CN116746485B - Induction method and application of aronia pestilence tetraploid - Google Patents
Induction method and application of aronia pestilence tetraploid Download PDFInfo
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses a method for inducing a tetraploid of a Sorbus pohuashanensis and application thereof. The method comprises the following steps: 1) Layering Sorbus pohuashanensis seeds at 0-5deg.C for 14-28 days; 2) Soaking and inducing the low-temperature laminated Sorbus pohuashanensis seeds in the colchicine solution with the mass percentage concentration of 0.2% for 48 hours; and then, continuously carrying out low-temperature lamination at 0-5 ℃ to ensure that the total low-temperature lamination days reach 28 days, 3) sowing the seeds treated in the step 2), and screening the tetraploid seedlings of the Sorbus pohuashanensis in the seedling stage. The tetraploid induction rate of the method can reach 24.75%, and the seed source and the induction of polyploid mountain ash tree are not influenced. The length and width of the tetraploid Sorbus pohuashanensis air holes and the quantity of chloroplasts in the air holes are obviously improved, the plant height is reduced, the plant ground diameter is increased, and the widths of the top and middle small leaves are increased.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for inducing a tetraploid of a Sorbus pohuashanensis and application thereof.
Background
The Sorbus plant (SorbusL.) belongs to the Malvaceae (Rosaceae) and is mainly deciduous trees or shrubs, the Sorbus plant is naturally distributed in Asia, europe, north America and other areas of northern hemisphere, about 258 types of Sorbus (Phipps J B,1988.A checklist of the subfamily Maloideae (Rosoceae) [ EB/OL ] (1988-08-22) [2022-09-08 ]), hybridization, polyploidy and apomixis phenomena are more common in the Sorbus, the condition ,Nelson-Jones(Nelson-Jones E,Briggs D,Smith A,2002.The origin of intermediate species of the genus Sorbus[J/OL].Theoretical and Applied Genetics,105(6):953-963.) that the coexistence of more ploidy forms in the genus is generated is found, and the conditions that 2×,3×, 4× ploidy ;Pellier(Pellicer J,Clermont S,Houston L,et al.,2012.Cytotype diversity in the Sorbus complex(Rosaceae)in Britain:sorting out the puzzle[J/OL].Annals of Botany,110(6):1185-1193) exist in the Sorbus of Europe are proved in the research of the ploidy of Sorbus sample of the Sorbus, and the conditions that the ploidy coexistence exists in part of the population; in the study of the native Sorbus plants in China, the genome size of the Aronia melanocarpa ((also called as "Lianlian et al)") proves that the Aronia melanocarpa (S.villmorini) is tetraploid plant ;Li(Li J,Zhu K,Wang Q,et al.,2021.Genome size variation and karyotype diversity in eight taxa of Sorbus sensustricto(Rosaceae)from China[J/OL].Comparative Cytogenetics,15(2):137-148), and two tetraploid Sorbus plants distributed in China, namely S.files and S.ovalis, are found in 8 natural taxonomic groups. At present, the polyploidy research in the Sorbus is mainly concentrated in the Sorbus plant classification, but no report related to polyploid breeding is found.
The Aronia melanocarpa (s. Pohuashanensis (Hance) hedl.) is a known diploid tree species of the genus Aronia, mainly distributed in northwest, north China and northeast China, and the distribution range spans 2 climatic zones of warm temperate zones (Zheng Jian, 2008. Evaluation, preservation and utilization of genetic resources of the Aronia melanocarpa [ D/OL ]. National institute of forestry science [2022-09-10 ]). The natural resources of the Sorbus pohuashanensis are intensively distributed in the altitude of 1200-2000m, and the Sorbus pohuashanensis has extremely high ornamental value due to colorful autumn leaves and fruits, so that the Sorbus pohuashanensis is favored by urban landscaping; in addition, the fruits are rich in flavonoid compounds, which is beneficial to heart protection, so that the fruit has a very high medicinal prospect. However, the Sorbus pohuashanensis is restricted by abiotic stress such as high temperature, drought, weak alkaline soil and the like in summer in low-altitude domestication, and the ornamental value of the Sorbus pohuashanensis is affected by (Zhao D,Qi X,Zhang Y,et al.,2022.Genome-wide analysis of the heat shock transcription factor gene family in Sorbus pohuashanensis(Hance)Hedl identifies potential candidates for resistance to abiotic stresses[J/OL].Plant Physiology and Biochemistry,175:68-80), of leaf sunburn (sunburn) in summer; in addition, the natural variation range in the Sorbus pohuashanensis is limited, meanwhile, the breeding improvement work is less and the means are single (Gu Yanpeng, zhang Zeren, sun Tao, etc. 2022. Hybrid identification and genetic relationship analysis [ J ] of Sorbus pohuashanensis and less She Huaqiu hybrid F_1 generation group based on EST-SSR markers, 31 (4): 65-73), the intra-species variation is required to be increased by other breeding means, the ornamental value is improved while the resistance of the Sorbus pohuashanensis is enhanced, and a wide variation basis is provided for the breeding of new varieties.
