Method for creating pop corn inbred line

Abstract

The invention provides a method for creating a pop corn inbred line, and belongs to the technical field of new variety cultivation. The invention provides a method for creating a pop corn inbred line, which comprises the following steps: selecting pop corn germplasm to hybridize with other types of corns to obtain F1-generation seeds; burst corn germplasm is used as a female parent for backcrossing, then BC1F2 or BC2F2 seeds are obtained through planting and selfing, and bursting type grain seeds are selected; hybridizing the burst type grain seeds with a maize haploid induction line containing Ga1-s; hybridized clusters are taken after pollination, embryos are separated and cultured, and haploid embryos with haploid characteristics are selected; inducing the haploid embryo into a seedling, then inducing chromosome doubling, and selfing to obtain the pop corn selfing line. By utilizing the method, the pop corn inbred line can be efficiently, accurately and quickly created, the generation probability of haploid false positive is greatly reduced, the identification efficiency is remarkably improved, and the breeding cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of new variety breeding, and in particular relates to a method for creating a popcorn inbred line. Background Art

[0002] Popcorn (Zea mays L. var. everta Sturt.) is a special type of corn that is distinguished from other types of corn by its compact cobs and smaller kernels. The notable feature of its kernels is that they are almost entirely composed of hard keratin starch and come in a variety of colors, including white, yellow, or purple. They are mainly divided into two types: rice-type and pearl-type. At the right moisture content, popcorn kernels can expand rapidly during heating, and the rapid increase in internal pressure causes the seed coat to rupture, eventually forming a butterfly-shaped or spherical puffed structure. This characteristic makes it the only raw material for producing popcorn. Popcorn has a high nutritional value and is rich in protein, dietary fiber, inorganic salts, and multiple vitamins.

[0003] The cultivated varieties of popcorn are generally corn hybrids, and in the field of breeding, the creation and selection of excellent inbred lines are the basis for the preparation of high-quality popcorn hybrids. Haploid breeding technology can accelerate the acquisition of pure corn, but in popcorn, since the general popcorn germplasm contains the Ga1-s gene, it leads to partial reproductive isolation. In addition, the expression of the commonly used haploid induction line marker R1-nj in popcorn is completely different from that of other types of corn, which makes the efficiency of obtaining pure popcorn by haploid breeding extremely low. In the production process, a large number of false positive materials are also easily produced, which consumes a lot of costs. Therefore, breeders usually need to use the traditional two-ring system breeding method, and after more than seven generations of screening and selfing, they can obtain homozygous popcorn inbred lines. This process is not only time-consuming and labor-intensive, but also inefficient. In view of this, it is particularly important to improve the accuracy of popcorn haploid identification, which can significantly improve the breeding efficiency of popcorn. By improving the accuracy of identification technology, ineffective work can be reduced, resources can be saved, and the breeding process of excellent varieties can be accelerated. Summary of the invention

[0004] The invention provides a method for creating a popcorn inbred line, which can efficiently, accurately and quickly create a popcorn inbred line, and can more quickly realize the improvement of germplasm and the development of new varieties.

[0005] The present invention provides a method for creating a popcorn inbred line, comprising the following steps: (1) hybridizing popcorn germplasm with non-popcorn germplasm to obtain F1 generation seeds;

[0006] (2) using the popcorn germplasm as the female parent to perform backcrossing for 1 to 2 generations, planting and self-pollinating to obtain BC1F2 or BC2F2 seeds, and selecting popcorn seeds from the BC1F2 or BC2F2 seeds;

[0007] (3) hybridizing the bursting-type seeds selected in step (2) with a haploid induction line of maize containing Ga1-s, taking hybrid ears 28 to 33 days after pollination, separating embryos for culture, and selecting haploid embryos showing haploid characteristics;

[0008] (4) Cultivating the haploid embryos of step (3) into seedlings, doubling their chromosomes, and then self-pollinating to obtain popcorn inbred lines.

[0009] In a preferred embodiment of the present invention, in step (1), popcorn germplasm is used as the male parent, and non-popcorn germplasm is used as the female parent.

