A method for constructing an in vitro callus regeneration system of common wild rice

Abstract

The application discloses a construction method of a common wild rice callus in-vitro regeneration system, and comprises the following steps: (1) obtaining an explant; (2) callus induction, wherein the induction culture medium is MS 4.74g / L+3% sucrose+0.3% plant gel+6-BA 0.5mg / L+2,4-D 2mg / L; (3) callus subculture; (4) induction of adventitious buds, wherein the differentiation culture medium is MS 4.74g / L+3% sucrose+0.3% plant gel+NAA 2mg / L+KT 1.5mg / L+6-BA 0.5mg / L; (5) strong seedling culture; (6) rooting culture; and (7) seedling transplanting. The application improves the induction germination rate and the proliferation coefficient. The test proves that the regenerated seedlings obtained by the application have the advantages of strong buds, strong main roots and high survival rate of seedling transplanting. The method for constructing the common wild rice regeneration system provided by the application provides strong technical support for the differentiation of common wild rice into seedlings through seed tissue culture and rapid propagation, and also provides an effective method for the protection and utilization of common wild rice.

Description

Technical Field

[0001] This invention relates to the field of plant propagation technology, and in particular to a method for constructing an in vitro regeneration system of common wild rice callus. Background Technology

[0002] China boasts diverse wetland types and a vast distribution area, nurturing a rich variety of wetland wildlife. However, due to its large population and rapid industrialization, urbanization, and agricultural modernization, wetland ecosystems have faced immense pressure from human society. Wetlands have become one of the most threatened ecosystems globally. Wetland degradation, restoration, and reconstruction are current research hotspots in wetland science and core issues in Chinese wetland research. Scientific wetland protection and restoration are of great significance for protecting and restoring rare and endangered species and biodiversity.

[0003] Wild rice is a close wild relative of cultivated rice, with more than 25 species identified (Gowda et al., 2003). Having grown in natural environments for a long time and undergone natural selection under various adverse conditions, it has developed an extremely rich genetic base, containing numerous excellent germplasm resources and accumulating many superior genes, making it a natural gene pool for cultivated rice germplasm innovation and variety improvement. Among them, common wild rice (Oryza rufipogon Griff.) is a close ancestor of Asian cultivated rice, mostly living in various wetland environments such as ponds, ditches, and swamps. Common wild rice contains rich phenotypic and genetic variations, including excellent genes for disease and pest resistance, cold resistance, and stress resistance. It also has a good purifying effect on eutrophic water bodies, making it a promising plant for wetland restoration.

[0004] However, due to human activities and environmental damage, coupled with factors such as land reclamation, aquaculture, and a lack of conservation awareness, the native habitat of wild rice in my country is rapidly shrinking, with some distribution points on the verge of extinction or even already extinct. Against this backdrop, my country has gradually begun to emphasize the protection of the native habitat of wild rice, designating it as a national second-class protected endangered plant and including it in the China Plant Red Data Book. Therefore, establishing an efficient and rapid in vitro regeneration system for wild rice is urgently needed. This system can not only protect valuable material resources in rice genetic breeding but also provide significant restoration and landscape benefits for wetland environments.

[0005] There are numerous reports on the rapid propagation of wild rice through tissue culture. For example, Wang Lingxian et al. (2018) constructed a rapid propagation method for *O. eichlingeri* A. Peter using roots, buds, stems, leaves, leaf sheaths, and mature seeds as explants. Yan Chengqi et al. (2009 and 2016) obtained callus tissues from *O. meyeriana* and *O. officinalis* using young shoots and mature embryos, respectively. Ni Wenjin et al. (2007) obtained in vitro regenerated plants of *O. alta* using leaves as explants, but the efficiency of callus induction was relatively low (27.37%). Research on the rapid propagation of common wild rice (O. rufipogon) is mainly found in the 2017 study by Qiu Yongfu et al., who used sterile in vitro seedlings obtained from the germination of common wild rice seeds as explants to construct a tissue culture system. However, obtaining sterile in vitro seedlings and adding extracts such as kelp extract, aloe extract, wheat germ extract, and Chlorella extract during tissue culture is not only difficult due to the availability of these extracts, but the tissue culture process is also cumbersome and time-consuming. Therefore, in order to better protect and utilize common wild rice resources, it is essential to develop a more convenient, efficient, and rapid in vitro regeneration system. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for constructing an in vitro regeneration system of common wild rice callus, which can rapidly propagate a large number of high-quality common wild rice seedlings suitable for transplanting.

