CN111713403B - Method for doubling corn haploid seedlings - Google Patents
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- CN111713403B CN111713403B CN202010756858.6A CN202010756858A CN111713403B CN 111713403 B CN111713403 B CN 111713403B CN 202010756858 A CN202010756858 A CN 202010756858A CN 111713403 B CN111713403 B CN 111713403B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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- A—HUMAN NECESSITIES
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses a method for doubling corn haploid seedlings. The invention provides a plant haploid seedling doubling method, which comprises the following steps: 1) soaking roots of plant haploid seedlings growing to 1-4 leaf stages in podophyllotoxin solution to obtain treated plant seedlings; 2) transplanting the treated plant seedlings to a field to realize plant haploid seedling doubling. The corn haploid seedling doubling technology provided by the invention can obviously improve the powder scattering rate of the corn haploid, and the powder scattering rate is up to more than 70% and far higher than the powder scattering rate of a contrast (generally about 10%). The drug podophyllotoxin used by the method has weak volatility, and can be operated by an operator only by wearing a disposable glove and a mask; and simultaneously, haploid doubling is carried out by adopting a method of raising seedlings and transplanting in an arched shed or open field, and the method is independent of laboratory operation and suitable for large-scale batch production of doubled haploid.
Description
Technical Field
The invention relates to the field of agricultural plant breeding, in particular to a method for doubling maize haploid seedlings.
Background
Corn is the grain crop with the largest planting area and the highest total yield in China, corn varieties applied to production at present are almost all hybrid seeds formed by self-bred line combination, and in long-term practice, corn breeding workers have the consensus: "difficult to select and re-assemble, the center is the problem of combining ability". The method for breeding the inbred line by the traditional pedigree method adopts a continuous inbred method, at least more than 8 generations are needed from material composition to stability, and two generations are highly homozygous and stable by a method of inducing haploid to be doubled, so the method is called a double haploid breeding technology, because the method has the advantages of stable and fast line selection, high degree of purity, accurate test result of matched combination, simple project management and the like, and is very suitable for commercial breeding.
As the haploid only has one set of chromosome group, the female ear is normal and can be silked, the female ear can fruit after pollination, but most of the materials have abortive tassel, no anther or shriveled anther, and can not normally loose powder, and the breeding requirement can be met only by carrying out chemical doubling treatment. At present, the doubling method mainly comprises natural doubling, bud tip cutting, growth point injection, immature embryo culture and the like, the natural doubling efficiency is low, the bud tip cutting, growth point injection and immature embryo culture technologies need to be very skilled, colchicine needs to be used in the doubling process, the medicament has high toxicity to people, and great care needs to be taken during operation.
Therefore, it is very meaningful to explore a method which is simple in operation method, high in doubling efficiency and suitable for large-scale operation.
Disclosure of Invention
The invention aims to provide a method for doubling haploid seedlings of plants.
The method provided by the invention comprises the following steps:
1) soaking roots of plant haploid seedlings growing to 1-4 leaf stages in podophyllotoxin solution to obtain treated plant seedlings;
2) transplanting the treated plant seedlings to a field to realize plant haploid seedling doubling.
It is yet another object of the present invention to provide a method for increasing the rate of fines in a plant.
The method provided by the invention comprises the following steps:
1) soaking roots of plant haploid seedlings growing to 1-4 leaf stages in podophyllotoxin solution to obtain treated plant seedlings;
2) transplanting the treated plant seedlings to a field to realize the improvement of the powder scattering rate of plants.
In the above method, the concentration of the podophyllotoxin solution is 0.001-1 mg/ml; further preferably, the concentration of the podophyllotoxin solution is 0.05 mg/ml.
The solute of the podophyllotoxin solution is podophyllotoxin, and the preparation method comprises the following steps: dissolving 0.1g of podophyllotoxin (Bailingwei science and technology Co., Ltd., product number: 131142) in 100ml of 95% ethanol aqueous solution to obtain a podophyllotoxin mother liquor; then 10ml of the mother liquor is diluted with purified water to the target concentration of 0.05 mg/ml.
