CN114766353A - Rapid screening method and application of male pollination plants in actinidia arguta orchard - Google Patents
Rapid screening method and application of male pollination plants in actinidia arguta orchard Download PDFInfo
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Abstract
The invention provides a rapid screening method and application of male pollination plants in actinidia arguta orchard, and relates to the technical field of pollination tree configuration. According to the method, the influence of the actinidia arguta pollination tree selection and configuration on fruits is analyzed through methods such as flowering phase investigation, pollen preservation and activity determination, callose fluorescent dyeing and the like, a garden is scientifically built around actinidia arguta, the yield and quality are promoted to be improved, the efficiency of garden management is improved, and a foundation is laid for realizing efficient and standardized cultivation of actinidia arguta cultivation. The invention provides a pollination male plant rapid screening technology based on callose fluorescent staining, which is beneficial to scientific and reasonable configuration of pollination trees.
Description
Technical Field
The invention belongs to the technical field of pollination tree configuration, and particularly relates to a rapid screening method of male pollination plants in actinidia arguta orchard and application of the rapid screening method.
Background
In the cultivation research practice of actinidia arguta, the method for configuring the pollination tree is a basic means for obtaining high quality and high yield. At present, when the actinidia arguta is configured with the pollination trees, the method has great blindness, the pollination trees are often configured randomly, and a systematic and scientific pollination tree screening method is not available. From the aspect of morphology, the flower type of the actinidia plant is complete flower, but most of the actinidia plant belongs to functional male and female plants, and due to the special genetic background, the progeny is usually more male than female, and even the male and female plants have the situations of pollen inefficacy and self (hybrid) mating incompetence. Therefore, the pollination tree is reasonably configured during planting, and the main means for ensuring the yield and the quality of the fruits is provided. Therefore, the selection of proper pollination trees and the development of artificial supplementary pollination are an important way for obtaining high yield and high quality.
At present, when a pollination variety is configured, the pollination variety is determined according to the pollination fruit setting rate among different varieties in the field. Although the method is visual and effective, the workload is large, the period is long, and the method has great blindness in variety selection.
Disclosure of Invention
In view of the above, the invention aims to provide a screening method of actinidia arguta garden pollination trees and application thereof, which solve the problem that pollination trees are not properly applied in industrial development and realize scientific screening of male plants in garden building period or garden reconstruction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a screening method of actinidia arguta orchard pollination male plants, which comprises the following screening standards: flowering phase survey, pollen viability determination and pollen affinity detection.
Preferably, the florescence survey comprises: and (4) screening pollinating trees meeting the flowering phase of the fruiting tree.
Preferably, the pollen viability assay comprises assaying the viability of fresh pollen and assaying the viability of stored pollen.
Preferably, the pollen affinity assay comprises callose fluorescent staining visualization.
Preferably, the pollen affinity detection comprises early screening of callose fluorescent staining pollinated male plants and in-situ reaction of pollen on stigma.
Preferably, the orchard comprises an orchard during orchard establishment and/or an orchard when the orchard is modified.
The invention also provides application of the screening method in male plant selection during orchard establishment or garden reconstruction.
The invention also provides the application of the screening method in variety selection and matching during orchard establishment or garden reconstruction.
Has the beneficial effects that: in order to overcome the defects of pollination tree configuration, the invention utilizes the fluorescence microscope technology to perform tabletting on the flower column after self-flower and cross-flower pollination treatment, observes the growth condition of a pollen tube, judges the affinity of different varieties and can provide a basis for garden building and scale production of the varieties, thereby providing a screening method of actinidia arguta garden pollination trees. In the specific embodiment of the invention, the fluorescence microscope technology is utilized to perform tabletting on the flower columns after the self-blooming and cross-pollination treatments, the growth condition of the pollen tube is observed, and the affinity of different varieties is judged so as to provide theoretical basis for garden building and mass production of the varieties; affinity difference of self-pollination is disclosed for the first time, a pollination male plant early-stage screening technology based on callose fluorescent staining is provided, and scientific and reasonable configuration of pollination trees is realized.
