阅读说明：本技术 棉花种子剥皮加倍的种植方法 (Cotton seed peeling and doubling planting method ) 是由 李燕 张永山 陈伟 姚金波 赵兰杰 房圣涛 吕有军 王俊宜 袁黎 于 2018-09-13 设计创作，主要内容包括：本发明公开了一种棉花种子剥皮加倍的种植方法。本发明将棉花种子剥皮后进行秋水仙素处理,然后利用组培方法播种于培养基上,待植株根系长大后,移植到营养土内种植。剥掉种皮,可以剔除部分不成熟的种子,而且棉花处理秋水仙素时排除种皮的影响,处理时间一致,长势一致性也比带种皮要好。培养基育苗在棉苗较弱时进行一定的保护,待渡过易损期后真叶长出,再移栽到营养土内,棉苗对外界有一定的抵抗力,提高苗的存活率进而提高诱变率。另外,培养基的通透性,可以观察到根系的生长情况。本发明对于棉花多倍体育种有重要意义。(The invention discloses a planting method for peeling and doubling cotton seeds. The invention peels the cotton seeds, carries out colchicine treatment, then seeds on a culture medium by a tissue culture method, and transplants the cotton seeds into nutrient soil for planting after the root systems of the plants grow up. The seed coat is peeled off, partial immature seeds can be removed, the influence of the seed coat is eliminated when the cotton is used for treating colchicine, the treatment time is consistent, and the growth consistency is better than that of the seed-carrying seed coat. The culture medium is used for raising seedlings, certain protection is carried out when cotton seedlings are weak, true leaves grow out after the cotton seedlings are worn out, the true leaves are transplanted into nutrient soil, the cotton seedlings have certain resistance to the outside, the survival rate of the cotton seedlings is improved, and the mutagenesis rate is further improved. In addition, the permeability of the medium allows the observation of the growth of the root system. The invention has important significance for cotton polyploid breeding.)

1. A cotton chromosome doubling method, comprising the steps of:

(1) peeling cotton seeds and then treating the peeled cotton seeds by using a mutagen; the mutagen contains colchicine.

(2) Inoculating the seeds treated in the step (1) into a seedling culture medium for culture;

the seedling culture medium consists of solute and solvent; the solute and the concentration thereof in the seedling culture medium are as follows: 0.90-1.0g/L of potassium nitrate, 0.80-0.90g/L of ammonium nitrate, 0.080-0.090g/L of monopotassium phosphate, 0.180-0.190g/L of magnesium sulfate, 0.20-0.30g/L of calcium chloride, 10-20g/L of glucose and 2.0-3.0mg/L of plant gel; the solvent is water.

2. The method of claim 1, wherein: the solute and the concentration thereof in the seedling culture medium are as follows: 0.95g/L of potassium nitrate, 0.85g/L of ammonium nitrate, 0.085g/L of monopotassium phosphate, 0.185g/L of magnesium sulfate, 0.22g/L of calcium chloride, 15g/L of glucose and 2.5mg/L of plant gel.

3. The method of claim 1 or 2, wherein: the mutagen contains 1.6-1.8g/L colchicine, and/or the treatment time is 16-18 h.

4. The method of claim 3, wherein: the mutagen contains 1.5g/L colchicine, and/or the treatment time is 16 h.

5. The method of any of claims 1 to 4, wherein: in the step (2), the culture condition is dark culture at 28 ℃.

6. The method of any of claims 1 to 5, wherein: the step (2) is followed by a step (3): when the seeds germinate and grow 1cm, the seedlings are supported, dark culture is carried out for 2-3 days at 28 ℃, and then culture conditions are changed for continuous culture for 20-40 days; the changed culture conditions are 28 ℃, 16h of light culture/8 h of dark culture, and the illumination intensity is 2000 Lx.

7. The method of any of claims 1 to 6, wherein: the method of claim 4, wherein: the steps (1), (2) and (3) are all operated under aseptic conditions.

8. The method of claim 6 or 7, wherein: the step (3) is followed by a step (4): after the step (3) is finished, transplanting the seedlings into nutrient soil and carrying out moisturizing treatment; before transplanting, seedlings are dipped in an aqueous solution containing 5 mg/LNAA.

9. Use of the method of any one of claims 1 to 8 in the breeding of polyploid cotton.

10. Use of the seedling culture medium as claimed in claim 1 or 2 for seed selection of polyploid cotton.

Technical Field

The invention relates to a planting method for peeling and doubling cotton seeds.

Background

The cotton doubling work was rarely studied, in the 80 s, by the dawn group of cotton and wheat at the Huanggang division, the Chinese academy of agriculture, Huazhong, by soaking cotton (Zn ═ 26) seeds in county with aqueous colchicine solutions of different concentrations for different treatment times and then taking out and sowing. After the variant seedlings come out of the soil, the cotyledons are folded and not easy to unfold, the lower axis of the cotyledons does not come out of the soil, the part from the lower axis of the cotyledons to the radicle is swollen, the root tip is often yellow brown, the growth is stopped for a long time, the stress resistance is weak, and the variant seedlings are extremely easy to be infected with diseases and die when meeting overcast and rainy at low temperature. The growth rate of the variant seedlings was much further behind that of the control and treated normal seedlings.

