An anther in vitro culture method for both wheat and rice

Abstract

This invention relates to the field of plant tissue culture technology, and more particularly to a method for in vitro anther culture of both wheat and rice. The method includes the following steps: S1: treating wheat / rice spikelets at low temperature for 14-28 days; S2: disinfecting and cleaning the spikelets to obtain the anthers; S3: treating the anthers with a pretreatment medium and an induction medium; S4: treating the callus or embryoids obtained in S3 with a differentiation medium and a seedling-strengthening and rooting medium; S5: subjecting the obtained regenerated plants to hardening and doubling treatment to obtain homozygous rice and wheat seeds. This invention effectively overcomes the problems of genotype restriction, low culture efficiency, and high cost in anther culture technology, improving culture efficiency and practicality.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for in vitro culture of anthers of both wheat and rice. Background Technology

[0002] Anther culture technology can accelerate the homozygosity process and shorten the breeding cycle, playing an important role in crop genetic improvement. However, existing technologies have the following drawbacks:

[0003] Strong genotype dependence: Different crops (such as rice and wheat) require special culture media (such as N6 / SK3 for rice and W14 for wheat), and screening is time-consuming; Low culture efficiency: Low callus induction rate, difficult green seedling differentiation, and easy browning; There is an urgent need to develop a cross-crop universal and simplified anther culture method. Summary of the Invention

[0004] Based on the above analysis, the present invention aims to provide a method for in vitro anther culture that can be used for both wheat and rice, in order to solve one of the problems of genotype limitation and low efficiency.

[0005] On one hand, embodiments of the present invention provide a method for in vitro culture of anthers used in both wheat and rice, comprising:

[0006] S1: Low-temperature cold storage treatment of wheat / rice ears for 14-28 days;

[0007] S2: Disinfect and clean the spikelets to obtain the anthers from the spikelets;

[0008] S3: Treat anthers with pretreatment medium and induction medium;

[0009] S4: Treat the callus or embryoids obtained in S3 with differentiation medium and seedling rooting medium;

[0010] S5: Harden and double the seedlings obtained from the regenerated plants to obtain homozygous rice and wheat seeds.

[0011] Furthermore, in S1, the temperature is refrigerated at 3-5°C, preferably at 4°C.

[0012] Furthermore, before the ear of grain is refrigerated, it is pretreated. The pretreatment method includes: before refrigeration, removing the root of the ear, keeping the top two leaves, about 2cm long, removing dust, dew and other dirt from the surface of the ear, and keeping the surface of the ear dry.

[0013] Furthermore, in S2, after the ears of wheat are subjected to low-temperature refrigeration, they are sprayed with 75% alcohol for 1 minute for disinfection, then soaked in a saturated sodium hypochlorite solution for 15 minutes for disinfection, then rinsed with sterile water 3-4 times, and the surface moisture of the ears of wheat is absorbed with sterile filter paper.

[0014] Remove the leaf sheaths from the spike, cut off the small, wet florets at both ends of the spike, peel off the glumes, and remove the anthers.

[0015] Furthermore, in S3, the pretreatment culture medium comprises: 180 g·L⁻¹ -1 Mannitol, 5.9 g·L -1 CaCl2·2H2O, 40 mg·L -1 Colchicine, 15 g·L -1 Agar, with a pH of 5.8;

[0016] One method for treating anthers using pretreatment medium is as follows: place the anthers evenly on the pretreatment medium and incubate them in the dark at 25°C for 5 days.

[0017] Furthermore, in S3, after treating the anthers with a pretreatment medium, the anthers are then treated with an induction medium.

[0018] The induction medium contains: N6, 80 g·L⁻¹ -1 Maltose, 55.6 mg·L -1 FeSO4·7H2O, 0.5 mg·L -1 KT, 2.0 mg·L -1 2,4-D, 0.5 mg / L NAA, 146 mg·L -1 Glutamine, 550 mg·L -1 Hydrolyzed casein, 5.0 mg·L -1 CuSO4·5H2O, 3 mg·L -1 Glycine, 80 mg·L -1 Inositol, 2 mg·L -1 Biotin, 2 mg·L -1 Niacin and 3.5 g·L -1 Plant-based gel with a pH of 5.8;

[0019] One method for treating anthers with induction medium is as follows: the anthers treated with pretreatment medium are placed alternately with callus tissue on induction medium, and 4-6 ovaries of other genotypes are added for co-culture.

[0020] At 25℃, in the dark and static culture for 3-5 weeks, some anthers can be induced to produce callus tissue, and callus tissue or embryoids of more than 5 mm can be obtained.

[0021] Once new callus tissue begins to emerge, the prepared callus tissue and ovary are removed.

[0022] Furthermore, in S4, when the callus grows to more than 5 mm on the induction medium, the callus or embryoid obtained by induction culture is transferred to the differentiation medium for further culture.

[0023] The differentiation medium contained: 2 / 3 MS, 30 g·L⁻¹ -1 Maltose, 100 mg·L -1 L-cysteine, 0.5 mg·L -1 6-BA, 1.5 mg·L -1 KT, 0.05 mg·L -1 NAA and 3.5 g·L -1 Plant-based gel with a pH of 5.8;

[0024] The method of treating callus or embryoids with differentiation medium is as follows: after transferring the callus or embryoids obtained by induction culture to differentiation medium, they are kept at 25°C with 12 hours of light per day and a light intensity of 8000 lux until the callus differentiates into regenerated shoots, and regenerated shoots of 3cm ± 0.1cm are obtained.

[0025] Among them, regenerated seedlings include green seedlings and white seedlings.

[0026] Furthermore, in step S4, the green seedlings are transferred to a strong seedling rooting medium to induce root development and obtain regenerated plants.

