CN110915656B - Female-line cucumber double haploid regeneration plant and preparation method and application thereof - Google Patents
Female-line cucumber double haploid regeneration plant and preparation method and application thereof Download PDFInfo
- Publication number
- CN110915656B CN110915656B CN201911320469.2A CN201911320469A CN110915656B CN 110915656 B CN110915656 B CN 110915656B CN 201911320469 A CN201911320469 A CN 201911320469A CN 110915656 B CN110915656 B CN 110915656B
- Authority
- CN
- China
- Prior art keywords
- cucumber
- female
- line
- culture
- double haploid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
-
- 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
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a female-line cucumber double haploid regeneration plant and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) introducing a female-line smooth fruit cucumber F1 commercial variety, culturing by using a cucumber unfertilized ovary culture screening culture medium, and screening commercial varieties with high frequency embryo occurrence rate; (2) respectively planting the high-frequency embryo incidence F1 commercial variety screened in the step (1) and other female-line smooth fruit cucumber F1 commercial products in the field, hybridizing and pollinating in the flowering period, and substituting a cucumber donor genotype material containing a high-frequency embryo incidence gene after hybridization; (3) obtaining a female-line cucumber double haploid regeneration plant by a cucumber unfertilized ovary culture technology. The method of the invention has high proportion of double haploid plants, does not need chromosome doubling treatment on the regeneration plants, avoids the death of the haploid plants through doubling, and improves the transplanting survival rate. Can efficiently obtain the excellent pure breeding line of the double haploid of the cucumber and can be directly used for breeding.
Description
Technical Field
The invention belongs to the field of plant cell engineering, and particularly relates to a double haploid regeneration plant of female-line cucumber, a preparation method and application thereof.
Background
The planting history of the cucumbers in China is long, the cucumbers are favored by Chinese people, and the planting types mainly include the tumor-shaped cucumbers. Cucumis sativus of Cucurbitaceae of Cucumis, hermaphrodite, and heterofloral plants, which can be classified into female line type and non-female line type according to female flower sex type. With the improvement of the living standard of people, the requirements on new, odd and special vegetable varieties are higher and higher, and the cucumber planting is in a diversified trend. The female-line smooth fruit cucumber has the advantages of beautiful fruit shape, fragrant taste, smooth surface and bright green color, is favored by consumers and becomes one of the main planting types of the cucumber in China. The fruit cucumber main cultivar is a foreign hybrid variety with high seed price, and with the development of fruit cucumber breeding research in China, new varieties cultivated by the self in China gradually replace foreign varieties, but because the fruit cucumber varieties in China are developed later, breeding materials are few, and the new varieties are urgently required to be improved in the aspects of quality, resistance and the like.
The haploid breeding technology is one of the important means of modern commercial breeding, an excellent breeding selection line can be created quickly, the breeding period is shortened, and the breeding efficiency is improved. The haploid plant is usually formed by the division and development of female gametophyte or male gametophyte, and the doubled haploid plant obtained by natural or artificial doubling of the pure genotype can be used as a breeding pure line to be directly used for variety breeding. Since the nineties of the last century, researchers have conducted a great deal of research into techniques for inducing haploid plants: anther or pollen microspore culture is a main method, researchers succeed in successively culturing vegetables such as Chinese cabbage, cauliflower, cabbage, hot pepper, eggplant and the like, and large-scale application is realized; the radiation pollen pollination method is another effective method, and has more researches, researchers have successful reports on wheat, potatoes, tomatoes, cucumbers, melons and the like, but the large-scale application is less; another major method is unfertilized ovary culture. The culture of the unfertilized ovary is successfully carried out on barley for the first time in 1976, and as the culture of cucumber microspores is difficult to succeed, a plurality of researchers develop the research of the culture method of the unfertilized ovary of cucumber.