[0007] To achieve the above objectives, the technical solution adopted in this invention is: a method for constructing an in vitro regeneration system of common wild rice callus, using mature seeds as explants to complete in vitro regeneration, comprising the following steps:

[0008] (1) Obtaining explants: Fresh mature seeds of common wild rice were taken as explants. The rice husks were removed to expose the rice grains. The seeds were rinsed and soaked in tap water for 10 minutes, rinsed twice with sterile water, soaked in 75% alcohol for 30 seconds, rinsed once with sterile water, and then transferred to 0.1% mercuric chloride and shaken for 5 minutes for disinfection. The seeds were then rinsed five times with sterile water and the surface moisture was dried with sterile filter paper to obtain sterile explants.

[0009] (2) Induction of callus: The sterile explants obtained in step (1) were inoculated onto a solid culture medium for inducing callus. A small amount of callus was induced after about 15 days of culture. The induction medium was: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + 6-BA (6-benzyladenine) 0.5 mg / L + 2,4-D (2,4-dichlorophenoxyacetic acid) 2 mg / L.

[0010] (3) Callus subculture: When the callus tissue in step (2) grows to a diameter of about 0.3 cm, subculture is started to obtain embryogenic callus tissue. The subculture medium has the same composition as the induction medium. The subculture cycle is 7 days and the number of subcultures is 4. After 28 days of proliferation culture, a large amount of firm, transparent, and granular callus tissue is obtained.

[0011] (4) Induction of adventitious shoots: The callus cultured in step (3) was transferred to differentiation medium and cultured for 7 days to grow many adventitious shoots of common wild rice; the differentiation medium was: MS 4.74g / L + 3% sucrose + 0.3% plant gel + NAA (naphthaleneacetic acid) 2mg / L + KT (kinetin) 1.5mg / L + 6-BA (6-benzyladenine) 0.5mg / L;

[0012] (5) Seedling cultivation: The common wild rice seedlings obtained in step (4) are transferred to the seedling cultivation medium and cultured under light for 10 days to obtain common wild rice seedlings; the seedling cultivation medium has the same composition as the differentiation medium.

[0013] (6) Rooting culture: The robust seedlings obtained in step (5) were transferred to the rooting medium to induce rooting. After 15 days of culture, ordinary wild rice seedlings were obtained. The rooting medium was: 1 / 2 MS 2.47 g / L + 3% sucrose + 0.3% plant gel + IBA (indolebutyric acid) 0.5 mg / L.

[0014] (7) Hardening and transplanting: When the rooted seedlings in step (6) reach a height of 10-15cm, remove the bottle caps and harden the seedlings in the tissue culture room for 1-2 days. Take the regenerated seedlings out of the culture bottle, wash off the culture medium from the roots, and transplant them into a mixed substrate (by volume, peat moss: vermiculite = 2:1). Continue to cultivate until the seedlings reach a height of 30-50cm, and then transplant them normally.

[0015] Preferably, in step (1), after removing the husk from fresh, mature wild rice seeds, the seed coat is sanded off using 400-mesh kraft paper sandpaper.

[0016] Preferably, the culture conditions for steps (2)-(7) are: a constant temperature culture room with a temperature of 25±1℃, a humidity of 50%-60%, a light intensity of 2000 lux, and a light intensity of 12 hours / day.

[0017] Preferably, the pH of all culture media is 5.8-6.0.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) In this invention, a tissue culture rapid propagation system for common wild rice was established, using mature seeds as explants, which breaks through the limitations of season and growth stage when obtaining explants.

[0020] (2) The present invention eliminates the sterile test-tube seedling stage and the addition of various extracts, which greatly improves the convenience and effectiveness of rapid propagation of common wild rice tissue culture.

