Method for obtaining homogeneous mutant of kiwi fruit callus
阅读说明：本技术 一种获得猕猴桃愈伤组织的同质突变体的方法 (Method for obtaining homogeneous mutant of kiwi fruit callus ) 是由 苑平 卜范文 徐海 杨莉颖 程小梅 汤佳乐 张平 黄佳 何科佳 陈为峰 于 2021-09-03 设计创作，主要内容包括：本发明公开了一种获得猕猴桃愈伤组织的同质突变体的方法,该方法通过将猕猴桃愈伤组织进行～(60)Co-γ辐射处理后,所述猕猴桃愈伤组织按4分法分成4份,分别置于继代培养基上进行第一次继代培养后,所述猕猴桃愈伤组织按9分法分成9份,且每块所述猕猴桃愈伤组织只保留左侧边缘中间部位的1份用于继代培养,培养15-20d,接着按9分法取左侧边缘中间部位的1份用于预分化培养,培养20-30d后,每块愈伤组织按4分法取其中有芽点且生长状态好的1份,转移至分化培养基中进行分化培养,培养15-20d,取长度3-5cm的芽进行嫩枝扦插,得到猕猴桃愈伤组织的同质突变体。通过本发明方法,可以得到性状稳定的同质突变体。(The invention discloses a method for obtaining a homogeneous mutant of kiwi callus, which is implemented by carrying out the following steps on the kiwi callus 60 After Co-gamma radiation treatment, dividing the kiwi fruit callus into 4 parts according to a 4-division method, respectively placing the kiwi fruit callus on a subculture medium for first subculture, dividing the kiwi fruit callus into 9 parts according to a 9-division method, only reserving 1 part of the middle part of the left edge of each kiwi fruit callus for subculture, culturing for 15-20d, then taking 1 part of the middle part of the left edge of each kiwi fruit callus for pre-differentiation culture according to the 9-division method, after culturing for 20-30d, taking 1 part of each callus with bud points and good growth state according to the 4-division method, transferring the callus to a differentiation medium for divisionAnd (3) carrying out chemical culture, culturing for 15-20d, and carrying out twig cuttage on buds with the length of 3-5cm to obtain the homogeneous mutant of the callus of the kiwi fruit. By the method, the homogeneous mutant with stable characters can be obtained.)
1. A method for obtaining a homogeneous mutant of kiwi callus is characterized by comprising the following steps:
(1) subjecting Actinidia chinensis callus to60Co-gamma radiation treatment of said60The radiation dose of Co-gamma radiation treatment is 0-500 Gy;
(2) dividing each irradiated kiwi fruit callus into 4 parts by 4-division method (as shown in figure 1), and respectively placing on subculture medium for primary subculture for 15-20 days;
(3) dividing the callus of the kiwi fruit after the first subculture into 9 parts by a 9-division method (as shown in figure 1), and reserving only 1 part of the middle part of the left edge of each callus of the kiwi fruit for subculture for 15-20 days;
(4) repeating the step (3) for 3-5 times, and taking 1 part of the middle part of the left edge according to a 9-division method for pre-differentiation culture for 20-30 d;
(5) after differentiation and sprouting, taking 1 part of each callus with bud points and good growth state according to a 4-division method, transferring the callus into a differentiation culture medium for differentiation culture, and culturing for 15-20 d;
(6) repeating the step (5) for 3-5 times to obtain buds with the length of more than 3 cm;
(7) and (4) carrying out twig cuttage on the buds with the length of 3-5cm obtained in the step (6) to obtain the homogeneous mutants of the calluses of the kiwi fruits.
2. The method for obtaining the homogeneous mutant of kiwi callus according to claim 1, wherein in step (1), said kiwi callus is obtained by the following method; and (3) placing 2 pieces of filter paper in a culture dish, adding 5-10ml of liquid subculture medium to thoroughly wet the filter paper, placing the pre-cultured callus in the culture dish, and sealing with a sealing film to obtain the kiwi fruit callus.
3. Method for obtaining a homological mutant of kiwi callus according to claim 2, wherein said pre-cultured callus is obtained by the following method: taking annual non-lignified kiwi branches for callus induction, and carrying out amplification culture through a subculture medium to obtain callus; cutting the callus into 0.4 x 0.4cm small blocks, and placing on a subculture medium for further culture for 7-10 days to obtain pre-cultured callus.
