CN115777538A - Short-period cultivation method for cotton - Google Patents
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Abstract
The invention relates to the technical field of cotton cultivation, and provides a short-period cotton cultivation method, which comprises the following steps: step A, sampling: selecting the malformed seedlings as samples; step B, inducing embryogenic callus: placing the abnormal seedlings into an embryo maturation culture medium for induction culture and growth, and culturing for 1-2 months until 40-60% of the abnormal seedlings grow embryogenic callus; step C, culturing embryoid: putting the embryonic callus into a cotton immature embryo culture medium for culturing for 1-2 months, and then transferring the embryonic callus into an embryo maturation culture medium for culturing and growing until all the embryonic callus develops into embryoids; step D, cultivating normal plants: continuously culturing the embryoid for 2-3 months until 25-35% of embryoid grows into normal plant; and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, repeating the step A, the step B, the step C and the step D, and eliminating the rest deformed seedlings. Through the technical scheme, the problem of low callus seedling rate in the related technology is solved.
Description
Technical Field
The invention relates to the technical field of cotton cultivation, in particular to a short-period cotton cultivation method.
Background
Cotton is the most important natural fiber crop in the world, has no alternative position in human clothes and food activities, and with continuous improvement of spinning technology and improvement of living standard of people, higher requirements are put forward on the quality of cotton fibers, particularly on the fiber strength. Cotton is the first economic crop in China, plays an important role in national economy, cotton fiber is an important textile raw material, and cottonseed oil and cottonseed protein are important vegetable oil and protein resources. With the rapid development of economy and improvement of living standard of people in China, the demand on cotton is increasing day by day.
The plant tissue culture technology is a process of culturing explants in a culture medium under aseptic conditions to provide culture conditions, so that the explants become finished plants. Therefore, the technology can provide abundant receptor materials for plant genetic transformation. Tissue culture is widely applied to the fields of rapid propagation, artificial seeds, new species culture and the like, and plays an irreplaceable role in the field of plant genetic engineering improvement as a basic way and an important means for plant basic genetic engineering research.
The regeneration of cotton tissue culture plants is mainly to induce the appearance of embryoid, and the embryogenesis of cotton is the process of generating regeneration plants from radicles, cotyledons, hypocotyls or protoplasts of aseptic seedlings through callus induction, callus differentiation and somatic embryo development. The physiological state of the embryo callus is relatively consistent, so that a stable material source can be provided for research. However, abnormal seedlings inevitably appear in the callus culture, and the abnormal seedlings are difficult to grow into robust seedlings due to the dysplasia of individuals, so that the abnormal seedlings are usually picked and thrown away when the abnormal seedlings are observed to be generated. The generation of abnormal seedlings causes the problem of low seedling rate of callus, and the regenerated seedlings are obtained by culturing abnormal seedlings in the paper of research on cotton somatic embryogenesis mechanism and genetic transformation of p53 gene, but the specific technical scheme, culture conditions and time are not described.
Disclosure of Invention
The invention provides a short-period cotton cultivation method, which solves the problem of low callus seedling rate in the related technology.
The technical scheme of the invention is as follows: a short-period cotton cultivation method comprises the following steps:
step A, sampling: selecting abnormal seedlings generated in the cotton cultivation process as a sample, as shown in figure 2-A;
step B, inducing embryonic callus: placing the abnormal seedlings into an embryo maturation culture medium for induction culture and growth, culturing for 1-2 months until 40-60% of abnormal seedlings grow embryogenic callus, as shown in figure 2-B, controlling indoor temperature at 25-28 deg.C, indoor humidity at 45-60%, and indoor illumination time at 12-14 hr;
step C, embryoid cultivation: placing the selected embryogenic callus on cotton immature embryo culture medium, culturing for 1-2 months, transferring into embryo maturation culture medium, and culturing until about 80% of embryogenic callus develops into embryoid body as shown in FIG. 2-C;
step D, cultivating normal plants: continuing to culture the embryoid bodies for 2-3 months in step C until 25-35% of the embryoid bodies have grown into normal plants, as shown in FIG. 2-D;
and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, and repeating the step A, the step B, the step C and the step D.
As a further technical scheme, the cotton immature embryo culture medium comprises the following components in parts by weight: 825 parts of ammonium nitrate, 950 parts of potassium nitrate, 95 parts of magnesium sulfate, 85 parts of monopotassium phosphate, 165 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 part of cobalt chloride hexahydrate, 0.025 part of copper sulfate pentahydrate, 0.83 part of potassium iodide, 0.25 part of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, 1 part of glycine, 50 parts of inositol, 20 parts of proline, 500 parts of glutamine, 20000 parts of sucrose, 2500 parts of Phytagel, 0.1 part of IBA, 0.1 part of KT and 0.1 part of 6-BA.
