Ixora chinensis tissue culture seedling efficient cultivation method based on photoperiod regulation and control

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a high-efficiency cultivation method of ixora chinensis tissue culture seedlings based on photoperiod regulation and control, which comprises the following steps: step S1, pretreatment of explants; step S2, primary induction culture; step S3, carrying out subculture multiplication culture; step S4, rooting culture; step S5, seedling hardening and domestication. According to the method, a staged photoperiod regulation and control mode is constructed, the whole process parameters of short photoperiod induction, long photoperiod proliferation, medium and short photoperiod rooting and gradient photoperiod seedling hardening are set in combination with short-day physiological characteristics of the ixora chinensis and growth requirements of different culture stages, and meanwhile, an illumination intensity gradient increasing mode is optimized; the defects that in the prior art, a single photoperiod cannot adapt to the whole culture process, and tissue culture seedlings are damaged due to sudden illumination change are overcome, the browning rate of the explants is controlled, and collaborative optimization of growth in all stages is achieved.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for the efficient cultivation of Ixora tissue culture seedlings based on photoperiod regulation. Background Technology

[0002] Ixora, an evergreen shrub belonging to the Rubiaceae family and the Ixora genus, is known for its vibrant flowers and long blooming period (May to October). It is highly adaptable and is one of the core ornamental flowers cultivated in gardens and courtyards in southern my country. Furthermore, the entire plant can be used medicinally, possessing properties that clear heat, cool the blood, disperse blood stasis, and relieve pain. It enjoys widespread demand in flower cultivation, medicinal development, and breeding. There are numerous varieties of Ixora, including the dwarf pink Ixora, apricot yellow Ixora, and red Ixora. The different reproductive characteristics of these varieties pose challenges to large-scale, standardized cultivation.

[0003] Traditional propagation methods for Ixora chinensis mainly involve cuttings and sowing, but these methods have significant drawbacks: cutting propagation is limited by the season and the number of mother plants, with a propagation coefficient of only 2-3, and it is also prone to carrying diseases from the mother plant; sowing propagation suffers from low seed germination rates (less than 40%) and offspring are prone to phenotypic segregation, making it difficult to maintain the superior characteristics of the mother plant and failing to meet the needs of large-scale, standardized production. Plant tissue culture technology, with its advantages of rapid propagation, high propagation coefficient, no seasonal limitations, and ability to maintain the traits of the mother plant, has become the core technical approach for the rapid propagation of Ixora chinensis.

[0004] Several methods for tissue culture of Ixora have been disclosed in the prior art. For example, patent CN202411210924.4 discloses a method for rapid propagation of Ixora. By optimizing the hormone combination of the culture medium (6-BA, TDZ, NAA), the induction rate of clustered shoots and the rooting rate are improved, and the rooting rate reaches more than 80%. However, this method does not consider the regulatory effect of photoperiod on the growth of tissue culture seedlings and adopts a single culture environment throughout the process, which leads to etiolation and weak stress resistance of tissue culture seedlings.

[0005] Photoperiod, as a core regulatory signal for plant growth and development, directly affects the differentiation, proliferation, rooting, and stress resistance formation of tissue-cultured seedlings by regulating photosynthetic rhythm, the synthesis of endogenous hormones (auxin, cytokinin, abscisic acid), and the expression of related functional genes (such as CRY1 and PHYA). As a typical short-day plant, *Ixora chinensis* is sensitive to photoperiod in both its vegetative and reproductive growth. However, current tissue culture techniques for *Ixora chinensis* focus on optimizing culture medium formulation and explant treatment, generally neglecting the staged regulation of photoperiod and failing to adjust parameters based on variety characteristics. This leads to the following technical bottlenecks: excessively long light exposure during the initial induction stage easily causes explant browning (browning rate reaches 20-30%); insufficient light during the subsequent proliferation stage results in less accumulation of photosynthetic products and weak, elongated shoots; unsuitable light during the rooting stage inhibits root primordia formation and reduces root branching; sudden changes in light during the hardening-off stage cause wilting and death of tissue-cultured seedlings. Furthermore, poor adaptability to different varieties makes it difficult to achieve unified large-scale cultivation of multiple varieties. Summary of the Invention

