CN113632728A - Watermelon polyploid induction and identification method - Google Patents
Watermelon polyploid induction and identification method Download PDFInfo
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Abstract
The invention relates to a watermelon polyploid induction and identification method, which comprises the following steps: (1) polyploid induction; (2) and performing morphological identification; (3) and flow cytometry identification. The method utilizes a mutagen colchicine to treat watermelon cotyledons so as to obtain higher polyploidy induction rate of the watermelon and provide technical support for creating watermelon germplasm resources; meanwhile, the method for ploidy identification of the watermelon is improved, and a basis are provided for the next large-scale high-efficiency identification.
Description
Technical Field
The invention belongs to the technical field of agriculture, relates to a watermelon cultivation method, and particularly relates to a watermelon polyploid induction and identification method.
Background
Watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) belongs to Cucurbitaceae (Cucurbitaceae), is native to tropical grassland in southern Africa, and is an important melon and fruit crop with sweet and juicy flesh. The conventional production and seedling raising of the watermelon is realized through seed propagation, but the conventional genetic breeding is time-consuming and labor-consuming due to the narrow genetic basis and the relative lack of germplasm resources of the watermelon, so that a plurality of important excellent characters cannot be improved through a conventional breeding method.
Therefore, the adoption of biotechnology means to carry out germplasm resource innovation and the cultivation of new varieties with high quality, high yield and strong resistance become research hotspots of watermelon genetic breeding. Among them, the in vitro culture technique has become an important component of biotechnology.
In the isolated culture of the watermelon, polyploid induction and identification are very important. However, the existing polyploid induction method has certain defects, so that the germination rate and the polyploid induction rate are not high, and the requirement of the existing watermelon in vitro culture is difficult to meet.
In conventional experiments, chromosome identification is generally used for identifying watermelon polyploids. However, the chromosome identification method is time-consuming and labor-consuming, is not suitable for large-scale batch identification, and is difficult to meet the requirement of large-scale cultivation of the watermelon.
In view of the above technical defects in the prior art, there is an urgent need to develop a watermelon polyploid induction and identification method.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a watermelon polyploid induction and identification method, which utilizes a mutagen colchicine to treat watermelon cotyledons so as to obtain higher polyploid induction rate of watermelons and provide technical support for creating watermelon germplasm resources; meanwhile, the method for ploidy identification of the watermelon is improved, and a basis are provided for the next large-scale high-efficiency identification.
In order to achieve the above purpose, the invention provides the following technical scheme:
the watermelon polyploid induction and identification method is characterized by comprising the following steps:
(1) polyploid induction: selecting complete watermelon cotyledon as material, soaking in 0.06-0.08% colchicine solution for 3 days; after soaking, shaking and washing the soaked seeds for 5 times on a super-clean workbench by using sterile water, and then inoculating the seeds to an induction culture medium;
(2) and morphological identification: carrying out first-generation enrichment culture on the induced adventitious buds to obtain watermelon plants, carrying out morphological identification on the watermelon plants, and preliminarily screening polyploid plants;
(3) flow cytometry identification: selecting 0.2g of true leaves of the polyploid plants screened primarily, placing the true leaves in a culture dish, adding 0.5mL of LB01 dissociation solution, immersing the true leaves in the dissociation solution and cutting the true leaves with a blade, so that the cut true leaves absorb the dissociation solution in the culture dish to enable cell nucleuses in the true leaves to enter the dissociation solution; filtering with 400 mesh filter membrane after 10min, placing the filtrate into a centrifuge tube and into a 4 deg.C refrigerator, incubating for 5min, centrifuging for 5min, discarding the supernatant, adding 70 μ L LB01 dissociation solution and 130 μ L dye, placing into a 4 deg.C refrigerator, keeping out of the sun, dyeing for 10min, transferring to flow cytometer for detection, and identifying polyploidy by flow cytometer observation.
Preferably, in the step (1), the shaking table is placed on the shaking table for shaking during soaking, and the temperature during soaking is 28 ℃ and the rotation speed of the shaking table is 100 r/min.
Preferably, in the step (1), the induction medium is MS medium, and 0.5mg/L of 6-BA and 0.05mg/L of TDZ are added.
Preferably, in the step (3), the rotation speed during centrifugation is 1000r/min, and the temperature is 5 ℃.
Preferably, in the step (3), the dye is propidium iodide.
Preferably, in the step (3), the LB01 dissociation solution contains 15mmol/L Tris and 2mmol/L Na2EDTA, 0.5mmol/L spermine tetrahydrate, 80mmol/L KCl, 20mmol/L NaCl, 0.1% volume fraction TritonX-100 and 15mmol/L beta-mercaptoethanol, and the pH value is 7.0-8.0.
