CN117074450B - Method for screening kiwi fruit mutant based on pollen morphology - Google Patents
Method for screening kiwi fruit mutant based on pollen morphology Download PDFInfo
- Publication number
- CN117074450B CN117074450B CN202311049283.4A CN202311049283A CN117074450B CN 117074450 B CN117074450 B CN 117074450B CN 202311049283 A CN202311049283 A CN 202311049283A CN 117074450 B CN117074450 B CN 117074450B
- Authority
- CN
- China
- Prior art keywords
- pollen
- kiwi fruit
- screening
- morphology
- kiwi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000009436 Actinidia deliciosa Nutrition 0.000 title claims abstract description 118
- 244000298697 Actinidia deliciosa Species 0.000 title claims abstract description 106
- 238000000034 method Methods 0.000 title claims abstract description 42
- 238000012216 screening Methods 0.000 title claims abstract description 42
- 241000196324 Embryophyta Species 0.000 claims abstract description 50
- 238000010586 diagram Methods 0.000 claims description 12
- 230000000877 morphologic effect Effects 0.000 claims description 12
- 238000005259 measurement Methods 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 230000000007 visual effect Effects 0.000 claims description 5
- 238000004364 calculation method Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000002390 adhesive tape Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000010191 image analysis Methods 0.000 claims description 3
- 238000004544 sputter deposition Methods 0.000 claims description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 3
- 238000009395 breeding Methods 0.000 abstract description 27
- 230000001488 breeding effect Effects 0.000 abstract description 25
- 238000009396 hybridization Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000000463 material Substances 0.000 description 61
- 244000298715 Actinidia chinensis Species 0.000 description 21
- 235000009434 Actinidia chinensis Nutrition 0.000 description 16
- 230000008569 process Effects 0.000 description 11
- 244000298800 Actinidia arguta Species 0.000 description 10
- 235000016416 Actinidia arguta Nutrition 0.000 description 10
- 238000011160 research Methods 0.000 description 7
- 230000035784 germination Effects 0.000 description 6
- 235000014443 Pyrus communis Nutrition 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 238000000513 principal component analysis Methods 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 241000219068 Actinidia Species 0.000 description 3
- 244000144730 Amygdalus persica Species 0.000 description 3
- 235000006040 Prunus persica var persica Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- QZLYKIGBANMMBK-UGCZWRCOSA-N 5α-Androstane Chemical compound C([C@@H]1CC2)CCC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CCC[C@@]2(C)CC1 QZLYKIGBANMMBK-UGCZWRCOSA-N 0.000 description 2
- 241001164374 Calyx Species 0.000 description 2
- 244000046146 Pueraria lobata Species 0.000 description 2
- 235000010575 Pueraria lobata Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000282553 Macaca Species 0.000 description 1
- 241000731396 Pueraria montana var. thomsonii Species 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000013075 data extraction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000010196 hermaphroditism Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a method for screening kiwi fruit mutants based on pollen morphology, which comprises the following steps: step one, collecting P/E index data of morphology of kiwi fruit pollen; and secondly, visualizing the distribution range of the P/E index data of the morphology of the kiwi fruit pollen. The beneficial effects of the invention are as follows: the method can distinguish the morphology of the kiwi fruit pollen through index parameters under lower multiples, can evaluate female plant pollen and male plant pollen between and in the seeds simultaneously, effectively and rapidly screen kiwi fruit mutants intuitively, and provides a new method for screening kiwi fruit mutants, a hybrid parent in directivity and theoretical basis for kiwi fruit hybridization and breeding of new varieties, and the efficiency and precision of screening kiwi fruit mutants are improved.
Description
Technical Field
The invention relates to the technical field of variety breeding, in particular to a method for screening kiwi fruit mutants based on pollen morphology.
Background
The kiwi fruits belong to the genus kiwi fruits of the family kiwiaceae, have rich germplasm resources and various materials, the genetic difference of the germplasm resources directly affects the effect of character improvement, the variety breeding and germplasm innovation depend on the development of excellent genes, and the method is represented by the screening of mutant materials and the selection of hybrid parents. The material of different sources has large inter-species and intra-species difference, and has great potential for resource development and utilization in the aspects of new variety breeding and germplasm innovation. The existing breeding methods are various and comprise traditional seedling breeding, hybridization breeding, bud mutation selection, radiation mutagenesis, biotechnology breeding and the like, but the breeding of new varieties mainly comprises the step of screening materials from wild resources and the seedling filial generation of the wild resources, and the traditional screening method mainly aims at carrying out analysis and research on the appearance characteristics of tissue structures such as leaves, fruits, rhizomes and the like of seedlings. In recent years, along with the application of a scanning electron microscope technology in the sporopollen aspect of kiwi plants, related researches show that the genetic information carried by kiwi pollen has stable genetic commonality characteristics and also has diversity and diversity.
