Temperature-sensitive sterility characteristic of MIR162 transgenic maize and related application of temperature-sensitive sterility characteristic

Abstract

The invention relates to a new discovery of male flower temperature-sensitive sterility characteristics of a transgenic maize event MIR162 and application of the male flower temperature-sensitive sterility characteristics in parent breeding, backcross transformation, pre-breeding and two-line hybrid production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the field of corn biological trait development technology, and more particularly to MIR162 seed production technology. The present invention finds that the corn event MIR162 related gene loci are related to thermosensitive sterility and its application in parental reproduction, backcrossing, pre-breeding and two-line hybrid production. Background Art

[0002] Corn event MIR162 is a very important transgenic product that provides insecticidal activity against a broad spectrum of lepidopteran pests, providing primary protection for many of Syngenta’s best-selling hybrids against aboveground insect feeding damage, including but not limited to fall armyworm, cutworm, tobacco armyworm larvae, sugarcane borer, small corn borer, and cotton bollworm [1].

[0003] Corn event MIR162 is known in the art and is described in many patents. For example, CN101548011A states that its applicant has deposited corn seeds of event MIR162 with the American Type Microbiological Collection (ATCC) at 1801 University Boulevard, Manassas, VA 20110 on January 23, 2007 under the Budapest Treaty, with ATCC registration number PTA-8166. In addition, CN101548011A also describes how to identify corn event MIR162. Summary of the invention

[0004] The present invention confirms that the loci associated with the maize event MIR162 are associated with temperature sensitivity and sterility characteristics under a specific germplasm background. The present invention is further used to guide parental propagation, backcrossing, pre-breeding and two-line hybrid production, including: (1) selecting a suitable low-temperature ecological environment and an optimal sowing window will increase the success rate of parental propagation, backcrossing and pre-breeding of these MIR162 materials with a specific genetic background, and reduce the cost of seed production; (2) based on the high-temperature sterility associated with the loci associated with the maize event MIR62, these MIR162 materials with a specific genetic background can be used as maize two-line hybrid seed production technology as female parents. Parental propagation refers to the large-scale propagation of excellent inbred line parents, an important process for preparing seed sources for hybrid breeding and production of hybrid plants. Backcrossing refers to a breeding method in which certain specific traits of donor B are introduced into parent A through repeated backcrossing for multiple generations. Pre-breeding refers to the process of evaluating, innovating and improving plant germplasm resources before the formal breeding program begins; here it specifically refers to the breeding process of directly utilizing transgenic target traits using different superior parents of transgenic traits as basic materials, thereby eliminating the later breeding process of introducing transgenic target traits through backcrossing.

[0005] In the present invention, male flowers of the backcross donor inbred lines of a group of 12 transgenic events MIR162 covering multiple corn heterotic groups were confirmed to have temperature-sensitive sterility, that is, these MIR162 materials with a specific genetic background have no pollen or little pollen under high temperature conditions. The critical temperature for fertility conversion of MIR162, a corn material to be involved in the present invention, is 26±1°C, and the fertility fluctuation period is between 25 and 27°C, wherein 25°C and below are clearly fertile, and 27°C and above are clearly infertile. These MIR162 materials with a specific genetic background begin to show partial male sterility at 26°C, and the sterility rate increases significantly with increasing temperature. However, high temperature does not affect the fertility of female spike silk. Factors such as the duration of high temperature and the period of occurrence have a greater impact on the fertility of its male flowers. The present invention relates to a sensitive period for fertility conversion of a specific germplasm containing a gene locus related to the corn event MIR162, which is the middle and late stages (4-2 leaves) of the development and formation period of the male organs (anthers and pollens) of corn. During this period, if the average daily temperature is higher than 26.5°C, preferably higher than 27°C for 3 consecutive days, sterility can be induced; if the average daily temperature is not higher than 25.5°C, preferably higher than 25°C for 3 consecutive days, normal fruiting can be achieved. The gene locus is recessive, and the temperature-sensitive male sterility can only be manifested when the gene locus is homozygous in the inbred line. When heterozygous, pollen can be normally shed like the wild type. The present invention further relates to corn with a gene locus related to the corn event MIR162 in a specific genetic background, which can select a suitable low-temperature ecological environment (such as: Yunnan spring corn area with an altitude of more than 1,300 meters and 18-23°N winter corn area) and a sowing window, which can effectively restore the fertility of the MIR162 material, increase the success rate of parent reproduction, backcrossing and pre-breeding, and reduce the risk and cost of seed production. At the same time, high-temperature ecological environments (such as spring sowing in winter corn areas at 18-23°N, summer corn areas below 500 meters above sea level, 40-18°N, and some spring corn areas in Xinjiang) and optimal sowing windows can be selected. These MIR162 materials with specific genetic backgrounds can be used as female parents to develop new two-line hybrid seed production technologies using the characteristics of high-temperature sterility.

