Thermo-sensitive sterility characteristics of MIR162 transgenic corn and its related applications

Abstract

The present application relates to the new discovery of thermo-sensitive male flower sterility characteristics of transgenic corn event MIR162, and its application in parental reproduction, backcross breeding, pre-breeding and two-line hybrid production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the technology of corn biological trait development, and more particularly to MIR162 seed production technology. The present invention discovers that the maize event MIR162-related gene loci are associated with temperature-sensitive male sterility and its application in parental reproduction, backcross breeding, 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 against aboveground pests for many of Syngenta's best-selling hybrids, 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 the applicant deposited corn seeds of event MIR162 with the American Type Culture Collection (ATCC) at 1801 University Boulevard, Manassas, VA 20110 under the Budapest Treaty on January 23, 2007, 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 can be further used to guide parental propagation, backcross breeding, 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, backcross breeding and pre-breeding of these MIR162 materials with a specific genetic background, thereby reducing seed production costs; (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 female parents to develop maize two-line hybrid seed production technology. 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. Backcross breeding 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 excellent parents with transgenic traits as basic materials, thereby eliminating the later breeding process of introducing transgenic target traits through backcrossing.

[0005] In the present invention, the male flowers of the donor inbred lines of a group of 12 transgenic events MIR162 covering multiple corn heterotic groups were confirmed to have temperature-sensitive sterility characteristics, that is, these MIR162 materials with a specific genetic background had no pollen or little pollen under high temperature conditions. The critical temperature for fertility conversion of the corn material MIR162 involved in the present invention is 26±1°C, and the fertility fluctuation period is between 25~27°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 began to show partial male sterility at 26°C, and the sterility rate increased 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 in specific germplasm containing a locus associated with the maize event MIR162. The sensitive period is the mid-to-late developmental stage (4-2 leaves) of maize male organs (anthers and pollen). During this period, exposure to a critical high temperature of 26.5°C, preferably 27°C, for three consecutive days can induce sterility. Normal seed set occurs when the average daily temperature exceeds 25.5°C, preferably 25°C, for three consecutive days. This locus is recessive, and temperature-sensitive male sterility is manifested only when the inbred line is homozygous for this locus. When heterozygous, pollen can be shed normally, similar to the wild type. The present invention further relates to a method for maize plants carrying the locus associated with the maize event MIR162 in a specific genetic background. The method can effectively restore the fertility of MIR162 materials by selecting suitable low-temperature environments (e.g., spring maize areas above 1300 meters in Yunnan and winter maize areas between 18-23°N) and sowing windows, thereby increasing the success rates of parental reproduction, backcrossing, and pre-seed breeding, while reducing seed production risks and costs. 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 Uygur Autonomous Region) and optimal sowing windows can be selected. These MIR162 materials with specific genetic backgrounds can be used as female parents to exploit the characteristics of high-temperature sterility to develop new two-line hybrid seed production technologies.

[0006] From 2019 to 2022, multi-year, multi-site field testing and seed propagation were conducted using specific inbred lines containing loci associated with the maize event MIR162 (Tables 1 and 2). These lines exhibited male sterility under high-temperature testing conditions. This characteristic is that the florets and glumes of the tassels remain closed, and the pollen is mostly or completely sterile under high-temperature conditions. For example, batches 1-3 and 7-10, sown during the hot seasons of Sanya and Nanning, and batches 12-13 and 15-21, sown during summer in Beijing and spring in Yongji (during the 2021 high-temperature season in Jilin), all showed no pollen. However, under low-temperature testing conditions, the tassels shed pollen normally. For example, batch 5 was sown in spring in Pengpu, Yunnan; batches 4 and 6 were sown in winter in Binglang, Hainan; batch 14 was sown in early spring under film mulching in Shunyi; and batch 21, an extremely early maturing line, was sown in spring in Yongji, Jilin. Table 2 shows the pollen performance of six lines with similar genetic backgrounds and different traits under high-temperature conditions. BtGT (Bt11 / GA21) and GA21 were fertile under high temperature conditions, but only ZL (MIR162 / Bt11 / GA21) was sterile at high temperature. It can be inferred that the temperature-sensitive sterility was caused by a locus related to 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 discovered that specific MIR162 inbred lines exhibit temperature-sensitive male sterility. These inbred lines, containing loci associated with the maize event MIR162, become male sterile starting at 26°C, and the sterility rate increases significantly with increasing temperature. However, high temperatures do not affect silk fertility.