[0010] In a preferred embodiment of the present invention, when hybridization is performed in step (3), the plant grown from the selected bursting-type seeds is used as the female parent, and the maize haploid induction line containing Ga1-s is used as the male parent.

[0011] In a preferred embodiment of the present invention, the maize haploid induction line containing Ga1-s includes the maize haploid induction line SPIL01.

[0012] In a preferred embodiment of the present invention, the culturing in step (3) comprises inoculating the separated embryos onto MS culture medium for tissue culture and culturing them under full illumination for 48 hours.

[0013] In a preferred embodiment of the present invention, after the culture in step (3), the uncolored embryos are selected, which are haploid embryos with haploid characteristics.

[0014] In a preferred embodiment of the present invention, the culture medium used for culturing seedlings in step (1) comprises 1 / 2MS culture medium; and when culturing seedlings, the illumination time is 10 hours / day and the temperature is 26°C.

[0015] In a preferred embodiment of the present invention, step (4) comprises culturing the haploid embryos to the 2-3 leaf stage and inducing chromosome doubling using colchicine.

[0016] In a preferred embodiment of the present invention, the colchicine-induced chromosome doubling comprises soaking the roots of the 2-3 leaf stage haploid seedlings in a colchicine solution for 14 hours; then rinsing with clean water and transplanting to a field environment.

[0017] The present invention also provides the use of the popcorn inbred line created by the above method as a parent in breeding new popcorn varieties.

[0018] Beneficial effects: The present invention provides a method for creating a popcorn inbred line, comprising selecting popcorn germplasm and hybridizing it with other types of corn (including temperate or tropical non-popcorn) to obtain F1 generation seeds; using the popcorn germplasm as the female parent for 1 to 2 generations of backcrossing, planting and self-pollinating to obtain BC1F2 or BC2F2 seeds, and selecting popcorn seeds; hybridizing the popcorn seeds with a haploid induction line of corn containing Ga1-s; taking hybrid ears 28 to 33 days after pollination, separating embryos for cultivation, and selecting haploid embryos showing haploid characteristics; inducing the haploid embryos into seedlings and then inducing chromosome doubling to obtain the popcorn inbred line.

[0019] The method of the present invention can be used to efficiently, accurately and quickly create popcorn inbred lines. The method can be used to more quickly improve germplasm improvement and develop new varieties, and the probability of haploid false positives is greatly reduced, thereby significantly improving identification efficiency and reducing breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a color picture of the embryos 28 days after pollination of the separation and induction lines after 48 hours of culture under full light;

[0021] Figure 2 This is a picture of the young embryos 15 days after pollination for separation and hybridization with the induced line, which did not show color after being cultured in full light for 10 days. DETAILED DESCRIPTION

[0022] The present invention provides a method for creating a popcorn inbred line, comprising the following steps: (1) hybridizing popcorn germplasm with non-popcorn germplasm to obtain F1 generation seeds;

[0023] (2) using the popcorn germplasm as the female parent to perform backcrossing for 1 to 2 generations, planting and self-pollinating to obtain BC1F2 or BC2F2 seeds, and selecting popcorn seeds from the BC1F2 or BC2F2 seeds;

[0024] (3) hybridizing the bursting-type seeds selected in step (2) with a haploid induction line of maize containing Ga1-s, taking hybrid ears 28 to 33 days after pollination, separating embryos for culture, and selecting haploid embryos showing haploid characteristics;

[0025] (4) Cultivating the haploid embryos of step (3) into seedlings, doubling their chromosomes, and then self-pollinating to obtain popcorn inbred lines.