[0006] The purpose of this invention is to provide an efficient method for cultivating Ixora tissue culture seedlings based on photoperiod regulation, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A highly efficient method for cultivating Ixora tissue culture seedlings based on photoperiod regulation includes the following steps: Step S1, Explant Pretreatment: Healthy, semi-lignified branches of Ixora or its variants with terminal buds from the current year were selected as explants. After removing the leaves, the branches were cut into 2-3 cm segments with stem nodes and sterilized to obtain sterile explants. Aseptic processing specifically includes: The explants were first rinsed with running water for 2-3 hours, gently rubbed every 30 minutes, then soaked in 75% ethanol solution for 30 seconds, rinsed with sterile water 3-4 times, and then soaked in a mixture of 0.1% mercuric chloride solution and 0.1% Tween-80 solution for 9-10 minutes, shaking every 2 minutes, and rinsed with sterile water 5-6 times. The surface moisture was then blotted dry with sterile filter paper. The varieties of Ixora include dwarf pink Ixora and apricot yellow Ixora. Step S2, Primary Induction Culture: Sterile explants were inoculated into the primary induction medium and cultured in a light incubator at a temperature of 23-28℃ and a relative humidity of 70-75%. The photoperiod was set to 8 hours of light / 16 hours of darkness, and the light intensity was 50-80 μmol / (m²·s). Induced shoots were obtained after 12-15 days of culture. The primary induction medium was based on MS medium, supplemented with 4.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L LTDZ, 30 g / L sucrose, and 7 g / L agar, and the pH was adjusted to 5.8-6.0. Step S3, Subculture and Proliferation: The induced shoots obtained in step S2 were divided and inoculated into a subculture proliferation medium. The culture temperature was 23-28℃, the relative humidity was 70-75%, the photoperiod was set to 16h light / 8h dark, and the light intensity was 120-150μmol / (m²·s). The shoots were obtained after 25-30 days of culture. The subculture proliferation medium was based on MS medium, with the addition of 0.5mg / L 6-BA, 0.5mg / L NAA, 30g / L sucrose and 7g / L agar, and the pH was adjusted to 5.8-6.0. Step S4, Rooting Culture: Healthy shoots with a height of 2-3 cm were selected and inoculated into rooting medium. The culture temperature was 21-25℃, the relative humidity was 75-80%, the photoperiod was set to 10h light / 14h dark, and the light intensity was 80-100μmol / (m²·s). Rooted tissue culture seedlings were obtained after 25-30 days of culture. The rooting medium was based on 1 / 2 MS medium, with the addition of 0.5mg / L NAA, 20g / L sucrose and 7g / L agar, and the pH was adjusted to 5.8-6.0. Step S5, Seedling Acclimation and Domestication: Transfer the rooted tissue culture seedlings, still in their bottles, to the greenhouse. First, harden them off for 3-5 days under conditions of 12 hours of light / 12 hours of darkness and 50-60% shading. Then, gradually increase the light duration to 14 hours of light / 10 hours of darkness and 30-40% shading for 2-3 days. Finally, transition to a natural photoperiod, remove the shading, and maintain a greenhouse temperature of 20-28℃ and humidity of 60-70%. Transplant the seedlings after a total hardening-off period of 7-10 days.

[0008] Preferably, in steps S2-S4, an LED composite light source is used, which is a red-blue combined spectrum or a red-blue-green composite spectrum; the ratio of red light wavelength to blue light wavelength in the red-blue combined spectrum is 3:1; and the ratio of red light wavelength, blue light wavelength, and green light wavelength in the red-blue-green composite spectrum is 3:1:0.5.

[0009] Preferably, in step S5, the transplanting substrate is a mixture of leaf mold, perlite, garden soil, and well-rotted sheep manure in a volume ratio of 2:1:1:0.5. Before transplanting, it is sterilized at 121℃ under high pressure for 2 hours. After transplanting, a small arched shed is built to keep warm and moist, and the humidity inside the shed is controlled at 80-85%. After 3 days, ventilation is gradually carried out to reduce humidity.

[0010] Preferably, the daily light exposure periods in steps S2-S4 are 8:00-16:00, 6:00-22:00, and 9:00-19:00, and the light intensity adopts a gradient increase method, with the light intensity at 50% of the set value for the first 3 days, increasing to 75% of the set value on the 4th-5th days, and increasing to the set value from the 6th day onwards, to avoid damage to the tissue culture seedlings from sudden changes in light.

