CN106900536A - Photo-thermo sensitive male nuclear sterile rice line orients transformation into the method for protection cytoplasmic male sterile line - Google Patents

Abstract

The present application provides a method for directional transformation of a rice photothermosensitive genic male sterile line into a broad-preserving cytoplasmic male sterile line, which includes: using the rice photothermosensitive genic male sterile line as the backcross female parent, cultivating rice as the male parent, Through hybridization, backcrossing and selfing, the same-type fertile line of the rice photothermosensitive genic male sterile line is selected and bred; then the same type fertile line of the rice photothermosensitive genic male sterile line is used as the backcross male parent, and the rice The wide-preserving cytoplasmic male sterile line is the female parent. Through hybridization and backcrossing, a new rice line with changed genetic properties is obtained, that is, the newly bred wide-preserving cytoplasmic male sterile line, which retains the original light and temperature sensitivity to the greatest extent. The excellent genetic background, comprehensive traits and breeding potential of the nuclear male sterile line, the pollen sterility is high, the sterility is stable, and it is not disturbed by the natural light length and temperature changes. It still guarantees the hybrid rice in complex and changeable climate conditions. Production of seeds of high purity.

Description

Method for Directed Transformation of Rice Photothermosensitive Genic Male Sterile Line into Widely Preserved Cytoplasmic Male Sterile Line

technical field

The application belongs to the technical field of hybrid rice breeding, and in particular relates to a method for directional transformation of a rice photothermosensitive nuclear sterile line into a cytoplasmic male sterile line of wide-protected type (CMS-FA).

Background technique

In 1973, Hubei Shimingsong discovered a male sterile plant in the japonica rice Nongken 58 population. The plant was male sterile when heading in summer, and male fertile when heading in autumn. The relationship between pollen fertility and temperature and light was studied. There is a close relationship between length and length, that is, heading under the climate of high temperature and long sunshine in summer is male sterile, while heading under the climate of low temperature and short sunshine in autumn is male fertile. Genetic sterile rice.

Since Shi Mingsong (1981) proposed the concept of rice natural dual-purpose lines, many dual-purpose rice photothermo-sensitive genic male sterile lines have been selected and bred, and the combination of two-line hybrid rice combinations has passed the examination and promotion, opening up rice heterosis. It has gradually developed into an important type of hybrid rice breeding technology, and has made great contributions to my country's hybrid rice technology innovation and continued to maintain the world's leading level.

The breeding research of rice photothermosensitive genetic male sterile line is the core technology in this field. The representative literatures related to the research and discussion of rice photothermosensitive genetic male sterile line breeding technology are as follows:

(1) "Super Hybrid Rice Research", edited by Yuan Longping, 2006, Shanghai Science and Technology Press, Shanghai.

(2) "Hybrid Rice Science", edited by Yuan Longping, 2002, China Agricultural Press, Beijing.

(3) "Theory and Technology of Two-line Hybrid Rice", edited by Chen Liyun, 2001, Shanghai Science and Technology Press, Shanghai.

(4) "Rice Male Sterility Biology", edited by Zhu Yingying, 2000, Wuhan University Press, Wuhan.

(5) "Heterosis and Hybrid Rice Breeding" Yang Rencui, translated by Chen Shunhui, 1996, Fujian Science and Technology Press, Fuzhou (Heterosis and Hybrid Rice Breeding S.S.Virmani, Springer-Verlay Berlin Heidelberg Publishing House, 1994, Germany) .

(6) "Rice Breeding", edited by Min Shaokai, Shen Zongtan, Xiong Zhenmin, 1996, China Agricultural Press, Beijing.

(7) "Research on the Mechanism and Application of Fertility Transformation of Photosensitive GMS Line Rice", edited by Li Zebing, 1995, Hubei Science and Technology Press, Wuhan.

(8) "Research and Application of Two-line Hybrid Rice", edited by He Haohua, Yuan Shengchao, 1993, Jiangxi Science and Technology Press, Nanchang.

(9) "Research and Utilization of Photoperiod Sensitive Genic Male Sterile Rice", edited by Zhu Yingying and Yang Daichang, 1992, Wuhan University Press, Wuhan.

(10) "Chinese Rice Varieties and Their Genealogy", edited by Lin Shicheng and Min Shaokai, 1991, Shanghai Science and Technology Press, Shanghai.

(11) "Discovery and Preliminary Study of Recessive Male Sterile Rice Sensitive to Light Length", Shi Mingsong, 1985, "Chinese Agricultural Science" (2): 44-48.

(12) "Research and Practice of Hybrid Rice", edited by Li Zebing et al., 1982, Shanghai Science and Technology Press, Shanghai.

1. Advantages and achievements of rice photothermosensitive male sterile line and two-line hybrid rice breeding technology

Breeding two-line hybrid rice by using photothermosensitive male sterile line has many advantages in breeding technology: First, no maintainer line is needed for photothermosensitive male sterile line breeding, as long as it is planted in the rice-growing area in autumn or spring heading in Hainan , temperature and light can meet the conditions for inducing pollen to become fertile, realize the purpose of self-propagating sterile lines, and reduce the link of propagating sterile lines that are planted and pollinated at intervals with maintainer lines (such as the three-line wild type) in production. French CMS), which can reduce production costs and improve economic benefits; the second is that the light-temperature-sensitive CMS can be combined freely, and existing studies have shown that 98% of common rice cultivars can restore the pollen fertility of dual-purpose CMS , the probability of matching high-quality hybrid rice is greatly improved. However, only about 20% of wild-type cytoplasmic male sterile lines can be used as restorer lines. In contrast, the utilization rate of restorer lines of two-line hybrid rice is much higher than that of wild-type cytoplasmic hybrid rice; Third, it is generally believed in the academic circles that photothermosensitivity sterility is inherited by nuclear genes and has nothing to do with cytoplasm, which can overcome the negative effect of cytoplasm and the potential danger of single cytoplasm in wild-type three-line male sterile lines; The fourth is to be able to follow and utilize the progress of conventional rice breeding. In addition to rapidly improving rice quality, it can also take advantage of the heterosis between indica and japonica subspecies in order to achieve the breeding goal of higher yield levels of hybrid rice.

