CN112243862A - Method for separating and purifying ploidy stable regeneration plant from polyploid sweet potato - Google Patents
Method for separating and purifying ploidy stable regeneration plant from polyploid sweet potato Download PDFInfo
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Abstract
A method for separating and purifying ploidy stable regeneration plants from a polyploid ginseng potato mainly comprises the following steps: 1) stem segment disinfection, 2) callus induction, 3) differentiation culture, 4) rooting culture, 5) root tip chromosome number identification, and 6) biological character and chromosome ploidy stability re-identification. The method has ideal design, and by performing tissue culture on the stem segments of the hybrid (such as tetraploid and hexaploid) sweet potatoes serving as explants, not only can a regenerated plant with stable single ploidy and biological characters be obtained, but also strains with different chromosome ploidy and biological characters can be obtained, so that the hybrid sweet potatoes are used in a breeding plan. But also enriches the germplasm resources of the ginseng and the sweet potato, shortens the breeding time, opens up a new way for breeding the ginseng and the sweet potato, and lays a foundation for the later-stage variety breeding.
Description
Technical Field
The invention relates to a technique for separating and purifying a polyploid plant, in particular to a method for separating and purifying a ploidy stable regeneration plant from polyploid sweet potatoes.
Background
The ginseng potato, also called big potato, is a dioscoreaceae, belongs to perennial vine herbaceous plant, and the tuber of the ginseng potato is rich in starch and crude protein, can be eaten as grain and vegetable, and can be used for preparing starch and medicine. Produced mainly in Zhejiang, Guangdong, Guangxi, Hunan, Hubei, Fujian, Sichuan and Jiangxi provinces in China. Except for eating, the ginseng and sweet potato are medicinal plants, and according to records in Shen nong's herbal Jing and Ben Cao gang mu, the ginseng and sweet potato have the effects of strengthening spleen and nourishing stomach, promoting the production of body fluid and benefiting lung, tonifying kidney and replenishing vital essence, and benefiting brain and beautifying, and are commonly used for resisting aging, treating diabetes, cardiovascular diseases, dyspepsia and the like.
In Fujian, the ginseng and sweet potato are mainly used for dishes and special snacks, the planting history is long, and the development prospect is wide, so that the research on breeding of the ginseng and sweet potato is further developed, and the breeding of a new excellent variety of the ginseng and sweet potato has important significance for promoting the development of the ginseng and sweet potato industry. In 2013, the ploidy identification of the yam cultivated in Fujian was carried out by the project group of Chinese yam of the agricultural academy of Sanming City. The result shows that part of the ginseng potatoes cultivated in Fujian is a polyploid, when the polyploid is planted in the field, the plant leaves, vines and underground tubers of the ginseng potatoes are alternate green and purple, the potato blocks are alternate white and purple, the biological properties are extremely unstable, and a plant line with stable properties cannot be obtained through multiple planting and purification, so that the polyploid ginseng potatoes are difficult to use in a breeding plan.
Disclosure of Invention
The invention provides a method for separating and purifying a ploidy stable regeneration plant from a polyploid ginseng potato, and aims to overcome the defects that the existing method for separating and purifying the ginseng potato is not ideal enough, the biological properties of the obtained strain are extremely unstable, and the strain is difficult to popularize and use as breeding and the like.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for separating and purifying ploidy stable regeneration plants from the polyploid sweet potatoes comprises the following steps: a) stem section disinfection: cutting stem segments with axillary buds of the same polyploid ginseng potato stem and tendril into a strain, washing the stem segments with water, filling the stem segments into a bottle, washing the bottle with flowing tap water for 1 to 2 hours, soaking the dried stem segments with 75 percent alcohol on a super clean workbench for 15 s after being dried, washing the bottle with sterile water for 2 to 4 times, putting the dried stem segments into 0.1 percent mercuric chloride, continuously shaking the stem segments, taking the stem segments out after the stem segments are continuously washed for 8 to 10 min, and washing the stem segments with the sterile water for 5 to 8 times for later use;
b) and (3) callus induction, namely inoculating the disinfected stem segments to an induced callus culture medium for induction treatment, wherein the induced callus culture medium comprises the following components: 1/2MS +2, 4-D0.5 mg/L +6-BA 2.0 mg/L + NAA 0.2 mg/L + coconut milk 70 mL/L;
c) differentiation culture: transferring the well-induced callus (without cutting) to a differentiation culture medium for culture treatment, wherein the differentiation culture medium comprises: 1/2MS +6-BA 2.0 mg/L + NAA 0.1 mg/L;
d) rooting and strengthening seedlings: the callus tissue culture differentiated seedling (no re-proliferation culture is carried out), when the differentiated seedling grows to 3 cm high, the seedling is directly cut off and inoculated into a rooting culture medium for culture treatment, and the rooting culture medium is as follows: 1/2MS +6-BA 0.5 mg/L + NAA 0.5 mg/L + AC 0.5 g/L.
