CN111699970A - Pear immature embryo culture method - Google Patents
Pear immature embryo culture method Download PDFInfo
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- CN111699970A CN111699970A CN202010410106.4A CN202010410106A CN111699970A CN 111699970 A CN111699970 A CN 111699970A CN 202010410106 A CN202010410106 A CN 202010410106A CN 111699970 A CN111699970 A CN 111699970A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for culturing pear immature embryos, and relates to a method for culturing the somatic embryos of pears (pear bretschneideri) by a plant cell culture technology so as to break dormancy of pear seeds and realize higher seed germination rate. The method takes immature embryos or mature embryos of pears as explants, establishes a cultivation technical system of in vitro embryos of pears through explant disinfection, embryo cultivation, rooting cultivation and seedling hardening and transplanting, lays a foundation for establishing a pear regeneration system and improving pear varieties by using genetic engineering and cell engineering, can obviously shorten breeding process, improves breeding efficiency and opens up a new breeding way.
Description
Technical Field
The invention relates to a breeding method for in vitro rapid propagation in plant biotechnology, in particular to a pear immature embryo culture method.
Background
The pear is one of the main fruits in the world and is the third largest fruit which is second to apples and oranges in China. Pears are one of the longest fruit trees in the planting history in China, and as early as the early days of the Qin, Shijing has records of 'mountain with skipper and low marshy land with '. The pear is one of the fruit trees with the widest planting range in China, and according to the latest statistical data of the Food and Agriculture Organization (FAO) of the United nations in 1 month in 2019, the cultivation area of the pear in China is about 96 million hectares, the total yield is about 1653 million tons, the pear occupies 69.1 percent and 68.4 percent of the total cultivation area and the total yield in the world respectively, and the pear stably stays in the first place in the world.
Through long-term natural selection and production development, China has gradually established dominant production areas of autumn pears, white pears, Chinese pears and the like. According to the national pear key area development plan issued by the Ministry of agriculture in 2009, the traditional pear dominant producing area is divided into three areas and four points, and stronger regional characteristics and production advantages are embodied. In recent years, with the rapid development of new early-medium-maturing varieties such as Cuiguan and Huangguan, the southern early-maturing pear industry in Yangtze river basin and south areas thereof has a new and more diverse appearance, and the quality and economic benefit of pears are remarkably improved.
The pears can be propagated by adopting methods such as sowing, grafting, cuttage and the like, but the methods have certain limitations. The two modes of sowing and propagation are spring sowing and autumn sowing respectively, wherein the seeds sowed in spring need to be subjected to pregermination, and the seeds sowed in autumn need to be soaked and washed, so that the propagation time is longer. In the cutting mode, robust branches without pest and disease damage are required to be selected as cutting slips, so that the material consumption is high and the weather influence is easily caused. The grafting mode needs thinner manual operation, the propagation efficiency is low, and the scions are not easy to survive. These factors have greatly limited the development of the pear industry. Under natural conditions, the seeds of the pear fruits can germinate after ripening generally in the next year, and the water obstruction and the endosperm wrapping effect of lignified seed shells can be the reasons for dormancy of the pear seeds. At present, few research reports about the culture of pear immature embryos exist, and mature and complete theories and method systems are not formed. Therefore, it is necessary to establish a pear embryo culture technical system and accumulate experience for constructing a pear regeneration system.
Disclosure of Invention
The invention aims to provide a pear immature embryo culture method, which takes immature embryos or mature embryos of pears as explants, establishes a culture technical system of in vitro immature embryos of pears through explant disinfection, immature embryo culture, rooting culture and hardening and transplanting, can obviously shorten the breeding process, improves the breeding efficiency and opens up a new breeding way.