In the process of variety breeding, the existing kiwi fruit mutant screening technology has the problems or defects that:
1. in the traditional screening method, the tissue structures such as leaves, fruits, rhizomes and the like are required to be observed according to seasons or years, the performance of the mutant character is not shown in the F1 generation in many times, the mutant character is likely to be shown only by repeated breeding for several generations, the workload of screening the mutant in many seedlings is very large, the screening process is complex and slow, and the screening efficiency and accuracy are greatly reduced.
2. The scanning electron microscope observation technology has two defects in the aspect of pollen morphology application, on one hand, the method is characterized in that multiple indexes of pollen morphology, namely polar axis length (P), equatorial axis length (E), germination ditch length, polar axis length-equatorial axis length ratio (P/E), pollen grain size (P multiplied by E), germination ditch width, outer wall texture and the like are required to be measured, and average value and standard deviation analysis, variation coefficient calculation or clustering analysis are carried out according to the multiple indexes, so that the relationship is studied. The disadvantage of this aspect is that firstly, in actual breeding work and scientific research, under the condition that the variety of the plant species and the species resources of the kiwi fruit is based on the variety of the pollen species and the quantity, the workload is too large due to the fact that the observation and measurement of various indexes are too many, and a great deal of complicated manpower and time are required to carry out. Secondly, the observation of the pollen outer wall texture has high requirements on the sample and shooting conditions of the machine, under the prior art condition, the observation is generally carried out under the condition that the magnification of a scanning electron microscope is more than 10000 times, and if the water content or the conductivity of the sample is poor, the sample needs to be treated in advance, thereby being time-consuming and labor-consuming; meanwhile, as the structure of the outer wall texture is complex and various, related researches are all dependent on subjective judgment of observers, quantitative indexes are not available, and standardized execution and improvement of working efficiency are not facilitated in a large number of screening works. Thirdly, the final presented results are mostly mutation coefficients, cluster analysis and the like, the data cannot be visualized, screening personnel are required to have a statistical basis for interpretation of experimental results, and the requirements of the analysis personnel are high. The other aspect has the defects that the related research is limited by the prior method, the related research is carried out on pollen of male kiwi fruits, but pollen is generated by female kiwi fruits, and Wang Mingzhong and the like screen out an amphiprotic flower (hermaphrodite) variety Longshan red with production and utilization value from delicious kiwi fruits mutant color kiwi fruits in the south of Longmenshan, and the research report of evaluating and screening female pollen and male pollen together is not found in the prior method.
Therefore, the method for screening the kiwi fruit mutants based on the pollen morphology is provided, the kiwi fruit pollen morphology can be distinguished through index parameters under lower multiples, the kiwi fruit mutants can be screened efficiently, quickly and intuitively in the breeding process of new varieties of kiwi fruits, hybrid parents can be bred directionally, and a theoretical basis is provided for kiwi fruit hybridization and breeding of the new varieties.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a method for screening kiwi fruit mutants based on pollen morphology, which can distinguish kiwi fruit pollen morphology through index parameters under lower multiples, effectively, quickly and intuitively screen kiwi fruit mutants in the breeding process of new kiwi fruit varieties, provides a novel method for solving the problems of complicated and slow process in the traditional screening of kiwi fruit mutants, improves the screening efficiency and precision of kiwi fruit mutants, selectively breeds hybrid parents in directivity, and provides theoretical basis for kiwi fruit hybridization and breeding of new varieties.
The aim of the invention is realized by the following technical scheme:
a method for screening kiwi fruit mutants based on pollen morphology, comprising the following steps:
step one, collecting P/E index data of morphology of kiwi fruit pollen
Collecting female plant pollen and male plant pollen of different kiwi fruits respectively, carrying out morphological observation and photographing of pollen grain samples by using a scanning electron microscope, measuring polar axis length (P) of pollen grains and equatorial axis length (E) corresponding to the same pollen grain by using a software imageJ, and then carrying out ratio (P/E) measurement calculation of the polar axis length and the equatorial axis length to obtain P/E index data of female plant pollen and male plant pollen;
step two, visualizing distribution range of P/E index data of morphology of kiwi fruit pollen
And (3) simultaneously using software Origin software to manufacture a normal distribution curve line whisker graph according to the P/E index data of the female pollen and the male pollen obtained in the step (I).
Further, in the present embodiment, the method for screening kiwi fruit mutants based on pollen morphology further comprises a step three;
step three, image analysis
And analyzing the difference condition of the normal distribution range of the normal distribution curve line whisker graph, and screening out the kiwi fruit mutants.