[0021] (3) This invention significantly improves the callus induction rate and seedling rate of mature seeds of common wild rice by optimizing and adjusting plant growth regulators. It effectively solves the problem of rapid propagation of wild rice, promotes the protection and utilization of common wild rice, and provides an efficient method for obtaining large quantities of common wild rice on a large scale. Attached Figure Description

[0022] To clearly present the objectives, technical solutions, and beneficial effects of this invention, the following figures are provided for illustration:

[0023] Figure 1 Common wild rice seeds were inoculated onto an induction medium;

[0024] Figure 2 The germination and whitening of common wild rice seeds on an induction medium;

[0025] Figure 3 This refers to the formation of callus tissue in common wild rice.

[0026] Figure 4 A small amount of callus tissue was induced in common wild seeds;

[0027] Figure 5 Subculture of callus tissue from common wild rice;

[0028] Figure 6 This is the induced formation of adventitious buds in common wild rice;

[0029] Figure 7 Cultivate robust seedlings of common wild rice for 5 days;

[0030] Figure 8 Cultivate robust seedlings of common wild rice for 10 days;

[0031] Figure 9 Inducing root growth in robust seedlings of common wild rice;

[0032] Figure 10 The rooting and growth of vigorous seedlings of common wild rice;

[0033] Figure 11 Common wild rice seedlings were hardened off and transplanted into a mixed substrate. Detailed Implementation

[0034] To better illustrate the purpose, technical solution, and advantages of the present invention, the following detailed description of embodiments of the present invention will be provided in conjunction with the accompanying drawings.

[0035] This embodiment provides a method for constructing an in vitro regeneration system of common wild rice callus, comprising the following steps:

[0036] (1) Obtaining explants: Fresh mature seeds of common wild rice were taken as explants. The rice husks were removed to expose the rice grains. The seed coat was removed with 400-mesh kraft paper and wet sandpaper. The seeds were rinsed and soaked in tap water for 10 minutes, rinsed twice with sterile water, soaked in 75% alcohol for 30 seconds, rinsed once with sterile water, and then transferred to 0.1% mercuric chloride and shaken for 5 minutes for sterilization. The seeds were then rinsed five times with sterile water and the surface moisture was dried with sterile filter paper to obtain sterile explants.

[0037] (2) Induction of callus: The sterile explants obtained in step (1) were inoculated onto a solid culture medium for inducing callus. Figure 1 Seeds germinate and show white sprouts in about 3 days. Figure 2 A small amount of callus tissue can be induced after about 15 days of culture. Figure 3 , Figure 4 The induction medium was: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + 6-BA 0.5 mg / L + 2,4-D 2 mg / L.

[0038] (3) Callus subculture: When the callus tissue from step (2) grows to a diameter of approximately 0.3 cm, subculture begins to obtain embryogenic callus tissue. The subculture medium has the same composition as the induction medium: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + 6-BA 0.5 mg / L + 2,4-D 2 mg / L. The subculture cycle is 7 days, and the number of subcultures is 4. After 28 days of proliferation culture, a large amount of firm, milky white, and plump callus tissue is obtained. Figure 5 ).

[0039] (4) Induction of adventitious shoots: The callus tissue cultured in step (3) was transferred to differentiation medium and cultured for 7 days to grow many adventitious shoots of common wild rice. Figure 6 Differentiation medium was: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + NAA 2 mg / L + KT 1.5 mg / L + 6-BA 0.5 mg / L.

[0040] (5) Seedling cultivation: The common wild rice seedlings obtained in step (4) were transferred to a seedling cultivation medium and cultured under light for 10 days to obtain common wild rice seedlings. Figure 7 , Figure 8 The seedling growth medium and differentiation medium have the same composition: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + NAA 2 mg / L + KT 1.5 mg / L + 6-BA 0.5 mg / L.

[0041] (6) Rooting culture: The robust seedlings obtained in step (5) are transferred to a rooting culture medium to induce rooting. After 15 days of culture, ordinary wild rice seedlings are obtained. Figure 9 The rooting medium was: 1 / 2 MS 2.47 g / L + 3% sucrose + 0.3% plant gel + IBA 0.5 mg / L.

[0042] (7) Hardening off and transplanting: The rooted seedlings from step (6) should be 10-15cm tall. Remove the bottle caps and harden off in the tissue culture room for 1-2 days. Remove the regenerated seedlings from the culture bottles and wash off the culture medium from the roots. Figure 10 Transplant to a mixed substrate (by volume, peat moss: vermiculite = 2:1) Figure 11 Continue cultivating until the seedlings reach a height of 30-50cm, then transplant normally.

