A method to improve the survival rate of dragon fruit tissue culture seedlings

Abstract

This invention discloses a method for improving the survival rate of dragon fruit tissue culture seedlings, belonging to the field of plant cultivation technology. The invention first decouples the tissue culture process into two independent stages: proliferation culture in a first culture medium dominated by cytokinins and rooting culture in a second culture medium dominated by auxins, to obtain robust buds and well-developed root systems, respectively. Subsequently, the rooted seedlings undergo programmed hardening-off, including gradient light and temperature stress and exogenous addition of abscisic acid (ABA), as well as gradual dehumidification after opening the bottle, to actively induce their stress resistance. This method overcomes the shortcomings of existing "synchronous culture" strategies, such as uneven seedling quality and unsystematic hardening-off. Through the synergistic effect of each step, the transplant survival rate of dragon fruit tissue culture seedlings is stably increased to over 97%, achieving an efficient and stable transition from the laboratory to the field, suitable for industrialized seedling production.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, and specifically relates to a method for improving the survival rate of dragon fruit tissue culture seedlings. Background Technology

[0002] Tissue culture for rapid propagation of dragon fruit is a core technology for achieving virus-free, vigorous seedling production and year-round industrialized production. Compared to traditional cuttings, it has significant advantages in maintaining seed uniformity and production efficiency. Therefore, optimizing the tissue culture process, especially overcoming the bottleneck of transplant survival rate, is crucial for the development of the dragon fruit industry.

[0003] Existing technologies have explored various aspects of dragon fruit tissue culture. For example, Chinese patent CN103931497B (hereinafter referred to as D1) discloses a method aimed at improving seedling survival rate. Its core technology lies in the use of specific explants (stem segments with spiny bases) and a culture strategy of "synchronous proliferation and rooting". By simultaneously regulating the development of buds and roots in the seedling culture medium (such as MS + 6-BA 2~4 mg / L + NAA 0.1 mg / L), a single explant produces multiple lateral branches with aerial roots in the bottle, thereby significantly increasing the "seedling quantity" (i.e., propagation coefficient), and it has been reported that the transplant survival rate can reach over 95%.

[0004] However, through in-depth analysis and repeated practice, the inventors have discovered that the "synchronization" strategy and its technical solution advocated by D1 have the following deep-seated and insufficiently revealed technical bottlenecks when applied to industrial-scale applications, resulting in insufficient stability and universality of its final transplant survival rate:

[0005] (1) The synchronous culture strategy presents a physiological contradiction: the single culture medium used in D1 is difficult to simultaneously meet the optimal requirements of shoot proliferation (requiring higher cytokinin) and root differentiation (requiring appropriate auxin). This compromise in hormone ratio can easily lead to uneven development of culture products, or weakened roots and "leggy seedlings" in pursuit of a higher proliferation coefficient; or weak shoots in order to ensure rooting. This inherent contradiction results in uneven quality of tissue culture seedlings, which poses a risk for subsequent transplanting.

[0006] (2) Lack of stress-resistant physiological induction in the hardening-off stage: The hardening-off program of D1 is relatively simple and fails to systematically address the physiological challenges faced by tissue culture seedlings when transitioning from a sterile, heterotrophic, constant temperature and humidity bottle environment to a field environment with bacteria, autotrophic conditions, and variable temperature and humidity (such as thin cuticle and weak stomatal regulation). The lack of procedural physiological acclimatization measures in existing technologies results in poor adaptability of tissue culture seedlings after transplanting, and the survival rate is easily affected by environmental fluctuations.

[0007] (3) Lack of systematic integration of technical solutions: D1 and similar technologies mostly focus on improving a certain intermediate index (such as proliferation coefficient and rooting rate) in the in-bottle stage, and fail to systematically design explant treatment, culture stage optimization, stress resistance induction and post-transplant management as an organic whole. This fragmented improvement leads to insufficient stability and reproducibility of technical solutions, making it difficult to meet the stringent requirements of consistency for industrial seedling production.