The female plant pollen and the male plant pollen of the kiwi fruits have large shape difference, the female plant pollen grains are nearly spherical or oblate spherical, the male plant pollen grains are nearly ultra-long spherical or prolate spherical, the numerical difference of the ratio (P/E) of polar axis length to equatorial axis length is obvious, the normal distribution curve axis whisker graph does not have coincidence intersection, in the new kiwi fruit variety breeding process, the normal distribution diagram of the female plant kiwi fruit mutant is coincident with the same kind of male flowers, and the normal distribution diagram of the male plant kiwi fruit mutant is coincident with the same kind of female flowers. In the axial whisker graph, the normal distribution range of the kiwi fruit mutant is wider than that of the unmutated kiwi fruit pollen.
In the new variety breeding of kiwi fruits, when a large number of wild kiwi fruits and actual offspring samples thereof need to be screened and evaluated, the prior method is either required to be observed according to seasons or years, or is too high in requirements on samples and operators, or is too large in workload due to more indexes of observation and measurement, and often cannot meet the requirements of the actual new variety breeding screening mutant. The scheme can distinguish the morphology of the kiwi fruit pollen through index parameters under lower multiples, and can realize the screening and evaluation of the actual generation offspring samples in the breeding of new kiwi fruit varieties.
Because this scheme is going on under lower number multiple, can show the pollen particle count of a lot of in the electron microscope picture to this scheme need not to take many photos and carries out data extraction, has reduced work load, and electron microscope scanning is taken time less, has improved the efficiency that kiwi fruit mutant was screened greatly, and this scheme is visual with data, can high-efficient quick and screen kiwi fruit mutant directly perceivedly, and screening precision is high.
In a preferred embodiment, in the step one, the scanning electron microscope performs morphological observation photographing of pollen grains at a magnification of 300 to 800 times.
In a preferred embodiment, in the first step, the software ImageJ is used to measure the polar axis length (P) and the equatorial length (E) corresponding to each pollen grain, and the software Excel is used to calculate the corresponding polar red ratio (P/E), and the number of each pollen grain sample is not less than 100.
In a preferred embodiment, in the step one, the specific operation of collecting the female plant pollen and the male plant pollen of the kiwi fruit is as follows:
picking flower buds to be opened in the initial flowering period of kiwi fruits, peeling anthers into a covered culture dish by using tweezers, placing the culture dish in a constant temperature oven at 25 for 24 hours, slightly shaking the culture dish, placing the culture dish in a glass sample bottle after pollen is completely dispersed, and hermetically preserving the culture dish at-20 for later use.
In a preferred embodiment, in the step one, the specific operation of using a scanning electron microscope to perform morphological observation photographing of pollen grains is as follows:
and (3) adhering the dried pollen grain sample to a sample carrying platform adhered with a conductive adhesive tape, spraying metal by an ion sputtering instrument, forming a layer of conductive film on the surface, spraying metal for 1min, and selecting typical pollen grains in a visual field by an upper machine for observation and photographing.
The beneficial effects of the invention are as follows:
the method can utilize a scanning electron microscope to observe and photograph the morphology of pollen grains at a lower multiple, uses software imageJ to measure the polar axis length (P) of the pollen grains and the equatorial axis length (E) corresponding to the same pollen grains, then performs the ratio (P/E) measurement calculation of the polar axis length and the equatorial axis length, and collects the P/E index data of female plant pollen and male plant pollen; and then, a normal distribution curve axis whisker graph is manufactured by using software Origin software, the distribution range of the P/E index data of the kiwi fruit pollen morphology is visualized, the kiwi fruit pollen morphology is distinguished by analyzing the normal distribution curve axis whisker graph, and the kiwi fruit mutants can be efficiently, quickly and intuitively screened in the new kiwi fruit variety breeding process, so that a new method is provided for solving the problems of complicated and slow processes in the traditional screening of kiwi fruit mutants and improving the screening efficiency and precision of the kiwi fruit mutants, and a theoretical basis is provided for directionally breeding hybridized parents of kiwi fruits and breeding new varieties.