Polyploids are cells or organisms possessing more than two sets of intact chromosomes, typically a ploidy change in the chromosome set caused by mitotic or meiotic errors, which occur widely in angiosperms. The event of multiple or whole genome replication (WGD) is an important impetus for plant evolution. After chromosome doubling, it often shows trait variation far exceeding that of non-doubled individuals, such as faster growth rate in polyploid plants; larger organs; the setting rate is increased, and the yield is improved; reinforcement of plant resistance, and the like. The improvement of the forest is always an important work of the genetic research of the forest, the induction of polyploidy is helpful for the generation of new species and promotes the development of plant diversity, so that the example of ploidy breeding as a germplasm innovation means is not rare, such as the use of wood species, the heterogenic triploid poplar species has faster growth speed and higher growth quantity; in economic tree species, the polyploidization brings higher fruit yield and increases the contents of substances such as total sugar, soluble solids and the like in the fruits; and the method can also utilize ploidy breeding to select and breed excellent multipurpose tree species with strong adaptability, quick growth and large biomass. In addition, a plurality of researches prove that the triploid forest has the growth advantage and excellent character of far super diploid, and the induction and cultivation of tetraploid plants are initiated, so that not only is germplasm innovated, but also the possibility is provided for the creation of triploid varieties.
After successful induction of stramonium tetraploid with colchicine in 1937 Blakesle, scholars began to explore artificially induced polyploids. The method for artificially inducing polyploidy can be divided into physical induction means (such as mechanical injury, extreme temperature, rays and the like) and chemical induction (such as colchicine, huang Caoxiao, trifluralin, rylene and the like) according to treatment means, for example, physical means are adopted in poplar to induce the plant to successfully double pear test-tube plantlet by means of extreme high temperature or low temperature, gamma ray irradiation is utilized, and polyploidy plant can be obtained after subculture. At present, most polyploid induction is obtained by treating plant (in vitro) organs (seeds, terminal buds, seedlings or explants) with chemical agents, so exploration of factors related to different treatment agents, chemical agent concentrations, polyploid induction effects and the like is one of the key points of polyploid research. In contrast to many woodvarieties, which have been artificially induced to obtain polyploid plants and to search for factors influencing induction and optimal induction methods, studies on polyploid induction methods in Sorbus pohuashanensis have not been reported yet, and ploidy breeding is still in a blank stage.
Disclosure of Invention
The invention aims to provide a method for mutagenesis of Sorbus pohuashanensis seeds by adopting colchicine, and the method is used for artificially inducing and cultivating Sorbus pohuashanensis tetraploid, so as to lay a foundation for research on Sorbus pohuashanensis artificial homologous polyploid and provide new germplasm resources for selection of Sorbus pohuashanensis stress-resistant high-quality new varieties.
The specific technical scheme of the invention is as follows:
An induction method of a tetraploid of a Sorbus pohuashanensis comprises the following steps:
1) Layering Sorbus pohuashanensis seeds at 0-5deg.C (specifically 4 deg.C) for 14-28 days;
2) Soaking and inducing the low-temperature laminated Sorbus pohuashanensis seeds in the colchicine solution with the mass percentage concentration of 0.2% for 48 hours; then, continuously laminating at 0-5 ℃ to ensure that the total number of days of low-temperature lamination reaches 28 days;
3) Sowing the seeds treated in the step 2), and screening the tetraploid seedlings of the Sorbus pohuashanensis in the seedling stage.
In one embodiment of the invention, the Sorbus pohuashanensis seeds are treated with 1g/L gibberellin (GA 3) solution for 24 hours prior to low temperature stratification.
In yet another embodiment of the present invention, the method of screening the tetraploid seedlings of Sorbus pohuashanensis is by chromosome ploidy analysis of the seedlings using a flow cytometer.