In the method, the soaking time is 1-10 hours; more preferably, the soaking time is 5 hours.
Further, in the soaking step of the method of the present invention, the white part of the root is completely soaked in the solution, and the green part of the leaf is completely exposed out of the solution.
In the above method, the plant is a monocotyledon, and in the examples of the present invention, corn is used as an example.
The application of the method in improving the doubling efficiency of the plant haploid seedlings is also within the protection scope of the invention;
or the application of the method in promoting the doubling of the plant haploid seedlings is also the protection scope of the invention;
or the application of the method in improving the plant loose powder rate is also within the protection scope of the invention.
The application of the podophyllotoxin solution in the method for improving the doubling efficiency of the plant haploid seedlings is also within the protection range of the invention;
or the application of the podophyllotoxin solution in the method in promoting the doubling of plant haploid seedlings is also within the protection scope of the invention;
alternatively, the application of podophyllotoxin solution in the method for improving the powder scattering rate of plants is also within the protection scope of the invention.
The realization of doubling of the plant haploid seedlings or the promotion of the plant haploid seedling polyploidy now improves the plant haploid doubling efficiency, and the improvement of the plant haploid doubling efficiency is specifically embodied in the improvement of the plant pollen scattering rate.
The plant loose powder rate is improved in such a way that the plant loose powder rate obtained by the method is higher than that obtained by the method without adding podophyllotoxin solution for soaking treatment.
In the method, all the operations are performed in the field, so laboratory equipment is not needed, the doubling effect is good, and the method has a good application prospect.
The corn haploid seedling doubling technology provided by the invention can obviously improve the powder scattering rate of the corn haploid, and the powder scattering rate is up to more than 70% and far higher than the powder scattering rate of a contrast (generally about 10%). The drug podophyllotoxin used by the method has weak volatility, and can be operated by an operator only by wearing a disposable glove and a mask; and simultaneously, haploid doubling is carried out by adopting a method of raising seedlings and transplanting in an arched shed or open field, and the method is independent of laboratory operation and suitable for large-scale batch production of doubled haploid.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Unless otherwise specified, the chemical agents used in the examples are all conventionally available, seeds can be pollinated with an inducing line and selected, and the technical means used in the examples are conventional means well known to those skilled in the art.
The corn haploid kernel in the following example is obtained as follows:
1. cultivation of corn haploid grains
The research on induction frequency of different varieties and generation haploids of corn by Nongda high inducing No. 1 (Lizhongliang, Sujun, Lichuxia, Gongcheng, Song-xi, Yan shuqin, Huhuhui, Wangmquan. Nongda high inducing No. 1 [ J ]. corn science, 2008(05):3-6) is taken as a male parent, Vitaceae 702 (Henan Jinyuan breed Ltd.) is taken as a female parent for hybridization, after grouting is finished, fruit ears are harvested, and seeds with purple top embryos and colorless tops at the tops of the seeds are selected to be taken as the haploidal seeds.
2. Identification of corn haploid grains
Identifying the haploid-like seeds by the following methods 1) or 2) to obtain purple apical white embryo corn haploid seeds obtained by hybridization of Nonggaoqiao No. 1 and Vitaceae 702:
1) identification of genetic marker
The method comprises the following specific steps of recording in the following documents, Lizhongliang, Sujun, Lichuxia, Gorghen, Song tin chapter, Ducheng qin, Huguanghui and Wangmuan, and research on different qualities and generation haploid induction frequencies of corn by Nongda GaoJing No. 1 [ J ] corn science, 2008(05): 3-6.
