CN117461558A - Method for inducing diverse population of canna and identification - Google Patents
Method for inducing diverse population of canna and identification Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
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Abstract
The invention provides a method for inducing a diversity population of canna and identification thereof, wherein the ultrasonic wave induced diversity population of canna has obvious influence on the phenotypic characteristics of canna by an ultrasonic wave mutagenesis technology, and the mutation types of canna comprise flowers, wide leaves, high stems and narrow leaves, and the mutation ratios of different mutation types are different: the floral leaf mutation rate is 16.5%, the wide leaf mutation rate is 3.4%, the high stem mutation rate is 4.5%, and the narrow leaf mutation rate is 3.4%.
Description
Technical Field
The invention belongs to the technical field of wild banana breeding, and particularly relates to a method for inducing a wild banana diversity population.
Background
The canna (Musa balbsiana) is used as one of parents of modern cultivated species of bananas, and has the characteristics of strong self-compatibility, easy fruiting and the like. However, the number of genetic groups of the canna is small, the characteristics are single, the diversity of the canna is limited, and the available canna parent resources are relatively deficient. The research aims at constructing a wild banana ultrasonic mutagenesis technology by using an ultrasonic mutagenesis method, creating a wild banana mutant library with a larger scale so as to enrich available germplasm resources and carrying out preliminary identification on the obtained mutant germplasm materials. The research aims at solving the problems of insufficient diversity of the wild banana, scarcity of genetic groups and the like, and provides powerful support for multi-generation group selection, stable inheritance mutant screening, banana genetic breeding and functional genome research in the future.
Disclosure of Invention
In view of the above, the present invention proposes a method for identifying a diverse population of musa indiana, solving the above-mentioned problems.
The technical scheme of the invention is realized as follows:
the method for inducing the diversity population of the canna is ultrasonic induction.
Further, the method for inducing the diverse population of the canna comprises the following steps:
(1) Collecting the canna seed: picking canna when the canna is ripe, crushing pulp, putting the crushed pulp into a triangular flask containing water, shaking the triangular flask on a shaking table for 1-2 days, putting seeds on gauze for rubbing, and collecting the canna seeds;
(2) The seed of Canada is placed in a plastic test tube with the capacity of 15ml, 15ml of water is added, and ultrasonic induction is carried out.
Further, the frequency of the ultrasonic induction is 38-42KHz.
Further, the temperature of the ultrasonic induction is 22-32 ℃.
Further, the ultrasonic induction time is 1-14min.
Further, the frequency of the ultrasonic induction is 40KHz.
Further, the temperature of the ultrasonic induction is 28 ℃.
Further, the time of ultrasonic induction is 9min.
Compared with the prior art, the invention has the beneficial effects that:
the germination rate of the canna is up to 33.3% by the mutagenesis method. The ploidy level of the wild banana mutagenesis pool remained unchanged, and both wild-type and mutant wild bananas exhibited diploid chromosome numbers. Ultrasonic mutagenesis techniques have a significant impact on the phenotypic characteristics of canna, resulting in considerable phenotypic diversity. The observed mutation types include flowers, leaves, stems and leaves, and the mutation ratios of different mutation types are different: the floral leaf mutation rate is 16.5%, the wide leaf mutation rate is 3.4%, the high stem mutation rate is 4.5%, and the narrow leaf mutation rate is 3.4%.
Secondly, the ultrasonic mutation Mutant (MT) has obvious difference with the wild banana variety (WT) in field photosynthetic efficiency, and the difference is mainly represented by Photo, trmmol, cond, ci and other parameters. In addition, the intercellular carbon dioxide concentration, the transpiration rate, the leaf chamber water content and the leaf chamber relative humidity are positively correlated with the stomatal conductance, the leaf chamber water content is positively correlated with the transpiration rate, the leaf chamber carbon dioxide concentration is positively correlated with the reference chamber carbon dioxide concentration, and the reference chamber relative humidity and the leaf chamber relative humidity are negatively correlated with the air temperature and the leaf temperature.
The construction of the wild banana mutant library provides a foundation and a platform for future genetic improvement and gene function research. Through preliminary identification and screening of mutants, excellent mutants with stable genetic characters can be obtained, and powerful support is provided for genetic improvement of bananas. In addition, the functional genome research of the mutant is helpful to reveal the molecular mechanism and regulation network of important agronomic characters of bananas, and provides a new theoretical basis for further gene function analysis and variety improvement.