[0027] The seedling rooting medium contained: 1 / 2 MS, 0.05 mg·L⁻¹ -1 NAA and 4 mg·L -1 Paclobutrazol;

[0028] The method for promoting seedling rooting includes: transferring the green seedlings obtained from differentiation culture to a seedling rooting culture medium, inserting the base of the seedling into the rooting culture medium, and culturing at 25℃ with 12 hours of light per day and a light intensity of 3000 lux for 15-20 days until the seedlings develop roots and regenerated plants are obtained.

[0029] Furthermore, in S5, the regenerated plants obtained in S4 are subjected to hydroponic hardening. During the hardening period, samples of the regenerated plants are taken for chromosome ploidy identification. Diploid plants are directly transplanted into pots and placed in artificial climate chambers for cultivation until the plants mature and bear fruit.

[0030] Among them, after the haploids undergo chromosome doubling, they continue to be hydroponically hardened off for 3-4 weeks. During the first week, they are shaded and transplanted after new leaves grow. They are then placed in an artificial climate chamber for cultivation together with the previously identified diploids.

[0031] Furthermore, in S5, the cultivation conditions in the artificial climate chamber are: 12 hours of light per day, light intensity of 8000 lux, and humidity of 65%.

[0032] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0033] This invention optimizes cultivation conditions to achieve dual cultivation of wheat and rice, improves cultivation efficiency, promotes the practical application of haploid breeding technology, reduces cultivation costs, simplifies operation methods, and avoids the time wasted by constantly adjusting and screening multiple culture medium formulas during the anther culture process. It significantly increases the difficulty of crop anther culture and simplifies operation methods; it effectively overcomes the problems of genotype restriction, low cultivation efficiency, and high cost in anther culture technology, and improves cultivation efficiency and practicality.

[0034] This invention improves the adaptability of materials by optimizing the pretreatment time and temperature at low temperatures, specifically by pretreating at 3-5℃ for 2-4 weeks.

[0035] In the anther culture of this invention, a universal culture medium formula for wheat / rice was developed, reducing the time and cost of culture medium selection. The induction medium uses N6 as the basal medium, and the differentiation medium uses MS as the basal medium. By adding specific components, the culture efficiency was improved.

[0036] This invention adds compound growth regulators, such as CuSO4, KT, 2,4-D, and NAA, to the culture medium to promote the formation of callus or embryogenic tissue. In the differentiation medium, the addition of 6-BA, KT, and NAA promotes shoot differentiation, while the addition of L-cysteine ​​reduces tissue browning and increases the differentiation rate of green shoots. Furthermore, this invention adds a small amount of ovaries of other genotypes for co-culture during the early induction stage, and adds previously induced callus tissue, spaced apart from newly extracted anthers, which increases the probability of microspore-induced callus formation and improves induction efficiency. Attached Figure Description

[0037] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0038] Figure 1 This is a schematic diagram of rice anther culture in this invention;

[0039] Figure 2 This is a schematic diagram of the anther culture of *Ophiopogon japonicus* in this invention;

[0040] Figure 3This is a schematic diagram of the results of the experiment to reduce browning in this invention. A and B are controls, and C and D are differentiation media supplemented with 100 mg·L⁻¹. -1 L-cysteine;

[0041] Figure 4 This is a schematic diagram illustrating the hydroponic seedling hardening and transplanting process of the present invention;

[0042] Figure 5 For the identification of chromosome ploidy in the present invention, the left image is a haploid plant and the right image is a diploid plant;

[0043] Figure 6 This is a schematic flowchart of the in vitro anther culture method for both wheat and rice according to the present invention. Detailed Implementation

[0044] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0045] Anther culture plays a crucial role in variety selection, DH genetic population construction, mutant creation, and transgenic research. Traditional breeding methods for wheat / rice have long cycles, while anther culture offers significant advantages in improving plant breeding efficiency. Firstly, anther culture can rapidly obtain homozygous double haploid (DH) plants, avoiding severe segregation of traits in hybrid offspring, thus accelerating trait recombination and shortening the breeding cycle. Secondly, anther culture allows for screening of superior genes at the gametophyte level, greatly improving selection efficiency. Furthermore, regenerated double haploid (DH) lines are ideal materials for molecular markers and gene mapping. Therefore, anther culture significantly improves plant breeding efficiency by increasing homozygosity, enhancing mutant screening efficiency, and optimizing culture conditions, providing an important tool for modern crop improvement.

[0046] Rice, as one of the world's most important food crops, has always been a key area of ​​agricultural scientific research in its breeding. Naked barley, also known as barley stalks, was once one of the main grain crops in Jiangsu Province, alongside wheat and hulled barley, collectively known as the "Three Wheats." Naked barley is also the only crop that can mature normally in the Qinghai-Tibet Plateau region, serving as a staple food for local residents. Because naked barley matures earlier than wheat, it is often used as a preceding crop for high-quality rice in the middle and lower reaches of the Yangtze River and coastal areas, and its ample growing time is beneficial for improving rice yield and quality. Anther culture technology, as an effective method for obtaining haploid plants, has been applied in the breeding research of various plants. Especially for rice and naked barley, researchers can quickly obtain a large number of genetically homozygous double haploid (DH) plants through anther culture, providing a powerful tool for rapid breeding.

