Method of Xinjiang early yellow fig tissue culture and rapid propagation system

Abstract

The invention discloses a method of a Xinjiang early yellow fig tissue culture and rapid propagation system, and belongs to the technical field of plant tissue culture. The method comprises the steps of explant selection and treatment, induction culture, subculture, rooting culture and domestication and transplanting. In the explant pretreatment, a bacitracin-ursolic acid-abamectin conjugate is used for disinfection, so that the bacitracin and abamectin cooperate to resist bacteria and prevent microbial infection, and ursolic acid is used for inhibiting the activity of polyphenol oxidase to prevent explant browning; hormone-enriched microspheres with chitosan as a core layer and calcium alginate-lignosulfonate as a shell layer are added into the culture medium, BA or NAA can be stably stored and accurately released, nutrient substances can also be adsorbed, meanwhile, mannitol, L-proline and betaine loaded on the microspheres can adjust osmotic pressure, enhance cell stress resistance and reduce vitrification, and the hormone-enriched culture medium is suitable for being used as a culture medium. The rapid propagation of the fig explant can be efficiently promoted, and a feasible technical path is provided for the tissue propagation of the fig explant.

Description

Technical Field

[0001] This invention belongs to the field of rapid propagation technology of plant tissue culture, specifically relating to a method for rapid propagation system of Xinjiang early yellow fig through tissue culture. Background Technology

[0002] Xinjiang Early Yellow Fig is a high-quality fig variety unique to Xinjiang. It gets its name from its early ripening time and pale yellow skin, and is mainly produced in Turpan and Kashgar. The fruit is oblate, with a yellowish skin tinged with a faint red blush when ripe. The flesh is soft and delicate, and when peeled, it is amber in color. It is sweet and juicy with a unique fruity aroma and has no obvious seed, offering an excellent eating experience. It is also rich in glucose, fructose, vitamin C, dietary fiber, and various minerals, making it a popular local specialty fruit.

[0003] In vitro rapid propagation systems using plant tissue culture technology have been widely established in figs. However, research shows that tissue culture regeneration systems are not compatible between different fig varieties, and even between different explants of the same variety. Research on rapid propagation technology for Xinjiang Early Yellow figs using tissue culture is still in its early stages. Therefore, it is urgent and important to further summarize rapid propagation technology for figs using tissue culture based on existing research. The application of rapid propagation technology for figs using tissue culture not only enables rapid propagation of seedlings but also finds effective use in the preservation of fig germplasm resources, plant virus elimination, and the breeding of superior varieties. Micropropagation and shoot tip culture techniques have been used to eliminate fig mosaic virus, improving the yield and quality of fig fruits.

[0004] The establishment of a rapid propagation system for fig tissue culture provides a technical foundation for further developing regeneration and doubling systems, and offers a new method for the innovation of fig germplasm resources. Current rapid propagation techniques for fig tissue culture involve explant pretreatment, explant disinfection and inoculation, adventitious bud induction, seedling strengthening culture, and rooting culture. Explant disinfection often uses chemical reagents such as ethanol, mercuric chloride, bleaching powder, and sodium hypochlorite. However, these chemical disinfectants easily cause residues and environmental pollution, and prolonged disinfection time can increase the browning rate of explants. Furthermore, during rapid propagation of fig tissue culture, the abundant phenolic substances in the explants are prone to oxidation, leading to browning of the culture medium and affecting the cultivation of fig explants. Vitrification is also likely to occur during the adventitious bud induction stage. In addition, fig explants themselves contain a large number of endophytic bacteria. During tissue culture, due to the infection of microorganisms such as bacteria and fungi, a large number of bacterial plaques are generated during culture, which prevents the tissue culture material from growing and developing normally. Although appropriate sterilization methods can be used to reduce the contamination rate of fig explants, it will lead to an increase in the probability of browning. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention discloses a method for rapid propagation of early-ripening figs from Xinjiang through tissue culture, belonging to the field of plant tissue culture technology. This method includes explant selection and treatment, induction culture, subculture, rooting culture, and acclimatization transplantation. Explant pretreatment involves disinfection with a bacitracin-ursolic acid-avermectin conjugate, which utilizes the synergistic antibacterial and antimicrobial effects of bacitracin and avermectin to prevent microbial infection, while ursolic acid inhibits polyphenol oxidase activity, preventing explant browning. Hormone-enriched microspheres with a chitosan core and calcium alginate-lignin sulfonate shell are added to the culture medium. These microspheres can stably store and precisely release BA or NAA, and can also adsorb nutrients. Simultaneously, the mannitol, L-proline, and betaine loaded on the microspheres can regulate osmotic pressure, enhance cell resistance, and reduce vitrification, effectively promoting rapid propagation of fig explants and providing a feasible technical pathway for their tissue propagation.