[0008] In summary, existing technologies for optimizing seedling survival rate are mostly limited to improving intermediate indicators during the laboratory culture stage. The inventors recognize that the core bottleneck for industrialization lies in achieving the ecological adaptability leap from "laboratory survival" to "stable survival in the field" for tissue culture seedlings. Therefore, there is an urgent need in this field for an innovative cultivation method that can fundamentally solve the above problems, ensuring high yields of tissue culture seedlings in bottles and stable survival in the field. Summary of the Invention

[0009] In view of the above, it is necessary to provide a method to improve the survival rate of dragon fruit tissue culture seedlings. This method overcomes the shortcomings of uneven seedling quality and unsystematic hardening of existing "synchronous cultivation" strategies. Through the synergistic effect of each step, the transplant survival rate of dragon fruit tissue culture seedlings can be stably increased to over 97%, achieving an efficient and stable transition from the laboratory to the field, and is suitable for factory-scale seedling production.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] A method for improving the survival rate of dragon fruit tissue culture seedlings includes the following steps:

[0012] (1) Obtaining aseptic clustered shoots: The stem segments of dragon fruit with areoles were used as explants, and after disinfection, they were inoculated into the starter culture medium to obtain aseptic clustered shoots for later use;

[0013] (2) Bud proliferation and rooting: The clustered buds obtained in step (1) are first transferred to the first culture medium dominated by cytokinin for proliferation culture; then transferred to the second culture medium dominated by auxin for rooting and seedling strengthening culture; rooted seedlings are obtained and ready for use.

[0014] (3) Hardening and acclimatization: For the rooted seedlings obtained in step (2), perform the following operations in sequence:

[0015] a. In-bottle acclimatization treatment: Under conditions where the culture container is not opened, physiological acclimatization of tissue culture seedlings is carried out by gradually increasing light intensity, applying day-night temperature differences, and adding exogenous plant-inducing substances;

[0016] b. Adaptation after opening the container: Open the culture container and spray with a low concentration of nutrient solution while gradually reducing the ambient humidity;

[0017] (4) Transplanting: Transplant the tissue culture seedlings after hardening in step (3) into a special substrate.

[0018] Preferably, the starting medium is a solid medium containing MS basic components, 1.0-2.0 mg / L of 6-benzylaminopurine (6-BA), 0.05-0.1 mg / L of naphthaleneacetic acid (NAA), and 50-100 mg / L of vitamin C.

[0019] Preferably, the first culture medium is a solid culture medium containing MS basic components, 3.0-5.0 mg / L of 6-benzylaminopurine (6-BA) and 0.1-0.3 mg / L of naphthaleneacetic acid (NAA).

[0020] Preferably, the first culture medium also contains 10-20 mg / L of adenine sulfate.

[0021] Preferably, the second culture medium is a solid culture medium based on 1 / 2 MS solution, with the addition of indolebutyric acid (IBA) 0.5-1.0 mg / L, naphthaleneacetic acid (NAA) 0.1-0.3 mg / L and activated carbon 0.5-1.0 g / L.

[0022] Preferably, in step (3)a, the gradient increase of light intensity is to gradually increase the light intensity from 2000-2500 lux to 5000-6000 lux over 7-10 days; the application of diurnal temperature difference is to control the daytime temperature at 28-32℃ and the nighttime temperature at 18-22℃; the plant inducing substance is abscisic acid (ABA), and its concentration is 50-100μM.

[0023] Preferably, in step (3)b, the gradual reduction of ambient humidity means gradually reducing the humidity from over 90% to 70%-80% over 5-7 days; the low-concentration nutrient solution refers to an aqueous solution containing only the macro-elements of MS culture medium and with a concentration of 1 / 4 of its conventional concentration.

[0024] Preferably, the low-concentration nutrient solution also contains 0.05%-0.1% trehalose.

[0025] Preferably, in step (4), the special substrate is a mixture of peat moss, vermiculite, perlite and biochar in a volume ratio of (3-4):(1-2):(1-2):(0.5-1).

[0026] Another object of the present invention is to provide a dragon fruit tissue culture seedling, which is cultivated by the method described above.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] 1. This application decomposes the traditional "synchronous proliferation and rooting" process into asynchronous optimized steps: a first culture medium dominated by cytokinin (6-BA) for proliferation culture, and a second culture medium dominated by auxin (IBA) for rooting culture. This approach provides the most suitable hormonal environment for the two physiologically distinct stages of bud proliferation and root development. Specifically, cytokinin mainly promotes bud differentiation and proliferation, while auxin dominates root induction and growth; simultaneously meeting the needs of both in a single culture medium inevitably leads to compromises. This invention, through physically separated culture stages, allows buds to proliferate fully in a high-concentration 6-BA environment, yielding a large number of robust bud clusters. These robust buds can then be induced to form well-developed and fully functional root systems under appropriate auxin ratios. This achieves superior innate traits of "strong buds and vigorous roots" and uniform quality in tissue culture seedlings produced from the source, overcoming the problems of "leggy seedlings" or "weak root seedlings" caused by hormonal conflicts in existing technologies, and laying a solid material foundation for high transplant survival rates.