The technical problem solved by the invention is realized by adopting the following technical scheme:
a method for culturing pear immature embryos comprises the following steps:
(1) and (3) explant disinfection treatment: selecting immature pear fruits which are pollinated for 5-6 weeks, performing vernalization treatment for 3-5d at a low temperature after picking, washing the vernalized fruits with water, peeling off peels and pulps to obtain immature embryos or mature embryos with seed coats, fully washing the immature embryos or the mature embryos with the seed coats with water (preferably washing the immature embryos or the mature embryos with tap water for 1-2 times and then washing the immature embryos or the mature embryos with the seed coats with distilled water for 5-7 times), and then sucking off surface water; soaking immature embryos or mature embryos with seed coats in 70% -75% of ethanol on a super-clean workbench for 10-30s, cleaning with sterile water (preferably 3-5 times), disinfecting with 3.3% sodium hypochlorite solution for 4-6min, cleaning with sterile water, and sucking water on the surfaces of the immature embryos or mature embryos with seed coats for later use;
(2) culturing the young embryo: splitting the seed coats of the mature immature embryos with the seed coats treated in the step (1) by using sterilized tweezers and a splitting knife to take out mature immature embryos, and inoculating the immature embryos or the mature immature embryos on an immature embryo culture medium to perform immature embryo culture; after inoculation, the mixture is placed in the illumination for 10 to 14 hours every day with the illumination intensity of 800--2s-1Culturing at relative humidity of 70-75% and temperature of 22-28 deg.C until young embryo germinates, and continuously culturing to obtain young seedling with 2-3 pairs of true leaves;
(3) rooting culture: cutting off the main root of the seedling growing 2-3 pairs of true leaves obtained in the step (2) at a position 0.6-1.2cm below the root neck, and inoculating the cut seedling to the rootRoot-promoting culture is carried out on the root culture medium, after inoculation, the culture is carried out for 1 to 3 days in full dark at the temperature of between 25 and 28 ℃, and then the culture is placed under the illumination for 12 to 14 hours every day with the illumination intensity of 800--2s-1Culturing at relative humidity of 70-75% and temperature of 22-28 deg.C until rooting;
(4) hardening and transplanting seedlings: after the seedlings grow to 3-5cm high, the seedlings are placed under natural illumination to be acclimatized for 5-7d, a root culture medium is cleaned, the seedlings are transplanted into a turfy soil matrix, the matrix is covered with a film, 1/4MS macroelement nutrient solution is applied to the matrix and the leaves, the humidity is kept, the film is gradually removed after 7d, the application frequency of the nutrient solution is gradually reduced, and the seedlings are transplanted into a field after survival.
The immature embryo culture medium in the step (2) is as follows: MS +1.8-2.0 mg/L6-BA +0.1-0.2mg/L IBA + 0.5% -0.7% agar, and pH value is 5.6-5.9.
The rooting culture medium in the step (3) is as follows: 1/2MS +0.4-0.5mg/L IBA + 0.5% -0.7% agar, and pH value is 5.6-5.9.
The low temperature condition in the step (1) is 1-7 ℃.
The invention has the beneficial effects that:
(1) the invention adopts a biotechnology method to take immature embryos or mature embryos from young fruits for sterile culture, so that the breeding process can be shortened and the breeding efficiency can be improved.
(2) The in vitro culture method provided by the invention can be combined with the traditional field crossbreeding to open up a new breeding way.
Drawings
FIG. 1 is a pictorial representation of sample sizes of immature young fruits and immature, mature embryos;
FIG. 2 is a diagram showing the propagation of immature and mature embryos in the rapid propagation process (immature and mature embryo culture).
FIG. 3 is a diagram of a root seedling.
Detailed Description
The following describes embodiments of the present invention in detail. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
Example 1: pear immature embryo culture method
(1) And (3) explant disinfection treatment:
selecting immature pear fruits (figure 1) which are 5 weeks after pollination, performing low-temperature vernalization treatment at 4 ℃ after picking, taking out after 5 days, washing the fruits for 10min by using tap water, then peeling off peels and pulps, washing the obtained immature embryos (immature seeds of the seeds) or mature embryos (seeds) with the seeds coats by using tap water for 1 time, then washing the immature embryos or the mature embryos with the seeds coats by using distilled water for 5 times, standing the obtained immature embryos or the mature embryos on filter paper for 10min, and sucking the water on the surfaces of the immature embryos or the mature embryos with the seeds coats. Soaking immature embryos or mature embryos with seed coats in 75% ethanol for 20s on a super-clean workbench, cleaning with sterile water for 4 times, sterilizing with 3.3% sodium hypochlorite solution for 6min, cleaning with sterile water for 4 times, and sucking water on the surfaces of the immature embryos or the mature embryos by using sterile filter paper for later use;
(2) culturing the young embryo:
placing the mature embryo with the seed coat treated in the step (1) on sterile filter paper, using sterilized tweezers and a dissecting knife to dissect the seed coat and take out the mature embryo, and inoculating the immature embryo or the mature embryo on a embryo culture medium (figure 2) containing MS +2.0 mg/L6-BA +0.2mg/L IBA + 0.7% agar and having a pH value of 5.8 to culture the embryo. After inoculation, the mixture is placed in the daily illumination for 12 hours, and the illumination intensity is 800 mu mol-2s-1Culturing at relative humidity of 75% and culturing temperature of 22-28 deg.C until young embryo germinates, and continuously culturing to obtain young seedling with 2-3 pairs of true leaves;
(3) rooting culture:
cutting off main root of 2 pairs of true leaf seedlings at 1.0cm below root neck, inoculating to rooting culture medium containing 1/2MS +0.5mg/LIBA + 0.7% agar and pH of 5.8, culturing in dark at 25-28 deg.C for 2 days, and illuminating at 800 μmolm for 12 hr per day-2s-1Culturing at relative humidity of 75% and temperature of 22-28 deg.C until rooting (FIG. 3). After 35 days, the rooting and seedling rate can reach about 40%.
(4) Hardening and transplanting seedlings:
after the seedlings grow to 4cm high, the seedlings are placed under natural illumination for hardening for 7d, the root culture medium is cleaned, the seedlings are transplanted into a turfy soil matrix (a conventional matrix sold in the market), the matrix and the leaves are coated with 1/4MS macroelement nutrient solution (sold in the market), the humidity is kept, the coating is gradually removed after 7d, the application frequency of the nutrient solution is gradually reduced, and the seedlings are transplanted into a field after survival.