The application of the method in obtaining the Sorbus pohuashanensis plants with obviously improved length, width and chloroplast quantity in the air holes also belongs to the protection scope of the invention.
The application of the method in obtaining the Sorbus pohuashanensis plants with reduced plant height and increased plant ground diameter also belongs to the protection scope of the invention.
The application of the method in obtaining the mountain ash tree plant with the increased top and middle small leaf width also belongs to the protection scope of the invention.
The invention has the following beneficial effects:
The method has the advantages that the aronia melanocarpa autotetraploid is successfully induced by treating the aronia melanocarpa seeds by using colchicine, the colchicine solution concentration is found to be a key factor influencing the successful induction of polyploid aronia melanocarpa, the colchicine induction effect is better at the concentration of 0.2 percent for 48 hours, the tetraploid induction rate can reach 24.75 percent, and the seed source has no influence on the induction of polyploid aronia melanocarpa. Through morphological observation, correlation and principal component analysis for two years, the length and width of the tetraploid Sorbus pohuashanensis air holes and the quantity of chloroplasts in the air holes are obviously improved, the plant height is reduced, the plant ground diameter is increased, and the widths of the top and middle small leaves are increased.
Drawings
FIG. 1 is a flow cytometry detection result; and (3) injection: a. a diploid plant; b. tetraploid plants; c. mixed ploid plants; d. diploid and tetraploid plants.
FIG. 2 is a graph comparing diploid and tetraploid plants. And (3) injection: a. comparing the diploid with the tetraploid plant single plant, wherein the scale is 15cm; b. diploid versus tetraploid plant populations on a scale of 25cm.
FIG. 3 shows the comparison of tetraploid with diploid plant leaves, notes: panels a and b are tetraploid plant leaves, panels c and d are diploid plant leaves, scale 2cm.
FIG. 4 is a major component analysis of morphological traits of tetraploid and diploid plants. And (3) injection: the percentage of total variance explained for each principal component is shown in brackets. Red lines and red dots represent diploids; blue lines and blue dots represent tetraploids; the green arrows represent different shape variables.
FIG. 5 is a box plot of tetraploid and diploid plant traits; different colors represent different morphological traits; squares in the middle of the box represent the data mean.
FIG. 6 is a view of the stomata of tetraploid and diploid plants;
note that: a. diploid leaf pores, b. tetraploid leaf pores, scale 50 μm.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions of the embodiments of the present invention are clearly and completely described, and it is apparent that the embodiments described below are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Induction method of Sorbus pohuashanensis tetraploid of the present invention condition optimization and Effect identification
1 Test materials
The induction tests of the Sorbus pohuashanensis seeds are respectively carried out in 12 months in 2019, 1 month in 2020 and 11 months in 2021, the plant materials used for doubling treatment are wild Sorbus pohuashanensis seeds (TS 2018) collected in 2018 and domesticated and cultivated Sorbus pohuashanensis seeds (ZY 2021) collected in 2021 respectively, and the plant materials are stored in a refrigerator at 4 ℃ after being purified and dried. The Sorbus pohuashanensis seeds have dormancy characteristics, so that pure and full seeds are selected before the test, treated by 1g/L gibberellin (GA 3) solution for 24 hours, and then put into a refrigerator with the temperature of 4 ℃ for low-temperature lamination, so that the early-stage preparation work is completed.
2 Test design and results
2.1 Effect of colchicine concentration and treatment time on seed germination
In 12 months 2019, TS2018 seeds treated by low-temperature lamination treatment 28d are used as test materials, 0.01%, 0.05%, 0.1% and 0.2% colchicine solutions are respectively adopted for soaking treatment, the soaking time is respectively 12 hours, 24 hours and 48 hours, the soaking treatment with clear water is used as a comparison, and 150 seeds are treated each time and are repeated for 3 times. The treated seeds are sowed in a seedling raising tray (length: width: height: 54:28.5:4 cm), and the matrix is prepared by mixing turf and vermiculite (3:1 volume ratio). The germination percentage was counted at 20d after sowing, and the germination percentage% = normal germination seed number/sowing number x 100%.
