CN117898209A - Method for rapidly collecting pollen in pigment marigold seed production process - Google Patents
Method for rapidly collecting pollen in pigment marigold seed production process Download PDFInfo
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- CN117898209A CN117898209A CN202410312395.2A CN202410312395A CN117898209A CN 117898209 A CN117898209 A CN 117898209A CN 202410312395 A CN202410312395 A CN 202410312395A CN 117898209 A CN117898209 A CN 117898209A
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- 235000005881 Calendula officinalis Nutrition 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000000049 pigment Substances 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title claims description 14
- 240000000785 Tagetes erecta Species 0.000 title abstract description 5
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- 230000006698 induction Effects 0.000 claims abstract description 15
- 238000005520 cutting process Methods 0.000 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 9
- 241000736851 Tagetes Species 0.000 claims description 29
- 238000005286 illumination Methods 0.000 claims description 19
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 14
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 238000007789 sealing Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 230000035755 proliferation Effects 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 230000007480 spreading Effects 0.000 claims description 5
- 238000003892 spreading Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 239000012869 germination medium Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 3
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 2
- 230000035784 germination Effects 0.000 claims description 2
- 229960002523 mercuric chloride Drugs 0.000 claims description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 230000035899 viability Effects 0.000 description 14
- 238000009472 formulation Methods 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 4
- 239000012879 subculture medium Substances 0.000 description 4
- 235000012311 Tagetes erecta Nutrition 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 235000012308 Tagetes Nutrition 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 239000001656 lutein Substances 0.000 description 2
- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 description 2
- 229960005375 lutein Drugs 0.000 description 2
- ORAKUVXRZWMARG-WZLJTJAWSA-N lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C ORAKUVXRZWMARG-WZLJTJAWSA-N 0.000 description 2
- 235000012680 lutein Nutrition 0.000 description 2
- 230000010152 pollination Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 2
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 description 2
- 241001124076 Aphididae Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
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- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000026786 pollen maturation Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- 231100000747 viability assay Toxicity 0.000 description 1
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Classifications
-
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The application relates to a method for rapidly collecting pollen in the process of preparing pigment marigold seeds, which comprises the following steps: (1) seed pretreatment; (2) aseptic seedling acquisition; (3) adventitious bud induction culture: cutting the obtained leaves of the aseptic seedlings, and inoculating the cut leaves to an adventitious bud induction culture medium for culture; (4) adventitious bud bloom and pollen collection; (5) subculture; and (3) cutting off roots of the root seedlings after the pollen is collected, transferring the root seedlings into a new culture medium for continuous subculture, and allowing the root seedlings to continuously bloom and collecting the pollen. The method is quick and simple, and a large number of seedlings which are tidy, uniform and consistent in purity are obtained in a short period based on tissue culture, so that pollen can be collected conveniently and quickly and efficiently, and the activity of the obtained pollen is still high during subculture.
Description
Technical Field
The application relates to the technical field of plant breeding, in particular to a method for rapidly collecting pollen in a pigment marigold seed production process.
Background
Tagetes erecta (Tagetes erecta L.) is an annual herb of the genus Tagetes of the family Compositae. Tagetes erecta is cultivated in mexico, all places in China. The pigment marigold contains rich lutein, and the lutein can be used as raw materials of foods, feeds and medicines, meanwhile, the pigment marigold is usually a head-like inflorescence, the color is mainly orange, the flower is large, the flower petal is distinct in layering, and the shape is unique, so that the pigment marigold has high ornamental and medicinal values.
In the actual production of the pigment marigold hybrid (F1), pollen sucking devices are commonly used for collecting pollen of restorer lines. However, because of scattered pollen of the fertility line in the air, i.e. other pollen sources (such as male fertility plants) exist in the pollen collecting area, cross contamination of pollen is caused, which tends to cause the reduction of seed purity, and the risk of leakage of core germplasm resources exists; at the same time, pigment marigold is usually sown at the beginning of 3 months, transplanted at the beginning of 4 months and pollen is collected at the beginning of 6 months. About 130 days are required from sowing to collecting pollen, and the production period is long. In addition, the Yunnan enters the rainy season for 6 months, the air is relatively moist, which is unfavorable for collecting pollen, and the pollen storage life is greatly shortened, the pollen activity is reduced, and the pollination effect is affected. Thus, pollen collection, storage and pollination are significantly limited by seasons. In addition, in rainy season, marigold is more susceptible to various factors such as gray mold, brown spot, leaf blight and aphid, and the like, so that the problems of plant growth vigor weakness, pollen yield reduction and the like are caused.
