A rapid propagation method for inducing seedling of nitraria tangutorum leaves

Abstract

This invention discloses a rapid propagation method for *Nitraria tangutorum* seedlings induced from leaves, belonging to the field of tissue culture. The method includes: rooting culture of explant leaves, with a rooting medium of 1 / 2 MS + 0.5 mg / L indolebutyric acid; proliferation culture, with a proliferation medium of MS + 2.0 mg / L 6-benzylaminopurine + 1.0 mg / L zeatin + 2.0 mg / L indolebutyric acid; and rooting culture, with a rooting medium of 1 / 3 MS + 2.0 mg / L indolebutyric acid. This invention achieves rapid propagation of *Nitraria tangutorum* under artificially controlled conditions using tissue culture (from explant culture to seedling rooting takes only 35-45 days). Compared with previous tissue culture methods, this invention eliminates the callus culture step, avoiding browning, vitrification, and contamination that occur in callus culture, thus significantly accelerating the seedling formation rate and greatly improving seedling quality.

Description

A rapid propagation method for inducing seedlings from leaves of *Nitraria tangutorum*. Technical Field

[0001] This invention relates to the field of tissue culture, and in particular to a rapid propagation method for inducing seedlings from leaves of *Nitraria tangutorum*. Background Technology

[0002] *Nitraria tangutorum* Bobr., belonging to the genus *Nitraria* in the family Zygophyllaceae, is a perennial shrub mainly distributed in northern Shaanxi, central and western Inner Mongolia, Hexi Corridor in Gansu, and Xinjiang in my country. It commonly grows in desert and semi-desert lacustrine sandy areas, river terraces, sandy plains at the foot of mountains, and clay soils with aeolian deposits. *Nitraria tangutorum* Bobr. exhibits strong adaptability to extreme desert habitats, being drought-resistant, salt-tolerant, wind-eroded, sand-buried, heat-resistant, and tolerant of poor soil. Its fruit is sweet and sour, and has spleen-strengthening, digestive, lactation-promoting, and calming effects; it can be used to treat spleen and stomach weakness, indigestion, insufficient lactation after childbirth, and neurasthenia. Propagation is generally done by seed sowing.

[0003] Currently, domestic and international research on *Nitraria tangutorum* mainly focuses on drought and salt tolerance mechanisms and seedling cultivation techniques. There are a few reports on tissue culture, such as CN103250643B, a method for in vitro rooting culture of *Nitraria tangutorum* clonal lines. While this patent successfully induces rooting in *Nitraria tangutorum*, it employs conventional tissue culture methods: callus induction, proliferation culture, adventitious bud culture, and rooting culture. This process, including the conventional callus culture step, is prone to browning, vitrification, and contamination, and is time-consuming and complex, making seedling formation difficult. Shortening the tissue culture growth cycle and improving the rooting rate are fundamental to rapidly obtaining large quantities of *Nitraria tangutorum*. Therefore, seeking new tissue culture methods remains a crucial task for *Nitraria tangutorum* tissue culture. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid propagation method for inducing seedlings from leaves of *Nitraria tangutorum*, in order to solve the problems existing in the prior art. This rapid propagation method, from the perspective of plant tissue culture, screens culture medium formulas, optimizes culture conditions and tissue culture processes, and obtains explants by directly using *Nitraria tangutorum* leaves for rooting culture, followed by proliferation culture and rooting culture, without the callus culture step, thus avoiding phenomena such as browning and vitrification that are prone to occur during callus culture, and can achieve rapid propagation of *Nitraria tangutorum*.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a rapid propagation method for inducing seedlings from leaves of *Nitraria tangutorum*, comprising the following steps:

[0007] Using young leaves of *Nitraria tangutorum* from the current year as explants, after disinfection, they were inoculated with leaf rooting medium for rooting culture, and young plants grew out; the components of the leaf rooting medium were: 1 / 2 MS + indolebutyric acid.

[0008] The young plants were cut into small segments and transferred to a proliferation medium for further culture to obtain adventitious buds; the components of the proliferation medium were: MS + 6-benzylaminopurine + zeatin + indolebutyric acid;

[0009] The adventitious buds were transferred to rooting medium for rooting culture, and then subjected to conventional culture to obtain seedlings induced by leaves of *Nitraria tangutorum*. The rooting medium consisted of 1 / 3 MS + indolebutyric acid.