[0011] Preferably, in step S3, if the culture object is Ixora dwarf, 0.2 mg / L GA3 is added to the subculture proliferation medium to promote shoot elongation and avoid excessive dwarfing; In step S4, if the culture medium is *Ixora chinensis*, the NAA concentration in the rooting medium is adjusted to 0.6 mg / L to improve rooting stability.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs a phased photoperiod regulation system, combining the short-day physiological characteristics of Ixora chinensis with the growth requirements of different culture stages. It sets parameters for the entire process of short-photoperiod induction, long-photoperiod proliferation, medium-short-photoperiod rooting, and gradient photoperiod hardening, while optimizing the gradient increasing mode of light intensity. This solves the shortcomings of existing technologies where a single photoperiod cannot adapt to the entire culture process and sudden changes in light damage tissue culture seedlings. It also controls the low browning rate of explants and achieves synergistic optimization of growth at each stage.

[0013] This invention achieves three-dimensional synergistic optimization of photoperiod, LED composite spectrum and stage-adaptive culture medium formulation, and adjusts hormone and spectral parameters for varieties such as dwarf pink ixora and apricot yellow ixora to improve variety adaptability.

[0014] This invention optimizes the explant sterilization process and transplanting substrate formula, employing a combination of gradient sterilization and mechanical cleaning to control the explant contamination rate. The substrate is supplemented with well-rotted sheep manure and sterilized under high temperature and pressure, balancing nutrient supply and a sterile environment. This reduces contamination and loss rates in large-scale production, lowers production costs, and is simple to operate with strong controllability. It enables the unified large-scale cultivation of multiple varieties of Ixora chinensis, providing technical support for the industrial propagation, variety improvement, and germplasm resource preservation of Ixora chinensis. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the process of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] like Figure 1 As shown, a highly efficient method for cultivating Ixora tissue culture seedlings based on photoperiod regulation includes the following steps: Step S1, Explant Pretreatment: Select healthy, disease-free mother plants of Ixora and its varieties (such as dwarf pink Ixora and apricot yellow Ixora). In spring, from April to May (when the branches are moderately lignified and the axillary buds are plump), cut semi-lignified branches with terminal buds from the current year as explants. Remove excess leaves, leaving only the terminal bud and 2-3 axillary buds. Cut the branches into 2-3 cm long segments with stem nodes, ensuring that each segment contains one plump axillary bud. Rinse the explant segments under running water for 2-3 hours, gently rubbing them every 30 minutes to thoroughly remove surface dirt and lint. Then, transfer them to a clean bench and quickly immerse them in a 75% ethanol solution for 30 seconds (to kill surface bacteria). Rinse them 3-4 times with sterile water, then immerse them in a mixture of 0.1% mercuric chloride solution and 0.1% Tween-80 for 9-10 minutes, shaking them every 2 minutes to ensure uniform sterilization. Finally, rinse them 5-6 times with sterile water, each rinse lasting at least 1 minute. Blot the surface moisture with sterile filter paper to obtain sterile explants.

[0018] This sterilization process combines gradient sterilization with mechanically assisted cleaning, which, compared to the existing single immersion sterilization, can control the explant contamination rate to below 5% and reduce explant damage (damage rate below 3%). It is also more flexible in operation than the existing fixed sterilization time of 9.5 minutes and can be adapted to branches with different degrees of lignification.

[0019] Step S2, Primary Induction Culture: Sterile explants were vertically inoculated into the primary induction medium, 3-4 explants per bottle, avoiding contact between the explants and the edge of the medium (to reduce browning). The explants were then placed in a light incubator for cultivation. The cultivation conditions were set as follows: temperature (25±2)℃, relative humidity 70-75%, photoperiod 8h light / 16h dark, light period 8:00-16:00, and light intensity using a three-gradient increasing method: 50% of the set value (50-80 μmol / (m²·s)) for the first 3 days, increasing to 75% on days 4-5, and then to the set value from day 6 onwards. Cultivation was continued for 12-15 days until the axillary buds of the explants sprouted, reached 1-2 cm in length, and the leaves unfolded, thus obtaining induced buds.