2) Cytological characterization
And cutting root tips for cytological tabletting when the similar haploid seeds are sowed and grow to sprouts, and observing the number of chromosomes. Cutting corn root tip 2-3mm, soaking in saturated p-dichlorobenzene water solution for 3-4 hr, and fixing with Carnot fixing solution (95% ethanol: glacial acetic acid: 3: 1) for 24 hr. Transferring into 70% ethanol, and storing in refrigerator at 4 deg.C. Washing with ddH2O for several times, adding a mixture of cellulase (2%) and pectinase (0.5%), and dissociating at 28 deg.C for 4-5 h. Tabletting, observing the chromosome number under a microscope, and photographing.
The normal number of maize chromosomes is 2 n-20, and the number of haploid chromosomes is n-10.
Example 1 corn haploid doubling method
The corn haploid doubling method provided by the embodiment comprises the following steps:
1. seeding and seedling raising
In 11 th month in winter, in a south shore farm breeding base in the san city, cliff, hainan province, 960 grains of the purple-top white-embryo corn haploid seeds obtained by hybridization of the Dagaoqiao No. 1 and Vitaceae 702 are selected and dibbled in a commercial 96-hole seedling tray using river sand as a medium for seedling cultivation, the river sand needs to be screened out, stones are removed, the sowing depth is 2cm, and 960 grains of the haploid seeds are sown in 10 seedling trays together. Laying a micro-spraying belt on every two rows of seedling raising trays, and immediately watering thoroughly after sowing; spraying water for 2 minutes every morning, keeping river sand moist, and obtaining haploid seedlings;
2. haploid doubling
And (3) in the 3-leaf stage, taking a seedling tray containing haploid seedlings, washing river sand with water in the morning, carefully taking out the haploid seedlings, avoiding breaking off main roots of the seedlings, cleaning and drying the seedlings by using clear water, bundling each 50 seedlings by using nylon grass, stacking the seedlings in 50mm 25mm 60mm plastic boxes, placing the plastic boxes in shady positions, and obtaining 900 haploid seedlings in total, wherein part of the haploid seeds do not emerge, so that part of the haploid seedlings with too small ages are eliminated, and each plastic box can contain 900 seedlings.
Preparing a 0.05mg/ml podophyllotoxin solution: dissolving 0.1g of podophyllotoxin (Bailingwei science and technology Co., Ltd., product number: 131142) in 100ml of 95% ethanol aqueous solution to obtain a podophyllotoxin mother liquor; then 10ml of podophyllotoxin mother liquor is added into 200ml of purified water to be diluted to the concentration of 0.05 mg/ml.
Pouring 0.05mg/ml podophyllotoxin solution from one corner of a plastic box at 9 am, so that the podophyllotoxin solution can submerge the roots of all haploid seedlings, the liquid level is most suitable for the white and green junction of the roots and the leaves, and soaking for 5 hours at normal temperature to obtain the treated seedlings.
3. Transplanting to field
Draining the liquid medicine, repeatedly washing the treated seedlings with running water for 30 minutes, transplanting the seedlings into a field in the afternoon with row spacing of 60cm and plant spacing of 12cm, and immediately watering. The first week is watered once every morning and afternoon, new leaves grow out after one week, and normal management (enough fertilizer and water supply and good cultivation management measures are ensured, fertilizer is applied in times, irrigation is carried out timely and properly, and insect and mouse damage is prevented seriously) is carried out (described in the following documents: Lijiwen, Liuchang rainbow, Liu Zhi Xian, Yanghe, Li Shun, Sunxou, Yangli, Dingzhuhua, efficient doubling technical specification of corn haploid [ J. Shandong agricultural science, 2013,45(10):121 + 124+130.), and finally 850 plants survive normally.
4. Detection of
The powder scattering rate is a main index for measuring the doubling effect of the corn haploid.
And (5) surveying the number of scattered powder plants in the powder scattering period, wherein the scattered powder plants are haploid doubled plants. The powder scattering period of the whole group lasts for 5-25 days, the number of new powder scattering plants is surveyed and recorded every day, the powder scattering rate of the corn haploid plants is calculated after the powder scattering period is finished, and the formula is the percentage of the number of the powder scattering plants in the total number of the plants.