[0047] Anther culture and haploid breeding in rice and wheat represent one of the most widely applied and fruitful areas of biotechnology in agricultural breeding. Successful anther culture depends to a certain extent on the choice of culture medium; different media and treatment methods have different effects on different crops. Although anther culture technology has been used for many years, it still suffers from high workload and concentration, complex operation, high culture costs, low differentiation efficiency, and the effectiveness of anther culture is highly dependent on the growth and development conditions and physiological state of the donor plants, pretreatment methods, and culture medium composition. In the anther culture process, the selection and optimization of the culture medium are crucial for improving plant regeneration rates. The induction medium is particularly important, directly affecting callus yield, quality, and green seedling differentiation rate. This, to some extent, limits the application of anther culture methods.

[0048] First, there are genotype limitations: different crops or genotypes require specific culture media, and screening is time-consuming; second, the culture efficiency is low: callus induction rate is low, green seedling differentiation is difficult, and the operation is complicated; third, the cost is high: multiple screening of culture media and repeated experiments increase time and economic costs.

[0049] Therefore, there is an urgent need to develop a method for cultivating anthers for different crops.

[0050] To address the above problems, this invention provides a method for in vitro anther culture suitable for both wheat and rice, comprising:

[0051] S1: Low-temperature cold storage treatment of wheat / rice ears for 14-28 days;

[0052] S2: Disinfect and clean the spikelets to obtain the anthers from the spikelets;

[0053] S3: Treat anthers with pretreatment medium and induction medium;

[0054] S4: Treat the callus or embryoids obtained in S3 with differentiation medium and seedling rooting medium;

[0055] S5: Harden and double the seedlings obtained from the regenerated plants to obtain homozygous rice and wheat seeds.

[0056] Specifically, in S1, the material is subjected to low-temperature refrigeration treatment at 3-5℃, preferably at 4℃.

[0057] The wheat / rice mentioned herein is grown in the field under normal water and fertilizer management; the panicles mentioned are vigorous and robust panicles collected when the microspores of wheat / rice have grown to the mid-to-late stage of uninucleate development (uninucleate edge stage).

[0058] In one possible embodiment, the ears are pretreated before being refrigerated. The pretreatment method includes: before refrigeration, removing the root of the ear, leaving the top two leaves about 2cm long, removing dust, dew and other dirt from the surface of the ear, and keeping the surface of the ear dry; further, the treated ear is placed in a freezer bag and then refrigerated.

[0059] Furthermore, during the low-temperature treatment, observe the condition of the ears and promptly separate any moldy or contaminated ears.

[0060] Specifically, in S2, after the ears of wheat are refrigerated at low temperature, they are sprayed with 75% alcohol for 1 minute for disinfection, then soaked in a saturated sodium hypochlorite solution for 15 minutes for disinfection, then rinsed with sterile water 3-4 times, and the surface moisture of the ears is absorbed with sterile filter paper.

[0061] The ears of grain are selected from fresh ears of grain after low-temperature refrigeration. Before disinfection, the ears of grain are pretreated. The pretreatment method includes: removing leaves and straw from the ears of grain, cutting off the awns at the top of the ears of grain, and keeping the leaf sheaths in the middle of the ears of grain.

[0062] During the process of immersing the wheat ears in a sterilizer with a saturated sodium hypochlorite solution, the ears are constantly shaken to ensure thorough sterilization.

[0063] Furthermore, after absorbing the moisture from the spike with sterile filter paper, the leaf sheaths are removed, the wetted florets at both ends of the spike are cut off, the glumes are peeled off with tweezers, and the anthers are removed, taking care not to damage them. The tweezers and surgical scissors, sterilized by scalding, are used to handle the spike.

[0064] Specifically, in S3, hormones and organic nitrogen sources are added to the pretreatment and induction media to promote callus formation.

[0065] The pretreatment culture medium contains: 180 g·L -1 Mannitol, 5.9 g·L -1 CaCl2·2H2O, 40 mg·L -1 Colchicine, 15 g·L -1 Agar, with a pH of 5.8.

[0066] The preparation method for the pretreatment culture medium is as follows: Weigh 180g mannitol, 5.9g CaCl2·2H2O, and 15g agar, dissolve in pure water and bring the volume to 1L. Adjust the pH to 5.8 with NaOH, and autoclave at 0.11 MPa and 121℃ for 15 min. Prepare colchicine at 1000X, i.e., 40 mg / ml. -1The mother liquor was sterilized by filtration through a 0.22-micron filter membrane. After the autoclaved culture medium cooled to about 55°C (warm but not hot to the touch), 1 ml of colchicine mother liquor was added to the clean bench, mixed evenly, and dispersed into a 6 cm petri dish. After cooling and solidification, it was ready for use.

[0067] In one possible implementation, the method of treating anthers with a pretreatment medium is as follows: the anthers are evenly placed on the pretreatment medium and incubated in the dark at 25°C for 5 days.

[0068] The pretreated culture medium was placed in Petri dishes, and the anthers were cultured in the Petri dishes; 40 anthers were placed in each Petri dish.

[0069] Colchicine can improve callus induction rate and promote microspore development.

[0070] Furthermore, after treating the anthers with a pretreatment medium, the anthers were then treated with an induction medium.

[0071] The induction medium contains: N6, 80 g·L⁻¹ -1 Maltose, 55.6 mg·L -1 FeSO4·7H2O, 0.5 mg·L -1 KT, 2.0 mg·L -1 2,4-D, 0.5 mg / L NAA, 146 mg·L -1 Glutamine, 550 mg·L -1 Hydrolyzed casein, 5.0 mg·L -1 CuSO4·5H2O, 3 mg·L -1 Glycine, 80 mg·L -1 Inositol, 2 mg·L -1 Biotin, 2 mg·L -1 Niacin and 3.5 g·L -1 Plant gel with a pH of 5.8.

