Method for culturing stem tip thin cell layer of paspalum vaginatum to obtain regenerated plant

Abstract

The invention discloses a method for culturing a stem tip thin cell layer of paspalum vaginatum to obtain a regenerated plant, and belongs to the technical field of tissue culture of paspalum vaginatum. According to the method, the stem tip thin cell layer of paspalum vaginatum is used as an explant material for induction to obtain a callus, then the callus is proliferated, then the callus is induced to differentiate and bud, and the regenerated plant is obtained. And finally, cutting bud clusters for seedling strengthening and rooting culture to obtain a complete plant. The method disclosed by the invention is simple and efficient, and can provide technical support for researches on germchit breeding, transgenosis and gene editing technologies, gene function verification, biotechnology breeding and the like of the paspalum vaginatum.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology of *Hymenochloa chinensis*, specifically to a method for obtaining regenerated plants through thin cell layer culture of *Hymenochloa chinensis* shoot tips. Background Technology

[0002] Aukia ( Paspalum vaginatum It is a perennial herbaceous plant of the Poaceae family, widely used for pasture, ecological restoration and lawn establishment in tropical / subtropical regions. Its excellent characteristics such as tolerance to trampling, low mowing, salt and heavy metals make it of important economic value and scientific research value for stress resistance molecular regulation mechanism.

[0003] The foundation for research on the molecular regulatory mechanisms of stress resistance lies in efficient tissue culture techniques. Currently reported tissue culture techniques for *Agrostis arvensis* mainly use young inflorescences, seeds, and stolons as explants. However, the sources of young inflorescences and seeds are limited, restricted by season or variety, while the callus induction efficiency of stolons still needs further improvement. Furthermore, *Agrostis arvensis* callus easily loses its ability to regenerate plants during subculturing, limiting the development of efficient genetic transformation techniques and gene function verification research, and hindering research on the molecular regulatory mechanisms of stress resistance.

[0004] Thin-layer culture (TLC) is a technique that uses longitudinally and transversely cut thin layers of cells and tissues to induce regeneration of plants. Since its invention in 1973, it has been widely used in the tissue culture of monocots and dicots, showing higher efficiency than traditional tissue culture. It has become an important tool in plant tissue culture and an effective model system for studying morphogenesis regulation mechanisms and genetic transformation. Currently, there are no reports on TLC studies of *Paspalum notatum*. Summary of the Invention

[0005] In view of this, the present invention provides a method for obtaining regenerated plants by thin cell layer culture of the shoot tip of *Hippophae rhamnoides*, aiming to provide a more efficient tissue culture program for *Hippophae rhamnoides*.

[0006] To achieve the above objectives, the present invention specifically adopts the following technical solution: A method for obtaining regenerated plants through thin-cell layer culture of *Paspalum distichum* shoot tips includes the following steps: S1. Take the tips of the erect or creeping stems of the arugula, wash and disinfect them; S2. Trim the stem tip to a length of 1-2 cm, and then slice it crosswise into thin slices less than 1 mm thick; S3. Inoculate the thin slices into callus induction medium and culture in the dark to obtain callus tissue. The callus induction medium is N6 or MS + Dicamba (dicamba, chemical name is 3,6-dichloro-2-methoxybenzoic acid, CAS number is 1918-00-9) or 2,4-D (2,4-dichlorophenoxyacetic acid) 3 mg / L + TDZ (thiazidone) 0.1-0.5 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8. S4. Inoculate the obtained callus tissue into proliferation medium and culture in the dark. Subculture once every 10-15 days, for a total of 1-2 subcultures. S5. Differentiate and culture the callus tissue obtained in step S4 to differentiate into green shoot clusters; S6. Green shoot clusters are cultured to promote seedling growth and root development, thereby obtaining complete regenerated plants.

[0007] Preferably, in the above method, step S1 specifically involves: cutting the stem from the tip of the erect or creeping stem of the aralia to the third to fourth node from the base, washing it with water, trimming the base, leaving 2-3 cm from the tip of the stem to the base, then soaking and scrubbing it in detergent, washing it with water, then disinfecting it with alcohol and disinfectant in sequence, and finally rinsing it with sterile water.