Drawings
FIG. 1 is a graph showing the P/E ratio distribution axis of different pollens of actinidia chinensis;
FIG. 2 is a scanning electron microscope morphology diagram of different pollens of actinidia chinensis;
FIG. 3 is a graph showing the P/E ratio distribution axillary whiskers of different pollens of the delicious kiwi fruit;
FIG. 4 is a scanning electron microscope morphology of different pollens of the delicious kiwi fruit;
FIG. 5 is a graph of P/E ratio distribution axis whisker for different pollens of actinidia arguta;
FIG. 6 is a scanning electron microscope morphology of different pollens of the actinidia arguta;
FIG. 7 is a graph of P/E ratio distribution axillary whiskers of different pollens of eriodictyota, pueraria lobata, four calyx, beijing pear kiwi;
FIG. 8 is a scanning electron microscope morphology of different pollens of the Pueraria thomsonii, the four calyx and the Beijing pear kiwi fruit;
FIG. 9 is a scanning electron microscope morphology diagram of the female strain pollen of actinidia chinensis at different multiples;
FIG. 10 is a scanning electron microscope morphology of male Chinese goosebeery pollen at different magnification;
FIG. 11 is a scanning electron microscope morphology of the male plant pollen of Actinidia chinensis at 200X;
fig. 12 is a PCA analysis chart.
Detailed Description
The technical scheme of the present invention is described in further detail below with reference to specific embodiments, but the scope of the present invention is not limited to the following description.
Example 1
Referring to table 1, the method for screening kiwi fruit mutants based on pollen morphology comprises the following steps:
step one: pollen collection
11 parts of kiwi fruit pollen to be tested is numbered as samples 1-11, in the initial flowering period of kiwi fruit, buds to be opened are picked, anthers are peeled into a covered culture dish by tweezers, the culture dish is placed in a constant temperature oven at 25 for 24 hours, the culture dish is gently shaken, and after pollen is completely scattered out, the culture dish is placed in a glass sample bottle and is stored in a closed manner at-20 for standby;
step two: morphological observation of pollen grains with a scanning electron microscope at 500 Xmagnification
Taking out the pollen grain sample dried in the first step, adhering the pollen grain sample to a sample carrying platform stuck with a conductive adhesive tape, spraying metal by an ion sputtering instrument, forming a layer of conductive film on the surface, spraying metal for 1min, and selecting typical pollen grains in a visual field for observation and photographing after the sample carrying platform is put on a machine;
step three: P/E index data collection
Measuring polar axis length (P) and equatorial length (E) corresponding to each pollen grain in the photo obtained in the second step by using software imageJ, and calculating corresponding polar red ratio (P/E) by using software Excel, wherein the number of pollen grains of each sample is not less than 100, so as to obtain P/E index data of female plant pollen and male plant pollen;
step four: P/E index data distribution range visualization
P/E index data of female pollen and male pollen obtained in the third step are simultaneously used for manufacturing a normal distribution curve line whisker graph by software Origin software, and the normal distribution curve line whisker graph is shown in figure 1;
step five: image analysis
And analyzing the difference condition of the normal distribution range of the normal distribution curve line whisker graph, and screening out the kiwi fruit mutants.
In this embodiment, the normal distribution diagram of the sample No. 10 of the female kiwi fruit is overlapped with the same male flowers, the normal distribution diagram of the sample No. 4 of the male kiwi fruit is overlapped with the same female flowers, and the normal distribution ranges of the sample No. 4 and the sample No. 10 in fig. 1 are wider than those of the rest kiwi fruit pollen.
Specifically, in this embodiment, the X values of sample materials of the female pollen 7, 8, 9, and 11 of the actinidia chinensis shown in fig. 1 are approximately distributed between 0.60 and 1.25, the distribution range is narrow and concentrated, and the coincidence degree is high; the X values of the materials No. 1, 2, 3, 5 and 6 of the male plant pollen are distributed between about 1.60 and 2.60, the distribution range is also narrower and concentrated, the overlapping ratio is higher, and the distribution trend of the materials No. 7, 8, 9 and 11 in the female plant pollen is the same.
And the distribution trend of the material No. 10 in the female pollen and the material No. 4 in the male pollen is greatly different. The X distribution range of the material No. 10 is 0.60 to 1.80, the distribution range is very wide, the overlapping degree of the material with most female pollen is not high, partial material is overlapped with male pollen, the ratio distribution is concentrated at about 1.25, and the area almost does not contain the ratio of female pollen to male pollen. The distribution range of the material No. 4 in the male plant pollen is about 1.50 to 2.50, part of the distribution range is not overlapped with other male plant pollen, and is close to the side of the female plant pollen and partially overlapped with the material No. 10 in the female plant pollen, and the material No. 4 and the material No. 10 are different from other materials in a distribution axial whisker chart, so that the material No. 4 and the material No. 10 can be judged to be mutant plants in the breeding process of new varieties of kiwi fruits.
Further, in this embodiment, the P/E value corresponding to the highest point of the P/E normal distribution curve of the sample is taken as X of the sample m The method comprises the steps of carrying out a first treatment on the surface of the X of sample 4 in the Male population m Value and androstane population X m The difference in size of (2) is greater than 0.50; x of sample 10 in female population m Value and female population X m The difference in size is greater than 0.25. The difference is obvious.
