A method for inducing callus of quinoa microspore culture

Abstract

The application provides a culture method of quinoa microspore induced callus and belongs to the technical field of plant tissue culture. Young panicles in the flowering stage are subjected to low-temperature treatment, microcracked flower buds are selected from the young panicles, are mixed with an extraction liquid, are rotated and cut to obtain free microspores; the free microspores are inoculated into an induction culture medium and are cultured to obtain callus. The application establishes the external morphological characteristics of the flower buds in the optimum period of quinoa microspore culture, which is convenient for naked eye identification; and suitable induction culture media are screened to successfully obtain quinoa microspore callus.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and particularly relates to a method for culturing callus induced by quinoa microspores. Background Technology

[0002] Quinoa ( Chenopodium quinoa Quinoa, a pseudograin native to the Andes Mountains of South America, is a promising supergrain due to its nutritional value, versatility, and tolerance to harsh climates. Quinoa is rich in nutrients, containing protein (essential amino acids), dietary fiber, vitamins, phosphorus, potassium, polysaccharides, saponins, and flavonoids. The bioactive components in quinoa, such as ecdysterone, saponins, phytosterols, polysaccharides, and phenolic compounds, are significant for promoting health. Quinoa possesses various biological activities, including antioxidant, neuroprotective, immunomodulatory, lipid-lowering, anti-diabetic, anti-inflammatory, antibacterial, and anti-cancer properties. Despite its excellent characteristics, quinoa's seed yield is typically low (approximately 1-2 tons / hectare), and it has a high cross-pollination rate of about 15%, making it prone to seed contamination and degeneration during cultivation. In the context of rapid advancements in modern biotechnology, breeding techniques have become crucial for ensuring food security and increasing crop yields. Improving the speed of crop improvement is essential for achieving sustainable food production and meeting the other needs of a growing population.

[0003] Doubled haploid (DH) technology is a revolutionary technique for rapidly creating pure lines and accelerating plant breeding processes. This technology improves the efficiency of plant breeding programs, enables the selection of optimal genotypes, and the valuable pure lines obtained have been applied in breeding programs and genetic research. Microspore culture is one of the effective methods for producing double haploids. The basic culture process of this technique mainly includes donor plant collection, stress pretreatment, microspore isolation, and microspore-induced callus regeneration. Using microspore culture technology to induce haploids in quinoa, and subsequently double haploids, with all traits homozygous at once, plays an important role in accelerating quinoa breeding and genetic improvement. Currently, there are no reports on microspore culture of quinoa. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for culturing callus induced by quinoa microspores.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for culturing callus induced by quinoa microspores, comprising the following steps: (1) During the flowering period, young spikelets were treated at low temperature. Microcleft flower buds were selected from the young spikelets, sterilized, mixed with the extract, and rotary cut to obtain free microspores; (2) Inoculate the free microspores into the induction medium and culture them to obtain callus tissue.

[0006] Preferably, the free microspores in the microcleft buds described in step (1) are in the late uninucleate stage.

[0007] Preferably, the temperature of the low-temperature treatment in step (1) is 3~5℃, and the time of the low-temperature treatment is 5~7 days.

[0008] Preferably, the sterilization in step (1) uses 5-15% sodium hypochlorite.

[0009] Preferably, the extract in step (1) comprises the following components: 50~70 g / L mannitol, 0.8~1.5 g / L anhydrous calcium chloride, and 0.8~1.2 g / L 2-morpholine ethanesulfonic acid; Mix 30-50 microcleft flower buds with 10-20 mL of extract.

[0010] Preferably, the rotary cutting speed in step (1) is 800~1200 rpm, the rotary cutting time is 3~8s, and it is repeated 2~4 times.

[0011] Preferably, the density of the free microspores in step (2) is 0.5~2×10⁻⁶. 5 Induction medium (cFU / mL)

[0012] Preferably, the induction medium in step (2) is MS as the basic medium and also includes the following components at the following concentrations: 90 g / L sucrose, 2 mg / L 2,4-D, 1 mg / L KT, 1 mg / L NAA, 0.5 mg / L IAA and 0.976 g / L 2-morpholinoethanesulfonic acid.