Since the research on the characteristics of Nongken 58s photothermo-sensitive sterility and the development of dual-purpose genetic male sterile line and two-line hybrid rice breeding, so far, the National Rice Data Center (http://www.ricedata.cn/) has listed More than 70 (articles) of light-temperature-sensitive sterile lines have been identified at or above the provincial level, and more than 19 (articles) have passed the identification at or above the provincial level and obtained variety rights authorization. According to the National Rice Data Center, as of 2013, there were as many as 606 (articles) of two-line hybrid rice combinations approved at or above the provincial level. According to statistics in 2008, the planting area of two-line hybrid rice in my country accounts for about 25% of the total planting area of hybrid rice, and the planting area of two-line hybrid rice in Anhui, Hunan, Hubei and other provinces has exceeded that of three-line hybrid rice. From 1993 to 2009, the cumulative promotion area reached 315 million mu. Two-line hybrid rice has become an important type of rice production and application in my country, and has played an important role in ensuring food security and harvest.

The two-line hybrid rice breeding technology has also been extended to India, the United States, Vietnam, the Philippines, Japan and other countries, and is gradually going to the world.

2. Production risk of photothermosensitive male sterile line and two-line hybrid rice

While great achievements have been made in the breeding and popularization of rice photothermo-sensitive male sterile lines and two-line hybrid rice, the potential risks are unavoidable and increasingly prominent.

So far, scientists have designed photothermosensitive male sterile lines of rice to follow the changes of natural light and temperature to regulate pollen fertility. Seed production: Under short-day low-temperature conditions in autumn, the pollen of the photothermosensitive male sterile line is induced to be fertile, and the male sterile line is propagated.

However, due to the complex and changeable climate under natural conditions, unpredictable and uncontrollable low temperature and cold waves in summer often induce the pollen conversion of some photothermosensitive male sterile lines of rice into fertile or semi-fertile. Fertile pollen still produces light-temperature-sensitive sterile lines through self-pollination, which are mixed in hybrid rice seeds in the seed production field, resulting in a significant drop in the purity of hybrid rice seeds, which cannot meet the standards of commercial seeds and cause huge losses.

Due to uncertain climate change, the failure of seed production or the phenomenon of seed purity not up to standard often occurs. In addition to being affected by external climate change, the genetic drift phenomenon of the sterile line itself can also lead to unstable fertility. Since the genetic background of the sterile line is controlled by multiple genes, as the number of generations increases, its fertility transition temperature It will drift upwards, and the fertility will fluctuate.

To sum up, while the rice photothermosensitive male sterile line has made great achievements in breeding applications, there are huge potential risks in production. So far, the level of human science and technology is still unable to effectively predict the low temperature and cold wave in summer. Once the low temperature and cold wave in summer come, the existing production technology level and ability of human beings to resist and control the low temperature and cold wave in summer are very limited.

Therefore, for a long time, from the perspective of existing research ideas and production applications, it has been difficult to resolve the problem of low temperature in summer and cold waves attacking the pollen fertility of the sterile line in the two-line hybrid rice seed production field, and the potential risks have always threatened the two-line hybrid rice. Seed production.

Contents of the invention

In one aspect, the present invention provides a method for directional transformation of rice photothermosensitive genetic male sterile lines into broad-preserving cytoplasmic male sterile lines.

The method for directional transfection of rice photothermosensitive genic male sterile line into wide-preserving cytoplasmic male sterile line provided by the present invention comprises the following steps: using rice photothermosensitive genic male sterile line as backcross female parent, cultivating rice as male parent, Through hybridization, backcrossing and self-crossing, a homotype fertile line of rice photothermosensitive genetic male sterile line is obtained through selection.

On the other hand, the method for directional transformation of rice photothermosensitive genic male sterile line into wide-preserving cytoplasmic male sterile line provided by the present invention comprises the following steps: using rice photothermosensitive genic male sterile line as backcross female parent, cultivating rice As the male parent, through hybridization, backcrossing and selfing, selection and breeding to obtain the same-type fertile line of the rice photothermosensitive genic male sterile line; The male parent, the rice wide-protected cytoplasmic male sterile line is the female parent, and through crossing and backcrossing, a new rice line with changed genetic properties is obtained, that is, the newly bred wide-protected cytoplasmic male sterile line.

In one embodiment, the genetic attribute change includes rice sterility being insensitive to light and temperature.

In another embodiment, the step of selecting and obtaining the same-type fertile line of rice photothermosensitive genic male sterile line includes 2-8 times of backcrossing and 3-5 times of selfing, preferably 4-7 times of backcrossing and 4 times of Selfing, more preferably 6 backcrossings and 4 selfings.

In yet another embodiment, wherein the step of breeding a new rice line with altered genetic properties includes 2-8 backcrosses, preferably 4-7 backcrosses, more preferably 6 backcrosses.

In yet another embodiment, the rice photothermosensitive genic male sterile line is: long-light high-temperature sterile genic male sterile line, high-temperature sterile genic male sterile line, short-light low-temperature sterile genic male sterile line, Or low-temperature sterile nuclear sterile line. In an optional embodiment, the photothermosensitive genic male sterile line of rice is Guangzhan 63-4s, Fan 8s or Shuang8s, etc.; and/or the rice Guangbao cytoplasmic male sterile line is Xinyuan 5A , Jinnong 1A, Jinnong 2A or Jinnong 3A, etc.

