Improved rice multi-variety free microspore efficient culture method based on callus induction culture medium

Abstract

The invention relates to the technical field of rice microspore culture, in particular to an improved rice multi-variety free microspore efficient culture method based on a callus induction culture medium, which comprises the following steps: step 1, sampling and pretreatment, collecting young panicles of which floret microspores in the middle of the rice young panicles develop from a mononuclear middle-late stage to a binuclear early stage in the morning; 2, preparing an extracting solution and a culture medium: preparing the extracting solution, an induction culture medium, a differential culture medium and a seedling strengthening and rooting culture medium; step 3, separating microspores and inducing calluses, preparing microspore suspension by utilizing the induction culture medium and the microspores, and placing the microspore suspension in an incubator with the temperature of 26 DEG C and the relative humidity of 60-75% for dark culture; 4, plant regeneration, seedling strengthening and rooting; 5, field transplanting, through the culture method, the problem that free microspores are prone to pollution in the culture process can be effectively solved, and the induction effect of the induction culture medium is good.

Description

Technical Field

[0001] This invention relates to the field of rice microspore culture technology, and in particular to a high-efficiency method for culturing free microspores of multiple rice varieties based on a callus induction culture medium. Background Technology

[0002] Haploid breeding technology, also known as double haploid breeding technology, can obtain homozygous recombinants with stable traits in 1-2 generations, significantly shortening the breeding cycle and thus gaining popularity among breeders (Hale et al., 2022). Rice is one of the world's most important food crops, a staple food in Asian countries, and the main food source for more than half of the global population. Therefore, rice is crucial for ensuring global food security (Radanielson et al., 2018). Thus, research on rice haploid technology is of great significance.

[0003] Since Chinese scientists Chen Ying et al. (1980) first reported the culture of free microspores in rice in the last century, research progress on this technology in rice has been relatively slow, with only sporadic reports (Raina and Irfan, 1998; Islam et al., 2013; Rahman et al., 2022; Gao et al., 2024). However, it has been reported that microspore culture is more efficient than anther culture (Li and Devaux, 2005), and can also eliminate interference from anther wall and tapetum cells in anther culture. In addition, with the development of automation and intelligent technologies, free microspore culture can avoid the cumbersome process of inoculating anthers in anther culture, and is more likely to achieve culture automation; moreover, the large population of microspores from free microspore culture is easier and more representative for analyzing related molecular mechanisms than the single anthers from anther culture. Therefore, establishing an efficient rice free microspore culture system can not only accelerate the development of rice haploid breeding technology, but also better help to resolve a series of molecular mechanisms that pose challenges in rice microspore culture.

[0004] Previously, we established an efficient free microspore culture system for barley (Lu et al., 2016; Chen et al., 2023), and recently established an efficient anther culture system for rice (Guo et al., 2024), and preliminarily explored various aspects of rice microspore culture (Gao et al., 2024). In this study, based on our previous experience, we further optimized the key step of microspore callus induction and attempted to establish a free microspore culture system suitable for different rice genotypes, providing a feasible solution for the promotion and large-scale use of this technology. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a high-efficiency method for culturing free microspores of multiple rice varieties based on a callus induction culture medium.

[0006] A high-efficiency method for culturing free microspores of multiple rice varieties based on a callus induction culture medium includes the following steps:

[0007] Step 1, Sampling and Pretreatment: During the booting stage of rice, young panicles with microspores in the middle part of the panicle at the mid-to-late mononuclear to early binucleate stage were collected from 8:00 to 10:00 am. The collected young panicles were wiped with cotton balls soaked in 75% alcohol. After wiping, they were wrapped in clean, damp gauze and then wrapped in plastic film to keep them moist. They were then placed in a refrigerator at 2-5℃ for low-temperature pretreatment for 8-14 days.