Based on the above, in recent years, along with the development of plant tissue culture technology, tissue culture is adopted to rapidly reproduce pigment marigold with excellent variety characteristics, so that a large number of healthy and uniform seedlings can be produced in a short period. However, the applicant found that in the process of culturing pigment marigold by using a tissue culture technique, the pollen viability of plants after primary culture often decreases during the secondary culture, and the pollen viability decreases more and more with the increase of the number of secondary culture. That is, although a large amount of pollen can be obtained in a short time by tissue culture, the quality of pollen viability obtained in the subculture is not stable, and thus the seed production process is affected.
Disclosure of Invention
In order to solve or partially solve the problems existing in the related art, the application provides a method for rapidly collecting pollen in the process of preparing pigment marigold seeds, which is rapid and simple, obtains a large number of uniform and consistent seedlings in a short period based on tissue culture, thereby facilitating rapid and efficient pollen collection, and ensures the pollen activity due to higher pollen activity during subculture.
The method for rapidly collecting pollen in the process of preparing the pigment marigold seeds comprises the following steps:
(1) Seed pretreatment
Sun-drying pigment marigold restorer seeds in the sun, sterilizing the seeds, and washing with sterile water for later use;
(2) Acquisition of aseptic seedlings
Spreading the standby seeds obtained in the step (1) on a germination culture medium in an ultra-clean workbench, sealing, and transferring the seeds into a culture room for culture to obtain aseptic seedlings;
(3) Adventitious bud induction culture
Cutting the obtained sterile seedling leaves, inoculating the obtained sterile seedling leaves to an adventitious bud induction culture medium, sealing the culture medium, transferring the culture medium into a culture chamber, culturing for one week under the condition of 500lx illumination intensity, and transferring the culture medium into the culture chamber under the condition of 2500-3000lx illumination intensity until regenerated adventitious buds are obtained; the formula of the adventitious bud induction culture medium is as follows: MS powder 4.4g/L, 6-BA 10mg/L, IAA mg/L, silver nitrate 2mg/L, agar 8g/L, sugar 30g/L;
(4) Adventitious bud flowering and pollen collection
Inoculating the regenerated adventitious bud obtained by induction into a proliferation culture medium under the incision of an ultra-clean bench, sealing, transferring into a culture chamber for rooting culture until flowering, and collecting pollen;
(5) Cutting off roots of the collected rooting seedlings, transferring the rooting seedlings into a new culture medium for continuous subculture, and allowing the rooting seedlings to continuously bloom and collect pollen;
The formula of the culture medium for the secondary culture is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L, sugar 30g/L;
The conditions for the subculture are: culturing under illumination intensity of 2500-3000 lx; the culture temperature is 24+/-2 ℃, the illumination time is 16h/d, and the relative humidity is 65% -75%.
Alternatively, in some embodiments, the culture chamber conditions in step (3) are a culture temperature of 24.+ -. 2 ℃ and an illumination time of 16h/d, and a relative humidity of 65% -75%.
Optionally, in some embodiments, the step (1) of airing specifically includes: the drying time is 3d, the spreading thickness of the seeds is not more than 0.5mm, and the drying temperature is 18-28 ℃.
Optionally, in some embodiments, the germination medium formulation in step (2) is: MS powder 4.4g/L, TDZ 0.3.3 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
Optionally, in some embodiments, the proliferation medium formulation in step (4) is: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
The technical scheme provided by the application can comprise the following beneficial effects:
(1) By utilizing the method, the production period of the traditional seed production 130 d is shortened to about 80 days, the time required for pollen maturation is greatly shortened, the problems that the pollen yield and activity are easily affected by seasons and the pollen yield is reduced due to plant diseases and insect pests and other sudden weather before pollen collection are avoided, the pollen production is not limited by the seasons, the seed production process is fully ensured, and the preservation of germplasm resources and the popularization of good varieties are facilitated.
