in step S3, the rooting medium comprises 0.6 mg / L indolebutyric acid, 1.0 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0019] Based on the above scheme, the present invention has obtained an optimal rooting culture medium formula through experiments: 0.6 mg / L indolebutyric acid, 1.0 mg / L 6-benzylaminopurine and half-concentration MS culture medium. Under this formula, the rooting rate of tea trees reaches the highest, and the root system is white in color and there is no callus tissue at the base.
[0020] In a preferred embodiment, in step S3, the rooting medium comprises 0.5 mg / L indolebutyric acid, 1.5 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0021] In a preferred embodiment, in step S3, the rooting medium comprises 0.7 mg / L indolebutyric acid, 1.2 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0022] Based on the above scheme, the present invention has been proved through experiments that adding low concentration of 6-benzylaminopurine and indolebutyric acid to the culture medium can greatly increase the rooting rate to 96.85%, and the plants are in good condition, the roots are white in color, and there is no callus tissue at the base.
[0023] In a preferred embodiment, in step S4, the nutrient soil includes peat, perlite and vermiculite.
[0024] In a preferred embodiment, in the nutrient soil, the ratio of peat:perlite:vermiculite is 2:1:1.
[0025] In a second aspect, the present invention provides a culture medium for cultivating leaf-fruit dual-purpose tea tree seed embryos to generate regenerated plants in vitro, characterized in that: it comprises a proliferation culture medium and a rooting culture medium;
[0026] The proliferation culture medium comprises 0.9-1.1 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L 6-furfurylaminopurine, 0.4-0.6 mg / L indolebutyric acid, 1.0-1.5 mg / L zeatin and WPM culture medium;
[0027] The rooting medium comprises 0.5-0.7 mg / L indolebutyric acid, 0.8-1.5 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0028] The beneficial effects of the present invention are:
[0029] The present invention provides a method for in vitro regeneration of tea tree seed embryos for leaf and fruit dual-purpose plants, using embryos of tea seeds of tea trees for leaf and fruit dual-purpose plants as explants, and successfully screening suitable tea tree embryo induction and proliferation conditions for leaf and fruit dual-purpose plants by optimizing culture medium formulas of different concentrations of 6-benzylaminopurine, indolebutyric acid, zeatin and 6-furfurylaminopurine. This method can quickly obtain a large number of tea tree plants with a consistent genetic background, significantly shorten the traditional breeding cycle, and improve breeding efficiency, thereby screening tea tree varieties with excellent traits such as high yield, disease resistance, and insect resistance in a relatively short period of time, and improving genetic traits. By controlling the type and concentration of plant hormones in the culture medium, the present invention realizes the directional induction and regulation of tea tree embryos, and promotes their differentiation and growth. The proliferation culture medium provided by the present invention: 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L 6-furfurylaminopurine, 0.5 mg / L indolebutyric acid, 1.2 mg / L zeatin and WPM culture medium, the tea seedlings grown by proliferation are healthy, with dark green leaves, clustered buds, and a high proliferation rate, which can be proliferated 3.2 times. In addition, the rooting culture medium provided by the present invention: 0.6 mg / L indolebutyric acid, 1.0 mg / L 6-benzylaminopurine and half-concentration MS culture medium, the roots grown are white in color, without callus at the base, which helps to improve the survival rate of seedlings after rooting and transplanting, which can reach more than 90%. Therefore, this method not only provides a new way for the germplasm innovation of leaf and fruit dual-purpose tea trees, but also lays a foundation for the rapid propagation of tea trees and the selection of excellent varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.
[0031] Figure 1 This is a photo of the tea seed with embryo germination growth in the present invention.
[0032] Figure 2 It is a photograph of the tea plant seedling proliferation and rooting in the present invention.
[0033] Figure 3 It is a photo of the substrate seedlings grown after the rooted seedlings were transplanted in the present invention.
[0034] Description of the numbers in the figure:
[0035] A: Cotyledon of tea seed with embryo; B: in vitro embryo culture (embryo turns green); C: in vitro embryo culture (embryo sprouts and grows); D: multiplication seedlings; E: rooted bottle seedlings; F: rooted seedlings before transplanting; G: matrix seedlings. DETAILED DESCRIPTION
[0036] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.
