Method for creating homozygous tetraploid rice by using haploid anther

Abstract

The invention belongs to the technical field of crop variety breeding in modern agriculture, and discloses a method for creating homozygous tetraploid rice by using haploid anther, which makes full use of the characteristic that the number of chromosomes in cells subjected to meiosis is halved, and microspores formed by meiosis are subjected to anther culture at a proper development stage, so that the homozygous tetraploid rice is created. The method comprises the following steps of: a, selecting a diploid rice line with excellent characters as a parent material, and carrying out continuous secondary induction by colchicine to form a callus, and differentiating to emerge seedlings to form a stable homozygous tetraploid rice line; b, selecting anther with microspores in the late uninucleate stage during observation; c, inducing anther calluses; e, differentiating the anther calluses to emerge seedlings; f, seedling callus differentiation and rooting; and g, determining the authenticity of a homozygous tetraploid plant formed by anther culture through morphological and cytological technologies (flow cytometry). The method is simple, low in cost and easy to operate.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop variety breeding in modern agriculture, and discloses a method for creating homozygous tetraploid rice by using haploid anthers. Background Art

[0002] Rice is one of the most important food sources for humans, and more than 60% of the world's population feeds on rice. However, in recent years, with the rapid growth of the population, the reduction of arable land area, and the frequent occurrence of extreme weather such as drought and flood, food security is facing an overall challenge. At present, the rice planting in China is mainly conventional diploid hybrid rice, with an average yield per mu exceeding 1000 catties, and it is extremely difficult to further increase the yield. Polyploid rice has obvious advantages over its diploid counterparts in terms of growth potential, biological yield, and resistance, and thus has the potential to increase yield. However, due to various reasons, the problem of low seed setting rate of polyploid rice cannot be solved, and no breakthrough has been made. For example: First, polyploid rice is prone to produce multivalents during meiosis, forming aneuploid chromosomes, which increases the genetic instability of the genome (Zhang et al., 2013; Fu et al., 2021; Wang et al., 2023). Second, after chromosome doubling, the redundancy brought by whole-genome duplication increases the difficulty of domestication selection and prolongs the breeding cycle (Van de Peer et al., 2021; Qin et al., 2023). Third, compared with diploids, the pollen fertility of polyploid rice decreases, and the seed setting rate drops significantly (Ye et al., 2018; Ghen et al., 2023).

[0003] In response to the worldwide problem of low seed setting rate of polyploid rice, significant breakthroughs have been made by researchers in meiosis regulation, genome stability, and fertility improvement. For example, the pollen fertility can be improved by CRISPR editing of OsPSS1 or exogenous hormone treatment (Chen et al., 2023; Zhou et al., 2021). The team of Professor Cai Detian established a patented technology for high-frequency induction of polyploid rice through technological innovation, significantly increasing the induction rate from less than 5% to 46.7% (Cai Detian, et al., 2013), and creating more than 5000 polyploid rice resources (Cai et al., 2014); furthermore, by introducing the Ph-like gene and wide-compatibility gene, the PMeS line with stable meiosis was successfully selected, and its seed setting rate reached a breakthrough of 85% (Cai et al., 2016). Nevertheless, due to the problem of unstable meiosis not being completely solved, the seed setting rate is still generally low, which remains the main bottleneck restricting the breeding application of polyploid rice. In the future, it is necessary to further explore the precise regulation mechanism of meiosis to achieve a stable increase in the fertility of polyploid rice. Summary of the Invention

[0004] To solve the above-mentioned problems of the prior art, the present invention discloses a method for creating homozygous tetraploid rice using haploid anthers. The present invention makes full use of the characteristic that the chromosomes in cells are halved after meiosis. Through anther culture, microspores that have undergone meiosis can form callus at their appropriate developmental stage. After secondary induction with colchicine and other agents, and then through differentiation into seedlings and rooting, a stable homozygous tetraploid rice line is formed, thereby overcoming the problem of low seed setting rate of polyploid rice and providing a good foundation for promoting the theoretical research and breeding practice of polyploid rice.

