A tissue culture system of elaeagnus ruptrata and a breeding method thereof

Abstract

The present application relates to a kind of Siberian hackberry tissue culture system and its breeding method.The Siberian hackberry tissue culture system includes seed disinfection solution, seed germination medium, subculture rooting medium and cultivation matrix;Wherein, the seed disinfection solution includes sodium hypochlorite solution, and / or the seed germination medium includes N6 or MS medium, sucrose and agar, and / or the subculture rooting medium includes MS medium, IBA, MES, sucrose and agar, and / or the cultivation matrix includes grass carbon, vermiculite and perlite.The Siberian hackberry tissue culture system provided by the present application can be well used for Siberian hackberry seedling efficient rapid propagation production, test procedure is simple, wide genotype is adapted, and has good popularization, production application value.

Description

Technical Field

[0001] This invention relates to the field of Siberian white thorn propagation technology, and in particular to a Siberian white thorn tissue culture system and its propagation method. Background Technology

[0002] *Nitraria* L. is an ancient genus within the family Zygophyllaceae, representing Tertiary relict plants. There are 12 species worldwide, distributed across Asia, Africa, Europe, and Australia, with 7 species found in my country, primarily in the northwest region. *Nitraria siberiana* is the dominant species, thriving in exceptionally harsh environments with strong resilience, drought resistance, salt tolerance, and tolerance to poor soil conditions. It can grow in heavily saline-alkali soils and is one of the important tree species for establishing communities in saline-alkali desert regions. The tender stems, leaves, and fruits of *Nitraria siberiana* contain high levels of crude fat, crude protein, crude fiber, and vitamin C, making it a high-quality forage plant. For a long time, the fruits of *Nitraria siberiana* have been used in traditional medicine to treat spleen and stomach weakness, neurasthenia, indigestion, and colds, while its leaves are used as a folk remedy for spasms, arrhythmia, and neuralgia. Therefore, *Nitraria tangutorum* is a tree species with enormous ecological benefits and value for forage and medicinal purposes, making it highly valuable for development and utilization. Currently, seed propagation is commonly used in production, but this method suffers from low natural seed germination rates, uneven seedling growth, and low seedling propagation index, posing significant challenges to large-scale cultivation. Plant tissue culture propagation is unaffected by external environmental conditions, allowing for year-round production and rapid, efficient production of large quantities of seedlings, effectively promoting the development of the *Nitraria tangutorum* industry. However, plant tissue culture often exhibits limitations, with specific formulations only yielding ideal results for specific genotypes of the same plant species. While there are some reports on tissue culture techniques for *Nitraria tangutorum*, most are limited to scientific research, with formulations targeting specific genotypes. No reports have been found on the rapid propagation of widely adaptable *Nitraria tangutorum* through tissue culture, which to some extent limits the breeding and widespread application of *Nitraria tangutorum*.

[0003] Most studies on the rapid propagation techniques of Tissue Culture of Nitraria tangutorum have focused on Nitraria tangutorum (He Zhenglun et al., 1998; Zhang Hongxiao et al., 2003; Guo Yehong et al., 2009; Cheng Tielong et al., 2013). Research on Nitraria siberiana is relatively limited, with only Wang Chenxia and Chen Guilin (2007) and Zhang Qichang et al. (2008) reporting on tissue culture techniques for Nitraria siberiana.

