CN113498738A - Method for creating new interspecific allopolyploid germplasm of tobacco by utilizing horizontal genome transfer - Google Patents
Method for creating new interspecific allopolyploid germplasm of tobacco by utilizing horizontal genome transfer Download PDFInfo
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Abstract
The invention discloses a method for creating a new interspecific allopolyploid germplasm of tobacco by utilizing horizontal genome transfer, belonging to the technical field of polyploid breeding of tobacco. The invention adopts safflower cultivated tobacco and yellow flower tobacco as grafting parent materials, and realizes the horizontal transfer of tobacco genome by using the test tube in vitro grafting technology; the artificial tissue culture technology is utilized to realize the induction of grafting and jointing calluses and the regeneration of asexual strains of two grafting parents, overcome the sexual breeding obstacle between two kindred tobacco seeds and form fertile tobacco allopolyploid plants.
Description
Technical Field
The invention relates to the technical field of polyploid breeding of tobacco, in particular to a method for creating a novel interspecific allopolyploid germplasm of tobacco by utilizing horizontal genome transfer.
Background
The allopolyploid is a combination of genomes from two different species and is a main source of species evolution, and the excellent characters of a plurality of modern crop plants are obtained by an allopolyploid pathway, such as the formation of common tobacco, namely the allopetraploid formed by natural doubling of chromosomes after the hybridization of forest tobacco and villous tobacco. More and more studies have found that the close contact between cells of different species induces a horizontal transfer of genetic material, i.e. the process by which a recipient organism obtains genetic material from a donor organism in a asexual manner, in contrast to the sexual transmission during generational multiplication. Horizontal gene transfer is also called horizontal gene transfer, and the new gene plays an important role in the origin, evolution and environmental adaptability of species, and the horizontal gene transfer is an important way for introducing the new gene into the species, can span the interspecies and can carry out genetic information transfer between organisms with distant or close relativity. Can lead the receptor organism to bypass point mutation and recombination to quickly form a new species, accelerate the innovation and evolution of genome and improve the genetic diversity of organisms. The rootstock and the scion are accompanied with the transfer of genetic materials among cells in the process of the reconstruction of callus, cell walls and vascular tissues. Grafting between plants can result in the communication of DNA fragments or the entire plastid genome. Adventitious buds generated by inducing callus through in vitro culture of the grafting junction part are called as grafting chimeras; the chimera induction technology can overcome sexual breeding obstacle of distant or nearby plants and form fertile allopolyploid. The allopolyploid can occur by a asexual mechanism, and grafting can be used as a rapid method for creating a new allopolyploid.
At present, because a single cultivated variety is selected as a parent for cross breeding of a new variety among varieties in tobacco breeding for a long time, the genetic relationship among the germplasms of domestic cultivated tobacco is relatively close, the genetic basis is narrow, the genetic diversity is low, and the method is a prominent problem faced by the tobacco breeding at present. The tobacco distant hybridization is an effective way for directly transferring dominant disease and insect resistant genes in wild tobacco to common cultivated tobacco. Therefore, the genetic basis of tobacco cultivars is widened and enriched, the genetic potential of the wild species of the tobacco is explored, and the creation of backbone parent materials carrying excellent genes is a problem to be solved urgently in tobacco breeding. However, reproductive isolation is common between tobacco species, such as Nicotiana tabacum and Nicotiana flavicana, and severe reproductive isolation exists between Nicotiana tabacum and most of wild tobacco. Therefore, how to effectively overcome the incompatibility of sexual hybridization between common safflower tobacco and yellow flower tobacco or wild tobacco is a technical problem which needs to be solved urgently in the present tobacco distant hybridization breeding.
Disclosure of Invention
The invention aims to provide a method for creating a new interspecific allopolyploid germplasm of tobacco by utilizing horizontal genome transfer, wherein safflower cultivated tobacco and yellow flower tobacco are used as grafting parent materials, and the horizontal transfer of a tobacco genome is realized by utilizing a test tube in vitro grafting technology; the induction of grafting and jointing calluses and the regeneration of asexual strains of two grafting parents are realized by utilizing an artificial tissue culture technology; overcomes sexual breeding obstacle between two closely related tobacco species, and forms fertile allopolyploid.
