阅读说明：本技术 一种棉花染色体加倍的方法 (Cotton chromosome doubling method ) 是由 周宝良 陈于 于 2019-10-25 设计创作，主要内容包括：本发明公开一种棉花染色体加倍的方法,该方法为用滴加了二甲基亚砜的秋水仙碱溶液对从棉花植株上剪下来的嫩芽进行加倍处理,将加倍处理后的嫩芽嫁接到砧木上,嫁接苗长大后,叶片增大增厚者有可能为染色体加倍植株；待棉花长至现蕾时,取其花蕾进行常规的染色体数目观察,从细胞学上直接观察染色体数目,增加一倍者即为加倍植株；如果处理材料是棉花三倍体种间杂种,则育性正常或有提高者可能为染色体加倍植株,通过细胞学进行染色体数目确认得到染色体加倍植株。本发明方法通过该离体处理加倍技术,工作量小、无需组织培养过程,在实验室内处理不易受环境影响,成功率高,不存在原植株死亡的问题,一般当年处理、当年就可获得染色体加倍的植株。(The invention discloses a method for doubling cotton chromosomes, which is characterized in that a colchicine solution dropwise added with dimethyl sulfoxide is used for doubling tender shoots cut from cotton plants, the doubled tender shoots are grafted onto stocks, and after grafted seedlings grow up, leaves are enlarged and thickened to possibly double the plants for the chromosomes; when the cotton grows to bud, taking the bud to carry out conventional chromosome number observation, directly observing the chromosome number in cytology, and obtaining a doubled plant if the chromosome number is doubled; if the treated material is a cotton triploid interspecific hybrid, then a plant with normal or improved fertility may be a chromosome-doubled plant, and chromosome number confirmation is performed cytologically to obtain a chromosome-doubled plant. The method of the invention adopts the in vitro processing and doubling technology, has small workload, does not need a tissue culture process, is not easily influenced by the environment in a laboratory, has high success rate, does not have the problem of death of the original plant, and can obtain the plant with doubled chromosomes in the current year by processing in general.)

1. A method for doubling cotton chromosome is characterized in that the method is that colchicine solution added with dimethyl sulfoxide is used for doubling tender shoots cut from cotton plants, the tender shoots after doubling treatment are grafted to rootstocks, after the grafted seedlings grow up, the plants can be doubled for chromosome by increasing and thickening leaves; when the cotton grows to bud, taking the bud to carry out conventional chromosome number observation, directly observing the chromosome number in cytology, and obtaining a doubled plant if the chromosome number is doubled; if the treated material is a cotton triploid interspecific hybrid, then a plant with normal or improved fertility may be a chromosome-doubled plant, and chromosome number confirmation is performed cytologically to obtain a chromosome-doubled plant.

2. The cotton chromosome doubling method according to claim 1, which comprises the following steps:

(1) planting a strong cotton plant, removing a main stem growing point to enable the main stem growing point to grow lateral branches and form a plurality of tender buds;

(2) cutting lateral branch tender shoots with 2-3 leaves, removing larger leaves on the lateral branch tender shoots, and preventing water from evaporating and wilting; inserting the cut lateral branch tender shoots into colchicine solution dropwise added with dimethyl sulfoxide for doubling treatment;

(3) grafting the lateral branch tender bud subjected to colchicine doubling treatment onto the stock, and managing by a conventional grafting method;

(4) after the grafted seedling grows up, the plant with doubled chromosome is possible to be the plant with the thickened leaves; when the cotton grows to bud, taking the bud to carry out conventional chromosome number observation, directly observing the chromosome number in cytology, and obtaining a chromosome doubled plant if the chromosome number is doubled; if the treated material is a cotton triploid interspecific hybrid, then a plant with normal or improved fertility may be a chromosome-doubled plant, and chromosome number confirmation is performed cytologically to obtain a chromosome-doubled plant.

3. The method of cotton chromosome doubling according to claim 1 or 2, wherein the concentration of the colchicine solution is 0.1%; the dropping amount of the dimethyl sulfoxide stock solution is 5-8 drops added into 100ml of solution.

4. The cotton chromosome doubling method according to claim 1 or 2, wherein the doubling treatment time is 22-26 h.

5. The cotton chromosome doubling method of claim 4, wherein the doubling treatment is performed for 24 hours.

Technical Field

The invention belongs to the field of biotechnology breeding, and particularly relates to a cotton chromosome doubling method.

Background

Of the 52 cotton species, 45 are diploid species, 2 n-2 x-26 are important gene sources for improved cultivated cotton, however, cultivated cotton accounting for more than 95% of the world cotton yield is heterotetraploid, 2 n-4 x-52. Crossing the diploid with the tetraploid yields hybrid F1 which is triploid (2 n-3 x-39) and highly sterile. The main reasons are as follows: (1) the degree of homology between cotton species chromosomes is low. The chromosomes of hybrid F1 cannot pair into normal bivalents during meiosis, and chromosomes with poor homology mostly exist as monovalents; (2) differences in chromosome structure between cotton species, such as chromosomal translocations, formation of polyvalent and monovalent bodies, result in high sterility, heterozygosity and cytological variability of the hybrid gametes, even with somatic reductions and chimera formation. In order to overcome the sterility of triploid hybrid F1, Qian thinking (1988) considers that the normality of ovules may vary with the combination of crosses, the sunshine and temperature during the formation of floral organs, and the age of the storage of the perennial root, and can be overcome by direct backcross, however, the research results of a large number of cotton interspecific crosses indicate that direct backcross is difficult to succeed in most cases, even if backcross seeds are obtained, the number is very limited, generally only 1 to 2 seeds are obtained, the chromosome number may be incomplete, and the requirement of progeny research is difficult to meet. Therefore, the ideal method is to use colchicine for chromosome doubling to obtain the allopolyploid.