[0004] Currently reported formulas for rapid propagation of *Nitraria tangutorum* via tissue culture are all for stem segments from plants in specific regions; their broad applicability remains to be determined. Furthermore, obtaining sterile explants is fundamental to plant tissue culture and plays a crucial role in the entire process. However, the explants used in current *Nitraria tangutorum* tissue culture techniques are all young stem segments. During sterilization, issues arise such as short sterilization times, incomplete sterilization of stem segments, high contamination rates, and prolonged sterilization times causing significant damage to young stem segments, requiring a longer recovery time in the culture medium. Seeds have a closed seed coat. During sterilization, the disinfectant primarily disinfects the seed coat surface, causing minimal damage to the embryo inside. Therefore, even after thorough seed coat sterilization, the seeds retain strong viability, and seedlings grow rapidly. *Nitraria tangutorum* seedlings sometimes show weakened growth after a period of tissue culture, possibly due to changes in the pH of the culture medium as the seedlings grow. 2-Morpholine ethanesulfonic acid (MES) is a biological buffer that can stabilize the pH of a solution. Whether its addition to the culture medium of *Nitraria tangutorum* will promote the growth of tissue-cultured seedlings is unknown. Reported rapid propagation techniques for *Nitraria tangutorum* tissue culture can be broadly divided into five steps: obtaining sterile explants, primary culture, subculture proliferation culture, rooting culture, and hardening-off transplanting. These steps are numerous and result in slow seedling formation. The only simplified method is described by Zhang Yanping et al. (2015), who disclosed a method for direct rooting of single-bud stem segments. However, this method requires stem segments with terminal buds and lacks details on hardening-off transplanting, making it unsuitable for large-scale production of *Nitraria tangutorum*. Currently, there is an urgent need for a new, efficient propagation method for *Nitraria tangutorum* to address the aforementioned problems in its tissue culture propagation. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention has developed a tissue culture system and rapid propagation technology for Siberian white thorn seedlings that is simple to implement, adaptable to a wide range of genotypes, and applicable to production.

[0006] Specifically, the Siberian white thorn tissue culture system provided by the first aspect of this invention includes a seed disinfectant, a seed germination medium, a subculture rooting medium, and a cultivation substrate; wherein the seed disinfectant includes a sodium hypochlorite solution, and / or the seed germination medium includes N6 medium or MS medium, sucrose and agar, and / or the subculture rooting medium includes MS medium, IBA, MES, sucrose and agar, and / or the cultivation substrate includes peat moss, vermiculite and perlite. This invention utilizes the above-mentioned Siberian white thorn tissue culture system, enabling efficient and rapid propagation of Siberian white thorn seedlings through the synergistic cultivation of the seed disinfectant, seed germination medium, subculture rooting medium, and cultivation substrate. The entire production cycle can be shortened to approximately 70 days, demonstrating significant value for promotion and production application.

[0007] Preferably, the seed disinfectant is a sodium hypochlorite solution with a mass concentration of 8% to 12%, and more preferably a sodium hypochlorite solution with a mass concentration of 10%.

[0008] Preferably, the seed germination medium consists of N6 medium or MS medium, 20-30 g / L sucrose and 6-7 g / L agar, with the pH adjusted to 5.8.

[0009] Further preferably, the subculture rooting medium consists of MS medium, 0.5 mg / L IBA, 0.4–0.6 g / L MES, 15–25 g / L sucrose and 6–7 g / L agar, with the pH adjusted to 5.8.

[0010] Further preferably, the cultivation substrate is composed of peat moss, vermiculite and perlite in a volume ratio of 2:2:1, with a moisture content of 80% to 90%.

[0011] The application of any of the above-mentioned Siberian Nitraria tissue culture systems provided in the second aspect of the present invention, particularly in the efficient and rapid propagation of Siberian Nitraria seedlings.

[0012] Preferably, the application includes at least one of the following: applied to achieve efficient and rapid propagation of Siberian white thorn seedlings, shortening the production cycle to 70 days; the Siberian white thorn seeds used include seed sources from Hebei, Tianjin, Gansu, and Inner Mongolia; and the subculture rooting medium used replaces the culture medium for subculture proliferation, seedling strengthening, and rooting.

[0013] The second aspect of this invention provides a highly efficient Siberian white thorn propagation method independent of genotype, which uses any of the above-mentioned Siberian white thorn tissue culture systems for propagation; preferably, it includes disinfecting the seeds of Siberian white thorn with the seed disinfectant; culturing the disinfected seeds with the seed germination medium to obtain seedlings; culturing the seedlings in the subculture rooting medium to obtain rooted seedlings; and culturing the rooted seedlings in the cultivation substrate.