In a specific embodiment, the method for directed transformation of rice photothermosensitive male sterile line into Guangzhan cytoplasmic male sterile line provided by the present invention comprises the following steps: 1) Using rice photothermosensitive male sterile line Guangzhan 63- 4s is the female parent, and it is crossed with the cultivated rice Zhe 987 as the male parent to obtain a hybrid; 2) The rice photothermosensitive male sterile line Guangzhan 63-4s is used as the backcross female parent, and it is used as the male parent Step 1) backcross the obtained hybrid, and continue to backcross about 6 times to obtain a backcross; and 3) carry out 3-5 self-crosses to the backcross obtained in step 2), and obtain rice by selection The isotype fertile lines of photothermosensitive genic male sterile lines widely accounted for 63-4B.

In another specific embodiment, the method for directional transformation of rice photothermosensitive genic male sterile line into Guangzhan cytoplasmic male sterile line provided by the present invention comprises the following steps: 1) Using rice photothermosensitive male sterile line Guangzhan 63 -4s is the female parent, and it is crossed with the cultivated rice Zhe 987 as the male parent to obtain hybrids; In the present step 1) the hybrid species obtained is backcrossed, and the backcrossed species is continued for about 6 times to obtain the backcrossed species; and 3) the backcrossed species obtained in step 2) is self-crossed 3-5 times to obtain The isotype fertile line Guangzhan 63-4B of the rice photothermosensitive genic male sterile line; 4) The isotype fertile line Guangzhan 63-4B was used as the male parent, and it was used as the female parent of the rice Guangzhan type cytoplasmic male sterile line Line Xinyuan 5A for hybridization to obtain hybrids; and 5) use the same type of fertile line Guangzhan 63-4B as the backcross male parent, backcross it with the hybrid obtained in step 4) as the female parent, and continue Six times of backcrossing were carried out to obtain a new rice line with changed genetic properties, namely the newly bred Guangbao-type cytoplasmic male sterile line Guangzhan 63-4A.

In the above embodiment, the photothermosensitive genic male sterile line of rice, the homofertile line of the photothermosensitive genic male sterile line of rice, or the new rice line with changed genetic properties is selected from rice, upland rice, indica rice , japonica rice, early rice, late rice, sticky rice, glutinous rice. Optionally, the rice photothermosensitive genic male sterile line, the isotype fertile line of the rice photothermosensitive genic male sterile line, or the new rice line with changed genetic properties are selected from rice with hairy leaves and rice Rice glabrous.

It can be seen that the method for the directional transformation of the rice photothermosensitive male sterile line into a broad-preserving cytoplasmic male sterile line provided by this application has at least one of the following advantages: the comprehensive genetic background of the rice photothermosensitive male sterile line remains unchanged, Change the sterility genetic attributes of the sterile line, and breed it into a new line that is not disturbed by the length of light and the change of temperature, has high pollen sterility, and/or stable sterility, that is, the newly bred Guangbao type (CMS-FA) cytoplasmic male sterile line or new plasma source (CMS-FA) sterile line. Therefore, in hybrid rice seed production, using this new male sterile line method, even in the face of complicated and changeable low temperature and cold wave climate conditions, it is still possible to produce hybrid rice that maintains a high degree of male sterility and/or high purity Seeds that meet high-purity seed quality standards.

detailed description

As used herein, "rice" is any rice plant and includes all plant varieties that can be bred with rice. As used herein, "plant" or "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant bushes, and plants or plant parts Intact plant cells, such as plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. Rice as used herein includes paddy rice, upland rice, indica rice, japonica rice, early rice, late rice, glutinous rice, glutinous rice, rice with hairy leaves and rice with glabrous leaves (Oryza sativa).

As used herein, "rice photothermo-sensitive genic male sterile line" includes long-light high-temperature sterile type, high-temperature sterile type, short-light low-temperature sterile type, low-temperature sterile type genic male sterile line, etc., for example, Guangzhan 63- 4s, traditional 8s or double 8s etc.

As used in the symbols used in the breeding methods herein, "F" means cross; "B" means backcross; "n" means number of backcross generations.

As used herein, "high male sterility" refers to the pollen morphology and staining degree observed under a 100× optical microscope after mature rice pollen is stained with 1% iodine-potassium iodide (I ₂ -KI) solution, wherein the sterile pollen reaches More than 99.9% of the samples are said to be highly male sterile.

As used herein, "high-purity hybrid rice seeds" means that the purity of qualified commercial hybrid rice seeds must reach more than 96%.

As used herein, a "sterile line" is also known as a "male sterile line" or "cytoplasmic male sterile line", ie a rice line that is sterile in pollen and can transmit this characteristic to the offspring.

As used herein, "the same-type fertile line of the rice photothermosensitive genic male sterile line" refers to a rice photothermosensitive male sterile line with similar comprehensive genetic background, similar agronomic traits, and similar breeding potential, but pollen and spikelet selfing A completely fertile new line, because this strain can be further cultivated into a new plasmogen (CMS-FA) sterile line after being transferred into the cytoplasm of the new plasmogen through backcrossing, it can be called the isotype maintainer of the new plasmogen or The B line, which has just been bred, is called the new B line.

As used herein, "new quality source (CMS-FA) male sterile line", "new quality source (CMS-FA) male sterile line", "Guangbao type (CMS-FA) cytoplasmic male sterile line", " "Guangbao type (CMS-FA) male sterile line", or "Guangbao type (CMS-FA) male sterile line" can be used interchangeably, both refer to the successful research and development from Fujian wild rice (O.rufipogon) and contain Fujian wild rice The male sterile line of rice male sterile cytoplasm, that is, the sterile line with a specific CMS-FA cytoplasmic background, is referred to as the new quality source CMS-FA male sterile line or the new quality source sterile line. Because the resource utilization of the maintainer line of the cytoplasm reaches 55.5%, which is more extensive than that of the wild type (CMS-WA) maintainer line resource utilization of 20%, it is also called the broad-preservation cytoplasmic male sterile line, and the wide-preservation type male sterile line Line or wide-protected sterile line, for example, please refer to the Chinese invention patent document CN1954666B.