[0006] From 2019 to 2022, multi-year multi-point field tests and seed propagation were carried out using specific inbred line materials containing loci related to the maize event MIR162 (Tables 1 and 2). It was found that these materials were male sterile under high temperature test environments. Its main feature is that under high temperature conditions, the tassel florets and outer lemmas are closed, and most or completely aborted pollen. For example, batches 1-3 and 7-10 were all pollen-free in the high temperature seasons of Sanya and Nanning, and batches 12-13 and 15-21 were sown in summer in Beijing and spring in Yongji (high temperature in Jilin in 2021). Under low temperature test environments, the tassel showed normal pollen shedding. For example, test batch 5 was sown in spring in Pengpu, Yunnan, batches 4 and 6 were sown in winter in Betel nut, Hainan, batch 14 was sown in early spring with film covering in Shunyi, and batch 21 was sown in spring in Yongji, Jilin. Table 2 shows the pollen performance of different traits of six similar genetic backgrounds in high temperature environments. BtGT (Bt11 / GA21) and GA21 were fertile under high temperature, and only ZL (MIR162 / Bt11 / GA21) was sterile at high temperature, from which it can be inferred that the temperature-sensitive sterility was caused by a locus associated with the specific event MIR162 material.

[0007] Table 1: Fertility evaluation of inbred lines containing loci associated with maize event MIR162

[0008]

[0009] Table 2: Comparison of fertility of ZL, BtGT (Bt11 / GA21) and GA21 materials under high temperature environment

[0010]

[0011] The present invention found that the specific MIR162 material inbred lines have thermosensitive male sterility characteristics. These inbred lines containing the maize event MIR162 related loci begin to be male sterile at 26°C, and the sterility rate increases significantly with increasing temperature. However, high temperature does not affect the fertility of silk.

[0012] The sensitive period of fertility conversion is the middle and late stages of the development and formation of the male organs (anthers and pollen) of corn (4-2 leaves). The anther and pollen development period is usually 2 weeks before the pollination date. During this period, if high temperatures are encountered, the pollen will basically be sterile; under low temperature conditions, it can bear fruit normally. This characteristic is controlled by a recessive locus, and the temperature-sensitive sterility characteristic can only be manifested when the locus is homozygous for the inbred line. When heterozygous, it can shed pollen normally like the wild type.

[0013] Selecting a low-temperature ecological environment (such as the Yunnan spring corn area above 1,300 meters above sea level and the 18-23°N winter corn area) and an optimal sowing window will effectively restore the fertility of these specific MIR162 materials and increase the success rate of parental reproduction, backcrossing and pre-breeding.

[0014] Based on the work and discovery of the inventors, in one aspect, the present invention provides a method for producing corn plants or seeds (e.g., corn hybrid seed production), which comprises: hybridizing a corn plant containing a corn event MIR162-related locus having a thermosensitive male sterility characteristic as a female parent with another corn plant having a desired trait as a male parent, wherein the corn plant containing the corn event MIR162-related locus is homozygous with respect to the MIR162-related locus. In a further embodiment of this aspect, before the hybridization, the corn plant containing the corn event MIR162-related locus is placed in a temperature condition sufficient to cause pollen abortion in the middle and late stages of anther and pollen development (e.g., a temperature condition higher than the critical temperature for fertility conversion (e.g., 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is higher than 26.5°C for 3 consecutive days, more preferably higher than 27°C). In a further embodiment of this aspect, the corn plant containing the corn event MIR162 related locus is placed in the middle and late stages of anther and pollen development under temperature conditions sufficient for normal pollen development (for example, under temperature conditions below the critical temperature for fertility conversion (for example, 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is not greater than 25.5°C, more preferably greater than 25°C for three consecutive days) for parental breeding, thereby providing the required amount of parents for the hybridization.

[0015] In another aspect, the present invention provides a use of a corn plant comprising a corn event MIR162-related locus having a thermosensitive male sterility characteristic as a female parent in producing corn plants or seeds (e.g., corn hybrid seed production), wherein the corn plant comprising the corn event MIR162-related locus is homozygous for the MIR162-related locus. In a further embodiment of this aspect, before the process of producing corn plants or seeds is carried out, the corn plant comprising the corn event MIR162-related locus is placed in a temperature condition sufficient to cause pollen abortion in the middle and late stages of anther and pollen development (e.g., a temperature condition higher than the critical temperature for fertility conversion (e.g., 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is higher than 26.5°C for 3 consecutive days, more preferably higher than 27°C). In a further embodiment of this aspect, the corn plant containing the corn event MIR162 related locus is placed in the middle and late stages of anther and pollen development under temperature conditions sufficient for normal pollen development (for example, under temperature conditions below the critical temperature for fertility conversion (for example, 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is not greater than 25.5°C, more preferably greater than 25°C for three consecutive days) for parental breeding, thereby providing the required amount of parents for the process of producing corn plants or seeds.