[0006] Technical solution: A method for rapid propagation of early yellow figs from Xinjiang via tissue culture, comprising the following steps: S1. Explant selection and treatment: The tips of the new shoots of Xinjiang early yellow fig (Fig. 0.05) were cut into 1-2 cm sections as explants. After collection, the young leaves were removed and the shoots were disinfected to obtain pretreated fig shoots. S2. Induction culture: The pretreated fig shoots described in S1 are subjected to shoot tip peeling treatment under sterile environment to obtain 0.3-0.5 mm shoot tips, which are then inoculated on induction culture medium for induction culture to form adventitious buds; S3. Subculture: Divide the adventitious buds of S2 that have grown to 1.0cm into bud blocks of 3-5 small buds, transfer them to subculture proliferation medium for subculture, subculture once a month to obtain rootless seedlings. S4. Rooting culture: Select rootless seedlings with a length of 2-3cm and inoculate them onto rooting culture medium for rooting culture to obtain rooted tissue culture seedlings; S5. Acclimatization and Transplanting: Once the rooted tissue culture seedlings have developed complete root systems and grown to 6-8 leaves, they are moved to a greenhouse for acclimatization treatment for 7-10 days without opening the culture bottle or 1-2 days with the bottle opened. Then, they are removed from the culture bottle and the culture medium is washed off the roots. They are then transplanted into a sterilized vermiculite substrate and covered with a film to retain moisture. After one week, ventilation is gradually increased until the film is removed. When the rooted tissue culture seedlings grow to 10cm, they can be planted in the orchard soil.

[0007] Alternatively, young stem segments, leaves, or buds of early yellow figs from Xinjiang can be selected as explants. These explants are disinfected with 75% alcohol for 30 seconds, 2% sodium hypochlorite for 15 minutes, and rinsed three times with sterile water. Then, they undergo induction culture, subculture, rooting culture, and acclimatization transplantation in sequence. The induction medium described in S2 can be replaced with fig axillary bud germination medium (composed of MS + 1 mg / L 6-BA + 0.1 mg / L NAA), and the rooting medium described in S4 can also be composed of MS + 0.2 mg / L 6-BA + 0.5% activated carbon.

[0008] Furthermore, the disinfection method in S1 is to first soak in 75% ethanol solution for 10 seconds, then sterilize with a mixed solution of 2-3.5 wt% sodium hypochlorite and bacitracin-ursolic acid-avermectin conjugate or 2-4 wt% bacitracin-ursolic acid-avermectin conjugate solution for 20-25 minutes, and then rinse with sterile water 4-5 times.

[0009] Furthermore, the preparation method of the bacitracin-ursolic acid-avermectin conjugate is as follows: ① Ursolic acid and avermectin are dissolved in anhydrous ethanol at a mass ratio of 1:(1.5-2.5), reacted at 60-65℃ for 3-6 hours, and then concentrated under reduced pressure, washed and dried to obtain the ursolic acid-avermectin complex; ② Bacitracin is dissolved in water to prepare a bacitracin solution with a concentration of 3-5.5wt%, the pH is adjusted to 5.0-5.5, 4-6wt% ursolic acid-avermectin complex is slowly added to the bacitracin solution and reacted at room temperature for 2-4 hours, and then concentrated and purified to obtain the bacitracin-ursolic acid-avermectin conjugate.

[0010] Furthermore, the induction medium in S2 consists of MS + 0.2 mg / L BA + (0.1-0.15) mg / L hormone-enriched microspheres, and the induction culture conditions are a temperature of 23-25℃, a light intensity of 2000-2500 lx, and a photoperiod of 12-16 h / day; the subculture proliferation medium in S2 consists of MS + 0.2 mg / L BA + (0.15-0.3) mg / L hormone-enriched microspheres, and the subculture proliferation conditions are a temperature of 25-27℃, a light intensity of 2500-3000 lx, and a photoperiod of 15-16 h / day; the rooting medium in S2 consists of 1 / 2 MS + 0.2 mg / L BA. NAA+ (0.1-0.3) mg / L hormone-enriched microspheres; the rooting culture conditions are a temperature of 25-27℃, a light intensity of 2500-3000 lx, and a photoperiod of 15-16 h / day.

[0011] Furthermore, the preparation method of the hormone-enriched microspheres includes the following steps: Step 1. Dissolve chitosan in glacial acetic acid to prepare a chitosan solution with a concentration of 1-2.5 wt%. Then, mix the chitosan solution with a composition made of mannitol, L-proline and betaine at a ratio of 1 g: (4-8 mL) and stir at 200-300 rpm for 25-40 min to obtain chitosan microspheres. Step 2. Dissolve sodium alginate in water to prepare a sodium alginate solution with a concentration of 1-1.5wt%. Add 2-5% sodium lignin sulfonate to the sodium alginate solution and mix well to prepare a mixed solution. Then mix chitosan microspheres with the mixed solution at a ratio of 1g:(3-5mL) and add 1.5-2wt% calcium chloride solution dropwise at a uniform speed. Stir at 200-300rpm for 25-40min to obtain hormone-enriched microspheres.