Drawings
FIG. 1 ploidy analysis of wild-type canna (WT) and mutant canna (MT)
FIG. 2 analysis of significance of growth investigation of mutant canna (MT) and wild-type canna (WT)
FIG. 3 mutant canna (MT) is of different mutation types, A is of flower She Bianyi type; b is a broadleaf variation type; c is the high-pole variation type; d is the variation type of narrow leaf
FIG. 4 statistical plot of percent (%) of different variant types of mutant canna (MT)
FIG. 5 wild-type and mutant canna (WT) stomata observations, A represents the wild-type canna stomata observed under the conditions of an eyepiece magnification of 16 times and an objective magnification of 10 times, and B represents the mutant canna stomata observed under the conditions of an eyepiece magnification of 16 times and an objective magnification of 10 times; c represents the air holes of wild-type canna observed under the conditions of 16 times magnification of an eyepiece and 40 times magnification of an objective lens, and D represents the air holes of mutant canna observed under the conditions of 16 times magnification of an eyepiece and 40 times magnification of an objective lens
FIG. 6 mutant canna (MT) and wild canna (WT) field photosynthetic efficiency Principal Component Analysis (PCA)
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The invention will be further illustrated with reference to specific examples.
The plant material of the invention is derived from banana resources and wild banana (M.balbsiana) seed resources collected by a breeding subject group of a seaport experiment station of the national academy of tropical agriculture.
Experimental reagent: PI ploidy kit (purchased from Hainan and Instrument Co., ltd.), 1% gentian violet staining solution, carnot's fixative, pure alcohol, glacial acetic acid and other relevant chemical reagents are all purchased from Hainan sandisk trade Co., ltd.
Experimental instrument:
proper nouns and unit notes:
example 1
(1) Collecting the canna seed: picking canna when the canna is ripe, crushing pulp, putting the crushed pulp into a triangular flask containing water, putting the triangular flask on a shaking table to shake for 2 days, putting seeds on gauze to rub, and collecting the canna seeds;
(2) The seed of Canada is placed in a plastic test tube with the capacity of 15ml, 15ml of water is added, and ultrasonic induction is carried out.
(3) The frequency of the ultrasonic wave is fixed at 40KHz. The temperatures were set at 32 ℃, 28 ℃ and 22 ℃ and ultrasonic treatments were performed, respectively. The ultrasonic treatment time was set to 1 minute, 3 minutes, 6 minutes, 9 minutes, 12 minutes and 14 minutes, respectively. Three replicates were set for each treatment, 20 seeds per treatment.
Test example:
1. ploidy analysis
Performing ploidy analysis on mutant canna in a mutagenesis library by using a flow cytometer, selecting 100 cannas in the mutagenesis library for ploidy analysis, firstly preparing RNase A stock solution, adding 1.5 ml of distilled water into a test tube containing RNase, shaking uniformly, and storing the RNase in a dyeing solution at-20 ℃; secondly, preparing a dye solution (10 samples are satisfied in the following preparation work), adding 120ul of propidium chloride and 60ul of RNaseA stock solution into 20ml of dyeing buffer solution, stabilizing the freshly prepared dyeing solution for 24 hours, and storing in a refrigerator at 4 ℃ in a dark place; finally, preparing a sample and detecting on-machine, wherein the whole process of the sample preparation needs to wear gloves. The sample preparation steps are as follows:
(1) The blade is used to select about 0.5cm from the plants to be tested 2 Is placed in a plastic culture dish;
(2) Adding 500ul of cell nucleus lysate to the culture dish on the plant material;
(3) Vertically cutting the sample with a sharp blade for 30-60 seconds (blade replacement is required after 5-10 samples);
(4) Cutting the sample, filtering the sample with a 50um cell block filter, discarding filter residues after the filtration, and retaining the filtrate;
(5) Pouring the filtrate into a sample tube, adding 1500ul of prepared dye liquor, uniformly mixing, and then placing the mixture in a condition without illumination for incubation for 30 minutes (25 ℃);
the flow cytometer is used as follows:
(1) Firstly, debugging an instrument, checking the instrument before starting up, timely supplementing sheath fluid, checking whether waste liquid is poured out or not, and logging in a detection system after starting up;
(2) Detecting and analyzing by a machine, firstly pouring with distilled water, and clicking Prime to perform pouring;
(3) Clicking a required graph after filling, setting required parameters in Settings, clicking a start key in an acquisition to start analysis after loading a sample;
(4) Clicking to save the picture after the analysis is completed, dragging the picture according to Reports, clicking to save the Export in the menu in pdf format;
(5) After the pictures are stored, maintaining the instrument, clicking a Clean cleaning instrument, closing the detection system after cleaning, and then closing the device, and pouring out the waste liquid in the waste liquid bottle after closing the device.