[0014] Further preferably, the genotype-independent, high-efficiency Siberian white thorn breeding method includes:

[0015] 1) Seed disinfection: The seeds of Siberian white thorn were disinfected using the aforementioned seed disinfection solution;

[0016] 2) Seed germination culture: Sterilized seeds are placed in tissue culture bottles containing seed germination medium and cultured in a culture room to obtain seedlings;

[0017] 3) Subculture rooting culture: The sterile seedlings that have grown to the height of the bottle are cut into stem segments containing single buds and transferred to tissue culture bottles containing the subculture rooting medium to obtain rooted seedlings;

[0018] 4) Hardening off and transplanting: Place the rooted seedlings from the tissue culture room in the greenhouse, then take the rooted seedlings out of the tissue culture bottle, wash the culture medium with clean water, soak them in carbendazim solution, and then transplant them into the nutrient pots of the cultivation substrate. Water them thoroughly, cover them with plastic sheeting and shade netting to maintain humidity inside the greenhouse.

[0019] Further preferably, the seed disinfectant is an 8%–12% sodium hypochlorite solution; the seed germination medium consists of N6 medium or MS medium, 20–30 g / L sucrose, and 6–7 g / L agar, with the pH adjusted to 5.8; the subculture rooting medium consists of MS medium, 0.5 mg / L IBA, 0.4–0.6 g / L MES, 15–25 g / L (preferably 15–20 g / L) sucrose, and 6–7 g / L agar, with the pH adjusted to 5.8; and the cultivation substrate consists of peat moss, vermiculite, and perlite in a volume ratio of 2:2:1, with a humidity of 80%–90%.

[0020] Further preferably, the genotype-independent, high-efficiency Siberian white thorn breeding method includes:

[0021] 1) Seed disinfection: Soak the seeds in clean water overnight, disinfect them with 75% alcohol for 15±5 seconds, rinse them with sterile water 3 to 5 times, and then soak them in 8% to 12% sodium hypochlorite solution for 15 to 20 minutes, gently shaking the disinfectant solution during the process to ensure that the disinfectant solution fully contacts the seeds, and then rinse them with sterile water 3 to 5 times.

[0022] 2) Seed germination culture: Sterilized seeds are placed in tissue culture bottles containing seed germination medium and cultured in a culture room. The temperature of the culture room is controlled at 25℃, the light intensity is 2000-2500 Lx, and the light duration is 14h / d. Seedlings with a height of 6-8cm are obtained after 20 days.

[0023] 3) Subculture rooting culture: Cut the seedlings that have grown to the height of the bottle into stem segments containing single buds and transfer them to tissue culture bottles containing rooting and seedling strengthening culture medium; after 30 days, the stem segments will grow to a height of 7-9 cm, with a rooting rate of over 94% and robust seedlings with well-developed root systems.

[0024] 4) Hardening off and transplanting: Take robust seedlings from the tissue culture room to the greenhouse. The temperature in the greenhouse is 18-28℃ and the humidity is 40±5%. Place them for 3-4 days, unscrew the bottle caps, and place them for another 3-4 days. Then, take the rooted seedlings out of the tissue culture bottles, wash the culture medium with clean water, soak the seedlings in a 0.1±0.02g / L carbendazim solution for 1±0.5min, transplant the seedlings into the substrate nutrient pots, water them thoroughly, cover them with a small arched shed with plastic sheeting, and cover them with a shade net. Water them as needed according to the humidity of the small arched shed to maintain the humidity at 80%-90%. Remove the shade netting and plastic sheeting after half a month. The survival rate of transplanted seedlings is over 90%.