[0029] 2. This application also innovatively designs a seedling hardening program that combines in-bottle gradient stress with open-bottle adaptation, incorporating exogenous abscisic acid (ABA), transforming passive environmental exposure into active physiological acclimatization. The core reason for transplant failure of tissue culture seedlings is considered to be their physiological structure (e.g., thin cuticle, weak stomatal regulation) and metabolic pattern (heterotrophic) inability to adapt to the complex field environment. This invention simulates abiotic stress in the natural environment by gradually increasing light intensity and setting diurnal temperature differences within the bottle. Crucially, it purposefully adds ABA, a key signaling molecule in plants responding to adversity, which can actively initiate a series of stress-resistance physiological and biochemical reactions, such as promoting the accumulation of compatible solutes like proline to maintain cell osmotic balance, inducing stomatal closure to reduce water transpiration, and enhancing the antioxidant defense system. Subsequently, by gradually dehumidifying the bottle and spraying with a low-concentration nutrient solution, the seedlings are smoothly guided to complete the transition from heterotrophic to autotrophic. This procedural hardening process allows seedlings to complete the "pre-adaptation" of key physiological functions before transplanting, thereby significantly improving their ability to cope with drought, strong light and temperature fluctuations after transplanting, laying a solid physiological foundation for subsequent high survival rate cultivation.

[0030] 3. This application systematically integrates and seamlessly connects two core aspects: high-quality tissue culture seedling production (asynchronous optimized culture) and efficient stress resistance induction (programmed hardening-off), generating a synergistic effect. High-quality tissue culture seedlings are the material basis for successful hardening-off, while efficient hardening-off is the necessary means to transform the potential of high-quality seedlings into a high survival rate; neither can be dispensed with. The data from the examples and control examples fully demonstrate this synergy: while asynchronous culture or programmed hardening-off alone can improve the survival rate to some extent, the effect is limited; however, combining the complete solution of this invention can stably increase the transplant survival rate of dragon fruit tissue culture seedlings to over 97%, a figure that significantly surpasses any other improvement scheme with a single feature. This stable and reproducible high survival rate effectively solves the persistent industry problem of the disconnect between laboratory indicators and field performance, providing a reliable technical solution that can be directly applied to factory-scale, large-scale seedling production, and has outstanding industrial application value. Detailed Implementation

[0031] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Example 1:

[0033] This embodiment proposes a method to improve the survival rate of dragon fruit tissue culture seedlings. The cultivation process includes the following steps:

[0034] (1) Obtaining sterile seedlings: Select healthy, semi-lignified green stem segments from the current year of 'Da Hong Yi Hao' plants, remove surface thorns with a blade, and rinse under running water for 120 minutes. In a clean bench, immerse the stem segments in a 75% (v / v) ethanol solution for 30 seconds for surface disinfection, rinse once with sterile water, and then immerse them in a 0.1% (w / v) mercuric chloride (HgCl2) solution containing 0.05% (v / v) Tween-80 for 7 minutes, gently shaking during the process. After disinfection, rinse five times with sterile water. Cut the stem segments into slices approximately 1.0 cm thick with a complete areole and inoculate them onto the starter culture medium.

[0035] The start-up medium contains the following components: MS basal medium, 6-benzylaminopurine (6-BA) 1.5 mg / L, naphthaleneacetic acid (NAA) 0.08 mg / L, vitamin C 80 mg / L, sucrose 30 g / L, agar 6.8 g / L, and pH adjusted to 5.8.

[0036] The cultivation conditions were: temperature (25±2)℃, light intensity 1200 lux, and daily light duration 12 hours. After 30 days of cultivation, sterile clustered shoots with good growth were obtained.

[0037] (2) Rooting culture: The sterile bud clusters obtained in step (1) are divided into single buds or small clusters containing 2-3 buds and transferred to the first culture medium for proliferation culture.

[0038] The first culture medium contains the following components: MS basal medium, 6-BA 4.0 mg / L, NAA 0.2 mg / L, adenine sulfate 15 mg / L, sucrose 30 g / L, agar 6.8 g / L, pH 5.8.

[0039] Under the same temperature and light cycle, the light intensity was adjusted to 1500 lux, and the culture was carried out for 35 days. Subsequently, healthy single shoots with a height of not less than 2.5 cm were selected and transferred to a second culture medium for rooting culture.