[0008] Step 2, Preparation of Extract and Culture Medium: Prepare the extract, induction medium, differentiation medium, and seedling rooting medium;

[0009] Step 3: Microspore isolation and callus induction: Disinfect the pretreated young spikelets from Step 1 with 10% disinfectant for 10 minutes, rinse 4-5 times with sterile water, and inoculate anthers into test tubes (1000-1400 anthers per tube). Add 10-20 mL of extraction solution, homogenize at high speed using a homogenizer, filter the suspension through a sieve, centrifuge, discard the centrifuged liquid, and collect the microspores. Prepare a microspore suspension using induction medium and microspores, adjusting the microspore density in the suspension to 1.0 × 10⁻⁶. 5 Take 1.0 mL of microspore suspension in a culture dish, seal it with sealing film, and place it in an incubator at 26℃ and 60%–75% relative humidity for dark culture. After 4 to 10 weeks, weigh the callus tissue. After weighing, the callus tissue is immediately transferred to differentiation medium for plant regeneration.

[0010] Step 4, Plant regeneration and seedling rooting: After inoculating the callus tissue into the differentiation medium, place it in a culture room at 24℃, 60%-75% relative humidity and 16h photoperiod. Transfer the 1.5-2.5cm regenerated green plants to the seedling rooting medium for further culture.

[0011] Step 5, Field Transplanting: After the plant roots have developed, clean them and transplant them into the paddy field, and manage them as normal rice cultivation.

[0012] Preferably, the extract contains 58-62 g / L mannitol, with added CaCl2 0.9-1.3 g / L, colchicine 18-22 mg / L, and MES 0.95-1 g / L, and the pH of the extract is 5.6-6; the induction medium contains basal medium, with added glycogen 85-95 g / L, proline 0.45-0.55 g / L, glutamine 0.45-0.55 g / L, hydrolyzed casein 0.25-0.35 g / L, 2,4-D 1.8-2.2 mg / L, and KT 0.45-0.55 mg / L, and the pH of the induction medium is 5.6-6; the differentiation medium uses 1 / 2 MS as the basal medium, with added maltose 28-32 g / L, KT 1.5-2.5 mg / L, and 6-BA. The medium for promoting seedling growth and rooting consisted of 0.8–1.2 mg / L sucrose, 0.45–0.55 mg / L NAA, and 5.5–6.5 g / L agar powder, with the pH adjusted to 5.6–6. The medium for strengthening seedlings and promoting rooting consisted of 1 / 2 MS as the basic medium, with 28–32 g / L sucrose, 0.35–0.45 mg / L NAA, and 2.7–3.3 mg / L paclobutrazol added. The medium was then solidified with 5.6–6 g / L agar powder, with the pH adjusted to 5.6–6.

[0013] Preferably, the extract contains 60 g / L mannitol, with added CaCl2 1.1 g / L, colchicine 20 mg / L, and MES 0.976 g / L, and the pH of the extract is 5.8; the induction medium contains basal medium, with added glycogen 90 g / L, proline 0.5 g / L, glutamine 0.5 g / L, hydrolyzed casein 0.3 g / L, 2,4-D 2 mg / L, and KT 0.5 mg / L, and the pH of the induction medium is 5.8; the differentiation medium uses 1 / 2 MS as the basal medium, with added maltose 30 g / L, KT 2 mg / L, 6-BA 1 mg / L, and NAA 0.5 mg / L, and the medium is fixed with 6 g / L agar powder, and the pH is adjusted to 5.8; the seedling rooting medium uses 1 / 2 MS as the basal medium, with added sucrose 30 g / L, NAA... 0.4 mg / L and paclobutrazol 3 mg / L were used to solidify the culture medium with 6 g / L agar powder, and the pH was adjusted to 5.8.

[0014] Preferably, the glycogen in the induction medium is one or a mixture of sucrose and maltose.

[0015] Preferably, in step 2, after the extract and induction culture medium are prepared, they are filtered through a 0.22 μm pore size filter membrane, and after the differentiation culture medium and rooting culture medium are prepared, they are sterilized at 0.11 MPa and 121°C for 20 min.

[0016] Preferably, in step 3, before preparing the microspore suspension, the microspores are washed once with an induction medium.

[0017] Preferably, in step 3, young spikelets with anther length accounting for 1 / 3 to 1 / 2 of the total floret length are selected.

[0018] Preferably, in step 3, during centrifugation: the filtrate is centrifuged at a centrifugal force of 100×g to 200×g for 3 to 7 minutes.

[0019] Preferably, in step 3, the suspension is filtered through a 150-200 mesh sieve.

