CN112493120A - Embryo rescue method for distant hybridization of okra cultivars and wild okra - Google Patents
Embryo rescue method for distant hybridization of okra cultivars and wild okra Download PDFInfo
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- CN112493120A CN112493120A CN202011513957.8A CN202011513957A CN112493120A CN 112493120 A CN112493120 A CN 112493120A CN 202011513957 A CN202011513957 A CN 202011513957A CN 112493120 A CN112493120 A CN 112493120A
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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
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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 provides an embryo rescue method for distant hybridization of okra cultivars and wild okra, which comprises the following steps: carrying out distant hybridization by taking okra cultivars as female parents and wild okra as male parents, after pollination for 9-11 days, stripping ovaries, carrying out low-temperature treatment, then stripping ovules after disinfection treatment, and inoculating the ovules onto a development culture medium for culture; transecting the developed ovule, transferring to a germination medium, and culturing until the young embryo germinates into an embryo with cotyledons and radicles; transferring the germinated embryo to a seedling culture medium for culturing until the young embryo grows into a complete test-tube seedling; the test-tube plantlets are transplanted after hardening in a greenhouse for about 1 week. The invention adopts a three-step culture method to carry out embryo rescue on the okra distant hybridization seeds, designs proper culture mediums respectively according to different development stages of the embryos and optimizes culture conditions, can effectively promote the okra hybrid embryos to continue to grow and develop under the in vitro condition, promotes the young embryos to germinate into seedlings, and provides an effective technology for improving the success rate of the okra distant hybridization.
Description
Technical Field
The invention belongs to the field of vegetable biotechnology breeding, and particularly relates to an embryo rescue method for distant hybridization of okra cultivars and wild okra.
Background
Okra (academic name:Abelmoschus esculentus(Linn.) Moench, also known as coffee mallow and okra, is an annual herb of the genus Abelmoschus (Hibiscus Linn.) of the family Malvaceae (Malvaceae). The okra has wide application, and is an economic crop integrating edible, medicinal and ornamental functions. In the aspect of eating: okra is called vegetable king, and its tender pod has tender meat quality and contains viscous substance composed of pectin and polysaccharide, so that it has a special flavour. The okra is rich in protein, cellulose, unsaturated fatty acid, and minerals such as iron, calcium, manganese, potassium, and zincThe product has high nutritive value. In the aspect of medicine: the okra is light in taste and cold in nature, and has the effects of relieving sore throat, treating stranguria, promoting lactation, regulating menstruation, removing blood stasis, detoxifying and the like. In addition, the okra is rich in various bioactive components such as soluble polysaccharide, alkaloid, flavone and the like, and has the medicinal health-care effects of helping digestion, resisting cancer, resisting tumors, treating diabetes, diminishing inflammation, invigorating stomach, protecting liver, enhancing human body endurance and the like. In the aspect of appreciation: the okra plants stand upright, the leaves are large, the flowers are large and beautiful, the flowering period is long, the requirement on planting soil is not high, and the okra plant cultivation method is suitable for planting greening and beautifying plants in places such as roadsides and parks. Okra is generally considered to originate in africa and is currently cultivated around the world, but is most prevalent in tropical and subtropical regions. In recent years, the okra industry is developed rapidly in China, cultivated in various regions in China, and particularly planted in large areas in Hebei, Shandong, Yunnan, Zhejiang, Hunan, Hubei, Guangdong, Jiangxi, Fujian and other places.
The okra serving as a novel multifunctional vegetable with high nutritive value has great application value and development potential. Research work related to okra breeding is increased in many countries, and many excellent new varieties are provided. The beginning of the cultivation and breeding of the domestic okra is later compared with that of the domestic okra, and some good varieties which are planted in a commercialized mode are almost introduced from the foreign countries, so that the long-term development of the okra industry in China is extremely not facilitated.
