阅读说明：本技术 一种获得栽培烟草和花烟草杂交后代的方法 (Method for obtaining filial generation of cultivated tobacco and floral tobacco ) 是由 吕立堂 陈穗云 廖菊够 于 2021-06-28 设计创作，主要内容包括：本发明提供了一种获得栽培烟草和花烟草杂交后代的方法,包括步骤：取第一栽培烟草的花粉,然后用第一栽培烟草的花粉对第二栽培烟草的柱头进行诱导处理,得第一次处理后的柱头；将所述第一次处理后的柱头依次进行细胞破碎处理、干燥处理和捣碎处理,得第二次处理后的柱头；取花烟草的花粉,并将所述花烟草的花粉与所述第二次处理后的柱头按预设比例混合,得待处理混合物；通过所述待处理混合物对第三栽培烟草在始开期后的120h内进行多次重复授粉,得授粉后的栽培烟草；对授粉后的栽培烟草以及得到的子代进行检测操作和筛分操作,得具有抗逆性的烟草杂交品种。本发明能够显著提升栽培烟与花烟草亲的亲和性,可以获得约5-8％的真实杂交后代。(The invention provides a method for obtaining filial generation of cultivated tobacco and floral tobacco, which comprises the following steps: pollen of the first cultivated tobacco is taken, and then the pollen of the first cultivated tobacco is used for carrying out induction treatment on stigma of the second cultivated tobacco to obtain stigma after the first treatment; sequentially carrying out cell crushing treatment, drying treatment and mashing treatment on the stigma after the first treatment to obtain a stigma after the second treatment; pollen of the flower tobacco is taken, and the pollen of the flower tobacco and the stigma after the second treatment are mixed according to a preset proportion, so that a mixture to be treated is obtained; repeatedly pollinating the third cultivated tobacco for a plurality of times within 120h after the beginning period through the mixture to be treated to obtain the pollinated cultivated tobacco; and (4) carrying out detection operation and screening operation on the cultivated tobacco after pollination and the obtained filial generation to obtain the tobacco hybrid variety with stress resistance. The method can obviously improve the affinity of the cultivated tobacco and the flower tobacco, and can obtain about 5-8% of real filial generation.)

1. A method for obtaining filial generation of cultivated tobacco and floral tobacco is characterized by comprising the following steps:

s1, dividing the cultivated tobacco according to the purpose, and respectively providing a first cultivated tobacco for providing pollen, a second cultivated tobacco for providing stigma and a third cultivated tobacco for receiving cross pollination;

s2, pollen of the first cultivated tobacco is taken, and then the pollen of the first cultivated tobacco is used for carrying out induction treatment on stigma of the second cultivated tobacco to obtain stigma after the first treatment;

s3, sequentially carrying out cell crushing treatment, drying treatment and mashing treatment on the stigma after the first treatment to obtain a stigma after the second treatment;

s4, pollen of the nicotiana tabacum is taken, and the pollen of the nicotiana tabacum and the stigma after the second treatment are mixed according to a preset proportion to obtain a mixture to be treated;

s5, repeatedly pollinating the third cultivated tobacco for a plurality of times within 120h after the beginning of the pollination through the mixture to be treated to obtain the pollinated cultivated tobacco;

s6, detecting the pollinated cultivated tobacco and screening the obtained filial generation to obtain the tobacco hybrid variety with stress resistance.

2. The method for obtaining hybrid progeny of nicotiana tabacum and nicotiana tabacum as claimed in claim 1, wherein in S2, the inducing treatment comprises in vivo or ex vivo induction of stigma of the second cultivated tobacco; wherein the content of the first and second substances,

the in vivo induction comprises: directly inducing stigmas with pollen of the first cultivated tobacco on the second cultivated tobacco of a living body, and shearing the induced stigmas for later use;

the ex vivo induction comprises: and shearing the stigma of the second cultivated tobacco, and then inducing the stigma of the second cultivated tobacco obtained after shearing by using pollen of the first cultivated tobacco under a preset induction condition.

3. The method for obtaining hybrid progeny of nicotiana tabacum and nicotiana tabacum as claimed in claim 2, wherein the predetermined inducing conditions comprise: under the condition of air circulation, the temperature is kept at 25-35 ℃, the humidity is kept at 45-70%, and the oxygen concentration is kept at 20-25%.

4. The method for obtaining hybrid progeny of cultivated tobacco and nicotiana tabacum according to any one of claims 1 to 3, wherein the induction time in the induction treatment is 0.5 to 2 hours.