e) And (3) root tip chromosome number identification: selecting seedlings with more than 3 roots, flushing agar by tap water, taking root tips of the seedlings to perform chromosome observation for chromosome number identification treatment, and identifying single ploidy tissue culture seedlings;
f) and (3) re-identifying the biological character and the ploidy stability of the chromosome, namely planting the tissue culture seedling identified to be single ploidy into a matrix, domesticating for three months, transplanting into a field for planting, and identifying the stability of the single plant.
Further, the method of the chromosome number identification process in the step e): A. treating the root tips, namely treating the root tips of the seedlings by a tabletting method to prepare slices; B. microscopic examination: observing the prepared slide under a microscope, selecting cells with dispersed chromosomes for photographing, and counting the number of the chromosomes.
Further, the method for performing stability identification processing on the plants in the step f) comprises the steps of extracting 3 individual plants with different biological properties as test materials, performing propagation, planting and propagating the test materials for years, dividing each test material into 60 blocks, 20 blocks and 1 group, repeating the 3 groups, using the polyploid ginseng potatoes as a control, observing or measuring the biological properties and the root tip chromosome multiple of the test materials, and analyzing the biological properties and the chromosome number stability of filial generations.
Furthermore, 20 g/L of sucrose and 5 g/L of agar are added into the induction callus culture medium, the differentiation culture medium and the rooting culture medium, the pH values are controlled to be 5.6-5.9, the culture environment temperature is controlled to be 23-27 ℃, the illumination intensity is controlled to be 1500-2000lx, and the illumination time is controlled to be 11-13 h/d.
From the above description of the present invention, it can be seen that the advantages of the present invention over the prior art are: the method has ideal design, and by performing tissue culture on the stem segments of the hybrid (such as tetraploid and hexaploid) sweet potatoes serving as explants, not only can a regenerated plant with stable single ploidy and biological characters be obtained, but also strains with different chromosome ploidy and biological characters can be obtained, so that the hybrid sweet potatoes are popularized and used in breeding plans. But also enriches the germplasm resources of the ginseng and the sweet potato, shortens the breeding time, opens up a new way for breeding the ginseng and the sweet potato, and lays a foundation for the later-stage variety breeding.
Drawings
FIG. 1 is a diagram showing the growth effect of the callus differentiated into different colors of regenerated plants.
FIG. 2 is a diagram showing the effect of rooting on the regenerated plant of the present invention.
FIG. 3 is a diagram showing the growth effects of purple, purplish and green new leaves of the regenerated plants after domestication and cultivation.
FIG. 4 is a graph showing the effect of growth on aerial parts of the control.
FIG. 5 is a graph showing the effect of the growth of the aerial parts of the No. 1 regenerated plant of the present invention.
FIG. 6 is a graph showing the effect of growing the aerial parts of the No. 2 regenerated plant of the present invention.
FIG. 7 is a graph showing the effect of growing the aerial parts of the No. 3 regenerated plant of the present invention.
Fig. 8 is a graph showing the effect of the growth of the control potato pieces.