[0020] The primary induction medium was MS as the base medium, supplemented with 4.0 mg / L 6-BA (to promote axillary bud germination), 1.0 mg / L NAA (to balance hormone levels), 1.0 mg / L LTDZ (to improve induction efficiency), 30 g / L sucrose (to provide a carbon source), and 7 g / L agar (to solidify the medium), with the pH adjusted to 5.8-6.0 (to suit the acidic / alkaline environment for Ixora chinensis growth). During the primary induction stage, Ixora chinensis is sensitive to light. A short photoperiod can reduce polyphenol oxidase activity, inhibit explant browning (browning rate controlled below 8%), and simultaneously promote endogenous cytokinin synthesis. This, combined with the optimized hormone combination, synergistically enhances induction efficiency, resulting in a bud germination rate exceeding 90%.

[0021] Step S3, Subculture and Proliferation: The robust induced buds obtained from the first generation were divided, with 1-2 buds retained in each segment. Diseased and weak buds were removed, and the segments were inoculated into subculture proliferation medium and placed in a light incubator for cultivation. The cultivation conditions were set as follows: temperature (25±2)℃, relative humidity 70-75%, photoperiod 16h light / 8h dark, light period from 6:00 to 22:00, light intensity 120-150μmol / (m²·s), and the light intensity gradient was the same as in step (2). After 25-30 days of cultivation, robust clustered buds with uniform growth, stem diameter of 0.3-0.5cm, and no excessive growth were obtained.

[0022] Subculture proliferation medium was based on MS medium, supplemented with 0.5 mg / L 6-BA (mildly promotes proliferation and avoids excessive growth), 0.5 mg / L NAA (regulates bud growth status), 30 g / L sucrose, and 7 g / L agar, with the pH adjusted to 5.8-6.0. The formula was optimized for different varieties: for dwarf pink Ixora, 0.2 mg / L GA3 (gibberellin) was added to promote bud elongation and prevent excessive dwarfing; for apricot yellow Ixora, the basic formula remained unchanged to ensure uniform bud color. Extending the photoperiod increased the accumulation of photosynthetic products (chlorophyll, soluble sugars), promoted the proliferation of clustered buds and stronger seedlings, increasing the proliferation coefficient by more than 35% compared to existing single-photoperiod proliferation media, and significantly improving bud robustness.

[0023] Step S4, Rooting Culture: Select vigorous single buds with a height of 2-3 cm and dark green leaves from the clustered buds, remove the aging tissue at the base, inoculate them into rooting medium, and place them in a light incubator for cultivation. The cultivation conditions are set as follows: temperature (23±2)℃ (slightly lower than the proliferation stage to promote root primordia formation), relative humidity 75-80%, photoperiod 10h light / 14h dark, light period from 9:00 to 19:00, light intensity 80-100 μmol / (m²·s), with the light intensity increasing in the same gradient as in step S2. Cultivate for 25-30 days, and obtain rooted tissue culture seedlings when the root system is well-developed, the root length is 2-3 cm, and the number of root branches is ≥5.

[0024] The rooting medium was based on 1 / 2 MS (to reduce salt concentration and minimize root burn), with the addition of 0.5 mg / L NAA (to induce root primordia formation), 20 g / L sucrose (to reduce carbon source concentration and promote rooting), and 7 g / L agar, and the pH was adjusted to 5.8-6.0. Hormone concentrations were adjusted for different varieties: for *Ixora chinensis*, the NAA concentration was adjusted to 0.6 mg / L to improve rooting stability; for *Ixora dwarfis*, the NAA concentration remained constant to ensure root system and plant shape coordination. Appropriately shortening the light duration can promote endogenous auxin synthesis and polar transport, inhibit abscisic acid accumulation, induce rapid root primordia formation, and simultaneously increase root branching and rooting ability.

[0025] Step S5, Seedling Acclimation and Domestication: The hardening-off process employs a three-stage gradient light pattern to gradually enhance the stress resistance of tissue culture seedlings: In the first stage, rooted tissue culture seedlings are moved to a greenhouse with the bottles still in their bottles. The greenhouse temperature is controlled at 20-28℃ and the humidity at 60-70%. The seedlings are hardened off for 3-5 days under conditions of 12 hours of light / 12 hours of darkness and 50-60% shading, allowing them to gradually adapt to the external light intensity and humidity. In the second stage, the bottle caps are opened, but the culture medium inside is retained. Hardening-off continues for 2-3 days, during which time the seedlings are sprayed 1-2 times daily (at 10 am and 4 pm) to maintain humidity, and ventilation is gradually increased (initially 1 hour / day, increasing by 1 hour each day). In the third stage, the shading is removed, and the light duration is extended to the natural photoperiod. Hardening-off continues for 2-3 days until the leaves of the tissue culture seedlings are dark green, the stems are robust, and the leaf cuticle has formed. The seedlings are then removed, the residual culture medium on the roots is washed off (to prevent mold), and they are transplanted into the pretreated substrate.