[0025] The beneficial effects of this invention are at least as follows: Currently reported explants for tissue culture of Siberian white thorn generally use young stem segments. However, the sterilization of these young stem segments is often incomplete or results in their death. Even when sterilization is successful, the damage to the young stem segments is significant, requiring a prolonged period of growth in the culture medium to recover. The seeds used in this invention have a closed seed coat. During sterilization, the disinfectant primarily disinfects the surface of the seed coat, with minimal impact on the embryo inside. Therefore, using seeds as explants yields seedlings with strong vitality and rapid growth, reaching 6-7 cm in height in approximately 20 days. The subculture and rooting culture medium selected in this invention is a single formula, better replacing the three different culture medium formulas currently used for subculture proliferation, seedling strengthening, and rooting. Simultaneously, this technology system enables efficient and rapid propagation of Siberian white thorn seedlings, shortening the entire production cycle to approximately 70 days. The existing tissue culture formulas for Siberian white thorn are designed for germplasm from a specific region, and their applicability needs to be determined. The tissue culture formula used in this invention has good rapid propagation effects on germplasm from Hebei, Tianjin, Gansu, Inner Mongolia and other regions. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The plant height of Siberian white thorn seedlings from different germplasm sources provided by this invention;

[0028] Figure 2 The growth of *Nitraria tangutorum* on the germination medium provided by this invention;

[0029] Figure 3The growth of Siberian white thorn on the subculture rooting medium provided by the present invention;

[0030] Figure 4 The present invention provides for the transplantation of Siberian white thorns into a seedling tray;

[0031] Figure 5 Transplanted Siberian white thorn seedlings provided for this invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, specific techniques or conditions in the embodiments of this invention shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Devices, instruments, reagents, etc., whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. All raw materials used in this invention are readily available in the domestic market.

[0034] In this embodiment of the invention, N6 is N6 culture medium base salt with product model PM1311 from Coollab; MS is MS culture medium base salt with product model PM1201 from Coollab.

[0035] Example 1

[0036] 1) Seed disinfection: Soak the seeds (Gansu seed source) in clean water overnight. The next day, after the seeds have absorbed water and the seed coat has softened, disinfect them with 75% alcohol for 15 seconds on a clean bench, rinse them 5 times with sterile water, and then soak them in 10% sodium hypochlorite solution for 15 minutes. During this time, shake the disinfectant solution slightly to ensure that it fully contacts the seeds. Then rinse them repeatedly with sterile water 3 times. The germination rate of the seeds was 86.5%, and the contamination rate was 21.3%.

[0037] 2) Seed germination culture: Place the sterilized seeds in a tissue culture bottle containing seed germination medium and culture them in a culture room. The temperature of the culture room is controlled at 25℃, the light intensity is 2000-2500 Lx, and the light duration is 14h / d. Seedlings with an average plant height of 6.7cm can be obtained in about 20 days.

[0038] The seed germination medium was: N6 + 30 g / L sucrose + 6.8 g / L agar, with a pH of 5.8.

[0039] 3) Subculture rooting culture: Sterile seedlings that have grown to the height of the flask are cut into stem segments containing single buds and transferred to tissue culture flasks containing rooting and seedling strengthening medium. After about 30 days, the stem segments can grow into robust seedlings with an average height of 8.4 cm, a rooting rate of 94.444%, and well-developed root systems.

[0040] The subculture rooting medium is: MS + 0.5 mg / L IBA + 0.4 g / L MES + 20 g / L sucrose + 6.8 g / L agar, pH = 5.8.

[0041] 4) Hardening off and transplanting: Take the rooted seedlings from the tissue culture room to the greenhouse (temperature between 18 and 28°C, humidity around 40%) and leave them for 3 days. Unscrew the bottle caps and leave them for another 4 days. Then, take the rooted seedlings out of the tissue culture bottles, wash off the culture medium with clean water, soak the seedlings in a 0.1 g / L carbendazim solution for about 1 minute, and then transplant the seedlings into the substrate nutrient pots. Water them thoroughly, cover them with a plastic sheet and then cover them with a shade net. Observe the humidity of the small arched greenhouse every day and water it accordingly to maintain the humidity of the small arched greenhouse at 80% to 90%. After half a month, gradually remove the shade net and plastic sheet. The survival rate of the transplanted seedlings can reach 91.04%.

