Method for efficiently obtaining callus of aquatic plant eustylis chinensis

Abstract

The application discloses a method for efficiently obtaining callus of aquatic plant Sagittaria trifolia, and comprises the following steps: (1) obtaining and screening explants; (2) screening disinfectants for the explants; (3) exploring disinfection time of the explants; (4) exploring callus induction conditions; (5) exploring callus proliferation conditions; and obtaining callus; wherein the induction medium comprises 4.74 g / L MS medium, 6-BA with a concentration of 0.5-3 mg / L, 2,4-D with a concentration of 0.5-3 mg / L, IBA with a concentration of 0.5 mg / L, TDZ with a concentration of 0.5 mg / L, 30 g / L sucrose and 3 g / L plant gel. The application screens the most suitable explants, optimizes and adjusts the concentration ratio of the induction and proliferation medium, and improves the induction rate of the callus of Sagittaria trifolia. The method has the advantages of simple culture, large proliferation amount, short cycle and the like; and the callus of Sagittaria trifolia can be successfully induced by the method, and the method provides a method basis for the researches on the genetic transformation system of Sagittaria trifolia, the preservation of germplasm resources and the rapid propagation production and the like.

Description

Technical Field

[0001] This invention relates to the field of aquatic plant biotechnology, and in particular to a method for efficiently obtaining callus tissue from the aquatic plant Agave americana. Background Technology

[0002] Ottelia alismoides, an annual submerged plant belonging to the genus Ottelia (also known as the water lily) of the family Hydrocharitaceae, is a sister plant in the same genus as the "clear stream spirit" of Yunnan plateau lakes. Ottelia alismoides is also a delicious wild vegetable and has medicinal uses. It was once widely distributed in rivers, lakes, and ditches throughout China, but due to habitat destruction, eutrophication, and the discharge of other pollutants, its population has rapidly declined and it has been listed as a national second-class protected plant. Meanwhile, Shao et al. (2017) discovered that Ottelia alismoides is currently the only higher aquatic plant that simultaneously possesses three carbon concentration mechanisms: bicarbonate, C4 metabolism, and CAM metabolism. This discovery is of great significance for the study of plant photosynthetic mechanisms and evolution.

[0003] Agave agave has a simple vegetative structure and cannot reproduce vegetatively; seed propagation is its only means of reproduction. Furthermore, the seeds have a strong dormancy period, which presents certain difficulties for propagation. Callus is the most common form of tissue culture. By inducing callus to obtain regenerated plants, a large number of regenerated plants can be proliferated in a short period of time. In addition, callus can also be used as a material for gene transformation, germplasm resource preservation, and rapid propagation, while also providing reference and guidance for genetic engineering research on other aquatic higher plants.

[0004] Currently, plant tissue culture is widely and deeply studied in terrestrial plants, but relatively less so in aquatic plants. The fundamental reason is that tissue culture in aquatic plants is more difficult than in terrestrial plants. Characteristics of aquatic plants include degenerated vascular bundles in the stems, a lack of wood and fiber, and well-developed aerenchyma throughout the plant, leading to high permeability of the epidermis to fungicides, sensitivity to sterilization agents, and significant challenges in sterilization. This is especially true for submerged plants like *Agave agave*, which have fibrous roots, short stems, and thin leaves (typically only two to three cells thick). Explants are often over-sterilized or incompletely sterilized, making it difficult to determine the optimal sterilization ratio and induce callus tissue. This has become a major obstacle to the development of tissue culture and genetic transformation techniques in *Agave agave* research. Currently, there are no reports on callus induction in submerged *Agave agave*, either domestically or internationally. Therefore, developing a rapid and efficient method for inducing callus tissue in *Agave agave* has become an urgent technical challenge. Summary of the Invention

[0005] To address the challenges of sterilizing aquatic plant explants and inducing callus in existing technologies, this invention aims to provide an efficient method for obtaining callus from the aquatic plant Agave americana. By screening for the optimal explants and optimizing the concentration ratio of plant growth promoters in the induction and proliferation culture media, a large amount of callus can be obtained in a short time. This not only provides excellent experimental materials for further research on the genetic transformation of Agave americana's photosynthetic mechanism but also provides a methodological basis for its rapid propagation.

[0006] To achieve the above objectives, the present invention employs the following technical measures:

[0007] A method for efficiently obtaining callus tissue from the aquatic plant Agave americana includes the following steps: explant collection and screening, explant sterilization, and determination of callus induction culture conditions and callus proliferation conditions.

[0008] (1) Acquisition and screening of explants: Select agave plants with good growth, and cut off tender and robust leaves, stem tips and root tips of agave as explants; in addition, soak mature seeds that have broken dormancy in tap water to germinate, and use the embryos that grow to 0.5-1cm as explants; after sterilization, inoculate them on induction medium, and count the induction rate after 30 days.