The method for detecting the loose powder plant comprises the following steps 1) or 2):
1) and (3) phenotype observation: the small ear glumes on the tassels are opened, the anthers are fully exposed and split, the pollen grains are scattered by knocking with hands, and the plants with the anthers and the pollen are marked as pollen-scattered plants, namely the plants with doubled haploids.
It should be noted that although the anther of an individual plant is exposed, the anther is shriveled, and if the individual plant is knocked by hand, the plant can not be regarded as a pollen-scattered plant without pollen drifting.
2) And (3) dyeing detection: when the pollen is scattered, the tassel is beaten by hands, the fallen pollen is collected by a paper bag and placed on a glass slide, 1-2 drops of 0.2% I-KI solution are added, and the pollen is tabletted and observed under a low power microscope. The normal pollen contains starch which turns blue when meeting iodine, and the pollen with low activity turns yellow brown when meeting iodine. Blue pollen is fertile and is regarded as a loose pollen plant if the doubling is successful; the yellow brown pollen is sterile and is not a loose powder plant, i.e. the doubling is not successful.
The results are as follows: in 850 normal survival corn plants prepared by the method, the loose powder plants (namely, the doubled haploid plants) are 680 plants (verified by a dyeing microscopic examination, the doubling is successful), and the loose powder rate is 80%.
Control group: 960 grains of the purple apical white embryo corn haploid seeds obtained by the hybridization of Nongda high inducing No. 1 and Vitaceae 702 are directly sowed in a field, the planting density is the same, no medicament doubling treatment is needed, the management is normal, and finally 860 haploid seedlings grow normally.
The number of the scattered powder plants is recorded in daily investigation in the powder scattering period, the powder scattering rate of the corn haploid plants is detected, the number of the scattered powder plants (namely, the plants after haploid doubling) is 86 under the contrast of natural conditions, and the powder scattering rate is 10%.
Therefore, the powder scattering rate of the doubled corn prepared by the method is far higher than that of the control, and the method disclosed by the invention is used for solving the key technical problem of low powder scattering rate in the haploid doubling process, so that the haploid doubling efficiency is greatly improved.
Example 2 corn haploid doubling method
The corn haploid doubling method provided by the embodiment comprises the following steps:
1. seeding and seedling raising
In late spring 4 months, in a Jinyuan breeding base in a cisoid district in Beijing, the simple plastic arched shed is supported by using wood piles, 960 grains of white embryo corn haploid seeds obtained by hybridization of Ningda Gaoqiang No. 1 and Vitaceae 702 are dibbled and sown in a commercially available 96-hole seedling tray using river sand as a medium for seedling, the river sand needs to be screened, the stones are removed, the sowing depth is 1.5-2cm, and 960 grains of haploid seeds are sown in 10 seedling trays together. Laying a micro-spraying belt on every two rows of seedling raising trays, and immediately watering thoroughly after sowing; spraying water for 2 minutes every morning, keeping river sand moist, and obtaining haploid seedlings;
2. haploid doubling
And (3) in the 3-leaf stage, taking a seedling tray containing haploid seedlings, washing river sand with water in the morning, carefully taking out the haploid seedlings, avoiding breaking off main roots of the seedlings, cleaning and draining the seedlings with clear water, bundling each 50 seedlings with nylon grass, stacking the seedlings in 50mm 25mm 60mm plastic boxes, placing the boxes in shady places, and obtaining 890 haploid seedlings in total, wherein part of the haploid seeds do not emerge, so that part of the haploid seedlings with too small ages are eliminated, and each plastic box is provided with 890 haploid seedlings.
Preparing a 0.05mg/ml podophyllotoxin solution: dissolving 0.1g of podophyllotoxin (Bailingwei science and technology Co., Ltd., product number: 131142) in 100ml of 95% ethanol aqueous solution to obtain a podophyllotoxin mother liquor; then 10ml of podophyllotoxin mother liquor is diluted by 200ml of purified water to the concentration of 0.05 mg/ml.
