Method for reducing browning rate of doubled haploid from female parent and application of method
By using dishwashing liquid and running water rinsing combined with alcohol disinfection on the surface of tobacco explants, the browning rate and contamination rate in the induction process of tobacco haploid was solved, and the browning rate of less than 6% and the contamination rate of 2% was achieved, supporting the efficient induction of haploid haploid of cigar cigarettes.
Patent Information
- Application Number
- CN202510619360.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-14
- Publication Date
- 2025-07-01
AI Technical Summary
During the induction of tobacco haploids, there are higher browning and contamination rates in the prior art, and the impact is doubled.
The surface of the explant was cleaned by applying dishwashing liquid and rinsing with running water, followed by disinfection, including 75% alcohol disinfection and 0.1% liter of mercury sterilization, and then bud and root induction culture were carried out in a specific culture medium.
The browning rate of explants was significantly reduced to less than 6% and the contamination rate was lower than 2%, providing a basis for doubling batch haploid culture and ensuring a sterile environment.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of doubled haploid, and particularly relates to a method for reducing the browning rate of female parent-derived doubled haploid and its application. Background Art
[0002] In conventional cross-breeding, due to trait segregation, it takes 3 - 4 years to stabilize the target traits. By using the doubled haploid (DH) method, a homozygous diploid can be obtained through chromosome doubling, excluding the interference of dominant and recessive traits, and playing an increasingly important role in shortening the breeding process, accelerating the screening efficiency of target traits, and constructing quantitative trait locus mapping. There are two ways to induce tobacco haploid, one is from male gametes and the other is from female gametes. As early as 1967, Bourgin et al. first obtained haploid plants from tobacco pollen culture. After that, scholars at home and abroad also carried out relevant research, and using anther haploid, they successively cultivated haploid varieties from single-breeding No. 1 to No. 3. However, years of research have shown that the quality and agronomic traits of doubled haploid obtained by anther culture degenerate seriously (E.A. Wernsman 1989), and it is only suitable for constructing a doubled haploid population (DH line) to provide basic materials for constructing a molecular marker genetic map. Using the hybridization technique of Nicotiana africana to produce female parent-derived haploid is widely used in breeding in the United States. Currently, its main cultivated varieties such as NC196 and other varieties have adopted the above technology to obtain female parent-derived haploids.
[0004] At present, the breeding of cigar tobacco in China has just started, and there are few reports on its haploid breeding research. The high browning rate during the conventional tissue culture doubling of haploid is the key factor affecting the success of haploid doubling. Summary of the Invention
[0005] The present invention aims to solve at least one of the technical problems existing in the prior art or related technologies.
[0006] To this end, in the first aspect of the present invention, a method for reducing the browning rate of female parent-derived doubled haploid is provided, including:
[0007] Washing the explant, taking the tobacco leaves to be doubled, applying dishwashing liquid on the tobacco leaves, then rinsing off the dishwashing liquid with running water, and then selecting the midrib part of the tobacco leaves with a diameter of 6 mm - 8 mm, cutting off the excess mesophyll tissue, and cutting it into midrib segments with a length of 8 cm - 9 cm to obtain the washed explant;
[0008] Disinfecting the explant, performing disinfection treatment on the washed explant to obtain the disinfected explant;
[0009] Explant culture: performing shoot induction culture and root induction culture on the disinfected explants.
[0010] Further, the shoot induction culture includes:
[0011] Inoculating the disinfected explants into a medium of MS + KT 2.0 mg / L + NAA 0.5 mg / L for the shoot induction culture.
[0012] Further, the root induction culture includes:
[0013] After the shoot induction culture, when the shoots grow to 1.8 cm - 2.2 cm, cutting the shoots and inoculating them into a medium of 1 / 2MS + IBA 0.5 mg / L for the root induction culture.
[0014] Further, the explant disinfection includes: disinfecting with 75% alcohol for 1 min and washing twice with sterile water; sterilizing with 0.1% mercuric chloride for 13 min and rinsing twice with sterile water.