Using the method provided in this application, the rice photothermosensitive genic male sterile line was directionally bred into a wide-protected type (CMS-FA) cytoplasmic male sterile line. The isotype fertile line of the nuclear sterile line is similar, but the pollen and spikelets are completely sterile, also known as the new A line, or the newly bred new plasmogenic (CMS-FA) male sterile line.

The directional transfer method of rice photothermosensitive male sterile line provided by this application follows the following two basic principles: one is to basically not change the excellent genetic background, comprehensive agronomic traits and breeding of rice photothermosensitive male sterile line germplasm potential, and continue to play a huge role in hybrid rice breeding with the excellent rice photo-temperature-sensitive male sterile line germplasm resources accumulated over a long period of breeding; the second is to use innovative genetic breeding methods to change the sterile genetic properties of the sterile line, and to transform rice The characteristics of the photothermosensitive male sterile line, which is sensitive to environmental changes such as the length of light and temperature, have changed into a genetically controlled CMS-FA nuclear-plasma interaction that is insensitive to environmental changes such as light length and temperature. Male sterility, which stabilizes pollen sterility.

In this way, in the application of hybrid rice seed production, once encountering low temperature and cold wave in summer, the pollen fertility of the sterile line will still maintain a high degree of male sterility, and there will be no change in pollen fertility, ensuring the production of high-purity hybrid rice seeds , and fundamentally resolve this long-standing and far-reaching scientific and production problem through technological innovation.

It can be seen that this application utilizes the technical advantages of the new quality source (CMS-FA) hybrid rice technology platform, keeps the comprehensive genetic background of the rice photothermosensitive male sterile line unchanged, changes the sterile genetic attributes of the male sterile line, and breeds it A new quality source (CMS-FA) male sterile line with high degree of pollen sterility and stable sterility was developed without being disturbed by the length of light and the change of temperature. This new type of sterile line is used in the production of hybrid rice seeds. Even in the face of complex and changeable low temperature and cold wave climate conditions, it can still maintain a high degree of male sterility, produce high-purity hybrid rice seeds, and achieve high-purity seed quality. Standards, fundamentally solve the problem of low safety and purity of rice photothermosensitive male sterile line seed production in my country.

Specifically, in one embodiment, the application provides a breeding method for directional transformation of rice photothermosensitive genetic male sterile lines into wide-protected type (CMS-FA) male sterile lines, and the main technical area is two steps:

The first step is to transform the rice photothermosensitive male sterile line into a fertile line with a similar comprehensive genetic background, similar agronomic traits, and similar breeding potential to the rice photothermosensitive male sterile line, but the pollen and spikelet selfing are completely normal, Or called the B series.

The rice light-temperature-sensitive male sterile line is used as the female parent, and the conventional rice variety is used as the male parent, and they are crossed with each other to obtain hybrid seeds. The hybrid seeds are planted into hybrid F1 plants, and after earing, the rice light-temperature-sensitive male sterile line is used as the female parent, and the F1 is used as the male parent to backcross to obtain the backcross seed B1F0. Plant the backcross seeds into B1F1 plants. After earing, continue to use the rice photothermosensitive male sterile line as the female parent and B1F1 as the male parent. Continue to backcross 2-7 times in this way to obtain BnF0 backcross seeds. Plant BnF1 seeds, allow them to self-fertilize after heading, and harvest about 2000 self-fertilized seeds of fully fertile plants with pollen and spikelets. About 1,000 BnF2 plants were planted, and allowed to self-fertilize after earing. After maturity, plants with completely normal pollen and spikelet fertility were selected, and 50 self-fertilized seeds were harvested and stored in individual plants. 50 strains were planted into the BnF3 generation, and 100 strains were planted for each strain. Let them self-cross after earing, and select all plants in the plot when they are mature. The pollen and spikelets of all plants are completely normal and fertile, and no sterile plants are separated. The male sterile lines are similar, that is, they are the isotype normal fertile lines of rice photothermosensitive male sterile lines. Because this line can be further bred into a new quality source (CMS-FA) sterile line, it can be called the isotype maintainer line of the new quality source, or called the B line or the new B line.

In the second step, the CMS-FA male sterile line is used as the female parent, and the new B line bred by the above method is used as the male parent, and they are crossed with each other to obtain hybrid seeds. Plant F1 plants, use F1 as the female parent after heading, and backcross the new B line as the male parent to obtain backcross seeds. Plant into B1F1 plants, use the B1F1 plants as the female parent after heading, and use the new B line as the male parent to continue backcrossing. Continuously backcross 2-7 times in this way to obtain BnF1 plants, which are called the new A line. The plant traits of the new A line are neat and stable, the comprehensive genetic background is similar to the new B line, and the comprehensive agronomic traits are similar to the new B line, but the pollen and spikelets are completely sterile. The new A line is the newly bred new quality source (CMS -FA) male sterile line, that is, a wide-protection type (CMS-FA) male sterile line that is directedly transformed from a rice photothermosensitive genic male sterile line.

The Guangbao-type (CMS-FA) sterile line used in this paper refers to a new type of male sterile cytoplasmic male sterile line of Fujian wild rice (O. The pistil of the male sterile line develops normally, but the development of the stamen is degenerated or aborted, and it cannot self-pollination and fruit.

The CMS-FA sterile maintainer line used in this paper refers to the new cytoplasmic male sterile maintainer line of Fujian wild rice (O. rufipogon), referred to as the maintainer line. The stamens and stamens of the maintainer line develop normally, and the pollen is given to the pistils of the male sterile line, not only can mature and healthy seeds be obtained, but also the grown plants after sowing are still male sterile plants.