The method of the invention takes immature embryo or mature embryo of pear as explant, establishes the cultivation technical system of in vitro embryo of pear through explant disinfection, embryo cultivation, rooting cultivation, hardening and transplanting, and lays the foundation for establishing pear regeneration system, improving pear variety by gene engineering and cell engineering.
Claims (4)
1. A method for culturing pear immature embryos is characterized by comprising the following steps:
(1) and (3) explant disinfection treatment: selecting immature pear fruits which are pollinated for 5-6 weeks, performing vernalization treatment for 3-5 days at a low temperature after picking, washing the vernalized fruits with water, peeling off peels and pulps to obtain immature embryos or mature embryos with seed coats, fully cleaning the immature embryos or the mature embryos with the seed coats with water, and then sucking off surface water; soaking immature embryos or mature embryos with seed coats in 70% -75% of ethanol on a super-clean workbench for 10-30s, cleaning with sterile water, disinfecting with 3.3% of sodium hypochlorite solution for 4-6min, cleaning with sterile water, and sucking water on the surfaces of the immature embryos or the mature embryos with seed coats for later use;
(2) culturing the young embryo: splitting the seed coats of the mature immature embryos with the seed coats treated in the step (1) by using sterilized tweezers and a splitting knife to take out mature immature embryos, and inoculating the immature embryos or the mature immature embryos on an immature embryo culture medium to perform immature embryo culture; after inoculation, the mixture is placed in the illumination for 10 to 14 hours every day with the illumination intensity of 800--2s-1Culturing at relative humidity of 70-75% and temperature of 22-28 deg.C until young embryo germinates, and continuously culturing to obtain young seedling with 2-3 pairs of true leaves;
(3) rooting culture: cutting off the main root of the seedling growing 2-3 pairs of true leaves obtained in the step (2) at a position 0.6-1.2cm below the root neck, inoculating the seedling to a rooting culture medium for root-promoting culture, culturing the seedling in the dark at the temperature of 25-28 ℃ for 1-3 days after inoculation, and then placing the seedling in the illumination for 12-14 hours each day with the illumination intensity of 800--2s-1Culturing at relative humidity of 70-75% and temperature of 22-28 deg.C until rooting;
(4) hardening and transplanting seedlings: after the seedlings grow to 3-5cm high, the seedlings are placed under natural illumination to be acclimatized for 5-7d, a root culture medium is cleaned, the seedlings are transplanted into a turfy soil matrix, the matrix is covered with a film, 1/4MS macroelement nutrient solution is applied to the matrix and the leaves, the humidity is kept, the film is gradually removed after 7d, the application frequency of the nutrient solution is gradually reduced, and the seedlings are transplanted into a field after survival.
2. The method for culturing the young pear embryos of claim 1, wherein the young pear embryo culture medium in the step (2) is: MS +1.8-2.0 mg/L6-BA +0.1-0.2mg/L IBA + 0.5% -0.7% agar, and pH value is 5.6-5.9.
3. The method for culturing the immature pear embryos of claim 1, wherein the rooting medium in the step (3) is: 1/2MS +0.4-0.5mg/L IBA + 0.5% -0.7% agar, and pH value is 5.6-5.9.
4. The method for culturing young pear embryos of claim 1, wherein the low temperature condition in the step (1) is 1-7 ℃.
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| CN202010410106.4A CN111699970A (en) | 2020-05-15 | 2020-05-15 | Pear immature embryo culture method |
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| CN202010410106.4A CN111699970A (en) | 2020-05-15 | 2020-05-15 | Pear immature embryo culture method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663444A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | Culture method of immature embryos of osmamthus fragrans |
| CN105359961A (en) * | 2015-11-27 | 2016-03-02 | 浙江省农业科学院 | Method for obtaining Apple Blossom hybrid cultivar of Hippeastrum Herb through immature embryo in vitro rescue |
| KR20180053833A (en) * | 2016-11-14 | 2018-05-24 | 대한민국(농촌진흥청장) | Medium for rooting of wonhwang pear |
-
2020
- 2020-05-15 CN CN202010410106.4A patent/CN111699970A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663444A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | Culture method of immature embryos of osmamthus fragrans |
| CN105359961A (en) * | 2015-11-27 | 2016-03-02 | 浙江省农业科学院 | Method for obtaining Apple Blossom hybrid cultivar of Hippeastrum Herb through immature embryo in vitro rescue |
| KR20180053833A (en) * | 2016-11-14 | 2018-05-24 | 대한민국(농촌진흥청장) | Medium for rooting of wonhwang pear |
Non-Patent Citations (1)
| Title |
|---|
| 刘艳等: "早熟梨新梨7号胚培养直接成苗的影响因素探讨", 《江西农业学报》 * |
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Application publication date: 20200925 |