[0015] Furthermore, after the disinfected explants are subjected to shoot induction culture and root induction culture, the browning rate is less than 6%, and the contamination rate is lower than 2%.
[0016] In the second aspect of the present invention, an application of a method for reducing the browning rate of doubled haploids derived from female parents is provided. The above method for reducing the browning rate of doubled haploids derived from female parents is used to induce the doubling of haploids of the cigar tobacco female parent.
[0017] Further, the application method includes:
[0018] Under greenhouse conditions, taking the pollen of N. africana and hybridizing and pollinating with the hybrid variety, and counting the haploid induction rate 30 days after emergence;
[0019] Among them, the male parent of the hybrid variety is Anti-Imperialism No. 3 or Rock Tobacco 97, and the female parent is Yunxue series, Chuanxue series, Haiyan series or Chuxue series.
[0020] Further, in the hybrid combinations with the female parent being Yunxue, Chuanxue, Haiyan or Chuxue series and the male parent being Anti-Imperialism No. 3 or Rock Tobacco 97, the haploid induction rate of the hybrid combinations with Anti-Imperialism No. 3 as the male parent is higher than that of the hybrid combinations with Rock Tobacco 97 as the male parent.
[0021] Further, in the hybrid combinations with the male parent being Anti-Imperialism No. 3 or Rock Tobacco 97 and the female parent being Yunxue, Chuanxue, Haiyan or Chuxue series, the hybrid combinations of the Chuxue series varieties have the highest haploid induction rate.
[0022] Furthermore, the haploid induction rate of the hybrid combination of the Chuxue series varieties whose male parent is Fan Di No. 3 is 33.33%, and the haploid induction rate of the hybrid combination of the Chuxue series varieties whose male parent is Yan Yan 97 is 14.71%.
[0023] Compared with the prior art, the present invention has at least the following beneficial effects:
[0024] The method for reducing the browning rate of doubled haploids of maternal origin provided by the present invention can more thoroughly clean the wax layer on the surface of the midrib of the leaf, as well as microorganisms such as dust and insect eggs, by applying detergent on the midrib segment of the leaf and then washing it with running water during explant pretreatment, so that the browning rate and contamination rate of the explant are significantly reduced, providing a key technical guarantee for breeding independent excellent varieties that are adapted to the ecological conditions of the main cigar raw material production areas in my country. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered limiting of the present application.
[0026] Figure 1 The browning conditions of the explants after 15 days of culture in different explant pretreatment methods provided in Example 1 of the present invention.
[0027] Figure 2 The results of the effects of different explant pretreatment methods on the browning rate provided in Example 1 of the present invention.
[0028] Figure 3 The haploid induction rate comparison of the hybrids of the main varieties of cigar producing areas provided in Example 1 of the present invention is shown.
[0029] Figure 4 The leaf midribs of 11 materials that were simultaneously sterilized as provided in Example 1 of the present invention (3 midrib segments for each material, with a scale of 3 cm). DETAILED DESCRIPTION
[0030] In order to better understand the above-mentioned technical scheme, the technical scheme of the embodiments of the present application is described in detail below through the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical scheme of the embodiments of the present application, rather than limitations on the technical scheme of the present application. In the absence of conflict, the embodiments of the present application and the technical features in the embodiments may be combined with each other.
[0031] A first aspect of the embodiments of the present invention provides a method for reducing the browning rate of doubled haploids of maternal origin, comprising:
[0032] Explant cleaning: take a tobacco leaf to be doubled, apply detergent on the tobacco leaf, then rinse off the detergent with running water, then select the midrib of the tobacco leaf with a diameter of 6 mm to 8 mm, cut off the excess mesophyll tissue, and cut into midrib segments of 8 cm to 9 cm in length to obtain a cleaned explant;
[0033] Explant disinfection, disinfecting the cleaned explant to obtain a disinfected explant;
[0034] Explant culture: performing bud induction culture and root induction culture on the sterilized explant.