In order to explore the optimal treatment concentration and treatment time of colchicine, an influence test of colchicine on the germination of the Sorbus pohuashanensis seeds is carried out, and the result shows that the germination rate of the Sorbus pohuashanensis seeds is not obvious from the comparison difference between the treatment concentration of 0.01% and the treatment concentration of 0.05%, so that the germination of the Sorbus pohuashanensis seeds is not influenced by the colchicine with lower concentration; however, the germination rate of the Sorbus pohuashanensis seeds (33.51% and 28.98% respectively) was significantly reduced under the treatment of 0.1% and 0.2% colchicine, and the difference between the 2 treatment concentrations was not significant, which indicates that the germination of Sorbus pohuashanensis seeds was affected by the 0.1% and 0.2% colchicine concentrations (Table 1). The germination rate of seeds gradually decreased with the increase of colchicine treatment time, and the germination rate could still reach 29.33% after 48h of treatment, and the germination rates were very significantly different under 3 treatments (table 1). The study also found no interaction between colchicine concentration and treatment time (table 2). The analysis shows that under the condition of high-concentration long-term treatment time, the Sorbus pohuashanensis seeds still can obtain ideal germination rate, so that colchicine can be considered to exert the strongest effect under the treatment condition, and therefore, in order to ensure that considerable polyploid plants are obtained, in the subsequent Sorbus pohuashanensis tetraploid plant induction test, a treatment method of soaking the seeds for 48 hours by using colchicine solution with concentration of 0.1% and 0.2% is selected.
TABLE 1 Sorbus pohuashanensis seed germination Rate at different colchicine concentrations and treatment times
Note that: mean ± Standard Error (SE). Lowercase letters represent the significance of the Duncan's multiplex assay (p < 0.05), and different letters represent the presence of significant differences from one another.
TABLE 2 analysis of variance of seed germination rates of Sorbus pohuashanensis under different colchicine-induced conditions
| Variant source | df | MS | F | P-value |
| Processing time | 2 | 1167.485 | 80.528 | 0.000* |
| Treatment concentration | 4 | 336.601 | 23.217 | 0.000* |
| Treatment time x treatment concentration | 8 | 10.111 | 0.697 | 0.691 |
| Error of | 30 | 14.498 | ||
| Totals to | 45 |
Note that: df: degree of freedom; MS: mean square; sig. Significance; * Representing significance reaching p <0.05 levels.
2.2 Effect of colchicine concentration and Low temperature stratification time on the Induction Rate
To further explore the effects of low temperature stratification time and colchicine concentration on induction of aronia tetraploid, TS2018 seeds were treated at different concentrations (0.1% and 0.2%) and different low temperature stratification times (0 d, 14d, 21d and 28 d) in 2020 and 2021 respectively and tested for plant ploidy levels using FCM, wherein ploidy levels of aronia seedlings in 2020 were tested for 2 consecutive years. In month 1 of 2020, TS2018 seeds are selected as materials, taken out respectively when the low-temperature lamination time is 0d, 14d, 21d and 28d, soaked in colchicine solution with concentration of 0.1% and 0.2% for 48 hours, and treated 100 seeds with clear water as a control group are repeated for 3 times. After the treatment is finished, cleaning the residual solution with clear water, continuously placing the solution into a refrigerator with the temperature of 4 ℃ for low-temperature lamination, uniformly taking out the solution after the total low-temperature lamination days reach 28d, sowing the solution in a seedling tray, and mixing and configuring the matrix of turf and vermiculite (3:1 volume ratio). And (3) carrying out germination rate statistics on the 20 th day after sowing, transplanting the seedlings into a flowerpot (caliber: bottom diameter: height 25.5cm:22cm:29 cm) for 4 months, and uniformly managing water and fertilizer.
In 2021, 11 months, based on the experiment result in 2020, in order to verify the feasibility of the inducing method of the tetraploid of the Sorbus pohuashanensis, selecting TS2018 and ZY2021 seeds, and repeating the above experiment treatment process.
The polyploid mountain ash tree identification method comprises the following steps: to increase the screening efficiency, 5 seedlings are first used as a group, each of which is mixed with a part of leaves (with the size of about 1cm multiplied by 1 cm), the mixed sample is placed in a culture dish with the diameter of 90mm, 1.5mL of lysate (Kiwifruitbuffer) is added, the sample is cut up by the cutter, the cut-up is filtered by a 30 mu m nylon net (CELLTRICS mu m disposable filters), the filtrate is collected in a 3.5mL glass sample tube (sample tube), and 100 mu LDAPI (50 mu g/mL) of solution is added to the tube for dyeing, and then the sample is subjected to flow cytometry [Ploidy analyser (SysmexPartec, goerlitz, germany) ] for chromosome ploidy analysis (fig. 1). If obvious tetraploid peaks exist in the mixed sample, the ploidy level of 5 individuals in the group is further detected one by one, and finally the chromosome ploidy of each individual is determined. At least 2000 nuclei were counted per sample. Based on ploidy analysis, tetraploid induction rate, mixed ploidy induction rate and induction percentage were calculated. The plants induced in 2020 were twice ploidy levels measured in 2021 and 2022 respectively, and the ploidy levels were measured in 2022 for 2021-induced plants.