[0026] The present invention uses popcorn germplasm and non-popcorn germplasm as parents for hybridization to obtain F1 generation seeds. In a preferred embodiment of the present invention, popcorn germplasm is used as the male parent and non-popcorn germplasm is used as the female parent. The popcorn germplasm can be a hybrid, an inbred line or a local species; the non-popcorn germplasm can be a temperate or tropical non-popcorn. For example, in one embodiment of the present invention, the popcorn inbred line SPL02 is selected as the male parent to be hybridized with the temperate dent corn Mo17; in another embodiment, the popcorn hybrid Shenkebao No. 8 is selected as the male parent to be hybridized with the temperate dent corn B73; in another embodiment, the popcorn inbred line SPL02 is selected as the male parent to be hybridized with the tropical flint corn CML32. The various germplasms used in the embodiments of the present invention can be obtained by the following methods: corn inbred lines B73, CML32 and Mo17 can all be obtained from the National Crop Germplasm Bank; the popcorn inbred line SPL02 was bred by the Special Corn Research Center of Shanghai Academy of Agricultural Sciences and has been disclosed in the literature (Yu, D., Wang, H., Gu, W. et al. Genetic diversity and population structure of popcorn germplasm resources using genome-wide SNPs through genotyping-by-sequencing. Genet Resour Crop Evol 68, 2379-2389 (2021).).

[0027] The invention uses popcorn germplasm as a female parent, and after 1-2 generations of backcrossing with pillars grown from F1 generation seeds, planting and self-pollination to obtain BC1F2 or BC2F2 seeds, and selecting popcorn seeds. The selection of the invention includes selecting rice-grain or pearl-shaped popcorn seeds with relatively small embryos, high endosperm keratin starch content, and hard texture.

[0028] The present invention hybridizes the pillars grown from bursting-type seed with the maize haploid induction line containing Ga1-s to induce haploids, wherein the selected plants grown from bursting-type seed are used as the female parent, and the maize haploid induction line containing Ga1-s is used as the male parent. In one embodiment of the present invention, the maize haploid induction line containing Ga1-s includes the maize haploid induction line SPIL01, which is cultivated by the Special Maize Research Center of Shanghai Academy of Agricultural Sciences and has an R1-nj color mark.

[0029] In the present invention, when inducing haploids, hybrid ears are taken 28 to 33 days after the hybrid pollination, and the separated embryos are inoculated on MS culture medium for tissue culture, and cultured under full light conditions for 48 hours, and haploid embryos with haploid characteristics are selected. In one embodiment of the present invention, the separated embryos are inoculated on MS culture medium for tissue culture, and cultured under full light conditions for 48 hours. The embryos that do not show color are haploid embryos ( Figure 1 ). The area of ​​pigmentation greater than 0% is considered to be pigmented, and the area with no pigmentation at all is considered to be non-pigmented.

[0030] The present invention further cultured the screened haploid embryos into seedlings under normal light cycle conditions, wherein the normal light cycle was 10 hours of light / 14 hours of darkness, the light intensity was 1500-1800 lux, and the temperature was 26°C.

[0031] The present invention doubles chromosomes of cultivated seedlings, such as using colchicine to induce chromosome doubling. In one embodiment, the roots of haploid seedlings at the 2-3 leaf stage are soaked in a 0.04% colchicine solution for 14 hours, rinsed with clean water, and then transplanted to a field environment.

[0032] The present invention also includes genotyping the transplanted plants, such as molecular markers, using low-density liquid phase chips based on targeted sequencing genotyping (Genotyping by Targeted Sequencing, GBTS) technology. The present invention obtains popcorn inbred lines by self-pollination of the identified positive materials, which can be used as parent materials for breeding new popcorn varieties.

[0033] The present invention also provides the use of the popcorn inbred line created by the above method as a parent in breeding new popcorn varieties.

[0034] In order to further illustrate the present invention, a method for creating a popcorn inbred line provided by the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Select popcorn inbred line SPL02 as the male parent and cross it with temperate dent corn Mo17 to obtain F1 generation seeds;

[0037] 2. Popcorn inbred line SPL02 was used as the female parent to cross with the hybrid F1 generation to obtain BC1F1 seeds, which were planted and self-fertilized to obtain BC1F2 seeds, from which popcorn seeds were selected;

[0038] 3. Hybridize the popping kernel seeds with the maize haploid inducer line SPIL01 containing Ga1-s;

[0039] 4. Take the hybrid ears 28 days after pollination, separate the embryos and inoculate them into MS medium for tissue culture. Culture them under full light conditions for 48 hours, and select the uncolored embryos as haploid embryos;

[0040] 5. The haploid embryos were continued to be cultured into seedlings under normal photoperiod (10 hours light / 14 hours dark);

[0041] 6. The roots of the haploid seedlings at the 2-3 leaf stage were immersed in a 0.04% colchicine solution for 14 hours, rinsed with clean water, and transplanted to the field. The genotypes were detected using molecular markers, and the identified haploid materials were self-pollinated to obtain popcorn inbred lines, which can be used as parent materials for breeding new popcorn varieties.