[0035] It is understandable that the high browning rate during the doubling process of haploid tissue culture is the key to the success of haploid doubling. The present invention uses 16 varieties from four cigar tobacco producing areas as female parents, and Anti-Emperor No. 3 and Rock Tobacco 97 as male parents, and conducts a batch of African tobacco hybridization-induced haploid doubling research to provide basic data for accelerating breeding efficiency.
[0036] The embodiment of the present invention provides a pre-treatment method for reducing the browning rate of the doubled haploid of the maternal source, which is an explant pretreatment process: using detergent to directly apply to the surface of the midrib segment of the leaf, and coordinating with the coordinated treatment method of running water washing, compared with the traditional process of washing with a brush dipped in soapy water or simply washing with running water, on the one hand, physical damage control: by canceling the contact cleaning of the brush, the risk of mechanical damage to plant tissue caused by brush-type tools is completely avoided; on the other hand, in terms of surface treatment effect: the surfactant in the detergent can effectively remove the wax layer and dust on the surface of the midrib of the leaf, and at the same time completely remove the microbial community attached to the surface of the midrib of the leaf (including fungal spores, insect eggs and other pollutants), and establish a sterile culture environment. This method significantly reduces the browning rate and contamination rate of the explant, and provides a key technical guarantee for breeding independent excellent varieties that adapt to the ecological conditions of the main production areas of cigar raw materials in my country.
[0037] Optionally, the detergent can be the detergent used for cleaning dishes, fruits and vegetables in daily life, and is preferably a neutral detergent without fragrance or additives.
[0038] In a feasible embodiment, the sprout inducing culture includes: inoculating the sterilized explant into a culture medium of MS+KT2.0mg / L+NAA0.5mg / L for sprout inducing culture.
[0039] Specifically, the MS medium contains rich inorganic salts, vitamins and organic components, providing essential nutrients for the explants and ensuring the stability of cell metabolism during bud differentiation. KT (kinetin) and NAA (naphthaleneacetic acid) synergistically promote bud differentiation. Among them, KT, as a cytokinin, can effectively activate the cell division of explants and induce the formation of bud primordia. Its concentration is 2.0 mg / L, which meets the requirements of plant bud differentiation and can avoid excessive proliferation of callus caused by too high concentration. NAA is an auxin-like substance, and its concentration of 0.5 mg / L can balance the effect of cytokinin and promote the robust growth of buds.
[0040] In a feasible implementation manner, root induction culture includes:
[0041] After shoot induction culture, when the shoots grow to 1.8 cm - 2.2 cm, the shoots are cut and inoculated into a medium of 1 / 2MS + 0.5 mg / L IBA for root induction culture.
[0042] Specifically, when the shoots grow to 1.8 cm - 2.2 cm, they are cut to ensure that the shoot bodies have sufficient physiological activity, which can significantly improve the rooting success rate and avoid rooting inhibition caused by overly tender or aging shoot bodies. Using 1 / 2MS medium (with half the salt concentration) can reduce the inhibition of root differentiation by the high-salt environment, while providing necessary nutritional support to promote the balance of root development and nutrient absorption. IBA (indolebutyric acid) is an auxin-like substance, and its concentration of 0.5 mg / L can effectively induce the formation of root primordia. Compared with NAA (naphthaleneacetic acid), IBA has higher stability and less damage to the shoot bodies.
[0043] In a feasible implementation manner, explant disinfection includes: disinfecting with 75% alcohol for 1 min and washing twice with sterile water; sterilizing with 0.1% mercuric chloride for 13 min and rinsing twice with sterile water.
[0044] Specifically, through the synergistic effect of alcohol and mercuric chloride, efficient removal of surface and deep microorganisms on the explants is achieved. At the same time, the disinfection and sterilization time and the sterile water rinsing operation are controlled to balance the sterilization effect and cell activity, laying a foundation for establishing a low-pollution and high-survival-rate sterile system for subsequent tissue culture.
[0045] In the embodiment of the present invention, through the above explant pretreatment method (including explant cleaning and explant disinfection), after shoot induction culture and root induction culture, the browning rate is less than 6%, providing a basis for carrying out batch haploid doubling culture, and the contamination rate is lower than 2%, ensuring that the haploid materials from the female parent are free from microbial interference during culture.