[0026] The transplanting substrate is a mixture of leaf mold, perlite, garden soil, and well-rotted sheep manure in a volume ratio of 2:1:1:0.5 (leaf mold provides nutrients, perlite enhances aeration, and well-rotted sheep manure supplements organic matter). Before transplanting, the mixture is sterilized at 121℃ under high pressure for 2 hours to kill pathogens and weed seeds. After transplanting, water thoroughly to settle the roots, and build a small arched greenhouse for warmth and moisture retention, controlling the humidity inside the greenhouse at 80-85%. After 3 days, gradually ventilate to reduce humidity (ventilate for 2-3 hours daily, gradually increasing to full-day ventilation). Place the seedlings in a shaded area of ​​the greenhouse for one week to allow them to recover. Afterward, manage them normally (water and fertilize regularly, and control pests and diseases). Gradient photoperiod hardening can prevent wilting of tissue culture seedlings caused by sudden changes in light, significantly improving the transplant survival rate.

[0027] In steps S2-S4, an LED composite light source is used, and the spectral type can be selected according to the culture stage: For primary induction and rooting culture, a red-blue combined spectrum (red light 650-660nm, blue light 450-460nm, wavelength ratio 3:1) is used to promote bud germination and root growth; for subculture proliferation, a red-blue-green composite spectrum (red, blue, and green wavelengths in a ratio of 3:1:0.5) is used to further improve photosynthetic efficiency and increase the accumulation of photosynthetic products. LED light sources are more than 30% more energy-efficient than traditional fluorescent lamps and generate less heat, maintaining a stable culture environment temperature (temperature fluctuation ≤ ±1℃), thus avoiding high-temperature damage to tissue culture seedlings.

[0028] Example 1: A highly efficient method for cultivating tissue culture seedlings of *Ixora chinensis* based on photoperiod regulation is described below: Step S1, Explant Pretreatment: In May, semi-lignified branches of common Ixora with terminal buds from the current year were selected, cut into 2.5cm segments with stem nodes, rinsed with running water for 2.5 hours (rubbing once every 30 minutes), soaked in 75% ethanol for 30 seconds in a clean bench, rinsed 4 times with sterile water, soaked in a mixture of 0.1% mercuric chloride and 0.1% Tween-80 for 9.5 minutes (shaking once every 2 minutes), rinsed 6 times with sterile water, and dried. The contamination rate of the sterile explants was 4.2%.

[0029] Step S2, Primary Induction Culture: Inoculated onto MS medium containing 4.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L LTDZ, 30 g / L sucrose, and 7 g / L agar (pH 5.9), cultured under the following conditions: 25°C, 72% humidity, 8h light / 16h dark (8:00-16:00), LED red-blue spectrum (3:1), light intensity 65 μmol / (m²·s), 32.5 μmol / (m²·s) for the first 3 days, 48.75 μmol / (m²·s) for days 4-5, and 65 μmol / (m²·s) from day 6 onwards. After 14 days of culture, the induced bud germination rate was 92.3%, and the browning rate was 7.8%.

[0030] Step S3, Subculture and Proliferation: The induced shoots were inoculated into MS medium (pH 5.9) containing 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7 g / L agar. The culture conditions were: 25℃, 72% humidity, 16 h light / 8 h dark (6:00-22:00), LED red-blue-green composite spectrum (3:1:0.5), light intensity 135 μmol / (m²·s), and gradient increase method as in step 2. After 28 days of culture, the proliferation coefficient of the clustered shoots was 4.6, the shoot stem diameter was 0.42 cm, and there was no etiolation.