Compared with the prior art, the watermelon polyploid induction and identification method has the following beneficial technical effects:
1. the watermelon cotyledon is treated by the mutagen colchicine, and the higher polyploidy inductivity and survival rate of the watermelon can be obtained through proper concentration and treatment time, so that technical support is provided for creating watermelon germplasm resources.
2. The method for identifying the ploidy of the watermelon is improved, and the identification of the polyploid of the watermelon can be quickly and accurately realized by proper treatment and a flow cytometry identification method, so that a basis and a basis are provided for the next large-scale efficient identification.
Drawings
FIG. 1 is a flow chart of the method for inducing and identifying watermelon polyploidy of the present invention.
FIG. 2 is a diagram showing the results of flow cytometry ploidy analysis of diploid and tetraploid watermelon leaves. Wherein 2n is diploid, 4n is tetraploid.
FIG. 3 is a comparison of diploid and tetraploid watermelon regenerated seedlings. Wherein, the left picture is diploid, and the right picture is tetraploid.
Detailed Description
The present invention is further described with reference to the following drawings and examples, which are not intended to limit the scope of the present invention.
The invention provides an in vitro culture method of watermelon, which utilizes mutagen colchicine to treat cotyledon of the watermelon so as to obtain higher polyploidy inductivity of the watermelon and provide technical support for creating germplasm resources of the watermelon; meanwhile, the method for ploidy identification of the watermelon is improved, and a basis are provided for the next large-scale high-efficiency identification.
In the invention, 2 small fruit type watermelon inbred lines of W-19-30W-19-66 are selected as watermelon varieties for experimental verification. These small fruit watermelon varieties are provided by vegetable breeding houses at the horticultural research institute of the academy of agricultural sciences, Anhui province. And the test material in the test is the complete cotyledon of the sterile seedling with the seedling age of 3-4 d.
FIG. 1 shows a flow chart of the method for inducing and identifying watermelon polyploidy of the invention. As shown in figure 1, the watermelon polyploid induction and identification method of the invention comprises the following steps:
first, polyploidy induction
The whole cotyledons of sterile seedlings with the seedling age of 3-4 d of two small fruit watermelons are selected as materials, are respectively soaked in colchicine (the concentration, namely the mass fractions are respectively 0.02%, 0.04%, 0.06% and 0.08%) solutions, and are placed on a shaking table to be shaken (the temperature is 28 ℃, the rotating speed is 100r/min) for treatment time (1d, 2d, 3d and 4 d). After soaking, shaking and washing with sterile water (temperature 28 deg.C, rotation speed 100r/min) on a clean bench for 5 times, inoculating to induction culture medium MS +0.5 mg/L6-BA (6-benzylaminopurine) +0.05mg/L TDZ (thidiazuron), and repeating for 1 time and 3 times when 20 whole cotyledons are treated. And after 25 days, counting the survival rate. The induced adventitious bud is subjected to first generation propagation culture for ploidy identification.
Survival (%) (number of surviving whole cotyledons/number of inoculated whole cotyledons) × 100 two, morphological identification
Carrying out first-generation enrichment culture on the induced adventitious bud to obtain a watermelon plant, carrying out morphological identification on the watermelon plant, and preliminarily screening out a polyploid plant.
Wherein, the diploid watermelon tissue culture seedlings and the polyploid watermelon tissue culture seedlings have larger difference in morphology. The polyploid plant is mainly characterized by slow growth, dark green leaf color, thick leaf and other obvious variations, and the variant plant is screened out by taking the external morphological characteristics as the standard, so that the initially screened polyploid plant can be obtained.
And thirdly, identifying by flow cytometry.
Selecting 0.2g of true leaves of the polyploid plants which are primarily screened out, placing the true leaves in 2 precooled culture dishes, and respectively adding 0.5mL of precooled dissociation liquid of LB01 and mGb to screen proper dissociation liquid. The leaves are immersed in the dissociation liquid and rapidly chopped at one time with a blade (i.e., without repeated chopping, the leaves are chopped once). And (4) sucking the dissociation solution in the culture dish after cutting, so that the cell nucleuses in the true leaf blades enter the dissociation solution. After 10min, the mixture was filtered through a 400-mesh filter membrane into a 1.5mL centrifuge tube, which was placed in a 4 ℃ refrigerator, incubated for 5min, centrifuged for 5min (1000 r/min, temperature: 5 ℃), the supernatant was discarded, and then 70. mu.L of the pre-cooled dissociation solution and 130. mu.L of the pre-cooled dye propidium iodide were added, for a total of about 200. mu.L each. Then placing in a refrigerator at 4 ℃, keeping out of the light and dyeing for 10 min. Moving to a flow cytometer for detection.