Tetraploid induction%times tetraploid plant number%times plant total number x 100%; the% of mixed ploidy induction =the number of mixed ploidy plants/(the total number of plants × 100%; percent induction = (number of tetraploid plants + number of mixed ploid plants)/(total number of plants x 100%; tetraploid induction efficiency% = survival (%) x tetraploid induction (%).
Statistical analysis: the significance and correlation were analyzed using SPSS 21.0 (SPSS Inc, chicago, USA) statistical analysis software. The data is subjected to a normalization test and a variance alignment test before analysis, and the percentage data is subjected to an arcsine conversion, wherein the formula is as follows: (Θ, angle, P, percent) significance between different treatment combinations was analyzed using t-test, kruskal-WALLIS TEST test, analysis of variance (ANOVA) and Duncan (Duncan) multiplex test. Correlation between phenotypic traits was analyzed using Pearson correlation coefficients. Arcsine conversion and tabulation were performed using microsoft excel 2019 (microsoft corp., washington.DC, USA). Principal component analysis was performed using Origin2022 (Origin Lab, northampton, USA).
Polyploid mountain ash tree induction and identification results thereof find that when the colchicine is lower than Wen Cengji d before treatment, the survival rate, the induction rate and the induction efficiency are higher than those of the colchicine which is laminated for 14d, but all indexes of the two time periods are not obviously different (table 3 and table 4), and the seed which is not laminated is directly subjected to colchicine induction treatment and then laminated for 28d treatment, the tetraploid induction rate and the tetraploid induction efficiency are respectively 0 and are obviously lower than the corresponding values of the colchicine which is laminated for 14d and 21 days before treatment (table 3). The survival rate, induction rate and induction efficiency of the seedlings of Sorbus pohuashanensis exhibited significant differences between the 2 colchicine treatment concentrations, wherein the index was higher than that of the treatment of 0.1% at the treatment concentration of 0.2% (Table 5), and the analysis of variance (Table 4) showed that the colchicine concentration significantly affected the induction effect of polyploid Sorbus pohuashanensis (Table 5). In conclusion, the dormancy characteristics influence the induction effect of colchicine on the polyploid of the Sorbus pohuashanensis, during the layering treatment, but the low temperature layering is needed for 14-28 days before the colchicine treatment, in the interval, the layering days do not have obvious influence on the induction of the tetraploid of the Sorbus pohuashanensis, the concentration is a key influence factor, the effect of the colchicine induction for 48 hours under the concentration of 0.2 percent is better, the tetraploid induction rate can reach 24.75 percent, and finally, 3-year-old tetraploid Sorbus pohuashanensis 25 plants and 2-year-old tetraploid Sorbus pohuashanensis 52 plants (comprising 24 plants TS2018 and 28 plants ZY 2021) are obtained.
TABLE 3 influence of days of low-temperature stratification on Sorbus pohuashanensis seed Induction results (colchicine concentration 0.1 and 0.2 data analysis of the two data sets were taken separately)
Note that: mean ± Standard Error (SE). Lowercase letters represent the significance of the Duncan's multiplex assay between each set of data.
TABLE 4 analysis of variance of the results of low-temperature stratification days and colchicine concentration effects on TS2018 Sorbus seed induction in different years
Note that: df: degree of freedom; MS: mean square; sig. Significance; * Representing significance reaching p <0.05 levels.
TABLE 5 influence of colchicine concentration on the results of Sorbus pohuashanensis seed induction (first stratification 28 Natural followed by colchicine induction)
Note that: mean ± Standard Error (SE). Lowercase letters represent the significance of the Duncan's multiplex assay between each set of data.
Subsequently, we further compare and analyze the difference in seed induction effect of colchicine between the natural seed source of the Sorbus pohuashanensis (TS 2018) and the domesticated cultivar source (ZY 2021), and found that the seed of the natural seed source of the Sorbus pohuashanensis has a higher germination rate (p < 0.05) than the domesticated cultivar source under colchicine solution treatment, but unfortunately, the tetraploid induction rate is not improved and the tetraploid induction rate is not significantly different between the two seed sources (Table 6); further analysis also shows that the mountain ash seeds of the natural seed source and the domesticated cultivar source have higher tetraploid induction rate under the treatment of 0.2% colchicine. Furthermore, analysis of variance found that there was no obvious interaction between colchicine treatment concentration and seed source (table 7), and that colchicine concentration at 0.1% and 0.2% had no significant effect on germination, which was consistent with previous experimental results of colchicine affecting seed germination (table 1).