3. Transplanting to field
Draining the liquid medicine, repeatedly washing the treated seedlings with running water for 30 minutes, transplanting the seedlings into a field in the afternoon with row spacing of 60cm and plant spacing of 12cm, and immediately watering. Watering with micro-spraying tape once every two afternoons of the ten morning in the first week, growing new leaves after one week, and performing normal management, wherein 840 plants grow normally.
4. Detection of
The number of the scattered powder plants is recorded in daily investigation of the powder scattering period, the powder scattering rate of the corn haploid plants is calculated, and the result is as follows: in the method of the invention, the number of pollen scattering plants (namely, the doubled haploid plants) in 840 plants is 630 (the doubling is successful after the verification of the microscopic dyeing inspection), and the pollen scattering rate is 75%.
Control group: 960 grains of the purple apical white embryo corn haploid seeds obtained by the hybridization of Nongda Gaoqiang No. 1 and Vitaceae 702 are directly sown in a field, the planting density is the same as the density of medicament treatment, normal management is carried out, and finally 850 haploid seedlings normally develop. The number of new scattered powder plants is recorded in daily investigation at the powder scattering period, the powder scattering rate of the corn haploid plants is calculated, the number of the scattered powder plants (namely the plants after haploid doubling) under the contrast natural condition is 68 (cell line verification shows that the doubling is successful), and the powder scattering rate is 8%.
Therefore, the powder scattering rate of the doubled corn prepared by the method is far higher than that of the control, and the method disclosed by the invention is used for solving the key technical problem of low powder scattering rate in the haploid doubling process, so that the haploid doubling efficiency is greatly improved.
Example 3 optimization of corn haploid doubling method
First, searching podophyllotoxin solution with different concentration
1. Seeding and seedling raising
960 pieces of purple apical white embryo corn haploid kernels obtained by crossing Nongda high inducing No. 1 with Vitaceae 702 are sown and raised according to the step 1 in the embodiment 1;
2. haploid doubling
Preparing podophyllotoxin solutions with different concentrations: dissolving 0.1g of podophyllotoxin in 100ml of 95% ethanol water solution to obtain a podophyllotoxin mother solution; then, 10ml of the podophyllotoxin mother liquor is diluted with 20L, 10L, 200ml and 10ml of purified water to the concentrations of 0.0005mg/ml, 0.001mg/ml, 0.05mg/ml and 1mg/ml respectively.
Substantially the same procedure as in step 2 of example 1, except that the 0.05mg/ml podophyllotoxin solution was replaced with the podophyllotoxin solution of different concentration.
3. Transplanting to a field: same as in example 1 or 2, step 3.
4. And (3) detection: as in step 4 of example 1 or 2, the loose powder rates of 20%, 70%, 80% and 75% were obtained after treatment with the podophyllotoxin solutions of 0.0005mg/ml, 0.001mg/ml, 0.05mg/ml and 1 mg/ml.
As can be seen, the podophyllotoxin solution concentration of 0.001-1mg/ml has good powder-dispersing rate, with 0.05mg/ml being the most preferred.
Secondly, the soaking time of the podophyllotoxin solution is groped
1. Seeding and seedling raising
960 pieces of purple apical white embryo corn haploid kernels obtained by crossing Nongda high inducing No. 1 with Vitaceae 702 are sown and raised according to the step 1 in the embodiment 1;
2. haploid doubling
Basically the same as in step 2 of example 1, except that the haploid seedling roots were soaked in the 0.05mg/ml podophyllotoxin solution for 0.5, 1, 5, 10 hours, respectively.
3. Transplanting to a field: same as in example 1 or 2, step 3.
4. And (3) detection: as a result, the soaking time of podophyllotoxin solution in seedling roots was 0.5, 1, 5, 10 hours, and the pollen-dispersing rate of corn plants prepared was 15%, 60%, 70%, 65% as in step 4 of example 1 or 2.