Mutagenesis rate (%) (polyploid plant number/treated explant number) x 100
Wherein, the LB01 dissociation liquid is: 15mmol/L Tris, 2mmol/L Na2EDTA, 0.5mmol/L spermine tetrahydrate, 80mmol/L KCl, 20mmol/L NaCl, TritonX-100 with the volume fraction of 0.1 percent, 15mmol/L beta-mercaptoethanol and the pH value of 7.0-8.0.
mGbThe dissociation liquid is: 45mmol/L MgCl220mmol/L MOPS, 30mmol/L sodium citrate, 10g/L PVP-40, 0.2% volume fraction Triton X-100, 10mmol/L Na2EDTA, 20. mu.L/mL beta-mercaptoethanol, pH 7.0.
The results obtained by the above polyploid induction and flow cytometry identification tests are shown in table 1 below.
TABLE 1 polyploidy induction and identification result table for small-fruited watermelon
As can be seen from Table 1, the overall survival rate of the W-19-30 of the small-fruit watermelon inbred line W-19-30 is higher than that of the W-19-66 under the same treatment conditions. Except that the two small-fruit watermelons are treated for 1 day at the concentration of 0.02 percent and 0.04 percent of colchicine, polyploid plants can be induced under other treatment time and concentration. Meanwhile, the survival rate and the mutagenesis rate of the two varieties in a certain range are positively correlated with the concentration of the mutagen and the treatment time, but when the concentration reaches 0.08% and the treatment time is too long, the survival rate and the mutagenesis rate begin to show a descending trend, which may be that the explants are poisoned by colchicine. The optimal treatment concentration of the mutagenic agent induced by polyploidy of the inbred line numbered W-19-30 can be obtained by integrating the survival rate and the mutagenesis rate, and the treatment time is 3 d. The optimum treatment concentration of the mutagenic agent for polyploidy induction of the inbred line numbered W-19-66 was 0.06% for a treatment time of 3 days.
According to the research, the morphological comparison between the polyploid watermelon and the diploid watermelon is shown, the leaves are thick, the leaf color is dark, and the stem nodes are shortened. As can be seen from the comparison of FIG. 3, the mutant plants after colchicine treatment are significantly different from the diploid plants without treatment. It can be identified as polyploid.
40 watermelon regeneration plants which accord with the variation characteristics are screened out through morphology, leaves of the watermelon regeneration plants are taken as materials, ploidy identification is carried out on the plants by using a flow cytometer, and as a result, 34 watermelon regeneration plants are found to be tetraploid plants, 4 watermelon regeneration plants are found to be heteroploid plants, and 2 watermelon regeneration plants are found to be diploid plants. As can be seen from FIG. 2, the diploid plant has a peak at a position with a relative fluorescence intensity of about 30X 104, and the mutagenized plant has a peak at a position with a relative fluorescence intensity of about 60X 104, and the ratio thereof is about 2, which indicates that the mutagenized plant is a tetraploid plant, and the coincidence rate of the two polyploid identification methods is 85%.
It is difficult to obtain a complete and large number of nuclei by merely cutting the explant by physical means such as cutting, and therefore, a dissociation solution suitable for the genus of this family is also required. The dissociation of LB01 was used in this experimentLiquid and mGbThe dissociation liquid is repeatedly tested under the same condition, and the result shows that mG is usedbAfter the dissociation liquid, a large number of fragment peak values appear in the identification result image, and the ploidy of the plant cannot be judged, but an obvious peak value image can be obtained by using the LB01 dissociation liquid. Therefore, the LB01 dissociation solution is more suitable for the identification of watermelon flow cytometry, mGbThe dissociation liquid is not suitable for the identification of watermelon flow cytometry.
Therefore, under the same conditions, the inductivity of the small-fruit watermelons of different varieties is different, wherein W-19-30 is higher than W-19-66. The colchicine solution is soaked for 1 day, the concentration of the solution is 0.02 percent and 0.04 percent, polyploidy cannot be induced, and the survival rate and the mutagenesis rate in a certain range are increased along with the increase of the colchicine solution. When the treatment time is 4 days when the concentration reaches 0.08%, the survival rate and the mutagenesis rate obviously start to decline. The optimum treatment concentration of colchicine in the inbred line W-19-30 is 0.08%, the treatment time is 3d, the mutagenesis rate reaches 25.00%, the optimum treatment concentration of mutagen in the inbred line W-19-66 is 0.06%, the treatment time is 3d, the mutagenesis rate is 25.00%
Meanwhile, the ploidy identification is carried out on the watermelon regenerated seedlings by using a flow cytometer after the morphological initial screening of the regenerated plants, the result coincidence rate of the two identification methods reaches 85 percent, and a basis is provided for the early large-scale identification of the regenerated seedlings. Meanwhile, the application of the two dissociation liquids in the flow cytometry is compared, and the LB01 dissociation liquid is more suitable for being applied to ploidy identification of watermelon regeneration plants.