[0012] Furthermore, in step 1, the mass ratio of mannitol, L-proline, and betaine is 1:(0.5-3):(1-2). Beneficial effects

[0013] 1. In the pretreatment of fig explants, this invention uses a bacitracin-ursolic acid-avermectin conjugate for explant disinfection and sterilization, inhibiting polyphenol oxidase activity and preventing the oxidation of phenolic substances in fig explants, thus preventing browning. On the one hand, the synergistic antibacterial effect of bacitracin and avermectin can kill harmful organisms in fig explants, forming a broad-spectrum antibacterial coating, effectively inhibiting the reproduction of microorganisms on and inside the explants, and preventing microbial infection from damaging the explant cell structure, accelerating the release and contact of polyphenol oxidase and phenolic substances, thus aggravating browning. On the other hand, ursolic acid can chelate copper ions at the active center of polyphenol oxidase, destroying the spatial structure of polyphenol oxidase, inhibiting the activity of polyphenol oxidase, and blocking the formation of quinone substances. This not only protects the physiological function of the explants but also maintains the chemical structure and efficacy of bacitracin and avermectin, ensuring the continuous effectiveness of the antibacterial defense. Meanwhile, the auxiliary stabilizing effect of bacitracin and avermectin in the conjugate on the cell membrane can further reduce the release of phenolic substances and indirectly enhance the anti-browning effect of ursolic acid.

[0014] 2. During the induction and proliferation culture of the hormone-enriching microspheres described in this invention, the chitosan polysaccharide backbone contains hydrophobic segments, which can adsorb BA through hydrophobic interactions, providing initial binding sites for BA; at the same time, lignin sulfonate contains a large number of aromatic ring structures, which can form a π-π stacking effect with BA, further capturing unadsorbed BA in the culture medium through hydrophobic interactions; in addition, the porous structure of the hormone-enriching microspheres can physically retain adsorbed BA, reduce its desorption in the system, and significantly improve the BA enrichment efficiency and stability.

[0015] During the rooting culture process, the hormone-enriching microspheres of this invention exhibit strong electrostatic interactions between the amino groups of chitosan and the carboxyl groups of NAA, while the hydroxyl groups of chitosan provide the main binding sites for NAA. Sodium alginate can assist the electrostatic binding of NAA and chitosan through "charge bridging," and the polar groups of lignin sulfonate can form secondary hydrogen bonds with NAA, further supplementing the adsorption sites. This achieves the superposition of dual polar action sites, significantly improving the enrichment capacity and selectivity for NAA.

[0016] 3. The hormone-enriched microspheres of this invention contain mannitol, L-proline, and betaine. Mannitol, as a non-metabolistic osmotic regulator, can form a stable osmotic pressure gradient in the culture medium, balancing the intracellular osmotic pressure and directly reducing excessive water absorption by cells, thus preventing cell structural disorder. On the other hand, proline and betaine are compatible solutes that not only help regulate osmotic pressure but also accumulate intracellularly to protect enzyme activity and cell membrane integrity, thereby jointly enhancing the stress resistance of fig explant tissue cells and inhibiting vitrification.

[0017] 4. This invention uses chitosan as the core layer and calcium alginate-lignin sulfonate as the shell layer to prepare hormone-enriched microspheres, which are added to the culture medium involved in the rapid propagation method of early yellow fig tissue culture in Xinjiang. These microspheres are loaded with mannitol, L-proline, and betaine, which can reduce the vitrification problem caused by excessive water absorption by fig tissue cells, efficiently deliver plant hormones to fig explants or adventitious seedlings, enhance nutrient absorption capacity, promote rapid propagation of fig explants through tissue culture, and provide a practical and feasible technical path for fig tissue propagation. Attached Figure Description

[0018] Figure 1 Images of adventitious buds during the rapid propagation of early yellow figs from Xinjiang via tissue culture. Figure 2 Figure showing the growth of adventitious buds during the rapid propagation of early yellow figs from Xinjiang via tissue culture. Figure 3 Image of rooted tissue culture seedlings during the rapid propagation of early yellow figs from Xinjiang. Figure 4 This image shows the growth of rooted tissue culture seedlings during the rapid propagation of early yellow figs from Xinjiang. Figure 5 This image shows the growth of tissue culture seedlings of early yellow fig from Xinjiang after transplanting. Figure 6 Photo of tissue culture seedlings of early yellow fig from Xinjiang transplanted to a greenhouse. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: Example 1

