CN112616649B - Rapid cultivation method of transgenic maize inbred line by efficient nitrogen utilization - Google Patents
Rapid cultivation method of transgenic maize inbred line by efficient nitrogen utilization Download PDFInfo
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Abstract
The invention discloses a method for quickly cultivating a transgenic maize inbred line with high nitrogen utilization efficiency, which comprises the steps of evaluating the nitrogen utilization efficiency of a first filial generation under the condition of nitrogen stress of a nutrient pool, selecting a transformant with the optimal nitrogen utilization efficiency and a single hybrid plant thereof, carrying out backcross transformation and inbred line cultivation in a targeted manner, and eliminating low-efficiency transformant materials and backcross progeny thereof by evaluating the nitrogen utilization efficiency of the transformant in a preposed manner, thereby greatly reducing the workload, simultaneously enlarging the planting scale of selected transformant materials and facilitating subsequent selection. The method comprises the steps of performing assignment according to the similarity degree of a certain character and recurrent parent characters by adopting 'nitrogen stress + large group + phenotype-genotype correlated selection', accumulating the assignments of a plurality of characters, performing phenotype-genotype correlated selection on a single plant according to the integral height, performing gene locus homozygous selection after continuous 3-4 representative type comprehensive identification, and obtaining backcross offspring with the background recovery rate of more than 99% 2 years in advance.
Description
Technical Field
The invention belongs to the field of genetic breeding, and particularly relates to a rapid breeding method of a transgenic maize inbred line by efficient nitrogen utilization.
Background
Corn belongs to high-yield crops and plays an important strategic position in food production in China and even the world. However, in recent years, to ensure high and stable yield of corn, the amount of fertilizer used in corn production in China is large, and ecological environmental problems such as soil hardening and acidification, water eutrophication, acid rain increase and the like are caused. Improves the nitrogen utilization efficiency of the corn, can effectively reduce the application amount of the fertilizer, reduces the environmental pollution and reduces the production cost. The existing nitrogen high-efficiency utilization type corn germplasm resources in China are extremely deficient, and the novel nitrogen high-efficiency utilization type corn variety is cultivated through a transgenic technology, so that very remarkable economic, social and ecological benefits can be generated.
The cultivation of the new transgenic corn variety with high nitrogen utilization efficiency comprises two links: firstly, the breeding of transgenic inbred line by utilizing nitrogen with high efficiency and secondly, the preparation of transgenic hybrid by utilizing nitrogen with high efficiency. In the breeding link of the transgenic maize inbred line with high nitrogen utilization, breeders need to adopt gene transformation methods such as an agrobacterium-mediated method, a gene gun method, a pollen tube channel method and the like to randomly introduce a nitrogen high-efficiency utilization gene (target gene) into receptor maize with higher conversion power, but common receptor plants do not have excellent agronomic and economic characters, the combining ability is low, and the direct application to the combination of hybrid seeds is difficult, therefore, the nitrogen high-efficiency utilization gene (target gene) is introduced into the corn of a receptor and then needs to be transferred by backcross, the nitrogen high-efficiency utilization gene (target gene) is introduced into a corn bone dry line (a parent of the corn hybrid seeds applied in large area in production), and the field verification shows that, after the high-efficiency nitrogen utilization performance evaluation is carried out on the transgenic maize skeleton line obtained by backcross transformation, hybrid combination is preferentially carried out and finally maize varieties with production value are screened out.
The existing backcross transformation process usually needs more than 6-7 generations of backcross and then 3 generations of selfing to obtain the transgenic version bone stem line with the genetic background highly similar to the backbone line, and the process is time-consuming and labor-consuming, and usually takes more than 10 seasons (about 5-5.5 years).
In the prior art, different transformant materials of a transgene (N) are efficiently utilized as male parents (A1 to An) and a backbone line is taken as a female parent (B) to carry out hybridization to obtain a plurality of F1(B×A1),F1(B×A2),……F1(BxAn) plants, and screening the marker (target) genes, wherein the screening mode comprises the following steps: screening herbicide resistance, test paper strips, PCR amplification and the like; taking the bone stem line (B) as a recurrent parent to carry out continuous multi-generation backcross; meanwhile, in each generation of backcross, the marker (target) gene is screened, and after 6-7 generations of backcross and screening, 3 generations of selfing are carried out to obtain the nitrogen high-efficiency utilization transgenic bone stem line (B + N) of different transformant materials from A1 to An, and because each generation of backcross and screening comprises backcross progeny of different transformant materials from A1 to An, the workload is increased. Theoretically, in F1In the generation hybrid, the genetic materials of the female parent and the male parent respectively account for 50 percent, the backcross method is adopted, the genetic materials of the recurrent parent in the filial generation are increased by 50 percent every backcross generation, and the genetic background of the recurrent parent continuously rises along with the increase of the backcross generation times. In generalTransgenic backcross transfer requires selfing to begin after the background recovery rate reaches 99%, i.e., backcrossing to BC7When the generation is finished, the inbreeding homozygosity is started. However, in breeding practice, the backcross generations have a significant correlation with the genetic background recovery rate, but the genetic background recovery rate of the recurrent parent is not completely increased by 50%, that is, the background recovery rate of a part of backcross progeny may be higher than the 50% increased rate in the same backcross generations; while the background recovery rate of another portion of backcross progeny may be less than or equal to a 50% incremental ratio. The existing method lacks the effective selection of positive plants with high background recovery rate in the backcross materials of each generation by a phenotype selection method, and has long backcross period and time and labor consumption.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the method solves the problems of time and labor consumption of restoring to be consistent with the genetic background after the existing nitrogen efficiently utilizes the transgenic receptor to hybridize with the backbone line.