[0009] (2) Screening of explant disinfectants: Explants such as leaves, stem tips, root tips and embryos were soaked in soapy water for 5 minutes, then rinsed under running tap water for half an hour, and then rinsed twice with sterile water. They were then placed in sterilized tissue culture bottles and placed in a clean bench. They were sterilized with 75% alcohol for 15 seconds and rinsed twice with sterile water. Then they were sterilized with different disinfectants such as 10% H2O2, 4% NaClO, 84 disinfectant, benzalkonium chloride and 0.1% HgCl2 for 2, 5, 10 and 15 minutes respectively. Finally, they were rinsed 5 times with sterile water, dried with sterile filter paper, and inoculated on solid culture medium without plant growth regulators. Each treatment had 30 bottles, repeated 3 times. The survival rate and the rate of non-contamination were counted after 7 days.

[0010] (3) Exploration of disinfection time for explants: Explants such as leaves, stem tips, root tips and embryos were soaked in soapy water for 5 minutes, then rinsed under running tap water for half an hour, and then rinsed twice with sterile water. They were then placed in sterilized tissue culture bottles and placed in a clean bench for 0-30 seconds of disinfection with 75% alcohol, then rinsed twice with sterile water, then disinfected with 0.1% HgCl2 for 2-5 minutes, and finally rinsed 5 times with sterile water. They were then inoculated onto solid culture medium without plant growth regulators. Each treatment consisted of 30 bottles, repeated 3 times. The survival rate and the rate of no bacterial contamination were counted after 7 days.

[0011] (4) Exploration of callus induction conditions: Sterilized explants (leaves, shoot tips and embryos) were placed in induction medium, which included 4.74 g / L MS medium, 0.5-3 mg / L 6-BA (6-benzyladenine), 0.5-3 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L IBA (indolebutyric acid), 0.5 mg / L TDZ (sepiandronate), 30 g / L sucrose and 3 g / L plant gel. The explants were cultured in a constant temperature culture room at 25±1℃, light intensity of 2000 lux, and light intensity of 12 hours / day for 30 days. The callus induction rate was then calculated.

[0012] (5) Exploration of callus proliferation conditions: The induced callus tissue was transferred to the proliferation medium, which included 2.47 g / L 1 / 2 MS medium, plant growth regulators (6-BA, 2,4-D, IBA and TDZ) at the same concentration as the optimal formula in step (4), 15 g / L sucrose and 3 g / L plant gel. The subculture cycle was 7 days and the number of subcultures was 4. After 28 days of proliferation culture, a large amount of callus tissue was obtained.

[0013] Preferably, in step (1), the optimal explant for inducing callus from Agave americana is a 0.5-1 cm long immature embryo. The induction medium formula is 4.74 g / L MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 30 g / L sucrose + 3 g / L plant gel.

[0014] Preferably, in step (2), the optimal sterilization agent for the selected explants is 0.1% HgCl2. The formulation of the solid culture medium without plant growth regulators is: 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel.

[0015] Preferably, in step (3), the optimal treatment times for sterilizing and disinfecting leaves, stem tips, and embryos with 0.1% HgCl2 are 3, 4, and 3 minutes, respectively. The formulation of the solid culture medium without plant growth regulators is the same as in step (2), which is: 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel.

[0016] Preferably, in step (4), the callus induction culture medium consists of: 4.74 g / L MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 30 g / L sucrose + 3 g / L plant gel.

[0017] Preferably, in step (5), the callus proliferation culture medium consists of: 2.47 g / L 1 / 2 MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 15 g / L sucrose + 3 g / L plant gel.

[0018] The pH value of all the above culture media is 5.8-6.0.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention uses Agave agave embryos as explant material for the first time. After culturing in an induction medium for 30 days, a small amount of callus tissue is obtained. The small amount of callus tissue is then transferred to a selected proliferation medium. The process is repeated 4 times in 7-day cycles, or 28 days of culture to obtain a large amount of callus tissue.

[0021] (2) This invention has the advantages of simple operation, short cycle and high efficiency. The method can successfully obtain Agave callus tissue, which provides good experimental material for the genetic transformation research on the photosynthetic mechanism of Agave and also provides a methodological basis for its rapid propagation.

[0022] (3) This invention breaks through the bottleneck of callus induction in aquatic plants and constructs an efficient method for obtaining callus from Agave americana, providing important reference and guidance for obtaining callus from other aquatic higher plants and subsequent genetic engineering research. Attached Figure Description

[0023] To clearly present the objectives, technical solutions, and beneficial effects of this invention, the following figures are provided for illustration:

[0024] Figure 1 These are agave seeds that have germinated after being soaked for 3 days.