[0008] Preferably, in the above method, the callus induction medium is N6 + Dicamba 3 mg / L + TDZ 0.1-0.5 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8. Compared with the callus induction medium of MS + 2,4-D 3 mg / L + TDZ + sucrose + agar, this medium has a relatively higher callus induction rate and is more conducive to subsequent proliferation culture.

[0009] Preferably, in the above method, step S1 involves cutting the stem tip of the stolon; compared to the stem tip of the erect stem, the thin cell layer obtained from the stem tip of the stolon has a significantly higher induction efficiency under the same induction culture conditions, and also has better effects in subsequent proliferation and differentiation.

[0010] Preferably, in the above method, the proliferation medium is MS + 2,4-D 0.5 mg / L + 6-BA (6-benzyladenine) 0.1 mg / L + hydrolyzed casein 1 g / L + sorbitol 100 mg / L + CuSO4 3 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8.

[0011] Preferably, in the above method, step S5 involves culturing using a differentiation medium, wherein the differentiation medium consists of MS + KT (kinetin) 0.5 mg / L + 6-BA 0.5 mg / L + NAA (naphthaleneacetic acid) 0.5 mg / L + hydrolyzed casein 1 g / L + sorbitol 100 mg / L + CuSO4 3 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8; and the culture conditions include: 25±2℃, light intensity 2000 lx, and a light / dark cycle of 12h / 12h.

[0012] Preferably, in the above method, step S6 uses a seedling rooting medium for cultivation, wherein the seedling rooting medium is 1 / 2 MS + 30 g / L sucrose + 6.5 g / L agar, pH 5.8; and the cultivation conditions for this step include 25±2℃, light intensity 2000 lx, and light / dark ratio of 12h / 12h.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes the thin cell layer of *Paspalum notatum* shoot tips as explant material for tissue culture propagation. Compared to other explants, it can generate callus tissue in a shorter time and exhibits higher callus induction rate, proliferation rate, and differentiation rate. Furthermore, this method is simple and easy to operate. This invention can provide technical support for research on *Paspalum notatum* seedling propagation, transgenic and gene editing technologies, and gene function verification, and has practical significance for the study of *Paspalum notatum* biotechnology breeding and the molecular regulatory mechanism of salt and alkali tolerance. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0015] Figure 1 The flowchart of the method for obtaining regenerated plants by thin cell layer culture of *Paspalum notatum* stem tip provided by the present invention is shown in the following figure: a is the cut stolon stem tip, b is the thin cell layer explant obtained by transverse section of the stem tip, c is the induced callus tissue, d is the callus tissue obtained by proliferation culture, e is the cluster bud obtained by differentiation culture, and f is the rooted tissue culture seedling. Detailed Implementation

[0016] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0018] Existing tissue culture techniques for *Hymenochloa argentea* have many limitations, hindering research on the plant. To address this issue, this invention develops a method for obtaining regenerated plants through thin-layer culture of *Hymenochloa argentea* shoot tips using shoot tips as explants, which can significantly improve the efficiency of *Hymenochloa argentea* tissue culture.

[0019] Reference Figure 1 The method for obtaining regenerated plants by thin cell layer culture of *Paspalum notatum* shoot tips provided in this embodiment of the invention includes the following steps: (1) Cut the erect stem or stolon from the tip of the stem to the third or fourth node from the base. Then, cut the tip of the cut erect stem or stolon. Figure 1 a) Clean and disinfect; (2) Trim the stem tip to a length of 1-2 cm, then cross-cut it into thin slices less than 1 mm thick. Figure 1 b) (3) The thin slices were inoculated into the callus induction medium and cultured in the dark to obtain callus with larger clumps. Figure 1 c) (4) The callus obtained in step (3) is inoculated into proliferation medium and cultured in the dark. Subculture is performed every 10-15 days, and the subculture is repeated 1-2 times to obtain white or light yellow, granular, and granular callus. Figure 1 d); (5) The callus tissue obtained in step (4) is transferred to a differentiation medium to differentiate into green shoots. Figure 1 e); (6) Take green shoots and inoculate them into a rooting and seedling culture medium to obtain complete regenerated plants. Figure 1 f).

[0020] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0021] Example 1 This example provides a method for obtaining regenerated plants through thin-layer cell culture of *Paspalum notoginseng* shoot tips, including the following steps: (1) Explant collection and disinfection.