The technical scheme of the invention is as follows: a method for quickly cultivating a transgenic maize inbred line by efficiently utilizing nitrogen comprises the following steps:
(1) hybridization of nitrogen high-efficiency transgenic transformant material and recurrent parent
Nitrogen high-efficiency utilization transgenic transformant materials A1-An and a recurrent parent B with stable heredity are planted in the field, the transgenic transformant materials A1-An are used as male parents, the recurrent parent B is used as female parents for hybridization, and the hybridized fruit ears are respectively selected for seed reservation;
(2)F1generation: low nitrogen stress, identification of key characters, screening of optimal transformant material Ax and excellent strain thereof
And (3) fertilizer and water management: planting in nutrient pond, pair F1Carrying out nitrogen stress treatment on the plants;
sowing and final singling: the ears of seeds left after pollination by taking A1-An as male parents are planted according to ear rows, and F is planted in the tasseling period1The plants are subjected to marker gene screening, and the negative and positive of the plants are marked;
chlorophyll relative content and fluorescence kinetic parameter determination: in the tasseling period, measuring the relative chlorophyll content and fluorescence kinetic parameters of the ear position of the positive plant by taking a single plant as a unit;
pollination: in the pollen-scattering period, the recurrent parent B is taken as a male parent, and the positive plants are pollinated;
harvesting and selecting seeds: after the fruit cluster is mature, obtaining backcross progeny materials of the positive plants by taking the nitrogen high-efficiency transgenic transformant materials A1 and A2 … … An as a unit individual plant; respectively counting the relative chlorophyll content, fluorescence kinetic parameters and the single plant yield mean value of positive plants according to a formula: the yield of each plant is multiplied by 50% + the chlorophyll relative content value is multiplied by 50% + the fluorescence kinetic parameter PI value is integrated score; respectively calculating the comprehensive score of each individual plant and the average score of each pollination combination from A1 xB to An xB, taking the Ax corresponding to the Ax xB combination with the highest score as the medium selection transformant material, and simultaneously taking the first BC with the highest comprehensive score of each individual plant in Ax B1The fruit cluster is bred in the next season;
(3)BC1F1generation: low nitrogen stress, large population + phenotype-genotype associated selection, excellent plant backcrossing
And (3) fertilizer and water management: field planting BC1F1Nitrogen stress treatment;
sowing and final singling: planting the selected material in the previous generation, performing marker gene screening on plants in the seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
pollination: pollinating the positive plants by taking the recurrent parent B as a male parent;
and (3) character investigation: investigating main agronomic and economic properties with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each property which is the same as the recurrent parent, recording 7-9 points for very similar properties, recording 4-6 points for similar properties, recording 1-3 points for basically similar properties, recording 0 point for different properties, and calculating a total score according to each property;
harvesting and selecting seeds: harvesting a single plant after the fruit cluster is mature, and breeding in the next season according to the first few clusters with the highest scores;
(4)BC2F1generation: normal nitrogen application, large group + phenotype-genotype association selection, excellent plant backcross
And (3) fertilizer and water management: field planting BC2F1Normal management of fertilizer and water;
sowing and final singling: planting the selected material in the previous generation, performing marker gene screening on plants in the seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
pollination: pollinating the positive plants by taking the recurrent parent B as a male parent;
and (3) character investigation: investigating main agronomic and economic properties with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each property which is the same as the recurrent parent, recording 7-9 points for very similar properties, recording 4-6 points for similar properties, recording 1-3 points for basically similar properties, recording 0 point for different properties, and calculating a total score according to each property;
harvesting and selecting seeds: harvesting a single plant after the fruit cluster is mature, and breeding in the next season according to the first few clusters with the highest scores;
(5)BC3F1generation: large group + phenotype-genotype correlation selection, excellent plant selfing and back crossing with backbone system B
And (3) fertilizer and water management: field planting BC3F1Normal management of fertilizer and water;
sowing and final singling: planting the selected material in the previous generation, performing marker gene screening on plants in the seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
character investigation and pollination: investigating main agronomic and economic properties with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each property which is the same as the recurrent parent, recording 7-9 points for very similar properties, recording 4-6 points for similar properties, recording 1-3 points for basically similar properties, recording 0 point for different properties, and calculating a total score according to each property; selfing the plants which score 20 th, and pollinating the recurrent parent B by the highest-scoring plant;
harvesting and selecting seeds: harvesting the inbred plant and the recurrent parent B for reverse crossing;
(6)BC3F2and BC4F1Generation: under normal nitrogen application conditions, BC4F1Phenotype-genotype associated selection; selfing the excellent plants; BC3F2Identification of homozygote, selfing of excellent plant, and testing and matching
And (3) fertilizer and water management: field planting BC3F2Generation and BC4F1Generation, fertilizer and water normal pipeC, processing;
sowing and final singling: mixing BC3F2Planting according to ear row, BC4F1Planting according to ear rows; carrying out marker gene screening on plants in the seedling stage, carrying out uniform seedling and final singling after one week, and removing weak plants and negative plants;
and (3) character investigation:
BC3F2materials: to BC3F2Investigating the screening result, wherein the positive results of the panicle plants indicate that the nitrogen is homozygous at the high-efficiency utilization gene locus, otherwise, the locus is not homozygous;
BC4F1：investigating main agronomic and economic characters with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each character which is the same as the recurrent parent, recording 7-9 points for very similar characters, recording 4-6 points for similar characters, recording 1-3 points for basically similar characters, recording 0 point for different characters, and calculating the comprehensive score of each single plant;
pollination:
BC3F2materials: strictly selfing ears which are homozygous for nitrogen and have high-efficiency utilization gene loci and excellent agronomic and economic characters, and simultaneously carrying out test cross by taking the ears as male parents;
BC4F1: calculating total scores according to the phenotype of each character, and performing selfing on the plants with the highest scores;
harvesting and selecting seeds:
BC3F2: nitrogen efficiently utilizes homozygous ear rows of the gene loci, and fruit ears are preferentially reserved;
BC3F2test crossing combination: harvesting and reserving, and measuring the yield of the field in the next season;
BC4F1：breeding in the next season according to the first few ears with the highest score;
(7)BC3F3and BC4F2Generation:
and (3) fertilizer and water management: field planting BC3F3、BC4F2Generation, normal management of fertilizer and water; BC3F2Test crossing combined low-nitrogen planting;
sowing and final singling: BC3F3Planting according to ear rows，BC4F2Planting according to ear rows, screening marker genes of plants in a seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
and (3) character investigation:
BC3F3: investigating agronomic and economic characters and uniformity;
BC3F2test crossing combination: and (5) investigating yield traits.
BC4F2: investigating the screening result, wherein all positive panicle lines show that nitrogen is homozygous at the high-efficiency utilization gene locus, grading the panicle lines with homozygous loci for main agronomic and economic characters, marking 10 points for each character which is the same as the recurrent parent, 7-9 points for each character, 4-6 points for each character, 1-3 points for each character and 0 point for each character, and calculating the comprehensive score of each single plant;
pollination:
BC3F3: strict selfing is carried out;
BC4F2: strictly selfing the nitrogen efficient gene locus homozygous panicle;
harvesting and selecting seeds:
BC3F3materials: selecting the optimal plants according to the comprehensive character investigation result and the test cross combination yield performance to obtain a nitrogen high-efficiency utilization transgenic inbred line B' + N with the recurrent parent B genetic background of more than 95%;
BC4F2: and (3) selectively selecting the clusters which are close to the front score for the plants homozygous at the nitrogen high-efficiency gene locus to obtain the nitrogen high-efficiency utilization transgenic inbred line B + N with the background recovery rate of more than 99%.