5. The method for obtaining filial generation of cultivated tobacco and nicotiana tabacum according to claim 1, wherein in S4, the preset ratio is 1:2-20 by mass.

6. The method for obtaining filial generation of cultivated tobacco and nicotiana tabacum according to claim 5, wherein the preset ratio is 1:10 by mass.

7. The method for obtaining filial generation of cultivated tobacco and nicotiana tabacum according to claim 1, wherein in S3, the drying time is 24-48h, and the drying temperature is 20-40 ℃.

8. The method for obtaining hybrid progeny of nicotiana tabacum and nicotiana tabacum as claimed in claim 1, wherein said repeatedly pollinating a third tobacco variety in sequence by said mixture to be treated for a plurality of times within 120h after the beginning of pollination in said S5 comprises:

and (3) repeatedly pollinating the third cultivated tobacco for 72h, 108h and 120h after the beginning period by using the mixture to be treated in the same proportion.

9. The method for obtaining filial generation of nicotiana tabacum and nicotiana tabacum according to claim 1, wherein in S6, the detecting operation comprises: counting the number of pollen tubes growing into an ovary and the seed setting rate through callose fluorescence;

the screening operation comprises: and (4) carrying out variety screening through chromosome content.

10. The method for obtaining hybrid progeny of nicotiana tabacum and nicotiana tabacum as claimed in claim 9, wherein said screening for varieties by chromosome content comprises:

s61, numbering plants to be detected, then respectively sampling from the leaves of each plant to be detected, and respectively chopping the sampled leaves;

s62, respectively adding pre-cooled cell nucleus extraction buffer solution into the chopped leaves, filtering, and then sequentially performing ice incubation treatment, centrifugal treatment and PI staining treatment on the filtrate obtained by the filtering treatment; wherein, a preset volume of RNase is added to prevent double-stranded RNA from being simultaneously colored while the PI staining treatment is carried out;

and S63, analyzing the products after PI staining treatment by using a flow cytometer to analyze the content of the nuclear DNA.

Technical Field

The invention relates to the technical field of tobacco hybridization, in particular to a method for obtaining a filial generation of cultivated tobacco and floral tobacco.

Background

The tobacco flower (N.alata, n ═ 9) of the Bidong Nicotiana has strong disease resistance and good resistance to wildfire, powdery mildew, tomato leaf spot and the like. In prior studies, attempts to introduce the superior trait of n.alata into cultivated tobacco species using conventional pollination methods have been explored, but no good results have been obtained, and prior studies have not systematically investigated the timing, location of and method of breaking through the cross-incompatibility that occurs.

In order to solve the problems, the chinese patent publication No. CN104996295A discloses a method for improving the affinity of hybrid between cultivated tobacco and flower tobacco and the seed setting rate, which comprises mixing the stigma of the female parent and the pollen of the male parent, and then pollinating 120h after the beginning of the female parent, although the method can improve the affinity of hybrid between cultivated tobacco and flower tobacco, the method directly mixes the stigma of the female parent and the pollen, and does not realize the significant improvement of the affinity between cultivated tobacco and flower tobacco.

In view of the above, there is a need for a method for obtaining hybrid progeny of cultivated tobacco and floral tobacco, which solves or at least alleviates the above technical drawback that the affinity of cultivated tobacco and floral tobacco is not significantly improved.

Disclosure of Invention

The invention mainly aims to provide a method for obtaining filial generations of cultivated tobacco and flower tobacco, and aims to solve the technical problem that the affinity of the cultivated tobacco and the flower tobacco is not obviously improved in the prior art.

In order to achieve the above object, the present invention provides a method for obtaining hybrid progeny of cultivated tobacco and nicotiana tabacum, comprising the steps of:

s1, dividing the cultivated tobacco according to the purpose, and respectively providing a first cultivated tobacco for providing pollen, a second cultivated tobacco for providing stigma and a third cultivated tobacco for receiving cross pollination;

s2, pollen of the first cultivated tobacco is taken, and then the pollen of the first cultivated tobacco is used for carrying out induction treatment on stigma of the second cultivated tobacco to obtain stigma after the first treatment;

s3, sequentially carrying out cell crushing treatment, drying treatment and mashing treatment on the stigma after the first treatment to obtain a stigma after the second treatment;

s4, pollen of the nicotiana tabacum is taken, and the pollen of the nicotiana tabacum and the stigma after the second treatment are mixed according to a preset proportion to obtain a mixture to be treated;

s5, repeatedly pollinating the third cultivated tobacco for a plurality of times within 120h after the beginning of the pollination through the mixture to be treated to obtain the pollinated cultivated tobacco;

s6, detecting the pollinated cultivated tobacco and screening the obtained filial generation to obtain the tobacco hybrid variety with stress resistance.

