CN116656593A - Culture medium for improving germination rate of pigment marigold pollen - Google Patents

Culture medium for improving germination rate of pigment marigold pollen Download PDF

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Publication number
CN116656593A
CN116656593A CN202310773943.7A CN202310773943A CN116656593A CN 116656593 A CN116656593 A CN 116656593A CN 202310773943 A CN202310773943 A CN 202310773943A CN 116656593 A CN116656593 A CN 116656593A
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pollen
culture medium
germination rate
pigment
marigold
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许凤
张艺萍
王丽花
黄望启
张丽芳
杨秀梅
苏艳
蒋亚莲
钱绍方
雷正松
邹凌
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Flower Research Institute of YAAS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0025—Culture media for plant cell or plant tissue culture
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Abstract

The application relates to a culture medium for improving germination rate of pigment marigold pollen, which comprises the following components: caCl (CaCl) 2 2H2O 0.3-1.0g/L, boric acid 0.2-0.5g/L, mgSO 4 ·7H 2 O 0.3‑1.0g/L、KNO 3 0.2-0.5g/L, 60-100g/L, PEG400050-100g/L of sucrose and pH value of 6.0-7.5. The culture medium can enable the germination rate of the marigold pollen to reach more than 20 percent, which is about 10 percent higher than other culture media, thereby increasing the pollination success rate, reducing the planting area of male parent, improving the use efficiency of pollen and further improving the seed yield of unit area.