[0040] The second culture medium contains the following components: 1 / 2 MS basal medium (i.e., the macro-elements and micro-elements are half the MS standard concentration), indolebutyric acid (IBA) 0.8 mg / L, NAA 0.2 mg / L, activated carbon 0.8 g / L, sucrose 20 g / L, agar 6.8 g / L, pH 5.8.

[0041] The cultivation conditions were adjusted to: temperature (25±2)℃, light intensity 2000 lux, daily light duration 14 hours, and cultivation for 30 days to obtain rooted tissue culture seedlings with well-developed root systems.

[0042] (3) Seedling hardening and domestication:

[0043] a. In-bottle acclimatization treatment: Place the rooted tissue culture seedlings and their culture containers in a controlled acclimatization room. Without opening the bottle caps, gradually (linearly) increase the light intensity from 2500 lux to 5500 lux over 7 days, setting the daytime temperature to 30℃ and the nighttime temperature to 20℃. On the 3rd day after the acclimatization begins, use aseptic techniques to inject 1.5 mL of 80 μM sterile abscisic acid (ABA) aqueous solution into each bottle of culture medium. This stage lasts for a total of 9 days.

[0044] b. Acclimation after opening the container: Open the lid of the culture container and spray the stems and leaves of the tissue culture seedlings with a low-concentration nutrient solution once a day. Simultaneously, gradually reduce the relative humidity of the air in the hardening-off room from over 95% to 75% over 6 days. The low-concentration nutrient solution is an aqueous solution containing only the macroelements of MS medium (ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate heptahydrate, and potassium dihydrogen phosphate) at a concentration of 1 / 4 of the MS standard formulation, with an additional 0.08% (w / v) of trehalose. This stage lasts for 6 days.

[0045] (4) Transplanting: Remove the hardened tissue culture seedlings from the container, gently wash off the agar adhering to the roots with clean water, and then soak the roots for 5 minutes in a mixture containing carbendazim (500 times diluted solution) and low-concentration rooting powder (at the lowest concentration recommended in the product instructions). Plant the seedlings in a pre-sterilized special substrate, which is a uniform mixture of peat moss, vermiculite, perlite and biochar in a volume ratio of 4:2:2:1. After transplanting, place the seedbed in a greenhouse, erect a small plastic arched shed to maintain a high temperature (initial relative humidity 85%-90%), and provide 50% shade, maintaining the ambient temperature at (26±2)℃. After 7 days of transplanting, gradually increase ventilation daily, and completely remove the arched shed after 10 days, switching to regular water management. On the 30th day after transplanting, count the number of surviving seedlings and calculate the survival rate.

[0046] Example 2:

[0047] This embodiment proposes a method to improve the survival rate of dragon fruit tissue culture seedlings. The cultivation process includes the following steps:

[0048] (1) Obtaining sterile seedlings: Select healthy, semi-lignified green stem segments from the current year of 'Da Hong Yi Hao' plants, remove surface thorns with a blade, and rinse under running water for 120 minutes. In a clean bench, immerse the stem segments in a 75% (v / v) ethanol solution for 30 seconds for surface disinfection, rinse once with sterile water, and then immerse them in a 0.1% (w / v) mercuric chloride (HgCl2) solution containing 0.05% (v / v) Tween-80 for 7 minutes, gently shaking during the process. After disinfection, rinse five times with sterile water. Cut the stem segments into slices approximately 1.0 cm thick with a complete areole and inoculate them onto the starter culture medium.

[0049] The start-up medium contains the following components: MS basal medium, 6-benzylaminopurine (6-BA) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.08 mg / L, vitamin C 50 mg / L, sucrose 30 g / L, agar 6.8 g / L, and pH adjusted to 5.8.

[0050] The cultivation conditions were: temperature (25±2)℃, light intensity 1200 lux, and daily light duration 12 hours. After 30 days of cultivation, sterile clustered shoots with good growth were obtained.

[0051] (2) Rooting culture: The sterile bud clusters obtained in step (1) are divided into single buds or small clusters containing 2-3 buds and transferred to the first culture medium for proliferation culture.

[0052] The first culture medium contains the following components: MS basal medium, 6-BA 3.0 mg / L, NAA 0.2 mg / L, adenine sulfate 15 mg / L, sucrose 30 g / L, agar 6.8 g / L, pH 5.8.

[0053] Under the same temperature and light cycle, the light intensity was adjusted to 1500 lux, and the culture was carried out for 35 days. Subsequently, healthy single shoots with a height of not less than 2.5 cm were selected and transferred to a second culture medium for rooting culture.

