Combined culture medium for portulaca oleracea anther culture and method for culturing portulaca oleracea by using culture medium

Abstract

The invention provides a combined culture medium for portulaca oleracea anther culture and a method for culturing portulaca oleracea by using the culture medium, and belongs to the technical field of plant tissue culture. The combined culture medium comprises an induction culture medium, a differential culture medium and a rooting culture medium. By adopting the combined culture medium provided by the invention, the regeneration plant of the purslane is obtained through anther culture for the first time, and an effective technical means is provided for breeding and genetic improvement of the purslane.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and particularly relates to a combined culture medium for culturing purslane anthers and a method for culturing purslane using the culture medium. Background Technology

[0002] purslane( Portulaca oleracea Purslane (L.) is an annual succulent herb belonging to the Portulacaceae family, mainly distributed in temperate regions. Purslane possesses various medicinal properties, such as anti-diabetic, anti-inflammatory, anti-cancer, antioxidant, and antibacterial effects. It is rich in nutrients, containing omega-3 fatty acids, vitamins A, C, and E, as well as essential minerals. The tender stems and leaves can be eaten as a vegetable. Furthermore, purslane exhibits strong resilience to adverse conditions, making it an excellent plant for ecological restoration. Therefore, given its combined edible, medicinal, and ecological value, further research and development of purslane are necessary. Utilizing biotechnology to breed superior, high-yielding varieties has become an imperative task.

[0003] Double haploid (DH) technology is a valuable tool in modern breeding programs. It can rapidly generate pure lines, significantly reducing the time required for developing new varieties. DH lines possess homozygosity and uniformity, making double haploids ideal materials for genetic mapping. Furthermore, haploids and DH are important tools in mutation breeding, enabling the rapid fixation of mutant traits. Anther culture is one of the commonly used techniques for producing haploids. This process involves aseptically inoculating anthers at a certain developmental stage onto a specific culture medium, inducing the formation of callus or embryoids, which then redifferentiate to form complete plants. However, limitations imposed by the genotype of the culture material and culture conditions make anther culture regeneration difficult for some species, becoming a bottleneck problem that urgently needs to be overcome in haploid breeding.

[0004] The explants used in purslane tissue culture are mostly leaves, stems, roots, and seeds, primarily forming regenerated plants through somatic embryogenesis. Anther culture technology can induce haploids in purslane, and subsequently double haploids, with all traits exhibiting stable homozygosity at a single stage. This technique plays a crucial role in accelerating the breeding process and genetic improvement of purslane. Currently, no reports have been found regarding purslane anther culture technology. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a combined culture medium for culturing purslane anthers and a method for culturing purslane using the combined culture medium of the present invention. The combined culture medium of the present invention is used to culture purslane anthers to obtain callus tissue, which can be further differentiated to form regenerated plants. This establishes a method for culturing purslane anthers.

[0006] To achieve the above-mentioned purposes, the present application provides the following technical solutions. The present application provides a combined medium for culturing anther of Portulaca oleracea, which comprises an induction medium, a differentiation medium and a rooting medium. The induction medium comprises the following components in the final concentration based on MS medium: 28~32g / L sucrose, 0.5~1.5mg / L 6-BA, 1~2mg / L NAA, 5~9g / L agar; The differentiation medium comprises the following components in the final concentration based on MS medium: 28~32g / L sucrose, 0.5~1.5mg / L 6-BA, 0.2~0.6mg / L IAA, 5~9g / L agar; The rooting medium comprises the following components in the final concentration based on 1 / 2MS medium: 28~32g / L sucrose, 0.3~0.7mg / L NAA, 5~9g / L agar.

[0007] The present application provides a method for culturing Portulaca oleracea using the combined medium, which comprises the following steps: (1) low-temperature pretreatment of the flower bud of Portulaca oleracea, sterilization and anther extraction; (2) placing the anther in the induction medium for induction culture to obtain callus; (3) placing the callus in the differentiation medium for differentiation culture to obtain a cluster of buds; (4) placing the cluster of buds in the rooting medium for rooting culture to obtain Portulaca oleracea plants.

[0008] Preferably, the pretreatment temperature is 2~6℃ and the pretreatment time is 1~3d.

[0009] Preferably, the induction culture time is 19~25d and the induction culture temperature is 22~27℃.

[0010] Preferably, the differentiation culture time is 2~3 months and the differentiation culture temperature is 22~27℃.

[0011] Preferably, the rooting culture time is 25~30d and the rooting culture temperature is 22~27℃.

[0012] The present application also provides the use of the combined medium in culturing Portulaca oleracea.