[0020] Preferably, the rice is selected from one or more of the following varieties: Nipponbare, Wuyujing 7, 93-11, Zhonghua 11, Nanjing 46, Zhongzao 39, Zhongjiazao 17, Xiushui 134, Nanjing 9108, Nanjing 5055, Shanghai Normal University 19, 7375, Hudao 89, Huruan 1212 Kang, and Huxiangruan 450.

[0021] The beneficial effects of this invention are:

[0022] 1. The culture method proposed in this invention can effectively reduce the problem of easy contamination encountered in the culture of free microspores, and the induction medium has a good induction effect.

[0023] 2. The cultivation method proposed in this invention can not only effectively promote callus induction in different rice varieties, but also help with subsequent green seedling regeneration.

[0024] 3. In most cases, when using the cultivation method proposed in this invention to induce haploid rice, there is no need to carry out additional artificial chromosome doubling treatment to obtain a high proportion of diploid plants. Detailed Implementation

[0025] The present invention will be further explained below with reference to specific embodiments.

[0026] Example 1 describes a method for efficient culture of free microspores from multiple rice varieties based on a callus induction culture medium, comprising the following steps:

[0027] Step 1, Sampling and Pretreatment: During the booting stage of rice, young panicles with microspores in the middle part of the panicle at the mid-to-late mononuclear to early binucleate stage were collected from 8:00 to 9:00 am. The collected young panicles were wiped with cotton balls soaked in 75% alcohol. After wiping, they were wrapped in clean, damp gauze and then wrapped in plastic film to keep them moist. They were then placed in a 2°C refrigerator for low-temperature pretreatment for 8 days.

[0028] Step 2, Preparation of Extract and Culture Medium: Prepare the extract, induction medium, differentiation medium, and seedling rooting medium;

[0029] Step 3: Microspore isolation and callus induction: Disinfect the young spikelets after the pretreatment in Step 1 with 10% disinfectant (Yinyan brand disinfectant powder, the main component of which is sodium dichloroisocyanurate, with an effective chlorine content of 18-22%) for 10 minutes, rinse with sterile water 4-5 times, inoculate anthers in test tubes (1000-1100 anthers per test tube), add 10 mL of extraction solution, homogenize at high speed using a homogenizer, filter the suspension through a sieve, centrifuge, discard the centrifuged liquid, collect the microspores, and prepare a microspore suspension using induction medium and microspores. Adjust the microspore density in the microspore suspension to 1.0 × 10⁻⁶. 5 1.0 mL of microspore suspension was placed in a culture dish, sealed with sealing film, and placed in an incubator at 26℃ and 60% relative humidity for dark culture. After 4 weeks, the callus was weighed and immediately transferred to differentiation medium for plant regeneration.

[0030] Step 4, plant regeneration and seedling rooting: After inoculating the callus tissue into the differentiation medium, place it in a culture room at 24℃, 60% relative humidity and 16h photoperiod. Transfer the 1.5-1.8cm regenerated green plants to the seedling rooting medium for further culture.

[0031] Step 5, Field Transplanting: After the plant roots have developed, clean them and transplant them into the paddy field, and manage them as normal rice cultivation.

[0032] The extract contained 58 g / L mannitol, with added CaCl2 0.9 g / L, colchicine 18 mg / L, and MES (2-morpholinoethanesulfonic acid) 0.95 g / L. The pH of the extract was 5.6. The induction medium contained basal medium, with added glycogen 85 g / L, proline 0.45 g / L, glutamine 0.45 g / L, hydrolyzed casein 0.25 g / L, 2,4-D (2,4-dichlorophenoxyacetic acid) 1.8 mg / L, and KT (kinetin) 0.45 mg / L. The pH of the induction medium was 5.6. The differentiation medium used 1 / 2 MS (Murashige and Skoog medium) as the basal medium, with added maltose 28 g / L, KT 1.5 mg / L, 6-BA (6-benzylaminopurine) 0.8 mg / L, and NAA. 0.45 mg / L, solidified with 5.5 g / L agar powder, and pH adjusted to 5.6; the seedling rooting medium is based on 1 / 2 MS, with 28 g / L sucrose, 0.35 mg / L NAA (naphthaleneacetic acid) and 2.7 mg / L paclobutrazol added, solidified with 5.6 g / L agar powder, and pH adjusted to 5.6.