Abundant okra germplasm resources are an important basis for screening good varieties and are basic guarantees for sexual hybridization breeding. The region of China is broad, the distributed okra plants are relatively rich, and besides being used as the okra for cultivating vegetables, the okra also can be used as wild species resources of okra, abelmoschus esculentus, okra and the like. The resources contain a plurality of genes with potential utilization values, such as resistance, disease resistance, enrichment of certain special components and the like, and can provide abundant breeding materials for improving various agronomic traits of the cultivated varieties. However, the okra cultivars and the wild species belong to distant hybridization, and are often difficult to succeed due to serious reproductive disorders. Among them, in addition to the great influence of the reproductive disorders before fertilization caused by the factors such as sterility of parent, incompatibility of hybridization and failure of fertilization, the post-fertilization disorders such as hybrid collapse caused by embryo degradation or abortion or hybrid sterility due to the disharmony of hybrid embryo and endosperm tissue development are often the key factors causing the failure of distant hybridization.
Since the early success of embryo in vitro culture by Ehanning in 1904, embryo rescue is a very effective technology for overcoming embryo abortion in post-fertilization obstacles, and has been widely applied in the field of distant hybridization breeding of plants such as flowers, vegetables, fruit trees and the like. Depending on the culture object, the immature embryo rescue can be divided into ovary culture, ovule culture and immature embryo culture. Generally, the smaller the age and size of the embryo, the more difficult it is to induce, especially early in development, and the less successful the in vitro culture of young embryos in the heterotrophic stage. The young embryos of most plants must grow in the endosperm until the formation of cotyledon primordia and organs begins, and ex vivo rescue is possible successfully. The young embryo rescue technology is an important technical means for overcoming embryo abortion of distant hybridization, shortening the generation period of hybridization breeding and accelerating the breeding speed in crop hybridization breeding, and has great potential in the breeding and the improvement of the species property of new okra varieties.
There are many factors affecting embryo culture during the rescue of immature embryos, and researchers mainly focus on the age and inoculation period of the embryos, a minimal medium, phytohormones, exogenous additives, culture conditions, pretreatment modes and other aspects. The time from pollination to embryo stripping is called embryo age, the development stages of embryos of different plant species are different, and the maturation degree of the embryos and whether the embryos are developed or die before stripping can influence the success rate of embryo culture. Different explants are cultured in tissue culture, and the type of culture medium is different, so that the selection of a proper culture medium is also a key link for the success of immature embryo rescue. In addition to the above important factors, embryo rescue techniques are also affected by environmental conditions, such as light, temperature, etc.
At present, the research on the saving of distant hybridization young embryos of okra plants at home and abroad is not reported yet.
Disclosure of Invention
The invention aims to provide an embryo rescue method for okra cultivars and wild okra distant hybridization, which aims to establish a good and efficient okra distant hybridization embryo culture system by optimizing and analyzing factors such as culture medium types, phytohormones, exogenous additives, inoculation periods, low-temperature pretreatment and the like in the culture system.
In order to realize the purpose, the invention adopts the following technical scheme:
an embryo rescue method for the distant hybridization of okra cultivars and wild okra is characterized by comprising the following steps:
1) distant hybridization: taking okra cultivars as female parents and wild okra as male parents, manually castrating and bagging the female parents 1-2 days before flowering, pollinating by using collected pollen of the male parents when the stigma of the female parents is completely mature and can secrete viscous substances, and bagging the female parents in time after pollination is finished;
2) and (3) ovule development and culture: picking the ovary from the female parent plant after 9-11 days of distant hybridization pollination, putting the ovary into a refrigerator with the temperature of 4 ℃ for low-temperature treatment for 3-5 days, then performing disinfection treatment, peeling ovules out under the aseptic condition, inoculating the ovules onto a development culture medium, and culturing under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 1500-2000 lx and the illumination is 10-12 hours per day until the ovules develop;
3) germination and culture of immature embryos: transversely cutting the developed ovule, transferring the ovule to a germination culture medium, and culturing under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 2000-3000 lx and the illumination is 14-16 h every day until the young embryo germinates into an embryo with cotyledons and radicles;
4) and (3) young embryo seedling culture: transferring the germinated embryos onto a seedling culture medium, and culturing under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 3000-4000 lx and the illumination is 14-16 h every day until the embryos grow into complete test-tube seedlings;
5) hardening and transplanting seedlings: taking out the test-tube plantlet from the bottle when the test-tube plantlet grows stably and robustly, washing with clear water to remove the residual culture medium at the root of the test-tube plantlet, transplanting the test-tube plantlet into a nutrition pot filled with a matrix, placing the test-tube plantlet into a greenhouse, covering the test-tube plantlet with a transparent plastic cup, gradually uncovering until the test-tube plantlet is completely uncovered, enabling the test-tube plantlet to be completely adapted to the external environment, transplanting the test-tube plantlet with the matrix into greenhouse soil after.
