A tissue culture method of yannia

Abstract

This invention provides a method for tissue culture of *Acer buergerianum*, comprising the following steps: S1, collecting unopened flower buds from *Acer buergerianum* plants as explants; S2, irradiating the explants with ultraviolet light; S3, subjecting the sterilized explants to low-temperature treatment followed by alternating red and blue light irradiation to obtain pretreated explants; S4, placing the pretreated explants on a liquid culture medium for suspension culture, with ultrasonic stirring during the process, to obtain regenerated seedlings; S5, placing the regenerated seedlings on a solid culture medium for culture to obtain regenerated plants; S6, transplanting the regenerated plants into a transplanting substrate for further culture. The method of this invention is scientifically sound and reasonable. Through the selection and pretreatment of explants, as well as the optimization of culture methods and culture media, the obtained regenerated plants have robust root systems, a transplant survival rate of over 92%, and a propagation coefficient greater than 8, meeting the needs of production and experimentation, and broadening the scope of plant tissue culture methods for *Acer buergerianum*.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for tissue culture of bird's nest fruit. Background Technology

[0002] Dragon fruit, also known as bird's nest fruit, is rich in nutrients and has unique functions. It contains plant-based albumin and anthocyanins, which are rare in other plants, as well as abundant vitamins and water-soluble dietary fiber. Compared to ordinary dragon fruit, bird's nest fruit is lighter in weight, with a fibrous, smooth flesh resembling bird's nest, and is sweet, delicious, and juicy. However, due to a severe shortage of seedlings in China, production is difficult, resulting in low yields and reliance on imports. Its cultivation and management requirements are relatively high, and the fruit has a longer ripening period, thus commanding a higher price—more than ten times that of ordinary red-fleshed dragon fruit.

[0003] Plant tissue culture technology can rapidly propagate large quantities of high-quality, uniformly growing, trait-stable, and virus-free healthy seedlings, while also preserving excellent germplasm resources of *Acer buergerianum*. It requires less material, causes minimal damage to the mother plant, has a high propagation coefficient, and a fast propagation speed, making it an important method for industrialized seedling production. Currently, there are few reports on plant tissue culture of *Acer buergerianum*. Existing plant tissue culture methods for *Acer buergerianum* often use stem buds as explants to cultivate tissue culture seedlings, which suffers from low survival rates and low propagation coefficients. Summary of the Invention

[0004] In view of this, the present invention proposes a tissue culture method for bird's nest fruit to solve the above problems.

[0005] The technical solution of this invention is implemented as follows:

[0006] A method for tissue culture of bird's nest fruit, comprising the following steps:

[0007] S1. Explant selection: Unopened flower buds of the bird's nest fruit plant were collected as explants.

[0008] S2, Disinfection: Irradiate the explants from S1 with ultraviolet light;

[0009] S3. Explant pretreatment: The disinfected explants are subjected to low-temperature treatment and then irradiated with alternating red and blue light to obtain pretreated explants.

[0010] S4. Induction culture: The pretreated explants are placed on liquid culture medium for suspension culture. During the culture, ultrasonic stirring is maintained to obtain regenerated seedlings.

[0011] S5, Proliferation Culture: The regenerated seedlings of S4 were placed on a solid culture medium for culture to obtain regenerated plants of bird's nest fruit;

[0012] S6. Hardening off and transplanting: Transplant the regenerated plants from S5 into the transplanting substrate for cultivation.

[0013] Furthermore, in step S2, the ultraviolet irradiation uses 10-20 mJ / cm². 2 Irradiate the explant with ultraviolet light at a distance of 15-25 cm for 10-20 minutes.

[0014] Furthermore, in step S3, the low-temperature treatment is performed at 3-7°C for 4-6 hours; the alternating red and blue light irradiation is performed first with a wavelength of 650-700 nm and an intensity of 50-70 μmol / m². 2 Irradiate with red light at a wavelength of 400-500 nm and an intensity of 60-80 μmol / m for 12 hours, followed by irradiation with red light at a wavelength of 400-500 nm and an intensity of 60-80 μmol / m. 2 Irradiate with blue light for 12 hours, followed by alternating red and blue light irradiation twice.

[0015] Furthermore, in step S4, the liquid culture medium is: MS medium, 0.5-1.5 mg / L NNA, 0.1-0.5 mg / L BAP, 1.0-2.0 mg / L TDZ, 0.5-0.8 mg / L jasmonic acid, and 1.5-2.5 mg / L coconut water.

[0016] Furthermore, in step S4, the photocycle for the suspension culture is 16h / 8h light / dark, and the temperature is 25-27℃.