TABLE 1 morphological characteristics and size of different Kiwi pollen
Scanning electron microscope image observation experiment
In the experiment, pollen forms of materials 1, 2, 4, 7, 9 and 10 in the example 1 are observed by using a scanning electron microscope, and whether the samples are mutated or not is judged according to whether the pollen forms are consistent.
When the mutation condition of the sample is observed by using a scanning electron microscope, the mutation condition of the sample is usually observed under the magnification of 1000X or more, so that the scanning electron microscope is adopted to shoot the scanning electron microscope morphological images of different pollens of the actinidia chinensis under the magnification of 1000X, and the result is shown in figure 2. In the figure, A, B, C is the material of female pollen 7, 9 and 10 of Chinese goosebeery respectively, and D, E, F is the material of male pollen 1, 2 and 4 of Chinese goosebeery respectively.
As can be seen from the scanning electron microscope of FIG. 2, the morphology of A, B female pollen is consistent, the morphology of D, E male pollen is consistent, and almost no change occurs, while the morphology of pollen of No. 4 material and No. 10 material in the C, F image is inconsistent with that of other pollen grains (pollen grains indicated by arrows), for example, the polar axis length P of male pollen grains illustrated by F in FIG. 2 is shortened, and the equatorial axis length E is lengthened; in fig. 2, the polar axis length P of female pollen grains is longer, the equatorial axis length E is shorter, and the mutation is generated correspondingly on the pollen morphology, which is consistent with the judgment result in example 1, so that the index parameter of the scheme can distinguish the morphology of the kiwi pollen under a lower multiple, and the scheme can be used for screening kiwi mutant strains in the new variety breeding process of kiwi fruits.
In addition, in the experiment, the female peach strain pollen of Hua Mihou was photographed by a scanning electron microscope under the magnification of 500, 1000, 3500, 15000, respectively, and the result is shown in fig. 9; in the pair Hua Mihou peach male pollen, photographing is carried out when the magnification is 500, 1000, 3500, 10000, respectively, and the result is shown in fig. 10; and the male kiwi pollen of the Chinese goosebeery of the Hua Mihou peach was photographed at a magnification of 200 x, and the result is shown in fig. 11.
It can be seen from fig. 9 and 10 that, when the magnification is 500the number of pollen is large, the image of pollen grains is small, and the morphology of pollen is not easy to observe visually, and from fig. 11, when the magnification is 200the morphology of pollen grains cannot be observed clearly, but the granule profile of pollen grains is clear, and the polar axis length (P) and equatorial axis length (E) of pollen grains can still be measured clearly, so that when the magnification is 200 times, the experimental requirement of the scheme can be met, and when 200 times, the scheme can be used for realizing rapid screening of kiwi fruit mutants, and compared with the conventional breeding method of mutant strains, the scheme can be used for realizing mutant screening under lower times.
Example 2
The procedure of this example is the same as that of example 1, and the difference between this example and example 1 is that the kiwi pollen material tested in this example is different from example 1, and the kiwi pollen material in this example is numbered 12 to 25, referring to table 1.
The P/E ratio distribution axis of different pollens of the delicious kiwi fruit obtained in this example is shown in fig. 3, the pollen distribution range of the delicious kiwi fruit shown in fig. 3 is generally that the materials X of female pollen 21, 22, 23 and 24 are distributed about 0.50 to 1.50, the materials X of male pollen 12, 13, 14, 15, 16, 18, 19 and 20 are distributed about 1.50 to 2.75, the female pollen and the male pollen are hardly overlapped, and the materials X are distributed in two areas more intensively.
The distribution range of the material X of the male plant pollen No. 17 is about 0.60 to 2.00, the distribution range is wide, the distribution range comprises the distribution ranges of female plant pollen and male plant pollen, and the overlapping area of the material X and the female plant pollen is more; the No. 25 material is an amphoteric Hualongshan red sample, the ratio is about 0.50 to 1.75, the distribution range is wider, the amphoteric Hualongshan red sample comprises female plant pollen and male plant pollen, a large part of the area is overlapped with the No. 17 material, and the No. 17 material and the No. 25 material can be judged to be mutant plants in the breeding process of new kiwi fruit varieties.
Further, in this embodiment, the P/E value corresponding to the highest point of the P/E normal distribution curve of the sample is also taken as X of the sample m The method comprises the steps of carrying out a first treatment on the surface of the X of sample 17 in the Male population m Value and androstane population X m The difference in size of (2) is greater than 0.50; x of sample 25 m Value and female population X m The difference in size is greater than 0.25. The difference is obvious.