Further, in the step (2), for F1The nitrogen stress treatment of the plants means that the application of nitrogen fertilizer is halved and the application of phosphate fertilizer and potash fertilizer is normal.
Furthermore, phosphate fertilizer and potash fertilizer are used as base fertilizer for one-time application, and nitrogen fertilizer is applied by the base fertilizer, seedling fertilizer and jointing fertilizer in sections according to the proportion of 2:3: 5.
Further, the main agronomic and economic traits are growth period, leaf sheath color, plant height, plant leaf type, male flower shape, anther color, filament color, grain type, grain color or axial color; the scoring criteria were as follows:
further, the nitrogen-efficient transgenic transformant material is HiII-AtAAP1, and the recurrent parent B is a corn backbone inbred line in southwest region including but not limited to C8210.
The core of the invention is that:
the method comprises the following steps of 1, performing nitrogen stress, large group, phenotype-genotype correlated selection, assigning values according to the similarity degree of a certain character and recurrent parent characters, accumulating the assigned values of a plurality of characters, and performing phenotype-genotype correlated selection on a single plant according to the integral height.
The method specifically comprises the following steps: in backcross generations, the growth period, the color of leaf sheath, the plant height, the shape of plant leaves, the shape of male flowers, the color of anther, the color of filament, the grain type, the grain color, the axial color and the like are investigated, each character is the same as that of a recurrent parent, 10 points are marked, the character is very similar to that of the recurrent parent, 7 to 9 points are marked, 4 to 6 points are marked, the character is basically similar to that of the recurrent parent, 1 to 3 points are marked, 0 point is marked differently, and the total score is calculated according to the phenotype of each character.
2. The method is characterized in that the evaluation of the high-efficiency utilization efficiency of the nitrogen of the transformant is carried out in advance, under the condition of nitrogen stress of a nutrient pool, key indexes such as yield, chlorophyll relative content and fluorescence kinetic parameters are evaluated, and low-efficiency transformant materials and backcross progeny thereof are removed, so that the workload is greatly reduced, the planting scale of the selected transformant materials can be enlarged, and the subsequent selection is facilitated.
The concrete expression is as follows: f1And respectively counting the relative chlorophyll content, fluorescence kinetic parameters and the single plant yield mean value of the positive plants according to a formula: yield per plant (weight 50%) + chlorophyll relative content value (weight 50%) + fluorescence kinetic parameter (PI value) ═ comprehensive score; respectively calculating the comprehensive score of each individual plant and the average score of each pollination combination from A1 multiplied by B to An multiplied by B, taking Ax corresponding to the Ax multiplied by B combination with the highest score as the medium selection transformant material, and simultaneously, feeding the fruit clusters with the highest comprehensive score of each individual plant in Ax multiplied by B into the next season for propagation.
Compared with the prior art, the invention has the following beneficial effects:
1. the method comprises the steps of performing assignment according to the similarity degree of a certain character and recurrent parent characters by adopting 'nitrogen stress + large group + phenotype-genotype correlated selection', accumulating the assignments of a plurality of characters, performing phenotype-genotype correlated selection on a single plant according to the integral height, performing gene locus homozygous selection after continuous 3-4 representative type comprehensive identification, and obtaining backcross offspring with the background recovery rate of more than 99% 2 years in advance. Quickens the breeding process and shortens the breeding period.
2. By the estimation of the high-efficiency utilization efficiency of the nitrogen of the transformant, the low-efficiency transformant material and the backcross progeny thereof are removed in the early stage, the workload is greatly reduced (the cost and the labor are saved, the backcross efficiency is improved), the planting scale of the selected transformant material can be enlarged, and the subsequent selection is facilitated.
Drawings
FIG. 1 is a diagram of a hybridization route according to the present invention.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
Description of terms:
1. gene of interest (nitrogen-highly active gene): the letter "N" indicates that a favorable gene to be transferred into a plant usually requires PCR amplification for detection;
2. marker genes: the other gene closely linked with the target gene can be used for deducing the existence of the target gene through the detection of the existence of the marker gene in the field, and the marker gene can be rapidly tested and distinguished through a test strip and a herbicide;
3. transformant materials: when the same target gene is transferred into a corn plant receptor, multiple conversions are carried out to form different transformant materials, the high-efficiency utilization efficiency of nitrogen of the different transformant materials can be greatly different, in the technical scheme, A1, A2 and A3 … … An represent the different transformant materials, and Ax represents one or a few of selected transformant materials;
4. and (3) recurrent parent: parents for backcrossing (backbone material), indicated by the letter "B";
5.F1: the first filial generation of B and A.
The invention will be described in detail below with reference to the accompanying drawing 1
Example 1
1. Nitrogen high-efficiency transgenic transformant material is hybridized with recurrent parent (field planting)
Planting genetically stable nitrogen high-efficiency utilization transgenic transformant materials (A1 to An) and recurrent parent (B) in a field, hybridizing the nitrogen high-efficiency utilization transgenic transformant materials (A1 to An) serving as male parents and the recurrent parent (B) serving as female parents to ensure that the nitrogen high-efficiency utilization transgenic transformant materials (A1 to An) and the recurrent parent (B) respectively obtain F1The hybrid ears are more than 10 ears. And (3) respectively selecting 5 ears for reserving seeds for the ears pollinated by the male parents by using the A1 to An as male parents (the selection standard is that the growth vigor of plants is increased, the ears are typical, the ears have more grains, and the seeds are full).
The main agronomic and economic characters (such as growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color, grain type, grain color and axial color) with obvious difference between the transgenic transformant material and the recurrent parent material are investigated and recorded in the whole growth period.
2.F1Generations (nutrient pool low nitrogen stress, key character identification, screening best transformant material Ax and its excellent strain)
And (3) fertilizer and water management: planting in nutrient pond, pair F1Carrying out nitrogen stress treatment on the plants (half-reduced application of nitrogen fertilizer and normal application of phosphorus-potassium fertilizer);
sowing and final singling: selecting fruit ears from A1-An as male parents after pollination, planting according to ear rows, planting 4 rows of each ear, 20 holes in each row, sowing 2-3 seeds in each hole, fixing seedlings at 3-4 leaf stage, reserving plants in single hole to ensure that the seedlings are uniform and strong, and controlling the density at 3600-; in the period of emasculation, F1Plants are screened for marker genes (usually herbicide smear or dipstick tests, the same applies below) and plants are marked for negative and positive (negative plants are not removed, avoiding marginal effects).