(2) The method for rapidly collecting pollen in the process of preparing the pigment marigold seeds is characterized in that after primary culture, the primary culture is carried out through a specific secondary culture medium, and on the basis that part of pollen is adopted after flowers are already bloomed, a large number of pigment marigold regeneration buds are continuously induced at high frequency and flowers are formed in the original branches, so that a large number of pollen is obtained in a short time. Meanwhile, when the method is used for subculture, the generation of vitrified seedlings can be effectively avoided, the quality of tissue culture seedlings during subculture is ensured, and the activity of the obtained pollen is further ensured.
(4) By utilizing the method, the condition of pollen cross contamination is avoided, the purity of the hybrid seeds is ensured, the problem that the pigment marigold restorer is not homozygous in genotype and the color of the hybrid seeds is different is avoided, and the risk of core germplasm resource leakage is reduced.
Detailed Description
Embodiments of the present application will be described in more detail below with reference to examples. While embodiments of the present application have been illustrated in the examples, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.
Example 1
The method for rapidly collecting pollen in the process of preparing the pigment marigold seeds in the embodiment comprises the following steps:
(1) Pigment marigold restorer seeds (Bohao 3, from Yunnan Bohao biotechnology group Co., ltd.) were sun-dried for 3d at a seed thickness of not more than 0.5mm and a sun-drying temperature of 18-28 ℃. And (3) vibrating and sterilizing seeds in an ultra-clean workbench for 10min by using 2% sodium hypochlorite, vibrating and sterilizing the seeds for 10min by using 0.1% mercuric chloride, and flushing the seeds for 4 times by using sterile water for later use.
(2) Spreading the sterilized seed on germination medium in an ultra-clean workbench, sealing, and transferring into a culture room under the culture conditions of 24+ -2deg.C, 2500-3000lx illumination intensity, 16h/d illumination time, 65% -75% relative humidity, wherein the germination medium comprises the following formula: MS powder 4.4g/L, agar 8g/L and sugar 30g/L, the seeds germinate partially on the next day, and the aseptic seedlings are obtained after continuous culture until cotyledons grow out.
(3) Cutting the leaves of the aseptic seedling plants in an ultra-clean bench, inoculating the leaves into an adventitious bud induction culture medium, sealing the culture medium with the inoculated leaves, transferring the culture medium into a culture chamber, performing micro-light culture for one week (the illumination intensity is 500 lx), and transferring the culture medium into light for culture (the illumination intensity is 2500-3000 lx); other culture conditions of the culture chamber are that the temperature is 24+/-2 ℃, the illumination time is 16h/d, and the relative humidity is 65% -75%. The formula of the adventitious bud induction culture medium is as follows: MS powder 4.4g/L, 6-BA 10mg/L, IAA mg/L, silver nitrate 2mg/L, agar 8g/L, sugar 30g/L. And finally obtaining regenerated adventitious buds through adventitious bud induction culture.
(4) Inoculating the induced regenerated adventitious buds to a proliferation culture medium in an ultra-clean bench, sealing, transferring into a culture chamber for rooting culture until flowering, and collecting pollen from the bloomed flowers. The viability of pollen harvested after primary culture was examined.
Wherein, the formula of the proliferation culture medium is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L, sugar 30g/L; the culture conditions of the culture chamber are as follows: the illumination time is 16h/d at 24+/-2 ℃ and the relative humidity is 65-75 percent.
(5) And (3) culturing the rooting seedlings which are flowering and pollen collecting, cutting the roots of the rooting seedlings, transferring the rooting seedlings into a new culture medium, continuing to perform first subculture, and allowing the rooting seedlings to continuously flowering and pollen collecting. The viability of the pollen harvested after the first subculture was examined.
Wherein, the formula of the culture medium for the first subculture is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
The conditions for the subculture are: culturing under illumination intensity of 2500-3000 lx; the culture temperature is 24+/-2 ℃, the illumination time is 16h/d, and the relative humidity is 65% -75%.