It can be seen that the soaking time of 1-10 hours gives a good powder scattering rate, with 5 hours being the most preferred.
Thirdly, seedlings and seedling roots
1. Seeding and seedling raising
960 pieces of purple apical white embryo corn haploid kernels obtained by crossing Nongda high inducing No. 1 with Vitaceae 702 are sown and raised according to the step 1 in the embodiment 1;
2. haploid doubling
Substantially the same as in step 2 of example 1, except that the haploid seedling roots and the haploid whole seedlings were soaked in the podophyllotoxin solution at 0.05mg/ml for a time.
3. Transplanting to a field: same as in example 1 or 2, step 3.
4. And (3) detection: the same as the step 4 of the embodiment 1 or 2, the result shows that the powder scattering rate of the doubled corn plants prepared by soaking the haploid seedling roots in the podophyllotoxin solution is 70 percent; the powder dispersing rate of doubled corn plants prepared by soaking the whole haploid seedlings in podophyllotoxin solution is 50%.
It can be seen that the root soaking effect is good.
Fourthly, seedlings in different periods
1. Seeding and seedling raising
960 pieces of purple apical white embryo corn haploid kernels obtained by crossing Nongda high inducing No. 1 with Vitaceae 702 are sown and raised according to the step 1 in the embodiment 1;
2. haploid doubling
Substantially the same as in step 2 of example 1, except that haploid seedlings at 1, 2, 3, and 4 leaf stages and seedlings at 5 leaf stages were soaked in a podophyllotoxin solution of 0.05mg/ml, respectively.
3. Transplanting to a field: same as in example 1 or 2, step 3.
4. And (3) detection: the same as the step 4 of the example 1 or 2, the results show that the pollen scattering rate of the doubled corn plants prepared by haploid seedlings in the 1, 2, 3 and 4 leaf stages is 70%, 75%, 80% and 75% respectively; the pollen scattering rate of the doubled maize plants prepared from the 5-leaf stage seedlings was 30%.
It can be seen that the haploid seedlings in the 1, 2, 3 and 4 leaf stages have good effects, and the haploid seedlings in the 3 leaf stage are particularly the best.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for doubling haploid seedlings of corn comprises the following steps:
1) soaking roots of corn haploid seedlings growing to 1-4 leaf stages in podophyllotoxin solution to obtain treated corn seedlings;
2) transplanting the treated corn seedlings to a field to realize doubling of corn haploid seedlings;
the concentration of the podophyllotoxin solution is 0.05 mg/ml;
the solute of the podophyllotoxin solution is podophyllotoxin;
the soaking time is 5 hours.
2. The method for improving the haploid powder scattering rate of the corn comprises the following steps:
1) soaking roots of corn haploid seedlings growing to 1-4 leaf stages in podophyllotoxin solution to obtain treated corn seedlings;
2) transplanting the treated corn seedlings to a field to improve the haploid pollen scattering rate of the corn;
the concentration of the podophyllotoxin solution is 0.05 mg/ml;
the solute of the podophyllotoxin solution is podophyllotoxin;
the soaking time is 5 hours.
3. Use of the method of claim 1 or 2 to increase the doubling efficiency of maize haploid seedlings;
or the use of the method of claim 1 or 2 for promoting doubling of maize haploid seedlings;
or the use of the method of claim 1 or 2 for increasing the haploid corn pollen dispersal rate.
4. Use of a podophyllotoxin solution in the method of claim 1 or 2 to increase the doubling efficiency of corn haploid seedlings by soaking the roots of corn haploid seedlings grown to the 1-4 leaf stage for 5 hours;
or the use of the podophyllotoxin solution of the method of claim 1 or 2 to promote doubling of corn haploid seedlings by soaking the roots of corn haploid seedlings grown to the 1-4 leaf stage for 5 hours;
or, the use of podophyllotoxin solution in the method of claim 1 or 2 to increase corn haploid pollen dispersal rate by soaking the roots of corn haploid seedlings grown to 1-4 leaf stage for 5 hours.
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