[0046] In the second aspect of the embodiment of the present invention, an application of a method for reducing the browning rate of doubled haploids from the female parent is provided. The above method for reducing the browning rate of doubled haploids from the female parent is used to induce the doubling of haploids of the cigar tobacco female parent.
[0047] It is understandable that the agronomic traits and quality characteristics of the doubled offspring of the maternal haploids have no degradation, and have been widely used in tobacco breeding practice. Inducing haploids after hybridization of African tobacco and cultivated tobacco is one of the main ways to obtain haploids of maternal origin. The present invention conducts a comparative analysis of the methods of inducing haploids and doubling haploids by hybridizing the main varieties of cigar tobacco in the main producing areas of domestic cigars with African tobacco. The application method comprises: in a greenhouse environment, taking African tobacco N. africana pollen, cross-pollinating with the hybrid, and calculating the haploid induction rate 30 days after emergence; wherein the male parent of the hybrid is Anti-Emperor No. 3 or Yanyan 97, and the female parent is Yunxue series, Chuanxue series, Haiyan series or Chuxue series. The results showed that in the hybrid combinations with Yunxue, Chuanxue, Haiyan or Chuxue series as the female parent and Fandi No. 3 or Yanyan 97 as the male parent, the haploid induction rate of the hybrid combination with Fandi No. 3 as the male parent was higher than that of the hybrid combination with Yanyan 97 as the male parent. The haploid induction rate of the hybrids of Chuxue variety in Hubei production area and two male parents (Fandi No. 3 male parent and Yanyan 97 male parent) was the highest, reaching 33.33% and 14.71% respectively; while the haploid induction rate of the hybrids of Chuanxue series and two male parents (Fandi No. 3 male parent and Yanyan 97 male parent) was the lowest, reaching 18.18% and 5.95% respectively.
[0048] In order to better understand the above technical solution, the technical solution of the present invention is described in detail below through specific embodiments.
[0049] Example 1 A method for reducing the browning rate of doubled haploids from maternal origin and its application
[0050] 1 Materials and Methods
[0051] 1.1 Materials: The required materials are shown in Table 1. Among them, the Yunxue series includes Yunxue 1, Yunxue 2, Yunxue 6, Yunxue 38 and Yunxue 39; the Chuanxue series includes Chuanxue 2, Chuanxue 3 and Dexue 4; the Haiyan series includes Hainan 1, Jianheng 2, Haiyan 103, Haiyan 201 and Haiyan 204; the Chuxue series includes CX07, CX14 and CX26.
[0052] Table 1 Test materials
[0053]
[0054] 1.2 Methods
[0055] 1.2.1 Pollination and identification of haploid morphology at the seedling stage
[0056] Under greenhouse conditions, pollen of N. africana was collected and used to pollinate 32 hybrid varieties (the female parent material was crossed with two male parents respectively) shown in Table 1. The pollination period, pollination method, and haploid identification were carried out according to the methods of Zeng Jianmin et al. (Zeng Jianmin, Jiao Fangchan, Tong Zhijun et al. (2016) Effects of pollination period and bagging method on the identification efficiency of tobacco maternal origin haploids. Tobacco Science & Technology, 49(12): 8-12). The haploid induction rate was counted 30 days after emergence.
[0057] Haploid induction rate = (number of haploid plants / number of initially screened plants) × 100%.
[0058] 1.2.2 Effect of pre-treatment before haploid doubling on browning
[0059] The pre-treatment methods of explants were compared to study the problem of severe browning during haploid tissue culture.
[0060] There were 3 treatments as follows. Each treatment was repeated 3 times, with 2 haploid plants in each repetition. For each plant, 3 middle leaves at the budding stage were taken, and a total of 18 leaf vein segments were obtained.