[0042] Example 2

[0043] 1. Select the popcorn hybrid Shenkebao 8 as the male parent and cross it with temperate dent corn B73 to obtain F1 generation seeds;

[0044] 2. After hybridizing the popcorn hybrid Shenkebao 8 as the female parent with the hybrid F1 generation, BC1F1 seeds were obtained, which were planted and then backcrossed with Shenkebao 8 as the parent to obtain BC2F1 seeds. After planting, BC2F1 seeds were self-pollinated and the popcorn seeds were selected;

[0045] 3. Hybridize the popping kernel seeds with the maize haploid inducer line SPIL01 containing Ga1-s;

[0046] 4. Take the hybrid ears 30 days after pollination, separate the embryos and inoculate them into MS medium for tissue culture. Culture them under full light conditions for 48 hours, and select the embryos that do not show color, which are haploid embryos;

[0047] 5. The haploid embryos were continued to be cultured into seedlings under normal photoperiod (10 hours light / 14 hours dark);

[0048] 6. The roots of the haploid seedlings at the 2-3 leaf stage were immersed in a 0.04% colchicine solution for 14 hours, rinsed with clean water, and transplanted to the field. The genotypes were detected using molecular markers, and the identified haploid materials were self-pollinated to obtain popcorn inbred lines, which can be used as parent materials for breeding new popcorn varieties.

[0049] Example 3

[0050] 1. Select the popcorn inbred line SPL02 as the male parent and cross it with tropical hard-kernel corn CML32 to obtain F1 generation seeds;

[0051] 2. Popcorn inbred line SPL02 was used as the female parent to cross with the hybrid F1 generation to obtain BC1F1 seeds, which were planted and self-fertilized to obtain BC1F2 seeds, from which popcorn seeds were selected;

[0052] 3. Hybridize the popping kernel seeds with the maize haploid inducer line SPIL01 containing Ga1-s;

[0053] 4. Take the hybrid ears 28 days after pollination, separate the embryos and inoculate them into MS medium for tissue culture. Culture them under full light conditions for 48 hours, and select the uncolored embryos as haploid embryos;

[0054] 5. The haploid embryos were continued to be cultured into seedlings under normal photoperiod (10 hours light / 14 hours dark);

[0055] 6. The roots of the haploid seedlings at the 2-3 leaf stage were immersed in a 0.04% colchicine solution for 14 hours, rinsed with clean water, and transplanted to the field. The genotypes were detected using molecular markers, and the identified haploid materials were self-pollinated to obtain popcorn inbred lines, which can be used as parent materials for breeding new popcorn varieties.

[0056] Comparative Example 1

[0057] 1. Select popcorn inbred line SPL02 as the male parent and cross it with temperate dent corn Mo17 to obtain F1 generation seeds;

[0058] 2. Popcorn inbred line SPL02 was used as the female parent to cross with the hybrid F1 generation to obtain BC1F1 seeds, which were planted and self-fertilized to obtain BC1F2 seeds, from which popcorn seeds were selected;

[0059] 3. Hybridize the popping kernel seeds with the maize haploid inducer line SPIL01 containing Ga1-s;

[0060] 4. According to the traditional method, the immature embryos 12 to 21 days after pollination were peeled off (Chen, C., Zhang, Y., Fu, X. et al. Influential factors and transcriptome analyses of immature diploid embryoanthocyanin accumulation in maize. BMC Plant Biol 22, 609 (2022). https: / / doi.org / 10.1186 / s12870-022-03971-5), and the immature embryos 15 days after pollination were selected and isolated, inoculated into MS medium for tissue culture, and cultured under full light conditions for 48 hours. The embryos did not show color. After continuing to culture for 10 days ( Figure 2), select embryos that have not developed color;

[0061] 5. The embryos that did not develop color continued to be cultured into seedlings under normal light cycle (10 hours light / 14 hours dark);

[0062] 6. The roots of the seedlings at the 2-3 leaf stage were soaked in a 0.04% colchicine solution for 14 hours, rinsed with clean water, and transplanted to the field. The genotypes were detected using molecular markers, and the identified haploid materials were self-pollinated to obtain popcorn inbred lines, which can be used as parent materials for breeding new popcorn varieties.