4. The method for obtaining the homogeneous mutants of kiwi callus according to claim 1, wherein in step (7), said shoot cutting is performed by the following method: taking buds with the length of 3-5cm, soaking the bases of the tender buds in rooting solution for 1-2h, then directly cutting the buds in vermiculite, and covering with a film for heat preservation.
5. Method for obtaining the homological mutants of kiwi callus according to claim 4, characterized in that said vermiculite is preferably vermiculite with a diameter of 0.1-0.3 cm.
6. The method for obtaining the homogeneous mutant of kiwi callus according to claim 1, wherein the subculture medium in step (2) comprises: MS minimal medium, sucrose with the concentration of 32g/L, agar with the concentration of 7.5g/L, 6-benzylaminopurine with the concentration of 0.25mg/L and naphthylacetic acid with the concentration of 1 mg/L; the illumination culture conditions are as follows: 16h/d light and 8h/d dark, and the light intensity is 5000 lx.
7. The method for obtaining the homogeneous mutants of kiwi callus according to claim 1, wherein the pre-differentiation medium in step (4) comprises: MS minimal medium, sucrose with the concentration of 32g/L, agar with the concentration of 7.5g/L and zeatin with the concentration of 3 mg/L; the illumination culture conditions are as follows: 16h/d light and 8h/d dark, and the light intensity is 5000 lx.
8. The method for obtaining the homogeneous mutant of kiwi callus according to claim 1, wherein said differentiation medium in step (5) comprises: MS minimal medium, sucrose with the concentration of 32g/L, agar with the concentration of 7.5g/L and zeatin with the concentration of 0.5 mg/L; the illumination culture conditions are as follows: 16h/d light and 8h/d dark, and the light intensity is 5000 lx.
9. The method for obtaining the homogeneous mutants of kiwi callus according to claim 4, wherein said rooting solution comprises the following components: the concentration is 100 mg/L3-indolebutyric acid and 50mg/L naphthylacetic acid solution.
10. The method for obtaining the homogeneous mutant of kiwi callus according to claim 1, further comprising the step of (8) verifying said homogeneous mutant of kiwi callus by phenotypic observation and molecular marker combination.
Technical Field
The invention belongs to the technical field of agricultural breeding, and particularly relates to a method for obtaining a homotypic mutant of kiwi callus.
Background
In the perennial and gynandropathic kiwi fruit, the vegetative organs such as branches are generally adopted for mutagenesis, but the mutation is unicellular, while the bud points on the branches are composed of multiple cells, and the plants after mutagenesis are genetic heterozygous individuals containing different mutant cells and normal cells, often exist in the form of chimeras, so that mutant plants with stable characters are difficult to obtain. Moreover, after mutagenesis, the plant is subjected to somatic selection, namely, the mutant cell and the non-mutant cell compete in the growth and development process, the initial mitosis of the mutant cell is usually inhibited or delayed due to the physiological damage or chromosome damage of the mutant cell caused by radiation, so that the normal cell is always dominant, and in the top dominant plant, the mutant cell may not exist in the growth cone at the upper part of the branch, the plant is recovered to be normal, the mutant character disappears, and the homogeneous mutant with stable character is difficult to obtain. In the traditional method, mutants are separated by methods such as short-cut pruning, multiple times of grafting and the like, and the homogeneous mutants with stable characters can be obtained in 3-5 years, and the period is too long. And the radiation mutagenesis is carried out on the callus, the homogeneous mutant can be separated only in 5-7 months, the efficiency is improved by 7-8 times, the influence of seasons is avoided, and the mutagenesis can be carried out in different periods all the year round.
Disclosure of Invention
In order to solve the defects and shortcomings of the prior art, the invention aims to provide a method for obtaining a homotypic mutant of kiwi callus. According to the method, the kiwi callus is subjected to radiation mutagenesis and combined with the existing kiwi tissue culture system, so that a homogeneous mutant with stable characters can be obtained, and the problems of chimeric variation and somatic cell selection are solved.