Further, in the S2, the inducing treatment includes performing in vivo induction or ex vivo induction on stigma of the second cultivated tobacco; wherein the content of the first and second substances,

the in vivo induction comprises: directly inducing stigmas with pollen of the first cultivated tobacco on the second cultivated tobacco of a living body, and shearing the induced stigmas for later use;

the ex vivo induction comprises: and shearing the stigma of the second cultivated tobacco, and then inducing the stigma of the second cultivated tobacco obtained after shearing by using pollen of the first cultivated tobacco under a preset induction condition.

Further, the preset inducing conditions include: under the condition of air circulation, the temperature is kept at 25-35 ℃, the humidity is kept at 45-70%, and the oxygen concentration is kept at 20-25%.

Further, the induction time in the induction treatment is 0.5-2 h.

Further, in the S4, the preset ratio is a mass ratio of 1: 2-20.

Further, the preset ratio is a mass ratio of 1: 10.

Further, in the step S3, the drying time is 24 to 48 hours, and the drying temperature is 20 to 40 ℃.

Further, in S5, the performing, by the mixture to be treated, multiple repeated pollinations of a third cultivated tobacco in sequence within 120 hours after the beginning includes:

and (3) repeatedly pollinating the third cultivated tobacco for 72h, 108h and 120h after the beginning period by using the mixture to be treated in the same proportion.

Further, in the S6, the detecting operation includes: counting the number of pollen tubes growing into an ovary and the seed setting rate through callose fluorescence;

the screening operation comprises: and (4) carrying out variety screening through chromosome content.

Further, the screening of the variety through the chromosome content comprises the following steps:

s61, numbering plants to be detected, then respectively sampling from the leaves of each plant to be detected, and respectively chopping the sampled leaves;

s62, respectively adding pre-cooled cell nucleus extraction buffer solution into the chopped leaves, filtering, and then sequentially performing ice incubation treatment, centrifugal treatment and PI staining treatment on the filtrate obtained by the filtering treatment; wherein, while the PI staining treatment is performed, a predetermined volume of RNase is added to prevent simultaneous staining of double stranded RNA.

And S63, analyzing the products after PI staining treatment by using a flow cytometer to analyze the content of the nuclear DNA.

Compared with the prior art, the method for obtaining the filial generation of the cultivated tobacco and the flower tobacco has the following beneficial effects:

the method for obtaining the filial generation of the cultivated tobacco and the flower tobacco can obviously improve the affinity of the cultivated tobacco and the flower tobacco and can obtain about 5-8% of real filial generation; by dividing the cultivated tobacco into a first cultivated tobacco for providing pollen, a second cultivated tobacco for providing stigma and a third cultivated tobacco for pollination, the subsequent treatment of stigma and the differentiation during pollination are facilitated; by inducing the stigma of the second cultivated tobacco by using the pollen of the first cultivated tobacco, an induction system is formed at the stigma based on the female parent, so that the affinity to homologous plants can be increased; the cell disruption treatment is carried out on the stigma after the first treatment, so that the interference of maternal pollen on pollination is avoided, and the competitive condition is avoided; further, the possibility of obtaining true hybrid progeny is ensured by mixing the pollen of the nicotiana tabacum with the stigma after the second treatment in a specific ratio and repeatedly pollinating the third cultivated tobacco with the pollen for a plurality of times.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.

FIG. 1 is a schematic flow chart of a method for obtaining hybrid progeny of Nicotiana tabacum and Nicotiana tabacum in one embodiment of the present invention;

FIG. 2 is a DNA content chart of cultivated tobacco (a), floral tobacco (b), and tobacco hybrid (c) in one embodiment of the present invention.

The objects, features and advantages of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.

In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.

It should be noted that, in the present invention, the time of the patency of the flowers of the female parent is defined as the beginning period, and in the present invention, the cultivated tobacco is the female parent and the flower tobacco is the male parent; wherein, the cultivated tobacco is cultivated tobacco Nicotiana tabacum 'K326'.

As shown in FIGS. 1-2, the present invention provides a method for obtaining hybrid progeny of cultivated tobacco and nicotiana tabacum, comprising the steps of:

and S1, dividing the cultivated tobaccos according to purposes, namely a first cultivated tobacco for providing pollen, a second cultivated tobacco for providing stigma and a third cultivated tobacco for receiving cross pollination.

The above-mentioned division of the cultivated tobacco is mainly because the cultivated tobacco needs to be selected respectively for different treatments in the subsequent operations.

S2, pollen of the first cultivated tobacco is taken, and then the pollen of the first cultivated tobacco is used for carrying out induction treatment on stigma of the second cultivated tobacco to obtain stigma after the first treatment; wherein the induction time in the induction treatment can be 0.5-2 h.

The induction treatment is mainly used for obtaining an induction system generated after the initial reaction between the pollen of the female parent and the stigma, so that the induction system is applied to the subsequent hybridization process to generate affinity induction on hybridization; it is particularly noted that the induction treatment is preferably an induction system generated after the initial reaction (for example, reaction for 0.5-2h) between pollen of the first cultivated tobacco and stigma of the second cultivated tobacco.

During the induction treatment, the pollen of the first cultivated tobacco is not too much, and only slight adhesion is needed to be carried out on the stigma of the second cultivated tobacco (namely, the pollen of the first cultivated tobacco and the stigma of the second cultivated tobacco do not need to be measured in proportion, and the purpose of mutual adhesion can be achieved to play a corresponding role in the invention), and in addition, the induction time is not too long; controlling the pollen amount of the first cultivated tobacco and controlling the induction time to avoid over-induction, reducing substances generated by pollen and stigma in the induction system at the initial reaction stage, and reducing the affinity induction effect of the induction system in the subsequent hybridization process.

In addition, the inducing treatment may include in vivo inducing or ex vivo inducing stigma of the second cultivated tobacco.

The in vivo induction comprises: and directly inducing stigmas with pollen of the first cultivated tobacco on the second cultivated tobacco of the living body, and shearing the induced stigmas for later use. The living body treatment has the advantages of good induction effect, capability of obtaining a real induction system of the female parent and suitability for operation in an experimental verification stage.

The ex vivo induction comprises: and shearing the stigma of the second cultivated tobacco, and then inducing the stigma of the second cultivated tobacco obtained after shearing by using pollen of the first cultivated tobacco under a preset induction condition. The in vitro induction has the advantages that the large-scale induction treatment is convenient, and the respective induction of stigma of each second cultivated tobacco is avoided;

specifically, the preset inducing conditions for the ex vivo induction may include: under the condition of air circulation, the temperature is kept at 25-35 ℃, the humidity is kept at 45-70%, and the oxygen concentration is kept at 20-25%, so that a proper environment is provided for the induction treatment under the condition of separation.

In the in-vitro induction, pollen of the first cultivated tobacco and stigma of the second cultivated tobacco can be directly mixed to realize quick and effective induction; in the living body induction, pollen of the first cultivated tobacco needs to be smeared on stigma of each second cultivated tobacco, and the operation difficulty and the strength are high.

And S3, sequentially carrying out cell crushing treatment, drying treatment and mashing treatment on the stigma after the first treatment to obtain the stigma after the second treatment. The drying treatment is mainly used for removing redundant moisture, the drying treatment time is 24-48h, and the drying temperature of the drying treatment is 20-40 ℃; naturally, it can also be dried directly at room temperature. In addition, the cell disruption treatment is mainly used for inactivating pollen cells of the first cultivated tobacco adhered to the stigma of the second cultivated tobacco, so as to avoid interference of the pollen cells on later pollination and stop the continuous reaction of the stigma and the pollen; the trituration process is mainly intended to obtain the second processed stigmas in powder form, so as to facilitate the subsequent corresponding mixing operations.

S4, pollen of the nicotiana tabacum is taken, and the pollen of the nicotiana tabacum and the stigma after the second treatment are mixed according to a preset proportion to obtain a mixture to be treated; the preset ratio can be a mass ratio of 1:2-20, and further, the preset ratio can be a mass ratio of 1:10, so as to achieve an optimal mixing ratio. And mixing the pollen of the flower tobacco with the stigma after the second treatment according to a preset proportion, so that the pollen of the flower tobacco carries an induction system for cultivating tobacco when pollinating, thereby obviously improving the affinity of hybridization.

S5, repeatedly pollinating the third cultivated tobacco for a plurality of times within 120h after the beginning of the pollination through the mixture to be treated to obtain the pollinated cultivated tobacco;

specifically, the repeated pollination of the third cultivated tobacco for a plurality of times within 120 hours after the beginning of the pollination by the mixture to be treated comprises the following steps: and pollinating the third cultivated tobacco for 72h, 108h and 120h after the beginning period by using the mixture to be treated in the same proportion.