[0063] Comparative Example 2

[0064] 1. Select popcorn inbred line SPL02 as the male parent and cross it with temperate dent corn Mo17 to obtain F1 generation seeds;

[0065] 2. Popcorn inbred line SPL02 was used as the female parent to cross with the hybrid F1 generation to obtain BC1F1 seeds, which were planted and self-fertilized to obtain BC1F2 seeds, from which popcorn seeds were selected;

[0066] 3. Hybridize the popping kernel seeds with the maize haploid inducer line SPIL01 containing Ga1-s;

[0067] 4. Following the same tradition as above, after the grains are fully mature, the haploid grains are selected by color marking and germinated;

[0068] 5. The roots of the seedlings at the 2-3 leaf stage were soaked in a 0.04% colchicine solution for 14 hours, rinsed with clean water, and transplanted to the field. The genotypes were detected using molecular markers, and the identified haploid materials were self-pollinated to obtain popcorn inbred lines, which can be used as parent materials for breeding new popcorn varieties.

[0069] Data statistics were performed on Examples 1 to 3 and Comparative Examples 1 to 2, and the results are shown in Table 1. The method of the present invention can significantly reduce the haploid false positive rate, thereby reducing the cost of haploid breeding of popcorn and breaking the technical limitations of haploid breeding of popcorn.

[0070] Table 1 Data statistics of embodiments and comparative examples

[0071]

[0072] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for creating an inbred line of popcorn, characterized in that: The steps include: (1) hybridizing popcorn germplasm with non-popcorn germplasm to obtain F1 generation seeds; (2) using the popcorn germplasm as the female parent to perform backcrossing for 1 to 2 generations, planting and self-pollinating to obtain BC1F2 or BC2F2 seeds, and selecting popcorn seeds from the BC1F2 or BC2F2 seeds; (3) hybridizing the bursting-type seeds selected in step (2) with a haploid induction line of maize containing Ga1-s, taking hybrid ears 28 to 33 days after pollination, separating embryos for culture, and selecting haploid embryos showing haploid characteristics; (4) Cultivating the haploid embryos of step (3) into seedlings, doubling their chromosomes, and then self-pollinating to obtain popcorn inbred lines.

2. The method according to claim 1, characterized in that: In step (1), popcorn germplasm is used as the male parent, and non-popcorn germplasm is used as the female parent.

3. The method according to claim 1, characterized in that: When hybridization is performed in step (3), the plant grown from the selected bursting-type seeds is used as the female parent, and the maize haploid induction line containing Ga1-s is used as the male parent.

4. The method according to claim 3, characterized in that: The maize haploid induction line containing Ga1-s includes the maize haploid induction line SPIL01.

5. The method according to claim 1, characterized in that: The culturing in step (3) comprises inoculating the separated embryos onto MS medium for tissue culture and culturing them under full light conditions for 48 hours.

6. The method according to claim 5, characterized in that: After the culture in step (3), the embryos that have not developed color are selected, which are haploid embryos with haploid characteristics.

7. The method according to claim 1, characterized in that: The culture medium used for culturing the seedlings in step (1) includes 1 / 2MS culture medium; and when culturing the seedlings, the lighting time is 10 hours / day and the temperature is 26°C.

8. The method according to claim 1, characterized in that: Step (4) includes culturing the haploid embryos to the 2-3 leaf stage and inducing chromosome doubling using colchicine.

9. The method according to claim 8, characterized in that: The colchicine-induced chromosome doubling comprises soaking the roots of the 2-3 leaf stage haploid seedlings in a colchicine solution for 14 hours; then washing with clean water and transplanting to a field environment.

10. Use of the popcorn inbred line produced by the method according to any one of claims 1 to 9 as a parent in breeding new popcorn varieties.

Images