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.
Claims (6)
1. The watermelon polyploid induction and identification method is characterized by comprising the following steps:
(1) polyploid induction: selecting complete watermelon cotyledon as material, soaking in 0.06-0.08% colchicine solution for 3 days; after soaking, shaking and washing the soaked seeds for 5 times on a super-clean workbench by using sterile water, and then inoculating the seeds to an induction culture medium;
(2) and morphological identification: carrying out first-generation enrichment culture on the induced adventitious buds to obtain watermelon plants, carrying out morphological identification on the watermelon plants, and preliminarily screening polyploid plants;
(3) flow cytometry identification: selecting 0.2g of true leaves of the polyploid plants screened primarily, placing the true leaves in a culture dish, adding 0.5mL of LB01 dissociation solution, immersing the true leaves in the dissociation solution and cutting the true leaves with a blade, so that the cut true leaves absorb the dissociation solution in the culture dish to enable cell nucleuses in the true leaves to enter the dissociation solution; filtering with 400 mesh filter membrane after 10min, placing the filtrate into a centrifuge tube and into a 4 deg.C refrigerator, incubating for 5min, centrifuging for 5min, discarding the supernatant, adding 70 μ L LB01 dissociation solution and 130 μ L dye, placing into a 4 deg.C refrigerator, keeping out of the sun, dyeing for 10min, transferring to flow cytometer for detection, and identifying polyploidy by flow cytometer observation.
2. The method for inducing and identifying the watermelon polyploidy as claimed in claim 1, wherein in step (1), the watermelon is soaked in water by shaking on a shaking table at a temperature of 28 ℃ and a rotation speed of the shaking table of 100 r/min.
3. The method for inducing and identifying the watermelon polyploidy as claimed in claim 2, wherein in the step (1), the induction medium is MS medium, and 0.5mg/L of 6-BA and 0.05mg/L of TDZ are added.
4. The method for inducing and identifying the watermelon polyploidy as claimed in claim 3, wherein in the step (3), the rotation speed during centrifugation is 1000r/min and the temperature is 5 ℃.
5. The method for inducing and identifying watermelon polyploidy as claimed in claim 4, wherein in step (3), said dye is propidium iodide.
6. The method for inducing and identifying the watermelon polyploidy as claimed in claim 5, wherein in the step (3), the LB01 dissociation solution comprises 15mmol/L Tris, 2mmol/L Na2EDTA, 0.5mmol/L spermine tetrahydrate, 80mmol/L KCl, 20mmol/L NaCl, 0.1% volume fraction TritonX-100 and 15mmol/L beta-mercaptoethanol, and the pH value is 7.0-8.0.
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|---|---|---|---|---|
| US20030217394A1 (en) * | 2002-05-14 | 2003-11-20 | Xingping Zhang | Inbred tetraploid watermelon line 90-4194 |
| CN1806524A (en) * | 2006-02-24 | 2006-07-26 | 浙江大学 | Method for in-vitro inducing watermelon tetraploid by utilizing colchicine |
| CN1994065A (en) * | 2006-12-22 | 2007-07-11 | 西北农林科技大学 | Method for improving seed selection of watermelon polyploid |
| CN109122309A (en) * | 2017-06-27 | 2019-01-04 | 湖南博达隆生物科技有限公司 | A kind of breeding method of watermelon |
-
2021
- 2021-07-22 CN CN202110828318.9A patent/CN113632728A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030217394A1 (en) * | 2002-05-14 | 2003-11-20 | Xingping Zhang | Inbred tetraploid watermelon line 90-4194 |
| CN1806524A (en) * | 2006-02-24 | 2006-07-26 | 浙江大学 | Method for in-vitro inducing watermelon tetraploid by utilizing colchicine |
| CN1994065A (en) * | 2006-12-22 | 2007-07-11 | 西北农林科技大学 | Method for improving seed selection of watermelon polyploid |
| CN109122309A (en) * | 2017-06-27 | 2019-01-04 | 湖南博达隆生物科技有限公司 | A kind of breeding method of watermelon |
Non-Patent Citations (1)
| Title |
|---|
| 高优洋: "小果型西瓜离体再生体系建立及多倍体诱导研究", 《道客巴巴网》 * |
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