[0012] The sensitive period for fertility conversion occurs in the middle to late stages of maize male organ development (anthers and pollen) (from the fourth to second leaf). This anther and pollen development period typically occurs two weeks before pollination. Exposure to high temperatures during this period will cause pollen to abort; however, normal seed set can occur under low temperatures. This trait is controlled by a recessive gene, and thermosensitive sterility is only expressed in inbred lines homozygous for this locus. Heterozygous lines can shed pollen normally, similar to wild-type lines.

[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 findings of the inventors, in one aspect, the present invention provides a method for producing corn plants or seeds (e.g., corn hybrid seed production), comprising: hybridizing a corn plant comprising a corn event MIR162-related locus having temperature-sensitive male sterility characteristics as a female parent with another corn plant having desired traits as a male parent, 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, prior to the hybridization, 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, a condition in which the average daily temperature is higher than 26.5°C, more preferably higher than 27°C, for three consecutive days). 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 for three consecutive days, more preferably greater than 25°C) 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 temperature-sensitive male sterility trait 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, the average daily temperature is higher than 26.5°C, more preferably higher than 27°C, for three consecutive days). 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, at a temperature 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 for three consecutive days, more preferably greater than 25°C) for parental reproduction, 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 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 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 the middle and late stages of anther and pollen development under temperature conditions sufficient for normal pollen development (for example, at a temperature 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 for three consecutive days, more preferably greater than 25°C).

[0017] In another aspect, the present invention provides a use of a corn plant comprising a corn event MIR162-related locus having a temperature-sensitive male sterility trait for parental propagation, backcross breeding or pre-breeding, 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, 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, a condition in which the average daily temperature is not greater than 25.5°C, more preferably greater than 25°C for three consecutive days).

[0018] In another aspect, the present invention provides a method for growing corn plants, which comprises placing corn plants having thermosensitive male sterility characteristics and comprising a corn event MIR162-related locus under temperature conditions sufficient for normal pollen development in the middle and late stages of anther and 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), and / or placing corn plants having thermosensitive male sterility characteristics and comprising a corn event MIR162-related locus under 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 placed under temperature conditions sufficient to cause pollen abortion in the middle and late stages of anther and pollen development (for example, under temperature conditions higher 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 higher than 26.5°C, more preferably higher than 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 those aspects described above, the temperature is usually not higher than the upper temperature limit for anther and pollen grain development, such as 38°C; and the temperature is usually not lower than the lower temperature limit for anther and pollen grain development, such as 16°C.

[0020] Locus: refers to a chromosomal region or chromosomal location where a polymorphic nucleic acid, trait-determining factor, 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 encompasses one or more polymorphisms in a population (e.g., the presence of alternative alleles in certain individuals).

[0021] Corn Event MIR162-Associated Locus: refers to the DNA sequence involved in the recombinant construct inserted into the corn genome that produces the MIR162 event and the corn 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 2020-2021.

[0025] Figure 1d . Average daily temperature in Pengpu 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 2022 Pengpu and Areca AX5707ZL has loose powder and firmness.

[0029] Figure 3 . The loose powder and strong texture of the 2022 KFX5360ZL of Pengpu and Areca.

[0030] Figure 4 . 2022 Ponpu and Areca MFA7191ZL has a loose powder and a strong finish.

[0031] Figure 5 . The loose powder and firmness of GAB2601ZL of 2022 Pengpu and Areca.

[0032] Figure 6 . The loose powder and firmness of IAF3318ZL of 2022 Pengpu and Areca.

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

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

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

[0036] Figure 10 . The loose powder and strong texture of the THD28ZL of 2022 Pengpu and Areca.

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

[0038] Figure 12 . 2022 Pengpu and Areca ID3606ZL has loose powder and firmness.

[0039] Figure 13 . The loose powder and firmness of the 2022 MFA7273ZL of Pompon and Areca.

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

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

[0042] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0043] Example 1. Fertility Study of 12 Different Germplasm ZL Inbred Lines in Areca and Pengpu

[0044] 1. Purpose

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

[0046] 1.2. Verify whether the thermosensitive male sterility trait of the MIR162-related loci can be reproducibly and stably inherited in this group of materials with different genetic backgrounds

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

[0048] 2. Materials and Methods

[0049] 2.1. A set of 12 donor inbred lines from the transgenic event MIR162, covering multiple maize heterotic groups, were backcrossed: AX5707ZL, FF6097ZL, GAB2601ZL, IAF3318ZL, ID3264ZL, ID3477ZL, ID3606ZL, IFA3351ZL, KFX5360ZL, MFA7191ZL, MFA7273ZL, and THD28ZL. ZL is a trivalent stacked trait, derived from a single donor line for the traits MIR162, Bt11, and GA21, transferred to a single target inbred line through conventional backcrossing.