The purpose of the invention is realized by the following technical scheme: a method for obtaining a homogeneous mutant of kiwi callus comprises the following steps:
(1) subjecting Actinidia chinensis callus to60Co-gamma radiation treatment of said60The radiation dose of Co-gamma radiation treatment is 0-500 Gy;
(2) dividing each irradiated kiwi fruit callus into 4 parts by 4-division method (as shown in figure 1), and respectively placing on subculture medium for primary subculture for 15-20 days;
(3) dividing the callus of the kiwi fruit after the first subculture into 9 parts by a 9-division method (as shown in figure 1), and reserving only 1 part of the middle part of the left edge of each callus of the kiwi fruit for subculture for 15-20 days;
(4) repeating the step (3) for 3-5 times, and taking 1 part of the middle part of the left edge according to a 9-division method for pre-differentiation culture for 20-30 d;
(5) after differentiation and sprouting, taking 1 part of each callus with bud points and good growth state according to a 4-division method, transferring the callus into a differentiation culture medium for differentiation culture, and culturing for 15-20 d;
(6) repeating the step (5) for 3-5 times to obtain buds with the length of more than 3 cm;
(7) and (4) carrying out twig cuttage on the buds with the length of 3-5cm obtained in the step (6) to obtain the homogeneous mutants of the calluses of the kiwi fruits.
In the step (1), the step (c),
the kiwi callus is obtained by the following method; and (3) placing 2 pieces of filter paper in a culture dish, adding 5-10ml of liquid subculture medium to thoroughly wet the filter paper, placing the pre-cultured callus in the culture dish, and sealing with a sealing film to obtain the kiwi fruit callus.
The pre-cultured callus is obtained by the following method: taking annual non-lignified kiwi branches for callus induction, and carrying out amplification culture through a subculture medium to obtain callus; cutting the callus into 0.4 x 0.4cm small blocks, and placing on a subculture medium for further culture for 7-10 days to obtain pre-cultured callus.
In the step (7), the step (c),
the twig cuttage is carried out by the following method: taking buds with length of 3-5cm, soaking tender bud base with rooting solution for 1-2h, directly cutting in vermiculite (diameter of 0.1-0.3cm), and coating with film for heat preservation.
The vermiculite is preferably 0.1-0.3cm in diameter.
After film covering and heat preservation, rooting can be carried out after 10-20 days, film uncovering and seedling hardening can be carried out after 30-40 days, and the seedlings can be transplanted to 3 gallon flowerpots after 40-50 days for seedling character observation.
Preferably, the method for obtaining the homogeneous mutant of the kiwi fruit callus further comprises the step (8) of verifying the homogeneous mutant of the kiwi fruit callus through phenotype observation and molecular markers such as SRAP.
In the step (8), the step (c),
the phenotypic observations are mainly counted by macroscopic phenotypes such as changes in leaf morphology and changes in internodal distances.
The molecular marker is mainly used for preliminarily verifying the generation of variation from the molecular level, and the mutation of radiation metabolism mostly belongs to large-fragment deletion variation of DNA, so that the variation is easily detected by using a molecular marker method.
The primers adopted by the molecular marker are shown in table 1, wherein 10 upstream primers, 10 downstream primers, 1 upstream primer and 1 downstream primer can be combined into a pair of identification primer pairs; the random combination between the upstream primer and the downstream primer can form 100 pairs of identifying primer pairs; the PCR reaction program is: 5min at 95 ℃; 1min at 94 ℃, 1min at 35 ℃, 2min at 72 ℃ and 5 cycles; 1min at 94 ℃, 1min at 55 ℃, 2min at 72 ℃ and 35 cycles; 6min at 72 ℃; the PCR products were separated by electrophoresis on a 3% agarose gel.
Compared with the prior art, the invention has the following advantages and effects: according to the invention, the radiation mutagenesis is carried out on the kiwi fruit callus, the existing kiwi fruit tissue culture system is combined, the homogeneous mutant with stable characters can be obtained, the problems of chimeric variation and somatic cell selection are solved, the homogeneous mutant can be separated only in 5-7 months, the efficiency is improved by 7-8 times, the method is not influenced by seasons, and the method can be carried out in different time periods all year round.
Drawings
FIG. 1 is a view of the 4-division method and the 9-division method of the subculture of the kiwi fruit callus in the embodiment of the invention, wherein A is a view of the 4-division method of the kiwi fruit callus; b is a 9-division view of the calluses of the kiwi fruits, and the part drawing the square root is the selection result of each division;
FIG. 2 is the PCR reaction result of the partial amplification of 11 homogeneous mutants of the callus of Actinidia chinensis planch by molecular marker in the example of the present invention;
FIG. 3 is a graph comparing the results of the leaflet mutants isolated from the homozygote mutants of kiwi callus with the control group in the present example;
FIG. 4 is a graph showing the comparison of phenotype and internode distance between the dwarf mutants isolated from the homogeneous mutants of kiwi callus and the control group in the present example.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
As used herein, the term "room temperature" or "ambient temperature" means a temperature of 4 to 40 ℃, preferably 25. + -. 5 ℃.