In the step 1), the pollination time is 8:00-10:00, and the pollination is repeated for 1 time after the same flower bud is pollinated for 2 days for the first time.
In the step 2), the method for disinfection treatment comprises the following steps: rinsing ovary with clear water, soaking in 75% alcohol for 30s, shaking, rinsing with sterile water for 1 time, soaking in 0.1% mercuric chloride for 10min, shaking, and rinsing with sterile water for 3 times.
In the step 2), the formula of the development culture medium is as follows: MS + IAA1.5mg/L +6-BA 0.5mg/L + paclobutrazol 0.5mg/L + casein hydrolysate 500mg/L + silver nitrate 8.0 mg/L.
In the step 2), ovule development refers to turning green and expanding ovules.
In the step 3), the formula of the germination culture medium is as follows: KNO3 1200~1400mg/L，NH4NO3 750~950mg/L，KH2PO4 177~225mg/L，MgSO4·7H2O 310~360mg/L，CaCl2·2H2O 450~500mg/L，Na2-EDTA 37~38mg/L，FeSO4·7H2O 27.4~28.2mg/L，H3BO3 3.0~3.4mg/L，KI 0.8~1.2mg/L，MnSO4·4H2O 7.8~8.4mg/L，ZnSO4·7H2O 2.7~3.3mg/L，Na2MoO4·2H2O 0.2~0.3mg/L，CuSO4·5H2O 0.02~0.03mg/L，CoCl2·6H20.04-0.06 mg/L of O, 90-110 mg/L of inositol, 0.4-0.6 mg/L of pyridoxine hydrochloride, 0.9-1.1 mg/L of thiamine hydrochloride, 1.0-2.0 mg/L of calcium pantothenate, 2.0-3.0 mg/L of tryptophan, 25-35 mg/L of proline, 240-280 mg/L of glutamine, 0.6-1.4 mg/L of D-biotin, 4.0-6.0 mg/L of 5-aminolevulinic acid, 0.5-1.5g/L of polyvinyl alcohol, 130-170 ml/L of mulberry leaf juice, 0.5-1.5 mg/L of sodium naphthenate, 0.8-1.2mg/L of IBA, 0.8-1.2mg/L of 6-BA, GA30.4-0.6 mg/L, 5.0-7.0 mg/L ferulic acid, 35-45 g/L sucrose, 15-25 g/L sorbitol, 15-25 g/L mannitol and 5-7 g/L agar powder.
In the step 4), the formula of the seedling culture medium is as follows: 1/2MS + IBA0.5mg/L + activated carbon 1.0g/L + sucrose 30g/L + agar 6 g/L.
In the step 5), the matrix components are as follows: peat, perlite and vermiculite = 1: 1.
In the step 5), the temperature of the greenhouse is kept at 25-30 ℃, and the relative humidity is kept at 85-95%.
As can be seen from Table 1, the low temperature treatment has a promoting effect on ovule development and germination of the immature embryo, and shows a certain regularity. When the embryo is treated at the low temperature for 3d, the ovule growth rate is the highest and reaches 80.3%, and when the embryo is treated at the low temperature for 5d, the embryo germination rate is the highest and reaches 54.5%, and along with the extension of the treatment time, the ovule growth rate and the embryo germination rate are reduced, so that the embryo rescue effect is the best when the embryo is treated at the low temperature for 3-5 d.