[0005] The present invention includes the following technical solutions:

[0006] A method for creating homozygous tetraploid rice using haploid anthers, comprising the following steps:

[0007] a. Select a conventional diploid rice line with stable traits and a seed setting rate ≥ 95% as the parental material;

[0008] b. Select anthers with microspores at the late uninucleate stage, and determine their developmental stage by the KI solution staining method;

[0009] c. Inoculate the anthers into a callus induction medium for dark culture to induce the formation of callus;

[0010] d. Treat the callus with colchicine twice and perform recovery culture successively. The two treatments both include:

[0011] i. Culture in a liquid medium containing colchicine for 24 - 48 hours;

[0012] ii. Transfer to a recovery medium for culture;

[0013] e. Inoculate the callus treated twice into a differentiation medium for light culture until bud seedlings are differentiated;

[0014] f. Transfer the bud seedlings to a rooting medium to induce rooting and obtain complete plants;

[0015] g. Verify by cytological techniques that the plants are homozygous tetraploid rice with a chromosome number of 2n = 4x = 48.

[0016] Further, in the above method for creating homozygous tetraploid rice using haploid anthers, the parental material in step a is the diploid rice line Balilla - 2x.

[0017] Further, in the above method for creating homozygous tetraploid rice using haploid anthers, the concentration of the KI solution in step b is 0.3% - 0.5%.

[0018] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, the components of the callus induction medium in step c are: N6 basal medium, 2,4-D 1.5 mg / L, NAA 1 mg / L, 6-BA 0.5 mg / L, sucrose 4%, agar 0.75%, pH 6.0.

[0019] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, the components of the liquid medium in step d are: N6 basal medium, 2,4-D 1.5 mg / L, NAA 1 mg / L, 6-BA 0.5 mg / L, colchicine 0.5 g / L, sucrose 4%, pH 6.0.

[0020] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, the components of the recovery medium in step d are: N6 basal medium, sucrose 2.5%, agar 0.75%, 2,4-D 0.5 mg / L, NAA 0.8 mg / L, 6-BA 0.5 mg / L, pH 6.0.

[0021] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, the components of the differentiation medium in step e are: N6 basal medium, sucrose 30 g / L, casein hydrolysate 300 mg / L, glutamine 500 mg / L, proline 500 mg / L, D-sorbitol 15 g / L, anhydrous CuSO4 2.5 mg / L, NAA 0.2 mg / L, 6-BA 2 mg / L, agar 0.75%, pH 6.0.

[0022] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, the components of the rooting medium in step f are: 1 / 2 MS medium, NAA 0.3 mg / L, 6-BA 0.5 mg / L, casein hydrolysate 200 mg / L, sucrose 2%, agar 0.75%, activated carbon 0.02%, pH 6.0.

[0023] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, the cytological technique in step g is chromosome preparation observation of root tips or DNA content detection by flow cytometry.

[0024] Further, for the method of creating homozygous tetraploid rice using haploid anthers as described above, for the tetraploid rice, its chromosome number is 2n = 4x = 48, and the single-plant yield, 1000-grain weight and nutritional quality are significantly higher than those of the parental diploid rice.

[0025] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0026] 1. The doubling efficiency is significantly improved

[0027] The efficiency of inducing polyploids by the traditional colchicine soaking method is usually less than 5% (Cai et al., 2014). However, through the optimized double-doubling treatment combination and culture medium formula in the present invention, the tetraploid induction rate has been increased to nearly 10 times. The key innovations are as follows:

[0028] Two-cycle treatment mode (24 - 48h colchicine treatment + recovery culture); optimized colchicine concentration (0.5g / L); precise regulation of 2,4-D in the recovery culture medium (0.5mg / L).

[0029] 2. Stable homozygosity and increased seed setting rate

[0030] Polyploid rice obtained by traditional methods generally has the problem of low seed setting rate (usually <50%). This invention solves this problem through the following innovations:

[0031] a) Strictly select diploid parents with high seed setting rate (>95%); b) Optimized method for determining the anther development stage (KI solution method); c) Innovative callus induction culture medium formula (N6 + 2,4-D 1.5mg / L + NAA 1mg / L + 6-BA 0.5mg / L).

[0032] 3. Broad application prospects

[0033] The induction method provided by this invention provides a large number of homozygous materials for polyploid rice breeding, solves the key technical bottleneck of low seed setting rate in polyploid rice, and provides a technical reference for polyploid breeding of other crops.