[0033] In step 2, after the extract and induction medium are prepared, they are filtered through a 0.22 μm pore size filter membrane. After the differentiation medium and rooting medium are prepared, they are sterilized at 0.11 MPa and 121℃ for 20 min.

[0034] In step 3, before preparing the microspore suspension, the microspores are washed once with induction medium.

[0035] In step 3, select young spikelets whose anther length accounts for 1 / 3 of the total floret length.

[0036] In step 3, during centrifugation: the filtrate is centrifuged at a centrifugal force of 100×g for 3 to 7 minutes.

[0037] In step 3, the suspension is filtered through a 150-mesh sieve.

[0038] Example 2 describes a method for efficient culture of free microspores from multiple rice varieties based on a callus induction culture medium, comprising the following steps:

[0039] Step 1, Sampling and Pretreatment: During the booting stage of rice, young panicles with microspores in the middle part of the panicle at the mid-to-late mononuclear to early binucleate stage were collected from 9:00 to 10:00 am. The collected young panicles were wiped with cotton balls soaked in 75% alcohol. After wiping, they were wrapped in clean, damp gauze and then wrapped in plastic film to keep them moist. They were then placed in a 5℃ refrigerator for low-temperature pretreatment for 14 days.

[0040] Step 2, Preparation of Extract and Culture Medium: Prepare the extract, induction medium, differentiation medium, and seedling rooting medium;

[0041] Step 3: Microspore isolation and callus induction: Disinfect the pretreated young spikelets from Step 1 with 10% disinfectant for 10 minutes, rinse 5 times with sterile water, and inoculate anthers into test tubes. Inoculate 1300–1400 anthers into each test tube, add 20 mL of extraction solution, and homogenize at high speed using a homogenizer. Filter the suspension through a sieve, centrifuge, discard the centrifuged liquid, and collect the microspores. Prepare a microspore suspension using induction medium and microspores, adjusting the microspore density in the suspension to 1.0 × 10⁻⁶. 5 1.0 mL of microspore suspension was placed in a culture dish, sealed with sealing film, and placed in an incubator at 26℃ and 75% relative humidity for dark culture. After 10 weeks, the callus was weighed and immediately transferred to differentiation medium for plant regeneration.

[0042] Step 4, Plant regeneration and seedling rooting: After inoculating the callus tissue into the differentiation medium, place it in a culture room at 24℃, 75% relative humidity and 16h photoperiod. Transfer the 2.3-2.5cm regenerated green plants to the seedling rooting medium for further culture.

[0043] Step 5, Field Transplanting: After the plant roots have developed, clean them and transplant them into the paddy field, and manage them as normal rice cultivation.

[0044] The extract contained 62 g / L mannitol, with added CaCl2 1.3 g / L, colchicine 22 mg / L, and MES 1 g / L; the pH of the extract was 6. The induction medium contained basal medium, with added glycogen 95 g / L, proline 0.55 g / L, glutamine 0.55 g / L, hydrolyzed casein 0.35 g / L, 2,4-D 2.2 mg / L, and KT 0.55 mg / L; the pH of the induction medium was 6. The differentiation medium used 1 / 2 MS as the basal medium, with added maltose 32 g / L, KT 2.5 mg / L, 6-BA 1.2 mg / L, and NAA. 0.55 mg / L, solidified with 6.5 g / L agar powder, and pH adjusted to 6; the seedling rooting medium is based on 1 / 2 MS, with 32 g / L sucrose, 0.45 mg / L NAA and 2.7–3.3 mg / L paclobutrazol added, solidified with 6 g / L agar powder, and pH adjusted to 6.

[0045] The glycogen in the induction medium is one or a mixture of sucrose and maltose.

[0046] In step 2, after the extract and induction medium are prepared, they are filtered through a 0.22 μm pore size filter membrane. After the differentiation medium and rooting medium are prepared, they are sterilized at 0.11 MPa and 121℃ for 20 min.

[0047] In step 3, before preparing the microspore suspension, the microspores are washed once with induction medium.

[0048] In step 3, select young spikelets whose anther length accounts for 1 / 3 to 1 / 2 of the total floret length.