As a further technical scheme, the embryo maturation medium comprisesThe following components in parts by weight: 3800 parts of potassium nitrate, 190 parts of magnesium sulfate, 170 parts of monopotassium phosphate, 330 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 parts of cobalt chloride hexahydrate, 0.025 parts of copper sulfate pentahydrate, 0.83 parts of potassium iodide, 0.25 parts of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, 1 part of glycine, 50 parts of inositol, 500 parts of glutamine, 500 parts of asparagine, 30000 parts of glucose, 2600 parts of Phytagel, 0.3-0.5 part of IBA and 0.1-0.15 part of KT.
The beneficial effects of the invention are as follows:
1. the deformed seedlings generated in the cotton cultivation process are applied to the cultivation method provided by the invention, and a part of the deformed seedlings can be cultivated into normal seedlings, so that the cost of a laboratory in the aspect of cotton seedlings is reduced;
2. the abnormal seedlings are developed into callus through the method, and then are cultured and developed into normal plants, so that the whole culture period of the cotton is greatly shortened, the stage from seed development to callus development of the cotton is avoided, the growth period of the normal plants in the culture method is further shortened, and a large number of normal plants can be provided for a laboratory in a short period;
3. the cultivation method provided by the invention can repeatedly utilize the abnormal seedlings generated in the self method, further reduce the dosage of the abnormal seedlings in the conventional cultivation method, and can perform one-round screening on the abnormal seedlings to ensure that each abnormal seedling can be sufficiently developed into a normal plant.
For reference, the method can be used for multiplying the number of transgenic regeneration plants in the process of obtaining transgenic over-expression regeneration plants by infecting hypocotyls with agrobacterium for genetic transformation. For reference, in the process of obtaining gene editing or RNAi regeneration plants through genetic transformation of agrobacterium-infected hypocotyls, the number of the gene editing or RNAi plants can be increased by times by using the method. For the characteristic of long transformation period of cotton, the increase of the number of over-expressed and gene-edited regenerated plants is particularly important.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic structural diagram of various stages in a conventional cotton cultivation method provided by the present invention;
FIG. 2 is a schematic diagram showing the structure of each stage of the short-cycle cotton training method according to the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.
Example 1
The embodiment provides a short-period cotton cultivation method, which comprises the following steps:
step A, sampling: selecting abnormal seedlings generated in the cotton cultivation process as a sample, as shown in figure 2-A;
step B, inducing embryonic callus: placing the abnormal seedlings into an embryo maturation culture medium for induction culture and growth, and culturing for 1-2 months until 40-60% of abnormal seedlings grow embryogenic callus, as shown in figure 2-B, wherein the indoor temperature is controlled at 25-28 deg.C, the indoor humidity is controlled at 45-60%, and the indoor illumination time is 12-14 hr;
step C, embryoid cultivation: placing the selected embryogenic callus on cotton immature embryo culture medium, culturing for 1-2 months, transferring into embryo maturation culture medium, and culturing until about 80% of embryogenic callus develops into embryoid body as shown in FIG. 2-C;
step D, cultivating normal plants: continuing to culture the embryoid bodies for 2-3 months in step C until 25-35% of the embryoid bodies have grown into normal plants, as shown in FIG. 2-D;
and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, and repeating the step A, the step B, the step C and the step D.
Comparative example 1
The comparative example provides a short-period cotton cultivation method, which comprises the following steps:
step A, sampling: selecting abnormal seedlings generated in the cotton cultivation process as samples;
step B, inducing embryogenic callus: placing the abnormal seedlings into an embryo maturation culture medium for induced culture growth, culturing for 1-2 months until 40-60% of the abnormal seedlings grow embryogenic callus, controlling indoor temperature at 25-28 deg.C, indoor humidity at 45-60%, and indoor illumination time for 3-4 hr;
step C, embryoid cultivation: placing the selected embryogenic callus on a cotton immature embryo culture medium for culturing for 1-2 months, and then transferring the embryogenic callus into an embryo maturation culture medium for culturing and growing until about 80% of the embryogenic callus develops into embryoid bodies;
step D, cultivating normal plants: continuing to culture the embryoid for 2-3 months in the step C until 25-35% of the embryoid grows into a normal plant;
and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, and repeating the step A, the step B, the step C and the step D.