[0042] The cultivation substrate is a mixture of peat moss, vermiculite, and perlite in a volume ratio of 2:2:1.

[0043] Example 2

[0044] 1) Seed disinfection: Soak the seeds (Gansu seed source) in clean water overnight. The next day, after the seeds have absorbed water and the seed coat has softened, disinfect them with 75% alcohol for 15 seconds on a clean bench, rinse them 5 times with sterile water, and then soak them in 10% sodium hypochlorite solution for 20 minutes. During this time, shake the disinfectant solution slightly to ensure that it fully contacts the seeds. Then rinse them repeatedly with sterile water 3 times. The germination rate of the seeds was 83.1%, and the contamination rate was 18.6%.

[0045] 2) Seed germination culture: Place the sterilized seeds in a tissue culture bottle containing seed germination medium and culture them in a culture room. The temperature of the culture room is controlled at 25℃, the light intensity is 2000-2500 Lx, and the light duration is 14h / d. Seedlings with an average plant height of 6.9cm can be obtained in about 20 days.

[0046] The seed germination medium was: MS + 30 g / L sucrose + 6.8 g / L agar, pH 5.8.

[0047] 3) Subculture rooting culture: Sterile seedlings that have grown to the height of the flask are cut into stem segments containing single buds and transferred to tissue culture flasks containing rooting and seedling strengthening medium. After about 30 days, the stem segments can grow to a height of 8.5cm, with a rooting rate of 95.26%, well-developed root systems, and robust seedlings.

[0048] The subculture rooting medium is: MS + 0.5 mg / L IBA + 0.6 g / L MES + 20 g / L sucrose + 6.8 g / L agar, pH = 5.8.

[0049] 4) Hardening off and transplanting: Take the rooted seedlings from the tissue culture room to the greenhouse (temperature between 18 and 28°C, humidity around 40%) and leave them for 4 days. Unscrew the bottle caps and leave them for another 3 days. Then, take the rooted seedlings out of the tissue culture bottles, wash off the culture medium with clean water, soak the seedlings in a 0.1 g / L carbendazim solution for about 1 minute, and then transplant the seedlings into the substrate nutrient pots. Water them thoroughly, cover them with a plastic sheet and then cover them with a shade net. Observe the humidity of the small arched greenhouse every day and water it in time to maintain the humidity of the small arched greenhouse at 80% to 90%. After half a month, gradually remove the shade net and plastic sheet. The survival rate of the transplanted seedlings can reach 90.98%.

[0050] The cultivation substrate is a mixture of peat moss, vermiculite, and perlite in a volume ratio of 2:2:1.

[0051] The following examples, along with various experimental treatments, illustrate the beneficial effects of this invention.

[0052] Experimental Example 1

[0053] This experiment investigated the effect of different NaClO disinfection times on the disinfection effect of Siberian white thorn seeds. Seeds were soaked in water overnight. The next day, after the seeds had absorbed water and the seed coat had softened, they were disinfected with 75% alcohol for 15 seconds on a clean bench, rinsed five times with sterile water, and then soaked in 10% sodium hypochlorite solution (5, 10, 15, 20, 25 min). After rinsing repeatedly with sterile water 3–5 times, the seeds were inoculated into Petri dishes containing N6 medium. Four Petri dishes were inoculated for each sodium hypochlorite treatment time, with 25 seeds inoculated per dish. After inoculation, the number of germinating seeds and the number of contaminated seeds were observed and recorded daily. After 10 days, the germination rate and contamination rate were calculated.

[0054] Table 1. Effects of different NaClO disinfection times on the disinfection effect of Siberian white thorn seeds.