In order to solve the problems, the invention adopts the following technical scheme:
a method for creating a novel germplasm of an interspecies heteropolyploid of tobacco using horizontal genome transfer, the method comprising the steps of:
(1) and (3) sterile seedling cultivation: pretreating safflower tobacco seeds and yellow flower tobacco seeds in a tissue culture room, respectively sowing the seeds in culture dishes containing a solid culture medium, transplanting seedlings growing to a small cross stage into a culture bottle containing the solid culture medium after culturing for two weeks, putting the culture bottle into the tissue culture room, performing illumination culture for 16 hours every day, performing dark culture for 8 hours every day, and taking the seedlings as grafting parents after the seedlings of the safflower tobacco plants and the yellow flower tobacco plants grow to the plant height of 5-10 cm;
(2) in vitro grafting: performing interactive grafting on the grafting parent obtained in the step (1) to obtain a grafted body, placing the obtained grafted body in an MS culture medium for culturing for 2-4 weeks until the grafting combination part is completely fused, and obtaining a scion plant with vascular tissues at the combination part, wherein the scion plant can normally grow;
(3) graft binding site callus induction: cutting off the grafting combination part on the scion plant obtained in the step (2), transversely cutting the grafting tight combination part into a plurality of slices by using a blade, respectively and horizontally placing each slice in a culture bottle containing a callus solid culture medium for culturing for 25d, and performing callus induction to obtain a plurality of callus blocks;
(4) adventitious bud induction and rooting culture: respectively transferring the plurality of callus blocks obtained in the step (3) to a culture bottle containing an MS culture medium to induce the callus blocks to generate adventitious buds, and respectively transplanting the formed adventitious buds to the culture bottle containing a solid culture medium for culture to obtain a regeneration plant; repeating the steps until no adventitious bud is generated;
(5) transplanting the regenerated plants: washing the root culture medium of the regenerated plant obtained in the step (4) with distilled water, transplanting the washed root culture medium into a nutrition pot filled with a sterilized substrate, hardening the seedling for 25-30 days, transplanting the hardened seedling into a flowerpot of a greenhouse, and performing management cultivation according to conventional tobacco to obtain a regenerated plant;
(6) phenotype screening of regenerated plants: performing phenotype screening on the regenerated plant obtained in the step (5) to screen out a plant with interspecific allopolyploid characteristics;
(7) ploidy identification: and (4) identifying the ploidy of the plant with the interspecific allopolyploid characteristic obtained in the step (6), and screening polyploids to obtain a plurality of tobacco interspecific allopolyploid new germplasm plants.
Because the method is adopted, the safflower cultivated tobacco and the yellow flower tobacco are used as grafting parent materials, and the safflower cultivated tobacco and the yellow flower tobacco are incompatible in sexual hybridization and cannot obtain filial generations, the invention realizes the horizontal transfer of tobacco genomes by utilizing a test tube in vitro grafting technology; the artificial tissue culture technology is utilized to realize the induction of grafting and jointing calluses and the regeneration of asexual strains of two grafting parents, overcome the sexual breeding obstacle between two kindred tobacco seeds and form a plurality of fertile tobacco allopolyploid plants. The fertile tobacco allopolyploid plants formed by the invention are tall and big, the stems are thick and strong, the leaves are large and deep in color, and the fruits and seeds are large.
Preferably, the pretreatment in the step (1) is to wash 3 times with sterile water after carrying out disinfection pretreatment with a sodium hypochlorite solution; the illumination intensity of the illumination culture is 50 mu Em-2 S-1The temperature of light culture is 24 ℃, and the temperature of dark culture is 22 ℃;
by adopting the method, the tobacco seeds are pretreated and then cleaned, so that the germination rate of the seeds is effectively improved; the healthy and strong tobacco seedlings can be conveniently obtained by culturing under specific illumination intensity and temperature, and the plant height of the tobacco seedlings can be influenced by the intensity of the illumination intensity and the temperature.
