CN111374048B

CN111374048B - Chromosome doubling method of pepper or eggplant haploid plant

Info

Publication number: CN111374048B
Application number: CN202010353750.2A
Authority: CN
Inventors: 邓晓梅; 张秦; 惠志明; 张树根; 张军民; 李春玲
Original assignee: Beijing Haihua Bio Tech Co ltd; BEIJING HAIDIAN TECHNOLOGY OF PLANT TISSUE CULTURE LABORATORY
Current assignee: Beijing Haihua Bio Tech Co ltd; BEIJING HAIDIAN TECHNOLOGY OF PLANT TISSUE CULTURE LABORATORY
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2022-03-08
Anticipated expiration: 2040-04-28
Also published as: CN111374048A

Abstract

The invention discloses a chromosome doubling method of a haploid plant of a hot pepper or an eggplant, belonging to the technical field of plant tissue culture. The method comprises the following steps: adding sterile colchicine solution and sterile DMSO into a sterile culture bottle, reserving or removing roots of a regenerated plant cultured by pepper or eggplant anther, placing the plant in the sterile culture bottle, and performing oscillation seedling soaking treatment; and after 24-48 h of treatment, taking out anthers to culture regenerated plants, transferring the regenerated plants to an MS minimal medium for illumination culture, and culturing for one month to obtain diploid plants. The method of the invention has the following advantages: (1) can ensure that all growing points on the plants are processed; (2) the treatment liquid can be always kept at the growing point of the plant during the treatment period, but the use amount of the treatment liquid is less, and the cost is reduced; (3) the doubling success rate is 60 to 100 percent.

Description

Chromosome doubling method of pepper or eggplant haploid plant

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a chromosome doubling method of a haploid plant of a hot pepper or an eggplant.

Background

Haploid breeding is one of plant breeding means, haploid plants can be generated by anther culture induction, and chromosome sets are doubled by artificial means such as colchicine treatment, so that the normal chromosome number of the plants is recovered, the plants become fertile homozygous Diploids (DH), the haploid breeding method is directly applied to breeding, the period is shortened, and the breeding process is greatly accelerated.

The establishment of DH population is a key technology of haploid breeding, including haploid induction and chromosome doubling. At present, haploid plants are obtained mainly by plant tissue culture techniques, through the androgenetic pathway, such as anther or microspore culture, and through the gynogenesis pathway, such as the culture of unpolished ovaries and ovules. Although the haploid plant can be naturally doubled to become a homozygous diploid plant, the natural multiplying power is 0-40% different due to different genotypes, and the artificial doubling of chromosomes is necessary for the application requirement of large-scale DH plants.

Since 1971 when Kaul and Zutshi treated wheat, barley and rye haploids with colchicine and succeeded in obtaining homozygous diploids, the haploid doubling technique has also been extensively studied in other families. At present, in agricultural breeding, the most commonly used chromosome doubling method is the colchicine doubling method. Colchicine is a tropolone alkaloid extracted from bulb and seed of colchicine of Liliaceae, and has mutagenesis effect on seed, bud, flower bud, pollen, and twig. Colchicine can inhibit the formation of spindle body during cell division, so that chromosome does not move to two poles, but is prevented in metaphase, and cell can not continue division, thereby generating chromosome-doubled nucleus. The colchicine haploid doubling technology is applied to mature plant species such as wheat, barley, corn, tobacco, beet, rice, pear, rape and the like.

The solanaceous vegetables such as hot pepper and eggplant are important fruit vegetables in China and are important research objects in the aspects of genetic breeding, cytology and the like. By utilizing the anther culture technology of hot pepper and eggplant, a large amount of haploids can be produced in a short time, and pure lines are obtained by artificial doubling, so that the selection efficiency of breeding is greatly improved, and the breeding period is shortened. At present, the haploid chromosome doubling of hot pepper and eggplant mainly adopts a method that absorbent cotton is used for embedding growing points of a haploid plant and dripping treatment liquid (a plurality of growing points are arranged on one plant and need to be wrapped), 0.2-0.4% colchicine solution is dripped on the absorbent cotton, and the solution is easy to volatilize, and the solution is dripped once every 4-8 hours to keep the absorbent cotton moist. The method is time-consuming and labor-consuming, and has a low one-time doubling success rate, which is generally 0-30% for hot pepper and eggplant.