[0013] Preferably, the culture temperature in step (2) is 24~26℃ and the culture time is 26~32 days.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention establishes the external morphological characteristics of flower buds at the optimal time for quinoa microspore culture, facilitating visual identification; and successfully obtains quinoa microspore callus tissue by screening suitable induction culture media. Attached Figure Description

[0015] Figure 1 The samples were obtained from quinoa microspores (where A is the young spikelet with bar=1cm; B is the size of the flower buds separated on the young spikelet; Type I flower buds are closed; Type II flower buds are slightly lobed; Type III flower buds are open; Type IV flower buds are fully open). Figure 2The images show the optimal morphological characteristics of flower buds cultured from quinoa microspores and the determination of the microspore stage – late uninucleate stage (where A represents the optimal morphological characteristics of flower buds cultured from quinoa microspores 25-192, B is a bright field image of quinoa microspores 25-192 stage – late uninucleate stage, C is a dark field image of quinoa microspores 25-192 stage – late uninucleate stage, D represents the optimal morphological characteristics of flower buds cultured from quinoa microspores 25-197, E is a bright field image of quinoa microspores 25-197 stage – late uninucleate stage, F is a dark field image of quinoa microspores 25-197 stage – late uninucleate stage, G represents the optimal morphological characteristics of flower buds cultured from quinoa microspores 25-198, H is a bright field image of quinoa microspores 25-198 stage – late uninucleate stage, and I is a dark field image of quinoa microspores 25-198 stage – late uninucleate stage). Figure 3 It is a microspore that initiates cell division; Figure 4 It is callus tissue produced by 25-192 in QQI-14 induction medium. Detailed Implementation

[0016] This invention provides a method for culturing callus induced by quinoa microspores, comprising the following steps: (1) During the flowering period, young spikelets were treated at low temperature. Microcleft flower buds were selected from the young spikelets, sterilized, mixed with the extract, and rotary cut to obtain free microspores; (2) Inoculate the free microspores into the induction medium and culture them to obtain callus tissue.

[0017] In this invention, young spikelets are subjected to low-temperature treatment during the flowering period. Microcleft buds are selected from the young spikelets, sterilized, mixed with the extract, and rotary excised to obtain free microspores. The low-temperature treatment temperature is 3-5℃, preferably 4℃; the low-temperature treatment time is 5-7 days, preferably 6 days; the free microspores in the microcleft buds are in the late uninucleate stage; sterilization uses 5-15% sodium hypochlorite, preferably 10% sodium hypochlorite; after sterilization with sodium hypochlorite, the buds are washed 3-5 times with sterile water and dried; the extract includes the following components: 50-70 g / L mannitol, 0.8-1.5 g / L anhydrous calcium chloride, and 0.8-1.2 g / L 2-morpholine ethanesulfonic acid; the mannitol... The concentration of mannitol is preferably 60 g / L, the concentration of anhydrous calcium chloride is preferably 1.1 g / L, and the concentration of 2-morpholine ethanesulfonic acid (MES) is preferably 0.976 g / L; 10-20 mL of extract is mixed with every 30-50 microcleft buds, preferably with every 40 microcleft buds and 15 mL of extract; the rotary cutting speed is 800-1200 rpm, the rotary cutting time is 3-8 s, and it is repeated 2-4 times, preferably the rotary cutting speed is 1000 rpm, the rotary cutting time is 5 s, and it is repeated 3 times.

[0018] In this invention, free microspores are inoculated into an induction culture medium and cultured to obtain callus tissue. The density of the free microspores is 0.5~2×10⁻⁶. 5 The induction medium is preferably 1×10⁶ cells / mL. 5 The induction medium is prepared by induction at a concentration of 1 / mL; the induction medium is based on MS medium and further includes the following components at concentrations: 90 g / L sucrose, 2 mg / L 2,4-D, 1 mg / L KT, 1 mg / L NAA, 0.5 mg / L IAA and 0.976 g / L 2-morpholine ethanesulfonic acid; the culture temperature is 24~26℃, preferably 25℃; the culture time is 26~32 days, preferably 28 days.