[0025] Figure 2 This refers to the growth of Agave americana embryos to 0.5-1cm.

[0026] Figure 3 Sterile agave leaves;

[0027] Figure 4 The stem tip of sterile Agave agave;

[0028] Figure 5 The root tip of sterile Agave americana;

[0029] Figure 6 It is a sterile Agave agave embryo;

[0030] Figure 7 The growth of embryos 7 days after inoculation into the induction medium;

[0031] Figure 8 The growth of embryos 12 days after inoculation into the induction medium;

[0032] Figure 9 The callus tissue was formed 25 days after inoculation with immature embryos and induction culture medium.

[0033] Figure 10 The callus tissue was obtained 40 days after inoculation with immature embryos and induction culture medium. Detailed Implementation

[0034] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0035] Example 1

[0036] Explant Acquisition and Screening

[0037] Select healthy agave plants and cut off tender, robust leaves, stem tips, and root tips as explants. Additionally, soak mature seeds that have broken dormancy in tap water to promote germination. Figure 1 Once the embryos grow to 0.5-1cm, they are also used as explants. Figure 2 All explants were soaked in soapy water for 5 minutes, then rinsed under running tap water for half an hour, and then rinsed twice with sterile water. They were then placed in sterile tissue culture bottles, placed in a clean bench, rinsed twice with sterile water, sterilized with 0.1% HgCl2 for 3 minutes, and finally rinsed five times with sterile water. Leaves, shoot tips, root tips, and immature embryos were then inoculated onto induction medium. The induction medium formula was 4.74 g / L MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 30 g / L sucrose + 3 g / L plant gel. Each treatment had 30 bottles, replicated three times. The induction rate was calculated after 30 days. Callus emergence rate = number of calluses / number of inoculated plants. The results showed that only immature embryos could successfully induce callus (76.58%), while no signs of callus were found in other explants (0%). Therefore, immature embryos are the best explants for inducing callus in Agave americana.

[0038] Example 2

[0039] Screening of disinfectants for explants

[0040] Explants, including leaves, stem tips, root tips, and young embryos, were soaked in soapy water for 5 minutes, then rinsed under running tap water for half an hour, and rinsed twice with sterile water. They were then placed in sterilized tissue culture bottles and placed in a clean bench for sterilization with 75% alcohol for 15 seconds, followed by rinsing twice with sterile water. Afterward, they were sterilized with 10% H₂O₂, 4% NaClO, 84 disinfectant, benzalkonium chloride, and 0.1% HgCl₂ for 2, 5, 10, and 15 minutes respectively. Finally, they were rinsed five times with sterile water, blotted dry with sterile filter paper, and inoculated onto solid culture medium without plant growth regulators. The formulation was: 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel. Each treatment had 30 bottles, replicated three times. The survival and non-contamination rate were counted after 7 days. Non-contamination rate = number of non-contaminants / number of inoculum. The results are shown in Table 1. Figure 3-6 As shown.

[0041] Table 1. Statistical results of explant survival and non-contamination after different sterilization and disinfection treatments.

[0042]

[0043]

[0044] As shown in Table 1, the fibrous roots of Agave sativus lost all activity after treatment with various disinfectants and were no longer used in subsequent experiments. For stem tips, leaves, and young embryos, 0.1% HgCl2 was the best disinfectant for sterilizing Agave sativus explants, as it achieved the highest rate of non-contamination.

[0045] Example 3

[0046] Exploration of disinfection time for explants

[0047] Explants, including stem tips, leaves, and young embryos, were soaked in soapy water for 5 minutes, then rinsed under running tap water for half an hour, and rinsed twice with sterile water. They were then placed in sterilized tissue culture bottles and placed in a clean bench for sterilization with 75% alcohol for 0-30 seconds, followed by rinsing twice with sterile water, sterilization with 0.1% HgCl2 for 2-5 minutes, and finally rinsed five times with sterile water. The culture was then inoculated onto solid culture medium without plant growth regulators, with the following formulation: 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel. Thirty bottles were used for each treatment, replicated three times. The survival and non-contamination rates were counted after 7 days. The results are shown in Table 2.

[0048] Table 2. Results of viable and uncontaminated specimens after sterilization with 75% alcohol and 0.1% HgCl2 at different times.

[0049]

[0050] As shown in Table 2, the best sterilization method for leaves, stem tips, and embryos is to treat the explants with 0.1% HgCl2 for 3, 4, and 3 minutes, and the sterilization effect is best without treatment with 75% alcohol.