[0020] A method for preparing a bacitracin-ursolic acid-avermectin conjugate includes the following steps: Step ①. Dissolve 1g of ursolic acid and 1.5g of avermectin in 100mL of anhydrous ethanol, react at 65℃ for 4h, then concentrate under reduced pressure to 1 / 3 of the original volume, wash and dry to obtain ursolic acid-avermectin complex. Step ②. Dissolve 4g of bacitracin in water to prepare a 4wt% bacitracin solution, adjust the pH to 5.0, slowly add 5g of ursolic acid-avermectin complex to 100mL of bacitracin solution and react at room temperature for 3h. Concentrate and purify to prepare bacitracin-ursolic acid-avermectin conjugate. Example 2

[0021] A method for preparing a bacitracin-ursolic acid-avermectin conjugate includes the following steps: Step ①. Dissolve 1g of ursolic acid and 2.5g of avermectin in 100mL of anhydrous ethanol, react at 60℃ for 4h, then concentrate under reduced pressure to 1 / 3 of the original volume, wash and dry to obtain ursolic acid-avermectin complex. Step ②. Dissolve 4g of bacitracin in water to prepare a 4wt% bacitracin solution, adjust the pH to 5.0, slowly add 5g of ursolic acid-avermectin complex to 100mL of bacitracin solution and react at room temperature for 3h. Concentrate and purify to prepare bacitracin-ursolic acid-avermectin conjugate. Example 3

[0022] A method for preparing a bacitracin-ursolic acid-avermectin conjugate includes the following steps: Step ①. Dissolve 1g of ursolic acid and 2g of avermectin in 100mL of anhydrous ethanol, react at 65℃ for 5h, then concentrate under reduced pressure to 1 / 3 of the original volume, wash and dry to obtain ursolic acid-avermectin complex. Step ②. Dissolve 4g of bacitracin in water to prepare a 4wt% bacitracin solution, adjust the pH to 5.0, slowly add 5g of ursolic acid-avermectin complex to 100mL of bacitracin solution and react at room temperature for 3h. Concentrate and purify to prepare bacitracin-ursolic acid-avermectin conjugate. Example 4

[0023] A method for preparing a bacitracin-ursolic acid-avermectin conjugate includes the following steps: Step ①. Dissolve 1g of ursolic acid and 2g of avermectin in 100mL of anhydrous ethanol, react at 65℃ for 4h, then concentrate under reduced pressure to 1 / 3 of the original volume, wash and dry to obtain ursolic acid-avermectin complex. Step ②. Dissolve 5g of bacitracin in water to prepare a 5wt% bacitracin solution, adjust the pH to 5.0, slowly add 5g of ursolic acid-avermectin complex to 100mL of bacitracin solution and react at room temperature for 3h. Concentrate and purify to prepare bacitracin-ursolic acid-avermectin conjugate. Example 5

[0024] A method for preparing a bacitracin-ursolic acid-avermectin conjugate includes the following steps: Step ①. Dissolve 1g of ursolic acid and 2g of avermectin in 100mL of anhydrous ethanol, react at 65℃ for 4h, then concentrate under reduced pressure to 1 / 3 of the original volume, wash and dry to obtain ursolic acid-avermectin complex. Step ②. Dissolve 5.5g of bacitracin in water to prepare a 5.5wt% bacitracin solution, adjust the pH to 5.5, slowly add 5g of ursolic acid-avermectin complex to 100mL of bacitracin solution and react at room temperature for 2h. Concentrate and purify to prepare bacitracin-ursolic acid-avermectin conjugate. Comparative Example 1

[0025] The difference between this comparative example and Example 3 is that bacitracin is not added; the remaining preparation methods are the same as in Example 3. Comparative Example 2

[0026] The difference between this comparative example and Example 3 is that ursolic acid is not added; the remaining preparation methods are the same as in Example 3. Comparative Example 3

[0027] The difference between this comparative example and Example 3 is that avermectin is not added; the remaining preparation methods are the same as in Example 3. Performance testing

[0028] (1) Browning rate The anti-browning effect of the couplings prepared in Examples 1-5 and Comparative Examples 1-3 was determined. The pretreated shoot tips of Xinjiang early yellow fig were cut into 1cm segments and placed on a culture medium (the culture medium composition was MS + 10mg / L couplings) for 15 days. The browning rate was then counted.

[0029] (2) Polyphenol oxidase inhibition rate Weigh 1g of bacitracin-ursolic acid-avermectin conjugate sample and dissolve it in 100mmol / L sodium acetate buffer (pH 5.5) containing 1% PVPP. Homogenize at low temperature and centrifuge at 8000r / min for 10min at 4℃. Mix the supernatant with 4mL of buffer substrate and 1mL of catechol. Measure the absorbance of the supernatant at 420nm. Set up a blank control.