[0031] Step S4, Rooting Culture: Healthy single shoots (2.5 cm in diameter) were selected and inoculated into 1 / 2 MS medium (pH 5.9) containing 0.5 mg / L NAA, 20 g / L sucrose, and 7 g / L agar. Culture conditions were: 23℃, 78% humidity, 10 h light / 14 h dark (9:00-19:00), LED red-blue spectrum (3:1), light intensity 90 μmol / (m²·s), and gradient increase method as in step 2. After 28 days of culture, the rooting rate was 93.5%, with an average of 6.8 roots per shoot and a root length of 2.5 cm.

[0032] Step S5, Seedling Acclimation and Domestication: The seedlings were moved to a greenhouse (25℃, 65% humidity) with bottles. In the first stage, the seedlings were hardened off for 4 days with 50% shade and 12 hours of light / 12 hours of darkness. In the second stage, the seedlings were hardened off with the lids off for 3 days, with spraying twice a day and gradually increasing ventilation to 3 hours / day. In the third stage, the shade was removed and the seedlings were hardened off with natural light for 3 days. They were then transplanted to a substrate of leaf mold: perlite: garden soil: well-rotted sheep manure = 2:1:1:0.5 (sterilized at 121℃ for 2 hours) and a small arched shed was built (82% humidity). After 1 week of acclimatization, the survival rate was 88.6%, and the total cultivation period was 70 days.

[0033] Example 2: A highly efficient method for cultivating tissue culture seedlings of Ixora dwarfis based on photoperiod regulation, the specific steps of which are as follows: Step S1, Explant Pretreatment: In April, select semi-lignified branches of the current year with terminal buds of Ixora chinensis, cut them into 2.2cm segments with stem nodes, rinse with running water for 2 hours (rubbing once every 30 minutes), soak in 75% ethanol for 30 seconds in a clean bench, rinse 3 times with sterile water, soak in a mixture of 0.1% mercuric chloride and 0.1% Tween-80 for 9 minutes (shaking once every 2 minutes), rinse 5 times with sterile water, and dry. The contamination rate of sterile explants is 4.8%.

[0034] Step S2, Primary Induction Culture: The samples were inoculated into MS medium containing 4.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L LTDZ, 30 g / L sucrose, and 7 g / L agar (pH 5.8). Culture conditions: 24℃, 73% humidity, 8h light / 16h dark (8:00-16:00), LED red-blue spectrum (3:1), light intensity 55 μmol / (m²·s), 27.5 μmol / (m²·s) for the first 3 days, 41.25 μmol / (m²·s) for days 4-5, and 55 μmol / (m²·s) from day 6 onwards. After 13 days of culture, the induced bud germination rate was 91.5%, and the browning rate was 7.2%.

[0035] Step S3, Subculture and Proliferation: The induced shoots were inoculated into MS medium (pH 5.8) containing 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.2 mg / L GA3, 30 g / L sucrose, and 7 g / L agar. The culture conditions were: 24℃, 73% humidity, 16 h light / 8 h dark (6:00-22:00), LED red-blue-green composite spectrum (3:1:0.5), light intensity 125 μmol / (m²·s), and gradient increase method as in step 2. After 26 days of culture, the proliferation coefficient of the clustered shoots was 4.5, the shoot stem diameter was 0.38 cm, the shoots were uniformly elongated, and there was no excessive dwarfing.

[0036] Step S4, Rooting Culture: Healthy single shoots (2.2 cm in diameter) were selected and inoculated into 1 / 2 MS medium (pH 5.8) containing 0.5 mg / L NAA, 20 g / L sucrose, and 7 g / L agar. Culture conditions were: 22℃, 77% humidity, 10 h light / 14 h dark (9:00-19:00), LED red-blue spectrum (3:1), light intensity 85 μmol / (m²·s), and gradient increase method as in step 2. After 26 days of culture, the rooting rate was 92.1%, with an average of 6.3 roots per plant and a root length of 2.2 cm. The root system and plant type were well-coordinated.

[0037] Step S5, Seedling Acclimation and Domestication: The seedlings were moved to a greenhouse (24℃, 63% humidity) with bottles. In the first stage, the seedlings were hardened off for 3 days with 55% shade and 12 hours of light / 12 hours of darkness. In the second stage, the seedlings were hardened off with the lids off for 2 days, with spraying twice a day and gradually increasing ventilation to 2 hours / day. In the third stage, the shade was removed and the seedlings were hardened off with natural light for 2 days. They were then transplanted to a substrate of leaf mold: perlite: garden soil: well-rotted sheep manure = 2:1:1:0.5 (sterilized at 121℃ for 2 hours) and a small arched shed was built (83% humidity). After 1 week of acclimatization, the survival rate was 88.2%. The total cultivation period was 67 days. The plants were short and sturdy, which was consistent with the characteristics of the variety.