[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 and serves as a selection marker in the screening process.

[0051] Corn event Bt11: This was obtained through a PEG-mediated method and contains the cry1Ab gene from the Bacillus thuringiensis strain HD-1. The expressed Cry1Ab protein is insecticidal against lepidopteran pests such as the European corn borer (Ostrinia nubillis) and the cotton bollworm (Helicoverpa armigera). It also contains the pat gene from the viridochromogenic Streptomyces strain Tü494, which confers tolerance to glufosinate-ammonium herbicides and can also serve as a selectable marker during genetic transformation. For example, corn event Bt11 is described in US Pat.

[0052] Corn event GA21: This event was obtained by biolistic bombardment and contains a modified mEPSPS gene from maize. The expressed mEPSPS protein has reduced affinity for glyphosate compared to the endogenous EPSPS, thus exhibiting tolerance to glyphosate-based herbicides. For example, corn event GA21 is described in US Pat. No. 6,762,344 B1.

[0053] Therefore, the ZL line (containing MIR162, Bt11 and GA21) has the following characteristics: resistance to fall armyworm, Asian corn borer, armyworm, cotton bollworm, black cutworm, tobacco armyworm larvae, sugarcane borer and small corn borer, while being tolerant to glyphosate and glufosinate ammonium.

[0054] 2.2. Experimental Design: Plot size: Two rows of each material, 5 m row length, one replicate.

[0055] 2.3. Experimental locations: Sanya Binglang (high temperature, 18.3°N, 0 m above sea level); Yunnan Pengpu (low temperature, 24°N, 1180 m 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 of anther number on a plot basis):

[0060] 1 = 100% anther extrusion from all tassel spikelets. Anthers are full and pollen count is the same or similar to that of the recurrent parent or other controls.

[0061] 2 = Only 50-75% of the anthers in the entire tassel are extruded from the spikelet. Pollen load is reduced.

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

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

[0064] 3. Experimental Results

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

[0066]

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

[0068]

[0069] Note: The date corresponding to the row where the corresponding ZL inbred line number is located belongs to the anther and pollen development period of the ZL inbred line.

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

[0071]

[0072] Note: The date corresponding to the row where the corresponding ZL inbred line number is located belongs to the anther and pollen development period of the ZL inbred line.

[0073] in conclusion:

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

[0075] Pengpu (24°N, 1100 m) is a potential candidate site for a MIR162 transformation breeding nursery, but there is a risk of annual temperature variability. Yunnan's spring maize region above 1300 m [2,3] and the winter maize region between 18°N and 23°N are recommended as candidate sites for seed propagation and breeding programs containing MIR162 material, backcrossing, and pre-breeding.

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

[0077] 1. Purpose

[0078] Under high temperature environment, the small-scale two-line field trial production of hybrid AZL was taken as an example to explore 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-GM ID3477 was used as the male parent and THD28ZL as the female parent. The male parent ratio was 1:4. The male parent was planted in two phases: 50% was planted 3 days later in the first phase; the remaining 50% was planted 5 days later. The area was 1 mu.

[0082] 2.3. Sowing Location and Window: Areca. Spring sowing ensures that the female plant's florets and anthers are exposed to high temperatures during development. Stagger sowing with other corn projects to reduce contamination from external pollen.

[0083] 2.4. Data Collection: Male fertility scoring, fruit set rate, and photos of peak flowering period, as well as 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 associated with the maize event MIR162 as female parents. The female parent, THD28ZL, produced pollen-free male flowers and was completely sterile. The anthers were few, withered, and shrunken. The number of male spikelets was reduced and thin. Silking was normal. The male parent, ID3477, exhibited normal pollen shedding and abundant pollen. Under both open pollination and artificial pollination (inappropriately delayed, premature male pollination), the female parent, THD28ZL, produced excellent seed set, with nearly full, clean spikes and a yield of 369 kg / mu. High temperatures did not affect the pollen receptivity of the ZL female silks and grain development (see [1]). Figure 14 ).

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

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

[0088] in conclusion:

[0089] In summary, the present invention takes the hybrid AZL two-line hybrid as an example to provide a method for producing two-line corn hybrids by 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 Uygur Autonomous Region, using MIR162 material as the female parent, providing a new option for future corn hybrid production models.