2. Phenotypic observation
For phenotypic observations we selected mutant canna (Mutants) and Wild-type canna (Wild type) in the mutagenesis pool. By using tape measure we measured the leaf length (leaf length), leaf width (leaf width) and Petiole length (Petiole length) of the third leaf of the plant. In addition, we also recorded the plant height (High) of the plants and measured the Diameter (Diameter) of the plants using vernier calipers.
3. Pore change identification
To observe the pore variation of the mutant and wild-type musa in the mutagenesis pool, we used the following experimental procedure. The measurement time was fixed at 11:00 am. First, a punch was used to sample the right side of the middle of the veins, and the obtained leaf was quickly placed in a prepared canola fixation solution (pure alcohol: glacial acetic acid=3:1). Then, the lower epidermis of the leaf was carefully torn off with forceps and spread out over the slide. Next, 1-2 drops of 1% gentian violet dye solution were added dropwise to the leaves and allowed to dye for 1-2 minutes. After the staining was completed, the coverslip was slowly covered and excess staining solution was aspirated using filter paper. In this way, we can place the sample under a microscope for observation and microscopy.
4. Determination of metrics such as net photosynthetic rate
Selecting a mutant type wild banana and a wild type wild banana from a wild banana mutagenesis library, measuring indexes such as net photosynthetic rate (Photo), air pore conductance (Cond), intercellular carbon dioxide concentration (Ci), transpiration rate (Trmmol) and the like of the mutant type wild banana and the wild type wild banana by using a Li-6400 portable photosynthetic measurement system, wherein the measured leaf is selected from the leaf surface in the middle of the right side of the leaf vein of the third leaf of the plant, and the measuring time is fixed to 15 pm on sunny days: 00.
the operation steps are as follows:
(1) Firstly, starting up a portable photosynthetic measurement system, selecting 2x3LED.xml, and entering a main interface after confirmation; (2) Establishing a folder, and then inputting a custom file name;
(3) Setting the flow rate, blowing air to the leaf chamber under the condition of closing the leaf chamber, observing whether the carbon dioxide content in the leaf chamber is rapidly increased, and indicating that the measuring system has no air leakage if the carbon dioxide content in the leaf chamber is not increased;
(4) Turning on the lamp of the leaf chamber for preheating for 10 minutes;
(5) When CO 2 R and CO 2 S is equal and then CO is carried out 2 R and CO 2 S matching;
(6) After preheating, opening a leaf chamber to clamp the leaf surface of the middle part of the right side of the leaf vein of the third leaf of the plant to be measured, and recording data after stabilizing;
(7) After all plants to be measured are measured, data are stored, and then file transmission is carried out after lamps of leaf chambers are turned off; (8) And after the file is transmitted, the portable photosynthetic measurement system is powered off.
5. Data arrangement and analysis
The study used a variety of specialized software including SigmaPlat 14.0, IBM SPSS Statistics 25.0.25.0, origin 2021, excel 2019, etc., to comprehensively process and analyze the resulting data. Aiming at different analysis purposes and statistical requirements, the following methods and indexes are adopted for data arrangement and analysis. Data sort and calculation were performed using SigmaPlot 14.0 software and a visual chart was made. To examine the significance level and correlation of the relevant data, we used the t-test and danken's new complex polar difference method (DMRT method).
8. Experimental results
Table 1 analysis of the significance of germination rates for different treatments
Note that: the letter A represents a temperature treatment of 32 ℃, B represents a temperature treatment of 28 ℃, C represents a temperature treatment of 22 ℃, the numbers at the back of the letter represent the corresponding ultrasonic treatment time, the significant level is 0.05, and the Fisher LSD analysis method is adopted
According to the experimental results in table 1, it was found that the average germination rate of the canna seed reached significantly 60% when the a12 treatment conditions were employed. In contrast, the average germination rate of the canna seed under the B9 treatment condition is the lowest, which is only 33.3%. The mutagenesis rate and degree of variation was higher under the conditions up to B9 treatment.
Referring to FIG. 1, the abscissa represents fluorescence intensity, the ordinate represents cell number, and the fluorescence intensity reflects intracellular DNA content, and the results of the present invention indicate that the ultrasonic mutagenesis technique does not cause the change of the ploidy of the canna.
