Culture medium and culture method for promoting germination of alkali reed sterile seedlings

Abstract

This invention relates to a tissue culture method for the germination of sterile Suaeda salsa seedlings. The method includes the following steps: (1) cleaning the harvested Suaeda salsa; (2) sterilizing the Suaeda salsa with 75% alcohol and 10% sodium hypochlorite in sequence; (3) selecting Suaeda salsa stem segments containing growth points and inoculating them in a culture medium; the culture medium consists of a basal culture medium and a growth inducer, and the growth inducer consists of a first growth inducer and a second growth inducer. The stem segments used in this invention for tissue culture of sterile Suaeda salsa seedlings are widely available and can be obtained directly from existing plants without relying on seed resources. The experimental steps of seed germination can be skipped, shortening the germination time to less than 15 days, increasing the germination rate, shortening the time to obtain propagated seedlings, and reducing propagation costs.

Description

Technical Field

[0001] This invention relates to the field of plant propagation, and more specifically to a cultivation method for promoting the germination of sterile seedlings of Suaeda salsa. Background Technology

[0002] Suaeda salsa ( Suaeda glauca (Bunge) Bunge Suaeda salsa is a salt-tolerant plant belonging to the genus Suaeda in the family Chenopodiaceae. It is widely distributed in salt-stressed environments such as coastal mudflats and saline-alkali lands. It possesses strong salt tolerance and adaptability, enabling it to grow in highly saline-alkali soils and absorb soil salts, thereby reducing soil salinity and improving soil structure and fertility. Its root system helps stabilize the soil and prevent soil erosion. As a pioneer plant in coastal wetlands, Suaeda salsa provides habitats and breeding grounds for birds and benthic organisms, effectively protecting biodiversity. Furthermore, it can absorb heavy metals from the soil and degrade organic pollutants, enhancing the carbon sequestration capacity of wetland ecosystems and playing a positive role in regional climate regulation.

[0003] In recent years, studies on the effective components and bioactivity of *Suaeda salsa* have revealed that its plants contain a variety of chemical components, including protein, dietary fiber, polysaccharides, pigments, flavonoids, terpenes, and coenzyme Q. The seeds are rich in unsaturated fatty acids, making it valuable for both food and medicine. To date, as many as 163 compounds have been isolated or detected from the *Suaeda* genus. Modern pharmacological studies have shown that *Suaeda salsa* possesses anti-inflammatory, antioxidant, antitumor, hypoglycemic, and lipid-lowering effects.

[0004] Plant tissue culture is an important technique for rapid propagation, resource preservation, and biotechnology breeding of plant germplasm. Currently, research on the establishment of tissue culture systems for *Suaeda salsa* is limited, and most studies primarily use seeds as explants, resulting in a narrow research focus and susceptibility to phenotypic segregation and variation. Furthermore, typically only one sterile seedling can grow from one seed, leading to low cultivation efficiency. Therefore, researching a method for cultivating *Suaeda salsa* sterile seedlings with rapid propagation and good seedling uniformity is of significant importance and has potential application value. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention includes the following aspects:

[0006] The first aspect of the present invention provides a tissue culture method for the germination of aseptic seedlings of *Suaeda salsa*, the method comprising the following steps:

[0007] (1) Clean the harvested Suaeda salsa and rinse it thoroughly with running water;

[0008] (2) The Suaeda salsa was disinfected and sterilized in sequence with 75% alcohol and 10% sodium hypochlorite.

[0009] (3) Select Suaeda salsa stem segments containing growth points and inoculate them into a culture medium. After culturing for 15 days, sterile Suaeda salsa seedlings in the budding stage are obtained.

[0010] The culture medium used in step (3) consists of a basal culture medium and a growth inducer. The growth inducer consists of a first growth inducer and a second growth inducer. The first growth inducer is selected from one or more of 6-BA and KT, and the second growth inducer is selected from one or more of NAA, IAA, IBA and 2,4-D.