[0037] Embodiment 1:
[0038] This embodiment provides a method for in vitro regeneration of tea plant seed embryos having both leaf and fruit functions, comprising:
[0039] S1. In vitro embryo disinfection: peel off the tea fruit peel, take out the tea seeds, and soak them in ethanol for 25-35 minutes, take out the tea seeds and ignite them until the ethanol on the surface of the tea seeds is completely burned, take out the seed kernels from the disinfected tea seeds, and take out the embryos and inoculate them on the induction medium to produce seedlings;
[0040] S2, in vitro embryo proliferation: removing the root system of the seedlings, selecting seedlings with tender stems larger than 0.5 cm, cutting them into stem segments of 0.4-1.2 cm, transferring them to proliferation medium, and culturing them under light to obtain proliferated seedlings;
[0041] The proliferation culture medium comprises 0.9-1.1 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L 6-furfurylaminopurine, 0.4-0.6 mg / L indolebutyric acid, 1.0-1.5 mg / L zeatin and WPM culture medium;
[0042] S3, in vitro embryonic rooting: transfer the proliferated seedlings with 2-4 leaves and 2.5-3.5 cm in size to rooting medium and culture for 28-32 days to obtain test tube seedlings;
[0043] The rooting medium comprises 0.5-0.7 mg / L indolebutyric acid, 0.8-1.5 mg / L 6-benzylaminopurine and half-concentration MS medium;
[0044] S4. Training and transplanting: Place the test tube seedlings in a climate box with a humidity of 55-65% for 2-4 days, transplant them into nutrient soil, and after culturing them under greenhouse conditions for 12-16 days, transplant them to outdoor cultivation to obtain leaf-fruit dual-purpose tea tree regeneration plants.
[0045] Among them, in step S2, a better scheme is that the proliferation culture medium includes 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L 6-furfurylaminopurine, 0.5 mg / L indolebutyric acid, 1.2 mg / L zeatin and WPM culture medium.
[0046] Alternatively, the proliferation medium includes 0.92 mg / L 6-benzylaminopurine, 1.0 mg / L 6-furfurylaminopurine, 0.4 mg / L indolebutyric acid, 1.5 mg / L zeatin and WPM medium.
[0047] Alternatively, the proliferation medium includes 1.1 mg / L 6-benzylaminopurine, 0.8 mg / L 6-furfurylaminopurine, 0.6 mg / L indolebutyric acid, 1.0 mg / L zeatin and WPM medium.
[0048] In addition, in step S3, the rooting medium includes 0.6 mg / L indolebutyric acid, 1.0 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0049] Alternatively, the rooting medium includes 0.5 mg / L indolebutyric acid, 1.5 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0050] Alternatively, in step S3, the rooting medium includes 0.7 mg / L indolebutyric acid, 1.2 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0051] In step S4, the nutrient soil includes peat, perlite and vermiculite.
[0052] Among them, the ratio of peat: perlite: vermiculite is 2:1:1.
[0053] Embodiment 2: This embodiment provides a culture medium for cultivating leaf-fruit dual-purpose tea tree seed embryos to generate regenerated plants in vitro, including a proliferation culture medium and a rooting culture medium;
[0054] The proliferation culture medium comprises 0.9-1.1 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L 6-furfurylaminopurine, 0.4-0.6 mg / L indolebutyric acid, 1.0-1.5 mg / L zeatin and WPM culture medium;
[0055] The rooting medium comprises 0.5-0.7 mg / L indolebutyric acid, 0.8-1.5 mg / L 6-benzylaminopurine and half-concentration MS medium.
[0056] The present invention is further explained below in conjunction with experiments:
[0057] In this experiment, the embryos of tea seeds of the dual-purpose tea tree for leaves and fruits were used as explants, and different concentrations of plant growth hormones were used to screen the conditions for embryo induction and proliferation of the dual-purpose tea tree for leaves and fruits, in order to provide a basis for the innovation and rapid propagation of excellent varieties of dual-purpose tea trees for seeds and leaves.
[0058] 1. Materials and methods
[0059] 1.1 Materials
[0060] The experimental materials are fresh tea seeds of Jincha 18, a tea variety with both leaves and fruits, collected from the experimental base of Jinhua Academy of Agricultural Sciences. Plant hormones: 6-BA (6-benzylaminopurine), IBA (indolebutyric acid), ZT (6-furfurylaminopurine), KT (6-benzylaminopurine), MS medium.
[0061] 1.2 Methods
[0062] 1.2.1 Culture conditions
[0063] In vitro embryo germination was carried out using MS medium, carrageenan 7g / L, sucrose 30g / L, pH 5.6-5.8, culture temperature (25±2)℃, light duration 12h / d, light intensity 60μmol / m2·s.