[0049] In step 3, during centrifugation: the filtrate is centrifuged at a centrifugal force of 200×g for 7 minutes.

[0050] In step 3, the suspension is filtered through a 200-mesh sieve.

[0051] In Example 3, a method for high-efficiency culture of free microspores of multiple rice varieties based on a callus induction culture medium includes the following steps:

[0052] Step 1, Sampling and Pretreatment: During the booting stage of rice, young panicles with microspores in the middle part of the panicle at the mid-to-late mononuclear to early binucleate stage were collected from 8:30 to 9:30 am. The collected young panicles were wiped with cotton balls soaked in 75% alcohol. After wiping, they were wrapped in clean, damp gauze and then wrapped in plastic film to keep them moist. They were then placed in a 5°C refrigerator for low-temperature pretreatment for 12 days.

[0053] Step 2, Preparation of Extract and Culture Medium: Prepare the extract, induction medium, differentiation medium, and seedling rooting medium;

[0054] Step 3: Microspore isolation and callus induction: Disinfect the pretreated young spikelets from Step 1 with 10% disinfectant for 10 minutes, rinse 5 times with sterile water, and inoculate anthers into test tubes. Inoculate 1250–1350 anthers into each test tube, add 15 mL of extraction solution, and homogenize at high speed using a homogenizer. Filter the suspension through a sieve, centrifuge, discard the centrifuged liquid, and collect the microspores. Prepare a microspore suspension using induction medium and microspores, adjusting the microspore density in the suspension to 1.0 × 10⁻⁶. 5 1.0 mL of microspore suspension was placed in a culture dish, sealed with sealing film, and placed in an incubator at 26℃ and 65% relative humidity for dark culture. After 4 weeks, the callus was weighed and immediately transferred to differentiation medium for plant regeneration.

[0055] Step 4, Plant regeneration and seedling rooting: After inoculating the callus tissue into the differentiation medium, place it in a culture room at 24℃, 65% relative humidity and 16h photoperiod. Transfer the 1.8-2.2cm regenerated green plants to the seedling rooting medium for further culture.

[0056] Step 5, Field Transplanting: After the plant roots have developed, clean them and transplant them into the paddy field, and manage them as normal rice cultivation.

[0057] The extract contained 60 g / L mannitol, with added CaCl2 1.1 g / L, colchicine 20 mg / L, and MES 0.976 g / L; the pH of the extract was 5.8. The induction medium contained basal medium, with added glycogen 90 g / L, proline 0.5 g / L, glutamine 0.5 g / L, hydrolyzed casein 0.3 g / L, 2,4-D 2 mg / L, and KT 0.5 mg / L; the pH of the induction medium was 5.8. The differentiation medium used 1 / 2 MS as the basal medium, with added maltose 30 g / L, KT 2 mg / L, 6-BA 1 mg / L, and NAA. 0.5 mg / L, solidified with 6 g / L agar powder, and pH adjusted to 5.8; the seedling rooting medium is based on 1 / 2 MS, with 30 g / L sucrose, 0.4 mg / L NAA and 3 mg / L paclobutrazol added, solidified with 6 g / L agar powder, and pH adjusted to 5.8.

[0058] The glycogen in the induction medium is one or a mixture of sucrose and maltose.

[0059] In step 2, after the extract and induction medium are prepared, they are filtered through a 0.22 μm pore size filter membrane. After the differentiation medium and rooting medium are prepared, they are sterilized at 0.11 MPa and 121℃ for 20 min.

[0060] In step 3, before preparing the microspore suspension, the microspores are washed once with induction medium.

[0061] In step 3, select young spikelets whose anther length accounts for 1 / 3 to 1 / 2 of the total floret length.

[0062] In step 3, during centrifugation: the filtrate is centrifuged at a centrifugal force of 100×g for 3 to 7 minutes.

[0063] In step 3, the suspension is filtered through a 150-mesh sieve.

[0064] The rice varieties selected are one or more of the following: Nipponbare, Wuyujing 7, 93-11, Zhonghua 11, Nanjing 46, Zhongzao 39, Zhongjiazao 17, Xiushui 134, Nanjing 9108, Nanjing 5055, Shanghai Normal University 19, 7375, Hudao 89, Huruan 1212 Kang, and Huxiangruan 450.