[0011] Preferably, in step (1), the Suaeda salsa is soaked in soapy water for 1 hour and then rinsed clean with running water.

[0012] Preferably, the disinfection and sterilization process in step (2) is carried out in a clean bench.

[0013] Preferably, in step (2), the sample is first soaked in 75% alcohol for 10-60 seconds, and then soaked in 10% sodium hypochlorite solution for 5-30 minutes. More preferably, in step (2), the sample is first soaked in 75% alcohol for 20-40 seconds, and then soaked in 10% sodium hypochlorite solution for 10-20 minutes. Even more preferably, in step (2), the sample is first soaked in 75% alcohol for 30 seconds, and then soaked in 10% sodium hypochlorite solution for 15 minutes.

[0014] Preferably, the basal culture medium used in step (3) comprises MS medium, NaCl, sucrose, and agar. More preferably, the basal culture medium comprises 1×MS medium, 0.05-0.5 g / L NaCl, 10-50 g / L sucrose, and 1-10 g / L agar. Further preferably, the basal culture medium comprises 1×MS medium, 0.05-0.2 g / L NaCl, 20-40 g / L sucrose, and 4-8 g / L agar. Most preferably, the basal culture medium comprises 1×MS medium, 0.1 g / L NaCl, 30 g / L sucrose, and 6 g / L agar.

[0015] Preferably, the first growth inducer in step (3) is 6-BA or KT, and the second growth inducer is NAA, IAA, IBA, or 2,4-D. More preferably, the first growth inducer is KT, and the second growth inducer is IBA or 2,4-D.

[0016] Preferably, the growth inducer in step (3) is a combination of 6-BA and NAA, a combination of 6-BA and IAA, a combination of 6-BA and IBA, a combination of KT and IBA, or a combination of KT and 2,4-D.

[0017] More preferably, the growth inducer in step (3) is a combination of 0.5-2 mg / L 6-BA and 0.5-2 mg / L NAA, a combination of 0.5-2 mg / L 6-BA and 0.5-2 mg / L IAA, a combination of 0.5-2 mg / L 6-BA and 0.5-2 mg / L IBA, a combination of 0.5-2 mg / L KT and 0.5-2 mg / L IBA, or a combination of 0.5-2 mg / L KT and 0.5-2 mg / L 2,4-D.

[0018] More preferably, the growth inducer in step (3) is a combination of 1 mg / L 6-BA and 1 mg / L NAA, a combination of 1 mg / L 6-BA and 1 mg / L IAA, a combination of 1 mg / L 6-BA and 1 mg / L IBA, a combination of 1 mg / L KT and 1 mg / L IBA, or a combination of 1 mg / L KT and 1 mg / L 2,4-D.

[0019] A second aspect of the present invention provides a culture medium for the germination of sterile seedlings of Suaeda salsa, the culture medium comprising a basal culture medium and a growth inducer, the growth inducer comprising a first growth inducer and a second growth inducer, the first growth inducer being selected from one or more of 6-BA and KT, and the second growth inducer being selected from one or more of NAA, IAA, IBA and 2,4-D.

[0020] Preferably, the growth inducer is a combination of 6-BA and NAA, a combination of 6-BA and IAA, a combination of 6-BA and IBA, a combination of KT and IBA, or a combination of KT and 2,4-D.

[0021] More preferably, the growth inducer is a combination of 0.5-2 mg / L 6-BA and 0.5-2 mg / L NAA, a combination of 0.5-2 mg / L 6-BA and 0.5-2 mg / L IAA, a combination of 0.5-2 mg / L 6-BA and 0.5-2 mg / L IBA, a combination of 0.5-2 mg / L KT and 0.5-2 mg / L IBA, or a combination of 0.5-2 mg / L KT and 0.5-2 mg / L 2,4-D.

[0022] More preferably, the growth inducer is a combination of 1 mg / L 6-BA and 1 mg / L NAA, a combination of 1 mg / L 6-BA and 1 mg / L IAA, a combination of 1 mg / L 6-BA and 1 mg / L IBA, a combination of 1 mg / L KT and 1 mg / L IBA, or a combination of 1 mg / L KT and 1 mg / L 2,4-D.