TABLE 6 influence of different colchicine concentrations on the percent induction and germination of Sorbus pohuashanensis polyploids
Note that: mean ± Standard Error (SE). Lowercase letters represent the significance of the Duncan's multiplex test (p < 0.05) between each column of data, and different letters represent the presence of significant differences from one another.
TABLE 7 analysis of variance of the induced results of Sorbus pohuashanensis seeds affected by different seed sources and treatment concentrations
Note that: df: degree of freedom; MS: mean square; sig. Significance; * Representing significance reaching p <0.05 levels.
Example 2 characterization of induced tetraploid Sorbus pohuashanensis
1. Morphological feature observations
Phenotypic character observation is carried out on the aronia pestis tetraploid plants induced in 2020 in 2021 and 2022 respectively, and the leaf sunburn percentage is observed in 2022 and 8 respectively, and diploid plants are used as a control. Leaf phenotypic traits include: the complex leaf length (Compoundleaflength, CLL), complex leaf width (Compound leafwidth, CLW), complex leaf stem length (Commonpetiolelength, CPL), small She Jianju (DBL) Distancebetweenleaflets, tip leaflet length (ALL) APICALLEAFLETLENGTH, tip leaflet width (ALW) APICALLEAFLETWIDTH, tip small She Shexing index (ALSI) ApicalleafletShapeIndex, the calculation formula is: alsi=all/ALW, middle Leaflet Length (MLL) MIDDLELEAFLETLENGTH, middle Leaflet Width (MLW) MIDDLELEAFLETWIDTH, middle small She Shexing index (MLSI) MIDDLELEAFLETSHAPE INDEX, calculated as: mlsi=mll/MLW, petiole middle Diameter (DMP) Diameterof middlepetiole, plant morphology traits including Plant Height (PH) PLANTHEIGHT, ground diameter (BD) Basaldiameter, annual growth (ABL) Annualbranchlength, leaf "sunburn" Percentage (PS) PERCENTAGE OFSUNBURN, calculated as: percent "sunset" (PS) =number of sunset leaves/(total leaves x 100%).
We observed the phenotype of 25 tetraploid plants obtained by induction in 2020 and 20 diploid plants randomly sampled, and found that the tetraploid plants were lower in plant height than the diploid plants (Table 8, FIG. 2), and the difference between the leaves of Sorbus pohuashanensis (FIG. 3) was found. The results showed that the tetraploid Plant Height (PH) was significantly lower than that of the diploid plants in the two consecutive years of observation, while the Annual Branch Length (ABL) of the tetraploid plants was observed significantly shorter than that of the diploid individuals in the second year (table 8), indicating that the slow growth of new branches was one of the reasons for the difference in tetraploid plant height. Furthermore, we noted that tetraploid plants were observed with ground diameters (BD) greater than diploids in the first year, but the difference between the two ploidy was not significant; the ground diameter observations in the second year were similar to those in the previous year, while tetraploid plants BD were larger, and analysis of variance showed a very significant difference between them (table 8), in addition to the plant growth differences, we found that top leaflet width (ALW), middle Leaflet Width (MLW), top small She Shexing index (ALSI) and middle small She Shexing index (MLSI) all exhibited very significant differences between 2 ploidy, with tetraploid ALW and MLW being wider than diploid plants, and tetraploid ALSI and MLSI being smaller than diploid individuals (table 8).
In addition, we increased the index of the middle Diameter (DMP) and sunburn Percentage (PS) of the leaf stalks in 2022 observation (Table 8), found that the leaves of the aronia pestis tetraploid showed higher PS in addition to thicker DMP, and all showed very significant differences from diploid leaves, indicating that the fold change had a certain effect on plant resistance in addition to changing plant organ size. In conclusion, the aronia pestis tetraploid and diploid plants are considered to show obvious morphological differences in plant height, ground diameter, annual branch growth, top and middle small leaf width, top and middle small She Shexing index and leaf stalk middle diameter, and meanwhile, the aronia pestis ploidy change is found to influence leaf 'sunburn' resistance.