Chlorophyll relative content and fluorescence kinetic parameter determination: in the tasseling period, measuring the chlorophyll relative content and fluorescence kinetic parameters of the leaves (the leaf tip part, the middle part and the leaf end part) at the ear position of the positive plant by taking a single plant as a unit;
pollination: in the pollen-dispersing period, the recurrent parent (B) is taken as a male parent, and pollination is carried out on each row of positive plants for 2 times, so that full pollination of the clusters is ensured. In general, the first pollination is carried out 3-4 days after the female ear of the female parent is bagged, and the second pollination is carried out 6-7 days after the bagging.
Harvesting and selecting seeds: after the fruit cluster is mature, the backcross progeny material of the positive plant is obtained by taking the nitrogen high-efficiency transgenic transformant material as a unit (A1, A2 … … An) for a single plant. Respectively counting the relative chlorophyll content, fluorescence kinetic parameters and the single plant yield mean value of positive plants according to a formula: yield per plant (weight 50%) + chlorophyll relative content value (weight 50%) + fluorescence kinetic parameter (PI value) ═ comprehensive score; respectively calculating the comprehensive score of each individual plant and the average score of each pollination combination from A1 xB to An xB, taking the Ax corresponding to the Ax xB combination with the highest score as the medium selection transformant material, and simultaneously taking 3-5 BC with the highest comprehensive score of each individual plant in Ax B1The fruit ears are bred in the next season.
3.BC1F1Generations (Low Nitrogen stress, Large population + phenotype-genotype associated selection, excellent backcross)
And (3) fertilizer and water management: field planting BC1F1Nitrogen stress treatment (nitrogen fertilizer half-reduced application, phosphorus potassium fertilizer normal application);
sowing and final singling: selecting materials in the previous generation, planting the materials in units of ear rows, 8-10 rows of ears, 20 holes in each row, 1-2 seeds in each hole, and controlling the density at 3600-; the plants were subjected to marker gene screening (positive plants resistant to herbicides) at the seedling stage (3-4 leaves), and after one week, the seedlings were homogenized and fixed (weak and negative plants were removed).
Pollination: and (3) pollinating each row of positive plants for 2 times by taking the recurrent parent (B) as a male parent to ensure full pollination of the clusters.
And (3) character investigation: the main agronomic and economic characters (growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color, grain type, grain color, axial color and the like) with obvious difference between the transformant material and the recurrent parent material are investigated, each character is the same as the recurrent parent, 10 points are marked, 7-9 points are marked very similar, 4-6 points are marked similarly, 1-3 points are marked basically similar, 0 point is marked differently (table 1), and the total score is calculated according to each character.
Harvesting and selecting seeds: after the fruit cluster is mature, harvesting a single plant, and breeding in the next season according to the first 5 clusters with the highest score.
4.BC2F1Generation: (Normal nitrogen application, large population + phenotype-genotype associated selection, excellent backcross)
And (3) fertilizer and water management: field planting BC2F1And normally managing the fertilizer and water.
Sowing and final singling: selecting materials in the previous generation, planting the materials in units of ear rows, 6-10 rows of ears, 20 holes in each row, 2-3 seeds in each hole, and controlling the density at 3600-; marker gene screening is carried out on the plants in the seedling stage (3-4 leaves), and seedlings are homogenized and fixed after one week (weak plants and negative plants are removed).
Pollination: and (3) pollinating each row of positive plants for 2 times by taking the recurrent parent (B) as a male parent to ensure full pollination of the clusters.
And (3) character investigation: the main agronomic and economic characters (growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color, grain type, grain color, axial color and the like) with obvious difference between the transformant material and the recurrent parent material are investigated, each character is the same as the recurrent parent, 10 points are marked, 7-9 points are marked very similar, 4-6 points are marked similarly, 1-3 points are marked basically similar, 0 point is marked differently, and the total score is calculated according to the phenotype of each character.
Harvesting and selecting seeds: after the fruit cluster is mature, harvesting a single plant, and breeding in the next season according to the first 5 clusters with the highest score.
5.BC3F1Generation: (Large population + phenotype-genotype related selection, excellent plant selfing, and backcrossing with the skeletal stem line (B))
And (3) fertilizer and water management: field planting BC3F1And normally managing the fertilizer and water.
Sowing and final singling: selecting materials in the previous generation, planting the materials in units of ear rows, wherein more than 300 plants are planted in each ear, and the density is controlled at 3600-3800 plants/mu; the plants were subjected to marker gene screening (positive plants resistant to herbicides) at the seedling stage (3-4 leaves), and after one week, the seedlings were homogenized and fixed (weak and negative plants were removed).
Character investigation and pollination: investigating the characters with obvious difference between the transformant material and the recurrent parent material, such as growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color and the like, marking 10 points for each character which is the same as the recurrent parent, marking 7-9 points for very similar characters, 4-6 points for similar characters, 1-3 points for basically similar characters and 0 point for different characters, calculating total score according to the phenotype of each character, selfing the 20-point plants, and pollinating the recurrent parent (B) with the highest score.
Harvesting and selecting seeds: and (4) harvesting the inbred plants and the recurrent parent (B) for backcrossing the ears.
6.BC3F2And BC4F1Generation: (under normal nitrogen application conditions, BC4F1Phenotype-genotype associated selection; selfing the excellent plants; BC3F2Homozygote identification, excellent plant selfing, test matching)
And (3) fertilizer and water management: field planting BC3F2Generation and BC4F1And (5) generation, normal management of fertilizer and water.
Sowing and final singling: mixing BC3F2Planting 3-5 rows of BC in each ear4F1Planting 10 rows in each ear; the plants were subjected to marker gene screening (positive plants resistant to herbicides) at the seedling stage (3-4 leaves), and after one week, the seedlings were homogenized and fixed (weak and negative plants were removed).
And (3) character investigation:
BC3F2materials: to BC3F2And (4) investigating herbicide screening results, wherein the panicle plants are homozygous at the indicated locus nitrogen high-efficiency utilization gene site for all herbicide-resistant plants, otherwise, the locus is not homozygous.