[0072] The induction medium was prepared as follows: Weigh 4g of the finished N6 medium and 80g of maltose, dissolve them in water, and add 1ml of 1000X (55.6 mg / ml). -1 FeSO4·7H2O, 1 ml 1000X, i.e. 5 mg·ml -1 CuSO4·5H2O, 1 ml = 1000X, which is 0.5 mg·ml -1 KT, 1 ml 1000X, which is 2 mg·ml -1 2,4-D, 1 ml 1000X, i.e. 0.5 mg / ml -1Weigh 3.5g of plant gel and add pure water to make up to 1L. Adjust the pH to 5.8 with NaOH. Autoclave at 0.11 MPa and 121 ℃ for 15 min. After the autoclaved medium cools to about 55℃ (warm but not hot to the touch), add 10ml each of glutamine and hydrolyzed casein 100X stock solution (filtered and sterilized with a 0.22-micron filter membrane) and 1ml each of glycine, inositol, biotin, and nicotinic acid 1000X stock solution in a clean bench. Mix well, disperse and pour into a 6cm petri dish. Let cool and solidify before use.

[0073] The induction medium used N6 as the basic medium (Sigma C 1416, containing macro and micro elements); maltose was used as the carbon source, KT as the hormone, and glutamine as the organic nitrogen source.

[0074] One method for treating anthers with induction medium is to place the pretreated anthers and callus tissue alternately on the induction medium, and add 4-6 ovaries of other genotypes for co-culture to increase the probability of microspore-induced callus tissue.

[0075] The callus tissue was prepared by aseptic culture and consisted of 4-6 pieces of callus tissue, each 5 mm in size; the ovaries of other genotypes were ovaries of different varieties or strains of Yuanmai; each petri dish contained 40 anthers and was a vessel for holding the culture medium.

[0076] Furthermore, by inducing callus formation in the dark at 25°C for 3-5 weeks, some anthers can be induced to produce callus or embryoids larger than 5 mm. Once new callus begins to emerge, the prepared callus and ovary are removed.

[0077] Specifically, in S4, when the callus grows to more than 5 mm on the induction medium, the callus or embryoid obtained from the induction culture is transferred to the differentiation medium for further culture.

[0078] The differentiation medium contained: 2 / 3 MS, 30 g·L⁻¹ -1 Maltose, 100 mg·L -1 L-cysteine, 0.5 mg·L -1 6-BA, 1.5 mg·L -1 KT, 0.05 mg·L -1 NAA and 3.5 g·L -1 The plant gel has a pH of 5.8; the differentiation medium is based on 2 / 3 MS, and the plant gel is used to solidify the components of the differentiation medium.

[0079] The differentiation medium was prepared as follows: Weigh 3.17g of finished MS medium (excluding agar and sucrose, 4.74g / L), 20g of sucrose, and 30g of maltose, dissolve in pure water, and add 1ml of 1000X, i.e., 1.5 mg / ml. -1 KT, 0.5ml 2000X, is equivalent to 0.1mg·ml -1 NAA, 1 ml 1000X, which is 0.5 mg / ml -1 6-BA, weigh 3.5g of plant gel, add pure water to make up to 1L, adjust the pH to 5.8 with NaOH, autoclave at 0.11 MPa and 121 ℃ for 15 min, and wait for the autoclaved culture medium to cool to about 55℃ (warm but not hot to the touch), disperse it into the sterilized culture bottle, cover it with a breathable cap, and let it cool and solidify before use.

[0080] The method of treating callus or embryoids with differentiation medium is as follows: after transferring the callus or embryoids obtained by induction culture to differentiation medium, they are kept at 25°C with 12 hours of light per day and a light intensity of 8000 lux until the callus differentiates into regenerated seedlings, and regenerated seedlings of 3cm±0.1cm are obtained.

[0081] Among them, regenerated seedlings include green seedlings and white seedlings.

[0082] Furthermore, the green seedlings were transferred to a strong seedling rooting culture medium to induce root development and obtain regenerated plants.

[0083] The seedling rooting medium contained: 1 / 2 MS, 0.05 mg·L⁻¹ -1 NAA and 4 mg·L -1 Paclobutrazol; the rooting medium was based on 1 / 2 MS.

[0084] The preparation method of the seedling rooting medium is as follows: Weigh 2.37g of finished MS medium (excluding agar and sucrose, 4.74g / L), 30g of sucrose, and 15g of agar, dissolve them in pure water, and make up to 1L with pure water. Adjust the pH to 5.8 with NaOH, and autoclave at 0.11Mpa and 121℃ for 15 min. After the autoclaved medium cools to about 55℃ (warm but not hot to the touch), add 0.5ml of 2000X, i.e., 0.1mg·ml. -1 NAA, 1 ml 1000X, which is 4 mg / ml -1 Disperse paclobutrazol into sterilized culture flasks, cover with breathable caps, and let cool and solidify before use.

[0085] Methods for promoting seedling rooting: Transfer the green seedlings obtained from differentiation culture to a seedling rooting medium. Insert the base of the seedling into the rooting medium to induce root development. In an artificial climate chamber at 25°C, with 12 hours of light per day and a light intensity of 3000 lux, culture for 15-20 days until the seedlings develop roots and obtain complete regenerated seedlings (regenerated plants).

[0086] Specifically, in S5, the regenerated plants obtained in S4 are washed with tap water to remove the culture medium from the roots and then hydroponically hardened off for 7 days.

[0087] Furthermore, during the hardening-off period, samples of regenerated plants were taken for chromosome ploidy identification. Diploid plants were directly transplanted into pots and placed in artificial climate chambers for cultivation until the plants matured and bore fruit.

[0088] Among them, after the haploids undergo chromosome doubling, they continue to be hydroponically hardened off for 3-4 weeks. During the first week, they are shaded and transplanted after new leaves grow. They are then placed in an artificial climate chamber for cultivation together with the previously identified diploids.