Fig. 4 is a scanning electron microscope morphological diagram (magnification is 1000) of different pollens of the delicious kiwi fruit, wherein A, B is respectively material number 22 and 23 of female strain pollens of the delicious kiwi fruit, C is material number 25 of amphoteric flower of the mutant of the delicious kiwi fruit, and D, E, F is respectively material number 16, 18 and 17 of male strain pollens of the delicious kiwi fruit; in the scanning electron microscope, the material No. 17 and the material No. 25 are greatly different in morphology (shown by arrows), and the pollen morphology is mutated, so that the judgment conclusion is consistent with the judgment conclusion of the scheme.
Example 3
The procedure of this example is the same as that of example 1, and the difference between this example and example 1 is that the kiwi pollen material tested in this example is different from example 1, and the kiwi pollen material in this example is numbered 26 to 39, referring to table 1.
In the embodiment, the materials 26 to 30 are used as a group of samples for mutant screening, the materials 31 to 39 are used as a group of samples for mutant screening, and the P/E ratio distribution axial whisker diagrams of different pollens of the obtained delicious kiwi fruits are respectively shown in fig. 5 and 6.
The actinidia arguta of fig. 5 has a female plant X ranging from about 0.60 to about 1.50 and a male plant ranging from about 1.60 to about 2.60. The female plants of actinidia arguta, actinidia kudzuvine, actinidia quasimilis and actinidia jingli shown in fig. 7 are distributed in a range of about 0.50 to 1.50, and the male plants are distributed in a range of about 1.50 to 2.75.
From fig. 5 and 7, the ratio distribution is shown as a concentrated distribution range and a large overlapping area, female and male pollen is distributed in different areas, and the areas are hardly overlapped, so that no mutant exists in the 26-39 kiwi fruit pollen material.
Fig. 6 is a scanning electron microscope morphological diagram (magnification is 1000) of different pollens of actinidia arguta and Mao Hua actinidia arguta, wherein A2 and B2 are respectively material number 29 and 30 of actinidia arguta female strain pollens, C2 is material number 32 of actinidia arguta, D2 and E2 are respectively material number 27 and 28 of actinidia arguta male strain pollens, and F2 is material number 31 of actinidia arguta.
Fig. 8 is a scanning electron microscope morphological diagram (magnification is 1000) of different pollens of the kiwi fruit, the four-calyx kiwi fruit and the Beijing pear kiwi fruit, wherein A3 is a kiwi fruit female plant pollen 34 # material, B3 is a kiwi fruit female plant pollen 36 # material, C3 is a Beijing pear kiwi fruit female plant pollen 39 # material, D3 is a kiwi fruit male plant pollen 33 # material, E3 is a kiwi fruit male plant pollen 35 # material and F3 is a Beijing pear kiwi fruit 37 # material.
From the scanning electron microscope images of fig. 6 and 8, it was also seen that the male pollen and the female pollen were identical in morphology, hardly different, and not mutated, and the judgment result was identical with the judgment result of the present scheme.
Principal component analysis ((PCA, principal component analysis)) experiment
The purpose of this experiment was to verify the method of the invention using PCA analysis
In the test materials of 39 parts in total in the examples 1 to 3, including 7 kinds of kiwi fruit pollen, in the polar axis length (P), the equatorial axis length (E), the germination ditch length, the polar axis length to equatorial axis length ratio (P/E), the pollen grain size (PE), the germination ditch width and the outer wall texture 7 pollen characteristic indexes, in order to reduce the operation requirement of a scanning electron microscope, the shooting observation under a lower multiple (500) is selected to improve the working efficiency, and the two characteristics of the germination ditch width and the outer wall texture observed under a high multiple are omitted, so that the number of observation groups is increased and the number of measurement indexes is reduced. Then, in order to explore whether the polar axis length (P), the equatorial axis length (E), the germination ditch length, the polar axis length to equatorial axis length ratio (P/E), the pollen grain size (PE) 5 indexes (under low multiples) are feasible or not and the dimension reduction of related indexes, software Grapad Prism 9.5 is used for carrying out Principal Component (PCA) analysis, the PCA analysis is shown in a graph, wherein A0 is a PCA steep slope graph, B0 is a PCA variance ratio graph, C0 is a PCA score graph, D0 is a PCA load graph, the steep slope graph and the variance ratio graph show that the contribution degree total variance of the first two principal components is 96.27 percent, the information of original variables can be almost covered, a certain degree of clustering exists between samples in the score graph, the female plant pollen and the male plant pollen, the similarity between the two is not large, the load graph shows that the polar axis length to equatorial axis length ratio (P/E) in the 5 indexes is the maximum with the included angle between the two is less than the equatorial axis length (E), the redundant information is provided, and the coefficient of the two is low, and the coefficient of the score is lower than the coefficient of the index can distinguish the pollen form by the coefficient of the macaque map.