S6, detecting the pollinated cultivated tobacco and screening the obtained filial generation to obtain the tobacco hybrid variety with stress resistance.

Wherein the detecting operation comprises: counting the number of pollen tubes growing into an ovary and the seed setting rate through callose fluorescence; the screening operation comprises: and (4) carrying out variety screening through chromosome content.

By way of further illustration, said screening for cultivars by chromosome content comprises:

s61, numbering plants to be detected, then respectively sampling from the leaves of each plant to be detected, and respectively chopping the sampled leaves;

s62, respectively adding pre-cooled cell nucleus extraction buffer solution into the chopped leaves, filtering, and then sequentially performing ice incubation treatment, centrifugal treatment and PI staining treatment on the filtrate obtained by the filtering treatment;

and S63, analyzing the products after PI staining treatment by using a flow cytometer to analyze the content of the nuclear DNA.

It is noted that, while the PI staining treatment is performed, a predetermined volume of RNase is added to prevent simultaneous staining of double-stranded RNA.

To facilitate a further understanding of the invention for those skilled in the art, reference will now be made to the following examples:

example 1

1. Taking mature pollen of the first cultivated tobacco, and then carrying out the living body induction treatment on stigma of the second cultivated tobacco for 1h by using the mature pollen of the first cultivated tobacco to obtain stigma after the first treatment; after the induction treatment, the stigma after the first treatment contains an induction system capable of enhancing hybridization affinity.

2. And immediately after the induction treatment, sequentially carrying out cell crushing treatment, drying treatment (drying at room temperature) and mashing treatment on the stigma after the first treatment to obtain the stigma after the second treatment.

3. And (3) taking pollen of the flower tobacco, and mixing the mature pollen of the flower tobacco with the stigma after the second treatment according to the mass ratio of 1:10 to obtain a mixture to be treated, wherein the mixture to be treated carries an induction system for cultivating the tobacco, so that the hybridization affinity can be obviously improved.

4. Pollinating the third cultivated tobacco for 72h, 108h and 120h after the beginning period through the mixture to be treated to obtain pollinated cultivated tobacco;

5. counting the number of pollen tubes growing into an ovary and the seed setting rate data by callose fluorescence after 48 hours of the last pollination; then, the variety screening of the offspring is carried out through the chromosome content.

Wherein, through callose fluorescence statistics, the number of pollen tubes in the ovary reaches about 234 pollen tubes/ovary, and the seed setting rate reaches 11.5 percent.

In addition, the process of screening the offspring varieties through the chromosome content specifically comprises the following steps:

numbering the parents and filial generation, and taking 1cm of each plant of the parents and the filial generation²The leaves were washed with deionized water and minced with a sharp scalpel, and 1mL of pre-chilled cell nuclear extraction Buffer (Galbraith's Buffer: 45mM MgCl)₂30mM sodium citrate, 20mM MOPS, 0.10% V/V Triton X, pH 7.0) and filtered into a 1.5mL centrifuge tube using a 50 μm nylon mesh.

After 5min incubation on ice, centrifugation at 1100rpm (5min, 4 ℃) was performed at low speed, the majority of the supernatant was discarded, 50. mu.L remained, PI staining was performed on the nuclei while 1. mu.L of RNase was added to prevent double stranded RNA staining at the same time, the samples were resuspended and incubated in the dark for 20 min.

Nuclear DNA content was analyzed using a BD Accuri-C6 flow cytometer, the FL2-a channel was run at medium speed (60 μ L/min), and data were acquired and analyzed using BD FACS Suite software.

The parameter evaluated in each sample was the relative Fluorescence intensity of the PI stained nuclei (Fluorescence intensities of PI-stabilized nuclei, FL).

As shown in fig. 2, after the flow test, the DNA content of the female parent is the highest, the DNA content of the male parent is the lowest, and the DNA content of the filial generation is half of the total of the DNA content of the male parent and the female parent, if the filial generation is the selfing and fructification of the female parent, the DNA content is the same as that of the female parent, but the probability of obtaining the true filial generation is 8% in this embodiment.

As an extension of the above example 1, under the same other conditions, only the mature pollen of the flower tobacco and the stigma after the second treatment are mixed in a mass ratio of 1: 20; the rate of obtaining true filial generation is 5% by detecting the chromosome content.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the technical spirit of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.