[0010] Preferably, the disinfection includes the following steps: soaking in 75% ethanol for 20 seconds, and then soaking in 0.1% HgCl2 solution for 3 minutes.

[0011] Preferably, the leaf rooting medium consists of 1 / 2 MS + 1.5 mg / L indolebutyric acid; the conditions for rooting the young leaves are: temperature 23±2℃, light intensity 1800-2000 lx, and light duration 14 h / d.

[0012] Preferably, the proliferation medium consists of: MS + 2.0 mg / L 6-benzylaminopurine + 1.0 mg / L zeatin + 2.0 mg / L indolebutyric acid; and the conditions for proliferation culture of the young plants are: temperature 23±2℃, light intensity 1800-2000 lx, and photoperiod 12 h / d.

[0013] Preferably, the rooting medium consists of 1 / 3 MS + 2.0 mg / L indolebutyric acid; and the conditions for rooting the adventitious buds are: temperature 23±2℃, light intensity 1800-2000 lx, and light duration 12 h / d.

[0014] Preferably, the conventional cultivation includes: cultivating seedlings in a greenhouse at a temperature of 23±2℃ under natural light.

[0015] The present invention also provides an application of the method in improving the rooting rate or seedling rate of *Nitraria tangutorum*.

[0016] The present invention discloses the following technical effects:

[0017] This invention achieves rapid propagation of *Nitraria tangutorum* seedlings under artificially controlled conditions through tissue culture. Compared to previous seed and cutting propagation methods, this invention uses the rooting culture of current-year young leaves of *Nitraria tangutorum* as explants. Compared to seed germination propagation, this method has the main advantage of maintaining the superior traits of the parent plant and preventing variation, while also eliminating the pretreatment steps required for seed propagation. The fruit traits of *Nitraria tangutorum* exhibit extremely complex variation; seed propagation easily leads to trait segregation and loss of superior traits. Furthermore, *Nitraria tangutorum* fruits are berries, and the dried berries surrounding the seeds after harvesting and drying can cause incomplete sterilization, resulting in a high probability of contamination after sterilization. Additionally, the seeds have a hard outer shell, typically requiring treatment with diluted sulfuric acid or warm water before germination. These processes are time-consuming, complex, and result in slow seedling formation. Compared to cutting propagation, this method achieves a higher survival rate.

[0018] Compared with previous tissue culture methods, this invention eliminates the callus culture step, thus avoiding phenomena such as browning, vitrification, and contamination that can occur in callus culture. Furthermore, it significantly accelerates the seedling emergence rate of *Nitraria tangutorum* and greatly improves seedling quality. Therefore, using current-year leaves as explants for propagation in this invention offers advantages such as rapid seedling emergence, low cost, and high survival rate. In particular, it avoids the plant variation problems associated with sexual reproduction, yielding a large number of test-tube seedlings. This provides technical support for the widespread cultivation of this species in arid regions, and also supports its introduction and domestication, selection of superior strains, germplasm resource protection, and desertification control. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0020] Figure 1 shows the rooting of the inoculated young leaves in the culture medium and the root growth of the inoculated young leaves in the culture medium.

[0021] Figure 2 shows the rooting status of leaves in the culture media of group 9 (A) and group 14 (B);

[0022] Figure 3 shows the tissue culture seedlings of *Nitraria tangutorum* after 7 days (A), 10 days (B), and 14 days (C) of rooting culture in the 14th group of rooting medium;

[0023] Figure 4 shows the growth status of roots in the 14th group of rooting medium and the growth status of rooted seedlings in the rooting medium. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The mechanism of tissue culture propagation in this invention is as follows: different explants require different types and concentrations of hormones to induce callus, and the callus induction rate of the same explant varies under different hormone and concentration conditions. This invention directly uses the current year's young leaves of naturally grown *Nitraria tangutorum* as explant material, allowing the leaves to root, and directly using the young plants that grow from the rooted leaves as explant material for the next stage of culture. This not only avoids the pollution problems and genetic variations in new plants caused by conventional seed germination as explants, but also improves the seedling growth rate and the number of seedlings. During tissue culture, most plants use 1 / 2 MS medium as the basic culture medium, while this invention uses 1 / 3 MS as the basic culture medium, resulting in a high adventitious bud rooting rate and rapid root emergence. This achieves rooting while saving on raw materials used in culture medium preparation, reducing culture costs. Specific examples are provided below for further explanation.