In the lower multiple 5 indexes, the index of polar axis length to equatorial axis length ratio (P/E) plays a key role in pollen morphology, particularly has important reference value in pollen morphology of mutants, the changed pollen morphology can be different from that of groups to a certain extent, therefore, after a plurality of groups of polar axis length to equatorial axis length ratio (P/E) are rapidly measured by using imageJ software, a normal curve distribution axial whisker graph of polar axis length to equatorial length ratio (P/E) is drawn by adopting software Origin2021, all data are simply and clearly visualized, 4, 10, 17 and 25 materials are large in difference from normal distribution intervals of P/E ratio of different kiwi fruit pollens, the material No. 4 is Chinese kiwi fruit male plant pollens, the material No. 10 is Chinese kiwi fruit female plant pollens, the material No. 17 is Chinese kiwi fruit male plant pollens, the material No. 25 is Chinese kiwi fruit female flower red flower pollens, and corresponding scanning electric pictures (1000) are adopted, all data are easily and clearly visualized, and the large difference between the mutant pollen and the mutant pollen can be simultaneously evaluated in a large visual way.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (5)
1. A method for screening kiwi fruit mutants based on pollen morphology, which is characterized by comprising the following steps:
step one, collecting P/E index data of morphology of kiwi fruit pollen
Collecting female plant pollen and male plant pollen of different kiwi fruits respectively, carrying out morphological observation and photographing of pollen grain samples by using a scanning electron microscope, measuring polar axis length (P) of pollen grains and equatorial axis length (E) corresponding to the same pollen grain by using a software imageJ, and then carrying out ratio (P/E) measurement calculation of polar axis length and equatorial axis length to obtain P/E index data of female plant pollen and male plant pollen;
step two, visualizing distribution range of P/E index data of morphology of kiwi fruit pollen
P/E index data of female pollen and male pollen obtained in the first step are simultaneously used for manufacturing a normal distribution curve line whisker graph by software Origin software;
step three, image analysis
Analyzing the difference condition of the normal distribution range of the normal distribution curve line whisker graph, and screening out kiwi fruit mutants;
in the third step, the normal distribution diagram of the female kiwi fruit mutant is overlapped with the same male flowers, and the normal distribution diagram of the male kiwi fruit mutant is overlapped with the same female flowers; in the axial whisker graph, the normal distribution range of the kiwi fruit mutant is wider than that of the unmutated kiwi fruit pollen.
2. The method for screening kiwi fruit mutants based on pollen morphology according to claim 1, wherein in the first step, the magnification of morphological observation photographing of pollen grains by a scanning electron microscope is 300-800 times.
3. The method for screening kiwi fruit mutants based on pollen morphology according to claim 1, wherein in the first step, the polar axis length (P) and the equatorial axis length (E) corresponding to each pollen grain are measured by using software ImageJ, and the corresponding polar to red ratio (P/E) is calculated by using software Excel, wherein the number of each pollen grain sample is not less than 100.
4. The method for screening kiwi fruit mutants based on pollen morphology according to claim 1, wherein in the first step, the specific operations of collecting kiwi fruit female pollen and male pollen are as follows:
picking flower buds to be opened in the initial flowering period of kiwi fruits, peeling anthers into a covered culture dish by using tweezers, placing the culture dish in a constant temperature oven at 25 for 24 hours, slightly shaking the culture dish, placing the culture dish in a glass sample bottle after pollen is completely dispersed, and hermetically preserving the culture dish at-20 for later use.