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

[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 the discovery of unstable male flower fertility in a specific MIR162 accession during seed propagation of MIR162, Bt11, and GA21 in 2019. ID3461MIR162 produced normal pollen (scored 1) in the 2019 Shunyi early spring sowing, but significantly reduced pollen (scored 3) in the Yongji backup late sowing, which was exposed to high temperatures. However, ID3461Bt11 and ID3461GA21, both genetically similar, produced normal pollen in both environments (either Shunyi early spring sowing or Yongji backup late sowing), comparable to the non-GM control ID3461 (scored 1). See Table 4 below.

[0094] Table 6

[0095]

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

[0097] Based on extensive experiments, the critical temperature for fertility transition has been determined to be 26 ± 1°C, with a fertility fluctuation period between 25 and 27°C. Temperatures of 25°C and below are clearly fertile, while temperatures of 27°C and above are clearly sterile. Plants containing the locus associated with the maize event MIR162 become male sterile at 26°C, and the sterility rate increases significantly with increasing temperature. Different germplasms have a small influence on the critical fertility temperature.

[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. Therefore, the sterility condition is roughly three consecutive days of average daily temperatures above 26.5°C, preferably 27°C.

[0099] Regarding the 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, respectively (pollen was abundant); and the anther and pollen development period of ID3461MIR162 in Shunyi in 2019 was from 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, respectively. Therefore, it was determined that the fertile condition is roughly no three consecutive days with an average daily temperature greater than 25.5°C, preferably 25°C.

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

[0101] 1. AX5707ZL backcross breeding process (including parental combination, parental sources, breeding methods, generations, and characterization)

[0102] AX5707ZL: A maize inbred line AX5707 (described previously as AX5707 and as NP2222 in US Pat. No. 6710233B2, ATCC Accession No. PTA-3967) was hybridized with DWFA4651ZL (described in US Pat. No. 11051473B2, ATCC Accession No. PTA-126339) as the trait donor, followed by continuous backcrossing with AX5707 as the recurrent parent. After three generations, AX5707 (Bt11×GA21×MIR162) was obtained and self-pollinated for one generation. Genotype testing revealed AX5707(GA21), AX5707(Bt11×GA21) and AX5707(Bt11×GA21×MIR162). After another self-pollination generation, the stable inbred lines AX5707(GA21), AX5707(Bt11×GA21) and AX5707(Bt11×GA21×MIR162) were obtained.

[0103] 2. FF6097ZL backcross breeding process (including parental combination, parental sources, breeding methods, generations, and characterization)

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

[0105] 3. GAB2601ZL backcross breeding process (including parental combination, parental sources, breeding methods, generations, and characterization)

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

[0107] 4. IAF3318ZL backcross breeding process (including parental combination, parental origin, 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 for one generation of hybridization. Then, IAF3318 was used as the recurrent parent for three consecutive backcross generations to obtain IAF3318 (Bt11×GA21×MIR162), which was then self-pollinated for one generation. Genotype testing revealed IAF3318(GA21), IAF3318(Bt11×GA21), and IAF3318(Bt11×GA21×MIR162). After self-pollinated for one generation, the stable inbred lines IAF3318(GA21), IAF3318(Bt11×GA21), and IAF3318(Bt11×GA21×MIR162) were obtained.

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

[0110] ID3264ZL: The ID3264 maize inbred line (described in US Pat. No. 7,323,626B1 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 backcrossed for three consecutive generations with ID3264 as the recurrent parent to obtain ID3264ZL (Bt11×GA21×MIR162). After one generation of self-pollination, genotype testing revealed ID3264(GA21), ID3264(Bt11×GA21), and ID3264(Bt11×GA21×MIR162). 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 parental combination, parental origin, 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 (Bt11×GA21×MIR162) was obtained by backcrossing for three consecutive generations with ID3477 as the recurrent parent. After one generation of self-pollination, ID3477(GA21), ID3477(Bt11×GA21) and ID3477(Bt11×GA21×MIR162) were obtained through genotype testing. After one generation of self-pollination, the stable inbred lines ID3477(GA21), ID3477(Bt11×GA21) and ID3477(Bt11×GA21×MIR162) were obtained respectively.

[0113] 7. ID3606ZL backcross breeding process (including parental combination, parental origin, breeding method, generations, and characterization)

[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 was used as the trait donor. After one generation of hybridization, ID3606 was used as the recurrent parent by backcrossing for three consecutive generations to obtain ID3606 (Bt11×GA21×MIR162), which was then self-pollinated for one generation. Genotype testing revealed ID3606(GA21), ID3606(Bt11×GA21), and ID3606(Bt11×GA21×MIR162). 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 parental combination, parental origin, breeding method, generations, and characterization)

[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 (Bt11×GA21×MIR162) was obtained by backcrossing for three consecutive generations with IFA3351 as the recurrent parent. After one generation of self-pollination, IFA3351(GA21), IFA3351(Bt11×GA21) and IFA3351(Bt11×GA21×MIR162) were obtained through genotype testing. After one generation of self-pollination, the stable inbred lines IFA3351(GA21), IFA3351(Bt11×GA21) and IFA3351(Bt11×GA21×MIR162) were obtained respectively.