The CMS-FA sterile restorer line used in this paper refers to the Fujian wild rice (O. rufipogon) cytoplasmic male sterile restorer line, referred to as the restorer line. The pollen of the restorer line is given to the pistil of the sterile line, and after the produced seeds are sown, the grown plants regain male (pollen) and normal fertility.

Among the symbols used in the breeding method for the directional transformation of the above-mentioned rice photothermosensitive genic male sterile line into the CMS-FA male sterile line, F represents hybridization; B represents backcrossing; n represents the number of backcrossing generations.

The rice referred to in the breeding method of the above-mentioned rice photothermosensitive genital male sterile line directedly transformed into a broad-spectrum (CMS-FA) male sterile line includes paddy rice, upland rice, indica rice, japonica rice, early rice, late rice, sticky rice, glutinous rice, rice Rice with hairy leaves and rice with glabrous leaves (Naked rice) rice.

The rice photothermosensitive genic male sterile line referred to in the breeding method of the above-mentioned rice photothermosensitive genic male sterile line being directedly transformed into a broad-spectrum type (CMS-FA) male sterile line includes long-light high-temperature sterile genic male sterile line, High-temperature sterile GMS line, short-light low-temperature sterile GMS line, low-temperature sterile GMS line.

The photothermosensitive genic male sterile line referred to in the breeding method of the above-mentioned rice photothermosensitive genic male sterile line being directedly transformed into a broad-type (CMS-FA) male sterile line includes rice, upland rice, indica rice, japonica rice, early rice, late rice, Sticky rice, glutinous rice, rice with hairy rice leaves and rice-leaf glabrous (Naked rice) rice-type photothermosensitive genetic male sterile lines.

Therefore, the rice photothermosensitive genic male sterile line provided by the application is directed to the breeding method of the wide-protected type (CMS-FA) sterile line, and a new product is obtained, that is, the newly bred new quality source (CMS-FA) sterile line. The technical features of the new A series (referred to as the new A series, hereinafter the same) are prominently manifested in at least one of the following aspects:

(1) The newly bred new quality source male sterile line relatively completely retains the good genetic background, comprehensive agronomic traits and comprehensive breeding potential of the original photothermosensitive genic male sterile line: the new A line adopts the photothermosensitive genic male sterile line of rice as the The reincarnated parent is bred through 2-7 crosses. Therefore, the new A line relatively completely retains the comprehensive genetic background, comprehensive agronomic traits and comprehensive breeding potential of the original photothermosensitive GMS line.

(2) The pollen sterility of the new A line is jointly controlled by the interaction between the nucleus and the cytoplasm: the new A line has the cytoplasmic background of the new quality source (CMS-FA) male sterility, and is the new type of source (CMS-FA) male sterile The result of the interaction between the cytoplasmic fertility gene and the nuclear fertility gene of the new B lineage, the inheritance and expression of male sterility traits are completely controlled by the nuclear-cytoplasmic interaction. The pollen sterility of the new A line has nothing to do with the natural sunlight and temperature.

(3) The ecological reflection and expression mechanism of male sterility in the new A line is stable: Compared with the parent of the original photothermosensitive genic male sterile line, the new A line also shows pollen sterility, but the ecological reflection and expression of pollen fertility Mechanisms have produced changes of a completely different nature. The pollen sterility of the new A line will not change with the change of natural light and temperature, but will always maintain a high degree of stable male sterility under any climatic conditions.

(4) The production and application system of the new A line is mature and stable: The new A line and the newly bred new quality source maintenance line or the new B line () are planted at intervals. The solid seeds on the plants are still the new A line, and this method is used to propagate the new A line; the new A line and the male sterile restorer line of the new quality source (CMS-FA) system are planted at intervals, and the pollen of the restorer plants is transferred to the new A line , the seeds that are solid on the new A line plants are the hybrid rice seeds. The production and application methods of the new A-series male sterile line are similar to those of the wild aborted male sterile line. Therefore, the production and application system of the new A line can be mature and stable with the help of the traditional wild-type three-line hybrid rice production technology system, providing a solid new technology guarantee for hybrid rice seed production.

The following examples are for the purpose of illustration only and not limiting the scope of the application.

The rice plant material information used in this application can be found in the Chinese rice variety and its pedigree database (http://www.ricedata.cn/variety/index.htm).

Example 1 Orientation of Guangzhan 63-4s to Guangzhan 63-4A

In this example, taking the photothermosensitive GMMS line Guangzhan 63-4s as an example, the method of directing it into a new plasma-derived male sterile line Guangzhan 63-4A is explained in detail as follows.

The first step is the breeding of the isotype fertile line of the photothermosensitive genic male sterile line of rice.

Guangzhan 63-4s was used as the female parent and the early indica variety Zhe 987 was used as the male parent to cross each other to obtain hybrid seeds. Hybrid seeds were planted to obtain F1 generation plants.

Guangzhan 63-4s was used as the female parent, and backcrossed with the F1 generation plant as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B1F1 generation plants.

Guangzhan 63-4s was used as the female parent, and the B1F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B2F1 generation plants.

Guangzhan 63-4s was used as the female parent, and the B2F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B3F1 generation plants.

Guangzhan 63-4s was used as the female parent, and the B3F1 generation plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B4F1 generation plants.

Guangzhan 63-4s was used as the female parent, and the B4F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B5F1 generation plants.

Guangzhan 63-4s was used as the female parent, and the B5F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B6F1 generation plants.

After the B6F1 generation plants were eared, they were allowed to self-cross, and 2000 self-seeds of the fully fertile plants with pollen and spikelets were harvested. Plant selfed seeds to obtain 1000 B6F2 generation plants.

After the B6F2 generation plants were eared, they were allowed to self-fertilize. After maturity, plants with completely normal pollen and spikelet fertility were selected, and 50 self-fertilized seeds were harvested from individual plants. Plant selfing seeds, obtain 50 strains of B6F3 generation plants, and plant 100 strains for each strain.