[0061] Treatment A: Take the leaves to be doubled. After cleaning the midrib leaves with a writing brush dipped in soapy water, then rinse with running water for 2 minutes. Select the midrib part of the leaf with a diameter of 6 mm - 8 mm, cut off the excess mesophyll tissue, and cut it into midrib segments 8 cm - 9 cm long. Then disinfect the midrib segments. The specific disinfection method is: disinfect with 75% alcohol for 1 minute, wash with sterile water 2 times; sterilize with 0.1% mercuric chloride (an aqueous solution of mercuric chloride with a concentration of 0.1% (w / v)) for 13 minutes, and rinse with sterile water 2 times before use.
[0062] Treatment B: Take the leaves to be doubled. Wear gloves and apply dishwashing liquid on the leaves, cancel the brushing with a writing brush in the conventional operation, rinse with running water for 2 minutes, take the midrib part of the leaf with a diameter of 6 mm - 8 mm, cut off the excess mesophyll tissue, and cut it into midribs 9 cm long. Then disinfect the midribs. The disinfection method is the same as that of Treatment A.
[0063] Treatment C: Take the leaves to be doubled, directly rinse with running water, take the midrib part of the leaf with a diameter of 6 mm - 8 mm, cut off the excess mesophyll tissue, and cut it into midribs 9 cm long. After rinsing with running water for 2 minutes, directly disinfect it. The disinfection method is the same as that of Treatment A.
[0064] The midribs of the leaves after the above three disinfection treatments were inoculated onto the shoot induction medium of MS + KT 2.0 mg / L + NAA 0.5 mg / L. After 20 days, multiple shoots grew on the leaves. When the shoots grew to 2 cm high, the shoots were cut and inoculated into the 1 / 2MS medium (the macronutrients were halved) + IBA 0.5 mg / L for root induction culture.
[0065] The browning rate was calculated after 15 days of culture, and the haploid doubling rate was calculated after 70 days of culture.
[0066] Browning rate = (browning explants / total explants) × 100%.
[0067] Contamination rate = (contaminated explants / total explants) x 100%.
[0068] 2 Results and analysis
[0069] 2.1 Effect of different pretreatments on browning rate
[0070] Previous studies have shown that leaves in the flowering period contain many impurities such as microorganisms and must be cleaned before disinfection. The present invention's research shows that if Figure 1 and Figure 2 As shown, among the three different explant pre-treatment methods: the conventional treatment A, the explant browning rate was as high as 50%, which was significantly higher than that of treatments B and C, and the contamination rate was about 5%; the explant browning rate of treatment B was 5%, and the contamination rate was less than 1%; although the explant browning rate of treatment C was 0, the contamination rate was 50%.
[0071] 2.2 Haploid induction rate of cigar tobacco
[0072] like Figure 3 As shown in the figure, the haploid induction results of 32 hybrid combinations of the main varieties in four cigar tobacco producing areas showed that the haploid induction frequency of the hybrid combinations with Fan Di No. 3 as the male parent was higher than that of the hybrid combinations with Yan Yan 97 as the male parent; among all the haploids induced by the hybrid combinations, the haploid induction rate of the hybrids of the Chuxue series varieties was the highest, which were 33.33% and 14.71% respectively. The haploid induction rate of the hybrids of the Chuxue series varieties was the lowest, which were 18.18% and 5.95% respectively.
[0073] 3 Discussions
[0074] The comparison of haploid induction rates of batch cigar tobacco showed that the induction of haploids in African tobacco hybrids was related to different genotypes of parents, such as Figure 3 As shown in the figure, the haploid induction rate of hybrids with the same female parent and Anti-Emperor No. 3 as the male parent is higher than that of the combination with Yanyan 97 as the male parent. Moreover, regardless of which parent is the male parent of the hybrid, the haploid induction rate of hybrids derived from Hubei cigar tobacco as the female parent is higher than that of hybrids from other production areas. The specific reasons need further study.
[0075] In addition, in order to carry out batch haploid doubling culture, the project team used toothpicks, different colors and numbers of threads to distinguish explants of different materials ( Figure 4 ), for example, the midribs of 11 leaf samples were disinfected at the same time, which can be shown in Table 2.
