Preparation method of beta-elemene-containing distant hybridization tobacco material

Abstract

The invention discloses a preparation method of a beta-elemene-containing distant hybridization tobacco material, and belongs to the technical field of plant breeding. According to the method disclosed by the invention, the tobacco material containing beta-elemene, namely the hybrid variety F1094, is created by taking the stem-embraced tobacco as a male parent and taking the Chinese tobacco 300 as a female parent and utilizing a distant hybridization technology. The germination rate of the hybrid F1094 is normal, the survival rate is 100%, and offspring can be fertile; compared with a female parent Zhongyan 300, the hybrid F1094 is higher in plant height, larger in leaf length and leaf width, and contains volatile aroma components such as beta-elemene and phenylacetaldehyde. Experimental results show that according to the preparation method disclosed by the invention, a fertile distant hybridization breeding material containing beta-elemene and phenylacetaldehyde is successfully created, and a new technical support is provided for the improvement of tobacco characters.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, in particular to a method for preparing a distant hybridization tobacco material containing beta-elemene. Background Art

[0002] Distant hybridization refers to the hybridization method between different species, genera, or even species with more distant genetic relationships. Distant hybridization can break through reproductive isolation, combine the excellent traits of different species, and expand genetic variation. It is of great significance to variety improvement, new variety creation, and enrichment of germplasm resources. Due to the reproductive differences between different species, distant hybridization usually has difficulties such as hybrid incompatibility, difficulty in fruiting, early death or infertility of hybrid offspring, etc. The probability of successful hybridization is low and the difficulty is high. Currently, the commonly used means to overcome this include extensive test crosses to select effective female parents, repeated pollination, early or delayed pollination, mixed pollination, media method, chromosome doubling method, etc. The above distant hybridization techniques are difficult and have a low success rate. Once the hybridization is successful, new varieties with excellent specific traits that cannot be obtained by close hybridization can be obtained, providing diversified support for germplasm innovation and enrichment of breeding materials.

[0003] β-elemene is a sesquiterpene substance found in wild tobacco, burley tobacco, and oriental tobacco. Its molecular formula is C 15 H 24 , with a molecular weight of 204.35. β-elemene is not contained in flue-cured tobacco varieties. β-elemene has natural anti-cancer activity and is a type II non-cytotoxic broad-spectrum anti-tumor drug independently developed and manufactured in my country. It can induce apoptosis of tumor cells, inhibit the proliferation and metastasis of tumor cells, regulate human immunity, reverse the drug resistance of cancer cells, and increase the drug sensitivity of tumor cells. In existing literature, β-elemene has a good inhibitory effect on malignant tumors such as liver cancer, lung cancer, and breast cancer, and plays an important role in cancer treatment. In addition, because β-elemene has certain immunomodulatory functions, it may be developed into a health product to improve human immunity and enhance the body's resistance to disease. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a distant hybrid tobacco material containing β-elemene to solve the problems existing in the above-mentioned prior art. According to the preparation method provided by the present invention, a fertile hybrid material containing β-elemene and having a taller plant and larger leaves than the mother plant can be efficiently obtained.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a preparation method of a beta-elemene-containing distant hybridization tobacco material, comprising the steps of performing distant hybridization with a flue-cured tobacco variety as a female parent and Nicotiana amplexicaulis as a male parent.

[0007] Preferably, the flue-cured tobacco variety is Zhongyan 300.

[0008] Preferably, the distant hybridization includes: collecting pollen, emasculating the female parent, pollination and post-pollination management.

[0009] Preferably, collecting pollen comprises: collecting the anthers of the male parent when the anthers are mature but not cracked;

[0010] The anthers were placed in a culture dish and left uncovered at 25° C. until the anthers matured, and then the culture dish lid was closed.

[0011] Preferably, the pollination comprises: taking pollen from the anther just cracked and evenly applying it on the pistil of the female parent;

[0012] Marking, bagging.

[0013] Preferably, the post-pollination management includes: observing the flowering of the female parent every day, emasculating the female parent in time, and repeating the pollination of the flowers that have been pollinated.

[0014] The present invention also provides an application of clasping tobacco in cultivating tobacco materials containing beta-elemene.

[0015] Preferably, the method comprises the step of using the clasping tobacco as the male parent and obtaining the tobacco material containing β-elemene through distant hybridization technology.

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

[0017] The present invention uses Nicotiana amplexicaulis as the male parent and Zhongyan 300 as the female parent, and uses the distant hybridization technology to create a tobacco material containing β-elemene, namely, a hybrid F1094. The germination rate of the hybrid F1094 is normal, the survival rate is 100%, and the offspring are fertile; compared with the female parent Zhongyan 300, the hybrid F1094 has a higher plant height, a larger leaf length and a larger leaf width, and contains volatile aroma components such as β-elemene and phenylacetaldehyde. The experimental results show that according to the preparation method of the present invention, a fertile distant hybridization breeding material containing β-elemene and phenylacetaldehyde is successfully created, which provides new technical support for the improvement of tobacco traits. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0019] Figure 1 This is a morphological diagram of tobacco hybrid F1094. 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0025] The tobacco material "Zhongyan 300" used in the present invention has been disclosed in "Cheng Lirui, Wang Yuanying, Jiang Caihong, et al. Breeding and characteristics of a new flue-cured tobacco variety Zhongyan 300 [J]. Chinese Tobacco Science, 2019, 40(03): 1-7."; the stem-clasping tobacco (Nicotiana amplexicaulis) used in the present invention has been disclosed in "Wang Rengang, Wang Yunpeng, Ren Xueliang. New progress in the botanical classification of Nicotiana [J]. Chinese Journal of Tobacco Science, 2010, 16(02): 84-90.". The present invention promises to distribute the above-mentioned biological materials to the public within 20 years from the date of application.