[0023] Preferably, the basal culture medium comprises 1×MS medium, NaCl, sucrose, and agar. More preferably, the basal culture medium comprises 1×MS medium, 0.05-0.5 g / L NaCl, 10-50 g / L sucrose, and 1-10 g / L agar. Even more preferably, the basal culture medium comprises 1×MS medium, 0.05-0.2 g / L NaCl, 20-40 g / L sucrose, and 4-8 g / L agar. Most preferably, the basal culture medium comprises 1×MS medium, 0.1 g / L NaCl, 30 g / L sucrose, and 6 g / L agar.

[0024] The beneficial effects of this invention are:

[0025] 1. The method for tissue culture of aseptic seedlings of Suaeda salsa in this invention uses plant stem segments as explants. By optimizing the disinfection method, the contamination rate and browning rate of Suaeda salsa explants are reduced. By optimizing the growth inducer components in the tissue culture medium, the adventitious bud induction rate of Suaeda salsa is significantly improved, and the bud induction and cluster bud proliferation of Suaeda salsa are promoted, so that a large number of aseptic seedlings can be obtained in a short time.

[0026] 2. The stem segments used in this invention for tissue culture of aseptic seedlings of Suaeda salsa are widely available and can be obtained directly from existing plants without relying on seed resources. The experimental steps of seed germination can be skipped, shortening the germination time to less than 15 days, increasing the germination rate, shortening the time to obtain propagated seedlings, and reducing propagation costs. Detailed Implementation

[0027] Experiment Example 1: Screening Experiment for Disinfection Methods of Suaeda salsa

[0028] 1. Test Methods

[0029] Based on the physiological characteristics of *Suaeda salsa*, select appropriate harvesting sites and harvesting times to ensure robust explants with vigorous growth and a high germination rate. After harvesting, soak the *Suaeda salsa* in soapy water for 1 hour, then rinse thoroughly with running water. Conduct disinfection experiments in a clean bench. First, disinfect with 75% alcohol for 30 seconds, then rinse three times with ddH2O, followed by soaking in a 10% sodium hypochlorite solution. Disinfection times with sodium hypochlorite solution were set at 5 min, 10 min, 15 min, and 20 min, for a total of four experimental groups, with 30 plants in each group. Finally, rinse five times with ddH2O. Calculate the contamination rate (contamination is indicated by the appearance of colonies on the culture medium or explant surface) and browning rate (browning is indicated by the explant turning brown after inoculation) of each experimental group according to the following method. Pollution rate = Number of contaminated Suaeda salsa plants / Total number of inoculated Suaeda salsa plants * 100%; Browning rate = Number of browned Suaeda salsa plants / Total number of inoculated Suaeda salsa plants * 100%.

[0030] 2. Test Results

[0031] The contamination rate and browning rate of each experimental group after NaClO treatment with different disinfection times are shown in Table 1 below.

[0032] Table 1. Effects of different NaClO disinfection times on the contamination rate and browning rate of Suaeda salsa.

[0033]

[0034] As shown in Table 1 above, when the NaClO disinfection time was set to 5 min and 10 min, the contamination rates of experimental groups 1 and 2 were 83.3% and 60%, respectively. When the NaClO disinfection time was extended to 15 min, the contamination rate of experimental group 3 significantly decreased to 13.3%. Further extending the NaClO disinfection time to 20 min did not significantly reduce the contamination rate of experimental group 4, but its browning rate significantly increased. Considering both the contamination rate and browning rate of the explants, the optimal disinfection method was determined to be immersion in 75% alcohol for 30 s combined with immersion in 10% sodium hypochlorite solution for 15 min.