[0054] The second culture medium contains the following components: 1 / 2 MS basal medium (i.e., the macro-elements and micro-elements are half the MS standard concentration), indolebutyric acid (IBA) 0.5 mg / L, NAA 0.2 mg / L, activated carbon 0.8 g / L, sucrose 20 g / L, agar 6.8 g / L, pH 5.8.

[0055] The cultivation conditions were adjusted to: temperature (25±2)℃, light intensity 2000 lux, daily light duration 14 hours, and cultivation for 30 days to obtain rooted tissue culture seedlings with well-developed root systems.

[0056] (3) Seedling hardening and domestication:

[0057] a. In-bottle acclimatization treatment: Place the rooted tissue culture seedlings and their culture containers in a controlled acclimatization room. Without opening the bottle caps, gradually (linearly) increase the light intensity from 2500 lux to 5000 lux over 7 days, setting the daytime temperature to 30℃ and the nighttime temperature to 20℃. On the 3rd day after the acclimatization begins, use aseptic techniques to inject 1.5 mL of a 50 μM sterile aqueous solution of abscisic acid (ABA) into each bottle of culture medium. This stage lasts for a total of 9 days.

[0058] b. Acclimation after opening the container: Open the lid of the culture container and spray the stems and leaves of the tissue culture seedlings with a low-concentration nutrient solution once a day. Simultaneously, gradually reduce the relative humidity of the air in the hardening-off room from over 95% to 75% over 6 days. The low-concentration nutrient solution is an aqueous solution containing only the macroelements of MS medium (ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate heptahydrate, and potassium dihydrogen phosphate) at a concentration of 1 / 4 of the MS standard formulation, with an additional 0.05% (w / v) of trehalose. This stage lasts for 6 days.

[0059] (4) Transplanting: Remove the hardened tissue culture seedlings from the container, gently wash off the agar adhering to the roots with clean water, and then soak the roots for 5 minutes in a mixture containing carbendazim (500 times diluted solution) and low-concentration rooting powder (at the lowest concentration recommended in the product instructions). Plant the seedlings in a pre-sterilized special substrate, which is a uniform mixture of peat moss, vermiculite, perlite and biochar in a volume ratio of 4:2:2:1. After transplanting, place the seedbed in a greenhouse, erect a small plastic arched shed to maintain a high temperature (initial relative humidity 85%-90%), and provide 50% shade, maintaining the ambient temperature at (26±2)℃. After 7 days of transplanting, gradually increase ventilation daily, and completely remove the arched shed after 10 days, switching to regular water management. On the 30th day after transplanting, count the number of surviving seedlings and calculate the survival rate.

[0060] Example 3:

[0061] This embodiment proposes a method to improve the survival rate of dragon fruit tissue culture seedlings. The cultivation process includes the following steps:

[0062] (1) Obtaining sterile seedlings: Select healthy, semi-lignified green stem segments from the current year of 'Da Hong Yi Hao' plants, remove surface thorns with a blade, and rinse under running water for 120 minutes. In a clean bench, immerse the stem segments in a 75% (v / v) ethanol solution for 30 seconds for surface disinfection, rinse once with sterile water, and then immerse them in a 0.1% (w / v) mercuric chloride (HgCl2) solution containing 0.05% (v / v) Tween-80 for 7 minutes, gently shaking during the process. After disinfection, rinse five times with sterile water. Cut the stem segments into slices approximately 1.0 cm thick with a complete areole and inoculate them onto the starter culture medium.

[0063] The start-up medium contains the following components: MS basal medium, 6-benzylaminopurine (6-BA) 2.0 mg / L, naphthaleneacetic acid (NAA) 0.08 mg / L, vitamin C 100 mg / L, sucrose 30 g / L, agar 6.8 g / L, and pH adjusted to 5.8.

[0064] The cultivation conditions were: temperature (25±2)℃, light intensity 1200 lux, and daily light duration 12 hours. After 30 days of cultivation, sterile clustered shoots with good growth were obtained.

[0065] (2) Rooting culture: The sterile bud clusters obtained in step (1) are divided into single buds or small clusters containing 2-3 buds and transferred to the first culture medium for proliferation culture.

[0066] The first culture medium contains the following components: MS basal medium, 6-BA 5.0 mg / L, NAA 0.2 mg / L, adenine sulfate 20 mg / L, sucrose 30 g / L, agar 6.8 g / L, pH 5.8.