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

[0118] KFX5360ZL: The KFX5360 maize inbred line (described in US2019297814A1) was used as the female parent and DWFA4651ZL as the trait donor. After one generation of hybridization, KFX5360 (Bt11×GA21×MIR162) was obtained by backcrossing for three consecutive generations with KFX5360 as the recurrent parent and self-pollinating for one generation. Genotype testing revealed KFX5360(GA21), KFX5360(Bt11×GA21) and KFX5360(Bt11×GA21×MIR162), which were then self-pollinated for one generation to obtain the stable inbred lines KFX5360(GA21), KFX5360(Bt11×GA21) and KFX5360(Bt11×GA21×MIR162), respectively.

[0119] 10. MFA7191ZL backcross breeding process (including parental combination, parental origin, breeding method, generations and characteristics description, etc.)

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

[0121] 11. MFA7273ZL backcross breeding process (including parental combination, parental origin, breeding method, generations and characteristics description, etc.)

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

[0123] 12. THD28ZL backcross breeding process (including parental combination, parental sources, breeding methods, generations and characteristics description, etc.)

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

[0125] 13. NP5024ZL backcross breeding process (including parental combination, parental origin, breeding method, generations and characteristics 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 (Bt11×GA21×MIR162) was obtained by backcrossing with NP5024 as the recurrent parent for three consecutive generations. After one generation of self-pollination, NP5024(GA21), NP5024(Bt11×GA21) and NP5024(Bt11×GA21×MIR162) were obtained through genotype testing. After one generation of self-pollination, the stable inbred lines NP5024(GA21), NP5024(Bt11×GA21) and NP5024(Bt11×GA21×MIR162) were obtained respectively.

[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 in maize. Chinese Agricultural Science Bulletin, Vol. 15, No. 5, 1999: 4-10

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

Claims

1. A method of producing a corn plant or seed comprising: The event MIR162 corn plant or ZL inbred corn plant having temperature-sensitive male sterility as the female parent is hybridized with other corn plants having desired traits as the male parent, wherein the ZL inbred corn plant is a corn plant having only three events, MIR162, Bt11 and GA21, wherein the event MIR162 corn plant or ZL inbred corn plant is homozygous for the MIR162-related locus, and wherein before the hybridization, the event MIR162 corn plant or ZL inbred corn plant is placed under temperature conditions sufficient to cause pollen sterility in the middle and late stages of anther and pollen development, and the temperature conditions sufficient to cause pollen sterility 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 three consecutive days.

3. The method according to claim 1 or 2, wherein the event MIR162 corn plant or the ZL inbred corn plant is placed in the middle and late stages of the anther and pollen development period under temperature conditions sufficient for normal pollen development for parental reproduction, thereby providing the required amount of parents for the hybridization.

4. Use of event MIR162 corn plants or ZL inbred corn plants having thermosensitive male sterility as female parents in producing corn plants or seeds, wherein the ZL inbred corn plants are corn plants having only three events, MIR162, Bt11 and GA21, wherein the event MIR162 corn plants or ZL inbred corn plants are homozygous for the MIR162-related loci, and before the process of producing corn plants or seeds, the event MIR162 corn plants or ZL inbred corn plants are placed in a temperature condition sufficient to cause pollen sterility in the middle and late stages of anther and pollen development, and the temperature condition sufficient to cause pollen sterility refers to a condition in which the average daily temperature is higher than 26.5°C for three consecutive days.

5. The use according to claim 4, 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 three consecutive days.

6. The use according to claim 4 or 5, wherein the event MIR162 corn plant or ZL inbred corn plant is placed in the middle and late stages of anther and pollen development under temperature conditions sufficient for normal pollen development for parental propagation, 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 an event MIR162 corn plant or a ZL inbred corn plant having thermosensitive male sterility to a parental propagation, backcrossing or pre-breeding process, wherein the ZL inbred corn plant is a corn plant having only three events, MIR162, Bt11 and GA21, wherein the event MIR162 corn plant or the ZL inbred corn plant is homozygous for the MIR162-related locus, and wherein the event MIR162 corn plant or the ZL inbred corn plant 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 pollen development refers to a condition in which the average daily temperature is not greater than 25°C for three consecutive days.

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