Further combining with correlation analysis, it was found that plant ploidy exhibited a significant negative correlation with Plant Height (PH), annual Branch Length (ABL), top end small She Shexing index (ALSI) and middle small She Shexing index (MLSI); has a significant positive correlation with ground diameter (BD), tip leaflet width (ALW), middle Leaflet Width (MLW), petiole middle Diameter (DMP), and Percent Sunscald (PS) (Table 9). Phenotypes with correlation coefficients greater than 0.6 were MLSI, DMP, ALSI, ALW and MLW (0.865, 0.777, 0.729, 0.643, 0.605, respectively). Taken together, it was shown that the leaf trait of Sorbus pohuashanensis was more closely related to the polyploid, and that the major phenotypic variation of Sorbus pohuashanensis was concentrated in the leaf trait (Table 9).
Then, the principal component analysis is carried out on the observed data in 2022, and as can be known from fig. 4, scattered points representing plant individuals are mutually gathered in groups, and obvious separation exists among the groups, so that the plant ploidy difference can be well shown from morphological characters; taking whether the score exceeds 0.6 as an index for distinguishing the strength of contribution of the trait in the principal component, we find that the trait with the score exceeding 0.6 in the first principal component is: small She Jianju (DBL) (0.882), complex Leaf Length (CLL) (0.869), complex Leaf Width (CLW) (0.791), middle Leaflet Length (MLL) (0.784), middle Leaflet Width (MLW) (0.741), tip leaflet length (ALL) (0.728), complex leaf stem length (CPL) (0.664), tip leaflet width (ALL) (0.651); the second principal component has a score of greater than 0.6: middle small She Shexing index (MLSI) (0.915), tip small She Shexing index (ALSI) (0.891), petiole middle Diameter (DMP) (0.759), tip leaflet width (ALW) (0.675), middle Leaflet Width (MLW) (0.613) (table 10, fig. 4). In combination with correlation analysis, the traits that are highly correlated with ploidy (ALW, MLW, ALSI, MLSI and DMP) were found to be concentrated predominantly in the second principal component, and we also noted that the middle leaflet trait was found to be higher in both principal components 1 and 2 than in the apical leaflet (except for the leaflet width in the second principal component). The distribution of each character variable is shown by a green arrow in fig. 4, wherein ALW, MLW, DMP is highly consistent with the distribution direction of tetraploid scattered points; ALSI, MLSI are distributed in the same direction as diploid scatter. This is consistent with the correlation analysis and analysis of variance results previously described. In summary, it can be primarily thought that the tip, middle leaflet width, leaf shape index and petiole middle diameter can be used as morphological features for distinguishing tetraploids from diploids, wherein the relationship between middle leaflet trait variation and ploidy is tighter.
Fig. 5 is a box plot produced by normalizing the numerical values of the morphological properties. The box line graph reflects the distribution characteristics of each group of data, and the situation that the data distribution of ALSI, MLSI and DMP is extremely concentrated and almost no data intersection exists between tetraploid plants and diploid plants in the data distribution range is found; WAL and WML times, data distribution was relatively concentrated with few data intersections between different ploidy plants across the data range (fig. 5). The above results can explain the strong correlation between ALSI, MLSI and DMP and ploidy in the correlation analysis, and also agree with the results that the distribution direction of diploid scatter in the principal component analysis is highly similar to ALSI, MLSI directions (fig. 4); in contrast, the plant height, ground diameter and annual growth showed a large number of overlapping data between the two ploidy values, which are reflected in the correlation values (table 9), and the result of the smaller correlation coefficient between ploidy values was shown.
TABLE 8 analysis of variance of morphological traits of Sorbus pohuashanensis tetraploid and diploid plants
Note that: mean ± Standard Error (SE) × represents significance reaching p <0.01 level.
TABLE 10 analysis of the morphological Properties of tetraploid and diploid plants
2. Air holes on the lower epidermis of the leaf of the tetraploid plant of the Sorbus pohuashanensis
To further explore the cytological differences between the tetraploid and diploid of Sorbus pohuashanensis, we observed statistics of the subcuticular stomata characteristics of tetraploid and diploid plants. Randomly selecting 5 tetraploid plants and diploid plants from the plants with determined ploidy, selecting 5 healthy and flat leaves from each individual, peeling off epidermis from the back of the leaves along the veins, placing the epidermis on a glass slide with clear water, and tabletting for observation and statistics. The leaf stomata and guard cells were observed and photographed under a microscope (ZeissAxio scope. A1, germany), 2 fields were randomly observed per leaf, 5 stomata were randomly selected in each field to photograph the length, width and chloroplast number in the guard cells, and the number of stomata under a 40 x objective was recorded and the stomata density was calculated.
