Method for brown stain prevention and rapid propagation of Xanthoceras sorbifolia Bunge explants

Abstract

The invention discloses a shiny-leaved yellowhorn explant browning prevention and rapid propagation method which comprises the following steps: cutting an explant from a shiny-leaved yellowhorn stem section, and refrigerating the explant; the refrigerated explants are disinfected; inoculating the disinfected explants in a pretreatment culture medium, performing pretreatment culture under a dark condition, and sequentially transferring the pretreated and cultured explants to a callus induction culture medium, an adventitious bud induction culture medium and a rooting culture medium for culture until a complete plant is obtained. The method provided by the invention realizes efficient induction of callus, adventitious buds and roots while reducing the browning rate of the explants, thereby greatly improving the success rate and regeneration efficiency of vegetative propagation of xanthoceras sorbifolia bunge.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant tissue culture technology, and particularly relates to a method for preventing browning of Xanthoceras sorbifolia Bunge explants and rapid propagation. BACKGROUND

[0002] Xanthoceras sorbifolia Bunge, the scientific name of which is: Xanthoceras sorbifolium Xanthoceras sorbifolia Bunge, also known as Xanthoceras sorbifolia Bunge, Xanthoceras sorbifolia Bunge, Xanthoceras sorbifolia Bunge, and Xanthoceras sorbifolia Bunge, is a deciduous shrub or small tree belonging to the family Sapindaceae. It is distributed in the Ningxia Hui Autonomous Region and Gansu in the west, Liaoning in the northeast, Inner Mongolia Autonomous Region in the north, and Henan in the south. It is also distributed in Hebei, Shandong, Shanxi, and Shaanxi. Xanthoceras sorbifolia Bunge mainly grows in barren slopes, valleys, and hilly areas, and prefers sunlight. It has strong adaptability to drought, poor soil, salt-alkali, and cold. It is a unique woody oil tree species in the north.

[0003] Xanthoceras sorbifolia Bunge has attracted much attention due to its significant economic and medicinal value. The kernel of Xanthoceras sorbifolia Bunge contains up to 55% to 66% oil, making it an important source of high-quality edible oil. In addition, the kernel of Xanthoceras sorbifolia Bunge is rich in essential fatty acids such as linoleic acid, linolenic acid, and eicosanoic acid, which cannot be synthesized by the human body. These fatty acids are of great significance in improving skin health and preventing hair loss. Therefore, Xanthoceras sorbifolia Bunge oil has broad application potential in the fields of health food and medicine.

[0004] At the same time, Xanthoceras sorbifolia Bunge has excellent characteristics such as drought tolerance, cold tolerance, poor soil tolerance, and salt-alkali resistance, and has important application value in the treatment of desertification, ecological greening, soil and water conservation, and development of saline-alkali land in the north of China. However, although Xanthoceras sorbifolia Bunge has broad application prospects, the development of its tissue culture technology has been limited by various factors.

[0005] During the tissue culture process of Xanthoceras sorbifolia Bunge, explants are prone to browning due to the accumulation of phenolic compounds. Since mechanical damage exacerbates the oxidation of phenolic compounds, browning is particularly severe during the induction of callus from explants, which affects the induction of callus and the formation of adventitious buds, thereby severely restricting the application and promotion of rapid propagation technology. The browning phenomenon is mainly due to the reaction between amino compounds and reducing sugars during the culture process, resulting in the formation of brown polymers, which causes the discoloration and even death of the culture. Therefore, inhibiting the browning of explants is one of the key problems to be solved in the tissue culture of Xanthoceras sorbifolia Bunge.

[0006] Currently, in order to alleviate the problem of explant browning, the usual measures are to pretreat the explants. Antioxidants such as vitamin C, citric acid, and activated carbon are usually added to the pretreatment medium to prevent browning. However, these methods have limited effect in preventing browning, and the pretreated explants have the problem of unsatisfactory induction rate when used in plant tissue culture. SUMMARY

[0007] To solve the above problems, the present application provides a method for preventing browning and rapid propagation of Xanthoceras sorbifolia Bunge explants.

[0008] The present application is realized by the following technical solutions: A method for preventing browning and rapid propagation of Xanthoceras sorbifolia Bunge explants, comprising the following steps: An explant is cut from a stem segment of Xanthoceras sorbifolia Bunge, and the explant is refrigerated. The refrigerated explant is sterilized.

[0009] The sterilized explant is inoculated into a pretreatment medium and pretreated in the dark; the formula of the pretreatment medium is 5.5-7.5 g / L agar, 10-15 g / L sucrose, and 1 L water.

[0010] The pretreated explant is sequentially transferred to a callus induction medium, an adventitious bud induction medium, and a rooting medium for culture until a complete plant is obtained.

[0011] The callus induction medium is based on 1 / 2MS medium and added with auxin analogue 2,4-D and auxin analogue NAA; in the callus induction medium, the mass concentration of 2,4-D is 1.0-1.2 mg / L, and the mass concentration ratio of 2,4-D to NAA is 25-50:1.

[0012] The adventitious bud induction medium is based on MS medium and added with cytokinin analogue TDZ, cytokinin analogue 6-BA, and auxin analogue NAA; in the adventitious bud induction medium, the mass concentration of 6-BA is 1.0-1.4 mg / L, and the mass concentration ratio of 6-BA+TDZ to NAA is 5.5-7.5:1.

[0013] The rooting medium is based on 1 / 2MS medium and added with IBA; in the rooting medium, the mass concentration of IBA is 0.8-1.2 mg / L.

[0014] Preferably, the pH of the pretreatment medium is 6.0-7.0; the pH of the callus induction medium is 6.0-7.0; the pH of the adventitious bud induction medium is 6.5-7.0; and the pH of the rooting medium is 6.5-7.0.

[0015] Preferably, the temperature during refrigeration is 4-6°C, and the refrigeration time is 10-14 h.