After the B6F3 generation plants are eared, let them self-cross, and when they mature, select the pollen and spikelets of all plants in the plot that are completely normal and fertile, no sterile plants are separated, and the comprehensive genetic background and comprehensive agronomic characteristics are similar to those of rice photothermosensitive male sterile lines. The strain B6F3-1, which is the isotype fertile line of rice photothermosensitive genic male sterile line, was named Guangzhan 63-4B line.

The second step is the selection of Guangbao type (CMS-FA) cytoplasmic male sterile line.

The CMS-FA male sterile line Xinyuan 5A was used as the female parent and the Guangzhan 63-4B line was used as the male parent to cross each other to obtain hybrid seeds. Hybrid seeds were planted to obtain F1 generation plants.

The F1 generation plants were used as the female parent and backcrossed with Guangzhan 63-4B line as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B1F1 plants.

The B1F1 generation plant was used as the female parent to backcross with the Guangzhan 63-4B line as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B2F1 plants.

The B2F1 generation plants were used as the female parent and backcrossed with Guangzhan 63-4B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B3F1 plants.

The B3F1 generation plant was used as the female parent to backcross with the Guangzhan 63-4B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B4F1 plants.

The B4F1 generation plant was used as the female parent to backcross with the Guangzhan 63-4B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B5F1 plants.

The B5F1 generation plants were used as the female parent and backcrossed with Guangzhan 63-4B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B6F1 plants, which were named Guangzhan 63-4A line.

The plant traits of the Guangzhan 63-4A line are neat and stable. The comprehensive genetic background is similar to that of the Guangzhan 63-4B line. The comprehensive agronomic traits are similar to those of the Guangzhan 63-4B line. The 4A line is the newly bred new quality source (CMS-FA) male sterile line, that is, the new line of rice photothermosensitive genic male sterile line, that is, the Guangbao type (CMS-FA) cytoplasmic male sterile line Tie.

The main technical indicators of Guangzhan 63-4A line are as follows: 85 days from sowing to the first ear, which is equivalent to the growth period of Guangzhan 63-4s sown and transplanted at the same time; Education is the main thing. The sterile plant rate of more than 1,000 plants is 100%, the pollen sterility rate is 99.98%, and the bagging self-seed rate is 0%.

The quality test results of Guangzhan 63-4A rice are: brown rice rate 80.9%, polished rice rate 72.4%, whole rice rate 60.3%, grain length 6.3 mm, aspect ratio 2.9, chalky grain rate 14.2%, chalkiness 3.0%, The transparency is grade 1, the alkali digestion value is 6.0, the gel consistency is 87.3 mm, and the amylose content is 15.7%. The rice quality generally reaches the national high-quality grade 3 rice and the new ministerial grade high-quality rice 2.

Example 2 The photothermosensitive genic male sterile line Guangzhan 63-4s was directional transformed into Pursuit A

In this example, the rice photothermosensitive male sterile line Guangzhan 63-4s was directional transformed into a new quality source (CMS-FA) male sterile line Pursuit A, where Quest A is equivalent to Guangzhan 63 in Example 1 -4A, Pursuit B is equivalent to Guangzhan 63-4B in Example 1, except that the number of backcrosses is different from Example 1, and the breeding direction and results are consistent.

1. Guangzhan 63-4s directional transfer into Pursuit A:

Guangzhan 63-4s was crossed with Zhe 987, and Guangzhan 63-4s was used as the recurrent parent for multiple backcrosses and self-crosses. Introduce the cytoplasm of the CMS-FA CMS line Xinyuan 5A, and breed the CMS-FA CMS line Quest A with neat and stable traits, high sterility, high outcrossing rate, and excellent rice quality .

The specific process is shown in Table 1, in which the two sets of work of male parent (maintainer) selection and female parent (sterile line) selection are combined. See the fifth column for male parent selection and the second column for female parent selection. List.

Table 1 Breeding process for pursuit of A

Note: "/" or "×" means crossing or backcrossing; "/value/" means the number of crossing or backcrossing; F means crossing; B means backcrossing; n means the number of backcrossing generations.

2. Comparison of botanical characteristics between Pursuit A and Guangzhan 63-4s:

Compared with Guangzhan 63-4s, Pursuit A is basically similar in terms of plant height, panicle length, number of grains per panicle, 1000-grain weight, sowing-first panicle, rice quality and other botanical characteristics (Table 2).

Table 2 Comparison of botanical characteristics between Qiuqiu A and Guangzhan 63-4s

3. Comparison of biological characteristics between Pursuit A and Guangzhan 63-4s:

Compared with the control Guangzhan 63-4s, Pursuit A had obvious differences in biological characteristics such as male sterile line type, male sterile stability, need for maintainer line, and source of restorer line (Table 3).

Table 3 Comparison of biological characteristics between Pursuit A and Guangzhan 63-4s

Example 3 The photothermosensitive genetic male sterile line Fan 8s was directed to be bred into Fan 8A

In this example, taking the photothermosensitive GMMS line Fan 8s as an example, the method for directing it to the new plasma-derived male sterile line Fan 8A is specifically illustrated, as follows.

The first step is the breeding of the isotype fertile line of the photothermosensitive genic male sterile line of rice.

Using Fan 8s as the female parent and the early indica variety Zhe 987 as the male parent, they crossed each other to obtain hybrid seeds. Hybrid seeds were planted to obtain F1 generation plants.

Taking Fan 8s as the female parent, backcross with the F1 generation plant as the male parent to obtain backcross seeds. Plant backcross seeds to obtain B1F1 generation plants.

Taking Fan 8s as the female parent, backcross with the B1F1 plant as the male parent to obtain backcross seeds. Plant backcross seeds to obtain B2F1 generation plants.