BC4F1：Investigating the characters with obvious difference between the transformant material and the recurrent parent material, such as growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color and the like, recording 10 points for each character which is the same as the recurrent parent, recording 7-9 points for each character which is very similar to the recurrent parent, recording 4-6 points for each character which is basically similar to the recurrent parent, recording 1-3 points for each character which is different from the recurrent parent, and calculating the comprehensive score of each single plant.
Pollination:
BC3F2materials: the method is characterized in that the method strictly selfs the panicles which are homozygous for the nitrogen efficient utilization gene loci (non-negative plants) and have excellent agronomic and economic characters, and simultaneously carries out test cross by taking the panicles as male parents.
BC4F1: and calculating total scores according to the phenotype of each character, and scoring high-value plants for selfing.
Harvesting and selecting seeds:
BC3F2: nitrogen efficiently utilizes homozygous ear rows of gene loci, and preferentially retains 5 ear clusters.
BC3F2Test crossing combination: harvesting and reserving, and measuring the yield of the field in the next season.
BC4F1: and (4) breeding in the next season according to the top 20 ears with the highest score.
7.BC3F3、BC4F2Generation:
and (3) fertilizer and water management: field planting BC3F3、BC4F2Generation, normal management of fertilizer and water; BC3F2And (5) carrying out test crossing and low nitrogen planting.
Sowing and final singling: BC3F3One-row planting in ear rows, BC4F2Planting the spike row in 5 rows, and controlling the density at 3600-3800 plants/mu; the plants were subjected to marker gene screening (positive plants resistant to herbicides) at the seedling stage (3-4 leaves), and after one week, the seedlings were homogenized and fixed (weak and negative plants were removed).
And (3) character investigation:
BC3F3: and investigating the agronomic and economic characters and the uniformity thereof.
BC3F2Test crossing combination: and (5) investigating yield traits.
BC4F2: and (4) investigating herbicide screening results, and performing high-efficiency utilization of gene locus homozygosis on all herbicide-resistant expression locus nitrogen of the panicle plants. The homozygous ear rows at the locus are investigated for growth period, leaf sheath color, plant height, plant leaf shape, anther color, flower silk color and the like, each character is the same as that of the recurrent parent and is recorded for 10 minutes, and the characters are very similar to that of the recurrent parent and are recorded for 7-And 9 points, similarly scoring 4-6 points, basically similarly scoring 1-3 points, and differently scoring 0 point, and calculating the total score of each individual plant.
Pollination:
BC3F3: and (4) strict selfing.
BC4F2: and strictly selfing the nitrogen efficient gene locus homozygous panicle.
Harvesting and selecting seeds:
BC3F3materials: and selecting the optimal plants according to the comprehensive character investigation result and the test cross combination yield expression to obtain the nitrogen high-efficiency utilization transgenic inbred line B' + N with the genetic background of the bone stem line B of more than 95%.
BC4F2: and selecting the plants homozygous for the nitrogen efficient gene locus according to the clusters with the first score of 3 to obtain the nitrogen efficient utilization transgenic inbred line B + N with the background recovery rate of more than 99%.
TABLE A Main agronomic and economic character scoring TABLE
Test examples
The HiII-AtAAP1 transformant was provided by the academy of agricultural sciences of Jilin province.
C8210 is obtained by research institute of agricultural sciences of Sichuan province in 2015, and is subjected to protection authorization of new plant varieties in 2018 in 1 month and 2 days, and is marketed before application date.
Winter (Hainan) hybridization in 1.2016 years
The transgenic transformant materials HiII-AtAAP1-1 and HiII-AtAAP1-2 are efficiently utilized by genetically stable nitrogen, 2 parts in total are used as donors (numbers 16A1 and 16A2), a corn backbone inbred line C8210 in the southwest region is used as a recurrent parent, transgenic test bases of southern breeding bases of agricultural science institute of Sichuan province, Ongzhou province, Toxico county, Shandong province in 2016 are planted in winter, 1 row of each donor material is planted, 20 seedlings are left in each row, the recurrent parent C8210 is sown in 2 stages according to the growth period, and the sowing period is separated by 7 days to ensure that the flowering stages meet. The major traits of the parents were recorded throughout the entire growth period (Table 1). Hybridizing in the stage of drawing tassels and spitting, respectively selecting plants with normal growth vigor from 16A1 and 16A2 as male parents, strictly bagging the male parents by using special male flower bags one day before pollen scattering of the male ears, bagging female parent C8210 female ears before spitting, respectively hybridizing 20 ears with C8210 by using 16A 1-16A 2 after the female parent C8210 spits, and isolating during hybridization to avoid flying. After the hybrid ears are harvested, selecting the ear threshing numbers of typical ears, large ear number and full seeds (Table 2):
TABLE 1 survey table of major characters of parents
Table 2 retained ear information table
Spring (Sichuan) F in 2.2017 year1
And respectively carrying out low-nitrogen treatment and identification on the retained clusters of 16C1 and 16C2 series in a nutrient pool of a Pengzhou Mongolian Yangzhou high-tech breeding base. Planting 16C1-1 to 16C1-3 and 16C2-1 to 16C2-3 according to spike rows, planting 4 rows in each spike row, counting to 20 pits in each row, planting 2-3 seeds in each pit according to the seed quantity, and reserving the seedlings in single hole after the seedlings are formed. After sowing, the nitrogen fertilizer is applied by half of the normal application amount, namely 8 kg/mu, and the phosphate fertilizer and the potash fertilizer are 6 kg/mu. Wherein, the phosphate fertilizer and the potash fertilizer are applied at one time, and the nitrogen fertilizer is applied as the base fertilizer, the seedling fertilizer and the jointing fertilizer in a ratio of 2:3: 5. In the emasculation period, herbicide smearing test is carried out on F1 plants, and positive plants are hung on a red board. C8210 is taken as a male parent in the spinning period, the male parent is pollinated with all positive plants from 16C1-1 to 16C1-3 and 16C2-1 to 16C2-3, the relative content of chlorophyll at the ear position of the positive plants, fluorescence kinetic parameters, yield and other indexes of the individual plants are measured by taking the individual plants as a unit, the comprehensive score of 16C1-1 to 16C1-3 and 16C2-1 to 16C2-3 is calculated (table 3 and table 4), and according to the measured data, the average comprehensive score (116.3) of all the individual plants of the transformant HiII-AtAAP1-1 is higher than the average comprehensive score (105.8) of the individual plants of the transformant HiII-AtAAP1-2 and the average comprehensive score (121.2) of the individual plants of 16C1-1 is higher than the average comprehensive score (105.9) of the individual plants of 16C1-2(111.9) and 16C1-3(114.9), so that the individual plants of 16C 45-1-8233, 16C-25-854 and 16C-894 are selected to enter into the individual plants before the rotation.