[0016] In another aspect, the present invention provides a method for parental propagation, backcrossing or pre-breeding, which comprises subjecting a corn plant comprising a corn event MIR162-related locus having a thermosensitive male sterility characteristic to parental propagation, backcrossing or pre-breeding process, wherein the corn plant comprising the corn event MIR162-related locus is homozygous with respect to the MIR162-related locus. In a further embodiment of this aspect, the corn plant comprising the corn event MIR162-related locus is placed in a temperature condition sufficient for normal pollen development in the middle and late stages of anther and pollen development (for example, a temperature condition lower than the critical temperature for fertility conversion (for example, 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is not greater than 25.5°C for 3 consecutive days, more preferably greater than 25°C).

[0017] In another aspect, the present invention provides the use of a corn plant comprising a corn event MIR162-related locus having a thermosensitive male sterility characteristic for parental propagation, backcrossing or pre-breeding, wherein the corn plant comprising the corn event MIR162-related locus is homozygous with respect to the MIR162-related locus. In a further embodiment of this aspect, the corn plant comprising the corn event MIR162-related locus is placed in a temperature condition sufficient for normal pollen development in the middle and late stages of anther and pollen development (for example, a temperature condition lower than the critical temperature for fertility conversion (for example, 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is not greater than 25.5°C for 3 consecutive days, more preferably greater than 25°C).

[0018] In another aspect, the present invention provides a method for growing corn plants, comprising subjecting corn plants having thermosensitive male sterility characteristics and comprising a corn event MIR162-related locus to temperature conditions sufficient for normal pollen development in the middle and late stages of anther and pollen development (e.g., at a temperature below the critical temperature for fertility conversion (e.g., 26°C, or any temperature between 25 and 27°C); preferably, at a temperature without an average daily temperature greater than 25.5°C, more preferably greater than 25°C for three consecutive days), and / or subjecting a corn plant having thermosensitive male sterility characteristics and comprising a corn event MIR162-related locus to temperature conditions sufficient for normal pollen development in the middle and late stages of anther and pollen development. A corn plant containing a corn event MIR162-related locus with male sterility characteristics is exposed to temperature conditions sufficient to cause pollen abortion in the middle and late stages of anther and pollen development (for example, under temperature conditions above the critical temperature for fertility conversion (for example, 26°C, or any temperature between 25 and 27°C); preferably, under conditions where the average daily temperature is above 26.5°C, more preferably above 27°C for three consecutive days), wherein the corn plant containing the corn event MIR162-related locus is homozygous for the MIR162-related locus.

[0019] In the aspects described above, the temperature is usually not higher than the upper temperature suitable for anther and pollen grain development, such as 38°C; and the temperature is usually not lower than the lower temperature suitable for anther and pollen grain development, such as 16°C.

[0020] Locus: refers to a chromosomal region or chromosomal position where a polymorphic nucleic acid, trait determinant, gene or marker is located. A locus can represent a single nucleotide, a few nucleotides, or a large number of nucleotides in a genomic region. A locus in the present disclosure includes one or more polymorphisms in a population (e.g., the presence of alternative alleles in certain individuals).

[0021] Maize event MIR162-associated locus: refers to the DNA sequence involved in the recombinant construct inserted into the maize genome generating the MIR162 event as well as the maize genomic sequences flanking the insertion site. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1a . Summary of the average daily temperature in Pengpu and Binglang in 2022.

[0023] Figure 1b . Average daily temperature in Nanning in 2020-2021.

[0024] Figure 1c . Average daily temperature in Betel nut in 2020-2021.

[0025] Figure 1d . Average daily temperature in Pongpu in 2020-2021.

[0026] Figure 1e . Average daily temperature in Shunyi from 2019 to 2021.

[0027] Figure 1f . Average daily temperature in Yongji from 2019 to 2021.

[0028] Figure 2 . The loose powder and firmness of AX5707ZL of 2022 Pengpu and Areca.

[0029] Figure 3 . The loose powder and firmness of the 2022 KFX5360ZL of Punch and Betel nut.

[0030] Figure 4 . 2022 Ponpu and Areca MFA7191ZL is loose powder and firm.

[0031] Figure 5 . The loose powder and firmness of GAB2601ZL of 2022 Pomp and Betel nut.

[0032] Figure 6 . 2022 IAF3318ZL of Pompom and Areca is loose and firm.

[0033] Figure 7 . 2022 Pengpu and Areca ID3264ZL is loose powder and strong.

