A method for chromosome doubling in wild tobacco

Abstract

The present invention discloses a method for chromosome doubling in wild tobacco, relating to the field of biotechnology. The method comprises the steps of soaking wild tobacco explants in a trifluralin solution, followed by tissue culture to obtain polyploid wild tobacco plants. The present invention develops a method for chromosome doubling in wild tobacco. By soaking wild tobacco explants in a trifluralin solution for the first time, chromosome doubling in wild tobacco is achieved at a high doubling rate, with the doubling rate reaching 66% for Nicotiana tabacum. The present invention provides technical support for chromosome doubling in wild tobacco, which is conducive to promoting the development and utilization of polyploid wild tobacco plants.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for chromosome doubling on wild tobacco. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an annual herbaceous plant of the genus Nicotiana in the Solanaceae family and is one of the most important economic crops. Among the currently recognized Nicotiana plants, common tobacco and yellow tobacco, as cultivated species, have significant value in the agricultural economy. Although other wild tobacco species are not yet commercially valuable, wild tobacco has high research and application value in the agricultural field due to its outstanding disease resistance, insect resistance, and stress tolerance. Polyploid plants often show significant enlargement of organs such as roots, stems, and leaves, which can provide high-quality and high-yield plants for agricultural cultivation. Currently, there is much research on chromosome doubling in economic crops such as wheat, zucchini, blueberries, apples, and melons, but there are few reports on chromosome doubling in wild tobacco. To advance research on chromosome doubling technology in wild tobacco, the present invention proposes to establish an efficient wild tobacco chromosome doubling method to produce new polyploid plants at a high frequency through artificial induction, thereby shortening the breeding period. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for chromosome doubling of wild tobacco to solve the problems existing in the above-mentioned prior art. The method has a high doubling rate for chromosome doubling of wild tobacco, provides technical support for chromosome doubling of wild tobacco, and is conducive to promoting the development and utilization of polyploid wild tobacco.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The invention provides a method for chromosome doubling of wild tobacco, comprising the steps of soaking wild tobacco explants in trifluralin solution and then conducting tissue culture to obtain wild tobacco polyploid plants.

[0006] Furthermore, the trifluralin solution is a 0.01% trifluralin solution.

[0007] Furthermore, the soaking treatment time is 48 hours.

[0008] Furthermore, the immersion treatment adopts a light-proof immersion method.

[0009] Furthermore, the explant is a leaf.

[0010] Furthermore, the tissue culture includes screening culture and rooting culture;

[0011] The screening culture uses a differentiation medium, and the formula of the differentiation medium is: 4.43g / L MS+30g / L sucrose+1mg / L 6-BA+0.1mg / L NAA+7.8g / L agar+80mg / L kanamycin, pH 5.8;

[0012] The rooting culture adopts a rooting medium, and the formula of the rooting medium is: 4.43g / LMS+30g / L sucrose+7.8g / L agar, pH 5.8.

[0013] Furthermore, the culture conditions for the screening culture include: light intensity 2000 Lux, temperature 25-28° C., light duration 16 h, and dark duration 8 h.

[0014] Furthermore, the culture conditions for the rooting culture include: light intensity 2000 Lux, temperature 25-28° C., light duration 16 h, and dark duration 8 h.

[0015] Furthermore, the wild tobacco is forest tobacco or little-flowered tobacco.

[0016] The present invention discloses the following technical effects:

[0017] The present invention develops a chromosome doubling method for wild tobacco. For the first time, by soaking wild tobacco explants in a trifluralin solution, the chromosomes of wild tobacco are doubling at a high rate, reaching 66% for Nicotiana pauciflora. The present invention provides technical support for chromosome doubling in wild tobacco, and is conducive to promoting the development and utilization of polyploid wild tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 These are the flow cytometry results for polyploid and haploid detection. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Terminology Notes:

[0026] MS refers to MS medium;

[0027] 6-BA refers to 6-benzylaminopurine;

[0028] NAA refers to 1-naphthylacetic acid;

[0029] DMSO refers to dimethyl sulfoxide.

[0030] The experimental materials involved in the following examples and comparative examples are as follows:

[0031] 1. Plant materials:

[0032] Wild tobacco and little-flowered tobacco 30-day sterile seedlings, wild tobacco forest tobacco 30-day sterile seedlings.