TABLE 316C1 (corresponding transformant HiII-AtAAP1-1) Individual score
TABLE 416C 2 (corresponding transformant HiII-AtAAP1-2) Individual score
Winter (Hainan) BC of 3.17 years1F1
The selected ears of 16C1-1-33,16C1-1-4 and 16C1-1-23 are planted according to ear rows, 1-2 seeds are sowed, the density is 3600 plants/mu, and about 160 plants are left in each ear. Nitrogen stress treatment (nitrogen fertilizer is half-reduced and phosphorus potassium fertilizer is normally applied); spraying glufosinate (1:200) in the seedling stage. Before pollination, the female parent plant removes smallpox, and C8210 is used to complete pollination for more than 2 times on the positive plant. Main agronomic economic traits (growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color, grain type, grain color, axial color and the like) are investigated and scored (Table 5), and the single plants 16C1-33-23, 16C1-33-36, 16C1-4-36, 16C1-33-120 and 16C1-4-96 are totally classified as 5 before, and are selected and retained.
TABLE 5BC1F1Single plant agronomic economic character comprehensive scoring sequencing list (front 10)
Spring (Sichuan) at 4.2018, BC2F1
Planting single plants 16C1-33-23, 16C1-33-36, 16C1-4-36, 16C1-33-120 and 16C1-4-96 of the selected materials in the previous generation by taking spike rows as units, wherein each spike has 8 rows and 20 holes, each hole has 2-3 seeds, the density is controlled at 3600 plants/mu, and normal fertilization treatment is carried out; spraying glufosinate (1:200) in the seedling stage. Before pollination, the female parent plant removes smallpox, and C8210 is used to complete pollination for more than 2 times on the positive plant. Main agronomic economic traits (growth period, leaf sheath color, plant height, plant leaf shape, male flower shape, anther color, filament color, grain type, grain color, axial color and the like) are investigated and scored (Table 6), and the single plants 16C1-33-36-2, 16C1-33-36-45, 16C1-33-23-79, 16C1-4-36-7 and 16C1-33-23-49 are totally classified as 5 before, and are selected and retained.
TABLE 6BC2F1Single plant agronomic economic character comprehensive scoring sequencing list (front 5)
Winter (Hainan) 5.2018, BC3F1
Planting single plants of selected materials in the previous generation, namely 16C1-33-36-2, 16C1-33-36-45, 16C1-33-23-79, 16C1-4-36-7 and 16C1-33-23-49, in a unit of spike rows, wherein 15 rows of spikes are planted, 20 holes are formed in each row, 300 basic seedlings are planted, the density is controlled to be 3600 plants/mu, and normal fertilization treatment is carried out; spraying glufosinate (1:200) in the seedling stage. Main agronomic economic traits (growth period, leaf sheath color, plant height, plant leaf morphology, male flower morphology, anther color, filament color and the like) are investigated and scored, 20 individual plants with scores ranked first are listed in table 7, the 20 plants are strictly selfed, the selfed ears are numbered from ZC8210-1 to ZC8210-20, 16C1-33-23-2-58 with the highest score of the individual plant is taken as a male parent and pollinated with recurrent parent C8210 to obtain 5 ears, and the selfed ears are numbered as 16C1-33-23-2-58-1, 16C1-33-23-2-58-2, 16C1-33-23-2-58-3, 16C1-33-23-2-58-4 and 16C1-33-23-2-58-5 respectively.
TABLE 7BC3F1Single plant agronomic economic character comprehensive scoring sequencing list (front 20)
Spring (Sichuan) at 6.2019, BC3F2 and BC4F1
Single plant selfing cluster (BC) selected in previous generation3F2) ZC8210-1 to ZC8210-20, planting 3 rows per ear according to ear row, backcrossing ears BC4F1(16C1-33-23-2-58-1, 16C1-33-23-2-58-2, 16C1-33-23-2-58-3, 16C1-33-23-2-58-4, 16C1-33-23-2-58-5) planting 10 rows per spike; the plants were subjected to marker gene screening (positive plants resistant to herbicides) at the seedling stage (3-4 leaves), and after one week, the seedlings were homogenized and fixed (weak and negative plants were removed). To BC3F2And (4) investigating herbicide screening results, wherein the complete herbicide resistance of the panicle plants shows that the locus nitrogen is homozygous for the high-efficiency utilization gene, otherwise, the locus is not homozygous (Table 8). To BC4F1The major agronomic traits of the middle plant (growth period, leaf sheath color, plant height, plant leaf morphology, male flower morphology, anther color, filament color, grain type, grain color and axial color, etc.) were investigated and scored (table 9).
TABLE 8BC3F2 statistical Table of Single Positive plants (first 20)
| Serial number | Number in field | Total number of seedlings | Number of positive plants | Number of negative plants |
| ZC8201-1 | 60 | 31 | 60 | 0 |
| ZC8201-2 | 59 | 33 | 33 | 26 |
| ZC8201-3 | 60 | 60 | 60 | 0 |
| ZC8201-4 | 60 | 29 | 29 | 31 |
| ZC8201-5 | 60 | 27 | 27 | 33 |
| ZC8201-6 | 59 | 27 | 27 | 32 |
| ZC8201-7 | 60 | 31 | 31 | 29 |
| ZC8201-8 | 60 | 29 | 60 | 0 |
| ZC8201-9 | 60 | 33 | 33 | 27 |
| ZC8201-10 | 60 | 25 | 25 | 35 |
| ZC8201-11 | 60 | 60 | 29 | 31 |
| ZC8201-12 | 60 | 33 | 33 | 27 |
| ZC8201-13 | 60 | 31 | 31 | 29 |
| ZC8201-14 | 59 | 24 | 24 | 25 |
| ZC8201-15 | 60 | 29 | 29 | 31 |
| ZC8201-16 | 60 | 24 | 24 | 36 |
| ZC8201-17 | 60 | 60 | 31 | 29 |
| ZC8201-18 | 59 | 31 | 31 | 28 |
| ZC8201-19 | 60 | 29 | 29 | 31 |
| ZC8201-20 | 60 | 31 | 31 | 29 |
The investigation result shows that in the BC3F2 ear row, plants with ZC8210-3, ZC8210-11 and ZC8210-17 are all positive, which shows that the plants are homozygous at the transferred gene locus; for the above earExcellent plants typical in agronomic economic characters in rows are selfed for 5 ears to remain, (numbers are ZC8210-3-1 to ZC8210-3-5, ZC8210-11-1 to ZC8210-11-5, ZC8210-17-1 to ZC8210-17-5), and the excellent plants are subjected to test crossing with Y9614 for 2 ears by using ZC8210-3, ZC8210-11 and ZC8210-17 mixed powder respectively, wherein the test crossing ears are numbered as Y9614/ZC8210-3, Y9614/ZC8210-11 and Y9614/C8210-17; at BC4F1Plants were inbred strictly and the score for the ear retention of the top 20 plants (table 9).