[0051] Example 4

[0052] Exploration of callus induction conditions

[0053] Mature seeds that had broken dormancy were soaked in tap water to promote germination. When the embryos grew to 0.5-1 cm, they were used as explants. These explants were soaked in soapy water for 5 minutes, then rinsed under running tap water for half an hour, followed by two rinses with sterile water. They were then placed in sterile tissue culture bottles, placed in a clean bench, rinsed twice with sterile water, sterilized with 0.1% HgCl2 for 3 minutes, and finally rinsed five times with sterile water before being inoculated onto induction medium. The induction medium consisted of 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel as the basal medium, prepared according to the concentration ratio of plant growth regulators in Table 3. Each treatment had 30 bottles, replicated three times. The callus induction rate was calculated after 30 days. The results are shown in Table 3. Figure 7-10 As shown.

[0054] Table 3. Effects of different combinations of plant growth regulator concentrations on callus induction.

[0055]

[0056] From Table 3, Figure 7-10 The optimal callus induction medium composition shown is: 4.74 g / L MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 30 g / L sucrose + 3 g / L plant gel.

[0057] In Examples 1-4 above, the pH of the culture medium was controlled between 5.8 and 6.0. The culture conditions were all: temperature 25±1℃, humidity 50%-60%, light intensity 2000 lux, and light exposure 12 hours / day in a constant temperature culture room.

[0058] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for efficiently obtaining callus tissue from the aquatic plant Agave americana, characterized in that, Includes the following steps: (1) Acquisition and screening of explants: mature seeds that have broken dormancy were soaked in tap water to promote germination. When the embryos grew to 0.5-1 cm, they were used as explants. After sterilization, they were inoculated onto induction medium. (2) Screening of explant disinfectants: Immerse the embryonic explants in soapy water for 5 min, then rinse them under running tap water for half an hour, and then rinse them twice with sterile water. Place them in sterile tissue culture bottles, then put them in a clean bench, disinfect them with 75% alcohol for 15 s, rinse them twice with sterile water, and then treat them with 10% H2O2, 4% NaClO, 84 disinfectant, benzalkonium chloride and 0.1% HgCl2 for 2, 5, 10 and 15 min respectively. Finally, rinse them five times with sterile water, absorb the water with sterile filter paper, and then inoculate them on solid culture medium without plant growth regulators. (3) Exploration of explant disinfection time: Immature embryo explants were soaked in soapy water for 5 min, then rinsed under running tap water for half an hour, then rinsed twice with sterile water, placed in sterile tissue culture bottles, then placed in a clean bench, disinfected with 75% alcohol for 0-30 s, then rinsed twice with sterile water, then disinfected with 0.1% HgCl2 for 2-5 min, and finally rinsed 5 times with sterile water and inoculated on solid culture medium without plant growth regulators; (4) Exploration of callus induction conditions: Sterilized explant embryos were placed in induction medium, which consisted of 4.74 g / L MS medium, 0.5-2 mg / L 6-BA, 1-2 mg / L 2,4-D, 0.5 mg / L IBA, 0.5 mg / L TDZ, 30 g / L sucrose and 3 g / L plant gel. The embryos were cultured in a constant temperature culture room at 25±1℃, with a light intensity of 2000 lux and a light intensity of 12 hours / day. (5) Exploration of callus proliferation conditions: The induced callus tissue was transferred to the proliferation medium, which was 2.47 g / L 1 / 2 MS medium. The concentration of plant growth regulator was the same as the optimal formula in step (4), 15 g / L sucrose and 3 g / L plant gel. The subculture cycle was 7 days and the number of subcultures was 4. After 28 days of proliferation culture, a large amount of callus tissue was obtained.

2. The method for efficiently obtaining callus tissue of the aquatic plant Agave americana according to claim 1, characterized in that, In step (2), the optimal disinfectant for the selected explant is 0.1% HgCl2; The formulation of solid culture medium without plant growth regulators is: 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel.

3. The method for efficiently obtaining callus tissue of the aquatic plant Agave americana according to claim 1, characterized in that, In step (3), the optimal treatment time for sterilizing and disinfecting the embryos with 0.1% HgCl2 is 3 min; The formulation of the solid culture medium without plant growth regulators is the same as in step (2): 4.74 g / L MS medium + 30 g / L sucrose + 3 g / L plant gel.

4. The method for efficiently obtaining callus tissue of the aquatic plant Agave americana according to claim 1, characterized in that, In step (4), the induction medium consists of: 4.74 g / L MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 30 g / L sucrose + 3 g / L plant gel.

5. The method for efficiently obtaining callus tissue of the aquatic plant Agave americana according to claim 1, characterized in that, In step (5), the proliferation medium consists of: 2.47 g / L 1 / 2MS medium + 1 mg / L 6-BA + 2 mg / L 2,4-D + 0.5 mg / L IBA + 0.5 mg / L TDZ + 15 g / L sucrose + 3 g / L plant gel.

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