The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions. Unless otherwise indicated, percentages and parts are by weight.
The invention provides a method for obtaining a homogeneous mutant of kiwi callus, which comprises the following steps:
(1) subjecting Actinidia chinensis callus to60Co-gamma radiation treatment of said60The radiation dose of Co-gamma radiation treatment is 0-500 Gy;
(2) dividing each irradiated kiwi fruit callus into 4 parts by 4-division method (as shown in figure 1), and respectively placing on subculture medium for primary subculture for 15-20 days;
the subculture medium comprises MS minimal medium, sucrose with the concentration of 32g/L, agar with the concentration of 7.5g/L, 6-benzylaminopurine with the concentration of 0.25mg/L and naphthylacetic acid with the concentration of 1mg/L, wherein the light culture conditions are as follows: 16h/d light and 8h/d dark, the light intensity is 5000 lx.
(3) Dividing the callus of the kiwi fruit after the first subculture into 9 parts by a 9-division method (as shown in figure 1), and reserving only 1 part of the middle part of the left edge of each callus of the kiwi fruit for subculture for 15-20 days;
(4) repeating the step (3) for 3-5 times, and taking 1 part of the middle part of the left edge according to a 9-division method for pre-differentiation culture for 20-30 d;
(5) the pre-differentiation culture medium comprises MS minimal medium, sucrose with the concentration of 32g/L, agar with the concentration of 7.5g/L and zeatin with the concentration of 3mg/L, and the illumination culture conditions are as follows: 16h/d light and 8h/d dark, the light intensity is 5000 lx. After differentiation and sprouting, taking 1 part of each callus with bud points and good growth state according to a 4-division method, transferring the callus into a differentiation culture medium for differentiation culture, and culturing for 15-20 d;
the differentiation culture medium comprises MS minimal medium, sucrose with the concentration of 32g/L, agar with the concentration of 7.5g/L and zeatin with the concentration of 0.5mg/L, and the illumination culture conditions are as follows: 16h/d light and 8h/d dark, the light intensity is 5000 lx.
(6) Repeating the step (5) for 3-5 times to obtain buds with the length of more than 3 cm;
(7) and (4) carrying out twig cuttage on the buds with the length of 3-5cm obtained in the step (6) to obtain the homogeneous mutants of the calluses of the kiwi fruits.
In the step (1), the step (c),
the kiwi callus is obtained by the following method; and (3) placing 2 pieces of filter paper in a culture dish, adding 5-10ml of liquid subculture medium to thoroughly wet the filter paper, placing the pre-cultured callus in the culture dish, and sealing with a sealing film to obtain the kiwi fruit callus.
The pre-cultured callus is obtained by the following method: taking kiwi fruits (in the embodiment of the application, the 'Hongyang' kiwi fruits are adopted, and it can be understood that other kiwi fruit varieties are also suitable for the technical scheme of the embodiment of the application), carrying out callus induction on annual non-lignified branches, and carrying out amplification culture on the branches by using a subculture medium to obtain callus; cutting the callus into 0.4 x 0.4cm small blocks, and placing on a subculture medium for further culture for 7-10 days to obtain pre-cultured callus.
In the step (7), the step (c),
the twig cuttage is carried out by the following method: taking buds with length of 3-5cm, soaking tender bud base with rooting solution for 1-2h, directly cutting in vermiculite (diameter of 0.1-0.3cm), and coating with film for heat preservation.
The rooting solution comprises the following components: a solution containing 3-indolebutyric acid at a concentration of 100mg/L and naphthylacetic acid at a concentration of 50 mg/L. The amount of the rooting solution is just 1cm above the base of the tender shoot each time.
The vermiculite is preferably 0.1-0.3cm in diameter.
After film covering and heat preservation, rooting can be carried out after 10-20 days, film uncovering and seedling hardening can be carried out after 30-40 days, and the seedlings can be transplanted to 3 gallon flowerpots after 40-50 days for seedling character observation.
Preferably, the method for obtaining the homogeneous mutant of the kiwi fruit callus further comprises the step (8) of verifying the homogeneous mutant of the kiwi fruit callus through phenotype observation and molecular markers such as SRAP.
In the step (8), the step (c),
the phenotypic observations are mainly counted by macroscopic phenotypes such as changes in leaf morphology and changes in internodal distances.