Taking Fan 8s as the female parent, backcross with the B2F1 plant as the male parent to obtain backcross seeds. Plant backcross seeds to obtain B3F1 generation plants.

With Fan 8s as the female parent, backcross with the B3F1 generation plant as the male parent to obtain backcross seeds. Plant backcross seeds to obtain B4F1 generation plants.

Using Fan 8s as the female parent, backcross with the B4F1 plant as the male parent to obtain backcross seeds. Plant backcross seeds to obtain B5F1 generation plants.

Using Fan 8s as the female parent, backcross with the B5F1 plant as the male parent to obtain backcross seeds. Plant backcross seeds to obtain B6F1 generation plants.

The female parent of Fan 8s was backcrossed with the B6F1 plant as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B7F1 generation plants.

After the B7F1 generation plants were eared, they were allowed to self-cross, and 2500 self-seeds of fully fertile plants with pollen and spikelets were harvested. Plant selfed seeds and obtain 800 B7F2 generation plants.

After the B7F2 generation plants headed, they were allowed to self-fertilize. After maturity, plants with completely normal pollen and spikelet fertility were selected, and 50 self-fertilized seeds were harvested from individual plants. Plant selfing seeds, obtain 40 strains of B7F3 generation plants, and plant 100 strains for each strain.

After the B7F3 generation plants are eared, let them self-cross, and when they mature, select the pollen and spikelets of all plants in the plot that are completely normal and fertile, no sterile plants are separated, and the comprehensive genetic background and comprehensive agronomic characteristics are similar to those of rice photothermosensitive male sterile lines. The strain B7F3-1, which is the isotype fertile line of rice photothermosensitive male sterile line, was named Fan 8B line.

The second step is the selection of Guangbao type (CMS-FA) cytoplasmic male sterile line.

The CMS-FA male sterile line Xinyuan 5A was used as the female parent, and the Fan 8B line was used as the male parent to cross each other to obtain hybrid seeds. Hybrid seeds were planted to obtain F1 generation plants.

The F1 generation plants were used as the female parent and backcrossed with the Fan 8B line as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B1F1 plants.

The B1F1 generation plant was used as the female parent to backcross with the Fan 8B line as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B2F1 plants.

The B2F1 generation plant was used as the female parent to backcross with the Fan 8B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B3F1 plants.

The B3F1 generation plant was used as the female parent to backcross with the Fan 8B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B4F1 plants.

The B4F1 generation plants were used as the female parent to backcross with the Fan 8B line as the male parent to obtain backcross seeds. The backcross seeds were planted to obtain B5F1 plants, which were named as the Fan 8A line.

The plant traits of the Fan 8A line are neat and stable, the comprehensive genetic background is similar to the Fan 8B line, and the comprehensive agronomic traits are similar to the Fan 8B line, but the pollen and spikelets are completely sterile. The Fan 8A line is the newly bred Guangbao type (CMS -FA) cytoplasmic male sterile line or new cytoplasmic source (CMS-FA) male sterile line.

The main technical indicators of Fan 8A are as follows: 81 days from sowing to the first ear, which is equivalent to the growth period of Fan 8s sown and transplanted at the same time; the pollen fertility of Fan 8A was detected at the first ear stage, and the typical abortion was the main one. The rate of sterile plants in groups of more than 1000 plants is 100%, the degree of pollen sterility is 99.97%, and the rate of bagging self-fertilization is 0%.

The rice quality inspection result of Fan 8A is: the rice quality is average.

Example 4 Directional Transplantation of Photothermosensitive Genic Male Sterile Line Double 8s into Double 8A

In this example, taking the photothermosensitive GMMS line Shuang8s as an example, the method of directing it into a new plasma-derived male sterile line Shuang8A is specifically explained, as follows.

The first step is the breeding of the isotype fertile line of the photothermosensitive genic male sterile line of rice.

Shuang 8s was used as the female parent and the early indica variety Zhe 987 was used as the male parent to cross each other to obtain hybrid seeds. Hybrid seeds were planted to obtain F1 generation plants.

Double 8s was used as the female parent, and the F1 generation plants as the male parent were backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B1F1 generation plants.

Double 8s was used as the female parent, and the B1F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B2F1 generation plants.

Double 8s was used as the female parent, and the B2F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B3F1 generation plants.

Double 8s was used as the female parent, and the B3F1 generation plants as the male parent were backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B4F1 generation plants.

Double 8s was used as the female parent, and the B4F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B5F1 generation plants.

Double 8s was used as the female parent, and the B5F1 plant as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B6F1 generation plants.

After the B6F1 plants headed, they were allowed to self-cross, and 1800 self-seeds of the fully fertile plants with pollen and spikelets were harvested. Plant selfed seeds and obtain 1200 B6F2 generation plants.

After the B6F2 generation plants were eared, they were allowed to self-fertilize. After maturity, plants with completely normal pollen and spikelet fertility were selected, and 50 self-fertilized seeds were harvested from individual plants. Plant selfing seeds, obtain 50 lines of B6F3 generation plants, and plant 80 plants for each line.

After the B6F3 generation plants are eared, let them self-cross, and when they mature, select the pollen and spikelets of all plants in the plot that are completely normal and fertile, no sterile plants are separated, and the comprehensive genetic background and comprehensive agronomic characteristics are similar to those of rice photothermosensitive male sterile lines. The strain B6F3-1 is the isotype fertile line of rice photothermo-sensitive male sterile line, and it is named double 8B line.

The second step is the selection of Guangbao type (CMS-FA) cytoplasmic male sterile line.

The male sterile line Xinyuan 5A of Xinzhiyuan (CMS-FA) was used as the female parent, and the double 8B line was used as the male parent to cross each other to obtain hybrid seeds. Hybrid seeds were planted to obtain F1 generation plants.

The F1 generation plants were used as the female parent and backcrossed with the double 8B line as the male parent to obtain backcrossed seeds. Plant backcross seeds to obtain B1F1 plants.