[0067] Under the same temperature and light cycle, the light intensity was adjusted to 1500 lux, and the culture was carried out for 35 days. Subsequently, healthy single shoots with a height of not less than 2.5 cm were selected and transferred to a second culture medium for rooting culture.

[0068] The second culture medium contains the following components: 1 / 2 MS basal medium (i.e., the macro-elements and micro-elements are half the MS standard concentration), indolebutyric acid (IBA) 1.0 mg / L, NAA 0.2 mg / L, activated carbon 1.0 g / L, sucrose 20 g / L, agar 6.8 g / L, pH 5.8.

[0069] The cultivation conditions were adjusted to: temperature (25±2)℃, light intensity 2000 lux, daily light duration 14 hours, and cultivation for 30 days to obtain rooted tissue culture seedlings with well-developed root systems.

[0070] (3) Seedling hardening and domestication:

[0071] a. In-bottle acclimatization treatment: Place the rooted tissue culture seedlings and their culture containers in a controlled acclimatization room. Without opening the bottle caps, gradually (linearly) increase the light intensity from 2500 lux to 6000 lux over 7 days, setting the daytime temperature to 30℃ and the nighttime temperature to 20℃. On the 3rd day after the acclimatization begins, use aseptic techniques to inject 1.5 mL of a 100 μM sterile abscisic acid (ABA) aqueous solution into each bottle of culture medium. This stage lasts for a total of 9 days.

[0072] b. Acclimation after opening the container: Open the lid of the culture container and spray the stems and leaves of the tissue culture seedlings with a low-concentration nutrient solution once a day. Simultaneously, gradually reduce the relative humidity of the air in the hardening-off room from over 95% to 75% over 6 days. The low-concentration nutrient solution is an aqueous solution containing only the macroelements of MS medium (ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate heptahydrate, and potassium dihydrogen phosphate) at a concentration of 1 / 4 of the MS standard formulation, with an additional 0.10% (w / v) of trehalose. This stage lasts for 6 days.

[0073] (4) Transplanting: Remove the hardened tissue culture seedlings from the container, gently wash off the agar adhering to the roots with clean water, and then soak the roots for 5 minutes in a mixture containing carbendazim (500 times diluted solution) and low-concentration rooting powder (at the lowest concentration recommended in the product instructions). Plant the seedlings in a pre-sterilized special substrate, which is a uniform mixture of peat moss, vermiculite, perlite and biochar in a volume ratio of 4:2:2:1. After transplanting, place the seedbed in a greenhouse, erect a small plastic arched shed to maintain a high temperature (initial relative humidity 85%-90%), and provide 50% shade, maintaining the ambient temperature at (26±2)℃. After 7 days of transplanting, gradually increase ventilation daily, and completely remove the arched shed after 10 days, switching to regular water management. On the 30th day after transplanting, count the number of surviving seedlings and calculate the survival rate.

[0074] Compare with Example 1:

[0075] This comparative example simulates the closest existing technical method, and the main difference from Example 1 lies in the cultivation and hardening strategies.

[0076] Step (1) is the same as in Example 1.

[0077] In step (2), instead of the stepwise culture of the present invention, the obtained clustered buds are directly inoculated into a single culture medium for "synchronous proliferation and rooting" (component: MS + 6-BA 2.0 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 6.8 g / L, pH 5.8) and cultured for 60 days until aerial roots are produced at the base of some buds.

[0078] In step (3), the programmed hardening of seedlings according to the present invention is not performed. Instead, the bottle cap is opened before transplanting, and the seedlings are hardened for 3 days in a culture room environment.

[0079] The transplanting substrate in step (4) is a mixture of conventional peat moss and vermiculite in a volume ratio of 1:1, and the remaining management is the same as in Example 1.

[0080] Compare with Example 2:

[0081] This comparative example is used to verify the effect of using only the culture steps of the present invention while omitting a specific hardening-off step. The only difference from Example 1 is the hardening-off step.

[0082] Steps (1) and (2) are exactly the same as in Example 1.

[0083] In step (3), no gradient light and temperature regulation or ABA addition was performed; instead, the same routine of opening the bottle and hardening off the seedlings for 3 days as in control example 1 was used.

[0084] Step (4) is the same as in Example 1.

[0085] Compare with Example 3:

[0086] This comparative example is used to verify the effect of applying the seedling hardening steps of the present invention to tissue culture seedlings obtained by different culture methods. The difference between this example and Example 1 lies in the culture steps.

[0087] Step (1) is the same as in Example 1.