Example 2
Carrying out first subculture until the rooting seedlings are flowering and pollen is collected in the embodiment 1, cutting off roots of the rooting seedlings, transferring the rooting seedlings into a new culture medium, continuing second subculture, and collecting pollen after the rooting seedlings continue to flowering;
Wherein, the formula of the culture medium for the secondary subculture is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
Conditions for the subculture were the same as in example 1.
Subculturing the root seedlings after flowers and pollen are collected for the second time in the embodiment, cutting off the roots of the root seedlings, transferring the root seedlings into a new culture medium for further subculture for the third time, and collecting pollen after flowers are continued;
Wherein, the formula of the culture medium for the third subculture is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
And detecting the vitality of pollen harvested after secondary culture and tertiary culture.
Example 3
Carrying out first subculture until the rooting seedlings are flowering and pollen is collected in the embodiment 1, cutting off roots of the rooting seedlings, transferring the rooting seedlings into a new culture medium, continuing second subculture, and collecting pollen after the rooting seedlings continue to flowering;
Wherein, the formula of the culture medium for the secondary subculture is as follows: 6.0g/L, IAA 3.0.0 mg/L of vegetable gel, 3.0 mg/L of 6-BA and 30g/L of sugar;
Conditions for the subculture were the same as in example 1.
Subculturing the root seedlings after flowers and pollen are collected for the second time in the embodiment, cutting off the roots of the root seedlings, transferring the root seedlings into a new culture medium for continuing the third subculture, and allowing the root seedlings to continuously flower and collect pollen;
wherein, the formula of the culture medium for the third subculture is as follows: 6.0g/L, IAA 3.0.0 mg/L of vegetable gel, 3.0 mg/L of 6-BA and 30g/L of sugar;
and detecting the vitality of pollen harvested after secondary culture and tertiary culture.
Example 4
Subculturing the root seedlings obtained after flowering and pollen collection in the embodiment 3 for the second time, cutting off the roots of the root seedlings, transferring the root seedlings into a new culture medium for continuing the third subculture, and collecting pollen after the root seedlings continue flowering;
wherein, the formula of the culture medium for the third subculture is as follows: 6.0g/L, IAA 3.0.0 mg/L of vegetable gel, 3.0 mg/L of 6-BA and 30g/L of sugar;
The viability of pollen harvested after the third subculture was examined.
The method for detecting pollen viability according to the present application, specifically TTC staining, is well understood by those skilled in the art.
Meanwhile, as comparison, pollen viability is compared with pollen produced by greenhouse seedlings of pigment marigold and field seedlings which are obtained from the same variety but are respectively cultivated in a greenhouse and a field according to a conventional cultivation method. The results of pollen viability assay are shown in the following table:
The above pollen measurement results show that the tissue culture seedlings of example 1 have slightly lower pollen viability than greenhouse seedlings and field seedlings, but have no significant difference. In the first subculture using the subculture medium of the present invention, the results of the test unexpectedly found that the pollen viability after the first subculture (73.65%) was even higher than the pollen viability harvested after the first culture (69.52%).
Comparing example 2 with example 3, the common features are: the tissue culture seedlings obtained after the first subculture in example 1 were used as the raw materials for the second subculture, except that the formulation of the subculture medium was different between the second subculture and the third subculture. In addition, the formulation of the subculture medium in example 3 is selected from the prior art subculture formulations with good seedling formation performance when the subculture is performed, and the specific sources of the formulation are: zhao Zigang, zeng Li, tang Kexuan, quantum, sun Jia, yang Fan. Establishment of regeneration System for pigment Tagetes leaves [ J ]. Gardening school report 2010, 37 (4): 655-660. As can be seen from the results in example 2 and example 3, the secondary culture and the third secondary culture using the formulation of the secondary culture medium of the present application are significantly higher in pollen viability than the formulation of the prior art which shows better seedling formation when the secondary culture is performed. When the third subculture is carried out by utilizing the formula of the subculture medium, the activity (71.41%) of pollen obtained by the third subculture is even higher than that of pollen obtained by the second subculture (67.52%); in example 3, the viability of the pollen obtained by the third subculture (55.66%) was lower than that of the pollen obtained by the second subculture (62.37%).