[0064] The in vitro embryo turning green was counted as germination, and the growth of radicle was counted as sprouting. The contamination rate was investigated and counted 10 days after inoculation, the germination rate was investigated and counted 30 days later, and the germination rate was investigated and counted 50 days later.
[0065] Germination rate = number of germinated cells / number of uncontaminated cells × 100%
[0066] Germination rate = number of radicles grown / number of sprouts × 100%
[0067] 1.2.2 Explant disinfection
[0068] Wash the tea fruit, peel off the peel and take out the tea seeds, soak the dry and plump tea seeds in 95% ethanol for 30 minutes. Light the tea seeds on an alcohol lamp on a clean bench, and place them in a sterile inoculation tray for later use after the ethanol on the surface is burned out.
[0069] The sterilized outer seed coat of the tea seeds was smashed open to take out the kernels, the cotyledons were separated with a sterile scalpel, and the embryos were taken out and inoculated onto the induction medium.
[0070] 1.2.3 In vitro embryo proliferation
[0071] The roots of the seedlings grown during the in vitro embryo culture stage were removed, and the seedlings with a height of less than 0.5 cm were directly transferred, and the seedlings (tender stems) larger than 0.5 cm were cut into stem segments of about 0.5 to 1.0 cm and transferred to the proliferation medium formula. 15 bottles were inoculated for each treatment, and 5 tender stems or stem segments were inoculated in each bottle. This was repeated 3 times. After 60 days of light culture, the proliferation rate was calculated.
[0072] 2. Results and Analysis
[0073] 2.1 Effects of different light intensity on in vitro embryo germination
[0074] See also Figure 1The in vitro embryos were cultured in MS medium under light conditions. On the 10th day, the in vitro embryos turned green, on the 15th day, the radicles were visible, and on the 30th day, the hypocotyls were elongated. As shown in Table 1, most of the in vitro embryos were able to germinate and sprout after 15 days.
[0075] Table 1 Germination rate and germination rate of in vitro embryos
[0076] variety Number of vaccinations Number of buds Number of sprouts Germination rate (%) Germination rate (%) Golden Tea 18 60 52 44 86.6 73.3
[0077] 2.2 Effects of different hormone combinations on in vitro embryo proliferation
[0078] (1) Effects of cytokinin 6-BA: see Figure 1-Figure 2 In the proliferation medium, cytokinin 6-BA has a certain effect on the proliferation rate of tea plant seedlings in vitro embryo culture. However, when a higher concentration of 6-BA (such as 2.0 mg / L) is used alone, although the proliferation rate is improved, the growth rate is slower and vitrification occurs, which may be detrimental to the healthy growth of the seedlings.
[0079] (2) Synergistic effect of 6-BA and IBA: When a lower concentration of 6-BA (1.0 mg / L) was used in combination with the auxin IBA (0.5 mg / L), although the growth rate was still slow, the proliferation rate increased and the leaf color of the seedlings remained green, indicating that this combination was conducive to the growth and proliferation of the seedlings.
[0080] (3) Effect of the combination of ZT and KT hormones: The experiment found that when the cytokinin ZT and the auxin KT were used in combination, the proliferation rate could be significantly increased, and the seedlings grew healthily and clusters of buds appeared. This shows that the combination of ZT and KT has a positive effect on promoting the proliferation and healthy growth of tea tree seedlings in vitro embryo culture.
[0081] Among all the tested culture medium formulas, formula 6 (WPM+1.0mg / L6-BA+0.5mg / LKT+0.5mg / LIBA+1.2mg / LZT) showed the best proliferation effect, with a proliferation rate of 3.2, healthy growth of seedlings, dark green leaves, and clusters of buds. This shows that this formula provides a suitable growth environment for tea tree in vitro embryo culture seedlings.