Disclosure of Invention

The invention aims to disclose a chromosome doubling method of a haploid plant of a hot pepper or an eggplant.

The purpose of the invention is realized by the following technical scheme:

a method of chromosome doubling of a pepper or eggplant haploid plant, comprising:

(1) 10ml to 20ml of sterile doubling treatment fluid is added into a 250ml or 500ml sterile culture bottle, and the doubling treatment fluid consists of 0.1 percent to 0.4 percent of colchicine solution and 1 percent to 4 percent of DMSO by volume percentage;

(2) selecting pepper or eggplant anther with the plant height of 3-5 cm to culture a regenerated plant, reserving a root system or cutting off the root system, placing the plant in a sterile culture bottle, and preparing for induction;

(3) transversely placing an aseptic culture bottle, fixing the aseptic culture bottle on a rolling shaking table, fully and uninterruptedly infiltrating the doubled treatment liquid into plants at the speed of 20-30 rpm, and carrying out shaking seedling infiltration treatment for 24-48 h, wherein the induction culture conditions are that the temperature is 22-28 ℃, the humidity is 40-60%, the illumination time is 12-16 h, and the illumination intensity is 1000-2000 lx;

(4) after 24-48 h of doubling treatment in the step (3), taking out anthers to culture regenerated plants, washing the regenerated plants with sterile water for 3-5 times, transferring the regenerated plants to an MS basic culture medium for illumination culture, and culturing for one month to obtain diploid plants; the illumination culture conditions are 22-28 ℃, 40-60% of humidity, 12-16 h of illumination time and 2000-4000 lx of illumination intensity.

The method for doubling the chromosome of the haploid plant of the pepper or the eggplant, which is characterized in that the regenerated plant cultured by the anther of the pepper or the eggplant in the step (2) is obtained through the following steps:

(a) taking the flower buds of the hot pepper and the eggplant, wherein the development period of the hot pepper or the eggplant pollen is the mononuclear side period, sterilizing the surfaces of the flower buds of the hot pepper and the eggplant by 10 percent of sodium hypochlorite under the aseptic condition for 10 to 20 minutes, and washing the flower buds of the hot pepper and the eggplant by sterile water for 3 times;

(b) peeling off anthers, inoculating the anthers in an induction culture medium to obtain embryoids, and transferring the embryoids to an MS minimal medium without any growth regulator to grow into plantlets; then transferring the plantlets to a rooting and seedling strengthening culture medium to finally obtain anther culture regeneration plants;

the induction culture medium of the pepper pollen embryoid is MS culture medium added with 0.1 mg/L-1 mg/L BA, 0.1 mg/L-1 mg/L IAA and 0.01 mg/L-0.1 mg/L NAA;

the induction culture medium of the eggplant pollen embryoid is MS culture medium added with 0.05 mg/L-2 mg/L KT, 0.1 mg/L-1 mg/L IAA, 0.01 mg/L-0.1 mg/L NAA and 0.1% -0.3% of active carbon;

the rooting and seedling strengthening culture medium is an MS basic culture medium added with IBA 1 mg/L.

The invention has the following beneficial effects:

1. compared with the traditional method for wrapping the growing points of the plants by the absorbent cotton, the doubling efficiency is higher and can reach 60-100%. According to the method, the regeneration plants are directly placed into a culture bottle, 10ml to 20ml of colchicine solution is added into the bottle, 10 to 50 plants can be placed in one bottle, and the double treatment is carried out on a rolling shaking table, so that all bud points can be treated; and traditional use absorbent cotton wrapping method, when the plant growing point is more, can not all wrap up the processing, too waste time and energy, when the leaf bud is too little, difficult parcel.