[0043] In this embodiment, the pH value of all culture media was 6.0.

[0044] In this embodiment, the culture conditions for steps (2)-(7) are: temperature of 25±1℃, humidity of 50%-60%, light intensity of 2000 lux, and constant temperature culture room with light exposure of 12 hours / day.

[0045] In this embodiment, the seed germination rate is over 95%, the callus induction rate is over 98%, the adventitious bud induction rate is over 90%, the rooting rate is over 98%, and the survival rate of the transplanted seedbed with clustered buds is 100%. The plants grow rapidly and are robust. Statistics show that callus induced from a single common wild rice seed can differentiate into 30-40 seedlings within 2-3 months.

[0046] The above-mentioned technical methods have established a highly efficient tissue culture in vitro regeneration system for common wild rice, which has overcome the problem of obtaining regenerated seedlings from common wild rice seeds, expanded the propagation range of common wild rice, and has the advantages of simple operation, low pollution rate, high survival rate and short culture cycle. It is worth promoting widely.

[0047] 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 constructing an in vitro callus regeneration system of Oryza rufipogon, characterized in that, It comprises the following steps: (1) Obtaining of explants: Fresh mature seeds of common wild rice are selected as explants, the husks are removed to expose the rice grains, the rice grains are washed with tap water for 10 min, washed with sterile water for 2 times, soaked in 75% alcohol for 30 s, washed with sterile water for 1 time, transferred into 0.1% mercuric chloride for shaking sterilization for 5 min, washed with sterile water for 5 times, the surface water is absorbed with sterilized filter paper, and the sterile explants are obtained; (2) Induction of callus: the sterile explants obtained in step (1) are inoculated on a solid medium for inducing callus, and a small amount of callus can be induced after 15 days; the induction medium is: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + 6-BA 0.5 mg / L + 2,4-D 2 mg / L; (3) Subculture of callus: the callus in step (2) is grown to a size of 0.3 cm in diameter, then subculture is started to obtain embryogenic callus, the subculture medium is the same as the induction medium, the subculture period is 7 days, the subculture times are 4, and a large amount of compact, transparent and granular callus is obtained after 28 days of proliferation culture; (4) Induction of adventitious buds: the callus cultured in step (3) is transferred to a differentiation medium, and many common wild rice adventitious buds are grown after 7 days of culture; the differentiation medium is: MS 4.74 g / L + 3% sucrose + 0.3% plant gel + NAA 2 mg / L + KT 1.5 mg / L + 6-BA 0.5 mg / L; (5) Culture of strong seedlings: the common wild rice seedlings obtained in step (4) are transferred to a strong seedling culture medium, and the common wild rice strong seedlings are obtained after 10 days of culture under light; the strong seedling culture medium is the same as the differentiation medium; (6) Rooting culture: the strong seedlings obtained in step (5) are transferred to a rooting culture medium to induce rooting, and the common wild rice seedlings are obtained after 15 days of culture; the rooting culture medium is: 1 / 2MS 2.47 g / L + 3% sucrose + 0.3% plant gel + IBA 0.5 mg / L; (7) Seedling hardening and transplanting: the rooting seedlings in step (6) have a height of 10-15 cm, the bottle cap is removed for 1-2 days of seedling hardening in a tissue culture room, the regenerated seedlings are taken out from the culture bottle, the root culture medium is washed away, and the seedlings are transplanted to a mixed substrate for further cultivation until the seedlings have a height of 30-50 cm, and then the seedlings are transplanted normally.

2. The method of claim 1, wherein the method is characterized by, In step (1), the husks of the fresh mature seeds of common wild rice are removed, and the seed coat is ground off with 400-mesh kraft paper water sandpaper.

3. The method of claim 1, wherein the method is characterized by, The culture conditions of steps (2)-(7) are as follows: the temperature condition is 25±1℃, the humidity is 50%-60%, the light intensity is 2000 lux, and the seedlings are cultured in a constant-temperature culture room with 12 hours of light per day.

4. The method of claim 1, wherein the method is characterized by, In step (7), the mixed substrate is composed of grass charcoal soil and vermiculite, and the volume ratio is 2:

1.

5. The method of claim 1, wherein the method is characterized by, The pH value of all culture media is 5.8-6.0.

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