The B1F1 generation plant was used as the female parent, and the double 8B line as the male parent was backcrossed to obtain backcrossed seeds. Plant backcross seeds to obtain B2F1 plants.

The B2F1 generation plant was used as the female parent, and the double 8B line as the male parent was backcrossed to obtain backcrossed seeds. The backcross seeds were planted to obtain B3F1 plants.

The B3F1 generation plant was used as the female parent, and the double 8B line as the male parent was backcrossed to obtain backcrossed seeds. The backcross seeds were planted to obtain B4F1 plants.

The B4F1 generation plants were used as the female parent, and backcrossed with the double 8B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B5F1 plants.

The B5F1 generation plants were used as the female parent, and backcrossed with the double 8B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B6F1 plants.

The B6F1 generation plants were used as the female parent, and backcrossed with the double 8B line as the male parent to obtain backcrossed seeds. The backcross seeds were planted to obtain B7F1 plants, which were named double 8A lines.

The plant traits of the double 8A line are neat and stable. The comprehensive genetic background and comprehensive agronomic traits are similar to the double 8B line, but the pollen and spikelets are completely sterile. The double 8A line is the newly bred Guangbao type (CMS-FA) cytoplasmic male Male sterile line or new quality source (CMS-FA) male sterile line.

The main technical indicators of double 8A are as follows: 80 days from sowing to the first ear, which is equivalent to the growth period of double 8s sown and transplanted at the same time; the pollen fertility of double 8A is detected at the first ear stage, and the typical abortion is the main one. The sterile plant rate of more than 1,000 plants is 100%, the pollen sterility rate is 99.96%, and the bagging self-seed rate is 0%.

The result of double 8A rice quality test is: the rice quality is average.

Claims (10)

1. photo-thermo sensitive male nuclear sterile rice line orients transformation into the side of protection cytoplasmic male sterile line Method, it is comprised the following steps：

Maternal by backcrossing of photo-thermo sensitive male nuclear sterile rice line, rice-cultivating is male parent, by hybridizing, Backcrossing and selfing, seed selection obtain the homotype fertile line of photo-thermo sensitive male nuclear sterile rice line.

2. the method for claim 1, its is further comprising the steps of：

It is backcrossing male parent with the homotype fertile line of the photo-thermo sensitive male nuclear sterile rice line, paddy rice extensively protects Type cytoplasmic male sterile line is female parent, and by hybridizing and being returned, seed selection obtains genetic property and changes The rice new strain of change, i.e., the new protection cytoplasmic male sterile line being bred as.

3. method as claimed in claim 2, wherein the genetic property changes includes paddy rice Sterility is to illumination and temperature-insensitive.

4. method as claimed any one in claims 1 to 3, wherein the seed selection obtains water The step of homotype fertile line of rice photo-thermo-sensitive nucleic male sterile line, includes 2-8 backcrossing and 3-5 selfing, It is preferred that 4-7 backcrossing and 4 selfings, more preferably 6 times backcrossings and 4 selfings.

5. the method as any one of Claims 1-4, wherein the seed selection is lost The step of rice new strain for passing attribute change, is returned including 2-8 times, preferably 4-7 times backcrossing, More preferably 6 times backcrossings.

6. the method as any one of claim 1 to 5, wherein the rice photo-thermo-sensitive Line with genic sterile is：Light high temperature-induced sterility type line with genic sterile long, high temperature-induced sterility type line with genic sterile, short light Low temperature infertility type line with genic sterile or low temperature infertility type line with genic sterile；Optionally, the paddy rice light Temperature-sensitive sterile line is extensively to account for 63-4s, numerous 8s or double 8s.

7. the method as any one of claim 1 to 6, wherein the paddy rice protection Cytoplasmic male sterile line is new source 5A, peasants who dig gold 1A, peasants who dig gold 2A or peasants who dig gold 3A.

8. the method as any one of claim 1 to 7, wherein methods described include with Lower step：

1) 63-4s is extensively accounted for as maternal with rice photo-thermo-sensitive sterile line, using itself and the cultivation as male parent Training paddy rice Zhejiang 987 is hybridized, and obtains cenospecies；

2) extensively to account for 63-4s with rice photo-thermo-sensitive sterile line maternal as backcrossing, using its with as male parent The step of 1) cenospecies that is obtained is returned, continues to be returned about 6 times, obtains backcrossing kind； And

3) by step 2) the backcrossing kind that is obtained carries out 3-5 selfing, the paddy rice light that seed selection is obtained The homotype fertile line of temperature-sensitive sterile line extensively accounts for 63-4B.

9. method as claimed in claim 8, further comprising the steps of：

4) 63-4B is extensively accounted for as male parent with homotype fertile line, it is extensively protected with as maternal paddy rice The new source 5A of type cytoplasmic male sterile line is hybridized, and obtains cenospecies；And

5) 63-4B as backcrossing male parent is extensively accounted for homotype fertile line, using itself and the step as female parent 4) cenospecies for being obtained is returned, and continues to be returned about 6 times, and seed selection obtains genetic property and changes The rice new strain of change, i.e., the new protection cytoplasmic male sterile line being bred as extensively accounts for 63-4A.

10. method as claimed in any one of claims 1-9 wherein, wherein the paddy rice light temperature Quick line with genic sterile, the homotype fertile line of the photo-thermo sensitive male nuclear sterile rice line or the heredity category The rice new strain for sexually revising is selected from paddy rice, upland rice, long-grained nonglutinous rice, japonica rice, early rice, late rice, viscous Rice, glutinous rice；

Optionally, the photo-thermo sensitive male nuclear sterile rice line, the photo-thermo sensitive male nuclear sterile rice line The rice new strain that homotype fertile line or the genetic property change is selected from rice leaf hairiness rice and rice Leaf is without hair rice.