Description

Culture medium for improving germination rate of pigment marigold pollen
Technical Field
The application particularly relates to a culture medium for improving germination rate of pigment marigold pollen.
Background
Pigment marigold is an annual herb of the genus marigold of the family Compositae, and is one of the types of marigold. The original mexico and central america areas are also known as cellular chrysanthemum and cottonrose hibiscus. The marigold has strong resistance, cold resistance, drought resistance, low soil requirement, strong adaptability, easy cultivation and good management.
The pigment marigold is used as one of main plants for extracting natural lutein for economic crop cultivation. Lutein can be widely applied to various industries such as food, medicine, feed, cosmetics and the like, has an important effect on eye health care, is an important flower for processing, is called as soft gold, and has good prospect because the natural pigment is in short supply in the market. At present, pigment marigold is planted in a plurality of places in Yunnan of China, and the planting area of the pigment marigold planted in Yunnan province reaches more than 40 ten thousand mu by the end of 2022.
Pigment marigold for extracting lutein in the current market is an F1 generation variety bred by utilizing a male sterile dual-purpose line. In the F1 generation seed production, the planting ratio of the male parent to the female parent is 4-5 because the pollen of the male parent is less and the pollen viability is low: 1. the germination rate of pollen is a main mark for measuring the vigor condition of pollen, so if the germination rate of pigment marigold pollen can be improved, the pollination success rate can be increased, the planting area of male parent can be reduced, the use efficiency of pollen can be improved, and the seed yield per unit area can be improved.
Disclosure of Invention
Aiming at the problems of large pollen consumption, insufficient pollen and the like caused by low pollen viability in the process of preparing the pigment marigold seeds, the application provides the culture medium for improving the germination rate of the pigment marigold pollen.
In order to achieve the above purpose, the application is realized by the following technical scheme:
the culture medium for improving the germination rate of pigment marigold pollen comprises CaCl 2 ·2H 2 O0.3-1.0 g/L, boric acid 0.2-0.5g/L, mgSO 4 ·7H 2 O 0.3-1.0g/L、KNO 3 0.2-0.5g/L, sucrose 60-100g/L, PEG400050-100g/L, pH 6.0-7.5.
Further preferably, the culture medium for improving germination rate of pigment marigold pollen comprises CaCl2.2H 2 0.3-0.5g/L O, 0.2-0.3g/L, mgSO boric acid 4 ·7H2O 0.3-0.5g/L、KNO 3 0.2-0.3g/L, sucrose 60g/L, PEG4000100g/L, pH6.0-7.0.
Further preferably, the culture medium for improving germination rate of pigment marigold pollen comprises CaCl 2 ·2H 2 O0.3 g/L, boric acid 0.2g/L, mgSO 4 ·7H 2 O 0.3g/L、KNO 3 0.2g/L, sucrose 60g/L, PEG400050g/L, pH 7.0.
CaCl 2 ·2H 2 O is the dihydrate of calcium chloride, which upon dissolution in water completely dissociates into calcium ions (Ca 2+ ) And chloride ions (Cl-). This means that the pollen comes into contact with CaCl 2 When in solution, the solution can quickly absorb and utilize calcium and chloride ions in the solution to provide nutrient elements required by plants. Furthermore, caCl 2 ·2H 2 O promotes pollen germination by providing calcium ions and chloride ions. Calcium ions play an important role in cell division, cell wall formation, cell signaling, and the like. Chloride ions participate in physiological processes such as photosynthesis and ion balance. Ca (NO) 3 ) 2 ·4H 2 O can have problems that excessive application is easy, so that excessive nitrate ion supply is caused, and thus accumulation of nitrate ions in soil is caused, and soil acidification can be caused.
Boric acid: boron is a trace element and has an important effect on pollen tube growth. It promotes the elongation and directional growth of pollen tube and maintains its morphological stability. Boron is also involved in cell wall synthesis and cell division processes and is critical to pollen tube development.
MgSO 4 ·7H 2 O (magnesium sulfate): providing magnesium ions (Mg) 2+ ) Providing inorganic salts for pollen growth, which are also activators of many enzymes, play a key role in cellular metabolism and energy conversion processes. Magnesium ions participate in important steps such as carbon metabolism, RNA synthesis and the like in the growth process of the pollen tube, and promote the normal growth of the pollen tube.
KNO 3 (potassium nitrate): providing a nitrogen source, nitrate ions (NO 3- ) Providing necessary nitrogen element for pollen, and participating in protein synthesis and metabolic process. Potassium nitrate can also regulate osmotic regulating substances and ion balance, and maintain normal growth of pollen tube.
Sucrose: as a substrate for pollen metabolism, the preparation method provides a carbon source required by pollen, promotes pollen germination and pollen tube growth, and provides necessary carbon-based raw materials for metabolic processes such as cell division, cell wall synthesis and the like.
PEG4000: polyethylene glycol (PEG) is a osmolyte that helps maintain water balance in the culture medium and increases the moisture absorbing capacity of pollen. PEG4000 helps to accelerate the pollen germination process by adjusting the osmotic pressure of the culture medium.
The above components have synergistic effect, and cooperate with each other to promote germination and growth of pollen. They act synergistically to ensure normal development and growth of pollen by enhancing the effects on cell wall synthesis, energy supply and cell activity. The application also provides a method for detecting the germination rate of the pigment marigold pollen, which comprises the following steps:
(1) Preparation of the culture medium: preparing a culture medium according to the culture medium formula;
(2) Collecting pollen: and 11:00-13 a.m. on sunny days: 00, collecting pollen of tubular flowers, and loading the collected pollen into a culture dish;
(3) Culturing pollen: uniformly scattering the pollen on a culture medium, and culturing in an incubator at 30 ℃ for 20 hours;
(4) And (5) microscopic examination, and calculating germination rate.
The application has the beneficial effects that:
1. the culture medium for improving the germination rate of the pigment marigold pollen tests the activity under different pH conditions, and selects the pH suitable for the germination of the pigment marigold pollen.
2. The germination rate of the marigold pollen is more than 20%, which is about 10% higher than other culture media, so that the pollination success rate can be increased, the planting area of the male parent can be reduced, the pollen use efficiency can be improved, and the seed yield per unit area can be improved.
Drawings
FIG. 1 shows a view of pollen germination under a microscope at 25X40 magnification;
FIG. 2 is a schematic diagram of marigold pollen germination.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present application more apparent, preferred embodiments of the present application will be described in detail below to facilitate understanding by the skilled person.
Examples 1 to 5:
CaCl is adopted 2 ·2H 2 O, boric acid and MgSO 4 ·7H 2 OL、KNO 3 The above components of sucrose and PEG4000 are used for preparing culture medium, and 5 examples are provided, and the specific culture medium formulation and the dosage are shown in Table 1.
Table 1 examples 1-5 medium formulations and pH
Comparative example:
a comparative example was set up using Ca (NO 3 ) 2 ·4H 2 O, boric acid and MgSO 4 ·7H 2 OL、KNO 3 The components above sucrose and PEG4000 are used for preparing a culture medium, and the specific formula and germination rate are shown in Table 3.
Table 2 comparative example medium formulation and germination
11:00-13 am on sunny days: 00 collecting pollen of tubular flowers, uniformly scattering the collected pollen on each culture medium, and culturing in an incubator at 30 ℃ for 20 hours. And (5) microscopic examination, and calculating germination rate. The results are detailed in Table 3.
TABLE 3 germination of examples 1-5
As shown in Table 3, the culture medium provided by the application is used for culturing the pollen of the pigment marigold, so that the germination rate of the pollen is effectively provided, and is over 20 percent, and is about 10 percent higher than that of the comparative example
CaCl 2 ·2H 2 O is the dihydrate of calcium chloride, which upon dissolution in water completely dissociates into calcium ions (Ca 2+ ) And chloride ions (Cl) - ). This means that the pollen comes into contact with CaCl 2 When in solution, the solution can quickly absorb and utilize calcium and chloride ions in the solution to provide nutrient elements required by plants. Furthermore, caCl 2 ·2H 2 O promotes pollen germination by providing calcium ions and chloride ions. Calcium ions play an important role in cell division, cell wall formation, cell signaling, and the like. Chloride ions participate in physiological processes such as photosynthesis and ion balance. Ca (NO) 3 ) 2 ·4H 2 O can have problems that excessive application is easy, so that excessive nitrate ion supply is caused, and thus accumulation of nitrate ions in soil is caused, and soil acidification can be caused.
Boric acid: boron is a trace element and has an important effect on pollen tube growth. It promotes the elongation and directional growth of pollen tube and maintains its morphological stability. Boron is also involved in cell wall synthesis and cell division processes and is critical to pollen tube development.
MgSO 4 ·7H 2 O (magnesium sulfate): providing magnesium ions (Mg) 2+ ) Providing inorganic salts for pollen growth, which are also activators of many enzymes, contribute to cellular metabolism and energy conversion processesKey function. Magnesium ions participate in important steps such as carbon metabolism, RNA synthesis and the like in the growth process of the pollen tube, and promote the normal growth of the pollen tube.
KNO 3 (potassium nitrate): providing a nitrogen source, nitrate ions (NO 3- ) Providing necessary nitrogen element for pollen, and participating in protein synthesis and metabolic process. Potassium nitrate can also regulate osmotic regulating substances and ion balance, and maintain normal growth of pollen tube.
Sucrose: as a substrate for pollen metabolism, the preparation method provides a carbon source required by pollen, promotes pollen germination and pollen tube growth, and provides necessary carbon-based raw materials for metabolic processes such as cell division, cell wall synthesis and the like.
PEG4000: polyethylene glycol (PEG) is a osmolyte that helps maintain water balance in the culture medium and increases the moisture absorbing capacity of pollen. PEG4000 helps to accelerate the pollen germination process by adjusting the osmotic pressure of the culture medium.
In summary, with the culture medium of this configuration, the components mutually promote, enhance or regulate the functions of each other to achieve a synergistic effect, and by enhancing the influence on cell wall synthesis, energy supply and cell activity, the germination and growth of pollen are promoted to the greatest extent, and necessary nutrition and energy are provided for pollen to support the development process thereof.
Effects of different pH conditions on pollen germination:
the medium was prepared using the formulation of example 3, except that the pH values were set to 1, 5.5, 6, 6.5, 7, 7.5 in this order. The results are detailed in Table 4.
TABLE 4 germination under different pH conditions
As shown in Table 4, the effect was better when the pH was 7.
The pollen germination rate is a main mark for measuring the vigor condition of pollen, and can increase the pollen germination rate and the pollination success rate. TTC method and spore powder dyeing method are adopted to test the vitality of pigment marigold pollen, and although the pollen can be dyed, the vitality pollen and abortive pollen can not be well distinguished. By adopting the culture medium provided by the application, the vigor of the marigold pollen can be improved by the cooperation of the components, so that the pollination rate is increased, and meanwhile, the in-vitro germination method of the culture medium can intuitively and accurately reflect the vigor of the pigment marigold pollen.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the application, and that, although the application has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the application as defined by the appended claims.