Drawings
Fig. 1 is a schematic configuration diagram of male and female actinidia arguta plants, wherein: o represents a female plant; the left indicates male plants (or grafts);
FIG. 2 is a graph showing the method of determining pollen viability of actinidia arguta by using the agar method, culturing at room temperature overnight;
FIG. 3 shows the pollen collection and determination method for female plants (left) and the pollen viability determination for the early flowering stage of Longcheng No. 2 (right);
FIG. 4 shows fluorescence detection of self-selected male pollination (left) and fluorescence detection after self-pollination (right);
FIG. 5 shows the in situ fluorescence reaction of pollen of different ploidy male plants on the stigma of Longcheng No. 2.
Detailed Description
The invention provides a screening method of orchard pollinated trees, which comprises the following screening standards: flowering phase survey, pollen viability determination and pollen affinity detection.
The florescence survey of the present invention preferably comprises: and (4) screening pollinating trees meeting the flowering phase of the fruiting tree. The pollination tree is preferably actinidia arguta which is planted in open field and has the age of more than 5 years, statistics is started when female flowers or male flowers appear, when statistics is carried out, 10 healthy standard branches are preferably selected at random, and 8: 00-10: 00 the flowering process was observed. In the invention, the florescence is divided, for example, 5 to 25 percent of flowers are opened in the initial florescence, more than 25 percent of flowers are opened in the full florescence, and more than 50 percent of anthers on male inflorescences are withered and fallen in the final florescence. Moreover, the orchard is preferably an orchard in the orchard establishment period and/or an orchard in the field reconstruction period, the fruit trees in the orchard are preferably actinidia arguta, as plants of the actinidia arguta are usually male and female heterozygotes, and the optimal pollination period of the female flowers is 2 days after the actinidia arguta blooms; the fruits are respiratory catamorph, the storage period is short, especially the early-maturing variety, therefore, when selecting pollinator, the full-bloom period of the female flowers meet the blooming period, and the pollinator with the long blooming period is selected as far as possible.
The pollen viability assay of the present invention preferably comprises assaying the viability of fresh pollen and assaying the viability of stored pollen, said viability preferably comprising the pollen germination rate. In the invention, the pollen germination rate is used as a marker of pollen viability, high pollen germination rate indicates high pollen viability, and low pollen germination rate indicates low pollen viability. The method for measuring the pollen germination rate is not particularly limited, but preferably measures the pollen viability of actinidia arguta by referring to a muskroot-like muskmelon agar method, and expresses the pollen viability by the pollen germination rate. The medium used in the agar method of the present invention preferably includes: boric acid (H)3BO3)0.1 g, calcium nitrate (Ca (NO)3)2·4H2O)0.3 g, magnesium sulfate (MgSO)4·7H20.2 g of O), potassium nitrate (KNO)3)0.1 g, agar 0.5% and distilled water 100 ml. The prepared culture medium is filled in a 100 ml triangular flask, and the culture medium is preferably just formed into a thin layer. After the culture medium is solidified, a small amount of pollen is adhered by a toothpick and is evenly and elastically sowed on the culture medium for germination for 24 hours. And observing the pollen germination condition under a microscope, measuring the length of a pollen tube, counting the pollen germination rate, and taking the length of the pollen tube which is more than 2 times of the diameter of the pollen particle as the germination standard. 3-6 visual fields are observed each time, about 300 pollen grains are observed, and the pollen germination rate is calculated. The pollen germination rate is the total number of germinated pollen/observed pollen count multiplied by 100%.
The preservation of the pollen preferably comprises the collection and preservation of the pollen, wherein the collection preferably comprises the collection of male flowers 1 day before flowering, the collection is carried back to the destination by wrapping with 2 layers of newspaper, and the male flowers are removed after the laboratory, kept in shade and dried for 1 day; the male flower has high activity of pollen, and can fully meet the breeding requirement; no loose powder loss exists, and the collection amount of pollen is larger and more sufficient; the possibility of contamination by other pollen can be reduced more easily.