TABLE 9BC4F1Single plant agronomic economic character comprehensive scoring sequencing list (front 20)
Winter (Hainan) 7.2019, BC3F3And BC4F2
(1) Field planting BC3F3、BC4F2Generation, BC3F3One-row planting in ear rows, BC4F2Planting the seeds in 5 rows in a ear row, normally managing fertilizer and water, and keeping the density of 3600 plants/mu; (2) field planting BC3F2And (4) carrying out test crossing combination on the excellent strain and Y9614, and carrying out low nitrogen treatment. The plants were subjected to marker gene screening (positive plants resistant to herbicides) at the seedling stage (3-4 leaves), and after one week, the seedlings were homogenized and fixed (weak and negative plants were removed).
To BC3F3Investigating agronomic and economic characters and uniformity thereof, calculating a score, and strictly selfing high-quality plants with high scores (Table 10); to BC3F2Test cross combined low nitrogen yield trait survey (table 11). To BC4F2Investigating herbicide screening results, investigating the growth period, leaf sheath color, plant height, plant leaf morphology, anther color, filament color and the like of the ear rows homozygous for the locus, recording 10 points for each character as same as the recurrent parent, recording 7-9 points for each character as similar as the recurrent parent, recording 4-6 points for each character as similar as the recurrent parent, recording 1-3 points for each character as basically similar as the recurrent parent, recording 0 point for each character as different as the recurrent parent, calculating the total weight of each individual plantThe high scoring superior plants were strictly selfed (tables 12, 13).
TABLE 10BC3F3Single plant agronomic economic character comprehensive scoring ranking list (front 30)
TABLE 11 test-crossing combination yields and yield-constituting factors
TABLE 12BC4F2Statistical table of single positive plants
In a BC3F3 ear row, ZC8210-3-10, ZC8210-3-11, ZC8210-11-12, ZC8210-3-19 and ZC8210-17-11 are subjected to division value in the first 5 bits; the test crossing result shows that the comprehensive performance of the combined yield of the Y9614/ZC8210-3 and the yield composition factors thereof is better than that of the test crossing combination of the Y9614/ZC8210-11 and the Y9614/ZC 8210-17. Combining with the individual plant property score, selecting a ZC8210-3-10 and ZC8210-3-11 in BC3F3 as a nutrient high-efficiency utilization transgenic inbred line, wherein the genetic background is C8210, the generation is BC3F3, the background recovery rate is more than 95%, the transferred gene is AtAAP1, and the transferred gene is homozygous at the gene locus.
The screening result of the BC4F2 herbicide shows (Table 12), the plants of the 16C1-33-23-2-58-3-25, the 16C1-33-23-2-58-4-88 and the 16C1-33-23-2-58-1-33 panicle are all positive, which shows that the introduced gene locus is homozygous; and comprehensively scoring the panicle plants homozygous at the gene locus, reserving 16C1-33-23-2-58-3-25-57, 16C1-33-23-2-58-4-88-39, 16C1-33-23-2-58-3-25-69, 16C1-33-23-2-58-3-25-79 and 16C1-33-23-2-58-3-25-12 as nutrients according to the scoring condition, efficiently utilizing the nutrients from an inbred line to select single plants, wherein the genetic background is C8210, the generation is BC4F2, the background recovery rate is more than 99 percent, the transferred gene is AtAAP1, and the plants are homozygous at the gene locus. (watch 13)
Comprehensive evaluation of ear-row plants homozygous for table 13 gene locus
Claims (1)
1. A method for quickly cultivating a transgenic maize inbred line by efficiently utilizing nitrogen is characterized by comprising the following steps:
(1) hybridization of nitrogen high-efficiency transgenic transformant material and recurrent parent
Nitrogen high-efficiency utilization transgenic transformant materials A1-An and a recurrent parent B with stable heredity are planted in the field, the transgenic transformant materials A1-An are used as male parents, the recurrent parent B is used as female parents for hybridization, and the hybridized fruit ears are respectively selected for seed reservation;
(2)F1generation: low nitrogen stress, identification of key characters, screening of optimal transformant material Ax and excellent strain thereof
And (3) fertilizer and water management: planting in nutrient pond, pair F1Carrying out nitrogen stress treatment on the plants;
sowing and final singling: the ears of seeds left after pollination by taking A1-An as male parents are planted according to ear rows, and F is planted in the tasseling period1The plants are screened for marker genes, and the negative and positive of the plants are marked；
Chlorophyll relative content and fluorescence kinetic parameter determination: in the tasseling period, measuring the relative chlorophyll content and fluorescence kinetic parameters of the ear position of the positive plant by taking a single plant as a unit;
pollination: in the pollen-scattering period, the recurrent parent B is taken as a male parent, and the positive plants are pollinated;
harvesting and selecting seeds: after the fruit cluster is mature, obtaining backcross progeny materials of the positive plants by taking the nitrogen high-efficiency transgenic transformant materials A1 and A2 … … An as a unit individual plant; respectively counting the relative chlorophyll content, fluorescence kinetic parameters and the single plant yield mean value of positive plants according to a formula: the yield of each plant is multiplied by 50% + the chlorophyll relative content value is multiplied by 50% + the fluorescence kinetic parameter PI value is integrated score; respectively calculating the comprehensive score of each individual plant and the average score of each pollination combination from A1 xB to An xB, taking the Ax corresponding to the Ax xB combination with the highest score as the medium selection transformant material, and simultaneously taking the first BC with higher comprehensive score of each individual plant in Ax B1The fruit cluster is bred in the next season;
(3)BC1F1generation: low nitrogen stress, large population binding phenotype-genotype association selection, excellent plant backcrossing
And (3) fertilizer and water management: field planting BC1F1Nitrogen stress treatment;
sowing and final singling: planting the selected material in the previous generation, performing marker gene screening on plants in the seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
pollination: pollinating the positive plants by taking the recurrent parent B as a male parent;
and (3) character investigation: investigating main agronomic and economic properties with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each property which is the same as the recurrent parent, recording 7-9 points for very similar properties, recording 4-6 points for similar properties, recording 1-3 points for basically similar properties, recording 0 point for different properties, and calculating a total score according to each property;
harvesting and selecting seeds: harvesting a single plant after the fruit cluster is mature, and breeding in the next season according to the first few clusters with higher scores;
(4)BC2F1generation: normal nitrogen application, large population binding phenotype-genotype association selection, superior strainBackcrossing