FIG. 9 is a graph showing the effect of potato tuber growth on regenerated plant No. 1 according to the present invention.
FIG. 10 is a graph showing the effect of potato tuber growth on regenerated plant No. 2 of the present invention.
FIG. 11 is a graph showing the effect of potato tuber growth on regenerated plant No. 3 of the present invention.
FIG. 12 is a schematic representation of chromosomes of a control mixloid sweet potato.
FIG. 13 is a schematic diagram of chromosomes of a No. 1 regenerated plant according to the present invention.
FIG. 14 is a schematic diagram of chromosomes of a No. 2 regenerated plant according to the present invention.
FIG. 15 is a schematic diagram of chromosomes of a No. 3 regenerated plant according to the present invention.
Detailed Description
Reference is made to the description accompanying fig. 1-15. A method for separating and purifying ploidy stable regeneration plants from the polyploid sweet potatoes comprises the following steps:
a) stem section disinfection: cutting the same one hybrid ploid ginseng potato stem into 1cm stem segments with axillary buds in the middle ten days of 6 months, washing with water, filling into a bottle, washing with flowing tap water for 1-2 h, soaking on a clean bench with 75% alcohol for 15 s after being dried, washing with sterile water for 2-4 times, adding 0.1% mercuric chloride, shaking continuously for 8-10 min, taking out, and washing with sterile water for 5-8 times for later use;
b) and (3) callus induction, namely inoculating the disinfected stem segments to an induced callus culture medium for induction treatment, wherein the induced callus culture medium comprises the following components: 1/2MS +2, 4-D0.5 mg/L +6-BA 2.0 mg/L + NAA 0.2 mg/L + coconut milk 70 mL/L.
c) Differentiation culture: transferring the well-induced callus (without cutting) to a differentiation culture medium for culture treatment, wherein the differentiation culture medium comprises: 1/2MS +6-BA 2.0 mg/L + NAA 0.1 mg/L. The callus differentiation condition is shown in figure 1 of the specification.
d) Rooting and strengthening seedlings: the callus tissue culture differentiated seedling (no re-proliferation culture is carried out), when the differentiated seedling grows to 3 cm high, the seedling is directly cut off and inoculated into a rooting culture medium for culture treatment, and the rooting culture medium is as follows: 1/2MS +6-BA 0.5 mg/L + NAA 0.5 mg/L + AC 0.5 g/L. The rooting condition is shown in the attached figure 2 of the specification.
e) And (3) root tip chromosome number identification: selecting seedlings with more than 3 roots, flushing agar with tap water, taking root tips of the seedlings to perform chromosome observation for identification treatment, and identifying single ploidy tissue culture seedlings; chromosome observation was performed on the treated (tabletting treated) root tips, and the segregation rate of ploidy was counted, and 20 plants were repeated 3 times. Specifically, the method for identifying the number of chromosomes at the root tip comprises the following steps: A. treating the root tips, namely treating the root tips of the seedlings by a tabletting method to prepare slices; B. microscopic examination: observing the prepared slide under a microscope, selecting cells with dispersed chromosomes for photographing, and counting the number of the chromosomes. At least 20 cells are counted for each root tip during counting, and more than 85% of chromosomes with constant consistency are taken as the chromosome number of the plant.
f) And (3) re-identifying the biological characters and the ploidy stability of the chromosome, namely planting the tissue culture seedling identified to be single ploidy into a substrate (peat soil: sand =2: 1), applying nutrient solution for 1 time every 7d, domesticating for three months, transplanting to a field for planting, and obtaining the growth condition of the domesticated and cultivated regenerated plants shown in the attached figure 3 of the specification; and performing stability verification processing. In addition, agar was washed out from the seedlings with tap water, and the tip of the root was observed for chromosomes, and the rate of separation of the polyploids was counted, and 20 plants were repeated 3 times, as shown in table 1.