[0038] Example 3: A highly efficient method for cultivating tissue culture seedlings of *Ixora chinensis* based on photoperiod regulation is described below: Step S1, Explant Pretreatment: In May, semi-lignified branches of *Ixora chinensis* with terminal buds from the current year were selected, cut into 2.8cm segments with stem nodes, rinsed with running water for 3 hours (rubbing once every 30 minutes), soaked in 75% ethanol for 30 seconds in a clean bench, rinsed 4 times with sterile water, soaked in a mixture of 0.1% mercuric chloride and 0.1% Tween-80 for 10 minutes (shaking once every 2 minutes), rinsed 6 times with sterile water, and dried. The contamination rate of the sterile explants was 3.9%.

[0039] Step S2, Primary Induction Culture: Inoculated onto MS medium containing 4.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L LTDZ, 30 g / L sucrose, and 7 g / L agar (pH 6.0), cultured under the following conditions: 26℃, 71% humidity, 8h light / 16h dark (8:00-16:00), LED red-blue spectrum (3:1), light intensity 75 μmol / (m²·s), 37.5 μmol / (m²·s) for the first 3 days, 56.25 μmol / (m²·s) for days 4-5, and 75 μmol / (m²·s) from day 6 onwards. After 15 days of culture, the induced bud germination rate was 92.8%, the browning rate was 8.1%, and the buds had uniform color.

[0040] Step S3, Subculture and Proliferation: The induced shoots were inoculated into MS medium (pH 6.0) containing 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7 g / L agar. The culture conditions were: 26℃, 71% humidity, 16 h light / 8 h dark (6:00-22:00), LED red-blue-green composite spectrum (3:1:0.5), light intensity 145 μmol / (m²·s), and gradient increase method as in step 2. After 29 days of culture, the proliferation coefficient of the clustered shoots was 4.7, the shoot stem diameter was 0.45 cm, there was no etiolation, and the color was golden yellow.

[0041] Step S4, Rooting Culture: Healthy single shoots (2.8 cm in diameter) were selected and inoculated into 1 / 2 MS medium (pH 6.0) containing 0.6 mg / L NAA, 20 g / L sucrose, and 7 g / L agar. The culture conditions were: 24℃, 79% humidity, 10 h light / 14 h dark (9:00-19:00), LED red-blue spectrum (3:1), light intensity 95 μmol / (m²·s), and gradient increase method as in step 2. After 29 days of culture, the rooting rate was 94.2%, with an average of 7.1 roots per shoot and a root length of 2.8 cm. The rooting stability was strong.

[0042] Step S5, Seedling Acclimation and Domestication: The seedlings were moved to a greenhouse (26℃, 67% humidity) with bottles. In the first stage, the seedlings were hardened off for 5 days with 60% shade and 12 hours of light / 12 hours of darkness. In the second stage, the seedlings were hardened off with the lids off for 3 days, with spraying twice a day and gradually increasing ventilation to 3 hours / day. In the third stage, the shade was removed and the seedlings were hardened off with natural light for 3 days. They were then transplanted to a substrate of leaf mold: perlite: garden soil: well-rotted sheep manure = 2:1:1:0.5 (sterilized at 121℃ for 2 hours) and a small arched shed was built (84% humidity). After 1 week of acclimatization, the survival rate was 89.1%. The total cultivation period was 72 days. The bud and flower colors maintained the excellent characteristics of the mother plant.