[0035] Experiment Example 2: Screening Experiment of Components for Induction Culture Medium of Aseptic Seedlings of Suaeda salsa

[0036] 1. Test Methods

[0037] Following the method described in Experiment 1, the harvested Suaeda salsa was soaked in soapy water for 1 hour and then rinsed thoroughly with running water. In a clean bench, the Suaeda salsa was disinfected by soaking in 75% alcohol for 30 seconds, then rinsed three times with ddH2O, followed by soaking in 10% sodium hypochlorite solution for 15 minutes, and finally rinsed five times with ddH2O. The disinfected Suaeda salsa stem segments, approximately 1 cm in length, with growth points, were cut using tweezers and a scalpel. These stem segments were then inoculated into different bud-inducing media, with 30 stem segments selected for each experimental group. The culture media for each experimental group consisted of basal medium (containing 1×MS medium, 0.1 g / L NaCl, 30 g / L sucrose, and 6 g / L agar) and different types of growth inducers. The growth inducers used in experimental groups 1-5 were 1 mg / L 6-BA (6-benzylpurine) + 1 mg / L NAA (naphthaleneacetic acid), 1 mg / L 6-BA + 1 mg / L IAA (indoleacetic acid), 1 mg / L 6-BA + 1 mg / L IBA (indolebutyric acid), 1 mg / L KT (kinetin, 6-furfurylaminopurine) + 1 mg / L IBA (indolebutyric acid), and 1 mg / L KT + 1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid). The culture temperature for all experimental groups was 25℃, the light intensity was 2000 Lx, and the photoperiod was 8 h. Aseptic seedlings of *Suaeda salsa* were induced using the culture media of the above-mentioned experimental groups. The effects of different culture media on the growth of adventitious buds of *Suaeda salsa* were observed, and the adventitious bud induction rate was calculated according to the following formula: Adventitious bud induction rate = (Number of *Suaeda salsa* stem segments that induced adventitious buds / Total number of *Suaeda salsa* stem segments) * 100%.

[0038] 2. Test Results

[0039] Table 2 shows the effect of different growth inducers on the induction rate and growth of adventitious buds of Suaeda salsa in the culture medium of each experimental group.

[0040] Table 2. Effects of different growth inducers on the induction rate and growth of adventitious buds in Suaeda salsa.

[0041]

[0042] As shown in Table 2 above, compared with experimental groups 1-2, the adventitious bud induction rate of Suaeda salsa using growth inducers from experimental groups 3-5 was significantly improved, and the number of induced adventitious roots was also greater. Among them, experimental group 5 had the highest adventitious bud induction rate, and successfully induced not only adventitious roots, but also numerous and robust buds, producing an unexpectedly excellent proliferation-promoting effect.

[0043] Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

Claims

1. A tissue culture method for the germination of aseptic seedlings of *Suaeda salsa*, characterized in that, The method includes the following steps: (1) Clean the harvested Suaeda salsa and rinse it with running water; (2) The Suaeda salsa was disinfected and sterilized in sequence with 75% alcohol and 10% sodium hypochlorite. (3) Select Suaeda salsa stem segments containing growth points and inoculate them into a culture medium. After culturing for 15 days, sterile Suaeda salsa seedlings in the budding stage are obtained. The culture medium used in step (3) consists of a basal culture medium and a growth inducer, wherein the growth inducer is a combination of 0.5-2 mg / L KT and 0.5-2 mg / L IBA or a combination of 0.5-2 mg / L KT and 0.5-2 mg / L 2,4-D; In step (2), the first step is to soak in 75% alcohol for 30 seconds, and then soak in 10% sodium hypochlorite solution for 15 minutes.

2. The tissue culture method according to claim 1, characterized in that, The disinfection and sterilization process in step (2) is carried out in a clean bench.

3. The tissue culture method according to claim 1, characterized in that, The basal culture medium used in step (3) includes MS medium, NaCl, sucrose and agar.

4. The tissue culture method according to claim 1, characterized in that, The growth inducer in step (3) is a combination of 1 mg / L KT and 1 mg / L IBA or a combination of 1 mg / L KT and 1 mg / L 2,4-D.

Images