[0089] Furthermore, the cultivation conditions in the artificial climate chamber were: 12 hours of light per day, light intensity of 8000 lux, and humidity of 65%, until the plants flowered, bore fruit, and matured to harvest seeds, thus obtaining homozygous (i.e., DH, Double haploid) rice and wheat seeds.

[0090] Before hydroponically cultivating the regenerated plants obtained in S4, the yellow leaves of the regenerated plants with well-developed root systems are removed. Well-developed root systems refer to having more than 3 fibrous roots with a length of more than 2 cm. Then, the regenerated plants are hydroponically cultivated in Hoagland nutrient solution to harden off the seedlings and obtain robust plants, which are then transplanted into pots (containing soil).

[0091] The purpose of chromosome ploidy identification is as follows: Since both rice and wheat are diploid crops, plants obtained from anther culture are derived from haploid microspores. During the culture process, there is a certain probability of chromosome doubling, directly obtaining double haploid (i.e., diploid) plants. However, some plants remain haploid. Therefore, chromosome ploidy identification is needed to determine whether the experimentally obtained seedlings are haploid, diploid, or polyploid. Haploids are infertile and require chromosome doubling to produce seeds. The identification method is as follows: During the hydroponic hardening-off period, leaves are collected, and chromosome ploidy is identified using flow cytometry.

[0092] The method for doubling the obtained haploid plants was as follows: after cutting the roots of the haploid plants, they were treated with 0.2% colchicine + 1% dimethyl sulfoxide and kept in the dark at 20°C for 12 hours to increase the diploidization rate. The diploidization rate refers to the proportion of fertile diploid plants to all plants obtained in the experiment.

[0093] Compared with existing technologies,

[0094] This invention optimizes cultivation conditions to achieve dual cultivation of wheat and rice, improves cultivation efficiency, promotes the practical application of haploid breeding technology, reduces cultivation costs, simplifies operation methods, and avoids the time wasted by constantly adjusting and screening multiple culture medium formulas during the anther culture process. It significantly increases the difficulty of crop anther culture and simplifies operation methods; it effectively overcomes the problems of genotype restriction, low cultivation efficiency, and high cost in anther culture technology, and improves cultivation efficiency and practicality.

[0095] This invention improves the adaptability of materials by optimizing the pretreatment time and temperature at low temperatures, specifically by pretreating at 3-5℃ for 2-4 weeks.

[0096] In the anther culture of this invention, a universal culture medium formula for wheat / rice was developed, reducing the time and cost of culture medium selection. The induction medium uses N6 as the basal medium, and the differentiation medium uses MS as the basal medium. By adding specific components, the culture efficiency was improved.

[0097] This invention adds compound growth regulators, such as CuSO4, KT, 2,4-D, and NAA, to the culture medium to promote the formation of callus or embryogenic tissue. In the differentiation medium, 6-BA, KT, and NAA are added to promote shoot differentiation, and L-cysteine ​​is added to reduce tissue browning, thereby increasing the differentiation rate of green seedlings.

[0098] This invention involves adding a small amount of ovaries of other genotypes for co-culture during the initial induction stage, along with previously induced callus tissue, and placing them alternately with newly extracted anthers. This increases the probability of microspore-induced callus tissue and improves induction efficiency.

[0099] Most current methods employ different culture methods for different crops, or even different genotypes of the same crop, and are highly dependent on the culture medium formulation. This invention uses a universal culture medium that is easy to obtain and prepare, and has good culture results for most of the tested rice and wheat genotypes.

[0100] Existing technologies employ different treatment methods and culture techniques for rice and wheat anther culture media formulations. Induction media for rice include He5, N6, SK3, M8, and GM8, while media for wheat include N6, W14, and various modified formulations for different genotypes. The sheer number of media makes selection difficult for researchers, forcing them to experiment repeatedly, which is time-consuming and labor-intensive, hindering the widespread adoption of anther culture methods. This method uses a universal basic media and a universal culture method, with the addition of a compound growth regulator. It is suitable for anther culture of both rice and wheat, achieving good results. This method is easy to learn, with simple steps, reducing the time and cost of media selection, and is conducive to the widespread adoption of anther culture methods.

[0101] This invention improves induction efficiency by adding a small amount of ovaries of different genotypes to the induction medium during the initial induction stage, and by adding previously induced callus tissue and placing it alternately with newly extracted anthers. Yuanmai material Tong 0612 is difficult to induce successfully using existing culture methods. However, by adding a small amount of rice callus tissue during the initial induction stage, this variety also showed a small anther response, successfully inducing callus tissue and obtaining green seedlings, achieving a transformation from nothing to something. This demonstrates that the novel co-culture strategy of this invention enables the successful induction of even sluggish genotypes.

[0102] In existing culture methods, anther culture is subject to many genotype limitations and has a low success rate. However, this invention adds compound growth regulators such as KT, 2,4-D, and NAA to the induction medium. Experimental data show that the use of these growth regulators significantly improves callus formation and green seedling differentiation. Callus was successfully induced and green seedlings differentiated in both of the tested rice varieties, and callus was successfully induced and green seedlings differentiated in all five tested wheat materials (including three cultivated wheat varieties and two intermediate materials). The success rate is significantly higher than that of previous methods.

[0103] Example 1

[0104] A method for in vitro culture of anthers used in both wheat and rice crops, comprising:

[0105] S1: Wheat / rice spikes were subjected to low-temperature cold storage at 4℃ for 21 days;

[0106] Before refrigerating, remove the base of the spike, keep the top two leaves, about 2cm long, remove dust, dew and other dirt from the surface of the spike, keep the surface of the spike dry, put it in a plastic bag and refrigerate it.