[0065] The following is a demonstration of actual research based on Example 3:

[0066] 1) Donor plants and their growth

[0067] This study used 15 rice varieties from the region as test materials, including 3 model varieties and 12 main cultivated varieties or backbone parents. Among them, 11 were from the China National Rice Mid-term Bank, and the remaining 4 were from the Institute of Crop Breeding and Cultivation, Shanghai Academy of Agricultural Sciences (see Table 1). The seeds of these rice varieties were sown in the seedbeds of the Chonggu Experimental Base of Shanghai Academy of Agricultural Sciences, and then their seedlings were transplanted into paddy fields and managed as normal rice cultivation.

[0068] Table 1. Rice varieties used in this study

[0069]

[0070] 2) Sampling and Pretreatment

[0071] During the booting stage of rice varieties, young panicles of the above 15 rice varieties were taken and pretreated in accordance with the method in Example 3.

[0072] 3) Preparation of extract and culture medium

[0073] The extract and culture medium were prepared according to Example 3. Five induction culture media were prepared (numbered CIM I, II, III, IV, and V). Detailed components of the induction culture media are shown in Table 2. Table 2. Components of different callus induction culture media.

[0074]

[0075] 4) Microspore isolation and callus induction

[0076] Microspore isolation and callus induction were performed according to the method described in Example 3. The induction effects of different callus induction media are shown in Table 3. For most rice varieties, the callus tissue per dish was weighed after 4 weeks to determine the callus yield (mg), which was used to assess callus induction capacity. Because callus induction was slower in some rice varieties, the weighing was adjusted accordingly; for example, rice varieties Q12 and Q13 were weighed after 10 weeks, while rice variety Q14 was weighed after 8 weeks. Rice variety Q9 was more complex in CIM IV medium, and weighing was performed after 7 and 10 weeks, respectively. After weighing, the callus tissue was immediately transferred to differentiation medium for plant regeneration.

[0077] Table 3. Callus induction of different rice varieties on different callus induction media (continued below)

[0078]

[0079]

[0080] Table 3. Callus induction of different rice varieties on different callus induction media (continued from above)

[0081]

[0082] Note: Blank spaces in the table indicate areas where microspore culture was not performed, and "ND" indicates that callus tissue was not induced.

[0083] One of the biggest challenges in the culture of free microspores is contamination. This experiment showed that overall contamination control was very good, with only two rice varieties (a total of four cultures) severely affected by contamination (see Table 3). Therefore, the experiment was generally successful. In callus induction, the induction effects of different callus induction media varied significantly (see Table 3): CIM I medium successfully induced callus formation in 8 rice varieties; CIM II had a stronger induction capacity, inducing callus formation in 12 varieties; CIM III cultured 14 varieties, with 12 successfully inducing callus formation; CIM IV cultured 13 varieties, inducing callus formation in only 5 varieties; and CIM V also cultured 13 varieties, successfully inducing callus formation in 10 varieties. This indicates that CIM I and CIM III media had relatively outstanding overall induction effects, while CIM IV medium had lower induction efficiency.

[0084] From the perspective of rice varieties capable of callus induction, the only difference between CIM I, II, and IV is the carbon source used. Results show that maltose is the most effective, sucrose is slightly less effective, and a combination of both carbon sources is the least effective. This trend also seems to be observed in callus yield. Therefore, it is recommended to use callus induction media with a single carbon source, such as CIMI and II. Regarding the preferred carbon source, considering that the effect of CIM I can cover that of CIM I, it is recommended to use CIM I medium with maltose as the sole carbon source.

[0085] Furthermore, CIMII, III, and V all use maltose as the carbon source, differing only in their basal culture media, and their effects on callus induction were further compared. In rice varieties capable of callus induction, CIMII and III showed the best results, with CIM V also performing well, only slightly worse than the first two. This trend also seems to be reflected in callus yield. Moreover, the effect of CIMIII appears to encompass that of CIM V. Therefore, in callus induction from free microspore culture, it is recommended to prioritize CIMII or III, and then consider CIMV. It should also be noted that callus induction in indica rice varieties was only successfully achieved in CIMII and III. Therefore, these two callus induction media can also be considered as the primary choices for indica rice free microspore culture.