5. The method for screening kiwi fruit mutants based on pollen morphology according to claim 4, wherein in the first step, the specific operation of using a scanning electron microscope to perform morphological observation photographing of pollen grain samples is as follows:
and (3) adhering the dried pollen grain sample to a sample carrying platform adhered with a conductive adhesive tape, spraying metal by an ion sputtering instrument, forming a layer of conductive film on the surface, spraying metal for 1min, and selecting typical pollen grains in a visual field by an upper machine for observation and photographing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311049283.4A CN117074450B (en) | 2023-08-18 | 2023-08-18 | Method for screening kiwi fruit mutant based on pollen morphology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311049283.4A CN117074450B (en) | 2023-08-18 | 2023-08-18 | Method for screening kiwi fruit mutant based on pollen morphology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117074450A CN117074450A (en) | 2023-11-17 |
| CN117074450B true CN117074450B (en) | 2024-04-16 |
Family
ID=88714722
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311049283.4A Active CN117074450B (en) | 2023-08-18 | 2023-08-18 | Method for screening kiwi fruit mutant based on pollen morphology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117074450B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110122335A (en) * | 2019-06-27 | 2019-08-16 | 西南林业大学 | The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant |
| CN114451299A (en) * | 2022-02-17 | 2022-05-10 | 浙江省农业科学院 | Wild flower kiwi fruit male parent creation with high pollen forming capacity and application thereof |
| CN114766353A (en) * | 2022-05-09 | 2022-07-22 | 辽宁省经济林研究所 | Rapid screening method and application of male pollination plants in actinidia arguta orchard |
-
2023
- 2023-08-18 CN CN202311049283.4A patent/CN117074450B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110122335A (en) * | 2019-06-27 | 2019-08-16 | 西南林业大学 | The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant |
| CN114451299A (en) * | 2022-02-17 | 2022-05-10 | 浙江省农业科学院 | Wild flower kiwi fruit male parent creation with high pollen forming capacity and application thereof |
| CN114766353A (en) * | 2022-05-09 | 2022-07-22 | 辽宁省经济林研究所 | Rapid screening method and application of male pollination plants in actinidia arguta orchard |
Non-Patent Citations (3)
| Title |
|---|
| 3 个猕猴桃栽培种花粉形态扫描电镜观察;齐秀娟 等;《果树学报》;20171231;第34卷(第11期);第1365-1373页 * |
| High-density interspecific genetic maps of kiwifruit and the identification of sex-specific markers;Qiong Zhang et al.;《DNA Research》;20150914;第22卷(第5期);第367–375页 * |
| 野生毛花猕猴桃雄株居群花粉形态观察;钟敏 等;《果树学报》;20161231;第33卷(第10期);第1251-1258页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117074450A (en) | 2023-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ercisli et al. | Determination of size and shape features of walnut (Juglans regia L.) cultivars using image processing | |
| Brewer et al. | Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species | |
| Hay et al. | Developmental changes in the germinability, desiccation tolerance, hardseededness, and longevity of individual seeds of Trifolium ambiguum | |
| Rezaei et al. | The selection of superior walnut (Juglans regia L.) genotypes as revealed by morphological characterization | |
| EP2798612A1 (en) | Immature ear photometry in maize | |
| Genaev et al. | Extraction of quantitative characteristics describing wheat leaf pubescence with a novel image-processing technique | |
| CN111402199A (en) | Pear fruit stone cell phenotype detection method based on computer image processing | |
| Rodrigues et al. | Agronomic performance of arabica coffee genotypes in northwest Rio de Janeiro State | |
| Krause et al. | Digital phenotyping for quantification of genetic diversity in inbred guava (Psidium guajava) families | |
| WO2022264076A1 (en) | Methods of sorting matthiola seeds | |
| Ewédjè et al. | Species delimitation in the African tree genus Lophira (Ochnaceae) reveals cryptic genetic variation | |
| CN117074450B (en) | Method for screening kiwi fruit mutant based on pollen morphology | |
| Xue et al. | Pearprocess: A new phenotypic tool for stone cell trait evaluation in pear fruit | |
| Naima et al. | Univariate and multivariate analysis of agronomical traits of preselected argan trees | |
| Acarsoy Bilgin et al. | The pollen and fruit properties of Ficus carica Caprificus | |
| Yu et al. | A non-destructive coconut fruit and seed traits extraction method based on Micro-CT and deeplabV3+ model | |
| Kona et al. | AMMI and GGE biplot analysis of genotype by environment interaction for yield and yield contributing traits in confectionery groundnut | |
| CN108369192A (en) | Method and apparatus for measuring inflorescence, seed and/or seed production phenotype | |
| Amorim et al. | Self-, cross-and interspecific pollinations in Passiflora capsularis and P. rubra | |
| Gong et al. | GpemDB: A scalable database architecture with the multi-omics entity-relationship model to integrate heterogeneous big-data for precise crop breeding | |
| Karimi et al. | Quantitative and qualitative analyses of genus Pyrus L.(Pear Tree) in West Azerbaijan province, Iran | |
| Costa et al. | Chlorophyll fluorescence and X-ray analyses to characterise and improve paddy rice seed quality | |
| US8691575B2 (en) | General method of classifying plant embryos using a generalized Lorenz-Bayes classifier | |
| Poulsen Hornum et al. | Expression of internal reproductive barriers in a germplasm bank accession of the wild potato Solanum chacoense Bitter in three ex situ regeneration cycles | |
| Cai et al. | Pollen morphology of the genus Gossypium and its systematic implications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |