Breeding and identification method of double-low photo-thermo-sensitive dual-purpose genic male sterile line rice

Abstract

The invention discloses a breeding and identification method of double-low photo-thermo-sensitive dual-purpose genic male sterile line rice, and belongs to the field of rice breeding. The method specifically comprises the following steps: respectively carrying out constant-temperature treatment at 23.0 + / -0.1 DEG C and 19.0 + / -0.1 DEG C for 10 days from a pollen mother cell formation stage to a meiosis stage, taking out a sterile line material after the treatment, and placing the sterile line material under a natural light temperature condition; the identification method of the double-low photo-thermo-sensitive dual-purpose genic male sterile rice comprises the following steps: placing anther on a glass slide during flowering, mashing the anther with tweezers in 0.1% I2-KI liquid, observing pollen fertility under a microscope, and treating at 23.0 DEG C for 10d to obtain pollen dyeable rate lt; the dyeable rate gt of the pollen under the conditions that the pollen is treated at 19.0 DEG C for 10 days is 1%; 70% of the sterile line is a double-low dual-purpose genic sterile line. Hybrid seed production and sterile line seed propagation of the double-low photo-thermo-sensitive dual-purpose genic male sterile line bred by the method are both safe, and sustainable development of two-line method hybrid rice is promoted.

Description

Technical Field

[0001] This invention relates to a method for breeding and identifying a "double-low" photoperiod-temperature-sensitive dual-purpose nuclear male-sterile rice line, belonging to the field of rice breeding. Background Technology

[0002] In 1973, Shi Mingsong discovered a male-sterile plant in the Nongken 58 population of japonica rice. In 1985, he named it the photosensitive nuclear male-sterile line Nongken 58S. This line is sterile under long-day, high-temperature conditions and is used for hybrid seed production; it is fertile under short-day, low-temperature conditions and is used to propagate male-sterile lines, i.e., a dual-purpose nuclear male-sterile line. In 1988, Deng Huafeng et al. from Anjiang Agricultural School in Hunan Province bred the first indica-type dual-purpose nuclear male-sterile line, Anong S-1. Subsequent research showed that Anong S-1 is a high-temperature induced pollen-producing line. For thermosensitive nuclear male-sterile lines that induce pollen fertility at low temperatures, the sensitive period for pollen fertility is from the pollen mother cell to the meiotic division stage. Pollen development in Nongken 58S is also significantly affected by temperature; high temperatures promote pollen sterility, while low temperatures induce pollen fertility. In particular, the pollen fertility of indica male-sterile lines such as W6154S and Pei'ai 64S, bred from Nongken 58S, is mainly regulated by temperature, with the temperature-sensitive period for pollen development also being from the pollen mother cell to the meiotic division stage. Therefore, the key to determining whether dual-purpose nuclear male-sterile lines can be applied in production is maintaining stable sterility in the male-sterile lines during the long, hot summer months to ensure the safety of hybrid seed production. Based on historical temperature records of the middle and lower reaches of the Yangtze River during the summer, the frequency of consecutive 6-day average daily temperatures below 23.5℃ is zero. Therefore, a 6-day constant temperature treatment at 23.5℃ during the pollen mother cell to meiotic division stage is used as the technical standard for stable pollen sterility in practical dual-purpose male-sterile lines. This identification method and technical standard have promoted the development of two-line hybrid rice. However, after years of production application, two-line hybrid rice still faces two major problems: First, the safety of hybrid seed production. Research on high-yield two-line seed production technology shows that when the minimum temperature exceeds 28℃ and the maximum temperature exceeds 35℃, seed production yield is reduced due to high-temperature damage. Rice-growing areas south of the Yangtze River at an altitude of about 300m and areas such as Yancheng north of the Yangtze River are high-yield areas for two-line seed production, but the frequency of low temperatures in these high-yield areas has increased significantly, leading to frequent hybrid seed production failures. The solution is to further lower the sterility threshold temperature of the dual-purpose male-sterile lines and improve the sterility stability of the male-sterile lines, thereby improving the safety of two-line hybrid rice seed production. Second, the propagation of dual-purpose male-sterile lines is unsafe. Rice cultivation requires that the temperature during the reproductive growth period of rice should not be lower than 20℃, meaning that temperatures below 20℃ easily induce low-temperature physiological damage to pollen, resulting in sterility. While the dual-purpose nuclear male-sterile lines 1S and Tanzao S have low sterility initiation temperatures, they are extremely sensitive to temperatures as low as 20°C, resulting in a narrow temperature range for seed propagation, low yields, and high costs. Zhu S is a recently bred male-sterile line that produces high-quality, high-yield hybrid rice, but its inability to tolerate low temperatures during the reproductive period hinders seed propagation, limiting its widespread application. Therefore, selecting male-sterile lines that are tolerant to low temperatures during the reproductive growth period has become an urgent need for ensuring the safe propagation of dual-purpose nuclear male-sterile lines.

[0003] This invention addresses two major challenges in the two-line hybrid rice industry by developing a method for selecting and identifying low-erucic acid, low-carbohydrate, and dual-purpose nuclear male-sterile lines to meet the industry's needs for safe seed production and propagation. Summary of the Invention

[0004] To address or partially address the problems existing in the prior art, this invention provides a method for breeding and identifying "double-low" photoperiod and temperature-sensitive dual-purpose nuclear male-sterile rice lines, specifically including the following steps: (1) Germination and sowing of sterile rice are carried out from May 10 to 20 every year; transplanting is carried out at the 3-leaf and 1-heart stage. Photoperiod- and temperature-sensitive dual-purpose nuclear sterile rice is planted in pots and conventional water and fertilizer management and cultivation are carried out. (2) Between July 15 and August 15 each year, when the main panicle of the sterile rice cultured in step (1) enters the pollen mother cell formation period, the same rice is divided into two parts. One part is placed in a constant temperature water tank at 23.0±0.1℃ and the other part is placed in a constant temperature water tank at 19.0±0.1℃ for 10 days. After the treatment, the rice is taken out and cultured under normal management until the flowering period to collect rice pollen. (3) When the sterile line cultivated in step (2) enters the flowering period, rice pollen is taken and the pollen fertility is observed by iodine-potassium iodide staining method. The sterile rice with pollen staining rate <1% under 23.0±0.1℃ treatment for 10 days and pollen staining rate >70% under 19.0±0.1℃ treatment for 10 days is the "double low" dual-purpose nuclear sterile rice.

[0005] Preferably, in step (2), the water depth in the constant temperature water tank is such that it submerges the young panicles, which are sensitive to fertility, by 15-25 cm below the rice plant.

[0006] The sowing time in step (1) is from May 10 to 20 each year, which allows the sterile line to enter the fertility sensitive period from pollen mother cell to meiosis between July 15 and August 15. This period is in the high temperature season (the average daily temperature is higher than 25.0℃), and the day length will be close to the longest of the year, about 13 to 14 hours.

[0007] The beneficial effects of this invention are: (1) The photoperiod-temperature-sensitive dual-purpose nuclear male sterile line screened by the method described in this invention remains sterile at 23.0℃ and can be safely hybridized for seed production. Only when the average daily temperature is below 23.0℃ will the pollen of the male sterile line change from sterile to fertile and is not suitable for seed production. At the same time, as long as the average daily temperature is above 19.0℃, the male sterile line has a high fertile pollen rate and a high seed setting rate, which is conducive to efficient propagation of male sterile line seeds.

[0008] (2) The rice sterile lines screened by the method described in this invention that simultaneously possess low fertility conversion temperature and low temperature tolerance for pollen development are called "double-low" dual-purpose nuclear sterile lines. Such sterile lines are safe for hybrid seed production and sterile line propagation. Attached Figure Description

[0009] Figure 1 This is a graph showing pollen fertility under a 23.0℃ treatment.

[0010] Figure 2 This is a graph showing pollen fertility under a 19.0℃ treatment. Detailed Implementation

[0011] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0012] Example 1 This embodiment describes the selection and identification of 50 photoperiod- and temperature-sensitive dual-purpose nuclear male-sterile rice lines. The specific steps are as follows: (1) On May 10, 50 dual-purpose nuclear male sterile rice plants were potted (two pots of each type) and germinated and sown; transplanted at the 3-leaf and 1-heart stage. The photoperiod and temperature-sensitive dual-purpose nuclear male sterile rice was planted in pots and managed with conventional water and fertilizer. (2) Starting on July 17, when the sterile lines cultured in step (1) entered the pollen mother cell formation stage, the same rice was divided into two parts. One part was placed in a constant temperature water tank at 23.0℃ and the other part was placed in a constant temperature water tank at 19.0℃ for 10 days. After the treatment, the rice was taken out and placed under natural light and temperature conditions for cultivation. On August 12, the last batch of sterile line materials were placed in a constant temperature water tank for 10 days and then taken out and placed under natural light and temperature conditions. (3) When the rice male-sterile lines obtained in step (2) enter the flowering stage, pollen is collected and its fertility is observed using the iodine-potassium iodide staining method. Materials with high pollen fertility under 23.0℃ treatment for 10 days include Huading 623S and Jing 06S, while other materials show low fertility, such as Kenlong S and Huahui 217S, indicating that these materials pose a risk during hybrid seed production. Ten male-sterile materials, including Zhu1S and BYXA1S, have a pollen staining rate of <1%. Figure 1 (Table 1) These materials have high safety during seed production; 22 sterile lines with pollen staining rate >70% under 19.0℃ treatment for 10 days, such as Huading623S and BYXA1S, indicate that these materials have high safety during seed propagation; some materials have low fertility, such as Yunjing 214S and Heng 59S. Another group of materials are completely sterile, such as Liang 1S and BYXA14S. Figure 2Table 1 shows that there are safety risks associated with the propagation of these materials. The final selection and identification results of 50 dual-purpose nuclear male-sterile lines grown in pots are shown in Table 1. As can be seen from the table, BYXA1S, BYXA9S, 2021-27S, and 2021-13S are four materials with low sterility initiation temperatures and low-temperature tolerance for pollen development, meeting the selection criteria and thus qualifying as "double-low" photosensitive and temperature-sensitive dual-purpose nuclear male-sterile lines.

[0013] Table 1. Pollen fertility (%) of dual-purpose nuclear male sterile lines under 23℃ and 19℃ treatment for 10 days. The self-pollination set rates of these four materials were statistically analyzed after treatment at 23℃ and 19℃ for 10 days, respectively. The control plant 1S had zero fruit set at 23℃, indicating its seed production safety, but also zero fruit set after 10 days of treatment at 19℃, indicating difficulty in propagation. Annong S-1 showed no safety in either seed production or propagation. The four materials screened in this invention all had a self-pollination set rate of 0% at 23℃, indicating very safe seed production; simultaneously, their fruit set rates were all above 65% after 10 days of treatment at 19℃, indicating safe propagation as well, further confirming them as "double-low" photoperiod- and temperature-sensitive dual-purpose nuclear male-sterile lines.

[0014] Table 2. Self-pollination seed set rate (%) of the "double-low" dual-purpose nuclear male sterile line under 23℃ and 19℃ treatment for 10 days.

Claims

1. A method for breeding and identifying a "double-low" photoperiod- and temperature-sensitive dual-purpose nuclear male-sterile rice line, characterized in that: Specifically, the following steps are included: (1) Germination and sowing of sterile rice are carried out from May 10 to 20 every year. Transplanting is carried out when the rice has 3 leaves and 1 heart. Photoperiod- and temperature-sensitive dual-purpose nuclear sterile rice is planted in pots and is managed and cultivated in a conventional manner with water and fertilizer. (2) Between July 15 and August 15 each year, when the main panicle of the sterile rice cultured in step (1) enters the pollen mother cell formation period, the same rice is divided into two parts. One part is placed in a constant temperature water tank at 23.0±0.1℃ and the other part is placed in a constant temperature water tank at 19.0±0.1℃ for 10 days. After the treatment, the rice is taken out and cultured under routine management. (3) When the rice sterile line cultivated in step (2) enters the flowering period, pollen is taken and pollen fertility is observed by iodine-potassium iodide staining method. The sterile rice line with pollen staining rate <1% under 23.0±0.1℃ treatment for 10 days and pollen staining rate >70% under 19.0±0.1℃ treatment for 10 days is a "double low" dual-purpose nuclear sterile rice line.

2. The method for breeding and identifying the "double-low" photoperiod-temperature-sensitive dual-purpose nuclear male-sterile rice line as described in claim 1, characterized in that: In step (2), the water depth in the constant temperature water tank should be such that it submerges the young panicles, which are sensitive to fertility, by 15-25 cm below the rice plants.

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