[0088] Step (2) uses the same “synchronous proliferation and rooting” culture medium and culture method as Control Example 1.

[0089] Steps (3) and (4) are exactly the same as in Example 1.

[0090] Compare with Example 4:

[0091] This comparative example is used to examine the effect of the initial treatment of explants on the final effect. The difference between this example and Example 1 is the treatment of the explants before disinfection.

[0092] In step (1), the explants were taken from the same mother plant, but in accordance with some existing technical methods, the hairs and small thorns on the areoles were deliberately preserved before disinfection. Instead of using disinfectant containing Tween-80, only 0.1% HgCl2 was used for disinfection for 8 minutes.

[0093] From the start of the culture, all subsequent steps (2), (3), and (4) are exactly the same as in Example 1.

[0094] Compare with Example 5:

[0095] This comparative example is used to explore the effect limit of advanced seedling hardening on the basis of inferior tissue culture seedlings. The difference from Example 1 is that a different basal culture medium was used.

[0096] Step (1) is the same as in Example 1.

[0097] In step (2), both proliferation and rooting were carried out in a compromise single medium (MS + 6-BA 3.0 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 6.8 g / L, pH 5.8) for 50 days.

[0098] Steps (3) and (4) are exactly the same as in Example 1.

[0099] Compare with Example 6:

[0100] This comparative example is used to verify the necessity of adding exogenous ABA during the seedling hardening stage. The only difference from Example 1 is the in-bottle acclimatization treatment.

[0101] Steps (1) and (2) are exactly the same as in Example 1.

[0102] In step (3)a, only the same pattern of light intensity gradient increase and diurnal temperature difference setting is performed, but ABA or any other plant growth regulator is added to different culture media.

[0103] Steps (3)b and (4) are the same as in Example 1.

[0104] Effect verification

[0105] To objectively evaluate the effectiveness of this invention, after hardening off and before transplanting, samples were uniformly taken from all tissue culture seedlings in the embodiments and control examples to determine their proline content and chlorophyll fluorescence parameter Fv / Fm value. The survival rate was calculated 30 days after transplanting. Proline content was determined using sulfosalicylic acid extraction and acidic ninhydrin colorimetric method; Fv / Fm value was determined using a portable modulated chlorophyll fluorescence spectrometer after dark adaptation. The results are summarized in Table 1 below.

[0106] Table 1: Physiological indicators and transplant survival rate of tissue culture seedlings in each example and control case

[0107]

[0108] Note: FW stands for Fresh Weight.

[0109] Based on the results in Table 1, the technical effects of the present invention can be analyzed as follows:

[0110] The complete technical solution represented by Examples 1-3 of this invention achieves a stable transplant survival rate of over 97%, significantly better than all control examples. This excellent effect is closely related to the high proline content (usually positively correlated with stress resistance) and high Fv / Fm value (reflecting the potential efficiency of photosystem II) of the tissue culture seedlings before transplanting.

[0111] However, if the "synchronous culture" method, which is closest to the existing technology, is combined with the conventional hardening-off strategy (Comparative Example 1), the survival rate is the lowest, and the physiological stress resistance reserves (proline content) and photosynthetic potential of the seedlings are also the lowest. This confirms the point made in the background art that the compromise of synchronous culture is difficult to obtain physiologically balanced and robust high-quality seedlings. If only the stepwise culture strategy of the present invention is used but the programmed stress resistance hardening-off is omitted (Comparative Example 2), the survival rate is improved compared with Comparative Example 1, but is still less than 90%. This shows that even if tissue culture seedlings with good initial quality are obtained, their ability to cope with drastic changes in the transplanting environment is still limited if targeted stress resistance physiological induction is not performed. Conversely, if the programmed hardening-off of the present invention is applied to the inferior seedlings obtained by "synchronous culture" (Comparative Example 3), the survival rate (91.5%) and proline content are significantly improved compared with Comparative Example 1, proving that the hardening-off step is effective, but its upper limit is still significantly lower than the complete scheme of the present invention. This result demonstrates that the sequential proliferation and rooting culture laid an excellent initial quality foundation for the tissue culture seedlings, while the programmed hardening process, including ABA induction, further stimulated the stress-resistance physiological potential of the seedlings on this high-quality basis. The two processes exhibit a significant synergistic effect, working together to achieve a high survival rate. Furthermore, the results of Control Example 4 show that if the initial treatment of explants is improper, even with the same subsequent culture and hardening process, the final effect will be compromised. This underscores the necessity of the systematic optimization design of this invention, starting with explant disinfection. Control Example 5, using a different compromise culture medium, still failed to achieve the same final survival rate (92.8%) as the present invention, further illustrating that the culture medium formulation of the present invention (especially the differentiated design of the first and second culture media) is crucial for constructing high-quality basic seedlings. In Control Example 6, omitting the addition of ABA resulted in a significant decrease in proline accumulation and final survival rate of the tissue culture seedlings. This confirms that, under light and temperature stress, exogenous ABA addition is a crucial step that cannot be ignored in deeply activating the stress-resistance physiological response and achieving a survival rate exceeding 97%.