[0107] During the low-temperature treatment, the condition of the ears should be observed, and any ears that have become moldy or contaminated should be separated in a timely manner.

[0108] S2: Disinfect and clean the spikelets to obtain the anthers from the spikelets;

[0109] After low-temperature refrigeration, select fresh ears of wheat, remove excess leaves and stalks, trim the top awns of the wheat, and keep the leaf sheaths in the middle of the ear wrapped. Spray with 75% alcohol for 1 minute for disinfection, then soak the ears of wheat in a saturated sodium hypochlorite solution for 15 minutes for disinfection, shaking continuously to ensure that all ears of wheat are thoroughly disinfected. Then rinse with sterile water 3-4 times and use sterile filter paper to absorb excess water.

[0110] After disinfection and cleaning, remove the leaf sheaths from the spike, cut off the wet florets at both ends of the spike, peel off the glumes with tweezers, and gently remove the anthers, avoiding damage. The spike is handled with tweezers and surgical scissors that have been sterilized by cauterization.

[0111] S3: Treat anthers with pretreatment medium and induction medium;

[0112] Hormones and organic nitrogen sources were added to the pretreatment and induction media to promote callus formation.

[0113] The pretreatment culture medium contains: 180 g·L -1 Mannitol, 5.9 g·L -1 CaCl2·2H2O, 40 mg·L -1 Colchicine, 15 g·L -1 Agar, with a pH of 5.8.

[0114] The preparation method for the pretreatment culture medium is as follows: Weigh 180g mannitol, 5.9g CaCl2·2H2O, and 15g agar, dissolve in pure water and bring the volume to 1L. Adjust the pH to 5.8 with NaOH, and autoclave at 0.11 MPa and 121℃ for 15 min. Prepare colchicine at 1000X, i.e., 40 mg / ml. -1 The mother liquor was sterilized by filtration through a 0.22-micron filter membrane. After the autoclaved culture medium cooled to about 55°C (warm but not hot to the touch), 1 ml of colchicine mother liquor was added to the clean bench, mixed evenly, and dispersed into a 6 cm petri dish. After cooling and solidification, it was ready for use.

[0115] The method for treating anthers using pretreatment medium is as follows: place the anthers evenly on the pretreatment medium, with 40 anthers per petri dish, and incubate in the dark at 25°C for 5 days.

[0116] Furthermore, after treating the anthers with a pretreatment medium, the anthers were then treated with an induction medium.

[0117] The induction medium contains: N6, 80 g·L⁻¹ -1 Maltose, 55.6 mg·L -1FeSO4·7H2O, 0.5 mg·L -1 KT, 2.0 mg·L -1 2,4-D, 0.5 mg / L NAA, 146 mg·L -1 Glutamine, 550 mg·L -1 Hydrolyzed casein, 5.0 mg·L -1 CuSO4·5H2O, 3 mg·L -1 Glycine, 80 mg·L -1 Inositol, 2 mg·L -1 Biotin, 2 mg·L -1 Niacin and 3.5 g·L -1 Plant gel with a pH of 5.8.

[0118] The induction medium was prepared as follows: Weigh 4g of the finished N6 medium and 80g of maltose, dissolve them in water, and add 1ml of 1000X (55.6 mg / ml). -1 FeSO4·7H2O, 1 ml 1000X, i.e. 5 mg·ml -1 CuSO4·5H2O, 1 ml = 1000X, which is 0.5 mg·ml -1 KT, 1 ml 1000X, which is 2 mg·ml -1 2,4-D, 1 ml 1000X, i.e. 0.5 mg / ml -1 Weigh 3.5g of plant gel and add pure water to make up to 1L. Adjust the pH to 5.8 with NaOH. Autoclave at 0.11 MPa and 121 ℃ for 15 min. After the autoclaved medium cools to about 55℃ (warm but not hot to the touch), add 10ml each of glutamine and hydrolyzed casein 100X stock solution (filtered and sterilized with a 0.22-micron filter membrane) and 1ml each of glycine, inositol, biotin, and nicotinic acid 1000X stock solution in a clean bench. Mix well, disperse and pour into a 6cm petri dish. Let cool and solidify before use.

[0119] One method for treating anthers with induction medium is as follows: anthers treated with pre-treated medium are placed alternately with callus tissue on induction medium, and six ovaries of other genotypes are added for co-culture to increase the probability of microspore-induced callus tissue.

[0120] Each petri dish contains 40 anthers and is a container for the culture medium.

[0121] The callus tissue was prepared by aseptic culture and consisted of 6 callus tissues, each 5 mm in size; the other genotype ovaries were ovaries of different varieties or strains of Yuanmai.

[0122] Furthermore, at 25°C, in the dark and statically cultured for 3-5 weeks, some anthers can be induced to produce callus tissue, yielding callus tissue or embryoids longer than 5 mm. When new callus tissue begins to emerge, the prepared callus tissue and ovary are removed, and the culture continues to be carried out to obtain new callus tissue or embryoids longer than 5 mm.

[0123] S4: Treat the callus or embryoids obtained in step S3 with differentiation medium and seedling rooting medium;

[0124] In this process, when the callus grows to more than 5 mm on the induction medium, the callus or embryoid obtained from the induction culture is transferred to the differentiation medium for further culture.

[0125] The differentiation medium contained: 2 / 3 MS, 30 g·L⁻¹ -1 Maltose, 100 mg·L -1 L-cysteine, 0.5 mg·L -1 6-BA, 1.5 mg·L -1 KT, 0.05 mg·L -1 NAA and 3.5 g·L -1 The plant gel has a pH of 5.8; the differentiation medium is based on 2 / 3 MS, and the plant gel is used to solidify the components of the differentiation medium.