EXAMPLE 3 Effect of different concentrations of 5-aminolevulinic acid on immature embryo Germination
According to the method in example 1, the developed ovules were crosscut and transferred to germination medium supplemented with 5-aminolevulinic acid at different concentrations, 5 concentrations of 5-aminolevulinic acid were set to 0mg/L, 1.0mg/L, 2.5mg/L, 5.0mg/L and 10.0mg/L, and the germination rate of embryos was counted, and the results are shown in Table 2 below.
As can be seen from Table 2, compared with a control, the addition of 0.5-10.0 mg/L of 5-aminolevulinic acid can improve the germination rate of the embryo, the highest germination rate of the embryo is 55.9% when the concentration of the 5-aminolevulinic acid is 5.0mg/L, and the germination rate of the embryo is 50.0% when the concentration of the 5-aminolevulinic acid is 1.0mg/L, but the germination rate of the embryo is reduced when the concentration of the 5-aminolevulinic acid is increased to 10.0mg/L, which shows that the addition of the 5-aminolevulinic acid with a lower concentration has a better effect on promoting the germination of the immature embryo, and the 5.0mg/L is the optimal concentration of the 5-aminolevulinic acid.
EXAMPLE 4 Effect of different concentrations of sodium naphthenate on embryo Germination
The developed ovules were cross-sectioned and transferred to germination media supplemented with sodium naphthenate at various concentrations, 5 concentrations of 0mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L and 2.0mg/L, as described in example 1, and the germination rates were counted, and the results are shown in Table 3 below.
As can be seen from Table 3, when the concentration of sodium naphthenate is less than 1.0mg/L, the germination rate of embryos is improved along with the increase of the concentration of sodium naphthenate, when the concentration of sodium naphthenate is 1.0mg/L, the germination effect of the embryos is the best, the germination rate of the embryos reaches 52.3%, when the concentration of sodium naphthenate is 2.0mg/L, the germination rate of the embryos is obviously reduced, and is lower than that of a control, which indicates that the germination of the embryos can be inhibited by the high concentration of sodium naphthenate. Therefore, 1.0mg/L is the optimum concentration of sodium naphthenate.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (9)
1. An embryo rescue method for the distant hybridization of okra cultivars and wild okra is characterized by comprising the following steps:
1) distant hybridization: taking okra cultivars as female parents and wild okra as male parents, manually castrating and bagging the female parents 1-2 days before flowering, pollinating by using collected pollen of the male parents when the stigma of the female parents is completely mature and can secrete viscous substances, and bagging the female parents in time after pollination is finished;
2) and (3) ovule development and culture: picking the ovary from the female parent plant after 9-11 days of distant hybridization pollination, putting the ovary into a refrigerator with the temperature of 4 ℃ for low-temperature treatment for 3-5 days, then performing disinfection treatment, peeling ovules out under the aseptic condition, inoculating the ovules onto a development culture medium, and culturing under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 1500-2000 lx and the illumination is 10-12 hours per day until the ovules develop;
3) germination and culture of immature embryos: transversely cutting the developed ovule, transferring the ovule to a germination culture medium, and culturing under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 2000-3000 lx and the illumination is 14-16 h every day until the young embryo germinates into an embryo with cotyledons and radicles;
4) and (3) young embryo seedling culture: transferring the germinated embryos onto a seedling culture medium, and culturing under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 3000-4000 lx and the illumination is 14-16 h every day until the embryos grow into complete test-tube seedlings;
5) hardening and transplanting seedlings: taking out the test-tube plantlet from the bottle when the test-tube plantlet grows stably and robustly, washing with clear water to remove the residual culture medium at the root of the test-tube plantlet, transplanting the test-tube plantlet into a nutrition pot filled with a matrix, placing the test-tube plantlet into a greenhouse, covering the test-tube plantlet with a transparent plastic cup, gradually uncovering until the test-tube plantlet is completely uncovered, enabling the test-tube plantlet to be completely adapted to the external environment, transplanting the test-tube plantlet with the matrix into greenhouse soil after.
2. The embryo rescue method for the distant hybridization of the okra cultivar and the wild okra according to claim 1, characterized in that in the step 1), the pollination time is 8:00-10:00, and the pollination is repeated for 1 time after the same flower bud is primarily pollinated for 2 days.