[0034] Figure 8 . 2022 Pengpu and Areca ID3477ZL loose powder and firm.

[0035] Fig. 9 . The loose powder and firmness of IFA3351ZL of 2022 Pengpu and Areca.

[0036] Fig.10 . The loose powder and firmness of the THD28ZL of 2022 Punch and Betel nut.

[0037] Fig.11 . The loose powder and firmness of FF6097ZL of 2022 Pengpu and Areca.

[0038] Fig.12 . 2022 Pengpu and Areca ID3606ZL loose powder and firm.

[0039] Fig.13 . 2022 Ponpu and Areca MFA7273ZL is loose powder and firm.

[0040] Fig.14 . 2022 Areca nut hybrid AZL two-line hybrid trial.

[0041] Fig.15 . Seed production technology and application based on MIR162 DETAILED DESCRIPTION

[0042] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0043] Example 1. Fertility study of 12 different germplasm ZL inbred lines in Areca catechu and Ponci

[0044] 1. Purpose

[0045] 1.1. Observation of fertility, pollen shedding and seed setting of inbred lines containing MIR162 from different heterotic groups under high and low temperature field conditions

[0046] 1.2. Verify whether the thermosensitive sterility of the loci related to the maize event MIR162 can be repeatedly and stably inherited in this group of materials with different genetic backgrounds

[0047] 1.3. Identification of potential suitable geographical locations for MIR162 multiplication, breeding nurseries and two-line seed production

[0048] 2. Materials and Methods

[0049] 2.1. A set of 12 transgenic event MIR162 backcross donor inbred lines covering multiple maize heterotic groups: AX5707ZL, FF6097ZL, GAB2601ZL, IAF3318ZL, ID3264ZL, ID3477ZL, ID3606ZL, IFA3351ZL, KFX5360ZL, MFA7191ZL, MFA7273ZL and THD28ZL. ZL is a trivalent composite trait, and a donor of three traits, MIR162, Bt11 and GA21, was transferred to a target inbred line through conventional backcrossing methods.

[0050] Corn event MIR162: It was obtained through the Agrobacterium-mediated method. It contains the vip3Aa20 gene from the Bacillus thuringiensis strain A188. The expressed Vip3Aa20 protein has insecticidal effects on various lepidopteran pests such as Spodoptera frugiperda and Agrotis ipsilon. It also contains the pmi gene from the Escherichia coli strain K-12, which encodes the phosphomannose isomerase PMI protein, which is used as a selection marker in the screening process.

[0051] Corn event Bt11: It is obtained by PEG-mediated method, contains cry1Ab gene from Bacillus thuringiensis strain HD-1, and the expressed Cry1Ab protein has insecticidal effect on lepidopteran pests such as Ostrinia nubillis and Helicoverpaarmigera; it also contains pat gene from viridisch strain Tü494, which confers tolerance to glufosinate herbicides and can also be used as a selection marker in genetic transformation process. For example, corn event Bt11 is described in US6342660B1.

[0052] Corn event GA21: obtained by particle gun method, it contains a modified mepsps gene from corn, and the expressed mEPSPS protein has a reduced affinity for glyphosate compared to the endogenous EPSPS, thus showing tolerance to glyphosate-based herbicides. For example, corn event GA21 is described in US6762344B1.

[0053] Thus, the ZL line (containing MIR162, Bt11 and GA21) is resistant to fall armyworm, Asian corn borer, armyworm, cotton bollworm, cutworm, tobacco armyworm larvae, sugarcane borer and small corn borer, while being resistant to glyphosate and glufosinate.

[0054] 2.2. Experimental design: Plot area: two rows for each material, 5 m row length, one replicate.

[0055] 2.3. Experimental location: Sanya Binglang (high temperature, 18.3°N, 0m above sea level); Yunnan Pengpu (low temperature, 24°N, 1180m above sea level).

[0056] 2.4. Sowing date: Sanya Areca: June 17, 2022; Yunnan Pengpu: May 30, 2022.

[0057] 2.5. Data Collection:

[0058] Photos were taken of the daily average temperature, male fertility score, male spikelet flowering period, and female spikelet fruiting rate.

[0059] Male fertility scoring criteria (scoring anther number on a plot basis):

[0060] 1 = 100% anthers extruded from all tassel spikelets. Anthers are full and pollen numbers are the same or similar to the recurrent parent or other controls.

[0061] 2 = Only 50-75% of the anthers in the whole tassel can be squeezed out of the spikelet. Pollen quantity is reduced.

[0062] 3 = 25% anther extrusion. Anthers may appear shrunken and pollen count greatly reduced compared to the recurrent parent

[0063] 4 = 0% anther extrusion. No pollen, tassel sterile.