[0126] The differentiation medium was prepared as follows: Weigh 3.17g of finished MS medium (excluding agar and sucrose, 4.74g / L), 20g of sucrose, and 30g of maltose, dissolve in pure water, and add 1ml of 1000X, i.e., 1.5 mg / ml. -1 KT, 0.5ml 2000X, is equivalent to 0.1mg·ml -1 NAA, 1 ml 1000X, which is 0.5 mg / ml -1 6-BA, weigh 3.5g of plant gel, add pure water to make up to 1L, adjust the pH to 5.8 with NaOH, autoclave at 0.11 MPa and 121 ℃ for 15 min, and wait for the autoclaved culture medium to cool to about 55℃ (warm but not hot to the touch), disperse it into the sterilized culture bottle, cover it with a breathable cap, and let it cool and solidify before use.

[0127] The method of treating callus or embryoids with differentiation medium is as follows: after transferring the callus or embryoids obtained by induction culture to differentiation medium, they are kept at 25°C with 12 hours of light per day and a light intensity of 8000 lux until the callus differentiates into regenerated seedlings, and regenerated seedlings of 3cm±0.1cm are obtained.

[0128] Among them, regenerated seedlings include green seedlings and white seedlings.

[0129] Furthermore, the green seedlings were transferred to a strong seedling rooting culture medium to induce root development and obtain regenerated plants.

[0130] The seedling rooting medium contained: 1 / 2 MS, 0.05 mg·L⁻¹ -1 NAA and 4 mg·L -1 Paclobutrazol; the rooting medium was based on 1 / 2 MS.

[0131] The preparation method of the seedling rooting medium is as follows: Weigh 2.37g of finished MS medium (excluding agar and sucrose, 4.74g / L), 30g of sucrose, and 15g of agar, dissolve them in pure water, and make up to 1L with pure water. Adjust the pH to 5.8 with NaOH, and autoclave at 0.11Mpa and 121℃ for 15 min. After the autoclaved medium cools to about 55℃ (warm but not hot to the touch), add 0.5ml of 2000X, i.e., 0.1mg·ml. -1 NAA, 1 ml 1000X, which is 4 mg / ml -1 Disperse paclobutrazol into sterilized culture flasks, cover with breathable caps, and let cool and solidify before use.

[0132] The method for promoting strong seedling rooting is as follows: the green seedlings obtained from differentiation culture are transferred to the strong seedling rooting medium, the base of the seedling is inserted into the rooting medium to induce root development, and the artificial climate chamber is set at 25℃ with 12 hours of light per day and a light intensity of 3000 lux for 15-20 days. The seedlings will grow roots and obtain complete regenerated seedlings (regenerated plants).

[0133] S5: Harden and double the number of regenerated plants obtained;

[0134] The regenerated plants obtained in S4 were hydroponically hardened for 7 days. During the hardening period, samples of the regenerated plants were taken for chromosome ploidy identification. The diploid plants were directly transplanted into pots and placed in artificial climate chambers for cultivation until the plants matured and bore fruit.

[0135] Among them, after the haploids undergo chromosome doubling, they continue to be hydroponically hardened off for 3-4 weeks. During the first week, they are shaded and transplanted after new leaves grow. They are then placed in an artificial climate chamber for cultivation together with the previously identified diploids.

[0136] The cultivation conditions in the artificial climate chamber were: 12 hours of light per day, light intensity of 8000 lux, and humidity of 65% until the plants flowered, bore fruit, and matured to harvest seeds, thus obtaining homozygous (i.e., DH, Double haploid) rice and wheat seeds.

[0137] Before hydroponically cultivating the regenerated plants obtained in S4, the yellow leaves of the regenerated plants with well-developed root systems are removed. Well-developed root systems refer to having more than 3 fibrous roots with a length of more than 2 cm. Then, the regenerated plants are hydroponically cultivated in Hoagland nutrient solution to harden off the seedlings and obtain robust plants, which are then transplanted into pots (containing soil).

[0138] The method for doubling the obtained haploid plants was as follows: after cutting the roots of the haploid plants, they were treated with 0.2% colchicine + 1% dimethyl sulfoxide and kept in the dark at 20°C for 12 hours to increase the doubling rate.

[0139] Based on the above method, anther culture was performed on different varieties of rice and wheat to obtain homozygous (i.e., DH, Double haploid) rice and wheat seeds. The callus induction rate and green seedling differentiation rate of different varieties of rice and wheat were calculated. The callus induction rate and green seedling differentiation rate are shown in Table 1.

[0140] Wherein, callus induction rate = number of anther callus / number of inoculated anthers × 100%; green seedling differentiation rate = number of differentiated green seedlings / number of transferred anther callus × 100%.

[0141] Table 1. Anther response rate and green seedling differentiation rate of rice and wheat.

[0142] name crop callus induction rate % Green seedling differentiation rate % Nanjing 5055 rice 13.8 44.4 Nanjing9108 rice 16.4 45.9 Tongmai No. 8 Yuanmai 9.24 72.7 0612 Yuanmai 11.1 42.8 24Z63 Yuanmai 32.9 38.7 22-73-1 Yuanmai 46.9 72.4 24-61-1 Yuanmai 12.7 46.7 average value 20.4 51.9

[0143] As shown in the table above, good culture results were obtained for the tested rice varieties Nanjing 5055 and Nanjing 9108, wheat varieties Tongmai 8, Tong0612, and 24Z63, and intermediate materials 22-73-1 and 24-61-1 (intermediate materials 22-73-1 and 24-61-1 refer to offspring materials produced by hybridization of different parents during the hybridization breeding process. These offspring may exhibit new trait combinations, but have not yet undergone sufficient screening and identification). It can be seen that the callus induction rate and green seedling differentiation rate of the tested rice and wheat materials using the method of this invention are comparable to those of previously reported materials, and even the green seedling differentiation rate is higher than that of most previously reported materials. This indicates that this method achieves good culture results while taking into account both rice and wheat anther culture.