[0082] Table 2 Effects of different treatments on proliferation rate
[0083] Processing number Medium formulation Proliferation rate Growth status 1 WPM+1.0mg / L6-BA+0.2mg / LIBA 1 Growth arrest, no proliferation 2 WPM+1.5mg / L6-BA+0.2mg / LIBA 1.5 Slow growth and low proliferation rate 3 WPM+2.0mg / L6-BA+0.2mg / LIBA 1.8 Slow growth, vitrification 4 WPM+1.0mg / L6-BA+1.5mg / LKT+0.5mg / LIBA+1.2mg / ZT 2.3 Slow growth, green leaves 5 WPM+1.0mg / L6-BA+1.0mg / LKT+0.5mg / LIBA+1.2mg / ZT 2.8 Healthy growth, with buds 6 WPM+1.0mg / L6-BA+0.5mg / LKT+0.5mg / LIBA+1.2mg / ZT 3.2 Healthy growth, green leaves and buds
[0084] 2.3 Effects of different hormone combinations on in vitro embryonic rooting
[0085] See also Figure 2, using 1 / 2MS culture medium as the basis, adding different concentrations of IBA and 6-BA, transferring 3cm tea tree seedlings with 2-4 leaves to these culture media, observing and recording the rooting rate and root growth. When IBA was used alone, the rooting rate gradually increased with the increase in concentration, but the rooting speed was slow. Adding 6-BA on the basis of IBA significantly increased the rooting rate and improved the root growth.
[0086] The test results show that adding a certain concentration of cytokinin 6-BA to the proliferation medium in combination with auxin IBA can significantly increase the rooting rate of tea trees and improve the growth of the root system compared to using IBA alone. The most suitable medium formula is 1 / 2MS+0.6mg / LIBA+1.0mg / L6-BA. Under this formula, the rooting rate of tea trees reaches the highest, and the root system is white in color and there is no callus at the base, indicating that the plant is in good growth condition.
[0087] Table 3 Effects of different treatments on rooting rate
[0088] serial number Medium formulation Rooting rate Root growth 1 1 / 2MS+0.2mg / LIBA 38.76 The root system is green and thick, with a lot of callus at the base 2 1 / 2MS+0.4mg / LIBA 46.23 The root system is green and thick, with callus at the base 3 1 / 2MS+0.6mg / LIBA 51.64 The root system is green, with callus at the base 4 1 / 2MS+0.6mg / LIBA+0.5mg / L6-BA 76.88 The root system is light green with callus at the base 5 1 / 2MS+0.6mg / LIBA+1.5mg / L6-BA 87.34 The root system is white in color, with a small amount of callus at the base 6 1 / 2MS+0.6mg / LIBA+1.0mg / L6-BA 96.85 The root system is white in color and has no callus at the base
[0089] 2.4 Hardening and transplanting
[0090] See also Figure 3 , 30 days after the sterile seedlings have taken root, select the test tube seedlings with strong roots and good growth, open the bottle cap, and place them in an artificial climate box with a humidity of 60% for 3 days. Then take out the tea seedlings, wash the culture medium at the roots, and transplant them into a nutrient soil containing peat, perlite and vermiculite in a ratio of 2:1:1 in a glass greenhouse. After two weeks, transplant the surviving test tube seedlings outdoors for 30 days. Pay attention to maintaining proper humidity during outdoor cultivation, and the survival rate can reach more than 90%.
[0091] 3. Conclusion
[0092] This experiment systematically studied the effects of different hormone combinations on in vitro embryo culture of tea trees, and successfully explored two highly effective culture medium formulas for tea tree proliferation culture and rooting culture. In terms of proliferation culture medium, by comparing different concentrations of cytokinin 6-BA and its synergistic effect with auxin IBA, it was found that formula 6 (WPM+1.0mg / L6-BA+0.5mg / LKT+0.5mg / LIBA+1.2mg / LZT) can significantly increase the proliferation rate of tea tree in vitro embryos by 3.2%, while promoting the healthy growth of seedlings, as shown by dark green leaves and the formation of clustered buds. This result shows that the appropriate hormone combination can provide an ideal growth environment for tea tree in vitro embryos and promote their rapid proliferation.
[0093] In terms of rooting medium, the test shows that the combination of 6-BA and IBA can significantly increase the rooting rate of tea seedlings and improve the growth of the root system compared to using IBA alone. The optimal rooting medium formula is 1 / 2MS+0.6mg / LIBA+1.0mg / L6-BA. Under this formula, tea seedlings show the highest rooting rate, white root color, no callus at the base, and show good growth.
[0094] In addition, through appropriate seedling training and transplanting steps, the survival rate of tea seedlings successfully transplanted from test tube seedlings outdoors can reach more than 90%. This shows that the proliferation medium and rooting medium explored in this experiment can not only promote the rapid reproduction of tea trees, but also provide effective technical means for the selection and breeding of excellent tea varieties, and provide scientific basis and practical methods for the tissue culture and breeding of tea trees, which has important application value.