As a result, it was found that the number of chloroplasts in the stomata length, width and air holes of the tetraploid plant was significantly higher than that of the diploid plant, and the stomata density per unit area was significantly lower than that of the diploid plant, indicating that the tetraploid plant would possess larger stomata, smaller stomata density and greater chloroplast number (Table 11 and FIG. 6).
TABLE 11 analysis of variance of the pore length, density and chloroplast number of the leaf epidermis of the Aronia melanocarpa tetraploid and diploid plant
| Traits (3) | Diploid | Tetraploid |
| Air hole length (mum) | 28.53±0.41 | 37.10±0.46** |
| Air hole width (mum) | 19.56±0.27 | 24.44±0.31** |
| Density of air holes (number/mm 2) | 156.37±4.22** | 105.27±2.38 |
| Quantity of chloroplasts | 18.44±0.25 | 29.73±0.45** |
Note that: mean ± Standard Error (SE) × represents significance reaching p <0.01 level.
In conclusion, the aronia melanocarpa autotetraploid is successfully induced by using colchicine to treat the aronia melanocarpa seeds, the colchicine solution concentration is found to be a key factor influencing the successful induction of polyploid aronia melanocarpa, the colchicine is treated after 14-28 days of low-temperature lamination, the colchicine induction effect is better after 48 hours under the concentration of 0.2 percent, the tetraploid induction rate can reach 24.75 percent, and the seed source has no influence on the induction of polyploid aronia melanocarpa. Through morphological observation, correlation and principal component analysis for two years, the length and width of the tetraploid Sorbus pohuashanensis air holes and the quantity of chloroplasts in the air holes are obviously improved, the plant height is reduced, the plant ground diameter is increased, and the width of the top and middle small leaves is increased.
In summary, the above embodiments are only for illustrating the technical solution of the present invention, and are not limited thereto; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. An induction method of a tetraploid of a Sorbus pohuashanensis comprises the following steps:
1) Layering Sorbus pohuashanensis seeds at 0-5deg.C for 14-28 days;
2) Soaking the low-temperature laminated Sorbus pohuashanensis seeds in colchicine solution with the mass percentage concentration of 0.2% in the step 1) for induction for 48 hours, and then continuously laminating at the low temperature of 0-5 ℃ to ensure that the total low-temperature lamination days reach 28 days;
3) Sowing the seeds treated in the step 2), and screening the tetraploid seedlings of the Sorbus pohuashanensis in the seedling stage.
2. The method of claim 1, wherein the chokeberry seeds are treated with 1g/L gibberellin GA 3 solution for 24 hours prior to low temperature stratification.
3. The method of claim 1, wherein the method of screening the tetraploid seedlings of Sorbus pohuashanensis comprises chromosome ploidy analysis of the seedlings using a flow cytometer.
4. Use of the method according to any one of claims 1-3 for obtaining a plant of the Sorbus pohuashanensis having a significantly increased length, width and number of chloroplasts in the stomata.
5. Use of the method according to any one of claims 1-3 for obtaining a Sorbus pohuashanensis plant with reduced plant height and increased plant ground diameter.
6. Use of the method according to any one of claims 1-3 for obtaining a plant of the Sorbus pohuashanensis having an increased top, middle leaflet width.
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| CN105432464A (en) * | 2014-11-12 | 2016-03-30 | 河南农业大学 | Cultivation method for inducing autotetraploid of paulownia catalpifolia by colchicine |
| CN106718113A (en) * | 2017-01-19 | 2017-05-31 | 北京农学院 | A kind of Sorbus alnifloria tree engrafting method |
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| CN106718113A (en) * | 2017-01-19 | 2017-05-31 | 北京农学院 | A kind of Sorbus alnifloria tree engrafting method |
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| "Tetraploid Induction with Leaf Morphology and Sunburn Variation in Sorbus pohuashanensis (Hance) Hedl";Zeren Zhang等;《Forests》;20230804;第14卷(第8期);第1-18页 * |
| 秋水仙素浸泡法对黑果腺肋花楸多倍体的诱导及初步鉴定;李建勋;巴蕾;于欢;于敏;刘冰;吴荣哲;;延边大学农学学报;20170330(第01期);第1-8页 * |
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