[0016] Preferably, the stem segment of Xanthoceras sorbifolia Bunge is cut from a tender stem of a Xanthoceras sorbifolia Bunge seedling, and the length of the cut explant is 2.5-3.5 cm.

[0017] Preferably, the pre-culturing is carried out at a constant temperature of 24-26°C, and the pre-culturing time is 3-5 days.

[0018] Preferably, the callus induction culture is carried out using a callus induction medium, and the culture is carried out in the dark at a temperature of 23-27°C for 7-12 days.

[0019] Preferably, the adventitious bud induction culture is carried out using an adventitious bud induction medium, and the culture is carried out under a light cycle of 14-16 hours of light followed by 8-10 hours of darkness at a light intensity of 2200-2800 lux at a temperature of 24-26°C for 7-12 days.

[0020] Preferably, the rooting culture is carried out using a rooting medium, and the culture is carried out under a light cycle of 14-16 hours of light followed by 8-10 hours of darkness at a light intensity of 1300-1600 lux at a temperature of 24-26°C for 7-12 days.

[0021] Preferably, the method for disinfecting the explant is as follows: The explant is washed with Tween 20 for 1 hour, and then soaked in a 0.1% sodium hypochlorite solution, and after the soaking is completed, the explant is soaked in an ethanol solution with an ethanol volume fraction of 75%.

[0022] Preferably, the cut surface of the disinfected explant is treated for sealing, and then inoculated into the pre-culturing medium.

[0023] Preferably, the method for treating the cut surface of the disinfected explant for sealing is as follows: a) The cut surface of the disinfected explant is dipped into a 2.5wt% sodium alginate solution, and the M / G ratio of the sodium alginate is less than 1.

[0024] b) The cut surface of the explant is placed in a 100mM CaCl2 solution for reaction, and a calcium alginate gel layer is formed on the cut surface of the explant.

[0025] c) After the pre-culturing is completed, the explant is washed with sterile water, and the calcium alginate gel layer on the cut surface of the explant is removed.

[0026] The inventive concept of the present application is as follows: After the explant is taken, cold storage treatment is first carried out, which is advantageous for inhibiting the activity of various oxidative enzymes in the explant, reducing the metabolism of the explant, and preventing the explant from synthesizing and secreting a large amount of phenolic substances, which are oxidized to cause browning of the explant. If the temperature of the cold storage is too high or the time is too short, the explant cannot be effectively cooled, and the anti-browning effect is poor. If the temperature of the cold storage is too low or the time is too long, the explant is easily damaged by cold, which affects the subsequent callus induction culture process, and even causes the explant to die.

[0027] In the pre-culture stage, the explants are adapted to the culture conditions in a hormone-free environment, reducing the stress response to the wound and thus reducing the amount of phenolic substances released. Experiments have shown that the browning rate of explants placed in light conditions is much higher than that of explants cultured in the dark, because light can promote the synthesis of phenolic compounds, and dark conditions are conducive to reducing the browning rate.

[0028] "Green stems" refer to stem segments in seedlings that have not formed secondary tissues. These stem segments contain epidermis, cortex and primary vascular bundles, but no cambium. The content of phenolic substances in green stems is usually less than that in mature tissues, thus reducing the likelihood of being oxidized to quinones during culture and reducing the risk of browning. Green stem cells have strong division and callus capacity, and the wound on the explant is more likely to heal, reducing the release of phenolic substances and further reducing the likelihood of browning. In addition, the cell walls of green stem tissues are thin, the degree of lignification is low, and the metabolic activity is high, all of which make them more adaptable to the culture environment in tissue culture, reducing the stress response and thus reducing the incidence of browning.

[0029] When disinfecting the explants, 2-3 drops of Tween 20 are added to 500 mL of water, and then the explants are rinsed with the Tween 20-containing water flow for 1 h; after rinsing, the explants are transferred to a clean bench and soaked in 0.1% sodium hypochlorite solution under sterile conditions for 4 min, and the container containing the sodium hypochlorite solution and the explants is constantly shaken during the soaking process; after the soaking is completed, the explants are rinsed with sterile water for 4-5 times to wash off the sodium hypochlorite solution on the surface of the explants; then the explants are soaked in a 75% ethanol solution for 30 s; finally, the explants are rinsed with sterile water for 4-5 times to wash off the 75% ethanol solution on the surface of the explants.

[0030] The calcium alginate gel layer can prevent the exudation of phenolic substances from the wound, and by reasonably selecting the type and concentration of sodium alginate used to prepare the calcium alginate gel layer, the calcium alginate gel layer can be easily peeled off by hand. After the pre-culture is completed, the calcium alginate gel layer is removed manually, and the incision is exposed, which is conducive to ensuring the normal progress of callus induction culture. Through the linkage of incision sealing, pre-treatment, and hormone induction, the present application simultaneously inhibits the exudation and oxidation of phenolic substances from three aspects of physical inhibition, physiological regulation, and hormone regulation, effectively reducing the browning rate while ensuring a high induction rate during the callus induction culture and adventitious bud induction culture stages.

[0031] Compared with subculturing the callus, directly inducing adventitious buds from the callus can reduce the genetic and phenotypic variations that may occur during long-term culture of the callus, maintaining the genetic stability of the regenerated plants. By omitting the subculture step and directly inducing adventitious buds from the callus, the process of plant regeneration can be accelerated.

[0032] In the callus induction culture stage, although 2,4-D and 6-BA in the prior art belong to auxin and cytokinin plant hormones respectively, 2,4-D promotes cell division and expansion by activating auxin response factors and Aux / IAA proteins, promotes cell dedifferentiation to form callus, and the callus induction effect is better for woody plants and part of monocotyledonous plants. While 6-BA mainly promotes cell division and bud differentiation, and has weak effect on callus induction, and when used alone, it can only stimulate bud proliferation, and cannot effectively induce undifferentiated cell groups to form callus. In addition, 2,4-D has the effect of activating antioxidant enzymes in explants, and the ratio of 2,4-D and NAA will affect the browning rate. When the concentration ratio of cytokinin analogues and auxin analogues is higher or lower than this ratio in the adventitious bud induction culture stage, the induction rate of adventitious buds will decrease.