During the doubling work of cotton seeds, the cotton seeds are directly treated by colchicine and are sown in nutrient soil after being cleaned, but the cotton seedlings treated by the colchicine are easy to damage and rot cotyledons due to slow soil emergence, so that the normal growth of the cotton seedlings is seriously influenced. In addition, the seeds need to be treated with seed coats during the treatment, so that colchicine can act on the growth points of the seeds after soaking the seed coats, the treatment time and the treatment effect are greatly influenced, and if the seed coats are peeled off, the colchicine can rot and die after being sowed in nutrient soil.

Disclosure of Invention

The invention aims to provide a planting method for peeling and doubling cotton seeds.

The invention provides a cotton chromosome doubling method, which comprises the following steps:

(1) peeling cotton seeds and then treating the peeled cotton seeds by using a mutagen; the mutagen contains colchicine.

(2) Inoculating the seeds treated in the step (1) into a seedling culture medium for culture;

the seedling culture medium consists of solute and solvent; the solute and the concentration thereof in the seedling culture medium are as follows: 0.90-1.0g/L of potassium nitrate, 0.80-0.90g/L of ammonium nitrate, 0.080-0.090g/L of monopotassium phosphate, 0.180-0.190g/L of magnesium sulfate, 0.20-0.30g/L of calcium chloride, 10-20g/L of glucose and 2.0-3.0mg/L of plant gel; the solvent is water.

The solute and the concentration thereof in the seedling culture medium can be specifically as follows: 0.95g/L of potassium nitrate, 0.85g/L of ammonium nitrate, 0.085g/L of monopotassium phosphate, 0.185g/L of magnesium sulfate, 0.22g/L of calcium chloride, 15g/L of glucose and 2.5mg/L of plant gel.

The pH value of the seedling culture medium is 6.5.

The mutagen contains 1.6-1.8g/L colchicine.

The mutagen can specifically contain 1.5g/L colchicine.

The mutagen can be an aqueous solution containing 1.5g/L colchicine.

The treatment time is 16-18 h.

The treatment time may be 16 h.

In the step (1), after peeling the cotton seeds, sterilizing the cotton seeds and then treating the cotton seeds with a mutagen.

The sterilization method specifically comprises the following steps: sterilizing with one thousand L Hg for 8-10 min.

In the step (2), the culture condition is dark culture at 28 ℃.

The step (2) is followed by a step (3): when the seeds germinate and grow 1cm, the seedlings are supported, dark culture is carried out for 2-3 days at 28 ℃, and then culture conditions are changed for continuous culture for 20-40 days; the changed culture conditions are 28 ℃, 16h of light culture/8 h of dark culture, and the illumination intensity is 2000 Lx.

Any of the above steps (1), (2) and (3) is operated under aseptic conditions.

The step (3) is followed by a step (4): after the step (3) is finished, transplanting the seedlings into nutrient soil and carrying out moisturizing treatment; before transplanting, the seedlings are dipped in an aqueous solution containing 5mg/L NAA. After transplanting, the periphery of the seedling is covered with a preservative film for moisture preservation, and the seedling is removed after one week.

The step (4) is followed by a step (5): taking about 10 true-leaf seedlings, and screening the seedlings with multiplied chromosome number by flow cytometry.

The invention also protects the application of any one of the methods in polyploid cotton breeding.

The invention also protects the application of any one of the seedling culture media in the breeding of the polyploid cotton.

Any one of the above cotton may specifically be cotton No. 1.

The invention improves the seed doubling and planting method, peels the cotton seeds, treats the cotton seeds with colchicine, seeds the cotton seeds on a culture medium by a tissue culture method, and transplants the cotton seeds into nutrient soil after the root systems of the plants grow up. The seed coat is firstly peeled off, so that the isolation influence of the seed coat during the drug treatment of the seeds is solved, the drug is directly contacted with the growth point of the seeds, the drug action time can be determined, the drug action effect is improved, and the consistent treatment time of each seed is ensured. In addition, after the seed coats are peeled off, the seeds can be rotten and die after being sowed in nutrient soil, no pathogenic bacteria grow in a culture medium, and the problem can be well solved. Secondly, after the cotton seeds are treated by the medicament, the cotton seeds grow slowly, hypocotyls are shortened, the problem that cotyledons are damaged due to in-soil growth of the seeds is caused, the growth of plants is seriously influenced, a stable environment is provided for the growth of the plants through seedlings of the culture medium, and the growth of cotton seedlings is facilitated. In addition, due to the transparency of the culture medium, the growth condition of the root of each plant after the seeds are treated can be well observed, and an important judgment basis is provided for screening the treatment concentration and time. The invention has important significance for cotton polyploid breeding.

Drawings

FIG. 1 is a schematic view of the cotton seed peeling doubling and planting method at each stage in the planting method of example 1.

FIG. 2 shows the germination and growth of seeds in the first control method of example 1.

FIG. 3 shows the germination and rot of seeds in the second control method of example 1.

FIG. 4 shows the growth and damage of three neutron leaves according to the comparative method of example 1.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The cotton used in the following examples is Tonnong No. 1, reference: zhouyui, research on genetic diversity in China for storing Asian cotton [ D ]. Chinese academy of agricultural sciences, 2011. the public can be obtained from the Cotton research institute of Chinese academy of agricultural sciences.

The seedling culture medium consists of solute and solvent; the solute and the concentration thereof in the seedling culture medium are as follows: potassium nitrate 0.95g/L, ammonium nitrate 0.85g/L, potassium dihydrogen phosphate 0.085g/L, magnesium sulfate 0.185g/L, calcium chloride 0.22g/L, glucose 15g/L, plant gel Phytagel (Sigma)2.5mg/L, pH 6.5.