[0017] Furthermore, in step S4, the ultrasonic stirring is an intermittent stirring with a frequency of 20-40KHz and a power of 10-30W, with ultrasonic stirring for 5-10 minutes every 20-30 minutes.

[0018] Furthermore, in step S5, the solid culture medium is: MS medium, 0.1-0.5 mg / L IBA, 0.5-0.8 mg / L IAA, 10-15 mg / L ascorbic acid, and 1-3 g / L dextrin.

[0019] Furthermore, in step S5, the cultivation conditions are: cultivation at 25-27℃, with a photocycle of 16h light / 8h darkness.

[0020] Furthermore, in step S6, the transplanting substrate is composed of peat moss, perlite, vermiculite, and bentonite in a mass ratio of 1:1-3:1-3:3-5.

[0021] Furthermore, in step S6, the cultivation is carried out in an environment with humidity of 75%-90% and light intensity of 300-400 Lux for 3-5 days, with supplemental lighting to increase the light intensity by 50 Lux per day to 1000-1200 Lux, and then normal cultivation.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. The plant tissue culture method of bird's nest fruit of the present invention is scientific and reasonable. Through the selection and pretreatment of explants and the optimization of culture methods and culture media, the regenerated plants obtained have strong root systems, high transplant survival rate of over 92%, and a propagation coefficient of over 8, which meets the needs of production and experimentation and broadens the ideas for plant tissue culture methods of bird's nest fruit.

[0024] 2. The use of unopened flower buds as explants in this invention is based on the fact that cell division is active at this stage, resulting in high meristematic capacity and regeneration potential. The buds are of moderate size, easy to handle, and have relatively soft tissue, making them easy to disinfect and penetrate the culture medium. Ultraviolet physical disinfection of the explants avoids the damage caused by traditional chemical disinfection methods. Low-temperature treatment and alternating red and blue light irradiation increase the viability and resistance of the explants, improving the regeneration rate. In the induction culture stage, suspension culture is carried out using an optimized liquid culture medium, supplemented by intermittent ultrasonic stirring, to fully absorb nutrients, promote cell division, and efficiently induce regenerated seedlings, significantly improving the transplant survival rate and increasing the propagation coefficient, thus possessing certain application value. Detailed Implementation

[0025] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0026] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0028] Example 1

[0029] A method for tissue culture of bird's nest fruit, comprising the following steps:

[0030] S1. Explant selection: Unopened flower buds of the bird's nest fruit plant were collected as explants.

[0031] S2, Disinfection: Apply 15mJ / cm² disinfectant to the explants from S1. 2 Irradiate the explant with ultraviolet light at a distance of 20 cm for 15 minutes;

[0032] S3. Explant pretreatment: The sterilized explants were treated at 5℃ for 5 hours. After low-temperature treatment, they were first treated with a wavelength of 680 nm and an intensity of 60 μmol / m². 2 Irradiate with red light at a wavelength of 450 nm and an intensity of 70 μmol / m for 12 hours, followed by irradiation with red light at a wavelength of 450 nm and an intensity of 70 μmol / m. 2 The explants were pretreated by irradiating blue light for 12 hours with / s of light and then alternating between red and blue light twice.

[0033] S4. Induction Culture: The pretreated explants were placed on liquid culture medium, which consisted of MS medium, 1.0 mg / L NNA, 0.3 mg / L BAP, 1.5 mg / L TDZ, 0.7 mg / L jasmonic acid, and 2.0 mg / L coconut water. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h and 26 ± 1 °C. During the culture, intermittent ultrasonic stirring (frequency 30 kHz, power 20 W) was performed every 25 min for 8 min to obtain regenerated seedlings.

[0034] S5. Proliferation culture: The regenerated seedlings of S4 were placed on a solid culture medium, which was MS medium, 0.3 mg / L IBA, 0.7 mg / L IAA, 13 mg / L ascorbic acid, and 2 g / L dextrin. The culture was carried out at 26±1℃ under light / dark conditions for 16 h / 8 h to obtain the regenerated plants of the bird's nest fruit.

[0035] S6. Hardening and Transplanting: Transplant the regenerated plants from S5 into a transplanting substrate, which is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:2:2:4. The plants are cultivated for 4 days at 85% humidity and 350 Lux light, with supplemental lighting, increasing the light intensity by 50 Lux to 1100 Lux each day, and then cultivated normally.

[0036] Example 2

[0037] A method for tissue culture of bird's nest fruit, comprising the following steps:

[0038] S1. Explant selection: Unopened flower buds of the bird's nest fruit plant were collected as explants.