Comparative example 2
The comparative example provides a short-period cotton cultivation method, which comprises the following steps:
step A, sampling: selecting deformed seedlings generated in the cotton cultivation process as samples;
step B, inducing embryogenic callus: placing the abnormal seedlings into an embryo maturation culture medium for induction culture and growth, culturing for 1-2 months until 40-60% of the abnormal seedlings grow embryogenic callus, controlling indoor temperature at 25-28 deg.C, controlling indoor humidity at 70-80%, and controlling indoor illumination time at 12-14h;
step C, culturing embryoid: placing the selected embryogenic callus on a cotton immature embryo culture medium for culturing for 1-2 months, and then transferring the embryogenic callus into an embryo maturation culture medium for culturing and growing until about 80% of the embryogenic callus develops into embryoids;
step D, cultivating normal plants: continuing to culture the embryoid for 2-3 months in the step C until 25-35% of the embryoid grows into a normal plant;
and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, and repeating the step A, the step B, the step C and the step D.
Comparative example 3
The comparison example provides a short-period cotton cultivation method, which comprises the following steps:
step A, sampling: selecting deformed seedlings generated in the cotton cultivation process as samples;
step B, inducing embryonic callus: placing the abnormal seedlings into an embryo maturation culture medium for induction culture and growth, culturing for 1-2 months until 40-60% of the abnormal seedlings grow embryogenic callus, controlling indoor temperature at 20-22 deg.C, controlling indoor humidity at 45-60%, and controlling indoor illumination time at 12-14h;
step C, culturing embryoid: placing the selected embryogenic callus on a cotton immature embryo culture medium for culturing for 1-2 months, and then transferring the embryogenic callus into an embryo maturation culture medium for culturing and growing until about 80% of the embryogenic callus develops into embryoid bodies;
step D, cultivating normal plants: continuing to culture the embryoid for 2-3 months in the step C until 25-35% of the embryoid grows into a normal plant;
and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, and repeating the step A, the step B, the step C and the step D.
In example 1, comparative example 2 and comparative example 3, 10 normal plants in step D were randomly selected and numbered 1-1, 1-2, through 4-10 in order, and then the stem length and the leaf area of the true leaf of the plants were measured and recorded, finally forming table one and table two:
table one:
| serial number | Stem length (cm) | Serial number | Stem length (cm) | Serial number | Stem length (cm) | Serial number | Stem length (cm) |
| 1-1 | 7.5 | 2-1 | 4.3 | 3-1 | 4.9 | 4-1 | 4.2 |
| 1-2 | 8.2 | 2-2 | 4.2 | 3-2 | 4.7 | 4-2 | 4.1 |
| 1-3 | 8.1 | 2-3 | 3.9 | 3-3 | 5.1 | 4-3 | 3.3 |
| 1-4 | 7.9 | 2-4 | 4.1 | 3-4 | 4.7 | 4-4 | 3.4 |
| 1-5 | 8.3 | 2-5 | 3.7 | 3-5 | 5.2 | 4-5 | 3.5 |
| 1-6 | 8.2 | 2-6 | 3.8 | 3-6 | 5.1 | 4-6 | 3.9 |
| 1-7 | 7.6 | 2-7 | 3.9 | 3-7 | 5.3 | 4-7 | 4.3 |
| 1-8 | 7.9 | 2-8 | 4.0 | 3-8 | 5.1 | 4-8 | 3.4 |
| 1-9 | 7.7 | 2-9 | 4.1 | 3-9 | 4.8 | 4-9 | 4.0 |
| 1-10 | 7.8 | 2-10 | 3.8 | 3-10 | 4.6 | 4-10 | 4.1 |
As can be seen from the table I, the stem length of the normal plants cultured in example 1 is larger than that of comparative examples 1, 2 and 3, which indicates that the method provided in example 1 can improve the success rate of the abnormal seedlings to develop into normal plants.
Table two:
as can be seen from the table II, the true leaf area of the normal plant cultured in example 1 is larger than that of comparative examples 1, 2 and 3, which indicates that the method provided in example 1 can make the normal plant developed from the abnormal seedling have more vigor and can survive relatively more easily, and thus the method is more suitable for the cultivation concept of cotton in short period.
FIG. 2 is the process of developing a malformed seedling into a normal plant using the method of the present invention. Wherein (A) represents the state of a brown abnormal seedling in an embryo maturation medium, (B) represents the state of a yellow-green granular embryogenic callus growing from the abnormal seedling, (C) represents the state of the embryogenic callus developing into an embryoid body resembling an embryo, and (D) represents the state of the embryoid body developing into a normal plant.
FIG. 1 shows the cotton at various stages of a conventional growing process. The conventional cultivation method of the cotton comprises the following steps: planting aseptic seedlings (A) to grow for 5-6 days, cutting hypocotyl sections, placing the hypocotyl sections on a first callus induction culture medium (B), transferring the hypocotyl sections to a second callus induction culture medium after 1 month, growing embryonic callus (C) on about 20% of the hypocotyl sections after 4-5 months, placing the embryonic callus on an embryo maturation culture medium, growing embryoid (D) on about 80% of the embryonic callus in 2-3 months, and growing normal plants (E) on about 30% of the embryoid in 2-3 months.