[0033] Compared with the prior art, the present application has the following beneficial effects: The present application provides a method for preventing browning and rapid propagation of Xanthoceras sorbifolia Bunge explants, comprising the following steps: cutting explants from Xanthoceras sorbifolia Bunge stem segments, and cold storing the explants; disinfecting the cold-stored explants; inoculating the disinfected explants in a pretreatment medium, and performing pretreatment culture in the dark; the formula of the pretreatment medium is: 5.5g / L-7.5g / L agar, 10g / L-15g / L sucrose and 1L water; transferring the pretreatment cultured explants to callus induction medium, adventitious bud induction medium and rooting medium in turn for culture until complete plants are obtained; the callus induction medium is based on 1 / 2MS medium and adds 2,4-D and NAA; in the callus induction medium, the mass concentration of 2,4-D is greater than or equal to 1.0mg / L, and the mass concentration ratio of 2,4-D and NAA is 25-50:1; the adventitious bud induction medium is based on MS medium and adds TDZ, 6-BA and NAA; in the adventitious bud induction medium, the mass concentration of 6-BA is 1.0mg / L-1.4mg / L, and the mass concentration ratio of 6-BA+TDZ:NAA is 5.5-7.5:1; the rooting medium is based on 1 / 2MS medium and adds IBA; in the rooting medium, the mass concentration of IBA is 0.8mg / L-1.2mg / L.

[0034] 1、The present application selects the explant with better growth state, without obvious aging or lignification, to ensure that the explant has high division and callus capacity, thereby laying a foundation for subsequent induction culture. By low-temperature refrigeration and pretreatment culture of the explant, the cells are in a relatively "dormant" or "adapted" state, which reduces the activity of polyphenol oxidase in the explant, reduces the excretion and oxidation of phenolic substances, and plays a role in preventing browning. In addition, by controlling the addition of only auxin analogs 2,4-D and NAA during the callus induction stage, and controlling the concentration ratio of 2,4-D and NAA in the range of 20-50:1, and the concentration of 2,4-D being greater than or equal to 1.0 mg / L, on the basis of ensuring the callus induction rate, the activation of 2,4-D against oxidase is utilized to effectively prevent the browning phenomenon of the explant during the differentiation into callus. In addition, the low-temperature refrigeration and pretreatment culture method in the present application not only improves the initial physiological state of the explant, but also enables the explant and the callus formed by dedifferentiation of the explant to maintain good vitality at all times, thereby promoting the improvement of the adventitious bud induction rate. On this basis, during the induction of adventitious buds, the present application adopts the combined application of two cell division factors and one auxin, and by reasonably adjusting the concentration and proportion of the cell division factor analog 6-BA and TDZ and the auxin analog NAA, hormone balance is achieved, so that the cells rapidly and massively form adventitious buds during the division and differentiation process, and finally the dual beneficial effects of preventing the browning of Xanthoceras sorbifolia Bunge explants and rapid propagation are achieved.

[0035] 2、The present application also provides a method for physically sealing the incision of the explant, which utilizes the dense semi-transparent network of calcium alginate gel formed at the incision to initially block the excretion of phenols and PPO, effectively preventing the browning of the explant. Moreover, the calcium alginate gel is easy to remove after the end of the pretreatment culture stage, which can ensure that the callus induction culture, adventitious bud induction culture and rooting culture stages are not affected. In addition, in the present application, by optimizing the temperature and photoperiod conditions of the culture room, not only the normal metabolism of the cells is promoted, but also the oxidative stress caused by light is reduced to a certain extent, which further assists in reducing browning. The combined effect of various technical measures enables the explant to maintain high cell activity and reduce the generation of oxidized browning substances during callus induction and subsequent adventitious bud differentiation, and finally achieves high induction rate and rapid propagation effect.

[0036] 3、The method for preventing browning and rapid propagation of Xanthoceras sorbifolia Bunge explants provided by the present application can reduce the incidence of explant browning during tissue culture of Xanthoceras sorbifolia Bunge, and improve the induction rate of callus and adventitious buds, thereby improving the propagation efficiency of Xanthoceras sorbifolia Bunge during rapid propagation by tissue culture. Experiments show that by using the pretreatment method in the present embodiment, the browning rate of the explant can be reduced to 6% or even lower, which is much lower than the explant browning rate of more than 20% during conventional Xanthoceras sorbifolia Bunge tissue culture. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0038] Figure 1 The photo of part of callus obtained in Example 2 of the present application.

[0039] Figure 2 The photo of part of callus obtained in Comparative Example 1 of the present application. Figure 2 In the figure, A is the photo of part of callus obtained in Comparative Example 3, B is the photo of part of callus obtained in Comparative Example 2, and C is the photo of part of callus obtained in Comparative Example 1.

[0040] Figure 3 The photo of part of adventitious bud tissue obtained in Group 3-3 of Experimental Example 3 of the present application. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present application, the present application will be described more fully below, and preferred embodiments of the present application will be given. However, the present application can be realized in many different forms and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used in the present application have the same meanings as those commonly understood by those skilled in the art to which the present application belongs. The terms used in the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0043] The beneficial effects of the present application will be described below through specific embodiments.

[0044] In the present application, 6-BA represents 6-benzyladenine, TDZ represents thidiazuron, 2,4-D represents 2,4-dichlorophenoxyacetic acid, NAA represents naphthaleneacetic acid, and IBA represents indole-3-butyric acid.

[0045] Example 1 In this embodiment, a method for preventing browning and rapid propagation of Xanthoceras sorbifolia Bunge explants is provided. The method comprises the following steps: 1. Selection and pretreatment of explants Five-year-old Xanthoceras sorbifolia Bunge seeds, which were grown under the same conditions and in the same year as the Xanthoceras sorbifolia Bunge seed bank in Kundu Forest Farm, A'erkaimin Banner, Chifeng City, were selected for planting. After the seeds grew into seedlings, the tender stems of the seedlings were taken. The tender stems contained epidermis, cortex and primary vascular bundle, but did not contain cambium.