2. The method of the invention can ensure that the growing point of the plant always keeps the treatment solution during the treatment period, but the treatment solution has less dosage and reduced cost (colchicine is toxic and high in price).

Description of the drawings:

1. FIG. 1 is a DNA content distribution diagram of regenerated plants of Capsicum annuum L.

2. FIG. 2 is the DNA content distribution diagram of eggplant regenerated plant.

The specific implementation mode is as follows:

in order to facilitate understanding of the technical scheme of the invention, the method for doubling chromosomes of a pepper or eggplant haploid plant is further described below by combining specific embodiments.

Example 1:the method for doubling the chromosome of the pepper haploid plant comprises the following steps:

taking out the pepper buds with the pollen development period being the mononuclear border period from the planting greenhouse, sterilizing the surfaces of the pepper buds by 10 percent sodium hypochlorite for 20min under the aseptic condition, and washing the pepper buds by aseptic water for 3 times. Peeling off anther, inoculating in induction culture medium (MS minimal medium added with 0.1mg/L BA, 0.1mg/L IAA and 0.01mg/L NAA) to obtain embryoid, transferring embryoid into MS minimal medium without any growth regulator, and growing into plantlet; then, the plantlets are transferred to a rooting and seedling strengthening culture medium (MS basic culture medium is added with IBA 1mg/L), and finally, pepper pollen regeneration plants are obtained;

selecting 30 pepper pollen regenerated plants (haploid plants determined by a flow cytometer) with the plant height of 3-5 cm and 3-4 leaves, removing large leaves, reserving root systems, placing the plants in a 500ml sterile culture bottle, and adding 15ml of sterile solution consisting of 0.2% colchicine and 2% DMSO into the bottle;

the sterile culture bottle is transversely placed and fixed on a rolling shaking table, and the treatment fluid is enabled to fully and uninterruptedly vibrate and infiltrate the plants at the speed of 25rpm, so that the drug damage caused by long-time static soaking is avoided, and the cost is also saved;

the culture conditions comprise the temperature of 22-25 ℃, the humidity of 40-60%, the illumination time of 12h and the illumination intensity of 1000-2000 lx;

after 48h of doubling treatment, taking out the anther culture regenerated plant, washing with sterile water for 3-5 times, and transferring to an MS basic culture medium for illumination culture; the culture conditions are 22-28 ℃, the humidity is 40-60%, the illumination time is 12h, and the illumination intensity is 2000 lx-4000 lx;

after one month of culture, the DNA content of the newly grown plant leaves obtained in example 1 was determined using a flow cytometer Partec Space and Partec analysis software. And determining the regeneration plant to be haploid or diploid by taking the DNA content of the diploid of the pepper hybrid as a control according to the separation peaks with different intensities. When the relative fluorescence intensity value is 400, the plant is diploid, and when the relative fluorescence intensity value is 200, the DNA content in the cells of the anther culture regenerated plant is reduced by half compared with that of the diploid control plant, the plant is haploid plant (as shown in figure 1, figure 1 is a DNA content distribution diagram of a pepper plant, wherein the left figure is the haploid plant, and the right figure is the diploid plant). Through detection, 27 of 30 pepper haploid plants subjected to doubling treatment are doubled into diploid plants, and the doubling success rate is 90.0%.

Example 2: the chromosome doubling method of the eggplant haploid plant comprises the following steps:

taking out eggplant buds with pollen development period being the mononuclear border period from the planting greenhouse, sterilizing the eggplant buds with 10% sodium hypochlorite surface for 10min under aseptic condition, and washing with sterile water for 3 times. Peeling off anthers, inoculating the anthers in an induction culture medium (0.1-1 mg/L KT, 0.1-1 mg/L IAA, 0.01-0.05 mg/L NAA and 0.1-0.3% of activated carbon are added into an MS basic culture medium) to obtain embryoids, and transferring the embryoids to an MS0 culture medium without any growth regulator to grow into plantlets; then, the plantlets are transferred to a rooting and seedling strengthening culture medium (MS basic culture medium is added with IBA 1mg/L), and finally eggplant pollen regeneration plants are obtained;