[0034] In summary, through key step innovations and optimized combinations of culture media, this invention has successfully solved the technical problems such as low induction efficiency, poor homozygosity, and low seed setting rate existing in traditional polyploid breeding methods, and provides an efficient and reliable technical solution for polyploid rice breeding. Brief description of the drawings

[0035] Figure 1 . Callus is formed by anther culture;

[0036] Figure 2 . Doubling culture of callus;

[0037] Figure 3 . Shoots are differentiated from callus;

[0038] Figure 4 . Roots are differentiated from shoot seedlings;

[0039] Figure 5 . The root tip chromosomes of anther culture seedlings are 2n = 2X = 24, being true diploid plants;

[0040] Figure 6 .Comparison of agronomic traits between Balilla-4x and Balilla-2x;

[0041] Figure 7 .Comparison of grain shape and nutritional quality between Balilla-4x and Balilla-2x. DETAILED DESCRIPTION

[0042] A method for creating homozygous tetraploid rice using haploid anthers comprises the following steps:

[0043] a. Select conventional diploid rice lines with stable traits and a fruit setting rate of >95% as culture parent materials

[0044] Since all existing rice varieties, whether conventional rice or hybrid rice, are diploid, their fruit setting rate and traits are relatively stable and can be used as basic materials for the homozygous tetraploid anther culture of the present invention.

[0045] b. Sample collection and observation of anther development period (key step)

[0046] Generally, young panicles of diploid rice with inflorescence length less than 5 cm are taken, anthers are picked out, slices are made and observed using 0.3%-0.5% KI solution to determine that the anthers are in the microspore uninucleate marginal stage and are used as experimental materials.

[0047] Innovation: The use of 0.3%-0.5% KI solution can accurately determine the microspore mononuclear marginal stage. Comparative experiments show that the use of conventional acetic acid magenta staining method will lead to greater errors in period determination and reduce the induction efficiency by about 20%.

[0048] c. Callus induction

[0049] Select the pretreated young spikelets, pick the young spikelet anthers and let them fall on the callus induction medium to induce callus. Callus induction medium: N6+2,4-D 1.5mg / L+NAA1mg / L+6-BA 0.5mg / L+sucrose 4%+agar 0.75%, pH 6.0, culture at 26℃-30℃ in the dark for 3-4 weeks, callus can be gradually seen growing on the medium.

[0050] d. Double callus tissue and recovery culture (core innovation step)

[0051] The callus was inoculated into the doubling medium (liquid), placed in the dark, and cultured on a shaker at 26°C - 30°C for 24 - 48 h, then transferred to the recovery culture. This operation was repeated once, that is, doubling and recovery culture were carried out twice continuously. Doubling medium: N6 + 2,4-D 1.5 mg / L + NAA 1 mg / L + 6-BA 0.5 mg / L + colchicine 0.5 g / L + sucrose 4%, pH 6; Recovery medium: N6 + sucrose 2.5% + Agar 7.5‰ + 2,4-D 0.5 mg / L + NAA 0.8 mg / L + 6-BA 0.5 mg / L, pH 6.0.

[0052] e. The callus differentiates into seedlings

[0053] The callus after two doublings was inoculated into the differentiation medium and cultured under light at 26°C - 30°C. The callus would gradually show green spots from light yellow until it differentiated into seedlings. Differentiation medium: Differentiation medium: N6 + sucrose 30 g / L + casein hydrolysate 300 mg / L + glutamine 500 mg / L + proline 500 mg / L + D-sorbitol 15 g / L + anhydrous CuSO4 2.5 mg / L + NAA 0.2 mg / L + 6-BA 2 mg / L + agar 0.75%, pH 6.0.