As can be seen from table 1, after the tetraploid and hexaploid polyploid sweet potatoes are differentiated and rooted by the callus, the regeneration plants of the single ploid tetraploid and the hexaploid can be obtained, and the obtaining rates are respectively 28.33% and 20.00%. After differentiation, the probability of the occurrence of ploidy-unstable mixploids was 56.67%.
The inducing callus culture medium, the differentiation culture medium and the rooting culture medium are all added with 20 g/L of sucrose and 5 g/L of agar, the pH values are all controlled to be 5.6-5.9 (the optimal value is 5.8), the culture environment temperature is controlled to be 23-27 ℃ (the optimal temperature is 25 ℃), the illumination intensity is controlled to be 1500-2000lx, and the illumination time is controlled to be 11-13 h/d (the optimal temperature is 12 h/d).
Influence of different callus induction medium formulas on callus growth induction
The induced callus culture medium is used as a culture medium No. I; taking an induced callus culture medium with a formula of 2, 4-D0.5 +6-BA 2.0+ NAA 0.2 as a No. 2 culture medium; taking an induced callus culture medium with a formula of 2, 4-D0.5 +6-BA 2.0+ NAA 0.2 mg/L + KT 0.5 as a No. three culture medium; the induced callus culture medium with the formula of 6-BA 2.0+ NAA 0.2+ KT 0.5 is used as culture medium No. IV. The applicant adopts the four different induction callus culture media to perform the following experiments on the influence of the callus growth induction of the stem section, and concretely comprises the following experiment methods: the four different callus induction culture mediums are respectively processed into 20 stem segments which are repeated, 5 stem segments are inoculated in one bottle, the repetition is carried out for 3 times, and the callus induction rate and the callus weight are counted after 45 days. And then transferring the four groups of induced calluses (without cutting) to a differentiation medium in the application for culture treatment, and counting the differentiation rate of the calluses after 45 days. See table 2 for:
the algorithm for callus induction rate in table 2 is as follows: callus induction rate (%) = number of stem segments producing callus/number of stem segments inoculated + 100%; callus differentiation rate (%) = number of differentiated plantlets/number of inoculated calli 100%.
From the table 2, it can be seen that 2,4-D is added to the culture mediums (i.e., culture medium II) and culture medium III, callus is induced in the culture mediums, and no callus is induced in the culture medium II with KT added but no 2,4-D added, which indicates that the KT has no significant influence on the induction of the callus, 2,4-D plays a key role in inducing the callus, the culture medium II with the highest callus induction rate has the callus induction rate of 51.67%. The culture medium II and the culture medium III are added with more coconut milk, the weight and differentiation rate of the callus are obviously higher than those of the culture medium II and the culture medium III, and the weight and differentiation rate of the callus respectively reach 6.06g and 46.67%, so that the coconut milk can effectively promote the growth and development of the callus of the stem. In addition, the culture medium III is added with more KT than the culture medium II, but the difference between the weight of the callus and the differentiation rate of the culture medium II and the culture medium III is not obvious, which indicates that the KT has no obvious influence on the weight of the callus and the differentiation rate.
Second, the situation of character separation and ploidy identification of the regenerated plants
As can be seen from the attached figures 1-3 of the specification, the character separation of the regeneration plant occurs after the hybrid body of the ginseng potato is subjected to tissue culture. As can be seen in the attached figure 1, differentiated seedlings with different color leaves are grown from one callus. After the differentiated seedling is rooted and domesticated, more obvious different appearance characters are generated, for example, as shown in figure 3, the tissue culture seedling is domesticated for three months, and the color of the new leaf is purple, light purple and green.