selecting 40 eggplant flower culture regenerated plants (haploid plants determined by a flow cytometer) with the plant height of 3-5 cm and 3-4 leaves, removing large leaves and roots, placing the plants in a 500ml sterile culture bottle, and adding 15ml of sterile solution consisting of 0.4% colchicine and 2% DMSO into the bottle;

the sterile culture bottle is transversely placed and fixed on a shaking table, and the treatment fluid is enabled to fully and uninterruptedly vibrate and infiltrate the plants at the speed of 25rpm, so that the drug damage caused by long-time static soaking is avoided, and the cost is also saved;

the culture conditions are 22-25 ℃, the humidity is 40-60%, the illumination time is 12h, and the illumination intensity is 1000 lx-2000 lx;

after one month of culture, the newly grown plant leaves obtained in example 2 were subjected to DNA content determination using flow cytometry Partec Space and Partec analysis software. And determining the regeneration plant to be haploid or diploid by using the DNA content of the diploid of the eggplant hybrid as a control according to the separation peaks with different intensities. When the relative fluorescence intensity value is 200, the plant is diploid, when the relative fluorescence intensity value is 100, the DNA content in the cells of the anther culture regenerated plant is reduced by half compared with that of the diploid control plant, and the plant is haploid plant (as shown in figure 2, figure 2 is a DNA content distribution diagram of an eggplant plant, wherein the left figure is the haploid plant, and the right figure is the diploid plant). Through detection, 25 of 40 eggplant haploid plants subjected to doubling treatment are doubled into diploid plants, and the doubling success rate is 62.5%.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims; meanwhile, any equivalent changes, modifications and variations of the above embodiments according to the essential technology of the present invention are within the scope of the technical solution of the present invention.

Claims

1. A method of chromosome doubling of a pepper or eggplant haploid plant, comprising:

(1) 10ml to 20ml of sterile doubling treatment solution is added into a 250ml or 500ml sterile culture bottle, and the doubling treatment solution consists of 0.1 to 0.4 percent of colchicine solution and 1 to 4 percent of DMSO (dimethyl sulfoxide) by volume percentage;

(3) transversely placing an aseptic culture bottle, fixing the aseptic culture bottle on a rolling shaking table, fully and uninterruptedly infiltrating the plant with the doubled treatment solution at the speed of 20-30 rpm, and carrying out shaking seedling infiltration treatment for 24-48 h, wherein the induction culture condition is that the temperature is 22-28 ℃, the humidity is 40-60%, the illumination time is 12-16 h, and the illumination intensity is 1000-2000 lx;

2. The method for chromosome doubling of pepper or eggplant haploid plants as claimed in claim 1, characterized in that pepper or eggplant anther-cultured regenerated plants in step (2) are obtained by the following steps:

(a) taking pepper or eggplant buds with the development period of pepper or eggplant pollen being the mononuclear border period, sterilizing the surfaces of the pepper or eggplant buds by 10 percent of sodium hypochlorite under aseptic condition for 10-20 min, and washing the buds by aseptic water for 3 times;

(b) peeling off anthers, inoculating the anthers in an induction culture medium to obtain pollen embryoids, and transferring the embryoids to an MS minimal medium without any growth regulator to grow into plantlets; then transferring the plantlets to a rooting and seedling strengthening culture medium to finally obtain anther culture regeneration plants;

the induction culture medium of the pepper pollen embryoid is MS basic culture medium added with 0.1 mg/L-1 mg/L BA, 0.1 mg/L-1 mg/L IAA and 0.01 mg/L-0.1 mg/L NAA;

the induction culture medium of the eggplant pollen embryoid is an MS basic culture medium added with 0.05 mg/L-2 mg/L KT, 0.1 mg/L-1 mg/L IAA, 0.01 mg/L-0.1 mg/L NAA and 0.1% -0.3% of active carbon;