[0046] A 2.5 cm section was cut from the tender stem of the seedling as an explant. The length of the explant cut in this example was 3 cm. The obtained explant was refrigerated at 4°C for 10 h, and then the explant was sterilized.

[0047] When the explant was sterilized, 3 drops of Tween 20 were added to 500 mL of water, and then the explant was washed with the water flow added with Tween 20 for 1 h. After washing, the explant was transferred to a clean bench and immersed in a 0.1% sodium hypochlorite solution under sterile conditions for 4 min. During the immersion process, the container containing the sodium hypochlorite solution and the explant was constantly shaken. After the end of the immersion, the explant was washed with sterile water for 5 times to wash the sodium hypochlorite solution on the surface of the explant. Then the explant was immersed in a 75% ethanol solution for 30 s with shaking. Finally, the explant was washed with sterile water for 5 times to wash the 75% ethanol solution on the surface of the explant.

[0048] The sterilized explant was inoculated into a pretreatment medium and cultured at 24°C in the dark for 3 d. When the pretreatment medium was prepared, only 5.5 g / L agar and 10 g / L sucrose were added, and the pH was adjusted to 6.0.

[0049] 2. Callus induction The pretreated explant was transferred to a callus induction medium for callus induction culture. The callus induction medium used in this example was based on 1 / 2MS medium, and 1.0 mg / L 2,4-D and 0.04 mg / L NAA were added, and contained 6.5 g / L agar. The preparation method of 1 / 2MS medium is a conventional method in the prior art. After the callus induction medium was prepared, the pH was adjusted to 6.5, and after sterilization, it was poured into a plate.

[0050] When the callus induction culture was carried out, the culture was carried out in the dark, the culture temperature was 23°C, and the culture time was 12 d. After the culture was completed, the explant was dedifferentiated and callus was formed.

[0051] 3. Adventitious bud induction The callus was transferred to the adventitious bud induction medium under aseptic conditions for the adventitious bud induction culture. The adventitious bud induction medium used in this embodiment was based on MS medium and added with 0.1 mg / L TDZ, 1.0 mg / L 6-BA and 0.2 mg / L NAA, and contained 6.5 g / L agar. The preparation method of the MS medium was a conventional method in the prior art. After the adventitious bud induction medium was prepared, the pH was adjusted to 6.5, and then the medium was sterilized and poured into a plate to obtain the adventitious bud induction medium.

[0052] During the adventitious bud induction culture, the culture was performed under the artificial controlled photoperiod culture condition, the light intensity was 2200 lx, the photoperiod was 14 h light followed by 8 h dark, the culture temperature was 24°C, and the culture time was 12 d.

[0053] 4. Rooting culture The induced adventitious buds were transferred to the rooting medium for the rooting culture. The rooting medium used in this embodiment was based on 1 / 2MS medium and added with 0.80 mg / L IBA, and contained 6.5 g / L agar. The preparation method of the 1 / 2MS medium was a conventional method in the prior art. After the rooting medium was prepared, the pH was adjusted to 6.5, and then the medium was sterilized and poured into a plate to obtain the rooting medium.

[0054] During the rooting culture, the culture was performed under the artificial controlled photoperiod culture condition, the light intensity was 1300 lx, the photoperiod was 14 h light followed by 8 h dark, the culture temperature was 24°C, and the culture time was 12 d.

[0055] After the rooting culture, the complete plant was obtained.

[0056] The browning of the explants generally occurred in the callus induction stage. The browning rate of the explants before the callus induction stage in this embodiment was counted. The calculation formula was: browning rate = (number of browning explants / total number of explants) x 100%. In this embodiment, the browning rate of the explants was 6.17%.

[0057] In addition, the induction rate of the callus induction culture, the induction rate of the adventitious bud induction culture and the induction rate during the rooting culture in this embodiment were counted. The calculation formula of the callus induction rate was: number of explants differentiated into callus / total number of explants inoculated into the callus induction medium x 100%. The induction rate of the adventitious bud induction culture and the induction rate during the rooting culture were calculated in the same way. The final counting result was that the induction rate of the callus induction culture in this embodiment was 62.73%, the induction rate of the adventitious bud was (83.33 ± 5.77)%, and the induction rate during the rooting culture was 90%.

[0058] Example 2 The present embodiment provides a method for preventing browning of Xanthoceras sorbifolia Bunge explants and rapid propagation. The method comprises the following steps. 1. Selection and pretreatment of explants Seeds of Xanthoceras sorbifolia Bunge grown under the same conditions and in the same year as those in the Xanthoceras sorbifolia Bunge germplasm resource bank of Kundu Forest Farm, Arhorqin Banner, Chifeng City were selected and planted. After the seeds grew into seedlings, the tender stems of the seedlings were taken. The tender stems contained epidermis, cortex and primary vascular bundle, but did not contain cambium.

[0059] A 3 cm section was cut from the tender stems of the seedlings as the explants. The length of the explants cut in the present embodiment was 3 cm. The obtained explants were stored at 5°C for 12 h, and then the explants were sterilized.

[0060] When the explants were sterilized, 3 drops of Tween 20 were added to 500 mL of water, and then the explants were washed with the water flow added with Tween 20 for 1 h. After washing, the explants were transferred to a clean bench, and then the explants were immersed in 0.1% sodium hypochlorite solution under sterile conditions for 4 min. During the immersion, the container containing the sodium hypochlorite solution and the explants was continuously shaken. After the immersion, the explants were washed with sterile water for 5 times to wash the sodium hypochlorite solution on the surface of the explants. Then the explants were immersed in an ethanol solution with a volume fraction of 75% for 30 s. Finally, the explants were washed with sterile water for 5 times to wash the 75% ethanol solution on the surface of the explants.