[0039] S2, Disinfection: Apply 10mJ / cm² disinfectant to the explants from S1. 2 Irradiate the explant with ultraviolet light at a distance of 15 cm for 10 minutes;

[0040] S3. Explant pretreatment: The sterilized explants were treated at 3℃ for 4 hours. After low-temperature treatment, they were first treated with a wavelength of 650nm and an intensity of 50μmol / m². 2 Irradiate with red light at a wavelength of 400 nm and an intensity of 60 μmol / m for 12 hours, followed by irradiation with red light at a wavelength of 400 nm and an intensity of 60 μmol / m. 2 The explants were pretreated by irradiating blue light for 12 hours with / s of light and then alternating between red and blue light twice.

[0041] S4. Induction Culture: The pretreated explants were placed on liquid culture medium, which consisted of MS medium, 0.5 mg / L NNA, 0.1 mg / L BAP, 1.0 mg / L TDZ, 0.5 mg / L jasmonic acid, and 1.5 mg / L coconut water. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h and 26 ± 1 °C. During the culture, intermittent ultrasonic stirring (frequency 20 kHz, power 10 W) was performed every 20 min for 5 min to obtain regenerated seedlings.

[0042] S5. Proliferation culture: The regenerated seedlings of S4 were placed on a solid culture medium, which was MS medium, 0.1 mg / L IBA, 0.5 mg / L IAA, 10 mg / L ascorbic acid, and 1 g / L dextrin. The culture was carried out at 26±1℃ under light / dark conditions for 16 h / 8 h to obtain the regenerated plants of bird's nest fruit.

[0043] S6. Hardening and Transplanting: Transplant the regenerated plants from S5 into a transplanting substrate, which is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:1:1:3. Cultivation is carried out for 3 days in an environment with 75% humidity and 300 Lux of light, followed by supplemental lighting, increasing the light intensity by 50 Lux to 1000 Lux each day, and then normal cultivation.

[0044] Example 3

[0045] A method for tissue culture of bird's nest fruit, comprising the following steps:

[0046] S1. Explant selection: Unopened flower buds of the bird's nest fruit plant were collected as explants.

[0047] S2, Disinfection: Apply 20mJ / cm² disinfectant to the explants from S1. 2 Irradiate the explant with ultraviolet light at a distance of 25 cm for 20 minutes;

[0048] S3. Explant pretreatment: The sterilized explants were treated at 7℃ for 6 hours. After low-temperature treatment, they were first treated with a wavelength of 700nm and an intensity of 70μmol / m². 2 Irradiation with red light at a wavelength of 500 nm and an intensity of 80 μmol / m for 12 hours, followed by irradiation with red light at a wavelength of 500 nm and an intensity of 80 μmol / m. 2 The explants were pretreated by irradiating blue light for 12 hours with / s of light and then alternating between red and blue light twice.

[0049] S4. Induction Culture: The pretreated explants were placed on liquid culture medium, which consisted of MS medium, 1.5 mg / L NNA, 0.5 mg / L BAP, 2.0 mg / L TDZ, 0.8 mg / L jasmonic acid, and 2.5 mg / L coconut water. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h at 26 ± 1 °C. During the culture, intermittent ultrasonic stirring (frequency 40 kHz, power 30 W) was performed, with ultrasonic stirring for 10 min every 30 min to obtain regenerated seedlings.

[0050] S5. Proliferation culture: The regenerated seedlings of S4 were placed on a solid culture medium, which was MS medium, 0.5 mg / L IBA, 0.8 mg / L IAA, 15 mg / L ascorbic acid, and 3 g / L dextrin. The culture was carried out at 26±1℃ under light / dark conditions for 16 h / 8 h to obtain the regenerated plants of bird's nest fruit.

[0051] S6. Hardening and Transplanting: Transplant the regenerated plants from S5 into a transplanting substrate, which is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:3:3:5. Cultivation is carried out for 5 days in an environment with 90% humidity and 400 Lux of light. Supplemental lighting is then provided, increasing the light intensity by 50 Lux to 1200 Lux each day, and then normal cultivation is resumed.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 1 is that tender branches of the bird's nest fruit plant were selected as explants, while the rest is the same as Example 1.

[0054] Step S1 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0055] S1. Explant selection: collect tender branches (5cm) from the bird's nest fruit plant as explants.

[0056] Comparative Example 2

[0057] The difference between this comparative example and Example 1 is that the disinfection method used is a conventional disinfection method, while the rest is the same as Example 1.