Thirdly, analyzing the biological characters and ploidy stability of the regeneration plants
Performing stability identification treatment in the step f), wherein the stability identification treatment method comprises the following steps: 3 single plants with different biological characters are extracted as test materials (namely No. 1, No. 2 and No. 3) to be propagated, the test materials are planted and propagated for years, each part is divided into 60 blocks, 20 blocks and 1 group, 3 groups are repeated, and the polyploid ginseng potato is used as a control (namely ck). Observing or measuring the biological characters and root tip chromosome multiples of the test material, and analyzing the biological characters and chromosome number stability of the filial generation. Specific biological traits are observed with emphasis, including: leaf shape, leaf color, leaf vein color, leaf stem two-end color, leaf length, leaf width, potato skin color, and potato pulp color. The observation and recording time of the overground part of the plant is 6 months, and the investigation and recording time of the underground tuber part is 10 months. See the description in detail, figures 4-15 and table 3.
a. As can be seen from Table 3 and the accompanying figures 4-15 in the specification, the number, ploidy and quality of the chromosomes of the offspring are stable and consistent after the regeneration plant is amplified, and the difference of the quantitative characters is not obvious. Therefore, the regeneration plant with single ploidy and stable biological characters can be obtained after the tissue culture of the polyploid Shenshu.
b. As can be seen from Table 3 and the accompanying figures 4-11 of the specification, the biological characters between the regenerated plants are changed compared with the control, for example, the color of the potato pulp is changed into purple when the No. 1 regenerated plant is used; the biological characters of the No. 3 regenerated plant are almost different from the control, the color of leaves and veins is changed into green, the leaf shape is changed into regular triangle, the petiole is changed into light purple, the difference between the leaf length and the leaf width is obvious from the control and the No. 1 and No. 2 regenerated plants, and the color of potato pulp is also changed into white. Therefore, the regeneration plants with different biological characters can be obtained after the tissue culture of the polyploid Shenshu.
c. It can be seen from Table 3 and the accompanying FIGS. 12-15 of the specification that the number of stains significantly changed between individuals as compared with the control, i.e., 40 chromosomes were found in the regenerated plants No. 1 and No. 2, and 60 chromosomes were found in the regenerated plant No. 3. Therefore, the regeneration plants with different chromosome ploidy can be obtained after the tissue culture of the polyploid Shenshu.
In conclusion, by carrying out tissue culture on the polyploid ginseng potato, not only can a regenerated plant with stable single ploidy and biological characters be obtained, but also a plant line with different chromosome ploidy and biological characters can be obtained, so that not only are germplasm resources of the ginseng potato enriched, but also a foundation is laid for later-stage variety breeding.
The above description is only an embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing the present invention.
Claims (4)
1. A method for separating and purifying ploidy stable regeneration plants from the polyploid sweet potatoes is characterized by comprising the following steps:
a) stem section disinfection: cutting stem segments with axillary buds of the same polyploid ginseng potato stem and tendril into a strain, washing the stem segments with water, filling the stem segments into a bottle, washing the bottle with flowing tap water for 1 to 2 hours, soaking the dried stem segments with 75 percent alcohol on a super clean workbench for 15 s after being dried, washing the bottle with sterile water for 2 to 4 times, putting the dried stem segments into 0.1 percent mercuric chloride, continuously shaking the stem segments, taking the stem segments out after the stem segments are continuously washed for 8 to 10 min, and washing the stem segments with the sterile water for 5 to 8 times for later use;
b) and (3) callus induction, namely inoculating the disinfected stem segments to an induced callus culture medium for induction treatment, wherein the induced callus culture medium comprises the following components: 1/2MS +2, 4-D0.5 mg/L +6-BA 2.0 mg/L + NAA 0.2 mg/L + coconut milk 70 mL/L;
c) differentiation culture: transferring the well-induced callus (without cutting) to a differentiation culture medium for culture treatment, wherein the differentiation culture medium comprises: 1/2MS +6-BA 2.0 mg/L + NAA 0.1 mg/L;
d) rooting and strengthening seedlings: when the differentiated plantlets grow to 3 cm high, directly cutting the plantlets and inoculating the plantlets into a rooting culture medium for culture treatment, wherein the rooting culture medium comprises: 1/2MS +6-BA 0.5 mg/L + NAA 0.5 mg/L + AC 0.5 g/L;
e) and (3) root tip chromosome number identification: selecting seedlings with more than 3 roots, flushing agar by tap water, taking root tips of the seedlings to perform chromosome observation for chromosome number identification treatment, and identifying single ploidy tissue culture seedlings;
f) and (3) re-identifying the biological characters and the ploidy stability of the chromosome: and (3) planting the tissue culture seedlings identified to be single ploidy into a matrix, domesticating for three months, transplanting into a field for planting, and performing stability identification treatment on the single plants.