[0030] Table 1. Browning rate and polyphenol oxidase inhibition rate of Examples 1-5 and Comparative Examples 1-3

[0031] As shown in Table 1, the browning rate of Examples 1-5 was lower than that of Comparative Examples 1-3, while the polyphenol oxidase inhibition rate was higher than that of Comparative Examples 1-3. Furthermore, the bacitracin-ursolic acid-avermectin conjugate prepared in Example 3 exhibited good anti-browning effects and antibacterial activity. Examples 10-11 and Comparative Example 5 all used the bacitracin-ursolic acid-avermectin conjugate prepared in Example 3. Example 6

[0032] A method for preparing hormone-enriched microspheres includes the following steps: Step 1. Dissolve 2g of chitosan in glacial acetic acid to prepare a 2wt% chitosan solution. Then, mix the composition made of 5g mannitol, 5g L-proline and 5g betaine with 75mL of chitosan solution. Add 1.5wt% calcium chloride solution dropwise at a uniform rate and stir at 250rpm for 30min to obtain chitosan microspheres. Step 2. Dissolve 1g of sodium alginate in water to prepare a 1wt% sodium alginate solution. Add 2g of sodium lignin sulfonate to 100mL of sodium alginate solution and mix well to prepare a mixed solution. Then mix 10g of chitosan microspheres with 40mL of the mixed solution and add 1.5wt% calcium chloride solution dropwise at a uniform rate. Stir at 300rpm for 35min to obtain hormone-enriched microspheres. Example 7

[0033] A method for preparing hormone-enriched microspheres includes the following steps: Step 1. Dissolve 2g of chitosan in glacial acetic acid to prepare a 2wt% chitosan solution. Then, mix the composition made of 3g mannitol, 6g L-proline and 6g betaine with 75mL of chitosan solution. Add a 2wt% calcium chloride solution dropwise at a uniform rate and stir at 200rpm for 30min to obtain chitosan microspheres. Step 2. Dissolve 1g of sodium alginate in water to prepare a 1wt% sodium alginate solution. Add 2g of sodium lignin sulfonate to 100mL of sodium alginate solution and mix well to prepare a mixed solution. Then mix 10g of chitosan microspheres with 40mL of the mixed solution and add 1.5wt% calcium chloride solution dropwise at a uniform rate. Stir at 250rpm for 35min to obtain hormone-enriched microspheres. Example 8

[0034] A method for preparing hormone-enriched microspheres includes the following steps: Step 1. Dissolve 2g of chitosan in glacial acetic acid to prepare a 2wt% chitosan solution. Then, mix the composition made of 5g mannitol, 5g L-proline and 5g betaine with 105mL of chitosan solution. Add 1.5wt% calcium chloride solution dropwise at a uniform rate and stir at 250rpm for 30min to obtain chitosan microspheres. Step 2. Dissolve 1g of sodium alginate in water to prepare a 1wt% sodium alginate solution. Add 2g of sodium lignin sulfonate to 100mL of sodium alginate solution and mix well to prepare a mixed solution. Then mix 10g of chitosan microspheres with 40mL of the mixed solution and add 1.5wt% calcium chloride solution dropwise at a uniform rate. Stir at 300rpm for 35min to obtain hormone-enriched microspheres. Example 9

[0035] A method for preparing hormone-enriched microspheres includes the following steps: Step 1. Dissolve 2g of chitosan in glacial acetic acid to prepare a 2wt% chitosan solution. Then, mix the composition made of 5g mannitol, 5g L-proline and 5g betaine with 75mL of chitosan solution. Add 1.5wt% calcium chloride solution dropwise at a uniform rate and stir at 250rpm for 30min to obtain chitosan microspheres. Step 2. Dissolve 1.5g of sodium alginate in water to prepare a 1.5wt% sodium alginate solution. Add 4g of sodium lignin sulfonate to 100mL of sodium alginate solution and mix well to prepare a mixed solution. Then mix 10g of chitosan microspheres with 40mL of the mixed solution and add 1.5wt% calcium chloride solution dropwise at a uniform rate. Stir at 300rpm for 35min to obtain hormone-enriched microspheres. Comparative Example 4

[0036] The difference between this comparative example and Example 9 is that sodium alginate and sodium lignosulfonate are not added; the remaining preparation methods are the same as in Example 9. Performance testing

[0037] (1) Average particle size The hormone-enriched microspheres prepared in Examples 6-9 and Comparative Example 4 were diluted 250 times to prepare sample solutions, and the average particle size was determined using a laser particle size analyzer.

[0038] (2) Hormone enrichment efficiency 1g of hormone-enriched microspheres prepared in Examples 6-9 and Comparative Example 4 were immersed in 10mg / mL BA solution and 10mg / mL NAA solution for 1h, respectively, and the enrichment efficiency of the hormone-enriched microspheres was determined by enzyme-linked immunosorbent assay.

[0039] Table 2 shows the average particle size and enrichment efficiency of Examples 6-9 and Comparative Example 4.