Dirks (1996) reported U.S. patent, and the cucumber haploid plant is obtained by inducing the first unfertilized ovary culture method in the world by taking smooth fruit cucumbers in the netherlands as materials, and the average haploidy incidence rate is 50%. However, the technology has the following defects: 1. the haploid embryogenesis inducing medium needs to adjust the auxin and cytokinin concentrations according to the parthenocarpic capacity of the donor material, and obvious genotype differences exist. 2. Most regenerated plantlets are haploid, the haploid plants cannot normally seed and cannot be applied in breeding, and patent documents indicate that double haploid plants can be obtained only by inducing chromosome doubling by colchicine or other methods and can be used for breeding. Colchicine is a common reagent used for doubling plants at present, cucumber seedlings are sensitive to the colchicine, toxicity is easy to generate to cause dead seedlings, and the doubling process is long in time and low in efficiency.
Gemes Juhasz (2002) reports that cucumber single (double single) ploid regeneration plants are obtained by an unfertilized ovary culture method, which comprises the steps of inoculating unfertilized ovaries on a CBM basic culture medium added with 0.02 percent of TDZ and 4 percent of cane sugar, transferring the ovaries into a CBM basic culture medium added with 0.2mg/L of 6-BA, 0.05mg/LNAA and 3 percent of cane sugar after 2-4 days of high-temperature dark treatment at 35 ℃, observing embryos after 6-12 weeks, subculturing the embryos on the CBM basic culture medium without 3 percent of cane sugar containing hormone, and growing small plants. The method can obtain the haploid with the highest haploid embryo generation rate of 18.7%, the plant regeneration rate of 7.1% and 87.7% of the regenerated plants being haploid. Without an effective doubling method, only 24% of haploid plants were doubled to heterozygous haploid plants (Gemes Juhasz, 2006), which ultimately yielded fewer doubled haploid plants for breeding.
The Thailand scholars A.Sorntip (2017) report that commercial species Chai Lai and Big C of Thailand fresh cucumber F1 generation are used as donor materials for unfertilized ovary culture, the influence of an optimal culture medium on plant regeneration in the processes of induction, differentiation and regeneration of embryoids is researched, the influence of a genotype and an induction culture medium on the generation of the embryoids is considered to be small, and a D2 culture medium is used in the process of embryoid differentiation: the basic culture medium is MS, and comprises auxin NAA0.05mg/L, cytokinin BA0.2mg/L, Triacontanol (TRIA)0.02mg/L, proline (Pro)100mg/L, ABA 20mg/L, AgNO 3 2mg/L, Sucrose 30g/L, gelrite 3g/L, MST3+ medium used during regeneration: the basic culture medium is MS, and 0.05mg/L of auxin IBA, 1mg/L of cytokinin TDZ and gibberellin GA are added 3 0.5mg/L, ABA 0.1mg/L, TRIA, Polyvinylpyrolidone, Proline, Glutathionone, and AgNO 3 The highest embryoid induction rate of 59.89% is obtained by components such as Coconut water, Tomato, Khai banana and the like, and 6 double haploid plants are obtained from 10 regenerated plants. The method has the problems of low embryoid induction rate, less obtained regeneration plants and the like.
Researchers in China mostly use the tumor-like cucumbers as donor materials to carry out related research on the culture technology of the unfertilized ovary. Du Shengli (2001) uses domestic tumor-like cucumber (non-female line) as material, and develops a cucumber non-fertilized ovary culture technology to obtain single (double-haploid) ploid plants, wherein 80% of the single (double-haploid) ploid plants are double-haploid regeneration plants. In 2007, the technology was patented by the national invention. In subsequent researches, due to the genotype difference of donor materials, haploid embryo induction is difficult to perform on female-line smooth fruit cucumbers by applying the technology, and the embryo induction rate and the regenerated plant rate are low.