[0061] The sterilized explants were inoculated into a pretreatment medium and cultured at 25°C in the dark for 4 d. When the pretreatment medium was prepared, only 6.5 g / L of agar and 12.5 g / L of sucrose were added, and the pH was adjusted to 6.5.

[0062] 2. Callus induction The pretreated explants were transferred to a callus induction medium for callus induction culture. The callus induction medium used in the present embodiment was based on 1 / 2MS medium, and 1.20 mg / L of 2,4-D and 0.04 mg / L of NAA were added, and contained 6.5 g / L of agar. The preparation method of 1 / 2MS medium was a conventional method in the prior art. After the callus induction medium was prepared, the pH was adjusted to 6.7, and then the medium was poured into a plate after sterilization.

[0063] During the callus induction culture, the culture was carried out in the dark, the culture temperature was 25°C, and the culture time was 10 d. After the culture, the explants were dedifferentiated and callus was formed.

[0064] 3. Adventitious bud induction The callus was transferred to adventitious bud induction medium under sterile conditions for adventitious bud induction culture. The adventitious bud induction medium used in this example was based on MS medium and added with 0.1 mg / L TDZ, 1.20 mg / L 6-BA and 0.2 mg / L NAA, and contained 6.5 g / L agar. The preparation method of MS medium was a conventional method in the prior art. After the adventitious bud induction medium was prepared, the pH was adjusted to 6.7, and then the medium was poured into plates after sterilization.

[0065] During the adventitious bud induction culture, the culture condition was artificially controlled light period, the light intensity was 2500 lx, the light period was 15 h light followed by 9 h dark, the culture temperature was 25 °C, and the culture time was 10 d.

[0066] 4. Rooting culture The induced adventitious buds were transferred to rooting medium for rooting culture. The rooting medium used in this example was based on 1 / 2MS medium and added with 1.0 mg / L IBA, and contained 6.5 g / L agar. The preparation method of 1 / 2MS medium was a conventional method in the prior art. After the rooting medium was prepared, the pH was adjusted to 6.7, and then the medium was poured into plates after sterilization.

[0067] During the rooting culture, the culture condition was artificially controlled light period, the light intensity was 1500 lx, the light period was 15 h light followed by 9 h dark, the culture temperature was 25 °C, and the culture time was 10 d.

[0068] After the rooting culture, the complete plant was obtained.

[0069] The browning of the explants generally occurred in the callus induction stage. The browning rate of the explants before the callus induction stage in this example was counted. The calculation formula was: browning rate = (number of browning explants / total number of explants) x 100%. In this example, the browning rate of the explants was (6.0 ± 2.38)%.

[0070] In addition, the induction rate of the callus induction culture, the induction rate of the adventitious bud induction culture and the induction rate during the rooting culture in this example were counted. The calculation formula of the callus induction rate was: number of explants differentiated into callus / total number of explants inoculated onto the callus induction medium x 100%. The induction rate of the adventitious bud induction culture and the induction rate during the rooting culture were calculated in the same way. The final counting result was that the induction rate of the callus induction culture in this example was (67.2 ± 3.41)%, the induction rate of the adventitious bud was (90.00 ± 2.23)%, and the induction rate during the rooting culture was (91.0 ± 4.38)%.

[0071] Example 3 The present embodiment provides a method for preventing browning and rapid propagation of Xanthoceras sorbifolia Bunge explants. The method comprises the following steps. 1. Selection and pretreatment of explants Seeds of Xanthoceras sorbifolia Bunge grown under the same conditions and in the same year as five-year-old Xanthoceras sorbifolia Bunge in the Xanthoceras sorbifolia Bunge germplasm resource library of Kundu Forest Farm, Arhorqin Banner, Chifeng City were selected and planted. After the seeds grew into seedlings, the tender stems of the seedlings were taken. The obtained tender stems contained epidermis, cortex and primary vascular bundle, but did not contain cambium.

[0072] A 3.5 cm long section was cut from the tender stem of the seedling as an explant. In the present embodiment, the length of the explant was 3 cm. The obtained explant was stored at 6°C for 14 h, and then the explant was sterilized.

[0073] During sterilization of the explant, 3 drops of Tween 20 were added to 500 mL of water, and then the explant was washed with the water flow containing Tween 20 for 1 h. After washing, the explant was transferred to a clean bench and immersed in 0.1% sodium hypochlorite solution under sterile conditions for 4 min. During the immersion process, the container containing the sodium hypochlorite solution and the explant was constantly shaken. After the immersion was completed, the explant was washed with sterile water for 5 times to wash the sodium hypochlorite solution on the surface of the explant. Then the explant was immersed in a 75% ethanol solution for 30 s with shaking. Finally, the explant was washed with sterile water for 5 times to wash the 75% ethanol solution on the surface of the explant.

[0074] The sterilized explant was inoculated into a pretreatment medium and cultured at 26°C in the dark for 5 d. When the pretreatment medium was prepared, only 7.5 g / L agar and 15 g / L sucrose were added, and the pH was adjusted to 7.0.

[0075] 2. Callus induction The pretreated explant was transferred to a callus induction medium for callus induction culture. The callus induction medium used in the present embodiment was based on 1 / 2MS medium, and 1.0 mg / L 2,4-D and 0.02 mg / L NAA were added, and contained 6.5 g / L agar. The preparation method of 1 / 2MS medium was a conventional method in the prior art. After the callus induction medium was prepared, the pH was adjusted to 7.0, and after sterilization, it was poured into a plate.

[0076] During the callus induction culture, the culture was carried out in the dark, the culture temperature was 27°C, and the culture time was 7 d. After the culture was completed, the explant was dedifferentiated and callus was formed.

[0077] 3. Adventitious bud induction The callus was transferred to adventitious bud induction medium under sterile conditions for adventitious bud induction culture. The adventitious bud induction medium used in this example was based on MS medium and added with 0.1 mg / L TDZ, 1.4 mg / L 6-BA and 0.2 mg / L NAA, and contained 6.5 g / L agar. The preparation method of MS medium was a conventional method in the prior art. After the adventitious bud induction medium was prepared, the pH was adjusted to 7.0, and after sterilization, it was poured into a plate to obtain the adventitious bud induction medium.