[0144] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for in vitro culture of anthers used in both wheat and rice, characterized in that, Including the following steps: S1: Low-temperature cold storage treatment of wheat / rice ears for 14-28 days; S2: Disinfect and clean the spikelets to obtain the anthers from the spikelets; S3: Treat anthers with pretreatment medium and induction medium; S4: The callus obtained in S3 was treated with differentiation medium and seedling rooting medium; S5: Harden and double the obtained regenerated plants to obtain homozygous rice and wheat seeds. In S3, after treating the anthers with a pretreatment medium, the anthers are then treated with an induction medium. The induction culture medium components are as follows: N6, 80 g·L⁻¹ -1 Maltose, 55.6 mg·L -1 FeSO4·7H2O, 0.5 mg·L -1 KT, 2.0 mg·L -1 2,4-D, 0.5 mg / L NAA, 146 mg·L -1 Glutamine, 550 mg·L -1 Hydrolyzed casein, 5.0 mg·L -1 CuSO4·5H2O, 3 mg·L -1 Glycine, 80 mg·L -1 Inositol, 2 mg·L -1 Biotin, 2 mg·L -1 Niacin and 3.5 g·L -1 Plant-based gel with a pH of 5.8; One method for treating anthers with induction medium is as follows: the anthers treated with pretreatment medium are placed alternately with callus tissue on the induction medium, and 4-6 ovaries of other genotypes are added for co-culture. At 25℃, in the dark and static culture for 3-5 weeks, some anthers can be induced to produce callus tissue, and callus tissue larger than 5mm can be obtained. When new callus tissue begins to emerge, the pre-prepared callus tissue and ovary are removed. In S4, when the callus grows to more than 5 mm on the induction medium, the callus obtained from the induction culture is transferred to the differentiation medium for further culture. The differentiation medium components are as follows: 2 / 3 MS, 30 g·L⁻¹ -1 Maltose, 100 mg·L -1 L-cysteine, 0.5 mg·L -1 6-BA, 1.5 mg·L -1 KT, 0.05 mg·L -1 NAA and 3.5 g·L -1 Plant-based gel with a pH of 5.8; The method of treating callus with differentiation medium is as follows: after transferring the callus obtained by induction culture to differentiation medium, it is kept at 25°C with 12 hours of light per day and a light intensity of 8000 lux until the callus differentiates into regenerated shoots, and regenerated shoots of 3cm±0.1cm are obtained. Among them, regenerated seedlings include green seedlings and white seedlings; In step S4, the green seedlings are transferred to a strong seedling rooting medium to induce root development and obtain regenerated plants. The components of the seedling rooting medium are as follows: 1 / 2 MS, 0.05 mg·L⁻¹ -1 NAA and 4 mg·L -1 Paclobutrazol; The method for promoting seedling rooting includes: transferring the green seedlings obtained from differentiation culture to a seedling rooting culture medium, inserting the base of the seedling into the rooting culture medium, and culturing at 25℃ with 12 hours of light per day and a light intensity of 3000 lux for 15-20 days until the seedlings develop roots and regenerated plants are obtained.

2. The method according to claim 1, characterized in that: In S1, the material is subjected to low-temperature refrigeration at 3-5°C.

3. The method according to claim 1, characterized in that: In S1, the material is subjected to low-temperature refrigeration at 4°C.

4. The method according to any one of claims 1-3, characterized in that: Before refrigerating the ears, the ears are pretreated. The pretreatment method includes: before refrigerating at low temperature, removing the root of the ear, keeping the top two leaves, leaving about 2cm, removing dust, dew and other dirt from the surface of the ear, and keeping the surface of the ear dry.

5. The method according to claim 1, characterized in that: In S2, after the ears of wheat are subjected to low-temperature refrigeration, they are sprayed with 75% alcohol for 1 minute for disinfection, then soaked in a saturated sodium hypochlorite solution for 15 minutes for disinfection, then rinsed with sterile water 3-4 times, and the surface moisture of the ears of wheat is absorbed with sterile filter paper. Remove the leaf sheaths from the spike, cut off the small, wet florets at both ends of the spike, peel off the glumes, and remove the anthers.

6. The method according to claim 1, characterized in that: In S3, the pretreatment culture medium components are as follows: 180 g·L -1 Mannitol, 5.9 g·L -1 CaCl2·2H2O, 40 mg·L -1 Colchicine, 15 g·L -1 Agar, with a pH of 5.8; One method for treating anthers using pretreatment medium is as follows: place the anthers evenly on the pretreatment medium and incubate them in the dark at 25°C for 5 days.

7. The method according to claim 1, characterized in that: In S5, the regenerated plants obtained in S4 are hydroponically hardened off. During the hardening off period, samples of the regenerated plants are taken for chromosome ploidy identification. Diploid plants are directly transplanted into pots and placed in artificial climate chambers for cultivation until the plants mature and bear fruit. Among them, after the haploids undergo chromosome doubling, they continue to be hydroponically hardened off for 3-4 weeks. During the first week, they are shaded and transplanted after new leaves grow. They are then placed in an artificial climate chamber for cultivation together with the previously identified diploids.

8. The method according to claim 7, characterized in that: In S5, the cultivation conditions in the artificial climate chamber are: 12 hours of light per day, light intensity of 8000 lux, and humidity of 65%.

Images