[0086] 5) Plant regeneration and seedling rooting

[0087] After inoculating the callus tissue into the differentiation medium, it was placed in a culture room at 24℃, 60-75% relative humidity, and 16h photoperiod. The regenerated green plants of about 2cm were transferred to the seedling rooting medium for further culture. At the same time, the number of green seedlings and albino seedlings was recorded. The number of seedlings differentiated from 100mg of callus tissue was used to evaluate the regeneration frequency.

[0088] Table 4. Callus differentiation of different rice varieties obtained from different callus induction media

[0089]

[0090]

[0091] Note: The regeneration frequency is the number of regenerated seedlings (including regenerated green seedlings and white seedlings) produced per 100mg of callus tissue, while the regeneration green seedling frequency is the number of green seedlings produced per 100mg of callus tissue.

[0092] 6) Field transplanting and ploidy identification

[0093] Once the plant roots have developed, they are washed and transported to the Lingshui base in Hainan Province, where they are directly transplanted into paddy fields and managed as normal rice plants. During the grain-filling stage, the grain setting rate is investigated to assess ploidy based on whether the plants can set grains normally. Plant height and husk size are also used as reference factors.

[0094] After the regenerated seedlings were counted, considering that some green spots still had the potential to regenerate, the remaining callus tissue after the regeneration was not discarded, but continued to differentiate, in order to maximize the amount of green seedlings obtained. Therefore, the final number of regenerated green seedlings obtained was higher than the number counted in the initial period. Since regenerated green seedlings could not be successfully obtained for the three indica rice varieties Q3, Q6, and Q7, ploidy identification was only carried out for the remaining 12 japonica rice varieties. Except for Q8 and Q11, where the number of regenerated green seedlings was limited, about 50 regenerated green seedlings were randomly selected from the remaining rice varieties for subsequent ploidy identification (see Table 5). The identification results showed that the diploid ratio of the 12 rice varieties ranged from 14.3% to 98%. Among them, the diploid ratio of 8 rice varieties (accounting for 2 / 3) exceeded 60%, and the diploid ratio of 4 rice varieties (accounting for 1 / 3) even exceeded 90%. This data fully demonstrates that the regenerated plants obtained by this culture system have an extremely high diploid ratio. Therefore, in most cases, when using this system to induce haploid rice, there is no need to carry out additional artificial chromosome doubling treatment to obtain a high proportion of diploid plants.

[0095] Table 5. Pluripotency identification of regenerated plants from different rice varieties

[0096]

[0097] 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 equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A high-efficiency culture method of rice multi-varieties of free microspores based on callus induction medium modification, characterized by, The method comprises the following steps: Step 1, sampling and pretreatment: in the booting stage of rice, the middle young ear of rice is collected at 8:00-10:00 in the morning, and the young ear of rice is in the single nucleus middle-late stage to the double nucleus early stage. The collected young ear is wiped with a 75% alcohol cotton ball, and then wrapped with a clean wet gauze and a plastic film for moisturizing, and placed in a 2-5°C refrigerator for low-temperature pretreatment for 8-14 days; Step 2, preparation of extraction solution and culture medium: preparation of extraction solution, induction medium, differentiation medium and strong seedling rooting medium; Step 3, isolation of microspores and induction of callus: the pre-treated young panicles in step 1 were sterilized for 10 min with 10% sterilizing spirit, washed 4-5 times with sterile water, and then inserted into the anthers using test tubes, 1000-1400 anthers were inserted into each test tube, 10-20 mL of the extract was added, and the mixture was high-speed sheared using a homogenizer, the suspension was filtered using a screen, centrifuged, the centrifugal liquid was discarded, and the microspores were collected, the microspore suspension was prepared using the induction medium and the microspores, the density of the microspores in the microspore suspension was adjusted to 1.0 x 10 5 9 / mL, 1.0 mL of the microspore suspension was taken into a culture dish, the culture dish was sealed with a sealing film, and then placed in a 26°C, 60%-75% relative humidity incubator for dark culture, the callus was weighed after 4-10 weeks, and then immediately transferred into a differentiation medium for plant regeneration. Step 4, plant regeneration and strong seedling rooting: after the callus is inoculated into the differentiation medium, it is placed in a culture room at 24°C, 60-75% relative humidity and 16h light cycle for culture, and the 1.5-2.5cm regenerated green plants are transferred to the strong seedling rooting medium for continuous culture; Step 5, field transplanting: after the plant roots are developed, the plants are washed and transplanted into the rice field, and managed according to the normal rice planting.