2. The method of claim 1, wherein the polyploidy stable regenerated plant is isolated and purified from a polyploid panax pseudostellaria root, wherein the method comprises the following steps: the method of the chromosome number identification process in the step e): A. treating the root tips, namely treating the root tips of the seedlings by a tabletting method to prepare slices; B. microscopic examination: observing the prepared slide under a microscope, selecting cells with dispersed chromosomes for photographing, and counting the number of the chromosomes.
3. The method of claim 1, wherein the polyploidy stable regenerated plant is isolated and purified from a polyploid panax pseudostellaria root, wherein the method comprises the following steps: and f) extracting 3 single plants with different biological properties as test materials, carrying out propagation, planting and propagating the test materials for years, dividing each part into 60 blocks, 20 blocks and 1 group, repeating the 3 groups, taking the polyploid ginseng potato as a control, observing or measuring the biological properties and the root tip chromosome multiple of the test materials, and analyzing the biological properties of filial generations and the stability of chromosome number.
4. The method of claim 1, wherein the polyploidy stable regenerated plant is isolated and purified from a polyploid panax pseudostellaria root, wherein the method comprises the following steps: the inducing callus culture medium, the differentiation culture medium and the rooting culture medium are all added with 20 g/L of sucrose and 5 g/L of agar, the pH values are all controlled to be 5.6-5.9, the culture environment temperature is controlled to be 23-27 ℃, the illumination intensity is controlled to be 1500-2000lx, and the illumination time is controlled to be 11-13 h/d.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030226180A1 (en) * | 2002-06-03 | 2003-12-04 | Reliance Life Sciences Pvt. Ltd. | Development of a highly efficient in vitro system of micropropagation of solanum viarum |
| CN102388799A (en) * | 2011-07-19 | 2012-03-28 | 河南师范大学 | Method for regenerating dioscorea zingiberensis triploid plant |
| CN104782496A (en) * | 2015-05-02 | 2015-07-22 | 冯文杰 | Establishment method of Dioscorea alata L. tissue culture regeneration system |
| CN107750946A (en) * | 2017-10-18 | 2018-03-06 | 中国科学院亚热带农业生态研究所 | A kind of method cultivated suitable for medicinal plant sweet wormwood and Chinese yam polyploid variety |
-
2020
- 2020-11-09 CN CN202011235970.1A patent/CN112243862A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030226180A1 (en) * | 2002-06-03 | 2003-12-04 | Reliance Life Sciences Pvt. Ltd. | Development of a highly efficient in vitro system of micropropagation of solanum viarum |
| CN102388799A (en) * | 2011-07-19 | 2012-03-28 | 河南师范大学 | Method for regenerating dioscorea zingiberensis triploid plant |
| CN104782496A (en) * | 2015-05-02 | 2015-07-22 | 冯文杰 | Establishment method of Dioscorea alata L. tissue culture regeneration system |
| CN107750946A (en) * | 2017-10-18 | 2018-03-06 | 中国科学院亚热带农业生态研究所 | A kind of method cultivated suitable for medicinal plant sweet wormwood and Chinese yam polyploid variety |
Non-Patent Citations (3)
| Title |
|---|
| 曾德福: ""菊叶薯蓣愈伤组织诱导及毛状根系统的建立"", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 * |
| 李明军等: ""盾叶薯蓣胚乳再生体系的建立及其染色体倍性鉴定"", 《植物学报》 * |
| 王玉珍: "《现代植物组织培养原理及应用技术》", 31 January 2018, 中国原子能出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119666510A (en) * | 2025-02-21 | 2025-03-21 | 江西农业大学 | A method for rapidly obtaining chromosomes from root tips of ginseng potatoes and its application |
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