[0054] f. The seedlings differentiate into roots

[0055] When the anther callus differentiated buds and seedlings grew to a height of 10 cm - 15 cm, they were transferred to the rooting medium. After 3 - 4 weeks of culture, roots were differentiated to form complete plants. Rooting medium: 1 / 2MS + NAA 0.3 mg / L + 6-BA 0.5 mg / L + casein hydrolysate 200 mg / L + sucrose 2% + agar 0.75% + activated carbon 0.02%, pH 6.0;

[0056] g. Use cytological techniques to determine the authenticity of the tetraploid of the plants formed by anther culture: The root tips of the cultivated plants were observed after chromosome preparation. The diploid was 2n = 2x = 24; the tetraploid was 2n = 4x = 48; or the DNA content of the plants was measured by flow cytometry to determine the authenticity of the tetraploid.

[0057] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0058] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions. For example, when inducing callus, it is cultured in the dark, and the doubling medium is a liquid medium.

[0059] Example 1

[0060] A method for creating homozygous tetraploid rice using haploid anthers, comprising the following steps:

[0061] a. Select a stable conventional diploid rice line

[0062] Select the conventional diploid rice line Balilla-2x (Balilla) with stable traits and a seed setting rate > 95% as the culture parent material

[0063] b. Observe the anther development stage

[0064] At the booting stage of diploid rice, take young panicles with a distance of 0.5 - 1.5 cm between the flag leaf sheath and the next leaf sheath (young panicles with a rice inflorescence length less than 5 cm), take 1 - 2 spikelets, pick out the anthers from them, make slides with 0.3% - 0.5% KI solution, and observe and determine that the anthers are at the late uninucleate microspore stage as experimental materials.

[0065] c. Induce anther callus

[0066] After disinfecting the dissected young panicles with 75% alcohol, use forceps to pick out the anthers in the spikelets or knock down the anthers in about half-opened spikelets and place them on the callus induction medium. After culturing in the dark at 26°C for 3 - 4 weeks, gradually light yellow callus can be seen growing on the medium ( Figure 1 ).

[0067] d. Inoculate the callus into the doubling medium (liquid), place it in the dark, culture it on a shaker at 26°C - 30°C for 24 - 48 h and then transfer it to recovery culture ( Figure 2 ). Repeat this operation once, that is, double and recover culture continuously twice.

[0068] e. Differentiate anther callus into seedlings

[0069] Transfer the light yellow and compact anther callus to the differentiation medium, place it at 26°C, and culture it under the condition of light of 2000 / lux - 3000 / lux. Then gradually green spots can be seen on the callus until it differentiates into seedlings ( Figure 3 ). Differentiation medium: N6 + sucrose 30 g / L + casein hydrolysate 300 mg / L + glutamine 500 mg / L + proline 500 mg / L + D-sorbitol 15 g / L + anhydrous CuSO4 2.5 mg / L + NAA 0.2 mg / L + 6-BA 2 mg / L + agar 0.75%, pH 6.0.

[0070] f. The seedlings differentiate into roots

[0071] When the seedlings differentiated from the callus grow to a height of about 1.5 cm, transfer them to the rooting medium and culture them at 26 °C under the conditions of light intensity of 2000 / lux - 3000 / lux. Subsequently, it can be gradually observed that the callus shows roots and becomes a complete plant( Figure 4 ). Rooting medium: 1 / 2MS + NAA 0.3 mg / L + 6 - BA 0.5 mg / L + casein hydrolysate 200 mg / L + sucrose 2% + agar 0.75% + activated carbon 0.02%, pH 6.0.

[0072] g. Use morphological and cytological techniques to determine the diploid authenticity of the plants formed by anther culture

[0073] Plant the obtained homozygous tetraploid in the field. Observe whether there is trait segregation in the plants morphologically. If there is no trait segregation, then take samples and determine the homozygosity through PCR and sequencing. Meanwhile, use cytological techniques on the root tips of the seedlings. After chromosome preparation, observe to determine the tetraploid. If the chromosome number is 2n = 4X = 48, then the doubling is successful( Figure 5 Left). Or observe through a flow cytometer and measure the DNA content of the plants to determine the tetraploid authenticity( Figure 5 Right).