[0055]

[0056] The results are shown in Table 1. With increasing NaClO treatment time, the contamination rate of Siberian white thorn seeds gradually decreased. After 15 minutes of disinfection, the contamination rate had dropped to 21%, significantly lower than the rates after 5 and 10 minutes. With further extension of disinfection time, the contamination rate showed a decreasing trend, but the decrease was not significant. The germination rate of the seeds initially increased and then decreased with increasing NaClO treatment time. The germination rate reached its highest point of 86% after 15 minutes of treatment. With further extension of NaClO treatment time, the germination rate decreased, and at 25 minutes, the germination rate was significantly lower than that after 15 minutes. Therefore, disinfecting Siberian white thorn seeds with NaClO for 15-20 minutes is appropriate.

[0057] Experimental Example 2

[0058] This experimental example was used to study the effects of different culture media types on the growth of seedlings. The culture media types are shown in Table 2. Each liter of culture medium was supplemented with 30g of sucrose and 6.8g of agar.

[0059] The seeds were sterilized according to the method of Example 1 of this invention. The sterilized seeds were sown in tissue culture bottles containing seed germination medium. 50 bottles were sown for each culture medium, and one seedling was inoculated in each bottle. The seedling height on each germination medium was measured after 20 days, and the seedling growth was recorded.

[0060] Table 2 Effects of primary germination medium on seedling growth

[0061] MS 6.85±0.63a Green leaves, vigorous seedling growth N6 6.73±0.28a Green leaves, relatively strong seedlings WPM 5.17±0.14c Yellowing leaves, weak seedlings

[0062] The results are shown in Table 2. Siberian white thorn seeds showed good growth on both MS and N6 media. The plant height on the MS medium described in this invention was slightly higher than that on the N6 medium, but the difference was not significant. Seeds grew relatively poorly on WPM medium, with seedlings turning yellow and exhibiting less growth.

[0063] Experimental Example 3

[0064] This experimental example was used to study the effects of different culture medium formulations on the subculture rooting of Siberian white thorn stem segments. After primary culture of Siberian white thorn seeds according to the method in Example 1 of this invention, the seedlings were cut into stem segments containing single buds and transferred to subculture rooting media. The culture medium formulations are shown in Table 3. Each liter of medium contained 20g of sucrose and 6.8g of agar. Thirty bottles of each culture medium were inoculated, with four stem segments per bottle. After 30 days, the rooting rate of the stem segments and the plant growth were recorded.

[0065] Table 3. Effects of different subculture rooting medium formulations on the growth of *Nitraria tangutorum* stem segments.

[0066]

[0067]

[0068] (The culture medium formula for No. 1 is from the report by Wang Chenxia et al. (2007); the culture medium formula for No. 2 is from the report by Zhang Qichang et al. (2008).)

[0069] The experimental results showed that adding 0.5 mg / L IBA to MS medium or 1 / 2 MS medium resulted in a high rooting rate of over 87% for seedlings, but the seedlings were relatively weak and the root system was thinner. Adding MES to MS medium after adding 0.5 mg / L IBA resulted in a rooting rate that first increased and then decreased with increasing MES content. When 0.4 or 0.6 g / L MES was added, the rooting rate reached over 94%, and the seedlings were also relatively tall, exceeding 8.4 cm, with more roots and stronger growth.

[0070] Test Example 4

[0071] This experiment was used to study the effects of different cultivation substrates on the survival rate of transplanted Siberian white thorn seedlings. Siberian white thorn seedlings were hardened off according to the method in Example 2 of this invention, and then transplanted into cultivation substrates. The types of cultivation substrates are shown in Table 4. Each substrate was used to plant 3 seedlings in 3 plug trays, with 32 seedlings planted in each plug tray. The seedling survival rate was recorded after 20 days.

[0072] Table 4. Effects of cultivation substrate on seedling transplant survival rate

[0073] peat 77.08±4.77b vermiculite 82.29±6.51ab perlite 73.96±6.51b Peat moss:vermiculite:perlite = 2:2:1 90.63±3.13a

[0074] The results are shown in Table 4. Using the substrate formula described in this invention, the transplant survival rate of white thorn seedlings can reach 90.63%, which is significantly higher than the survival rate of seedlings when peat moss, vermiculite and perlite are used as cultivation substrates alone.