Preferably, the composition of the solid medium in the step (1) and the step (4) is as follows: 4.74g/L MS, 30g/L sucrose, 6g/L agar and distilled water, and the pH of the solid medium is 5.8.
By adopting the method, the elongation and growth of the tobacco seedling stem and the regenerated plant can be better promoted by selecting the culture medium, and the composition and components of the solid culture medium can more or less influence the growth of the seedling and the plant.
Preferably, the callus solid medium in the step (3) has the following composition: 4.74g/L MS, 5mg/L KT, 0.2mg/L NAA, 30g/L sucrose, 6g/L agar, and the pH of the callus solid medium is 5.8.
By adopting the method, the specific callus solid culture medium is selected, so that mineral nutrition required by tobacco tissue production can be ensured, cell division is induced, formation of roots is facilitated, differentiation of stems and leaves is inhibited, and rapid growth of induced callus can be accelerated; the composition and components of the solid callus culture medium are more or less, which influences whether the callus can be well induced.
Preferably, the interactive grafting method in the step (2) is as follows: selecting a seedling plant of the safflower tobacco as a scion plant, reserving 2 small leaves at the top of the scion plant, removing the rest leaves, symmetrically beveling the scion plant by 45 degrees along two sides of a stem to form a wedge-shaped interface; selecting a yellow tobacco seedling plant as a stock plant, reserving a stem section which is 3cm above the root system of the stock plant, cutting off the rest, and splitting an interface matched with a wedge-shaped interface of a scion plant from the middle part of the reserved stem by using a double-sided blade; inserting the wedge-shaped end of the scion plant into the stock plant interface, and fixing the interface by using a plastic sleeve to complete the interactive grafting.
Preferably, the interactive grafting method in the step (2) is as follows: selecting a yellow tobacco seedling plant as a scion plant, reserving 2 small leaves on the top of the scion plant, removing the rest leaves, symmetrically beveling the scion plant by 45 degrees along two sides of a stem to form a wedge-shaped interface; selecting a safflower tobacco seedling plant as a stock plant, reserving a stem section which is 3cm above the root system of the stock plant, cutting off the rest, and splitting an interface matched with a wedge-shaped interface of a scion plant from the middle part of the reserved stem by using a double-sided blade; inserting the wedge-shaped end of the scion plant into the stock plant interface, and fixing the interface by using a plastic sleeve to complete the interactive grafting.
By adopting the method, the two grafting parents of the safflower tobacco and the yellow flower tobacco can be mutually used as scions or stocks for interactive grafting, the grafting and the callus induction and the asexual line regeneration are carried out, the sexual breeding obstacle between two kindred tobacco seeds is overcome, and a multi-plant fertile tobacco allopolyploid plant is formed.
Preferably, the thickness of the slice cut from the grafting tight combination part is 1-2 mm.
Compared with the prior art, the invention has the advantages that:
(1) the invention realizes the horizontal transfer of the genome of the cultivated tobacco and the wild tobacco by using the test tube in vitro grafting technology for the first time, proves that the test tube in vitro grafting technology is an effective method for overcoming distant hybridization incompatibility, and the method can be applied to germplasm creation and variety breeding of other horticultural crops which are easy to graft and organize for culture.
(2) The invention adopts safflower cultivated tobacco and yellow flower tobacco as grafting parent materials, and realizes the horizontal transfer of tobacco genome by using the test tube in vitro grafting technology; the artificial tissue culture technology is utilized to realize the induction of grafting and jointing calluses and the regeneration of asexual strains of two grafting parents, overcome the sexual hybridization obstacle between two kindred tobacco seeds and obtain a plurality of fertile tobacco allopolyploid plants.
(3) The method has strong practicability and simple and easy operation, saves the time for obtaining the allopolyploid, can be widely applied to tobacco distant hybridization breeding with sexual hybridization incompatibility among tobacco seeds, and the allopolyploid material obtained by the method does not contain any transgenic component and can be directly applied to variety breeding.
(4) The invention effectively solves the problems of insufficient exploitation and utilization of wild tobacco resources and narrow genetic basis of common tobacco at present, creates a tobacco germplasm resource bank and has a guiding function on innovation of a tobacco breeding method.