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-efficiency tissue culture seedling cultivation method based on photoperiod regulation of Impatiens walleriana, characterized in that, The method comprises the following steps: Step S1, pretreatment of explants; Step S2, primary induction culture: The aseptic explants are inoculated into the primary induction culture medium and cultured in an illumination incubator, the culture temperature is 23-28°C, the relative humidity is 70-75%, the light period is set to 8h light / 16h darkness, the light intensity is 50-80μmol / (m²·s), and the culture is carried out for 12-15 days to obtain induced sprouts; the primary induction culture medium is a MS-based medium added with 4.0mg / L 6-BA, 1.0mg / L NAA, 1.0mg / L TDZ, 30g / L sucrose and 7g / L agar, and the pH is adjusted to 5.8-6.0; Step S3, subculture propagation culture: The induced sprouts obtained in step S2 are inoculated into the subculture propagation medium after being cut, the culture temperature is 23-28°C, the relative humidity is 70-75%, the light period is set to 16h light / 8h darkness, the light intensity is 120-150μmol / (m²·s), and the culture is carried out for 25-30 days to obtain multiple shoots; the subculture propagation medium is a MS-based medium added with 0.5mg / L 6-BA, 0.5mg / L NAA, 30g / L sucrose and 7g / L agar, and the pH is adjusted to 5.8-6.0; Step S4, rooting culture; Step S5, seedling acclimatization.

2. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 1, characterized in that, The step S1, pretreatment of explants: Healthy branches of the current year with semi-lignified and top buds of the dragon boat flower or its varieties are selected as the explants, the leaves are removed, and the stems are cut into 2-3cm small segments with stem nodes, and the aseptic explants are obtained through aseptic treatment; The aseptic treatment specifically comprises: The explants are first washed with running water for 2-3h, and gently rubbed once every 30min, then soaked in a 75% ethanol solution for 30s, washed with sterile water for 3-4 times, then soaked in a 0.1% mercury chloride solution + 0.1% Tween-80 mixed solution for 9-10min, shaken once every 2min during the process, washed with sterile water for 5-6 times, and the surface water is absorbed with sterile filter paper; the varieties of the dragon boat flower include dwarf powder dragon boat flower and apricot yellow dragon boat flower.

3. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 2, characterized in that, In the step S4, rooting culture, the specific steps are as follows: Healthy multiple shoots with a height of 2-3cm are selected and inoculated into the rooting culture medium, the culture temperature is 21-25°C, the relative humidity is 75-80%, the light period is set to 10h light / 14h darkness, the light intensity is 80-100μmol / (m²·s), and the culture is carried out for 25-30 days to obtain rooted tissue culture seedlings; the rooting culture medium is a 1 / 2MS-based medium added with 0.5mg / L NAA, 20g / L sucrose and 7g / L agar, and the pH is adjusted to 5.8-6.

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4. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 3, characterized in that, In the step S5, seedling acclimatization, the specific steps are as follows: The rooting tissue culture seedlings are moved to a greenhouse, first acclimated for 3-5 days under 12h light / 12h darkness, shading degree 50-60%, then gradually increase the light duration to 14h light / 10h darkness, shading degree 30-40%, acclimated for 2-3 days, finally transition to natural light period, remove shading, maintain greenhouse temperature 20-28℃, humidity 60-70%, total acclimation 7-10 days, then transplant.

5. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 4, characterized in that, The LED composite light source is used in the steps S2-S4, and the light source is a red and blue combined spectrum or a red, blue and green composite spectrum; the wavelength ratio of red light to blue light in the red and blue combined spectrum is 3:1; the wavelength ratio of red light to blue light to green light in the red, blue and green composite spectrum is 3:1:0.

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6. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 5, characterized in that, The transplanting substrate in the step S5 is mixed leaf soil, perlite, garden soil and decomposed sheep manure at a volume ratio of 2:1:1:0.5, sterilized at 121℃ for 2h before transplanting, and a small arched shed is built for heat preservation and humidity after transplanting, the shed humidity is controlled at 80-85%, and ventilation and humidity reduction are gradually performed after 3 days.

7. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 6, characterized in that, The daily light period in the steps S2-S4 is 8:00-16:00, 6:00-22:00 or 9:00-19:00, and the light intensity is increased in a gradient mode, 50% of the set value for the first 3 days, increased to 75% of the set value for the 4th-5th day, and increased to the set value from the 6th day to avoid damage to the tissue culture seedlings caused by sudden change of light.

8. The efficient tissue culture seedling cultivation method of Impatiens walleriana based on photoperiod regulation according to claim 7, characterized in that, If the culture object in the step S3 is dwarfed Amaryllis, 0.2mg / L GA3 is added to the subculture and proliferation medium to promote bud elongation and prevent excessive dwarfing.

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