[0058] Step S2 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0059] S2. Disinfection: The explants from S1 were first soaked in a 70% v / v ethanol solution for 60 seconds in a sterile environment, rinsed twice with sterile water, then soaked in a 0.1% w / v mercuric chloride solution for 10 minutes, and rinsed five times with sterile water.

[0060] Comparative Example 3

[0061] The difference between this comparative example and Example 1 is that the explant pretreatment step S3 is missing; otherwise, it is the same as Example 1.

[0062] The tissue culture method for the comparative example of bird's nest fruit includes the following steps:

[0063] S1. Explant selection: Unopened flower buds of the bird's nest fruit plant were collected as explants.

[0064] S2, Disinfection: Apply 15mJ / cm² disinfectant to the explants from S1. 2 Irradiate the explant with ultraviolet light at a distance of 20 cm for 15 minutes;

[0065] S3. Induction Culture: The sterilized explants were placed on liquid culture medium, which consisted of MS medium, 1.0 mg / L NNA, 0.3 mg / L BAP, 1.5 mg / L TDZ, 0.7 mg / L jasmonic acid, and 2.0 mg / L coconut water. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h and 26 ± 1 °C. During the culture, intermittent ultrasonic stirring (frequency 30 kHz, power 20 W) was performed every 25 min for 8 min to obtain regenerated seedlings.

[0066] S4. Proliferation culture: The regenerated seedlings of S3 were placed on a solid culture medium, which was MS medium, 0.3 mg / L IBA, 0.7 mg / L IAA, 13 mg / L ascorbic acid, and 2 g / L dextrin. The culture was carried out at 26±1℃ under light / dark conditions for 16 h / 8 h to obtain the regenerated plants of bird's nest fruit.

[0067] S5. Hardening and Transplanting: Transplant the regenerated plants from S4 into a transplanting substrate, which is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:2:2:4. The plants are cultivated for 4 days at 85% humidity and 350 Lux light, with supplemental lighting, increasing the light intensity by 50 Lux to 1100 Lux each day, and then cultivated normally.

[0068] Comparative Example 4

[0069] The difference between this comparative example and Example 1 is that the liquid culture medium in step S4 is lacking jasmonic acid, while the other two are the same as in Example 1.

[0070] Step S4 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0071] S4. Induction Culture: The pretreated explants were placed on liquid culture medium, which consisted of MS medium, 1.0 mg / L NNA, 0.3 mg / L BAP, 1.5 mg / L TDZ, and 2.0 mg / L coconut water. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h and 26 ± 1 °C. During the culture, intermittent ultrasonic stirring (frequency 30 kHz, power 20 W) was performed every 25 min for 8 min to obtain regenerated seedlings.

[0072] Comparative Example 5

[0073] The difference between this comparative example and Example 1 is that the liquid culture medium in step S4 lacks coconut water, while the rest is the same as in Example 1.

[0074] Step S4 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0075] S4. Induction Culture: The pretreated explants were placed on liquid culture medium, which consisted of MS medium, 1.0 mg / L NNA, 0.3 mg / L BAP, 1.5 mg / L TDZ, and 0.7 mg / L jasmonic acid. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h and 26 ± 1 °C. During the culture, intermittent ultrasonic stirring (frequency 30 kHz, power 20 W) was performed every 25 min for 8 min to obtain regenerated seedlings.

[0076] Comparative Example 6

[0077] The difference between this comparative example and Example 1 is that step S4 does not use ultrasonic stirring, while the rest is the same as Example 1.

[0078] Step S4 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0079] S4. Induction culture: The pretreated explants were placed on liquid culture medium, which consisted of MS medium, 1.0 mg / L NNA, 0.3 mg / L BAP, 1.5 mg / L TDZ, 0.7 mg / L jasmonic acid, and 2.0 mg / L coconut water. The explants were suspended and cultured under light / dark conditions of 16 h / 8 h at 26 ± 1 °C to obtain regenerated seedlings.

[0080] Comparative Example 7

[0081] The difference between this comparative example and Example 1 is that the culture medium in step S5 lacks ascorbic acid and dextrin, while the rest is the same as in Example 1.

[0082] Step S5 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0083] S5. Proliferation culture: The regenerated seedlings of S4 were placed on a solid culture medium, which was MS medium, 0.3 mg / L IBA, and 0.7 mg / L IAA. The culture was carried out at 26±1℃ under light / dark conditions for 16 h / 8 h to obtain the regenerated plants of the bird's nest fruit.

[0084] Comparative Example 8

[0085] The difference between this comparative example and Example 1 is that the transplanting substrate in step S6 is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:1:1:1, while the rest is the same as in Example 1.