The molecular marker is mainly used for preliminarily verifying the generation of variation from the molecular level, and the mutation of radiation metabolism mostly belongs to large-fragment deletion variation of DNA, so that the variation is easily detected by using a molecular marker method.
The primers adopted by the molecular marker are shown in the following table 1, wherein 10 upstream primers, 10 downstream primers, 1 upstream primer and 1 downstream primer can be combined into a pair of identification primer pairs; the random combination between the upstream primer and the downstream primer can form 100 pairs of identifying primer pairs; the PCR reaction program is: 5min at 95 ℃; 1min at 94 ℃, 1min at 35 ℃, 2min at 72 ℃ and 5 cycles; 1min at 94 ℃, 1min at 55 ℃, 2min at 72 ℃ and 35 cycles; 6min at 72 ℃; the PCR products were separated by electrophoresis on a 3% agarose gel.
TABLE 1 SRAP molecular marker primer sequences
FIG. 2 shows the results of the detection of the amplification of partial mutants (11) using molecular markers, in which: lane M, D2000plus marker; lane 1, control kiwifruit; lane 2-12, Kiwi mutant, arrow indicates the position of the cloned specific fragment.
FIG. 3 is a graph comparing the results of the leaflet mutants isolated from the homozygote mutants of kiwi callus with the control group in the present example. As can be seen from FIG. 3, the isolated leaflet mutant has stable shape, the leaves are significantly smaller than the control, and the shape of the leaflet can be stably maintained after many generations.
FIG. 4 is a graph showing the comparison of phenotype and internode distance between the dwarf mutants isolated from the homogeneous mutants of kiwi callus and the control group in the present example. As can be seen from FIG. 4, the dwarf mutant separated has the advantages of obviously reduced plant size, shortened internode distance and stable character.
In the range of 0-500Gy60The results of the effects on the lethality and differentiation rate of the callus of kiwi fruits, which are set in the Co-gamma radiation dose range by 11 radiation treatment doses, are shown in tables 2-1 and 2-2, and each experiment is repeated three times.
As can be seen from tables 2-1 and 2-2, the analysis of the lethality and differentiation rate of the kiwi callus at 11 different radiation doses showed that: the radiation dose is in positive correlation with the lethality, the lethality is gradually increased along with the increase of the radiation dose, the sensitivity of the kiwi fruit callus to radiation is low, the influence of low-dose radiation on the kiwi fruit callus is small, the lethality under 200Gy is 33.3%, and the lethality under 300Gy is 57.5%; the radiation dose and the differentiation rate are in negative correlation, the differentiation rate is gradually reduced along with the increase of the radiation dose, which is probably caused by the increase of harmful mutation, and the differentiation rate is 77.8 percent under 100Gy and 61.1 percent under 200 Gy; combining the lethality rate and the differentiation rate, and taking the radiation dose between 100Gy and 200Gy as the relatively proper mutagenesis dose of the kiwi callus.
And (3) detecting the obtained homogeneous mutant regeneration plant of the kiwi callus by using a molecular marker, and detecting the plant obtained by three experiments if a difference strip appears compared with a wild type control, wherein the induction rate result of the homogeneous mutant regeneration plant of the kiwi callus is shown in table 3.
As can be seen from Table 3, the analysis of the variability of the callus from Actinidia chinensis at 11 different radiation doses showed that: the radiation dose is in positive correlation with the variability, the variability is gradually increased along with the increase of the radiation dose, the variability is 35.9 percent under 100Gy and 78.2 percent under 200Gy, the time variability reaching more than 300Gy reaches 100 percent, but the dose is too high, most of the obtained regenerated seedlings are abnormal seedlings, and the production and utilization values are not large; combining lethality rate, differentiation rate and induction rate, the radiation dose between 100Gy and 200Gy is still the more appropriate mutagenesis dose for the kiwi callus.
According to the invention, the kiwi callus is subjected to radiation mutagenesis, and the existing kiwi tissue culture system is combined, so that a homogeneous mutant with stable characters can be obtained, and the problems of chimeric variation and somatic cell selection are solved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
TABLE 2-160Detection results of lethality and differentiation rate of callus after Co-gamma radiation treatment
Tables 2 to 260Detection results of lethality and differentiation rate of callus after Co-gamma radiation treatment
TABLE 3 detection results of the induction rate of the regeneration plants of the homogeneous mutants of the kiwi callus under different radiation doses
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