[0013] Compared with the prior art, the present application has the following beneficial effects: the present application provides a combined culture medium for anther culture of Portulaca oleracea and a method for culturing Portulaca oleracea by using the culture medium, the anther callus induction medium, differentiation medium and rooting culture medium provided by the present application can realize regeneration of Portulaca oleracea by anther culture, and provide an effective technical means for breeding and genetic improvement of Portulaca oleracea. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 1 is a diagram of the morphology of anther callus induction of Portulaca oleracea; Figure 2 Figure 2 is a diagram of the differentiation of anther callus of Portulaca oleracea, wherein the left side is a purple bud point just differentiated from the callus, and the right side is a cluster bud formed by the callus; Figure 3 Figure 3 is a diagram of the cluster bud of Portulaca oleracea; Figure 4 Figure 4 is a diagram of the formation of a complete plant from the cluster bud of Portulaca oleracea. DETAILED DESCRIPTION

[0015] The present application provides a combined culture medium for anther culture of Portulaca oleracea, which comprises an induction medium, a differentiation medium and a rooting medium; The induction medium is based on MS medium and further comprises the following components at the following final concentrations: sucrose 28~32g / L, preferably 29~31g / L, and further preferably 30g / L; 6-BA 0.5~1.5mg / L, preferably 0.7~1.2mg / L, and further preferably 1mg / L; NAA 1~2mg / L, preferably 1.2~1.8mg / L, and further preferably 1.5mg / L; agar 5~9g / L, preferably 6~8g / L, and further preferably 7g / L; The differentiation medium is based on MS medium and further comprises the following components at the following final concentrations: sucrose 28~32g / L, preferably 29~31g / L, and further preferably 30g / L; 6-BA 0.5~1.5mg / L, preferably 0.7~1.2mg / L, and further preferably 1mg / L; IAA 0.2~0.6mg / L, preferably 0.3~0.5mg / L, and further preferably 0.4mg / L; agar 5~9g / L, preferably 6~8g / L, and further preferably 7g / L; The rooting medium is based on 1 / 2MS medium and further comprises the following components at the following final concentrations: Sucrose, preferably 29~31g / L, further preferably 30g / L; NAA 0.3~0.7mg / L, preferably 0.4~0.6mg / L, further preferably 0.5mg / L; Agar 5~9g / L, preferably 6~8g / L, further preferably 7g / L.

[0016] The application provides a method for culturing Portulaca oleracea by using the combined medium, comprising the following steps: (1) low-temperature pretreatment of the flower buds of Portulaca oleracea, sterilization, and anther extraction; (2) placing the anther in the induction medium for induction culture to obtain callus; (3) placing the callus in the differentiation medium for differentiation culture to obtain a cluster of buds; (4) placing the cluster of buds in the rooting medium for rooting culture to obtain Portulaca oleracea plants.

[0017] In the application, the flower buds of Portulaca oleracea are subjected to low-temperature pretreatment, sterilization, and anther extraction. Preferably, young flower buds at the initial flowering stage are placed in a refrigerator at 2~6℃ for 1~3d of pretreatment, the pretreated flower buds are soaked in a 8%~12% sodium hypochlorite solution for 8~12min, rinsed with water for 3~5 times, the water on the surface of the flower buds is absorbed, and the anther is extracted. The length of the young flower buds is 2.5~3.5mm, the pretreatment temperature is preferably 3~5℃, further preferably 4℃, the concentration of the sodium hypochlorite solution is preferably 9%~11%, further preferably 10%, and the soaking time is preferably 9~11min, further preferably 10min.

[0018] In the application, the anther is placed in the induction medium for induction culture to obtain callus; the induction medium is based on MS medium and further comprises the following components at the following final concentrations: 28~32g / L sucrose, 0.5~1.5mg / L 6-BA, 1~2mg / L NAA, and 5~9g / L agar; the induction culture time is 19~25d, preferably 20~23d, further preferably 21d, and the induction culture temperature is 22~27℃, preferably 24~26℃, further preferably 25℃.

[0019] In the present application, the callus is placed in the differentiation culture medium, differentiation culture is carried out, and the cluster shoots are obtained; the differentiation culture medium further comprises the following components in the terminal concentration based on the MS culture medium: 28-32 g / L sucrose, 0.5-1.5 mg / L 6-BA, 0.2-0.6 mg / L IAA, and 5-9 g / L agar; the time of the differentiation culture is 2-3 months, and the temperature of the differentiation culture is 22-27℃, preferably 24-26℃, and further preferably 25℃.

[0020] In the present application, the cluster shoots are placed in the rooting culture medium, rooting culture is carried out, and the purslane plants are obtained; the rooting culture medium further comprises the following components in the terminal concentration based on the 1 / 2MS culture medium: 28-32 g / L sucrose, 0.3-0.7 mg / L NAA, and 5-9 g / L agar; the time of the rooting culture is 25-30 days, preferably 27-29 days, and further preferably 28 days, and the temperature of the rooting culture is 22-27℃, preferably 24-26℃, and further preferably 25℃.

[0021] The present application also provides the application of the combined culture medium in the culture of purslane.