Drawings
FIG. 1 is a diagram of an in vitro grafted plant of the present invention;
FIG. 2 is a diagram of callus induced from the grafting binding site according to the present invention;
FIG. 3 is a diagram of the morphological characteristics of the plants of the grafted parent nicotiana tabacum Yunyan 87, nicotiana tabacum G391 and the allopolyploid GY7 of the present invention;
FIG. 4 is a graph of the relative DNA content analysis of the grafted parent and the allopolyploid plant of the present invention;
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
a method for creating a novel germplasm of an interspecies heteropolyploid of tobacco using horizontal genome transfer, the method comprising the steps of:
because the carthamus tinctorius (Yunyan 87) and the yellow flower tobacco (G391) are incompatible in sexual hybridization and filial generation cannot be obtained, the carthamus tinctorius (Yunyan 87) and the yellow flower tobacco (G391) which are commonly cultivated are selected as two grafting parents, the carthamus tinctorius (Yunyan 87) seedling plant is taken as a scion plant, and the yellow flower tobacco (G391) seedling plant is taken as a stock plant.
(1) Cultivation of aseptic seedlings
Sterilizing the surfaces of safflower tobacco seeds (Yunyan 87) and yellow flower tobacco seeds (G391) by using a sodium hypochlorite solution in a tissue culture room, then repeatedly cleaning the surfaces by using sterile water for 3 times, respectively sowing the seeds into culture dishes containing solid culture media, transplanting seedlings growing to a small cross stage into a culture bottle containing the solid culture media after culturing for two weeks, putting the culture bottle into the tissue culture room, culturing for 16 hours every day by illumination, culturing for 8 hours every day in the dark, and taking the safflower tobacco plants and the yellow flower tobacco seedlings as grafting parents to perform subsequent tests after the safflower tobacco plants and the yellow flower tobacco seedlings grow to the plant height of 10 cm; the solid culture medium comprises the following components: 4.74g/L MS, 30g/L sucrose, 6g/L agar and distilled water, and the pH value of the solid culture medium is 5.8; the light intensity of the above light culture is 50 μ Em-2S-1The temperature for light culture was 24 ℃ and the temperature for dark culture was 22 ℃.
2) In vitro grafting:
and (2) carrying out interactive grafting on the grafting parent obtained in the step (1) to obtain a grafted body, and placing the obtained grafted body in an MS culture medium for culturing for 2-4 weeks until the grafting combination part is completely fused, so as to obtain a scion plant capable of normally growing. Selecting tobacco aseptic seedlings with similar stem circumferences as grafting parents, taking safflower tobacco seedling plants as scion plants, reserving 2 small leaves on the tops of the scion plants, removing the rest leaves, and symmetrically beveling the scion plants by 45 degrees along two sides of a stem to form a wedge-shaped interface, wherein the stem lengths are about 3 cm; selecting yellow tobacco seedling plants as stock plants, reserving stem sections which are 3cm above root systems of the stock plants, and cutting off the rest; splitting a 5mm interface from the middle part of the stem by using a double-sided blade; inserting the wedge-shaped end of the scion into a stock interface, beveling the stems of two grafting parents by about 45 degrees after cambiums are matched, connecting the stems to ensure that the cambiums are matched, and fixing the stems by using a plastic sleeve to complete the interactive grafting (as shown in figure 1); culturing the treated graft in an MS culture medium for 2-4 weeks until the grafting combination part is completely fused, and timely shearing axillary buds growing on the rootstock every week during the period; the sign of in vitro grafting survival is that the combination part of the rootstock and the scion forms functional vascular tissues, and the scion plant grows normally. The solid medium used was 4.74g/L MS +30g/L sucrose +6g/L agar, the remainder was distilled water, pH 5.8.