Claims (4)

1. A culture medium for improving germination rate of pigment marigold pollen is characterized in that: comprises CaCl 2 ·2H 2 0.3-1.0g/L O, 0.2-0.5g/L, mgSO boric acid 4 ·7H 2 O 0.3-1.0g/L、KNO 3 0.2-0.5g/L, 60-100g/L, PEG400050-100g/L of sucrose and pH value of 6.0-7.5.
2. The culture medium for improving germination rate of pigment marigold pollen as claimed in claim 1, wherein the culture medium is characterized by comprising the following components in percentage by weight: comprises CaCl 2 ·2H 2 0.3-0.5g/L O, 0.2-0.3g/L, mgSO boric acid 4 ·7H2O 0.3-0.5g/L、KNO 3 0.2-0.3g/L, sucrose 60g/L, PEG4000100g/L, pH6.0-7.0.
3. A culture medium for improving germination rate of pigment marigold pollen according to claim 1 or 2, characterized in that: comprises CaCl 2 ·2H 2 O0.3 g/L, boric acid 0.2g/L, mgSO 4 ·7H 2 O 0.3g/L、KNO 3 0.2g/L, sucrose 60g/L, PEG400050g/L, pH 7.0.
4. A method for detecting germination rate of pigment marigold pollen is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparation of the culture medium: preparing a medium according to the medium formulation of any one of claims 1-3;
(2) Collecting pollen: and 11:00-13 a.m. on sunny days: 00, collecting pollen of tubular flowers, and loading the collected pollen into a culture dish;
(3) Culturing pollen: uniformly scattering the pollen on a culture medium, and culturing for 20 hours in an incubator at 30 ℃;
(4) And (5) microscopic examination, and calculating germination rate.
CN202310773943.7A 2023-06-28 2023-06-28 Culture medium for improving germination rate of pigment marigold pollen Pending CN116656593A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117898209A (en) * 2024-03-19 2024-04-19 云南省农业科学院花卉研究所 Method for rapidly collecting pollen in pigment marigold seed production process

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117898209A (en) * 2024-03-19 2024-04-19 云南省农业科学院花卉研究所 Method for rapidly collecting pollen in pigment marigold seed production process
CN117898209B (en) * 2024-03-19 2024-06-11 云南省农业科学院花卉研究所 Method for rapidly collecting pollen in pigment marigold seed production process

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