[0030] Example 1

[0031] 1. Cleaning and disinfection of leaves:

[0032] Collect young leaves of *Nitraria tangutorum* that are free from disease, pests, and human damage and growing outdoors. After bringing them to the laboratory, place the leaves in a solution of laundry detergent and gently brush them with a soft brush. Then, place the leaves in a glass beaker, seal the beaker with white gauze, and then fill the beaker with tap water, allowing the leaves to rinse under running water for 5-6 hours.

[0033] After cleaning, place the leaves in a clean bench, soak them in 75% ethanol for 20 seconds, rinse them with sterile water 4-5 times, then soak them in 0.1% HgCl2 solution for 3 minutes, and rinse them with sterile water 5-6 times. During the soaking process, gently stir with a sterilized glass rod. Finally, use sterilized filter paper to absorb the moisture from the leaf surface, and then place the leaves in a sterilized petri dish for inoculation.

[0034] Table 1 Leaf disinfection time and reagent ratio

[0035]

[0036] As shown in Table 1, the disinfection method using a solution of 75% ethanol for 20 seconds followed by 0.1% HgCl2 solution for 3 minutes resulted in a leaf contamination rate of only 9.18%, demonstrating the best sterilization effect (Group 2).

[0037] 2. Leaf rooting induction:

[0038] Hold the leaf petiole on one side in the culture dish with tweezers, then inoculate it into the induction medium for culture. The induction medium formula is 1 / 2 MS + 1.5 mg / L indolebutyric acid (IBA). The culture conditions are: temperature 23±2℃, light intensity 1800-2000 lx, and photoperiod 12 h / d. After 10 days, white roots grow from the petiole cut, followed by rooted plantlets. As the plantlets grow, the leaves initially inoculated into the medium wither and fall off.

[0039] Table 2 Formulas for rooting induction medium from explant leaves

[0040]

[0041] Two explant leaves were inoculated into each bottle, and 30 replicates were performed for each hormone treatment.

[0042] Cultivation conditions: temperature 23±2℃, light intensity 1800-2000lx, light duration 14h / d.

[0043] After 14 days of cultivation, roots grew from the base of the leaves in all 16 groups of rooting media. The rooting and root growth of leaves in group 5 are shown in Figure 1, with a rooting rate of 63.38%. The rooting of leaves in group 9 is shown in Figure 2A, with a rooting rate of 11.64%, and the rooting of leaves in group 14 is shown in Figure 2B, with a rooting rate of 19.67%.

[0044] According to Table 2, the optimal rooting medium formula is 1 / 2 MS + 1.5 mg / L indolebutyric acid (Group 5). This formula results in the earliest rooting time (10 days) for explants, a high rooting rate, and early emergence of seedlings from the petiole cut, which are then used for the next step of propagation culture.

[0045] 3. Proliferation Culture

[0046] After the roots at the base of the leaf have grown for 20 days, the buds that have grown from the base of the leaf are cut into small segments of about 1 cm and transferred to different formulations of bud proliferation culture medium for continued cultivation.

[0047] The formulation of the proliferation medium is as follows:

[0048] Table 3 Formula for the culture medium for the proliferation of shoot clusters

[0049]

[0050] Three callus tissues were inoculated into each culture flask, and 30 replicates were performed for each culture medium.

[0051] Cultivation conditions: temperature 23±2℃, light intensity 1800-2000lx, light duration 12h / d.

[0052] After 14 days of culture, the adventitious shoot proliferation rate was the highest in group 7, at 73.38%. The culture medium formula was MS + 2.0 mg / L 6-benzylaminopurine + 1.0 mg / L zeatin + 2.0 mg / L indolebutyric acid. This combination of media resulted in the highest adventitious shoot proliferation rate, a large number of shoots, robust stems, and a tender green color, which is conducive to rooting culture.

[0053] Therefore, the optimal proliferation medium formulation of this invention is determined to be: MS + 2.0 mg / L 6-benzylaminopurine + 1.0 mg / L zeatin + 2.0 mg / L indolebutyric acid (Group 7).

[0054] Under this formula, the proliferation rate of clustered buds is 73.38%, the seedling height after 21 days of growth is 4.0cm, and there are many clustered buds, strong stems, tender green color, and vigorous growth.

[0055] 4. Rooting culture

[0056] Adventitious buds with leaf tips were cut and propagated, while adventitious buds without leaf tips were cut for comparison to determine the differences in rooting between the two types. The cut adventitious buds were then transferred to rooting media with different formulations for rooting culture.