[0022] Using sterile surgical instruments, cut the stolon tips of *Paspalum notatum* from the 3rd to 4th nodes from the base. Rinse under running water for 30 minutes to remove surface impurities, trim the base, leaving about 2 cm from the stolon tip. Then, soak the explants in a solution of Libai detergent, scrub for 3 minutes, rinse with running water for 5 minutes, soak the explants in 75% alcohol for 10 seconds, then quickly soak them in undiluted 84 disinfectant for 15 minutes. Finally, rinse three times with sterile water, each rinse lasting 2-3 minutes.

[0023] (2) Explant section.

[0024] The sterilized stem tips were trimmed to a length of 1-2 cm, and then transversely cut into thin slices less than 1 mm thick for use in callus induction.

[0025] (3) Callus induction culture.

[0026] The thin slices obtained in step (2) were inoculated into callus induction medium and cultured in the dark at 25±2℃ for 7 days to start generating callus. After 20 days of culture, larger callus clumps were obtained. The callus induction medium consisted of N6 + Dicamba 3 mg / L + TDZ 0.1 mg / L + sucrose 30 g / L + agar 6.5 g / L. The pH of the medium was adjusted to 5.8 before sterilization.

[0027] (4) Callus proliferation culture.

[0028] The induced callus aggregates were inoculated onto proliferation medium and cultured in the dark at 25±2℃. Subculture was performed every 10-15 days, for a total of two subcultures, resulting in firm callus aggregates with a granular surface. The proliferation medium consisted of MS medium containing 0.5 mg / L 2,4-D, 0.1 mg / L 6-BA, 1 g / L hydrolyzed casein, 100 mg / L sorbitol, 3 mg / L CuSO4, 30 g / L sucrose, and 6.5 g / L agar. The pH was adjusted to 5.8 before sterilization.

[0029] (5) Callus differentiation and culture.

[0030] The callus tissue obtained in step (4) was transferred to differentiation medium and cultured at 25±2℃, light intensity of 2000lx, and light / dark ratio of 12h / 12h to differentiate into green shoot clusters. The differentiation medium consisted of MS + KT 0.5 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + hydrolyzed casein 1 g / L + sorbitol 100 mg / L + CuSO4 3 mg / L + sucrose 30 g / L + agar 6.5 g / L. The pH of the medium was adjusted to 5.8 before sterilization.

[0031] (6) Seedling growth and rooting culture.

[0032] The green shoots obtained in step (5) were inoculated into a rooting and seedling strengthening medium. The seedlings were then cultured and strengthened under the conditions of 25±2℃, light intensity of 2000lx, and light / dark ratio of 12h / 12h to obtain complete regenerated plants. The rooting and seedling strengthening medium consisted of 1 / 2 MS + 30 g / L sucrose + 6.5 g / L agar. The pH of the medium was adjusted to 5.8 before sterilization.

[0033] Example 2 This example provides a method for obtaining regenerated plants by thin cell layer culture of the shoot tip of *Paspalum distichum*. The difference from Example 1 is that the callus induction medium used in step (3) of this example is N6 + Dicamba 3 mg / L + TDZ 0.5 mg / L + sucrose 30 g / L + agar 6.5 g / L (pH 5.8), while the rest is the same as in Example 1.

[0034] Example 3 This example provides a method for obtaining regenerated plants by thin cell layer culture of the shoot tip of *Paspalum notatum*. The difference from Example 1 is that the callus induction medium used in step (3) of this example is MS + 2,4-D 3 mg / L + TDZ 0.3 mg / L + sucrose 30 g / L + agar 6.5 g / L (pH 5.8), while the rest is the same as in Example 1.

[0035] Example 4 This example provides a method for obtaining regenerated plants by thin cell layer culture of the stem tip of *Hymenochloa chinensis*. Unlike Example 1, in step (1) of this example, the stem tip of the erect stem of *Hymenochloa chinensis* is cut from the 3rd to 4th node at the base, and the resulting erect stem tip is then processed. The specific operation is the same as in Example 1.

[0036] Comparative Example 1 This example provides a method for obtaining regenerated plants through thin-layer culture of *Paspalum notatum*, including the following steps: (1) Pretreatment of explants.