[0064] 3. Experimental Results

[0065] Table 3: Pollen shedding and seed setting performance of 12 ZL inbred lines of different germplasms

[0066]

[0067] Table 4: Anther and pollen development stages and their corresponding daily average temperatures in the ZL inbred lines of 12 different germplasms of Areca catechu

[0068]

[0069] Note: The row with the corresponding ZL inbred line number corresponds to the date of anther and pollen development period of the ZL inbred line.

[0070] Table 5: Anther and pollen development stages of 12 different germplasm ZL inbred lines in Pengpu and their corresponding daily average temperatures

[0071]

[0072] Note: The row with the corresponding ZL inbred line number corresponds to the date of anther and pollen development period of the ZL inbred line.

[0073] in conclusion:

[0074] All 12 different germplasm ZL inbred lines were male sterile in Areca (high temperature environment) and fertile in Pengpu (low temperature environment). The thermosensitive sterility characteristics of these 12 MIR162 inbred lines were reproducible and stable in Areca and Pengpu (see Table 3 and Figure 2-13 ).

[0075] Pengpu (24°N, 1100m above sea level) is a candidate site for MIR162 transformation breeding, but there is a risk of temperature variation from year to year. The Yunnan spring corn area above 1300m above sea level [2,3] and the 18-23°N winter corn area are recommended as candidate sites for breeding and pre-breeding projects containing MIR162 materials.

[0076] Example 2. Two-line seed production technology based on MIR162-containing materials

[0077] 1. Purpose

[0078] Under high temperature environment, taking the small-scale two-line field trial production research of hybrid AZL as an example, we explored whether MIR162 can be applied to two-line seed production.

[0079] 2. Materials and Methods

[0080] 2.1. Hybrid A parents: THD28ZL (female), ID3477 (father)

[0081] 2.2. Experimental design: Non-transgenic ID3477 was used as the male parent and THD28ZL was used as the female parent. The ratio of the male parent to the female parent was 1:4. The male parent was planted in two phases. In the first phase, 50% was planted 3 days later; and the remaining 50% was planted 5 days later. The area was 1 mu.

[0082] 2.3. Sowing location and window: Areca nut. Spring sowing ensures that the florets and anthers of the mother plant are under high temperature stress during development, and staggered sowing with other corn projects to reduce exogenous pollen contamination.

[0083] 2.4. Data collection: male fertility scoring, fruiting rate and flowering period photography, and meteorological data (daily maximum temperature, daily minimum temperature, daily average temperature, daily rainfall and humidity)

[0084] 3. Experimental Results

[0085] 3.1. For the first time, a small-scale two-line hybrid seed production was successfully delivered in Areca using plants containing loci related to the maize event MIR162 as female parents. When the female parent THD28ZL bloomed, the male flowers had no pollen and were completely sterile. The number of anthers was small and withered and shriveled. The number of male spike branches decreased and became thinner. Silking was normal. The male parent ID3477 showed normal pollen shedding and a large amount of pollen. Under open pollination and artificial assisted pollination (inappropriately delayed, the male parent was early), the female parent THD28ZL had good fruiting, almost full ears, very clean, and a yield of 369 kg / mu. The ability of the ZL female parent's silk to accept pollen and grain development were not affected by high temperature (see Fig.14 ).

[0086] 3.2. The production of two-line hybrids containing loci related to the maize event MIR162 under high temperature environment may be a new option for future maize hybrid production.

[0087] 3.3. Recommended candidate bases for two-line seed production in China: 18-23°N winter corn areas for spring sowing and below 500 meters above sea level, 40-18°N summer corn areas, and some spring corn areas in Xinjiang.

[0088] in conclusion:

[0089] In summary, the present invention takes the hybrid AZL two-line hybrid as an example to provide a method for selecting a suitable high-temperature field environment, such as spring sowing in China's 18-23°N winter corn area, below 500 meters above sea level, 40-18°N summer corn area and some spring corn areas in Xinjiang, by using MIR162 material as the female parent to carry out two-line corn hybrid production, providing a new choice for future corn hybrid production models.

[0090] Selecting a high-temperature ecological environment (such as spring sowing in winter corn areas at 18-23°N, summer corn areas below 500 meters above sea level, 40-18°N, and some spring corn areas in Xinjiang) and the best sowing window can allow the use of MIR162 material as the female parent to exploit the characteristics of high-temperature male flower sterility to develop two-line hybrid corn seed production.

[0091] Example 3. Confirmation of the thermosensitive sterility properties of MIR162-containing materials

[0092] Other experiments confirmed that, in the absence of Bt11 and GA21, the material containing MIR162 had thermosensitive sterility.