(3) Graft binding site callus induction: cutting off the grafting combination part on the scion plant obtained in the step (2), transversely cutting the grafting tight combination part into a plurality of slices by using a blade, respectively and horizontally placing each slice in a culture bottle containing a callus solid culture medium for culturing for 25d, and performing callus induction to obtain a plurality of callus blocks. Selecting a grafting body with better grafting survival, cutting off a grafting combination part, cutting off parts which are not adhered at two ends, only keeping the parts of two grafting parents which are tightly combined, transversely cutting the combination part into thin slices with the thickness of about 1mm by a blade, generally cutting one grafting part into about 4 slices, then horizontally placing the thin slices of the grafting combination part in a culture bottle containing a solid culture medium for about 25 days, and carrying out callus induction (as shown in figure 2).
(4) Adventitious bud induction and rooting culture: respectively transferring the plurality of callus blocks obtained in the step (3) to a culture bottle containing an MS culture medium to induce the callus blocks to generate adventitious buds, and respectively transplanting the formed adventitious buds to the culture bottle containing a solid culture medium for culture to obtain a regeneration plant; in the process, the callus can continuously generate adventitious buds, and the callus needs to be continuously transplanted until no adventitious buds are generated; the solid medium used was 4.74g/L MS +30g/L sucrose +6g/L agar, the remainder was distilled water, pH 5.8.
(5) Transplanting the regenerated plants: washing the root culture medium of the regenerated plant obtained in the step (4) with distilled water, transplanting the washed root culture medium into a nutrition pot filled with a sterilized substrate, hardening the seedling for 25-30 days, transplanting the hardened seedling into a flowerpot of a greenhouse, and performing management cultivation according to conventional tobacco to obtain a regenerated plant;
(6) phenotype screening of regenerated plants: performing phenotype screening on the regenerated plant obtained in the step (5) to screen out a plant with interspecific allopolyploid characteristics;
the external shape of the plant with the allopolyploid characteristic shows that the plant is tall and big, the stem is thick and strong, the leaves are large, the color of the leaves is dark, and the fruit and the seeds are large. According to the difference between the phenotype characters of the asexual regeneration plant and the two grafting parents, preliminarily screening out the plant line which is possible to be interspecific allopolyploid after investigating the phenotype characters. Morphological structures of pistils and stamens and pollen germination determination are observed, and fertility of the allopolyploid material is observed through selfing and backcrossing.
Compared with the grafted parent Yunyan 87 and G391, the leaf shape and the leaf tip of the allopolyploid GY7 plant are similar to the Yunyan 87, and the main botanical trait differences are that the leaf color is light green, the main vein is thick, the branch vein is thick and malformed, the branch vein at the leaf ear is densely distributed, and the leaf is thick (as shown in figure 3 and table 1)
Table 1: comparison of the botanical traits of the grafted parent Yunyan 87, G391 and the allopolyploid asexual Strain GY7
(7) Ploidy identification: and (4) identifying the ploidy of the plant with the interspecific allopolyploid characteristic obtained in the step (6), and screening polyploids to obtain a plurality of tobacco interspecific allopolyploid new germplasm plants.
The DNA content and ploidy level of the asexual regeneration plant are determined by adopting a flow cytometry, the tetraploid Yunyan 87 is taken as an external standard, and the relative fluorescence intensity values of G391, a cytoplasmic male sterile line (MS Yunyan 87) and G1 of various heterologous polyploid regeneration lines are determined by flow cytometry, according to the formula: sample ploidy × (sample G1 peak fluorescence intensity/external standard G1 peak fluorescence intensity value) for the external standard sample, the theoretical ploidy level for each unknown sample was calculated. The results show that the asexual strains obtained by the in vitro grafting combination part have allopolyploid such as pentaploid, hexaploid and octaploid (shown in figure 4 and table 2), and 8 new allopolyploid germplasm such as GY4, GY5, GY7, GY11, GY13, GY14, GY19 and GY38 are obtained in total.
Table 2: ploidy level of grafted parent and asexual strain
Example 2:
a method for creating a novel germplasm of an interspecies heteropolyploid of tobacco using horizontal genome transfer, the method comprising the steps of:
because the carthamus tinctorius (Yunyan 87) and the yellow flower tobacco (G391) are incompatible in sexual hybridization and cannot obtain filial generations, the carthamus tinctorius (Yunyan 87) and the yellow flower tobacco (G391) which are commonly cultivated are selected as two grafting parents, the yellow flower tobacco (G391) seedling plant is taken as a scion plant, and the safflower tobacco (Yunyan 87) seedling plant is taken as a stock plant.