The preservation of the invention preferably comprises low-temperature preservation, the low-temperature treatment is beneficial to the storage of the pollen, the lower the temperature is, the slower the pollen activity is reduced, and the longer the storage time is; if at 4 ℃, the reduction of the pollen viability is not obvious, the pollen viability is still over 50 percent after being stored for 30 days, and the pollen viability disappears after 120 days; under the preservation condition of 20 ℃ below zero, the viability of the pollen is slowly reduced along with the prolonging of the storage time, the viability of the pollen is still more than 50 percent after 120 days, and the pollen basically loses the viability after 300 days.
The pollen affinity detection comprises callose fluorescent staining observation and field investigation of fruit setting rate, more particularly comprises early screening of callose fluorescent staining pollination male plants and in-situ reaction of pollen on stigma, and preferably comprises detection of self-affinity and hybridization affinity of the pollen. The callose fluorescent staining observation of the invention preferably comprises that when a material fixed by FAA is observed by a fluorescent microscope, pollen tubes are green, for example, in the embodiment, a large number of pollen grains germinate on stigma of 4 self-pollination male plants subjected to affinity pollination, and the pollen tubes at action sites smoothly pass through mastoid cells and extend into the stigma and the style; in the case of self-incompatibility, pollen forms callose on stigma, but cannot form pollen tube. Preferably, the diploid, tetraploid and hexaploid resources are selected and identified by flow cytometry, and are verified by pollination affinity, and the pollen activity of the male plants of the four resources is determined to be more than 50%, so that the method can be used for subsequent affinity and fruit setting rate research. Although the ploidy of male plants is different, the male plants can be used as pollination male plants of actinidia arguta with good fruit setting. Fluorescence detection proves that the pollen can germinate on stigma and form a pollen tube. The invention uses the fluorescence microscope technology to perform tabletting on the flower columns after the self-flower and cross-flower pollination treatment, observes the growth condition of the pollen tube, judges the affinity of different varieties and provides a basis for the garden building and the scale production of the varieties.
By utilizing the screening method, the reasonability of pollination tree configuration can be detected before large-scale planting, the fruit quality and the yield are improved in an artificial pollination mode, the operation efficiency of field management is greatly improved, and a foundation is laid for realizing efficient and standardized cultivation of actinidia arguta.
The invention also provides application of the screening method in male plant selection during orchard establishment or garden reconstruction.
The application of the present invention is preferably the same as described above and will not be described further herein.
The invention also provides the application of the screening method in variety selection and matching during orchard establishment or garden reconstruction.
The application of the present invention is preferably the same as described above and will not be described further herein.
The following examples are provided to illustrate the screening method and the application of actinidia arguta garden pollination tree provided by the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Florescence survey
The method comprises the following steps: and (4) selecting the actinidia arguta planted in the open field with the age of more than 5 years for observation. Statistics was started when the female or male flowers appeared, and 10 healthy standard shoots were randomly selected, 8 in the morning of each day: 00-10: 00 the flowering process was observed. The initial flowering period is 5% -25% of flowers, the full flowering period is more than 25% of flowers, and the final flowering period is more than 50% of male inflorescences with anthers falling.
As a result: the actinidia arguta flower buds are mixed buds, and are subjected to physiological differentiation in autumn and morphological differentiation after sprouting in spring of the next year. The plants are usually hermaphrodite, and the optimal pollination period of the female flowers is 2 days after the blooming. The fruit is respiratory catamorph, and has short storage period, especially early maturing variety. Therefore, when selecting pollinator trees, the full-bloom stages of female flowers meet at the same flower stage, and pollinator trees with long flower stage are selected as far as possible.
Based on the research foundation, the phenological period of 16 domestic and foreign actinidia arguta resources in the continental region shown in the questionnaire 1 is shown in table 1. It can be seen that different varieties have certain differences in their flowering periods, the female plants have different flowering periods ranging from 7 days to 18 days, and the male plants have different flowering periods ranging from 7 days to 10 days. The male plant flowering period cannot fully cover all variety flowering periods in actual cultivation.
TABLE 1 survey of flowering phase of Actinidia arguta resource under facility cultivation conditions in Dalian region (2020)
2. Selection of pollen viability assay methods
After the pollen is cultured overnight by adopting an agar method, the pollen activity is quickly determined, and the result is shown in figure 2.