[0093] One example is that in the 2019 MIR162, Bt11 and GA21 seed propagation, a specific MIR162 material was found to have unstable male flower fertility. ID3461MIR162 had normal pollen (score of 1) in the 2019 Shunyi early spring sowing propagation, but greatly reduced pollen (score of 3) in the Yongji backup late sowing propagation under high temperature. However, ID3461Bt11 and ID3461GA21 of the same genetic background had normal pollen in the same two environments (either Shunyi early spring sowing or Yongji backup late sowing), the same as the non-transgenic control ID3461 (score of 1). See Table 4 below.

[0094] Table 6

[0095]

[0096] Example 4. Determination of the critical temperature for fertility conversion

[0097] Based on a large number of experiments, it can be determined that the critical temperature for fertility conversion is 26±1℃, and the fertility fluctuation period is between 25 and 27℃, among which 25℃ and below are clearly fertile, and 27℃ and above are clearly infertile. Plants containing the loci related to the maize event MIR162 begin to become male sterile at 26℃, and the sterility rate increases significantly with increasing temperature. Different germplasms have a certain slight effect on the critical temperature of fertility.

[0098] For example, regarding the critical high temperature, the anther and pollen development period of NP5024ZL in Nanning in 2020 was from September 13 to September 20, 2020, with daily average temperatures of 25.8, 26.7, 26.8, 26.5, 25.3, 20.7, 20.9 and 22.2 (basic sterility), respectively. It is determined that the sterility condition is roughly 3 consecutive days of daily average temperature above 26.5°C, preferably 27°C.

[0099] Regarding critical low temperature, the anther and pollen development period of ID3264ZL in Pengpu in 2022 was from June 28 to July 3, 2022, with daily average temperatures of 24.7, 25.1, 25.5, 24.1, 25.7 and 25.3 (sufficient pollen); and the anther and pollen development period of ID3461MIR162 in Shunyi in 2019 was about May 31 to June 7, 2019, with daily average temperatures of 25.4, 25.1, 25.7, 27.1, 24.1, 25.9, 22.3 and 25.2. It is determined that the fertile condition is roughly that there is no daily average temperature greater than 25.5℃ for 3 consecutive days, preferably 25℃.

[0100] The acquisition of some ZL inbred lines involved in this application (inbred line backcrossing breeding report):

[0101] 1. The backcross breeding process of AX5707ZL (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0102] AX5707ZL: A maize inbred line AX5707 (described in a previous article as AX5707 and described in US6710233B2 as NP2222, ATCC accession number PTA-3967) was used as the female parent and DWFA4651ZL (described in US11051473B2, ATCC accession number PTA-126339) was used as the trait donor. After one generation of hybridization, AX5707 was used as the recurrent parent. After three generations, AX5707 (Bt11×GA21×MIR162) was obtained and self-pollinated for one generation. After genotype detection, AX5707(GA21), AX5707(Bt11×GA21) and AX5707(Bt11×GA21×MIR162) were obtained. After another self-pollination, stable inbred lines AX5707(GA21), AX5707(Bt11×GA21) and AX5707(Bt11×GA21×MIR162) were obtained.

[0103] 2. FF6097ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0104] FF6097ZL: The FF6097 maize inbred line was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, FF6097 was used as the recurrent parent for three consecutive backcrossing generations to obtain FF6097 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype testing, FF6097(GA21), FF6097(Bt11×GA21) and FF6097(Bt11×GA21×MIR162) were obtained. After another generation of self-pollination, the stable inbred lines FF6097(GA21), FF6097(Bt11×GA21) and FF6097(Bt11×GA21×MIR162) were obtained.

[0105] 3. GAB2601ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0106] GAB2601ZL: The maize inbred line GAB2601 (described in US9332705B2, ATCC deposit number PTA-121333) was used as the female parent and DWFA4651ZL was used as the trait donor. After one generation of hybridization, GAB2601 was used as the recurrent parent for three consecutive backcrossing generations to obtain GAB2601 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype detection, GAB2601(GA21), GAB2601(Bt11×GA21) and GAB2601(Bt11×GA21×MIR162) were obtained. After one generation of self-pollination, the stable inbred lines GAB2601(GA21), GAB2601(Bt11×GA21) and GAB2601(Bt11×GA21×MIR162) were obtained.

[0107] 4. IAF3318ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0108] IAF3318ZL: The maize inbred line IAF3318 was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, IAF3318 was used as the recurrent parent for three consecutive backcrossing generations to obtain IAF3318 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype testing, IAF3318(GA21), IAF3318(Bt11×GA21) and IAF3318(Bt11×GA21×MIR162) were obtained. After another generation of self-pollination, the stable inbred lines IAF3318(GA21), IAF3318(Bt11×GA21) and IAF3318(Bt11×GA21×MIR162) were obtained.