[0033] 2. Culture medium preparation:

[0034] 2.1 Differentiation medium components:

[0035] 4.43 g / L MS + 30 g / L sucrose + 1 mg / L 6-BA + 0.1 mg / L NAA + 7.8 g / L agar + 80 mg / L kanamycin, pH 5.8.

[0036] 2.2 Rooting medium ingredients:

[0037] 4.43g / LMS+30g / L sucrose+7.8g / L agar, pH 5.8.

[0038] 2.3 Preparation of 0.01% trifluralin solution

[0039] Preparation of 10 mg / mL trifluralin solution: add 250 mg trifluralin to 25 mL DMSO and filter through a 0.22 μm filter.

[0040] Preparation of 0.01% trifluralin solution: Add 0.3 mL of 10 mg / mL trifluralin solution to 30 mL of sterile water and mix well.

[0041] 2.4 Colchicine solution preparation

[0042] Preparation of 10 mg / mL colchicine solution: Dissolve 100 mg of colchicine in 10 mL of 0.85% NaCl solution. Filter through 0.22 μm and aliquot into 5 mL tubes.

[0043] Preparation of 0.05% colchicine solution: Add 1.5 mL of 10 mg / mL colchicine solution to 30 mL of sterile water and mix well.

[0044] Preparation of 0.01% colchicine solution: Add 0.3 mL of 10 mg / mL colchicine solution to 30 mL of sterile water and mix well.

[0045] The method for detecting chromosome doubling plants by flow cytometry is as follows:

[0046] Chromosome ploidy of surviving treated tobacco plants was determined using flow cytometry. Regenerated plants soaked in sterile water served as controls. 0.3 g of fresh young leaves were placed in pre-chilled cell lysis buffer and quickly shredded and minced into very small particles using a blade. After filtering through 100 μm, 1 mL of the filtrate was placed in a centrifuge tube. 25 μL of RNase A was added, mixed by inversion, and allowed to stand for 5 minutes. Then, 50 μL of pre-chilled PI dye was added, mixed by inversion, and incubated at 4°C for 5 minutes.

[0047] The samples were analyzed using a flow cytometer manufactured by Backman, Germany. PI-stained samples were collected and excited by laser to emit fluorescence. The intensity of the fluorescence was detected in the FL2 channel and analyzed using the included software.

[0048] Doubling rate = number of doubled plants / number of surviving plants × 100%.

[0049] Example 1

[0050] (1) Preparation of tobacco explants

[0051] Transform tobacco by leaf disc method. When the plant grows to 6 cm in height, take the middle-sized and healthy leaf and cut it into 1 cm 2 The leaf discs were placed leaf side down on differentiation medium and cultured for 2 days at a temperature of 25-28°C.

[0052] (2) Soaking in trifluralin solution

[0053] After culturing the leaf discs on differentiation medium for 2 days, place them in a 0.01% trifluralin solution and protect from light for 48 hours. After absorbing any residual trifluralin solution with sterile filter paper, lay the discs flat on differentiation medium, leaf side down, with eight discs per dish. Culture conditions were a light intensity of 2000 Lux, a temperature of 25-28°C, a light cycle of 16 hours, and a dark cycle of 8 hours. Transfer the discs every two weeks, and continue the selection culture for 40 days.

[0054] (3) Tobacco rooting

[0055] After screening, clearly distinguishable shoots were excised, with all callus and basal leaves removed. The shoots were then planted on rooting medium, four seedlings per bottle, for 20 days. The culture conditions were a light intensity of 2000 Lux, a temperature of 25-28°C, a light period of 16 h, and a dark period of 8 h.

[0056] 4. Transplanting

[0057] After the roots of the chromosome-doubled plants after rooting culture grow to about 3 cm, they are transplanted and cultured to maturity, and the rate of deformed seedlings is counted.

[0058] Example 2

[0059] (1) Preparation of tobacco explants

[0060] Transform tobacco by leaf disc method. When the tobacco plant grows to 6 cm in height, take the middle-sized and healthy leaf and cut it into 1 cm 2 The leaf discs were placed leaf side down on differentiation medium and cultured for 2 days at a temperature of 25-28°C.

[0061] (2) Soaking in trifluralin solution

[0062] After culturing the leaf discs on differentiation medium for 2 days, place them in a 0.01% trifluralin solution and protect from light for 48 hours. After absorbing any residual trifluralin solution with sterile filter paper, lay the discs flat on differentiation medium, leaf side down, with eight discs per dish. Culture conditions were a light intensity of 2000 Lux, a temperature of 25-28°C, a light cycle of 16 hours, and a dark cycle of 8 hours. Transfer the discs every two weeks, and continue the selection culture for 40 days.