[0022] The technical solutions provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the protection scope of the present application.

[0023] Embodiment

[0024] The purslane seedlings provided by Jiangsu Coastal Region Agricultural Science Institute are used as raw materials, are placed in the greenhouse of Shanghai Academy of Agricultural Sciences Biotechnology Institute, and are subjected to conventional water and fertilizer management.

[0025] The culture method of purslane is as follows: (1) The flower buds of purslane in the initial flowering stage are collected, which are healthy, full, about 3 mm in length, and in the single nuclear side period, are placed in a 4℃ refrigerator for 3d to obtain the flower buds treated by low temperature, are prepared for use, and the oversize flower buds are removed in time to prolong the flowering period.

[0026] (2) The flower buds treated by low temperature are placed in 10% sodium hypochlorite for 10 min, are washed with sterile water for 3-5 times, and the surface water is absorbed by the water absorption paper.

[0027] (3) In the super-clean bench, the flower buds are peeled off with tweezers, the anthers are carefully picked, and the anther walls are avoided to be damaged as much as possible. Since the anthers are very small, the process can be peeled off with the aid of a magnifying glass.

[0028] (4) Put the obtained anther on the induction medium for culture, the medium component is MS medium as the basic medium, adding 30 g / L sucrose, 1 mg / L 6-BA, 1.5 mg / L NAA, 7 g / L agar solidification, pH is about 5.8. After dark culture for 3 weeks at 25±1℃, callus is obtained, the results are shown in Figure 1 .

[0029] (5) Put the induced callus on the differentiation medium, the callus turns green and expands rapidly, subculture once a month, when subculture, the callus is cut into small pieces, continue to grow on the same medium, culture for 3 months at 25±1℃, the light time is 16 h, and then the multiple shoots are obtained, the results are shown in Figure 2 . The callus differentiation medium is MS as the basic medium, adding 30 g / L sucrose, 1 mg / L 6-BA, 0.4 mg / L IAA, 7 g / L agar solidification, pH is about 5.8.

[0030] (6) Cut the multiple shoots ( Figure 3 ) from the callus, put on the strong seedling rooting medium, culture for 4 weeks at 25±1℃, the light time is 16 h, so that the multiple shoots continue to grow and root, and then the complete plant is formed ( Figure 4 ). The strong seedling rooting medium is 1 / 2MS medium, 30 g / L sucrose, 0.5 mg / L NAA, 7 g / L agar solidification, pH is about 5.8.

[0031] As shown in Figure 1 , in the callus culture process, most of the anthers are browned and dead, only a small number of anthers form callus, and the callus induction rate is 12.2%.

[0032] As shown in Figure 2 , the purple bud points grow on the surface of the callus first, and after 3 months of culture, the bud points on the surface of the callus can slowly grow into cluster multiple shoots.

[0033] As shown in Figure 3 and Figure 4 , cut the multiple shoots from the callus, put on the strong seedling rooting medium, the multiple shoots gradually elongate and root, and then the complete plant is formed.

[0034] The above only describes the preferred embodiments of the present application, and it should be noted that, for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A composite culture medium for culturing purslane anthers, characterized in that, The combined culture medium includes induction medium, differentiation medium, and rooting medium; The induction medium, based on MS medium, also includes the following components at the following final concentrations: 28~32g / L sucrose, 0.5~1.5mg / L 6-BA, 1~2mg / L NAA, 5~9g / L agar; The differentiation medium, based on MS medium, also includes the following components at the following final concentrations: 28~32g / L sucrose, 0.5~1.5mg / L 6-BA, 0.2~0.6mg / L IAA, 5~9g / L agar; The rooting medium, based on 1 / 2 MS medium, also includes the following components at the following final concentrations: 28~32g / L sucrose, 0.3~0.7mg / L NAA, 5~9g / L agar.

2. A method for culturing purslane using the combined culture medium according to claim 1, characterized in that, Includes the following steps: (1) Pre-treat the purslane flower buds at low temperature, disinfect them, and collect the anthers; (2) Place the anthers in the induction medium and induce culture to obtain callus tissue; (3) The callus tissue was placed in the differentiation medium and cultured to obtain clustered shoots; (4) Place the clustered buds in the rooting medium and root them to obtain purslane plants.

3. The cultivation method according to claim 2, characterized in that, The pretreatment temperature is 2~6℃, and the pretreatment time is 1~3 days.

4. The cultivation method according to claim 2, characterized in that, The induction culture time is 19-25 days, and the induction culture temperature is 22-27℃.

5. The cultivation method according to claim 2, characterized in that, The differentiation culture time is 2 to 3 months, and the differentiation culture temperature is 22 to 27℃.

6. The cultivation method according to claim 2, characterized in that, The rooting culture time is 25-30 days, and the rooting culture temperature is 22-27℃.

7. The application of the combined culture medium according to claim 1 in the cultivation of purslane.

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