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] 1. Materials: Shanyin No. 1, Shanyin No. 2, 25-192, 25-197, 25-198, materials provided by the Institute of Biotechnology, Shanghai Academy of Agricultural Sciences.

[0022] 2. Method: 2.1 Flower bud classification: Based on the degree of bud cracking, the buds of the two materials, Shanyin No. 1 and Shanyin No. 2, are divided into four types: buds that are not fully developed and have not cracked, buds that are fully developed and slightly cracked, buds that are fully developed and open, and buds that are fully open.

[0023] 2.2 Microspore viability identification

[0024] Different types of microspores were released, and the suspension was mixed with 4 µg / mL fluorescein diacetate (FDA) staining solution at a 1:1 ratio. The mixture was placed in the dark for 10 minutes and observed under a fluorescence microscope. The proportion of fluorescent microspores to the total number of microspores was counted.

[0025] 2.3 Identification of Microspore Development Stages

[0026] Different types of flower buds were placed in 0.2 ml EP tubes, and Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1) was added. After fixation for 10 min, the buds were cleaned and placed on a glass slide. 20 μL of ddH2O was added, and the flower buds were crushed with tweezers to release the microspores. The microspore suspension was mixed with 0.1 mg / mL of 4',6-diamidinyl-2-phenylindole (DAPI) staining solution at a ratio of 1:1 and observed under a fluorescence microscope.

[0027] 3. Results and Analysis

[0028] Quinoa was used to extract flower buds from young spikelets that were separated into different sizes. Figure 1 Based on the degree of bud splitting, they are divided into four types (Type I: closed buds; Type II: slightly split buds; Type III: open buds; Type IV: fully open buds). As the degree of bud splitting increases, the activity of free microspores continuously improves, and their developmental stage also changes from tetrad and uninucleate to multinucleate (Table 1).

[0029] Table 1. Different sizes of quinoa buds, activity of free microspores, and developmental stages in two quinoa samples.

[0030] Based on the correlation between microscopic examination results of microspore development stages and the degree of bud splitting, free microspores at the micro-split stage of type II buds were selected. Most microspores were in the uninucleate stage, and a few were in the binucleate stage, which can be used for free microspore culture.

[0031] To verify this result, the timing of three quinoa materials (25-192, 25-197, and 25-198) was determined. It was found that when the flower buds were slightly split, most of the free microspores were uninucleate, with a small number being binucleate, indicating that this was the optimal time for microspore culture. Figure 2 ).

[0032] Example 2

[0033] Material

[0034] Five quinoa samples (25-192, 25-197, 25-342, 25-343, and 25-346) were grown in greenhouses at the Fengxian base and managed with standard water and fertilizer. All materials were provided by the Institute of Biotechnology, Shanghai Academy of Agricultural Sciences.

[0035] method: During the flowering period, young spikelets were sampled from each material and placed in culture flasks. After 6 days of low-temperature treatment at 4°C, flower buds at the microcleft stage were picked from the young spikelets, sterilized with 10% sodium hypochlorite, washed four times with sterile water, and blotted dry with absorbent paper. Forty flower buds were placed in 50mL centrifuge tubes, and 15mL of extraction buffer was added. The extract consisted of 60g / L mannitol, 1.1g / L anhydrous calcium chloride, 0.976g / L MES, and pH 5.8. The buffer was then filtered and sterilized. The flower buds were rotary cut at 1000rpm for 5s each time, repeated three times to break the flower buds and release the microspores. The microspores were collected by filtration through a 300-mesh double-layer filter and the microspore density was adjusted to 1×10⁻⁶ using different induction media. 5 1 mL of microspore suspension was inoculated into a 30 mm × 15 mm culture dish, sealed with sealing film, and incubated in the dark at 25 °C for 28 days. Microscopic observation was performed during the incubation process.