[0086] Step S6 of the tissue culture method for the bird's nest fruit in this comparative example is as follows:

[0087] S6. Hardening and Transplanting: Transplant the regenerated plants from S5 into a transplanting substrate, which is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:1:1:1. Cultivation is carried out for 4 days in an environment with 85% humidity and 350 Lux of light, followed by supplemental lighting, increasing the light intensity by 50 Lux to 1100 Lux each day, and then normal cultivation.

[0088] Test case

[0089] The transplanted bird's nest fruit plants obtained using the plant tissue culture methods of Examples 1-3 and Comparative Examples 1-8 were used to calculate the transplant survival rate after 15 days.

[0090] Transplant survival rate (%) = (Number of surviving plants / Number of transplanted plants) × 100%

[0091] During the propagation process, more buds are obtained, and the propagation coefficient is calculated.

[0092] Propagation coefficient = (Total number of buds formed by regenerated seedlings after induction - Number of buds in the initial inoculated explant) / Number of buds in the initial inoculated explant

[0093] Unopened flower buds of the bird's nest fruit plant were used as explants, with only one initial bud point.

[0094] The results are shown in Table 1.

[0095] Table 1

[0096]

[0097]

[0098] As shown in Table 1, the plant tissue culture method of the present invention is scientific and reasonable. The transplant survival rate of the bird's nest fruit plants obtained in Examples 1-3 is high, reaching over 92%, and the propagation coefficient is greater than 8. Comparing Examples 1-8, it can be seen that Example 1, through the selection, disinfection, pretreatment, and optimization of the culture method and culture medium, yielded regenerated plants with robust root systems and a high transplant survival rate.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for tissue culture of bird's nest fruit, characterized in that, Includes the following steps: S1. Explant selection: Unopened flower buds of the bird's nest fruit plant were collected as explants. S2, Disinfection: Irradiate the explants from S1 with ultraviolet light; S3. Explant pretreatment: The disinfected explants are subjected to low-temperature treatment and then irradiated with alternating red and blue light to obtain pretreated explants. S4. Induction culture: The pretreated explants are placed on liquid culture medium for suspension culture. During the culture, ultrasonic stirring is maintained to obtain regenerated seedlings. The liquid culture medium is: MS medium, 0.5-1.5 mg / L NNA, 0.1-0.5 mg / L BAP, 1.0-2.0 mg / L LTDZ, 0.5-0.8 mg / L jasmonic acid, and 1.5-2.5 mg / L coconut water; S5, Proliferation Culture: The regenerated seedlings of S4 were placed on a solid culture medium for culture to obtain regenerated plants of bird's nest fruit; The solid culture medium is: MS medium, 0.1-0.5 mg / L IBA, 0.5-0.8 mg / L IAA, 10-15 mg / L ascorbic acid, 1-3 g / L dextrin; S6. Hardening off and transplanting: Transplant the regenerated plants from S5 into the transplanting substrate for cultivation.

2. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S2, the ultraviolet irradiation uses 10-20 mJ / cm². 2 Irradiate the explant with ultraviolet light at a distance of 15-25 cm for 10-20 minutes.

3. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S3, the low-temperature treatment is performed at 3-7°C for 4-6 hours; the alternating red and blue light irradiation is performed by first using a wavelength of 650-700 nm and an intensity of 50-70 µmol / m². 2 Irradiate with red light at a wavelength of 400-500 nm and an intensity of 60-80 µmol / m for 12 hours, followed by irradiation with red light at a wavelength of 400-500 nm and an intensity of 60-80 µmol / m. 2 Irradiate with blue light for 12 hours, followed by alternating red and blue light irradiation twice.

4. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S4, the photocycle for the suspension culture is 16h / 8h light / dark, and the temperature is 25-27℃.

5. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S4, the ultrasonic stirring is an intermittent stirring with a frequency of 20-40KHz and a power of 10-30W, with ultrasonic stirring for 5-10 minutes every 20-30 minutes.

6. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S5, the cultivation conditions are: cultivation at 25-27℃, with a photocycle of 16h light / 8h darkness.

7. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S6, the transplanting substrate is composed of peat moss, perlite, vermiculite and bentonite in a mass ratio of 1:1-3:1-3:3-5.

8. The method for tissue culture of bird's nest fruit as described in claim 1, characterized in that, In step S6, the cultivation is carried out in an environment with humidity of 75%-90% and light intensity of 300-400 Lux for 3-5 days, with supplemental lighting to increase the light intensity by 50 Lux per day to 1000-1200 Lux, and then normal cultivation.

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