3. The embryo rescue method for the okra cultivar distant hybridization with wild okra according to claim 1, wherein in the step 2), the sterilization treatment method comprises the following steps: rinsing ovary with clear water, soaking in 75% alcohol for 30s, shaking, rinsing with sterile water for 1 time, soaking in 0.1% mercuric chloride for 10min, shaking, and rinsing with sterile water for 3 times.
4. The embryo rescue method for the okra cultivar distant hybridization with wild okra according to claim 1, wherein in the step 2), the formula of the development medium is as follows: MS + IAA1.5mg/L +6-BA 0.5mg/L + paclobutrazol 0.5mg/L + casein hydrolysate 500mg/L + silver nitrate 8.0 mg/L.
5. The method for rescuing embryos of okra cultivars from distant hybridization with wild okra according to claim 1, wherein in the step 2), ovule development refers to green turning and expansion of ovules.
6. The embryo rescue method for the okra cultivar distant hybridization with wild okra according to claim 1, wherein in the step 3), the formula of the germination medium is as follows: KNO3 1200~1400mg/L，NH4NO3 750~950mg/L，KH2PO4 177~225mg/L，MgSO4·7H2O 310~360mg/L，CaCl2·2H2O 450~500mg/L，Na2-EDTA 37~38mg/L，FeSO4·7H2O 27.4~28.2mg/L，H3BO3 3.0~3.4mg/L，KI 0.8~1.2mg/L，MnSO4·4H2O 7.8~8.4mg/L，ZnSO4·7H2O 2.7~3.3mg/L，Na2MoO4·2H2O 0.2~0.3mg/L，CuSO4·5H2O 0.02~0.03mg/L，CoCl2·6H20.04-0.06 mg/L of O, 90-110 mg/L of inositol, 0.4-0.6 mg/L of pyridoxine hydrochloride, 0.9-1.1 mg/L of thiamine hydrochloride, 1.0-2.0 mg/L of calcium pantothenate, 2.0-3.0 mg/L of tryptophan, 25-35 mg/L of proline, 240-280 mg/L of glutamine, 0.6-1.4 mg/L of D-biotin, 4.0-6.0 mg/L of 5-aminolevulinic acid, 0.5-1.5g/L of polyvinyl alcohol, 130-170 ml/L of mulberry leaf juice, 0.5-1.5 mg/L of sodium naphthenate, 0.8-1.2mg/L of IBA, 0.8-1.2mg/L of 6-BA, GA30.4-0.6 mg/L, 5.0-7.0 mg/L ferulic acid, 35-45 g/L sucrose, 15-25 g/L sorbitol, 15-25 g/L mannitol and 5-7 g/L agar powder.
7. The embryo rescue method for the okra cultivar and wild okra distant hybridization according to claim 1, wherein in the step 4), the formula of the seedling culture medium is as follows: 1/2MS + IBA0.5mg/L + activated carbon 1.0g/L + sucrose 30g/L + agar 6 g/L.
8. The embryo rescue method for the okra cultivar distant hybridization with wild okra according to claim 1, wherein in the step 5), the matrix components are as follows: peat, perlite and vermiculite = 1: 1.
9. The embryo rescue method for the distant hybridization of the okra cultivar and the wild okra according to claim 1, wherein in the step 5), the temperature of a greenhouse is kept between 25 and 30 ℃, and the relative humidity is kept between 85 and 95%.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107295967A (en) * | 2017-07-10 | 2017-10-27 | 佛山科学技术学院 | A kind of gumbo combined hybrid method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107295967A (en) * | 2017-07-10 | 2017-10-27 | 佛山科学技术学院 | A kind of gumbo combined hybrid method |
Non-Patent Citations (2)
| Title |
|---|
| D.N.VLACHOSTERGIOS等: "In-vitro development of ovules obtained after pollination of cotton (Gossypium spp) flowers with pollen from okra (Abelmoschus esculentus L. Moench)", 《PLANT CELL,TISSUE AND ORGAN CULTURE》 * |
| 赵立宁等: "红麻与玫瑰麻种间杂交胚珠培养及杂种植株再生", 《植物生理学通讯》 * |
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Application publication date: 20210316 |