[0109] 5. ID3264ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0110] ID3264ZL: The ID3264 maize inbred line (described in US7323626B1 as G06-NP2623, ATCC deposit number PTA-8713) was used as the female parent and DWFA4651ZL was used as the trait donor. After one generation of hybridization, ID3264 was used as the recurrent parent for three consecutive backcrossing generations to obtain ID3264ZL (Bt11×GA21×MIR162), which was self-pollinated for one generation. After genotype detection, ID3264(GA21), ID3264(Bt11×GA21) and ID3264(Bt11×GA21×MIR162) were obtained. After one generation of self-pollination, the stable inbred lines ID3264(GA21), ID3264(Bt11×GA21) and ID3264(Bt11×GA21×MIR162) were obtained, respectively.

[0111] 6. ID3477ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0112] ID3477ZL: The maize inbred line ID3477 was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, ID3477 was used as the recurrent parent for three consecutive backcrossing generations to obtain ID3477 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype detection, ID3477(GA21), ID3477(Bt11×GA21) and ID3477(Bt11×GA21×MIR162) were obtained. After another generation of self-pollination, the stable inbred lines ID3477(GA21), ID3477(Bt11×GA21) and ID3477(Bt11×GA21×MIR162) were obtained.

[0113] 7. ID3606ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0114] ID3606ZL: The ID3606 maize inbred line (described as NPID3606 in US8003863B1, ATCC deposit number PTA-11894) was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, ID3606 was used as the recurrent parent for three consecutive backcrossing generations to obtain ID3606 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype detection, ID3606(GA21), ID3606(Bt11×GA21) and ID3606(Bt11×GA21×MIR162) were obtained. After one generation of self-pollination, the stable inbred lines ID3606(GA21), ID3606(Bt11×GA21) and ID3606(Bt11×GA21×MIR162) were obtained, respectively.

[0115] 8. IFA3351ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0116] IFA3351ZL: The maize inbred line IFA3351 was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, IFA3351 was used as the recurrent parent for three consecutive backcrossing generations to obtain IFA3351 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype testing, IFA3351(GA21), IFA3351(Bt11×GA21) and IFA3351(Bt11×GA21×MIR162) were obtained. After another generation of self-pollination, the stable inbred lines IFA3351(GA21), IFA3351(Bt11×GA21) and IFA3351(Bt11×GA21×MIR162) were obtained.

[0117] 9. KFX5360ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0118] KFX5360ZL: The KFX5360 corn inbred line (described in US2019297814A1) was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, KFX5360 was used as the recurrent parent for three consecutive backcrossing generations to obtain KFX5360 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype detection, KFX5360(GA21), KFX5360(Bt11×GA21) and KFX5360(Bt11×GA21×MIR162) were obtained. After another generation of self-pollination, the stable inbred lines KFX5360(GA21), KFX5360(Bt11×GA21) and KFX5360(Bt11×GA21×MIR162) were obtained.

[0119] 10. MFA7191ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0120] MFA7191ZL: The MFA7191 maize inbred line was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, MFA7191 was used as the recurrent parent for three consecutive backcrossing generations to obtain MFA7191 (Bt11×GA21×MIR162) and self-pollinated for one generation. Genotype testing revealed MFA7191(GA21), MFA7191(Bt11×GA21) and MFA7191(Bt11×GA21×MIR162). After another generation of self-pollination, the stable inbred lines MFA7191(GA21), MFA7191(Bt11×GA21) and MFA7191(Bt11×GA21×MIR162) were obtained.

[0121] 11. MFA7273ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0122] MFA7273ZL: The MFA7273 maize inbred line was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, MFA7273 was used as the recurrent parent for three consecutive backcrossing generations to obtain MFA7273 (Bt11×GA21×MIR162) and self-pollinated for one generation. Genotype testing revealed MFA7273(GA21), MFA7273(Bt11×GA21) and MFA7273(Bt11×GA21×MIR162). After another generation of self-pollination, the stable inbred lines MFA7273(GA21), MFA7273(Bt11×GA21) and MFA7273(Bt11×GA21×MIR162) were obtained.

[0123] 12. THD28ZL backcross breeding process (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0124] THD28ZL: The maize inbred line THD28 was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, THD28 was used as the recurrent parent for three consecutive backcrossing generations to obtain THD28 (Bt11×GA21×MIR162) and self-pollinated for one generation. Genotype detection revealed THD28 (GA21), THD28 (Bt11×GA21) and THD28 (Bt11×GA21×MIR162). After another generation of self-pollination, the stable inbred lines THD28 (GA21), THD28 (Bt11×GA21) and THD28 (Bt11×GA21×MIR162) were obtained.

[0125] 13. Backcross breeding process of NP5024ZL (including parent combination, parent source, breeding method, generation and characteristic description, etc.)