[0057] The formula for the rooting medium is as follows:

[0058] Table 4 Rooting Induction Culture Medium Formula

[0059]

[0060]

[0061] Two shoot clusters were inoculated into each culture flask, and 30 replicates were performed for each culture medium.

[0062] Cultivation conditions: temperature 23±2℃, light intensity 1800-2000lx, light duration 12h / d.

[0063] After 7-10 days of cultivation, roots began to grow in the rooting medium of each group. The growth status of roots in the rooting medium of group 14 at different times is shown in Figure 3, and the growth status of rooted seedlings is shown in Figure 4.

[0064] As shown in the figure, the tissue culture seedlings obtained by this step have an early rooting time, good root growth, a large number of hairy roots, and a distinct and robust taproot.

[0065] Therefore, the optimal rooting medium formulation of this invention is determined to be: 1 / 3 MS + 2.0 mg / L indolebutyric acid (Group 14).

[0066] Under this formula, the buds begin to root after 7 days, and the rooting rate can reach 91.26%.

[0067] After the rooting culture is completed, seedlings are cultivated using conventional culture methods (e.g., greenhouse, temperature 25℃, natural light conditions).

[0068] The experimental results above show that this invention, through tissue culture under artificially controlled conditions, achieves rapid propagation of *Nitraria tangutorum* seedlings (the entire culture process, from leaf rooting to seedling rooting, takes only 35-45 days, greatly improving the seedling growth rate). Compared to previous sexual reproduction methods, this invention demonstrates that both adventitious buds with and without leaf tips can successfully develop roots during the later rooting process (rooting rates of up to 91.26% and 74.26%, respectively). This significantly saves raw materials, increases the likelihood of genetic variation, and avoids the pretreatment procedures required when using seeds as explants for propagation. The fruit of *Nitraria tangutorum* is a berry. After the seeds are harvested and dried, the dried berries encase the seeds, making thorough sterilization difficult and increasing the risk of contamination after sterilization. Furthermore, the seeds have a hard outer shell, typically requiring treatment with diluted sulfuric acid or soaking in warm water before germination. These processes are time-consuming and complex. However, directly using current-year tender leaves as explants offers advantages such as rapid propagation, low cost, high survival rate, and fast seedling formation. This solves the problem of genetic variation in plants that occurs during sexual reproduction, resulting in a large number of high-quality test-tube seedlings. This greatly assists in the industrialized seedling production of *Nitraria tangutorum* and provides excellent technology for the production of high-quality seedlings in the process of desertification control.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rapid propagation method for inducing seedlings from leaves of *Nitraria tangutorum*, characterized in that, Includes the following steps: Using young leaves of *Nitraria tangutorum* as explants, after disinfection, they were inoculated into leaf rooting medium for rooting culture, resulting in the emergence of young plants. The leaf rooting medium consisted of 1 / 2 MS + 1.5 mg / L indolebutyric acid. The young plants were cut into small segments and transferred to proliferation medium for further culture to obtain adventitious buds. The proliferation medium consisted of MS + 2.0 mg / L 6-benzylaminopurine + 1.0 mg / L zeatin + 2.0 mg / L indolebutyric acid. The adventitious buds were then transferred to rooting medium for rooting culture. After rooting, they were cultured conventionally to obtain seedlings induced from *Nitraria tangutorum* leaves. The rooting medium consisted of 1 / 3 MS + 2.0 mg / L indolebutyric acid.

2. The method as described in claim 1, characterized in that, The disinfection process includes the following steps: soaking in 75% ethanol for 20 seconds, followed by soaking in 0.1% HgCl2 solution for 3 minutes.

3. The method as described in claim 1, characterized in that, The conditions for rooting the tender leaves were: temperature 23±2℃, light intensity 1800-2000lx, and light duration 14h / d.

4. The method as described in claim 1, characterized in that, The conditions for propagating the seedlings were: temperature 23±2℃, light intensity 1800-2000lx, and light duration 12h / d.

5. The method as described in claim 1, characterized in that, The conditions for rooting the adventitious buds were: temperature 23±2℃, light intensity 1800-2000lx, and light duration 12h / d.

6. The method as described in claim 1, characterized in that, The conventional cultivation process includes: cultivating seedlings in a greenhouse at a temperature of 23±2℃ under natural light.

7. The application of the method as described in any one of claims 1-6 in improving the rooting rate or seedling rate of *Nitraria tangutorum*.

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