[0112] In summary, this invention establishes an organically integrated and synergistic technical system by using first and second culture media with different compositions in a specific order for stepwise cultivation, combined with gradient stress hardening including the addition of exogenous ABA. This system not only optimizes the "innate constitution" and "acquired adaptability" of tissue culture seedlings separately, but also, through the organic combination of the two, produces unexpected technical effects beyond simple superposition, namely, a significant leap in the transplant survival rate of dragon fruit tissue culture seedlings from the conventional level of approximately 86-90% to over 97%.

[0113] The above embodiments are merely examples of several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A method for improving the survival rate of dragon fruit tissue culture seedlings, characterized in that, Includes the following steps: (1) Obtaining aseptic clustered shoots: The stem segments of dragon fruit with areoles were used as explants, and after disinfection, they were inoculated into the starter culture medium to obtain aseptic clustered shoots for later use; (2) Bud proliferation and rooting: The clustered buds obtained in step (1) are first transferred to the first culture medium dominated by cytokinin for proliferation culture; then transferred to the second culture medium dominated by auxin for rooting and seedling strengthening culture; rooted seedlings are obtained and ready for use. (3) Hardening and acclimatization: For the rooted seedlings obtained in step (2), perform the following operations in sequence: a. In-bottle acclimatization treatment: Under conditions where the culture container is not opened, physiological acclimatization of tissue culture seedlings is carried out by gradually increasing light intensity, applying day-night temperature differences, and adding exogenous plant-inducing substances; b. Adaptation after opening the container: Open the culture container and spray with a low concentration of nutrient solution while gradually reducing the ambient humidity; (4) Transplanting: Transplant the tissue culture seedlings after hardening in step (3) into a special substrate.

2. The method according to claim 1, characterized in that, The starting medium is a solid medium containing MS basic components, 1.0-2.0 mg / L of 6-benzylaminopurine (6-BA), 0.05-0.1 mg / L of naphthaleneacetic acid (NAA), and 50-100 mg / L of vitamin C.

3. The method according to claim 1, characterized in that, The first culture medium is a solid culture medium containing MS basic components, 3.0-5.0 mg / L of 6-benzylaminopurine (6-BA) and 0.1-0.3 mg / L of naphthaleneacetic acid (NAA).

4. The method according to claim 3, characterized in that, The first culture medium also contains 10-20 mg / L of adenine sulfate.

5. The method according to claim 1, characterized in that, The second culture medium is a solid culture medium based on 1 / 2 MS solution, with the addition of indolebutyric acid (IBA) 0.5-1.0 mg / L, naphthaleneacetic acid (NAA) 0.1-0.3 mg / L and activated carbon 0.5-1.0 g / L.

6. The method according to claim 1, characterized in that, In step (3)a, the gradient increase of light intensity means gradually increasing the light intensity from 2000-2500 lux to 5000-6000 lux over 7-10 days; the application of diurnal temperature difference means controlling the daytime temperature to 28-32℃ and the nighttime temperature to 18-22℃; the plant inducing substance is abscisic acid (ABA), and its concentration is 50-100μM.

7. The method according to claim 1, characterized in that, In step (3)b, the gradual reduction of ambient humidity means gradually reducing the humidity from over 90% to 70%-80% over 5-7 days; the low-concentration nutrient solution refers to an aqueous solution containing only the macro-elements of MS culture medium and with a concentration of 1 / 4 of its conventional concentration.

8. The method according to claim 7, characterized in that, The low-concentration nutrient solution also contains 0.05%-0.1% trehalose.

9. The method according to claim 1, characterized in that, In step (4), the special substrate is a mixture of peat moss, vermiculite, perlite and biochar in a volume ratio of (3-4):(1-2):(1-2):(0.5-1).

10. A dragon fruit tissue culture seedling, characterized in that, It is cultivated by the method described in any one of claims 1 to 9.

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