[0063] (3) Tobacco rooting

[0064] After screening, clearly distinguishable shoots were excised, with all callus and basal leaves removed. The shoots were then planted on rooting medium, four seedlings per bottle, for 20 days. The culture conditions were a light intensity of 2000 Lux, a temperature of 25-28°C, a light period of 16 h, and a dark period of 8 h.

[0065] 4. Transplanting

[0066] After the roots of the chromosome-doubled plants after rooting culture grow to about 3 cm, they are transplanted and cultured to maturity, and the rate of deformed seedlings is counted.

[0067] Comparative Example 1

[0068] The same as Example 1, except that in step (2), the 0.01% trifluralin solution is replaced with a differentiation medium containing 0.01% trifluralin. The differentiation medium containing 0.01% trifluralin is prepared by adding 0.3 mL of a 10 mg / mL trifluralin solution to 30 mL of the differentiation medium and mixing well.

[0069] In this comparative example, after pre-treating Nicotiana tabacum leaf discs with a differentiation medium containing 0.01% trifluralin, the leaf discs all turned yellow and died after two weeks of tissue culture, and could not grow normally.

[0070] Comparative Example 2

[0071] Same as Example 1, except that 0.01% trifluralin solution is replaced by 0.01% amoxicillin solution. The preparation method of 0.01% amoxicillin solution is the same as that of 0.01% trifluralin solution, except that trifluralin is replaced by amoxicillin.

[0072] Comparative Example 3

[0073] Same as Example 1, except that the 0.01% trifluralin solution is replaced by a 0.05% colchicine solution.

[0074] The results of chromosome doubling testing of the tobacco plants of Examples 1-2 and Comparative Examples 1-4 using flow cytometry are shown in Table 1. After transplanting, the rate of deformed seedlings was statistically analyzed, and the results are shown in Table 1. The present invention has discovered that directly immersing the explants in a 0.01% trifluralin solution enables normal subsequent tissue culture and effectively improves the doubling rate, compared to directly adding trifluralin to the differentiation medium.

[0075] During the chromosome doubling test, Nicotiana tabacum (diploid), Nicotiana pauciflora (diploid) and K326 (allotetraploid) were used as controls, the G1 phase peak was detected and recorded, and the regenerated plants of Nicotiana tabacum and Nicotiana pauciflora treated with trifluralin were detected. Figure 1 The flow cytometry results for haploid and polyploid plants are shown. The G1 peak of undoubled diploid Nicotiana pauciflora was 291, the G1 peak of undoubled tetraploid plants was 286, and the G1 peak of doubled plants was 495. The G1 peak of undoubled diploid Nicotiana tabacum was 114, the G1 peak of undoubled tetraploid plants was 271, and the G1 peak of doubled plants was 477. Comparing the G1 peaks of doubled plants with those of undoubled plants, the peaks were twice or more, indicating successful chromosome doubling.

[0076] Table 1 Statistics of flow cytometry test results

[0077]

[0078] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for chromosome doubling in wild tobacco, characterized in that: The method comprises the steps of soaking wild tobacco explants in trifluralin solution and then conducting tissue culture to obtain wild tobacco polyploid plants; The trifluralin solution is a 0.01% trifluralin aqueous solution; The soaking time is 48 hours; The soaking treatment adopts a light-proof soaking method; The explant is a leaf; The wild tobacco is forest tobacco or little-flowered tobacco.

2. The method according to claim 1, characterized in that The tissue culture includes screening culture and rooting culture; The screening culture uses a differentiation medium, and the formula of the differentiation medium is: 4.43g / LMS+30g / L sucrose+1mg / L 6-BA+0.1mg / L NAA+7.8g / L agar+80mg / L kanamycin, pH 5.8; The rooting culture adopts a rooting medium, and the formula of the rooting medium is: 4.43g / L MS+30g / L sucrose+7.8g / L agar, pH 5.

8.

3. The method according to claim 2, characterized in that The culture conditions of the screening culture include: light intensity of 2000 Lux, temperature of 25-28° C., light duration of 16 h, and dark duration of 8 h.

4. The method according to claim 2, characterized in that The culture conditions for the rooting culture include: light intensity of 2000 Lux, temperature of 25-28° C., light duration of 16 h, and dark duration of 8 h.

Images