And (3) fertilizer and water management: field planting BC2F1Normal management of fertilizer and water;
sowing and final singling: planting the selected material in the previous generation, performing marker gene screening on plants in the seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
pollination: pollinating the positive plants by taking the recurrent parent B as a male parent;
and (3) character investigation: investigating main agronomic and economic properties with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each property which is the same as the recurrent parent, recording 7-9 points for very similar properties, recording 4-6 points for similar properties, recording 1-3 points for basically similar properties, recording 0 point for different properties, and calculating a total score according to each property;
harvesting and selecting seeds: harvesting a single plant after the fruit cluster is mature, and breeding in the next season according to the first few clusters with higher scores;
(5)BC3F1generation: large group combines phenotype-genotype association selection, excellent plants are selfed and are backcrossed with recurrent parent B
And (3) fertilizer and water management: field planting BC3F1Normal management of fertilizer and water;
sowing and final singling: planting the selected material in the previous generation, performing marker gene screening on plants in the seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
character investigation and pollination: investigating main agronomic and economic properties with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each property which is the same as the recurrent parent, recording 7-9 points for very similar properties, recording 4-6 points for similar properties, recording 1-3 points for basically similar properties, recording 0 point for different properties, and calculating a total score according to each property; selfing the plants which score 20 th, and pollinating the recurrent parent B by the highest-scoring plant;
harvesting and selecting seeds: harvesting the inbred plant and the recurrent parent B for reverse crossing;
(6)BC3F2and BC4F1Generation: under normal nitrogen application conditions, BC4F1Phenotype-genotype associated selection; selfing the excellent plants; BC3F2The identification of homozygote and excellenceSelfing and testing
And (3) fertilizer and water management: field planting BC3F2Generation and BC4F1Generation, normal management of fertilizer and water;
sowing and final singling: mixing BC3F2Planting according to ear row, BC4F1Planting according to ear rows; carrying out marker gene screening on plants in the seedling stage, carrying out uniform seedling and final singling after one week, and removing weak plants and negative plants;
and (3) character investigation:
BC3F2materials: to BC3F2Investigating the screening result, wherein the positive results of the panicle plants indicate that the nitrogen is homozygous at the high-efficiency utilization gene locus, otherwise, the locus is not homozygous;
BC4F1: investigating main agronomic and economic characters with obvious difference between a transformant material and a recurrent parent material, recording 10 points for each character which is the same as the recurrent parent, recording 7-9 points for very similar characters, recording 4-6 points for similar characters, recording 1-3 points for basically similar characters, recording 0 point for different characters, and calculating the comprehensive score of each single plant;
pollination:
BC3F2materials: strictly selfing ears which are homozygous for nitrogen and have high-efficiency utilization gene loci and excellent agronomic and economic characters, and simultaneously carrying out test cross by taking the ears as male parents;
BC4F1: calculating total scores according to the phenotype of each character, and performing selfing on the plants with the highest scores;
harvesting and selecting seeds:
BC3F2: nitrogen efficiently utilizes homozygous ear rows of the gene loci, and fruit ears are preferentially reserved;
BC3F2test crossing combination: harvesting and reserving, and measuring the yield of the field in the next season;
BC4F1: breeding in the next season according to the first few ears with higher scores;
(7)BC3F3and BC4F2Generation:
and (3) fertilizer and water management: field planting BC3F3、BC4F2Generation, normal management of fertilizer and water; BC3F2Test crossing combined low-nitrogen planting;
sowing and final singling: BC3F3Planting according to ear row, BC4F2Planting according to ear rows, screening marker genes of plants in a seedling stage, homogenizing and fixing seedlings after one week, and removing weak plants and negative plants;
and (3) character investigation:
BC3F3: investigating agronomic and economic characters and uniformity;
BC3F2test crossing combination: investigating yield and character;
BC4F2: investigating the screening result, wherein all positive panicle lines show that nitrogen is homozygous at the high-efficiency utilization gene locus, grading the panicle lines with homozygous loci for main agronomic and economic characters, marking 10 points for each character which is the same as the recurrent parent, 7-9 points for each character, 4-6 points for each character, 1-3 points for each character and 0 point for each character, and calculating the comprehensive score of each single plant;
pollination:
BC3F3: strict selfing is carried out;
BC4F2: strictly selfing the nitrogen efficient gene locus homozygous panicle;
harvesting and selecting seeds:
BC3F3materials: selecting the optimal plants according to the comprehensive character investigation result and the test cross combination yield performance to obtain a nitrogen high-efficiency utilization transgenic inbred line B' + N with the recurrent parent B genetic background of more than 95%;
BC4F2: selectively selecting the clusters which are homozygous for the nitrogen efficient gene locus to obtain a nitrogen efficient utilization transgenic inbred line B + N with the background recovery rate of more than 99%;
the nitrogen stress treatment refers to half-reduced application of nitrogen fertilizer, normal application of phosphate fertilizer and potassium fertilizer, one-time application of phosphate fertilizer and potassium fertilizer as base fertilizer, and segmental application of nitrogen fertilizer, base fertilizer, seedling fertilizer and jointing fertilizer in a ratio of 2:3:5, wherein the main agronomic and economic characters refer to growth period, leaf sheath color, plant height, plant leaf type, male flower shape, anther color, flower filament color, grain type, grain color or axial color; the scoring criteria were as follows:
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| CN109576300A (en) * | 2018-12-12 | 2019-04-05 | 郝东云 | Corn transformation event HiII-AtAAP1-1 and its specificity identification method and application |
| CN111972286A (en) * | 2020-09-21 | 2020-11-24 | 河南省农业科学院粮食作物研究所 | Screening method of low-nitrogen-resistant corn variety |
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| CN109576300A (en) * | 2018-12-12 | 2019-04-05 | 郝东云 | Corn transformation event HiII-AtAAP1-1 and its specificity identification method and application |
| CN111972286A (en) * | 2020-09-21 | 2020-11-24 | 河南省农业科学院粮食作物研究所 | Screening method of low-nitrogen-resistant corn variety |
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