[0075] Experimental Example 5

[0076] This experiment was used to study the effect of this tissue culture system on the rapid propagation of *Nitraria tangutorum* from different seed sources. 400 seeds (collected from different plants in Gansu, Hebei, Inner Mongolia, Tianjin, etc., 100 seeds / seed source) were cultured using the method described in Example 2.

[0077] 1) The results of seed disinfection are shown in Table 1. There were no significant differences in the contamination rate and germination rate of seeds from different seed sources, indicating that the disinfection method screened in this application is universally applicable to seeds from these four seed sources.

[0078] Table 5. Effects of disinfection treatment on contamination and germination of *Nitraria tangutorum* seeds from different sources.

[0079] Gansu 20±3.27a 83±3.83a Hebei 19±3.83a 84±5.66a Inner Mongolia 21±2.00a 82±5.16a Tianjin 20±4.62a 85±5.03a

[0080] 2) Growth of different seedlings of *Nitraria tangutorum* on primary culture medium, as follows: Figure 1 As shown in the figure (dots represent outliers, upper and lower horizontal lines represent maximum and minimum values ​​respectively, upper and lower horizontal lines of the box represent upper and lower quartiles respectively, small squares inside the box represent the mean, and horizontal lines inside the box represent the median), the average plant height of seeds from Gansu, Hebei, Inner Mongolia, and Tianjin sources is 6.5, 6.7, 6.3, and 6.9 cm respectively, the maximum values ​​are 7.8, 7.8, 6.8, and 7.9 cm respectively, and the minimum values ​​are 6.1, 5.9, 5.4, and 5.7 cm respectively. The coefficients of variation for plant height are 0.05, 0.07, 0.05, and 0.08, respectively. The growth status of all sources is good and relatively uniform.

[0081] 3) Plant height of different seedlings of *Nitraria tangutorum* stem segments on subculture rooting medium, as shown in the figure. Figure 2 As shown: Sterile seedlings that have grown to the height of the flask are cut into stem segments containing single buds and transferred to tissue culture flasks containing rooting and seedling-strengthening medium. After approximately 30 days, the seedling height is measured as shown. Figure 1 As shown, the average plant heights of the seed sources from Gansu, Hebei, Inner Mongolia, and Tianjin were 8.4, 8.5, 8.3, and 8.5 cm, respectively, with maximum values ​​of 8.9, 8.9, 8.7, and 9.1 cm, and minimum values ​​of 7.6, 7.6, 7.4, and 8.1 cm, respectively. The coefficients of variation for plant height were 0.04, 0.04, 0.03, and 0.03, respectively, indicating relatively uniform growth. Furthermore, the rooting rates of the four seed sources were 94.99%, 94.06%, 95.86%, and 97.21%, respectively, and the root systems were all robust. The subculture seedlings from the stem segments of *Nitraria tangutorum* from all four seed sources showed good growth on this culture medium.

[0082] 4) After hardening off in a greenhouse, the transplant survival rates of tissue-cultured seedlings from the four seed sources of *Nitraria tangutorum* were as follows: 90.86%, 92.03%, 91.62%, and 93.95% for seedlings from Gansu, Hebei, Inner Mongolia, and Tianjin, respectively. Using this hardening-off transplanting method, the transplant survival rates of tissue-cultured *Nitraria tangutorum* seedlings from all four seed sources were satisfactory.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for breeding Siberian white thorns that is independent of genotype, characterized in that, The seeds of *Nitraria tangutorum* were disinfected and cultured using a tissue culture system. This system included a seed disinfectant, seed germination medium, subculture rooting medium, and a cultivation substrate. The seeds were disinfected using the seed disinfectant; the disinfected seeds were cultured in the seed germination medium to obtain seedlings; the seedlings were then cultured in the subculture rooting medium to obtain rooted seedlings; and the rooted seedlings were then cultured in the cultivation substrate. The seed disinfectant was a 10% sodium hypochlorite solution, and the seed disinfection time was 15-20 min. The seed germination medium consisted of N6 medium or MS medium, 20-30 g / L sucrose, and 6-7 g / L agar. The subculture rooting medium consisted of MS medium, 0.5 mg / L IBA, 0.4-0.6 g / L MES, and 15-25 mg / L agar. The cultivation substrate consists of g / L sucrose and 6-7 g / L agar, and is composed of peat moss, vermiculite and perlite in a volume ratio of 2:2:

1.