TABLE 2 growth investigation results of mutant canna (MT) and wild-type canna (WT)
Referring to Table 2 in combination with FIGS. 2-4, the mutant cannas generally have a higher plant height than the wild-type cannas, up to 6.65cm and a minimum of 4.55cm, whereas the wild-type cannas has a highest plant height of 4.32cm and a minimum of 2.53cm. There was a very significant difference in plant height between the mutant and wild type cannas, while there was no significant difference between leaf width, leaf stalk length and diameter. In addition, there was also a significant difference in leaf length between the mutant and wild-type cannas. Obvious variation of the phenotype of the mutant canna is found by observing mutant canna in a mutant library, and the phenotype of the mutant canna is mainly represented by four types of floral leaf type, wide leaf type, high stem type and narrow leaf type (figure 3). The variation rate of the flower leaf type is as high as 16.5%, the variation rate of the wide leaf type is 3.4%, the variation rate of the high-stalk type is 4.5%, and the variation rate of the narrow leaf type is 3.4%. The percentage of normal plants was 72.2% (fig. 4).
Referring to fig. 5, the ultrasonic mutagenesis technique had a significant effect on the stomatal characteristics of the canna plants. The action of the ultrasonic wave can promote the relaxation of cell walls and the expansion of cell gaps, so that the increase of the number of air holes and the alignment of the air holes are caused, the wild banana plants subjected to the ultrasonic wave mutagenesis treatment show more orderly and rich air hole alignment, and the number of the air holes is obviously increased compared with that of wild type wild bananas. In addition, ultrasonic mutagenesis also causes morphological changes in the guard cells of the canna, characterized by a short and broad profile, in contrast to the long and narrow guard cell morphology of wild-type canna.
TABLE 3 mutant canna (MT) and wild-type canna (WT) photosynthetic data
Referring to table 3 in conjunction with the analysis of fig. 6, field photosynthetic efficiency analysis was performed for wild banana variety (WT) and ultrasonic mutagenesis Mutant (MT) and various photosynthetic parameters including Ci, trmmol, cond and Photo were determined in the present invention. The PCA (principal component analysis) results showed that PC1 interpreted 62.6% of the total variance and PC2 interpreted 32.8% of the total variance. In the scatter plot of PCA results, the 95% elliptical confidence region for MT is minimal, comprising 10 MTs and 7WT samples. While the 95% elliptical confidence area of the WT is largest, comprising 10 MTs and 10 WT samples. In the first quadrant, the main loading is Photo (0.15,0.75), trmmol (0.62,0.15) and Cond (0.62,0.11), which means that these parameters are distinguished to a higher degree for the first quadrant, possibly with a difference between MT and WT. In the fourth quadrant, the main loading has Ci (3.5, -0.6), which indicates that Ci parameters have higher weights in the fourth quadrant, possibly with a significant difference between MT and WT. Ultrasound has the potential to trigger biochemical reactions inside and outside cells as a physical stimulus. The action of ultrasound may stimulate physiological and biochemical reactions within plant cells through its energy transfer and vibration effects. These reactions can lead to changes in pore conductance, affecting intercellular carbon dioxide concentration (Ci), transpiration rate (Trmmol) and leaf chamber water content (H 2 OS). In addition, the ultrasonic waves can influence the carbon dioxide (CO) in the reference chamber by adjusting the opening and closing degree of the air holes of the plants 2 R) and leaf chamber carbon dioxide (CO) 2 S) concentration difference. While the negative correlation between the reference chamber relative humidity (RH-R) and the leaf chamber relative humidity (RH-S) is related to the ultrasound induced temperature and the change in leaf temperature.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (7)
1. A method for inducing a diversity population of canna, which is characterized in that the method for inducing the diversity population of canna is ultrasonic induction.
2. The method of inducing a diverse population of canna as claimed in claim 1, wherein the frequency of ultrasonic induction is 38-42KHz.
3. The method of inducing a diverse population of canna as claimed in claim 1, wherein the temperature of ultrasonic induction is between 22-32 ℃.
4. The method of inducing a diverse population of canna as claimed in claim 1, wherein the time of ultrasonic induction is between 1 and 14 minutes.
5. The method of inducing a diverse population of canna as claimed in claim 2, wherein the frequency of ultrasonic induction is 40KHz.
6. The method of inducing a diverse population of canna of claim 1, wherein the temperature of ultrasonic induction is 28 ℃.
7. The method of inducing a diverse population of canna as claimed in claim 1, wherein the time of ultrasonic induction is 9 minutes.
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| CN102239799A (en) * | 2011-05-05 | 2011-11-16 | 中国农业科学院作物科学研究所 | Method for obtaining plant mutants |
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