3. Self-flower weak (or fruit) phenomenon
Through the mode of bagging isolation processing, carry out the survey of the self-pollination seed setting rate of 3 varieties, the result is seen in table 2, although the kiwi fruit of soft date part resource has the self-pollination phenomenon, adopt the measure of not configurating the pollination tree in production can produce huge loss to output, especially under the facility cultivation condition, more need to dispose suitable pollination tree to different varieties.
TABLE 2 Seedness survey of self-pollination (2021 year)
| Seedness in self-pollination (%) | Investigation region | Investigation time | |
| Longcheng No. 2 | 0.0 | Dandong (Chinese character of' Dandong | 6.8-9.10 |
| Anna | 0.0 | Big link | 5.30-9.1 |
| Kukuwa | 52.0 | Big link | 6.5-9.5 |
Therefore, flowers in the initial flowering period of 'Long Cheng No. 2' are collected, petals are removed, the flowers are placed on an agar culture medium in a mode shown in a figure 3 overnight, and after the pollen viability is measured the next day, the 'Long Cheng No. 2' pollen is found to have viability, which indicates that the actinidia arguta has different self-compatibility.
4. Research on pollen collection and preservation technology
(1) Collecting pollen
The test material is male actinidia arguta, and the sample is collected in Guandian county, Dandong City, in 6 Yue.2016. Collecting the male flowers 1 day before flowering, in the bud period and 1 day after flowering respectively, wrapping with 2 layers of newspaper, taking back to the laboratory, removing petals, keeping the stamens, and drying in the shade for 1 day. Collecting different pollen test materials, placing into different centrifuge tubes, marking, and wrapping with tinfoil paper.
As can be seen from Table 3, the germination rates of the pollen collected 1 day before flowering, at the bud growth stage and 1 day after flowering were 84.2%, 87.4% and 75.2%, respectively. Therefore, the vitality of 1 trichosanthes root is higher in the bud period and before and after blooming, and the trichosanthes root can be used as a pollination material. But by combining with the breeding practice process, the method has more advantages in that the male flowers are collected 1 day before the male flowers bloom and then isolated for pollen extraction: firstly, the pollen has higher activity, and can fully meet the breeding requirement; secondly, pollen is collected in the current period without loose pollen loss, and the pollen collection amount is larger and more sufficient; thirdly, the possibility of pollution by other pollen can be reduced more easily.
TABLE 3 pollen germination rates of Actinidia arguta at different pollen-picking periods
| Powder collecting period | Pollen germination rate (%) |
| 1 day before flowering | 85.2 |
| Bud period | 87.4 |
| 1 day after flowering | 75.2 |
(2) Preservation of pollen
As can be seen from Table 4, the germination rate of pollen rapidly decreases with the increase of the room temperature preservation time, and after 10 days, the germination rate of pollen decreases to below 10%, and the activity of 12 radix Trichosanthis disappears. Therefore, the effect of preserving pollen at room temperature for a long period of time is poor. But if the pollen is stored for a short time (within 4 days), the pollen activity can still be kept above 50 percent, and the pollen can still be used for cross pollination. At 4 ℃, the reduction of the pollen viability is not obvious, the pollen viability is still over 50 percent after being stored for 30 days, and the pollen viability disappears after 120 days. Under the preservation condition of 20 ℃ below zero, the viability of the pollen is slowly reduced along with the prolonging of the storage time, the viability of the pollen is still more than 50 percent after 120 days, and the pollen basically loses the viability after 300 days. Therefore, the low-temperature treatment is beneficial to storing the pollen, and the lower the temperature is, the slower the pollen activity is reduced, and the longer the storage time is.
TABLE 4 pollen germination rate of Actinidia arguta at different storage temperatures
The pollen still keeps high activity through the determination after being stored for 1 year at the lower storage temperature of 80 ℃ below zero, and the result is shown in the table 5, thereby providing reliable technical support for the subsequent pollen storage.