[0040] As shown in Table 2, the average particle size of Examples 6-9 was higher than that of Comparative Example 4, while the BA and NAA enrichment efficiencies were also higher than those of Comparative Example 4. This indicates that the hormone-enriched microspheres prepared in these examples can efficiently enrich BA and NAA, and can significantly improve the induction rate and rooting rate of *Figa arguta* var. *mongolica* during tissue culture rapid propagation in Xinjiang. Examples 10-11 and Comparative Example 6 all used the hormone-enriched microspheres prepared in Example 9. Example 10

[0041] A method for rapid propagation of early-ripening figs from Xinjiang via tissue culture includes the following steps: S1. Explant selection and treatment The tips of the new shoots of early yellow fig from Xinjiang were cut into 2cm sections as explants. After collection, the young leaves were removed. The shoots were first soaked in 75% ethanol solution for 10 seconds, then sterilized in a mixed solution of 2% sodium hypochlorite and bacitracin-ursolic acid-avermectin conjugate (sodium hypochlorite: bacitracin-ursolic acid-avermectin conjugate was 1:1) for 25 minutes. Finally, the shoots were rinsed 4 times with sterile water to obtain pretreated fig shoots. S2. Stem tip stripping Place sterilized filter paper on the dissecting microscope, pour in an appropriate amount of sterile water, place a steel ruler on top, adjust the dissecting microscope, and place the prepared fig shoot under the stereomicroscope to carefully observe the stem tip structure, facilitating the stem tip dissection operation; insert the dissecting needle tip along the center of the bud base, but do not touch the bud tip, and then place it under the binocular dissecting microscope, hold the dissecting needle handle with your left hand and the dissecting scalpel handle with your right hand, and peel off the leaves layer by layer to expose the bud until only 2-3 small leaf primordia remain and the growing point is exposed; use a sharp dissecting scalpel to cut off the 0.5mm stem tip; S3. Induction Culture The 0.5 mm shoot tips obtained from S2 were inoculated into induction medium and induced to form adventitious shoots at 25°C, light intensity of 2500 lx, and photoperiod of 10 h / day. The composition of the induction medium was MS + 0.2 mg / L BA + 0.1 mg / L hormone-enriched microspheres. S4. Subculture Adventitious buds of S3 that have grown to 1.0 cm were divided into bud blocks of 4 small buds and transferred to subculture proliferation medium. Subculture was carried out at 25℃, light intensity of 3000 lx, and photoperiod of 12 h / day, and was carried out once a month to obtain rootless seedlings. The composition of the subculture proliferation medium was MS + 0.2 mg / L BA + 0.2 mg / L hormone-enriched microspheres. S5. Rooting Culture Rootless seedlings with a length of 3cm were selected and inoculated onto rooting medium. Rooting culture was carried out at 25℃, light intensity of 3000lx, and photoperiod of 12h / day to obtain rooted tissue culture seedlings. S6. Domestication and Transplantation Once the rooted tissue culture seedlings have developed complete root systems and grown to 8 leaves, they are moved to a greenhouse for acclimatization treatment for 8 days without opening the culture bottle or 2 days with the bottle opened. Then, they are removed from the culture bottle and the culture medium is washed off the roots. They are then transplanted into a sterilized vermiculite substrate and covered with a film to retain moisture. After 1 week, ventilation is gradually increased until the film is removed. When the rooted tissue culture seedlings grow to 10cm, they can be planted in the orchard soil. Example 11

[0042] A method for rapid propagation of early-ripening figs from Xinjiang via tissue culture includes the following steps: S1. Explant selection and treatment The tips of the new shoots of early yellow fig from Xinjiang were cut into 2cm sections as explants. After collection, the young leaves were removed. The shoots were first soaked in 75% ethanol solution for 10s, then sterilized in a 2% bacitracin-ursolic acid-avermectin conjugate mixed solution for 25min, and then rinsed 4 times with sterile water to obtain pretreated fig shoots. S2. Stem tip stripping Place sterilized filter paper on the dissecting microscope, pour in an appropriate amount of sterile water, place a steel ruler on top, adjust the dissecting microscope, and place the prepared fig shoot under the stereomicroscope to carefully observe the stem tip structure, facilitating the stem tip dissection operation; insert the dissecting needle tip along the center of the bud base, but do not touch the bud tip, and then place it under the binocular dissecting microscope, hold the dissecting needle handle with your left hand and the dissecting scalpel handle with your right hand, and peel off the leaves layer by layer to expose the bud until only 2-3 small leaf primordia remain and the growing point is exposed; use a sharp dissecting scalpel to cut off the 0.5mm stem tip; S3. Induction Culture The 0.5 mm shoot tips obtained from S2 were inoculated into induction medium and induced to form adventitious shoots at 25°C, light intensity of 2500 lx, and photoperiod of 10 h / day. The composition of the induction medium was MS + 0.2 mg / L BA + 0.1 mg / L hormone-enriched microspheres. S4. Subculture Adventitious buds of S3 that have grown to 1.0 cm were divided into bud blocks of 4 small buds and transferred to subculture proliferation medium. Subculture was carried out at 25℃, light intensity of 3000 lx, and photoperiod of 12 h / day, and was carried out once a month to obtain rootless seedlings. The composition of the subculture proliferation medium was MS + 0.2 mg / L BA + 0.2 mg / L hormone-enriched microspheres. S5. Rooting Culture Rootless seedlings with a length of 3cm were selected and inoculated onto rooting medium. Rooting culture was carried out at 25℃, light intensity of 3000lx, and photoperiod of 12h / day to obtain rooted tissue culture seedlings. S6. Domestication and Transplantation Once the rooted tissue culture seedlings have developed complete root systems and grown to 8 leaves, they are moved to a greenhouse for acclimatization treatment for 8 days without opening the culture bottle or 2 days with the bottle opened. Then, they are removed from the culture bottle and the culture medium is washed off the roots. They are then transplanted into a sterilized vermiculite substrate and covered with a film to retain moisture. After 1 week, ventilation is gradually increased until the film is removed. When the rooted tissue culture seedlings grow to 10cm, they can be planted in the orchard soil. Comparative Example 5