Treatment of F with colchicine₁Seeds, for combinations that yield large quantities of hybrid seeds, using seed doubling to overcome F₁Sterility in (Pengzong, Qianshiying, 1989; Liu jin lan et al, 1990; Wang Shining et al, 1990) is advantageous, but in the case of difficulty in obtaining large numbers of hybrids F₁The seed treatment route is not realistic for the combination of (1). The Liangzhenlan et al (1978) for hybrid boll drop (spray) GA₃And NAA (ananas nakai) -hybrid embryo in vitro culture-in vitro chromosome doubling are combined to obtain a progeny of chromosome doubling, and a technical approach of chromosome doubling is provided, but obviously, the technology is more complicated, tissue culture and the like are needed, and the method is less applied at present.

Therefore, the predecessors usually obtained the chromosome-doubled plants directly by treating live shoots, but this method has many disadvantages: for example, the in vivo treatment of the tender shoots has a plurality of uncontrollable environmental factors, and the environmental conditions are not easy to control; secondly, too high concentration treatment time is too long, which easily leads to plant death or bud withering, and conversely, too low concentration treatment time is too short, which can not obtain plants with doubled chromosomes, and thus, the time and concentration are difficult to grasp. The difficulty of cotton chromosome doubling has become one of the important obstacles for breeding and utilizing diploid cotton gene resources. In order to overcome the problems, the invention uses isolated tender shoots to carry out colchicine treatment, and successfully obtains plant progeny with doubled chromosomes by debugging different concentrations, different times and the like, thereby eliminating the technical obstacle of triploid hybrid F1 for breeding.

Disclosure of Invention

The technical problems aimed at by the invention are as follows: in the traditional method, a large amount of seeds are treated by colchicine, so that the aim of doubling the chromosomes of a very small number of plants is fulfilled; however, for the hybrid combinations with strong hybridization incompatibility, it is difficult to obtain sufficient hybrid seeds for chromosome doubling treatment. Therefore, in general, the chromosome doubling treatment is performed on the basis of the obtained plants. For plant doubling treatment, tender shoots of the plants are generally selected to be directly doubled in vivo. However, in the living tender shoot treatment, firstly, the risk of plant death exists, secondly, the environment is not easy to control, the doubling efficiency is very low or even unsuccessful, and the method becomes one of the important obstacles for limiting the breeding and utilizing diploid cotton gene resources.

In order to solve the technical problem, the invention provides a cotton chromosome doubling method. The method adopts double treatment of tender shoot in vitro in a laboratory, and effectively overcomes the defects of the prior art.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

a method for doubling cotton chromosome, said method comprises using the colchicine solution dripping dimethyl sulfoxide (DMSO) to carry on the doubling to the tender bud that is cut off from the cotton plant, the tender bud after the doubling is grafted to the stock, after the grafted seedling grows up, the blade is enlarged and thickened probably for the chromosome doubles the plant; when the cotton grows to bud, taking the bud to carry out conventional chromosome number observation, directly observing the chromosome number in cytology, and obtaining a doubled plant if the chromosome number is doubled; if the treated material is a cotton triploid interspecific hybrid, then a plant with normal or improved fertility may be a chromosome-doubled plant, and chromosome number confirmation is performed cytologically to obtain a chromosome-doubled plant.

As a preferred technical solution, the method specifically comprises the following steps:

(1) planting a strong cotton plant, removing a main stem growing point to enable the main stem growing point to grow lateral branches and form a plurality of tender buds;

(2) cutting lateral branch tender shoots with 2-3 leaves, removing larger leaves on the lateral branch tender shoots, and preventing water from evaporating and wilting; inserting the cut lateral branch tender shoots into colchicine solution dropwise added with dimethyl sulfoxide for doubling treatment;

(3) grafting the lateral branch tender bud subjected to colchicine doubling treatment onto the stock, and managing by a conventional grafting method;

(4) after the grafted seedling grows up, the plant with doubled chromosome is possible to be the plant with the thickened leaves; when the cotton grows to bud, taking the bud to carry out conventional chromosome number observation, directly observing the chromosome number in cytology, and obtaining a chromosome doubled plant if the chromosome number is doubled; if the treated material is a cotton triploid interspecific hybrid, then a plant with normal or improved fertility may be a chromosome-doubled plant, and chromosome number confirmation is performed cytologically to obtain a chromosome-doubled plant.

Most preferably, the concentration of the colchicine solution is 0.1% (0.1g/100 ml); the dropping amount of the dimethyl sulfoxide stock solution is 5-8 drops in 100ml of solution. The time of the doubling treatment is 22-26 h, and the most preferable time is 24 h.

The invention has the beneficial effects that:

compared with the traditional chromosome doubling technology, the novel cotton chromosome doubling method provided by the invention has the following advantages: the traditional chromosome doubling technology has the defects of large processing workload, low success rate, easy environmental influence, easy death of cotton plants, and sometimes the plants which are successfully doubled cannot be obtained by colchicine solution treatment for many years. By the in vitro processing and doubling technology, the workload is low, the tissue culture process is not needed, the processing in a laboratory is not easily influenced by the environment, the success rate is high, the problem of death of the original plant does not exist, and the plant with doubled chromosomes can be obtained by processing in the same year.

Description of the drawings:

FIG. 1 shows a procedure for cotton chromosome doubling ex vivo.

Detailed Description