[0037] Wash the surface of the puffin stolon with water, cut it into 3-5 cm pieces and put it into an Erlenmeyer flask. Then put it into a 1% NaClO solution, add 1 drop of Tween-20, soak for 30 minutes, wash it with sterile water 5-7 times, and place it on sterile filter paper to dry excess water. Cut it into 2 cm sections with nodes.

[0038] (2) Callus induction culture.

[0039] The stem segments obtained in step (1) were inoculated into callus induction medium and cultured in the dark at 25±2℃ for 15 days to begin generating callus. After 30 days of culture, larger callus clumps were obtained. The callus induction medium consisted of MS medium salt + vitamin B5 + 2,4-D 3.0 mg / L + maltose 30 g / L + acid-hydrolyzed casein 1000 mg / L + sorbitol 20 mg / L + plant gel 2.5 g / L. The pH of the medium was adjusted to 5.8 before sterilization.

[0040] Subsequent callus proliferation culture, callus differentiation culture, seedling strengthening and rooting culture were the same as in Example 1.

[0041] The culture effects of each stage in Examples 1-4 and Comparative Example 1 were statistically analyzed, and the results are shown in Table 1. Compared with the induction culture of the jointed stolon segments in Comparative Example 1, the thin cell layer culture of the shoot tip of *Paspalum notatum* had a shorter callus induction time, a higher callus induction rate, and a slightly higher callus differentiation rate.

[0042] Table 1. Culture effects of different stages of *Pteris vittata* thin-layer culture.

[0043] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for obtaining regenerated plants through thin-cell layer culture of *Paspalum distichum* shoot tips, characterized in that... Includes the following steps: S1. Take the tips of the erect or creeping stems of the arugula, wash and disinfect them; S2. Trim the stem tip to a length of 1-2 cm, and then slice it crosswise into thin slices less than 1 mm thick; S3. The thin slices are inoculated into callus induction medium and cultured in the dark to obtain callus; wherein, the callus induction medium is N6 or MS+Dicamba or 2,4-D 3 mg / L+TDZ 0.1-0.5 mg / L+sucrose 30 g / L+agar 6.5 g / L, pH 5.8; S4. Inoculate the obtained callus tissue into proliferation medium and culture in the dark. Subculture once every 10-15 days, for a total of 1-2 subcultures. S5. Differentiate and culture the callus tissue obtained in step S4 to differentiate into green shoot clusters; S6. The green bud clusters are subjected to seedling strengthening and rooting culture to obtain complete regenerated plants.

2. The method according to claim 1, characterized in that, Step S1 is as follows: cut the stem from the tip of the erect or creeping stem of the aralia to the 3rd or 4th node from the base. After washing with water, trim the base, leaving 2-3 cm from the tip of the stem to the base. Then soak and scrub the stem in detergent, wash with water, disinfect with alcohol and disinfectant in turn, and finally wash with sterile water.

3. The method according to claim 1, characterized in that, The callus induction medium consisted of N6 + Dicamba 3 mg / L + TDZ 0.1 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.

8.

4. The method according to claim 1, characterized in that, Step S1: Cut off the stem tip of the stolon.

5. The method according to claim 1, characterized in that, The proliferation medium consisted of MS medium containing 2,4-D 0.5 mg / L, 6-BA 0.1 mg / L, hydrolyzed casein 1 g / L, sorbitol 100 mg / L, CuSO4 3 mg / L, sucrose 30 g / L, and agar 6.5 g / L, at pH 5.

8.

6. The method according to claim 1, characterized in that, Step S5 involves culturing the food using a differentiation medium, which is composed of MS + KT 0.5 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + hydrolyzed casein 1 g / L + sorbitol 100 mg / L + CuSO4 3 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.

8.

7. The method according to claim 6, characterized in that, Step S5 was performed at 25±2℃, light intensity of 2000lx, and light / dark ratio of 12h / 12h.

8. The method according to claim 1, characterized in that, Step S6 involves culturing the seedlings using a seedling rooting medium, which is 1 / 2 MS + 30 g / L sucrose + 6.5 g / L agar, pH 5.

8.

9. The method according to claim 8, characterized in that, Step S6 was performed at 25±2℃, light intensity of 2000lx, and light / dark conditions of 12h / 12h.

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