[0043] The difference between this comparative example and Example 10 is that the hormone-enriched microspheres prepared in Comparative Example 4 are used; the remaining methods are the same as in Example 10. Comparative Example 6

[0044] The difference between this comparative example and Example 10 is that the bacitracin-ursolic acid-avermectin conjugate prepared in Example 3 is not added; the remaining methods are the same as in Example 10. Comparative Example 7

[0045] The difference between this comparative example and Example 10 is that hormone-enriched microspheres are not added in the S3 induction culture; the remaining methods are the same as in Example 10. Comparative Example 8

[0046] The difference between this comparative example and Example 10 is that hormone-enriched microspheres are not added in the S4 subculture; the remaining methods are the same as in Example 10. Comparative Example 9

[0047] The difference between this comparative example and Example 10 is that hormone-enriched microspheres are not added in the S5 rooting culture; the remaining methods are the same as in Example 10. Performance testing

[0048] (1) Pollution rate The pretreated shoot tips from Examples 10-11 and Comparative Examples 5-9 were inoculated into MS medium. Thirty explant samples were inoculated into each example or comparative example and cultured at 25°C, light intensity of 2000 lx, and light duration of 16 h / day for 30 days. The contamination rate of each group was counted.

[0049] (2) Proliferation rate and rooting rate For the S4 subculture samples described in Examples 10-11 and Comparative Examples 5-9, 30 samples were prepared for each group and subcultured for 30 days at 25℃, light intensity 3000 lx, and photoperiod 12 h / day. The proliferation rate of each group was calculated. For the S5 rooting culture samples described in Examples 10-11 and Comparative Examples 5-9, 30 samples were prepared for each group and rooted for 30 days at 25℃, light intensity 3000 lx, and photoperiod 12 h / day. The rooting rate of each group was calculated.

[0050] Table 3 shows the contamination rate, proliferation rate, and rooting rate of Examples 10, 11, and Comparative Examples 5-9.

[0051] Figure 1 and Figure 2 This demonstrates the formation and growth of adventitious buds induced during the rapid propagation of early yellow figs from Xinjiang via tissue culture. Figure 3 and Figure 4 This reflects the status of tissue culture seedling cultivation during the rapid propagation of early yellow figs in Xinjiang via tissue culture. Figure 5 and Figure 6 The demonstration showed the transplanting of domesticated Xinjiang early yellow fig tissue culture seedlings into potted garden soil substrate or greenhouses.