[0078] During the adventitious bud induction culture, artificial controlled photoperiod culture conditions were adopted, the light intensity was 2800 lx, the photoperiod was 16 h light followed by 10 h dark, the culture temperature was 26°C, and the culture time was 7 d.

[0079] 4. Rooting culture The induced adventitious buds were transferred to rooting medium for rooting culture. The rooting medium used in this example was based on 1 / 2MS medium and added with 1.3 mg / L IBA, and contained 6.5 g / L agar. The preparation method of 1 / 2MS medium was a conventional method in the prior art. After the rooting medium was prepared, the pH was adjusted to 7.0, and after sterilization, it was poured into a plate to obtain the rooting medium.

[0080] During the rooting culture, artificial controlled photoperiod culture conditions were adopted, the light intensity was 1600 lx, the photoperiod was 16 h light followed by 1 h dark, the culture temperature was 26°C, and the culture time was 7 d.

[0081] After the rooting culture, complete plants were obtained.

[0082] Browning of the explants generally occurred in the callus induction stage. The browning rate of the explants before the callus induction stage in this example was counted. The calculation formula was: browning rate = (number of browning explants / total number of explants) x 100%. In this example, the browning rate of the explants was 8.03%.

[0083] In addition, the induction rate of the callus induction culture, the induction rate of the adventitious bud induction culture and the induction rate during the rooting culture in this example were counted. The calculation formula of the callus induction rate was: number of explants differentiated into callus / total number of explants inoculated onto the callus induction medium x 100%. The induction rate of the adventitious bud induction culture and the induction rate during the rooting culture were calculated in the same way. The final statistical results were that the induction rate of the callus induction culture in this example was 57.20%, the induction rate of the adventitious buds was (84.69 ± 3.54)%, and the induction rate during the rooting culture was 88%.

[0084] Comparative Example 1 In the present comparative example, the method of pretreatment was changed, tissue culture of Xanthoceras sorbifolia Bunge was carried out, and the browning rate of explants was counted.

[0085] In the tissue culture of Xanthoceras sorbifolia Bunge in the present comparative example, the difference between the method used and the method in Example 2 is only that, in the present comparative example, after the explants were refrigerated and sterilized, they were directly inoculated into the callus induction medium in Example 2. Other operations were exactly the same as in Example 2.

[0086] Comparative Example 2 In the present comparative example, the method of pretreatment was changed, tissue culture of Xanthoceras sorbifolia Bunge was carried out, and the browning rate of explants was counted.

[0087] In the tissue culture of Xanthoceras sorbifolia Bunge in the present comparative example, the difference between the method used and the method in Example 2 is only that, in the present comparative example, the explants were not refrigerated, but were directly inoculated into the pretreatment medium in Example 2 after sterilization. Other operations were exactly the same as in Example 2.

[0088] Comparative Example 3 In the present comparative example, the method of pretreatment was changed, tissue culture of Xanthoceras sorbifolia Bunge was carried out, and the browning rate of explants was counted.

[0089] In the tissue culture of Xanthoceras sorbifolia Bunge in the present comparative example, the difference between the method used and the method in Example 2 is only that, in the present comparative example, the explants were not refrigerated and pretreated, but were directly inoculated into the callus induction medium in Example 2 after sterilization. Other operations were exactly the same as in Example 2.

[0090] As Figure 1 The photos of part of callus obtained in Example 2. Figure 2 In the present comparative example, A is the photo of part of callus obtained in Comparative Example 3, B is the photo of part of callus obtained in Comparative Example 2, and C is the photo of part of callus obtained in Comparative Example 1. In Comparative Example 3, the callus browning was more obvious, and the callus volume was obviously smaller; in Comparative Examples 1 and 2, the callus also appeared slight browning, but was obviously slighter than in Comparative Example 3, and the callus obtained in Comparative Examples 1 and 2 was larger in volume, indicating that the explants in Comparative Examples 1 and 2 were relatively better in activity, and the browning affected the activity of the explants; the callus obtained in Example 2 had almost no browning, and the callus volume was larger, indicating that the activity of the explants in Example 2 was the best.

[0091] The browning rates of explants in Comparative Example 1, Comparative Example 2 and Comparative Example 3 were statistically calculated according to the statistical method in Example 2. The browning rates of explants in Comparative Example 1, Comparative Example 2 and Comparative Example 3 were 27.2%, 20.5% and 44.4% respectively, all of which were much higher than the browning rate of 6% in Example 2. The results showed that the pretreatment method in Example 2 could effectively reduce the browning rate of explants, and the reduction of the browning rate was the result of the combined action of low-temperature treatment and the selection of suitable pretreatment medium for pretreatment culture.

[0092] Example 4 In this example, the method proposed in Example 2 was improved to propose a treatment method with better anti-browning effect.

[0093] The difference between the method in this example and Example 2 is that, in this example, after the disinfection of the explants and before the pretreatment culture, the wound of the explants is sealed with alginate gel, and after the pretreatment culture is completed, the alginate gel layer is manually removed. The rest of the operations are the same as those in Example 2.

[0094] The specific operation method is as follows: Prepare a sodium alginate solution: weigh 2.5%, w / v sodium alginate, purchased from Macklin, product number S875337, M / G ratio = 1:2, dissolve in 100 mL distilled water, stir evenly, then filter sterilize with a 0.22 μm filter membrane for standby use.

[0095] Prepare a calcium ion solution: weigh CaCl2 and configure it as a 100 mM solution in distilled water, filter sterilize with a 0.22 μm filter membrane for standby use.