[0095] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.
Claims
1. A method for in vitro regeneration of tea plant seed embryos having both leaf and fruit properties, characterized in that: include: S1. In vitro embryo disinfection: peel off the tea fruit peel, take out the tea seeds, and soak them in ethanol for 25-35 minutes, take out the tea seeds and ignite them until the ethanol on the surface of the tea seeds is completely burned, take out the seed kernels from the disinfected tea seeds, and take out the embryos and inoculate them on the induction medium to produce seedlings; S2, in vitro embryo proliferation: removing the root system of the seedlings, selecting seedlings with tender stems larger than 0.5 cm, cutting them into stem segments of 0.4-1.2 cm, transferring them to proliferation medium, and culturing them under light to obtain proliferated seedlings; The proliferation culture medium comprises 0.9-1.1 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L 6-furfurylaminopurine, 0.4-0.6 mg / L indolebutyric acid, 1.0-1.5 mg / L zeatin and WPM culture medium; S3, in vitro embryonic rooting: transfer the proliferated seedlings with 2-4 leaves and 2.5-3.5 cm in size to rooting medium and culture for 28-32 days to obtain test tube seedlings; The rooting medium comprises 0.5-0.7 mg / L indolebutyric acid, 0.8-1.5 mg / L 6-benzylaminopurine and half-concentration MS medium; S4. Training and transplanting: Place the test tube seedlings in a climate box with a humidity of 55-65% for 2-4 days, transplant them into nutrient soil, and after culturing them under greenhouse conditions for 12-16 days, transplant them to outdoor cultivation to obtain leaf-fruit dual-purpose tea tree regeneration plants.
2. The method for in vitro regeneration of tea plant seed embryos having both leaf and fruit functions according to claim 1, characterized in that: In step S2, the proliferation culture medium includes 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L 6-furfurylaminopurine, 0.5 mg / L indolebutyric acid, 1.2 mg / L zeatin and WPM culture medium.
3. The method for in vitro regeneration of tea plant seed embryos with leaves and fruits according to claim 1, characterized in that: In step S2, the proliferation culture medium includes 0.92 mg / L 6-benzylaminopurine, 1.0 mg / L 6-furfurylaminopurine, 0.4 mg / L indolebutyric acid, 1.5 mg / L zeatin and WPM culture medium.
4. The method for in vitro regeneration of tea plant seed embryos with leaves and fruits according to claim 1, characterized in that: In step S2, the proliferation culture medium includes 1.1 mg / L 6-benzylaminopurine, 0.8 mg / L 6-furfurylaminopurine, 0.6 mg / L indolebutyric acid, 1.0 mg / L zeatin and WPM culture medium.
5. The method for in vitro regeneration of tea plant seed embryos with leaves and fruits according to claim 1, characterized in that: In step S3, the rooting medium includes 0.6 mg / L indolebutyric acid, 1.0 mg / L 6-benzylaminopurine and half-concentration MS medium.
6. The method for in vitro regeneration of tea plant seed embryos with leaves and fruits according to claim 1, characterized in that: In step S3, the rooting medium includes 0.5 mg / L indolebutyric acid, 1.5 mg / L 6-benzylaminopurine and half-concentration MS medium.
7. The method for in vitro regeneration of tea plant seed embryos having both leaf and fruit functions according to claim 1, characterized in that: In step S3, the rooting medium includes 0.7 mg / L indolebutyric acid, 1.2 mg / L 6-benzylaminopurine and half-concentration MS medium.
8. The method for in vitro regeneration of tea plant seed embryos having both leaf and fruit functions according to claim 1, characterized in that: In step S4, the nutrient soil includes peat, perlite and vermiculite.
9. The method for in vitro regeneration of tea plant seed embryos with leaves and fruits according to claim 8, characterized in that: In the nutrient soil, the ratio of peat: perlite: vermiculite is 2:1:
1.
10. A culture medium for cultivating leaf-fruit dual-purpose tea tree seed embryos to generate regenerated plants in vitro, characterized in that: Includes proliferation medium and rooting medium; The proliferation culture medium comprises 0.9-1.1 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L 6-furfurylaminopurine, 0.4-0.6 mg / L indolebutyric acid, 1.0-1.5 mg / L zeatin and WPM culture medium; The rooting medium comprises 0.5-0.7 mg / L indolebutyric acid, 0.8-1.5 mg / L 6-benzylaminopurine and half-concentration MS medium.