Further, as can be seen from the results of example 4, when the secondary subculture was performed using the formulation of the secondary culture medium of example 1 and the tertiary subculture was performed using the formulation of the secondary culture medium of example 3, the viability of pollen obtained after the tertiary subculture (51.45%) was significantly lower than that of pollen obtained by the tertiary subculture of example 2 (71.41%), which also illustrates the key effect of the formulation of the secondary culture medium of example 1 in the present application.
The foregoing description of embodiments of the application has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Claims (7)
1. A method for rapidly collecting pollen in the process of preparing pigment marigold seeds, which is characterized by comprising the following steps:
(1) Seed pretreatment
Sun-drying pigment marigold restorer seeds in the sun, sterilizing the seeds, and washing with sterile water for later use;
(2) Acquisition of aseptic seedlings
Spreading the standby seeds obtained in the step (1) on a germination culture medium in an ultra-clean workbench, sealing, and transferring the seeds into a culture room for culture to obtain aseptic seedlings;
(3) Adventitious bud induction culture
Cutting the obtained sterile seedling leaves, inoculating the obtained sterile seedling leaves to an adventitious bud induction culture medium, sealing the culture medium, and transferring the culture medium into a culture chamber for adventitious bud induction culture;
(4) Adventitious bud flowering and pollen collection
Inoculating the regenerated adventitious bud obtained by induction into a proliferation culture medium under the incision of an ultra-clean bench, sealing, transferring into a culture chamber for rooting culture until flowering, and collecting pollen;
(5) Subculture
Cutting off roots of the collected rooting seedlings, transferring the rooting seedlings into a new culture medium for continuous subculture, and allowing the rooting seedlings to continuously bloom and collect pollen;
the formula of the culture medium for the secondary culture is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L, sugar 30g/L;
The culture conditions for the subculture are as follows: culturing under illumination intensity of 2500-3000 lx; the culture temperature is 24+/-2 ℃, the illumination time is 16h/d, and the relative humidity is 65% -75%.
2. The method for rapidly collecting pollen during the process of preparing seeds of marigold pigment according to claim 1, wherein the culture condition of the culture chamber in the step (3) is that the temperature is 24+/-2 ℃, the illumination time is 16h/d, and the relative humidity is 65% -75%.
3. The method for rapidly collecting pollen during the process of preparing seeds of marigold pigment according to claim 1, wherein the step (1) of airing comprises the following specific operations: the drying time is 3d, the spreading thickness of the seeds is not more than 0.5mm, and the drying temperature is 18-28 ℃.
4. The method for rapidly collecting pollen during the production of marigold seeds according to claim 1, wherein the sterilization in step (1) is performed by: the mixture is sterilized by shaking with 2% sodium hypochlorite for 10min, and then is sterilized by shaking with 0.1% mercuric chloride for 10min.
5. The method for rapidly collecting pollen during the production of marigold seeds as claimed in claim 1, wherein the germination medium in the step (2) is formulated as follows: MS powder 4.4g/L, TDZ 0.3.3 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
6. The method for rapidly collecting pollen during the production of marigold seeds according to claim 1, wherein the proliferation medium formula in the step (4) is as follows: MS powder 4.4g/L, TDZ 0.2.2 mg/L, IBA 0.1.1 mg/L, agar 8g/L and sugar 30g/L.
7. The method for rapidly collecting pollen during the production of marigold seeds according to claim 1, wherein the adventitious bud induction medium in the step (3) is formulated as follows: MS powder 4.4g/L, 6-BA10 mg/L, IAA mg/L, silver nitrate 2mg/L, agar 8g/L, sugar 30g/L; the culture conditions of the adventitious bud induction culture are as follows: culturing for one week under 500lx illumination intensity, and transferring into 2500-3000lx illumination intensity for culturing; the culture temperature is 24+/-2 ℃, the illumination time is 16h/d, and the relative humidity is 65% -75%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410312395.2A CN117898209B (en) | 2024-03-19 | 2024-03-19 | Method for rapidly collecting pollen in pigment marigold seed production process |
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| CN202410312395.2A CN117898209B (en) | 2024-03-19 | 2024-03-19 | Method for rapidly collecting pollen in pigment marigold seed production process |
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| CN117898209A true CN117898209A (en) | 2024-04-19 |
| CN117898209B CN117898209B (en) | 2024-06-11 |
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