(1) Cultivation of aseptic seedlings
Sterilizing the surfaces of safflower tobacco seeds (Yunyan 87) and yellow flower tobacco seeds (G391) by using a sodium hypochlorite solution in a tissue culture room, then repeatedly cleaning the surfaces by using sterile water for 3 times, respectively sowing the seeds into culture dishes containing solid culture media, transplanting seedlings growing to a small cross stage into a culture bottle containing the solid culture media after culturing for two weeks, putting the culture bottle into the tissue culture room, performing illumination culture 16 every day, and performing dark culture 8 every day, and taking the safflower tobacco plants and the yellow flower tobacco seedlings as grafting parents to perform subsequent tests after the safflower tobacco plants and the yellow flower tobacco seedlings grow to the plant height of 8 cm; the solid culture medium comprises the following components: 4.74g/L MS, 30g/L sucrose, 6g/L agar and distilled water, and the pH value of the solid culture medium is 5.8; the light intensity of the above light culture is 50 μ Em-2 S-1The temperature for light culture was 24 ℃ and the temperature for dark culture was 22 ℃.
2) In vitro grafting:
and (2) carrying out interactive grafting on the grafting parent obtained in the step (1) to obtain a grafted body, and placing the obtained grafted body in an MS culture medium for culturing for 2-4 weeks until the grafting combination part is completely fused, so as to obtain a scion plant capable of normally growing. Selecting tobacco aseptic seedlings with similar stem circumferences as grafting parents, taking yellow tobacco (G391) seedling plants as scion plants, reserving 2 small leaves on the tops of the scion plants, removing the rest leaves, and symmetrically beveling the scion plants by 45 degrees along two sides of a stem to form a wedge-shaped interface, wherein the stem lengths are about 3 cm; selecting seedling plants of the tobacco (Yunyan 87) with safflower, reserving stem sections which are 3cm above root systems of the stock plants, and cutting off the rest; splitting a 5mm interface from the middle part of the stem by using a double-sided blade; inserting the wedge-shaped end of the scion into a stock interface, beveling the stems of two grafting parents by about 45 degrees after cambiums are matched, connecting the stems to ensure that the cambiums are matched, and fixing the stems by using a plastic sleeve to complete the interactive grafting (as shown in figure 1); culturing the treated graft in an MS culture medium for 4 times until the grafting combination part is completely fused, and timely cutting off axillary buds growing on the rootstock every week during the period; the sign of in vitro grafting survival is that the combination part of the rootstock and the scion forms functional vascular tissues, and the scion plant grows normally. The solid medium used was 4.74g/L MS +30g/L sucrose +6g/L agar, the remainder was distilled water, pH 5.8.
(3) Grafting binding site callus: cutting off the grafting combination part on the scion plant obtained in the step (2), transversely cutting the grafting tight combination part into a plurality of slices by using a blade, respectively and horizontally placing each slice in a culture bottle containing a callus solid culture medium for culturing for 25d, and performing callus induction to obtain a plurality of callus blocks. Selecting a grafting body with better grafting survival, cutting off a grafting combination part, cutting off parts which are not adhered at two ends, only keeping the parts of two grafting parents which are tightly combined, transversely cutting the combination part into thin slices with the thickness of about 1mm by a blade, generally cutting one grafting part into about 4 slices, then horizontally placing the thin slices of the grafting combination part in a culture bottle containing a solid culture medium for about 25 days, and carrying out callus induction (as shown in figure 2).
(4) Adventitious bud induction and rooting culture: respectively transferring the plurality of callus blocks obtained in the step (3) to a culture bottle containing an MS culture medium to induce the callus blocks to generate adventitious buds, and respectively transplanting the formed adventitious buds to the culture bottle containing a solid culture medium for culture to obtain a regeneration plant; in the process, the callus can continuously generate adventitious buds, and the callus needs to be continuously transplanted until no adventitious buds are generated; the solid medium used was 4.74g/L MS +30g/L sucrose +6g/L agar, the remainder was distilled water, pH 5.8.