2. The method according to claim 1, characterized in that, The pH of the seed germination medium was adjusted to 5.

8.

3. The method according to claim 1, characterized in that, The pH of the subculture rooting medium was adjusted to 5.

8.

4. The method according to any one of claims 1-3, characterized in that, The cultivation substrate has a moisture content of 80% to 90%.

5. The method according to claim 1, characterized in that, include: 1) Seed disinfection: The seeds of Siberian white thorn were disinfected using the aforementioned seed disinfection solution; 2) Seed germination culture: Sterilized seeds are placed in tissue culture bottles containing seed germination medium and cultured in a culture room to obtain seedlings; 3) Subculture rooting culture: The sterile seedlings that have grown to the height of the bottle are cut into stem segments containing single buds and transferred to tissue culture bottles containing the subculture rooting medium to obtain rooted seedlings; 4) Hardening off and transplanting: Place the rooted seedlings from the tissue culture room in the greenhouse, then take the rooted seedlings out of the tissue culture bottle, wash the culture medium with clean water, soak them in carbendazim solution, and then transplant them into the nutrient pots of the cultivation substrate. Water them thoroughly, cover them with plastic sheeting and shade netting to maintain humidity inside the greenhouse.

6. The method according to claim 5, characterized in that, The seed disinfectant is a 10% sodium hypochlorite solution; the seed germination medium consists of N6 medium or MS medium, 20-30 g / L sucrose and 6-7 g / L agar, with the pH adjusted to 5.8; the subculture rooting medium consists of MS medium, 0.5 mg / L IBA, 0.4-0.6 g / L MES, 15-20 g / L sucrose and 6-7 g / L agar, with the pH adjusted to 5.8; the cultivation substrate consists of peat moss, vermiculite and perlite in a volume ratio of 2:2:1, with a humidity of 80%-90%.

7. The method according to claim 1, characterized in that, include: 1) Seed disinfection: Soak the seeds in clean water overnight, disinfect with 75% alcohol for 15±5 s, rinse with sterile water 3~5 times, then soak the seeds in 10% sodium hypochlorite solution for 15~20 min, gently shaking the disinfectant solution during the process, and then rinse with sterile water 3~5 times. 2) Seed germination culture: Sterilized seeds are placed in tissue culture bottles containing seed germination medium and cultured in a culture room. The temperature of the culture room is controlled at 25±1℃, the light intensity is 2000~2500 Lx, the light duration is 14 h / d, and seedlings with a height of 6~8 cm are obtained after 20 days. 3) Subculture rooting culture: The seedlings that have grown to the height of the bottle are cut into stem segments containing single buds and transferred to tissue culture bottles containing subculture rooting medium; after 30 days, the stem segments grow to a height of 7-9 cm, and the rooting rate can reach more than 94%, forming robust seedlings with well-developed root systems. 4) Hardening off and transplanting: Take the healthy seedlings from the tissue culture room to the greenhouse. The temperature in the greenhouse is 18~28℃ and the humidity is 40±5%. Place them for 3~4 days, unscrew the bottle caps, and place them for another 3~4 days. Then take the rooted seedlings out of the tissue culture bottles, wash the culture medium with clean water, soak the seedlings in 0.1±0.02 g / L carbendazim solution for 1±0.5 min, transplant the seedlings into the substrate nutrient pots, water them thoroughly, cover them with plastic sheeting and a shade net, water according to the humidity of the small arched greenhouse, and maintain the humidity at 80%~90%. After half a month, remove the shade net and plastic sheeting. The survival rate of the transplanted seedlings is over 90%.

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