TABLE 5-80 deg.C pollen viability assay after 1 year storage
| (Resource) | 2020.6 pollen viability (%) | 2021.6 pollen Activity (%) | Collection ground |
| No. 1 pollen | 89.2 | 68.7 | Large connecting piece |
| No. 2 pollen | 96.4 | 72.3 | Dandong (Chinese character of' Dandong |
| Pollen commercially available | 93.6 | 63.5 | Saddle mountain |
5. Research on pollen affinity detection technology
(1) Pollen of 4 pollinated trees is selected for in vitro culture for 24 hours, and strong activity is shown, and the result is shown in a table 6. This indicates that the collection of fresh pollen can completely meet the requirement of pollination in the flowering period.
TABLE 6 pollen viability assay for self-selected males
When the FAA-immobilized material was observed by a fluorescence microscope, the pollen tube was green. A large amount of pollen grains on the stigma of 4 self-pollination male affinity pollinated plants germinate, and pollen tubes at action sites smoothly pass through mastoid cells and extend into the stigma and the style (left in figure 4); in the case of self-incompatibility, pollen forms callose on stigma, but fails to form pollen tube (FIG. 4 right).
(2) Pollination affinity verification of different ploidy resources
Through flow cytometry, diploid, tetraploid and hexaploid resources are selected and identified for pollination affinity verification, and the method comprises the following steps: wiki, Adam, male plant selected from male red-yang plant. As can be seen from Table 7, although the ploidy of the male plant is different, the male plant can be used as the pollinated male plant of Actinidia arguta with good fruit setting. Fluorescence detection proves that the pollen can germinate on stigma and form pollen tube, and the result is shown in figure 5.
TABLE 7 pollination seed setting ratio survey of different ploidy male plants (2018-2021)
5. Comparative field test
In 2021, the investigation of the artificial pollination and natural pollination of Actinidia arguta in Tokyo village in Guandian county, Dandong, shows the results in Table 8. And (4) selecting fruit branches at different parts in the surface pollination treatment, bagging and isolating the fruit branches before the female flowers bloom, and counting the number of pollinated flowers of each combination after pollination. And (5) marking by hanging, harvesting after the seeds are mature, and calculating the maturing rate. Setting rate (real number of setting/number of pollinated flowers) × 100%.
From the statistical results in table 8, the self-cross of 'long-grown 2' shows incompatibility, which is consistent with the result of fluorescence detection, and indicates that for actinidia arguta, the field does not allocate pollinator trees, which causes great reduction or no harvest of yield, but the pollinator trees should be selected in a targeted manner, and pollinator trees covered in a full-bloom stage are selected as the pollinator varieties as much as possible. Meanwhile, the artificial pollination of the pollen No. 2, 3 and 4 is obviously improved compared with the natural pollination.
TABLE 8 Actinidia arguta artificial pollination and natural pollination survey (2021 year)
7. In-field configuration
The configured male plants must cover the flowering phase of the resulting variety. The pollination mode of actinidia arguta takes natural pollination as the main mode, and pollination is accomplished mainly through modes such as insect pollination, anemophily. The ratio of male plants to female plants is 8: 1. The male plant standard is that the initial flowering phase is earlier than or equal to the female plant, the flowering phase is long, and the pollen amount is large. The planting is configured in a mode of referring to a figure 1.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A screening method of male pollination plants in actinidia arguta orchard is characterized by comprising the following screening standards: florescence survey, pollen viability assay and pollen affinity detection.
2. The screening method according to claim 1, wherein the florescence survey comprises: and (5) screening pollinated trees meeting the flowering period of the fruiting trees.
3. The screening method according to claim 1, wherein the pollen viability assay comprises assaying the viability of fresh pollen and assaying the viability of preserved pollen.
4. The screening method of claim 1, wherein the pollen affinity test comprises early screening of callose stained and pollinated male plants and in-situ reaction of pollen on stigma.
5. The screening method according to claim 1, wherein the actinidia arguta orchard comprises an orchard during orchard establishment and/or an orchard when a garden is modified.
6. Use of the screening method of any one of claims 1 to 5 for male selection during orchard establishment or garden renovation.
7. Use of the screening method of any one of claims 1 to 5 in selection and matching of varieties during orchard establishment or garden improvement.
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