[0074] Test example

[0075] 1. Polyploidization improves the plant height and yield of rice

[0076] Through the successfully constructed Balilla - 4x "Balilla - 4x" tetraploid, its agronomic traits (i.e., plant height, panicle length, tiller number, and 1000 - grain weight) were analyzed with those of "Balilla - 2x". The results show that compared with "Balilla - 2x", the plant height of "Balilla - 4x" is significantly higher, increasing by 19.35%( Figure 6 A–B, Table 1). The panicles of "Balilla - 4x" are longer and the stems are thicker. The panicle length increases by about 24.58%, although the difference in tiller number is not significant( Figure 6 C–D, Table 1). The number of grains per panicle of the transgenic plants of "Balilla - 4x" is also significantly higher than that of the "Balilla - 2x" plants, increasing by 37.60%( Figure 6 E, Table 1). The 1000 - grain weight of "Balilla - 4x" increases by 33.38% compared with that of "Balilla - 2x"( Figure 6 F, Table 1). The yield per plant of the "Balilla - 4x" plants increases by 25.10%( Figure 6 G, Table 1). These results indicate that compared with diploid rice, polyploid rice has significant potential in increasing yield.

[0077] Table 1: Phenotypic data of Balilla-2x and Balilla-4x plants

[0078] Characteristic Balilla-2x Balilla-4x Plant height (cm) 81.30±1.56 97.03±2.86＊＊ Panicle length (cm) 20.63±1.27 25.70±0.37＊＊ Tiller number 9.33±0.47 8.62±0.81 Grains per panicle 125.14±2.88 172.00±5.76＊＊ 1000-grain weight (g) 23.94±1.10 31.93±1.30＊＊ Yield per plant (g) 44.27±9.88 55.38±9.54＊＊

[0079] Note: Agronomic trait data are based on Balilla-2x and Balilla-4x plants in the natural growing season. The data were recorded in 2022 in Shanghai, China; agronomic trait data are expressed as mean ± standard deviation (n = 10). ** indicates extremely significant difference (P < 0.01) according to Student's t-test.

[0080] 2. Polyploidization alters grain shape and nutritional quality of rice

[0081] To determine whether polyploidization affects the grain shape of rice, we studied the grain length, grain width, and grain thickness of "Balilla-4x". The results showed that the grain length of "Balilla-4x" plants was longer, increasing by 9.43% compared with that of "Balilla-2x" plants ( Figure 7 A-B, Table 2). The grain width of "Balilla-4x" plants increased by 12.90% compared with that of "Balilla-2x" ( Figure 7 C-E, Table 2). The grain thickness of "Balilla-4x" plants increased by 15% compared with that of "Balilla-2x" ( Figure 7 D, Table 2). These results indicate that polyploidization of "Balilla" does affect the grain shape of rice.

[0082] Some studies have shown that polyploidization of rice can not only produce beneficial agronomic traits but also improve the nutritional quality of rice (Dhawan & Lavania, 1996; W. Wang et al., 2022b; H. Y. Zhang et al., 2016). Therefore, we detected the nutritional components of "Balilla-4x". The results showed that compared with "Balilla-2x", the grain protein content of "Balilla-4x" plants increased by 10.10%, and the lipid content increased significantly by 32.07% ( Figure 7 F-G, Table 2). These results indicate that polyploidization of "Balilla" not only affects the grain shape of rice but also indeed affects the nutritional quality of rice seeds.

[0083] Table 2 Grain shape data of Balila-2x and Balila-4x plants

[0084]

[0085] Note: The agronomic trait data are based on Balila-2x and Balila-4x plants in the natural growth season. WH represents Wuhan and SH represents Shanghai; the data in Wuhan were recorded in 2021 and the data in Shanghai were recorded in 2022, both in China. The agronomic trait data are expressed as mean ± standard deviation (n = 10). * indicates significant difference according to Student's t-test, P < 0.05; ** indicates extremely significant difference, P < 0.01.

[0086] In the embodiment of the present invention, the creation of homozygous tetraploid rice is achieved through the following steps: Select a diploid rice line with stable traits and a seed setting rate > 95% (such as Balilla-2x), precisely select anthers with microspores at the late uninucleate stage (judged by staining with 0.3%-0.5% KI solution), treat the callus with colchicine twice and optimize the medium formula (such as adding a specific hormone combination to the N6 basal medium), finally obtain tetraploid plants through differentiation culture and rooting, and verify their homozygosity (2n = 4x = 48) by chromosome observation or flow cytometry. The test examples show that the obtained tetraploid rice (Balilla-4x) has a significant increase in plant height, panicle length, 1000-grain weight and yield per plant by 19.35%, 24.58%, 33.38% and 25.10% respectively compared with the parental diploid, and at the same time, the grain protein and fat contents are increased by 10.10% and 32.07% respectively, fully verifying the dual advantages of this method in increasing yield and improving nutritional quality, and providing an efficient and reliable technical solution for polyploid rice breeding.