[0076] Table 2 Quick Distinguishing Method for Small-Batch Doubled Haploid Materials
[0077]
[0078]
[0079] Those skilled in the art can easily understand that, on the premise of no conflict, the above advantageous ways can be freely combined and superimposed. The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. The above is only the preferred implementation manner of the present application. It should be noted that for those of ordinary skill in the art in the technical field of the present application, several improvements and variations can be made without departing from the technical principle of the present application, and these improvements and variations should also be regarded as the protection scope of the present application.
Claims
1. A method for reducing the browning rate of doubled haploids of maternal origin, characterized in that: include: Explant cleaning: take a tobacco leaf to be doubled, apply detergent on the tobacco leaf, then rinse off the detergent with running water, then select the midrib of the tobacco leaf with a diameter of 6 mm to 8 mm, cut off the excess mesophyll tissue, and cut into midrib segments of 8 cm to 9 cm in length to obtain a cleaned explant; Explant disinfection, disinfecting the cleaned explant to obtain a disinfected explant; Explant culture: performing bud induction culture and root induction culture on the sterilized explant.
2. The method for reducing the browning rate of doubled haploids of maternal origin according to claim 1, characterized in that: The bud inducing culture comprises: The sterilized explants were inoculated into a culture medium of MS+KT 2.0 mg / L+NAA 0.5 mg / L for bud induction culture.
3. The method for reducing the browning rate of doubled haploids of maternal origin according to claim 1, characterized in that: The root inducing culture comprises: After the bud induction culture, when the buds grow to 1.8 cm to 2.2 cm, the buds are cut and inoculated into a medium containing 1 / 2MS+IBA 0.5 mg / L for the root induction culture.
4. The method for reducing the browning rate of doubled haploids of maternal origin according to claim 1, characterized in that: The explant disinfection comprises: disinfection with 75% alcohol for 1 minute, washing with sterile water twice; sterilization with 0.1% mercuric chloride for 13 minutes, and rinsing with sterile water twice.
5. A method for reducing the browning rate of doubled haploids of maternal origin according to any one of claims 1 to 4, characterized in that: After the sterilized explants are subjected to bud-inducing culture and root-inducing culture, the browning rate is less than 6% and the contamination rate is less than 2%.
6. Application of a method for reducing the browning rate of doubled haploids of maternal origin, characterized in that: The method for reducing the browning rate of doubled haploids of maternal origin as described in any one of claims 1 to 5 is used to induce doubling of maternal haploids of cigars.
7. The use of the method for reducing the browning rate of doubled haploids of maternal origin according to claim 6, characterized in that: Application methods include: In a greenhouse environment, N. africana pollen was taken and cross-pollinated with hybrids, and the haploid induction rate was counted 30 days after seedling emergence; Among them, the male parent of the hybrid is Fan Di No. 3 or Yan Yan 97, and the female parent is Yun Xue series, Chuan Xue series, Hai Yan series or Chu Xue series.
8. The use of the method for reducing the browning rate of doubled haploids of maternal origin according to claim 7, characterized in that: In the hybrid combinations with Yunxue, Chuanxue, Haiyan or Chuxue series as the female parent and Fandi 3 or Yanyan 97 as the male parent, the haploid induction rate of the hybrid combinations with Fandi 3 as the male parent was higher than that with Yanyan 97 as the male parent.
9. The use of the method for reducing the browning rate of doubled haploids of maternal origin according to claim 7, characterized in that: Among the hybrid combinations in which the male parent is Fan Di No. 3 or Yan Yan 97 and the female parent is Yun Xue, Chuan Xue, Hai Yan or Chu Xue series, the haploid induction rate of the hybrid combination of Chu Xue series varieties is the highest.
10. Use of the method for reducing the browning rate of doubled haploids of maternal origin according to claim 9, characterized in that: The haploid induction rate of the hybrid combination of Chuxue series varieties with Fan Di 3 as the male parent was 33.33%, and the haploid induction rate of the hybrid combination of Chuxue series varieties with Yan Yan 97 as the male parent was 14.71%.