[0026] Example 1

[0027] 1. Material creation method

[0028] 1. Parent selection

[0029] The product was produced by distant hybridization using the flue-cured tobacco variety Zhongyan 300 as the female parent and the wild species Nicotiana amplexicaulis as the male parent.

[0030] 2. Pollen collection and processing

[0031] Collect the anthers of male parent Nicotiana amplexicaulis when the anthers are mature but not dehiscent. Place them in a clean culture dish with the lid open and place them in a clean environment at 25°C until the anthers mature, dehiscent and release pollen. Cover the dish in time to prevent pollen contamination.

[0032] 3. Female emasculation

[0033] Before the flowers of the mother plant Zhongyan 300 bloom, select well-developed buds and carefully remove all anthers using tweezers to avoid damaging the pistil.

[0034] 4. Distant hybridization pollination

[0035] In sunny weather, use a cotton swab to dip into fresh pollen with high vitality (i.e. pollen when the anthers have just cracked), and apply the cotton swab to the pistil of the mother plant, applying it evenly throughout the pistil. After pollination, mark and bag the plants to prevent pollen from crossing.

[0036] 5. Post-pollination management

[0037] Observe the flowering of the female plant every day and remove the male plants in time to prevent pollination. Due to the incompatibility of distant hybridization, regularly re-pollinate the flowers that have been pollinated to increase the success rate of distant hybridization fertilization.

[0038] 2. Agronomic traits of distant hybrid offspring F1094

[0039] The seeds obtained after successful fertilization of distant hybridization were collected, named F1094, and planted according to conventional methods.

[0040] The germination rate of the distant hybrid F1094 was normal, the survival rate was 100%, and the offspring were fertile; the plants were tall and grew evenly, the plant height was significantly higher than the control Zhongyan 300, the leaf length and leaf width were significantly longer than the control Zhongyan 300, and the number of leaves did not change.

[0041] The agronomic traits of hybrid F1094 at full flowering stage were as follows: average plant height 187.60 cm, leaf length 73.60 cm, leaf width 35.60 cm, leaf angle 41.58°, main vein thickness 8.63 mm, stem girth 10.80 cm, leaf number 28, chlorophyll 39.48, no petiole, with leaf ears (see Table 1, Figure 1 shown).

[0042] The survey data of agronomic traits of the female parent Zhongyan 300 in the flowering period are as follows: average plant height is 166.0 cm, leaf length is 65.20 cm, leaf width is 29.375 cm, leaf angle is 43.9°, main vein thickness is 9.0 mm, stem girth is 11.0 cm, number of leaves is 28, chlorophyll is 36.42, there are no petioles, and there are leaf ears (as shown in Table 1).

[0043] Table 1 Statistics of agronomic traits of hybrid F1094

[0044]

[0045] Example 2

[0046] Detection and analysis of aroma components of distant hybrid F1094

[0047] 1. Method for determining aroma components of hybrid F1094 using solid phase microextraction (SPME-GCMS) technology:

[0048] Take the hybrid F1094 sample into a 15mL headspace bottle. Note that the sample volume does not exceed 1 / 2 of the bottle capacity, leaving space above the headspace bottle. Insert the SPME needle through the sample bottle injection septum into the headspace bottle. Gently push the handle bar to extend the extraction fiber head so that it is exposed to the headspace of the sample. Allow the adsorption to equilibrate for 20 minutes. Carefully push back the fiber head, pull out the needle, quickly insert it into the gas chromatography (GC) injection port, push out the fiber head, desorb the sample analytes adsorbed on the fiber head, and enter the chromatographic column for separation and analysis.

[0049] 2. Measurement results

[0050] The present invention uses solid phase microextraction SPME-GCMS technology to detect volatile components in the flower buds of the parents and distant hybrids. Analysis shows that the distant hybrid F1094 contains volatile aroma components such as β-elemene (CAS No.: 515-13-9) and phenylacetaldehyde (CAS No.: 122-78-1) (see Table 2).

[0051] The results show that the present invention successfully obtains fertile distant hybridization breeding materials containing beta-elemene and phenylacetaldehyde.

[0052] Table 2 Determination of aroma components of hybrid F1094

[0053]

[0054]

[0055] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a distant hybridization tobacco material containing β-elemene, characterized in that: The method comprises the steps of using a flue-cured tobacco variety as a female parent and Nicotiana amplexicaulis as a male parent for distant hybridization.

2. The preparation method according to claim 1, characterized in that The flue-cured tobacco variety is Zhongyan 300.

3. The preparation method according to claim 1, characterized in that: The distant hybridization includes: collecting pollen, emasculating the female plant, pollination and post-pollination management.

4. The preparation method according to claim 3, characterized in that: The pollen collection comprises: collecting the anthers of the male parent when the anthers are mature but not cracked; The anthers were placed in a culture dish and left uncovered at 25° C. until the anthers matured, and then the culture dish lid was closed.

5. The preparation method according to claim 3, characterized in that: The pollination comprises: taking pollen from the anther just cracked and evenly applying it on the pistil of the female parent; Marking, bagging.

6. The preparation method according to claim 3, characterized in that: The post-pollination management includes: observing the flowering condition of the female parent every day, emasculating the female parent in time, and repeating the pollination of the flowers that have been pollinated.

7. Use of clasping tobacco in cultivating tobacco materials containing β-elemene.

8. The use according to claim 7, characterized in that The method comprises the steps of using the clasping tobacco as the male parent and obtaining tobacco materials containing beta-elemene through distant hybridization technology.

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