2. The method according to claim 1, wherein the callus induction medium is modified. The extraction solution contains 58-62g / L mannitol, 0.9-1.3g / L CaCl2, 18-22mg / L colchicine and 0.95-1g / L MES, and the pH of the extraction solution is 5.6-6; the induction medium contains a basic medium, 85-95g / L glycogen, 0.45-0.55g / L proline, 0.45-0.55g / L glutamine, 0.25-0.35g / L hydrolyzed casein, 1.8-2.2mg / L 2,4-D and 0.45-0.55mg / L KT, and the pH of the induction medium is 5.6-6; the differentiation medium takes 1 / 2MS as the basic medium, and adds 28-32g / L maltose, 1.5-2.5mg / L KT, 0.8-1.2mg / L 6-BA and 0.45-0.55mg / L NAA, and the pH of the medium is adjusted to 5.6-6; the strong seedling rooting medium takes 1 / 2MS as the basic medium, and adds 28-32g / L sucrose, 0.35-0.45mg / L NAA and 2.7-3.3mg / L paclobutrazol, and the pH of the medium is adjusted to 5.6-6.

3. The method according to claim 2, wherein the medium is a callus induction medium. The extraction solution contains mannitol 60 g / L, CaCl2 1.1 g / L, colchicine 20 mg / L, MES 0.976 g / L, and the pH of the extraction solution is 5.8; the induction medium contains a basic medium, glycogen 90 g / L, proline 0.5 g / L, glutamine 0.5 g / L, hydrolyzed casein 0.3 g / L, 2,4-D 2 mg / L, KT 0.5 mg / L, and the pH of the induction medium is 5.8; the differentiation medium is based on 1 / 2MS, and contains maltose 30 g / L, KT 2 mg / L, 6-BA 1 mg / L, and NAA 0.5 mg / L, and the medium is solidified with 6 g / L agar powder, and the pH is adjusted to 5.8; the strong seedling rooting medium is based on 1 / 2MS, and contains sucrose 30 g / L, NAA 0.4 mg / L, and paclobutrazol 3 mg / L, and the medium is solidified with 6 g / L agar powder, and the pH is adjusted to 5.

8.

4. The method according to claim 3, wherein the medium is a callus induction medium. The glycogen in the induction medium is a mixture of one or both of sucrose and maltose, and the basic medium is one of N6, M8, and NB.

5. The method according to claim 3, wherein the medium is a callus induction medium. In step 2, after the preparation of the extraction solution and the induction medium is completed, the solution is filtered through a filter membrane with a pore size of 0.22 μm, and after the preparation of the differentiation medium and the rooting medium is completed, the medium is sterilized at 0.11 MPa and 121 ℃ for 20 min.

6. The method according to claim 1, wherein the callus induction medium is modified. In step 3, before the preparation of the microspore suspension, the microspores are washed once with the induction medium.

7. The method according to claim 1, wherein the medium is callus induction medium. In step 3, the young panicles with the anther length accounting for 1 / 3 to 1 / 2 of the total length of the small flowers are selected.

8. The method according to claim 1, wherein the callus induction medium is modified. In step 3, during the centrifugation, the filtrate is centrifuged at a centrifugal force of 100×g to 200×g for 3 to 7 min.

9. The method according to claim 1, wherein the callus induction medium is modified with 2,4-D and 6-BA. In step 3, the suspension is filtered through a sieve with a mesh size of 150 to 200 mesh.

10. The method according to claim 1, wherein the callus induction medium is modified with 2,4-D and 6-BA. The rice is selected from one or more of Nipponbare, Wuyujian No. 7, 93-11, Zhonghua 11, Nanjing 46, Zhongzao 39, Zhongjiaozao 17, Xiushui 134, Nanjing 9108, Nanjing 5055, Shangshida 19, 7375, Huroi 89, Huruan 1212, and Huxiangruan 450.

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