(5) Transplanting the regenerated plants: and (4) washing the root culture medium of the regenerated plant obtained in the step (4) with distilled water, transplanting the washed root culture medium into a nutrition pot filled with a sterilized substrate, hardening the seedling for 25-30 days, transplanting the hardened seedling into a flowerpot of a greenhouse, and managing and cultivating the hardened seedling according to conventional tobacco.
(6) Phenotype screening of regenerated plants: performing phenotype screening on the regenerated plant obtained in the step (5) to screen out a plant with interspecific allopolyploid characteristics; the external morphology of polyploids generally shows that plants are tall and big, stems are thick and strong, leaves are large, the color of the leaves is dark, and fruits and seeds are large. According to the difference between the phenotype characters of the asexual regeneration plant and the two grafting parents, preliminarily screening out the plant line which is possible to be interspecific allopolyploid after investigating the phenotype characters. Morphological structures of pistils and stamens and pollen germination determination are observed, and fertility of the allopolyploid material is observed through selfing and backcrossing.
(7) Ploidy identification: and (4) identifying the ploidy of the plant with the interspecific allopolyploid characteristic obtained in the step (6), and screening polyploids to obtain a plurality of tobacco interspecific allopolyploid new germplasm plants. The DNA content and ploidy level of the asexual regeneration plant are determined by adopting a flow cytometry, the tetraploid Yunyan 87 is taken as an external standard, and the relative fluorescence intensity values of G391, a cytoplasmic male sterile line (MS Yunyan 87) and G1 of various heterologous polyploid regeneration lines are determined by flow cytometry, according to the formula: sample ploidy × (sample G1 peak fluorescence intensity/external standard G1 peak fluorescence intensity value) for the external standard sample, the theoretical ploidy level for each unknown sample was calculated. The results show that the asexual strains obtained by the in vitro grafting and combining parts have allopolyploids such as pentaploid, hexaploid and octaploid, and 8 allopolyploid new germplasms are obtained in total.
The inventor finds that the two grafting parents of the safflower tobacco and the yellow flower tobacco can be mutually used as scions or stocks for interactive grafting, the grafting and grafting callus is induced and asexual strains are regenerated, and 8 new allopolyploid germplasm can be obtained through phenotype screening and ploidy identification of regenerated plants. Overcomes sexual breeding obstacle between two closely related tobacco species, forms fertile tobacco allopolyploid plants, and has guiding effect on innovation of tobacco breeding method. The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the equivalent replacement or change according to the technical solution and the modified concept of the present invention should be covered by the scope of the present invention.
Claims (7)
1. A method for creating a novel germplasm of an interspecies heteropolyploid of tobacco by using horizontal genome transfer, which is characterized by comprising the following steps: the method comprises the following steps:
(1) and (3) sterile seedling cultivation: pretreating safflower tobacco seeds and yellow flower tobacco seeds in a tissue culture room, respectively sowing the seeds in culture dishes containing a solid culture medium, transplanting seedlings growing to a small cross stage into a culture bottle containing the solid culture medium after culturing for two weeks, putting the culture bottle into the tissue culture room, performing illumination culture for 16 hours every day, performing dark culture for 8 hours every day, and taking the seedlings as grafting parents after the seedlings of the safflower tobacco plants and the yellow flower tobacco plants grow to the plant height of 5-10 cm;
(2) in vitro grafting: performing interactive grafting on the grafting parent obtained in the step (1) to obtain a grafted body, placing the obtained grafted body in an MS culture medium for culturing for 2-4 weeks until the grafting combination part is completely fused, and obtaining a scion plant with vascular tissues at the combination part, wherein the scion plant can normally grow;
(3) graft binding site callus induction: cutting off the grafting combination part on the scion plant obtained in the step (2), transversely cutting the grafting tight combination part into a plurality of slices by using a blade, respectively and horizontally placing each slice in a culture bottle containing a callus solid culture medium for culturing for 25d, and performing callus induction to obtain a plurality of callus blocks;
(4) adventitious bud induction and rooting culture: respectively transferring the plurality of callus blocks obtained in the step (3) to a culture bottle containing an MS culture medium to induce the callus blocks to generate adventitious buds, and respectively transplanting the formed adventitious buds to the culture bottle containing a solid culture medium for culture to obtain a regeneration plant; repeating the steps until no adventitious bud is generated;
(5) transplanting the regenerated plants: washing the root culture medium of the regenerated plant obtained in the step (4) with distilled water, transplanting the washed root culture medium into a nutrition pot filled with a sterilized substrate, hardening the seedling for 25-30 days, transplanting the hardened seedling into a flowerpot of a greenhouse, and performing management cultivation according to conventional tobacco to obtain a regenerated plant;
(6) phenotype screening of regenerated plants: performing phenotype screening on the regenerated plant obtained in the step (5) to screen out a plant with interspecific allopolyploid characteristics;
(7) ploidy identification: and (4) identifying the ploidy of the plant with the interspecific allopolyploid characteristic obtained in the step (6), and screening polyploids to obtain a plurality of tobacco interspecific allopolyploid new germplasm plants.