[0096] For the disinfected explants, under sterile conditions, quickly dip the incision into 2.5 wt% sodium alginate solution for 10 s to ensure that the incision surface is completely wetted; then place the incision into 100 mM CaCl2 solution and gently shake at room temperature for 30 s to form a stable calcium alginate gel layer at the incision. Then rinse with sterile water for 3 times to remove the surface residual Ca² + and uncrosslinked sodium alginate.

[0097] After the pretreatment culture is completed, the explants are taken out, the calcium alginate gel at the incision is rinsed with sterile water, and the calcium alginate gel layer remaining at the incision is peeled off with the aid of sterile tweezers to expose the complete incision.

[0098] The calcium alginate gel layer physically blocks the exudate of the incision, effectively inhibits the direct leakage of initial phenols and oxidases during the pretreatment stage, and effectively reduces the browning rate of the explants. Removing the calcium alginate gel layer before culture can ensure that the incision of the explants is in full contact with the hormone and nutrient components of the culture medium, promoting the formation of callus.

[0099] As shown in Table 1, the method provided in this embodiment further reduces the browning rate of explants in the callus induction culture stage to (4.2±0.83)%, while the callus induction rate, adventitious bud induction rate and rooting induction rate do not change significantly compared with Example 2. That is, the method provided in this embodiment reduces the browning rate of explants without affecting the callus induction culture and subsequent culture. The calcium alginate gel can prevent phenols and polyphenol oxidase from diffusing into the culture medium during the pretreatment stage, avoiding initial oxidation reaction; in addition, the M / G ratio is 1:2, i.e., the gel formed by high-G type sodium alginate has moderate toughness and is easy to mechanically peel off without residue, which does not affect callus induction culture.

[0100] Table 1 Browning rate, callus induction rate, adventitious bud induction rate and rooting induction rate of different pretreatment methods

[0101] Note: "-" means no such item.

[0102] Experimental Example 1 In this experimental example, the hormones added to the culture medium during callus induction culture were changed, callus induction culture was carried out, and the induction rate of callus was compared. Each experimental group was set up in triplicate, and the average value was taken as the result.

[0103] The method used in each experimental group in this experimental example for callus induction culture is different from the method in Example 2 only in that, in each experimental group of this experimental example, 2,4-D and NAA with concentrations shown in Table 2 were added to the callus induction medium in addition to the 1 / 2MS salt solution. The induction rate and browning rate under different hormone addition amounts are recorded in Table 2.

[0104] Table 2 Induction rate and browning rate of callus under different hormone addition amounts

[0105] As can be seen from the results in Table 2, under the same NAA concentration, the callus induction rate increases significantly with the increase of 2,4-D concentration; in contrast, under the same 2,4-D concentration, the increase of NAA concentration has a limited effect on the induction rate of callus; in addition, for the 1-1 and 1-6 groups, the 2,4-D / NAA concentration ratio is the same, but the 2,4-D concentration of the 1-6 group is higher, and the induction rate is also higher. When the NAA concentration of the 1-2 group is the same as that of the 1-6 group, and the 2,4-D concentration is halved, the induction rate of the 1-2 group is significantly lower than that of the 1-6 group. The above results show that there is no obvious correlation between the induction rate and the 2,4-D / NAA concentration ratio, and the induction rate of callus is mainly related to the 2,4-D concentration in the callus induction medium.

[0106] In addition, the browning rate comparison results between groups show that after the same pretreatment, when the concentration of 2,4-D and the concentration of NAA in the callus induction culture medium change, the browning rate of callus also changes in the callus induction culture stage, and reaches the minimum when the concentration ratio of 2,4-D / NAA is 30. However, the difference in browning rate between groups is relatively small, so when selecting the concentration of 2,4-D and NAA in the callus induction medium, the induction rate should be the dominant factor, and the browning rate should be the second. Therefore, based on the above experiments, when the concentration of 2,4-D is controlled to be 1.0 mg / L-1.2 mg / L, and the concentration ratio of 2,4-D and NAA is in the range of 25-50:1, the callus induction rate is greater than 55% on the basis of preventing the browning phenomenon of explants in the process of differentiating into callus based on the activation of 2,4-D to antioxidant enzymes.

[0107] Experimental Example 2 In this experimental example, the hormones added in the medium during callus induction culture are changed, callus induction culture is carried out, and the induction rate of callus is compared.

[0108] The difference between the method used in each experimental group in this experimental example and the method in Example 2 is only that in each experimental group of this experimental example, 6-BA and NAA with the concentration shown in Table 3 are added in the callus induction medium in addition to the 1 / 2MS salt solution. The induction rate under different hormone addition amounts is also recorded in Table 3.

[0109] Table 3 Different concentrations of 6-BA and NAA addition amount and the induction rate of callus

[0110] The results of Experimental Example 2 show that when the tender stems of Xanthoceras sorbifolia Bunge pretreated according to the pretreatment method provided in Example 2 are used as explants, the effect of 6-BA and NAA combination on inducing callus formation is not as ideal as that of 2,4-D and NAA combination in the callus induction stage.

[0111] Experimental Example 3 In this experimental example, the types and concentrations of hormones added in the medium during adventitious bud induction culture are changed, adventitious bud induction culture is carried out, and the induction rate of adventitious bud is compared. Adventitious bud induction rate = number of callus producing adventitious bud / total number of callus × 100%.

[0112] The difference between the method used in each experimental group in this experimental example and the method in Example 2 is only that in each experimental group of this experimental example, in addition to containing MS salt solution, the medium used in adventitious bud induction is added with hormones with the types and concentrations shown in Table 4.

[0113] Table 4: Different concentrations of TDZ, 6-BA and NAA and induction rate

[0114] The statistical results in Table 4 show that the adventitious bud induction rate increases first and then decreases with the increase of the 6-BA+TDZ:NAA concentration ratio, that is, when the 6-BA+TDZ:NAA concentration ratio is close to 6.5, the adventitious bud induction rate is the highest, and deviating from this ratio, whether increasing or decreasing, the adventitious bud induction rate decreases. Therefore, in the adventitious bud induction medium, the 6-BA+TDZ:NAA concentration ratio is selected to be 5.5-7.5.