[0087] It should be noted that the above description of the embodiments focuses on explaining the technical solutions of the present invention rather than precisely defining its protection scope. Professionals in the field should understand that based on the technical details disclosed in the embodiments of the present invention, appropriate adjustments and optimizations can be made, or equivalent substitutions can be made for individual or all technical elements. Such adjustments and substitution measures will not deviate from the core essence of the technical solutions of the present invention and should be included in the technical protection scope of the embodiments of the present invention. In short, the protection scope of the present invention should not be limited by the specific presentation of the above embodiments, but should widely cover all equivalent changes and improvements that do not deviate from its basic concept. In summary, the protection scope of the present invention should be based on the statements in the claims, and the above embodiments are only used as a reference for understanding the present invention.

Claims

1. A method for creating homozygous tetraploid rice using haploid anthers, characterized in that: The following steps are involved: a. Select conventional diploid rice lines with stable traits and a fruit setting rate of ≥95% as parent materials; b. Select anthers with microspores in the uninucleate marginal stage and determine their developmental period by KI solution staining; c. The anthers were inoculated into callus induction medium for dark culture to induce callus formation; d. The callus was treated with colchicine twice and recovered by culture, wherein the two treatments included: i. Cultivate in liquid medium containing colchicine for 24-48 hours; ii. Transfer to recovery medium for culture; e. The callus tissue after the two treatments was inoculated into a differentiation medium for light culture until the sprouts were differentiated; f. transferring the seedlings into a rooting medium to induce rooting and obtain complete plants; g. The plant was verified by cytological technology to be a homozygous tetraploid rice with a chromosome number of 2n=4x=48.

2. The method according to claim 1, characterized in that The parent material in step a is the diploid rice line Balilla-2x.

3. The method according to claim 1, characterized in that The concentration of the KI solution in step b is 0.3%-0.5%.

4. The method according to claim 1, characterized in that: The components of the callus induction medium in step c are: N6 basal medium, 2,4-D 1.5 mg / L, NAA 1 mg / L, 6-BA 0.5 mg / L, sucrose 4%, agar 0.75%, pH 6.

0.

5. The method according to claim 1, characterized in that The components of the liquid culture medium in step d are: N6 basal medium, 2,4-D 1.5 mg / L, NAA 1 mg / L, 6-BA 0.5 mg / L, colchicine 0.5 g / L, sucrose 4%, pH 6.

0.

6. The method according to claim 1, characterized in that The components of the recovery medium in step d are: N6 basal medium, sucrose 2.5%, agar 0.75%, 2,4-D 0.5 mg / L, NAA 0.8 mg / L, 6-BA 0.5 mg / L, pH 6.

0.

7. The method according to claim 1, characterized in that The components of the differentiation medium in step e are: N6 basal medium, sucrose 30 g / L, hydrolyzed casein 300 mg / L, glutamine 500 mg / L, proline 500 mg / L, D-sorbitol 15 g / L, anhydrous CuSO4 2.5 mg / L, NAA 0.2 mg / L, 6-BA 2 mg / L, agar 0.75%, pH 6.

0.

8. The method according to claim 1, characterized in that The components of the rooting medium in step f are: 1 / 2 MS medium, NAA 0.3 mg / L, 6-BA 0.5 mg / L, hydrolyzed casein 200 mg / L, sucrose 2%, agar 0.75%, activated carbon 0.02%, pH 6.

0.

9. The method according to claim 1, characterized in that: The cytological technique described in step g is root tip chromosome preparation and observation or flow cytometer detection of DNA content.

10. The method according to claim 1, characterized in that The tetraploid rice has a chromosome number of 2n=4x=48, and its single plant yield, thousand-grain weight and nutritional quality are significantly higher than those of its parent diploid rice.

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