Further, the callus induction culture medium comprises the following components in parts by weight: 1650 parts of ammonium nitrate, 1900 parts of potassium nitrate, 190 parts of magnesium sulfate, 170 parts of monopotassium phosphate, 330 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 part of cobalt chloride hexahydrate, 0.025 part of copper sulfate pentahydrate, 0.83 part of potassium iodide, 0.25 part of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, 1 part of glycine, 50 parts of inositol, 500 parts of glutamine, 500 parts of asparagineAmine, 30000 parts of glucose, 2600 parts of Phytagel, 0.1 part of 2,4-D and 0.1 part of KT.
Further, the second callus induction culture medium comprises the following components in parts by weight: 1650 parts of ammonium nitrate, 1900 parts of potassium nitrate, 190 parts of magnesium sulfate, 170 parts of monopotassium phosphate, 330 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 part of cobalt chloride hexahydrate, 0.025 part of copper sulfate pentahydrate, 0.83 part of potassium iodide, 0.25 part of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, 1 part of glycine, 50 parts of inositol, 500 parts of glutamine, 500 parts of asparagine, 30000 parts of glucose, 2600 parts of Phytagel, 0.5 part of IBA and 0.1 part of KT.
Further, the embryo maturation culture medium comprises the following components in parts by weight: 3800 parts of potassium nitrate, 190 parts of magnesium sulfate, 170 parts of monopotassium phosphate, 330 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 parts of cobalt chloride hexahydrate, 0.025 parts of copper sulfate pentahydrate, 0.83 parts of potassium iodide, 0.25 parts of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, 1 part of glycine, 50 parts of inositol, 500 parts of glutamine, 500 parts of asparagine, 30000 parts of glucose, 2600 parts of Phytagel, 0.1-0.5 part of IBA and 0.1-0.15 part of KT.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A short-period cotton cultivation method is characterized by comprising the following steps:
step A, sampling: selecting deformed seedlings generated in the cotton cultivation process as samples;
step B, inducing embryonic callus: placing the abnormal seedlings into an embryo maturation culture medium for induction culture and growth, culturing for 1-2 months until 40-60% of the abnormal seedlings grow embryogenic callus, controlling indoor temperature at 25-28 deg.C, controlling indoor humidity at 45-60%, and controlling indoor illumination time at 12-14h;
step C, culturing embryoid: placing the selected embryogenic callus on a cotton immature embryo culture medium for culturing for 1-2 months, and then transferring the embryogenic callus into an embryo maturation culture medium for culturing and growing until about 80% of the embryogenic callus develops into embryoids;
step D, cultivating normal plants: continuing to culture the embryoid for 2-3 months in the step C until 25-35% of the embryoid grows into a normal plant;
and E, selecting the deformed seedlings with the green buds in the step D, mixing the selected deformed seedlings with the conventionally cultured deformed seedlings, and repeating the step A, the step B, the step C and the step D.
2. The short-period cultivation method of cotton as claimed in claim 1, wherein the cotton immature embryo culture medium comprises the following components in parts by weight: 825 parts of ammonium nitrate, 950 parts of potassium nitrate, 95 parts of magnesium sulfate, 85 parts of monopotassium phosphate, 165 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 part of cobalt chloride hexahydrate, 0.025 part of copper sulfate pentahydrate, 0.83 part of potassium iodide, 0.25 part of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, 1 part of glycine, 50 parts of inositol, 20 parts of proline, 500 parts of glutamine, 20000 parts of sucrose, 2500 parts of Phytagel, 0.1 part of IBA, 0.1 part of KT and 0.1 part of 6-BA.
3. The short-period cultivation method of cotton as claimed in claim 1, wherein the embryo maturation medium comprises the following components in parts by weight: 3800 parts of potassium nitrate, 190 parts of magnesium sulfate, 170 parts of monopotassium phosphate, 330 parts of calcium chloride, 6.2 parts of boric acid, 16.9 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.025 parts of cobalt chloride hexahydrate, 0.025 parts of copper sulfate pentahydrate, 0.83 parts of potassium iodide, 0.25 parts of sodium molybdate dihydrate, 13.9 parts of ferrous sulfate heptahydrate, and 37.26 parts of Na 2 EDTA, 10 parts of thiamine, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid and 1 part of glycine50 parts of inositol, 500 parts of glutamine, 500 parts of asparagine, 30000 parts of glucose, 2600 parts of Phytagel, 0.3-0.5 part of IBA and 0.1-0.15 part of KT.
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