[0115] As shown in Figure 3 , the picture of the best induction rate group in the adventitious bud induction process is shown, that is, the adventitious bud photo of the 3-3 group.

[0116] Experimental Example 4 In order to explore whether the high adventitious bud induction rate of a specific hormone ratio in the adventitious bud induction culture stage is premised on "pre-treatment", an experiment was designed. The experimental design and the statistical results of the adventitious bud induction rate are shown in Table 5.

[0117] Based on the premise that the specific 2,4-D and NAA concentration ratio in the callus culture medium, the experiment was designed to inoculate the callus obtained by induction after pre-treatment and the callus obtained by induction without pre-treatment into the adventitious bud induction medium in Example 2, and the callus induction culture time of the two was the same, and the two calli did not brown at the time of inoculation. The adventitious bud induction rate was counted. The experimental design and the statistical results of the adventitious bud induction rate are shown in Table 5.

[0118] Table 5: Experimental design and statistical results of adventitious bud induction rate

[0119] Based on the above experimental design, the hormone dosage in the callus induction culture stage and the hormone dosage in the adventitious bud induction culture stage were changed, and the verification was carried out again. The experimental design and the statistical results of the adventitious bud induction rate are shown in Table 6.

[0120] Table 6

[0121] The comparison of the data in Table 5 and the data in Table 6 shows that the adventitious bud induction rate of the callus obtained by induction of the pre-treated explant, that is, the low-temperature refrigeration+pre-treatment culture, is higher, and even if the hormone dosage in the callus induction culture stage and the adventitious bud induction culture stage is not "optimal", the adventitious bud induction rate of the pre-treated explant is still higher than that of the non-pre-treated explant.

[0122] The reason for this phenomenon can be that low temperature treatment can reduce the key oxidation activity in the explants, slowing down the browning of the Xanthoceras sorbifolia explants. The accumulation of phenolic substances in the explants is delayed in the environment of low concentration of inorganic salts. In the two pretreatment methods, the browning degree and browning rate of the explants are reduced. Under the condition that the browning phenomenon is weakened, the induction of the explants into callus is more rapid and stable. The browning and death of the explants are reduced.

[0123] The technical features of the above-described embodiments can be combined in any manner. In order to make the description simple, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not contradict each other, they should be considered as falling within the scope of the present disclosure.

[0124] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. For those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preventing Xanthoceras sorbifolia Bunge explant browning and rapid propagation, characterized in that, The method comprises the following steps: cutting explants from Xanthoceras sorbifolia Bunge stem segments, and cold storing the explants; sterilizing the cold stored explants; inoculating the sterilized explants into a pretreatment medium, and performing pretreatment culture in dark conditions; the formula of the pretreatment medium is: 5.5g / L-7.5g / L agar, 10g / L-15g / L sucrose, and 1L water; transferring the pretreatment cultured explants into a callus induction medium, an adventitious bud induction medium, and a rooting medium in sequence for culture until complete plants are obtained; the callus induction medium is based on 1 / 2MS medium and added with 2,4-D and NAA; in the callus induction medium, the mass concentration of 2,4-D is 1.0mg / L-1.2mg / L, and the mass concentration ratio of 2,4-D to NAA is 25-50:1; the adventitious bud induction medium is based on MS medium and added with TDZ, 6-BA, and NAA; in the adventitious bud induction medium, the mass concentration of 6-BA is 1.0mg / L-1.4mg / L, and the mass concentration ratio of 6-BA+TDZ to NAA is 5.5-7.5:1; 2. The method of claim 1, wherein, the rooting medium is based on 1 / 2MS medium and added with IBA; in the rooting medium, the mass concentration of IBA is 0.8mg / L-1.2mg / L.

3. The method of claim 1, wherein, the temperature during cold storage is 4°C-6°C, and the cold storage time is 10h-14h.

4. The method of claim 1, wherein, the Xanthoceras sorbifolia Bunge stem segments are cut from tender stems of Xanthoceras sorbifolia Bunge seedlings; the length of the cut explants is 2.5cm-3.5cm.

5. The method of claim 1, wherein, during the pretreatment culture, constant temperature conditions of 24°C-26°C are adopted, and the pretreatment culture time is 3d-5d.

6. The method of claim 1, wherein, during the callus induction culture using the callus induction medium, culture is performed in dark conditions, the culture temperature is 23°C-27°C, and the culture time is 7d-12d.

7. The method of claim 1, wherein, during the adventitious bud induction culture using the adventitious bud induction medium, the light period is 14h-16h light followed by 8h-10h darkness, the light intensity is 2200lx-2800lx, the culture temperature is 24°C-26°C, and the culture time is 7d-12d.

8. The method of claim 1, wherein, during the rooting culture using the rooting medium, the light period is 14h-16h light followed by 8h-10h darkness, the light intensity is 1300lx-1600lx, the culture temperature is 24°C-26°C, and the culture time is 7d-12d. the sterilization method of the explants is:

9. The method of claim 1, wherein, the explants are washed with Tween 20 for 1h; the explants are soaked in 0.1% sodium hypochlorite solution, and after the soaking is completed, the explants are further soaked in an ethanol solution with an ethanol volume fraction of 75%.

10. The method of claim 9, wherein, the sterilized explants are subjected to incision sealing treatment, and then inoculated into the pretreatment medium. the incision sealing treatment method is: a) the incisions of the sterilized explants are dipped into a 2.5wt% sodium alginate solution; the M / G ratio of the sodium alginate is less than 1; b) the incisions of the explants are placed into a 100mM CaCl2 solution for reaction, and a calcium alginate gel layer is formed on the incisions of the explants; c) After the end of the pre-treatment culture, the explants are rinsed with sterile water; the layer of calcium alginate gel is removed from the cut of the explants. c) After the end of the pre-treatment culture, the explants are rinsed with sterile water; the layer of calcium alginate gel is removed from the cut of the explants. c) After the

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