2. The method of creating new germplasm of tobacco allopolyploids by horizontal genome transfer according to claim 1, wherein the new germplasm comprises: the pretreatment in the step (1) is to use a sodium hypochlorite solution for disinfection pretreatment and then use sterile water for cleaning for 3 times; the illumination intensity of the illumination culture is 50 mu Em-2S-1The temperature for light culture is 24 ℃, and the temperature for dark culture is 22 ℃.
3. The method of creating new germplasm of tobacco allopolyploids by horizontal genome transfer according to claim 1, wherein the new germplasm comprises: the solid culture medium in the step (1) and the step (4) comprises the following components: 4.74g/L MS, 30g/L sucrose, 6g/L agar and distilled water, and the pH of the solid medium is 5.8.
4. The method of creating new germplasm of tobacco allopolyploids by horizontal genome transfer according to claim 1, wherein the new germplasm comprises: the callus solid culture medium in the step (3) comprises the following components: 4.74g/L MS, 5mg/L KT, 0.2mg/L NAA, 30g/L sucrose, 6g/L agar, and the pH of the callus solid medium is 5.8.
5. The method of creating new germplasm of tobacco allopolyploids by horizontal genome transfer according to claim 1, wherein the new germplasm comprises: the interactive grafting method in the step (2) comprises the following steps: selecting a seedling plant of the safflower tobacco as a scion plant, reserving 2 small leaves at the top of the scion plant, removing the rest leaves, symmetrically beveling the scion plant by 45 degrees along two sides of a stem to form a wedge-shaped interface; selecting a yellow tobacco seedling plant as a stock plant, reserving a stem section which is 3cm above the root system of the stock plant, cutting off the rest, and splitting an interface matched with a wedge-shaped interface of a scion plant from the middle part of the reserved stem by using a double-sided blade; inserting the wedge-shaped end of the scion plant into the stock plant interface, and fixing the interface by using a plastic sleeve to complete the interactive grafting.
6. The method of creating new germplasm of tobacco allopolyploids by horizontal genome transfer according to claim 1, wherein the new germplasm comprises: the interactive grafting method in the step (2) comprises the following steps: selecting a yellow tobacco seedling plant as a scion plant, reserving 2 small leaves on the top of the scion plant, removing the rest leaves, symmetrically beveling the scion plant by about 45 degrees along two sides of a stem to form a wedge-shaped interface; selecting a safflower tobacco seedling plant as a stock plant, reserving a stem section which is 3cm above the root system of the stock plant, cutting off the rest, and splitting an interface matched with a wedge-shaped interface of a scion plant from the middle part of the reserved stem by using a double-sided blade; inserting the wedge-shaped end of the scion plant into the stock plant interface, and fixing the interface by using a plastic sleeve to complete the interactive grafting.
7. The method of creating new germplasm of tobacco allopolyploids by horizontal genome transfer according to claim 1, wherein the new germplasm comprises: the thickness of the slice cut from the grafting tight combination part is 1-2 mm.
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