[0126] NP5024ZL: The NP5024 maize inbred line was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, NP5024 was used as the recurrent parent for three consecutive backcrossing generations to obtain NP5024 (Bt11×GA21×MIR162) and self-pollinated for one generation. After genotype testing, NP5024(GA21), NP5024(Bt11×GA21) and NP5024(Bt11×GA21×MIR162) were obtained. After another generation of self-pollination, the stable inbred lines NP5024(GA21), NP5024(Bt11×GA21) and NP5024(Bt11×GA21×MIR162) were obtained.

[0127] References

[0128] [1] N. Long; D. Priam; J. Bottoms; M. Maggi; H. Hart; Q. Qu. Corn event MIR162. Patent application publication number CN101548011A

[0129] [2] He Zhongyou, Tan Shuyi, Lin Li, Hong Dekai, Bai Cuiyun, Hao Jingfeng, Gao Qunying, Tian Zhiguo. Ecological study on thermosensitive male sterility of maize. Chinese Agricultural Science Bulletin, Vol. 15, No. 5, 1999: 4-10.

[0130] [3] Liu Hai, Deng Linfeng, Deng Zhuqing, Deng Xiaolin. A breeding method for dual-purpose nuclear male sterile rice lines. Patent application publication number CN 109832126 A.

Claims

1. A method for producing a corn plant or seed, comprising: A corn plant containing a corn event MIR162-related locus and having temperature-sensitive male sterility characteristics is used as a female parent to be hybridized with another corn plant having desired traits as a male parent, wherein the corn plant containing the corn event MIR162-related locus is homozygous for the MIR162-related locus, and wherein prior to the hybridization, the corn plant containing the corn event MIR162-related locus is placed under temperature conditions sufficient to cause pollen abortion in the middle and late stages of anther and pollen development, wherein the temperature conditions sufficient to cause pollen abortion refer to conditions where the average daily temperature is higher than 26.5°C for three consecutive days.

2. The method according to claim 1, wherein the temperature condition sufficient to cause pollen abortion refers to a condition in which the average daily temperature is higher than 27°C for 3 consecutive days.

3. The method according to claim 1 or 2, wherein the corn plant containing the corn event MIR162 related locus is placed in a temperature condition sufficient for normal pollen development in the middle and late stages of anther and pollen development for parental breeding, thereby providing the required amount of parents for the hybridization.

4. Use of a corn plant comprising a corn event MIR162-associated locus having a temperature-sensitive male sterility characteristic as a female parent in producing corn plants or seeds, wherein the corn plant comprising a corn event MIR162-associated locus is homozygous for the MIR162-associated locus.

5. The use according to claim 4, wherein before the process of producing corn plants or seeds is carried out, the corn plants containing the corn event MIR162 related loci are placed under temperature conditions sufficient to cause pollen abortion in the middle and late stages of anther and pollen development.

6. The use according to claim 4 or 5, wherein the corn plant containing the corn event MIR162 related locus is placed in the middle and late stages of anther and pollen development under temperature conditions sufficient for normal pollen development for parental breeding, thereby providing the required amount of parents for the process of producing corn plants or seeds.

7. A method for parental propagation, backcrossing or pre-breeding, which comprises subjecting a corn plant comprising a corn event MIR162-related locus having a temperature-sensitive male sterility trait to a parental propagation, backcrossing or pre-breeding process, wherein the corn plant comprising the corn event MIR162-related locus is homozygous for the MIR162-related locus, and wherein the corn plant comprising the corn event MIR162-related locus is placed under temperature conditions sufficient for normal pollen development in the middle and late stages of anther and pollen development, and the temperature conditions sufficient for normal pollen development refer to conditions where the average daily temperature is not greater than 25.5°C for three consecutive days.

8. The method according to claim 7, wherein the temperature condition sufficient for normal development of pollen refers to the condition where the average daily temperature is not greater than 25°C for 3 consecutive days.

9. A method for growing corn plants, comprising subjecting corn plants containing corn event MIR162-related loci and having temperature-sensitive male sterility characteristics to temperature conditions sufficient for normal pollen development in the middle and late stages of anther and pollen development, and / or subjecting corn plants containing corn event MIR162-related loci and having temperature-sensitive male sterility characteristics to temperature conditions sufficient for pollen abortion in the middle and late stages of anther and pollen development, wherein the temperature conditions sufficient to cause pollen abortion refer to conditions where the average daily temperature is higher than 26.5°C for three consecutive days, wherein the corn plants containing the corn event MIR162-related loci are homozygous for the MIR162-related loci.

10. The method according to claim 9, wherein the temperature condition sufficient to cause pollen abortion refers to a condition in which the average daily temperature is higher than 27°C for 3 consecutive days.

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