[0052] Table 3 shows that the contamination rate of Examples 10 and 11 was lower than that of Comparative Examples 5-9, while the proliferation rate and rooting rate were higher. This indicates that the method described in this invention can effectively reduce the explant contamination rate while improving the proliferation efficiency and rooting rate of adventitious buds, demonstrating excellent tissue culture rapid propagation performance. Further analysis shows that Comparative Examples 5-9 showed a decrease in contamination rate, proliferation rate, and rooting rate, indicating that the hormone-enriched microspheres and the bacitracin-ursolic acid-avermectin conjugate played a synergistic role in improving the tissue culture effect. This not only verifies the scientific nature of the formulation design of this invention but also demonstrates its stability and reliability in practical applications. Furthermore, Figures 1 to 3 The results visually demonstrate the growth status of early yellow figs from Xinjiang at various stages of tissue culture, and corroborate the data results, further illustrating that the method of this invention can significantly optimize the rapid propagation system of fig tissue culture and has broad application value.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for rapid propagation system of Xinjiang early yellow fig through tissue culture, characterized in that, Includes the following steps: S1. Explant selection and treatment: The tips of the new shoots of Xinjiang early yellow fig (Fig. 0.05) were cut into 1-2 cm sections as explants. After collection, the young leaves were removed and the shoots were disinfected to obtain pretreated fig shoots. S2. Induction culture: The pretreated fig shoots described in S1 are subjected to shoot tip peeling treatment under sterile environment to obtain 0.3-0.5 mm shoot tips, which are then inoculated on induction culture medium for induction culture to form adventitious buds; S3. Subculture: Divide the adventitious buds of S2 that have grown to 1.0cm into bud blocks of 3-5 small buds, transfer them to subculture proliferation medium for subculture, subculture once a month to obtain rootless seedlings. S4. Rooting culture: Select rootless seedlings with a length of 2-3cm and inoculate them onto rooting culture medium for rooting culture to obtain rooted tissue culture seedlings; S5. Acclimatization and Transplanting: Once the rooted tissue culture seedlings have developed complete root systems and grown to 6-8 leaves, they are moved to a greenhouse for acclimatization treatment for 7-10 days without opening the culture bottle or 1-2 days with the bottle opened. Then, they are removed from the culture bottle and the culture medium is washed off the roots. They are then transplanted into a sterilized vermiculite substrate and covered with a film to retain moisture. After one week, ventilation is gradually increased until the film is removed. When the rooted tissue culture seedlings grow to 10cm, they can be planted in the orchard soil.

2. The method for rapid propagation system of Xinjiang early yellow fig tissue culture according to claim 1, characterized in that, The disinfection method in S1 is as follows: first, soak in 75% ethanol solution for 10 seconds, then sterilize with a mixed solution of 2-3.5 wt% sodium hypochlorite and bacitracin-ursolic acid-avermectin conjugate or 2-4 wt% bacitracin-ursolic acid-avermectin conjugate for 20-25 minutes, and then rinse with sterile water 4-5 times.

3. The method for rapid propagation system of Xinjiang early yellow fig tissue culture according to claim 2, characterized in that, The preparation method of the bacitracin-ursolic acid-avermectin conjugate is as follows: ① Dissolve ursolic acid and avermectin in anhydrous ethanol at a mass ratio of 1:(1.5-2.5), react at 60-65℃ for 3-6 hours, and then concentrate under reduced pressure, wash and dry to obtain ursolic acid-avermectin complex; ② Dissolve bacitracin in water to prepare a bacitracin solution with a concentration of 3-5.5wt%, adjust the pH to 5.0-5.5, slowly add 4-6wt% ursolic acid-avermectin complex to the bacitracin solution, react at room temperature for 2-4 hours, concentrate and purify to obtain bacitracin-ursolic acid-avermectin conjugate.

4. The method for rapid propagation system of Xinjiang early yellow fig tissue culture according to claim 1, characterized in that, The induction medium in S2 consists of MS + 0.2 mg / L BA + (0.1-0.15) mg / L hormone-enriched microspheres. The induction culture conditions are: temperature 23-25℃, light intensity 2000-2500 lx, and photoperiod 12-16 h / day. The subculture proliferation medium in S2 consists of MS + 0.2 mg / L BA + (0.15-0.3) mg / L hormone-enriched microspheres. The subculture proliferation conditions are: temperature 25-27℃, light intensity 2500-3000 lx, and photoperiod 15-16 h / day. The rooting medium in S2 consists of 1 / 2 MS + 0.2 mg / L BA. NAA+ (0.1-0.3) mg / L hormone-enriched microspheres; the rooting culture conditions are a temperature of 25-27℃, a light intensity of 2500-3000 lx, and a photoperiod of 15-16 h / day.

5. The method for rapid propagation system of Xinjiang early yellow fig tissue culture according to claim 4, characterized in that, The method for preparing the hormone-enriched microspheres includes the following steps: Step 1. Dissolve chitosan in glacial acetic acid to prepare a chitosan solution with a concentration of 1-2.5 wt%. Then, mix the chitosan solution with a composition made of mannitol, L-proline and betaine at a ratio of 1 g: (4-8 mL) at a uniform rate. Add 1.5-2 wt% calcium chloride solution dropwise at a uniform rate and stir at 200-300 rpm for 25-40 min to obtain chitosan microspheres. Step 2. Dissolve sodium alginate in water to prepare a sodium alginate solution with a concentration of 1-1.5 wt%. Add 2-5% sodium lignin sulfonate to the sodium alginate solution and mix well to prepare a mixed solution. Then mix chitosan microspheres with the mixed solution at a ratio of 1 g: (3-5 mL) and mix well. Add 1.5-2 wt% calcium chloride solution dropwise at a uniform speed and stir at 200-300 rpm for 25-40 min to obtain hormone-enriched microspheres.

6. The method for rapid propagation system of Xinjiang early yellow fig tissue culture according to claim 1, characterized in that, In step 1, the mass ratio of mannitol, L-proline, and betaine is 1:(0.5-3):(1-2).

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