[0036] Screening of induction media: Using MS, NLN, and N6 media as basal media (mainly MS), different hormone ratios (ZT+IAA, NAA+6-BA, 2,4-D+6-BA, 2,4-D+KT+NAA+IAA, 2,4-D+KT, NAA+6-BA, 2,4-D+KT), different carbon sources (maltose and sucrose), and carbon source concentrations (30g, 60g, 90g, 130g) were prepared, resulting in 22 induction media. The composition of the different induction media is listed in Table 2. All media were sterilized by filtration with 0.976g / L MES added and pH set to 5.8. Subsequently, the microspores of five quinoa materials were tested on different induction media.

[0037] Table 2. Number and composition of different induction culture media

[0038] Results and Analysis: Free microspores from five quinoa materials were placed in different induction media. After 6 days of culture, the development of microspores in all strains was observed. Obvious cell division was observed in a few media. Figure 3 After 4 weeks of dark incubation, these five materials produced 9 plates of callus. Among them, 25-192, 25-342, 25-343, and 25-346 all formed callus in QQI-14 medium, totaling 6 plates. 25-192 produced the most callus, with 11 cells. Figure 4 Callus formation was observed in QQI-21 in two dishes (25-197 and 25-343), and in QQI-8 in only one dish (25-343). No callus formation was observed in any of the other media. All three induction media that produced callus were based on MS, indicating that MS is the most suitable basal medium for quinoa microspore induction. The carbon source and concentration were 90 g / L maltose in QQI-8, and 90 g / L sucrose in QQI-14 and QQI-21. All eight dishes of callus formation originated from 90 g / L sucrose, demonstrating that quinoa microspore culture prefers sucrose as the carbon source, with an optimal concentration of 90 g / L. QQI-14 produced the most callus, and 25-192 produced the most callus in this medium, indicating that the hormone ratio of this medium is more suitable for quinoa microspore culture. The optimal hormone ratio is 2 mg / L 2,4-D, 1 mg / L KT, 1 mg / L NAA and 0.5 mg / L IAA.

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing callus induced by quinoa microspores, characterized in that, Includes the following steps: (1) During the flowering period, young spikelets were subjected to low temperature treatment. Microcleft flower buds were selected from the young spikelets, sterilized, mixed with the extract, and rotary cut to obtain free microspores; (2) Inoculate the free microspores into the induction medium and culture them to obtain callus tissue.

2. The cultivation method according to claim 1, characterized in that, The free microspores in the microcleft buds described in step (1) are in the late uninucleate stage.

3. The cultivation method according to claim 1, characterized in that, The temperature of the low-temperature treatment in step (1) is 3~5℃, and the duration of the low-temperature treatment is 5~7 days.

4. The cultivation method according to claim 1, characterized in that, The sterilization in step (1) uses 5-15% sodium hypochlorite.

5. The cultivation method according to claim 1, characterized in that, The extract in step (1) comprises the following components: 50-70 g / L mannitol, 0.8-1.5 g / L anhydrous calcium chloride, and 0.8-1.2 g / L 2-morpholinoethanesulfonic acid; Mix 30-50 microcleft flower buds with 10-20 mL of extract.

6. The cultivation method according to claim 1, characterized in that, The rotary cutting speed in step (1) is 800~1200 rpm, the rotary cutting time is 3~8s, and it is repeated 2~4 times.

7. The cultivation method according to claim 1, characterized in that, The density of the free microspores in step (2) is 0.5~2×10⁻⁶. 5 Induction medium (cFU / mL) 8. The cultivation method according to claim 1, characterized in that, The induction medium in step (2) uses MS as the basic medium and also includes the following components at the following concentrations: 90 g / L sucrose, 2 mg / L 2,4-D, 1 mg / L KT, 1 mg / L NAA, 0.5 mg / L IAA and 0.976 g / L 2-morpholinoethanesulfonic acid.

9. The cultivation method according to claim 1, characterized in that, The culture temperature in step (2) is 24~26℃, and the culture time is 26~32 days.

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