In Chenpeng (2005), the tumorous cucumber was used as the donor material, and the high embryogenic rate was obtained, but the regeneration of the single (double mono) ploid plant was not reported. The wang light (2008) studied the effect of high temperature pretreatment, different hormone types and concentrations on embryoid generation with two lunge-type cucumbers and 1 no-lunge type cucumber as materials, wherein the lunge-type cucumber obtained embryoid and the no-lunge type cucumber (fruit cucumber type) did not obtain embryoid. Cunwei ping (2008) takes thorn tumor type and smooth type cucumbers as materials, and obtains higher haploid embryogenesis rate which is more than 80% at most from the influences of an ovary development period, heat shock treatment time, TDZ and AgNO3 on embryogenesis of the cucumber in a non-pollinated ovary culture process, but does not report plant regeneration conditions. In subsequent researches, cunwei ping (2009) uses tumor-type cucumbers and south China cucumbers as materials to research embryoid differentiation and regenerated plant ploidy, 92.5% of 40 regenerated plants obtained are north China tumor-type cucumbers, and the regeneration condition of double haploid plants of female line fruit cucumbers is not reported. Li Jianxin (2012) reports that a tumor-type cucumber is used as a material, a regeneration plant is obtained by an unfertilized ovary culture method, the highest occurrence frequency of a haploid embryo is 87.5%, and the highest regeneration frequency of the plant is 10%.
In conclusion, although the relatively high embryogenic rate is reported in the culture of the female-line smooth fruit cucumber unfertilized ovary, the rate of double haploid regeneration plants is low, and a high-efficiency method is not yet seen.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a female-line cucumber double haploid regeneration plant.
The second purpose of the invention is to provide a method for preparing a female-line cucumber double haploid regeneration plant.
The third purpose of the invention is to provide an application of female-line cucumber double haploid regeneration plant as breeding pure line in cucumber breeding.
The technical scheme of the invention is summarized as follows:
a preparation method of a female-line cucumber double haploid regeneration plant comprises the following steps:
(1) introducing a female-line smooth fruit cucumber F1 commercial variety, culturing by using a cucumber unfertilized ovary culture screening culture medium, and screening commercial varieties with high frequency embryo occurrence rate;
(2) respectively planting the high-frequency embryo incidence F1 commercial variety screened in the step (1) and other female-line smooth fruit cucumber F1 commercial products in the field, hybridizing and pollinating in the flowering period, and substituting a cucumber donor genotype material containing a high-frequency embryo incidence gene after hybridization;
(3) obtaining a female-line cucumber double haploid regeneration plant by a cucumber unfertilized ovary culture technology.
The formula of the culture medium for culturing and screening the cucumber unfertilized ovary comprises the following components: KNO 3 750mg，NH 4 NO 3 300mg MgSO 4 .7H2O 75mg，CaCl 2 .2H 2 O 165mg，KH 2 PO 4 420mg，(NH 4 ) 2 SO 4 150mg，Mn SO 4 15mg，Zn SO 4 10mg，H 3 BO 3 7.2mg，KI 1.5mg，Fe SO 4 .7H 2 O 27.8mg，Na 2 EDTA·2H 2 O37.3 mg, inositol 150mg, vitamin B 1 1.0mg, vitamin B 6 2.0mg, 5.0mg of nicotinic acid, 5.0mg of glycine, 2.5mg of cytokinin, 0.1mg of vitamin H, 10mg of hydrolyzed milk protein, 30g of cane sugar and 7.0g of agar, and adding water to 1L, wherein the pH value is 5.8, and the cucumber non-fertilized ovary culture screening medium is abbreviated as S5.
The cucumber non-fertilized ovary culture technology in the step (3) comprises the following steps: planting the cucumber donor genotype material obtained in the step (2) in a greenhouse or a greenhouse, collecting unfertilized ovaries 2-4 days before flowering in the flowering period, sterilizing the surface, performing cross cutting or longitudinal cutting, inoculating the obtained material on an induction culture medium to perform unfertilized ovary culture, and after embryoid, plumule or embryonic callus appears, subculturing the culture into a differentiation culture medium to continue culturing to obtain the female-line cucumber double haploid regeneration plant.
The formula of the induction culture medium is as follows: KNO 3 300mg，NH 4 NO 3 600mg，MgSO 4 ·7H 2 O 135mg，CaCl 2 ·2H 2 O 100mg，KH 2 PO 4 90mg，NH 4 SO 4 50mg，Mn SO 4 3mg，Zn SO 4 3.5mg，H 3 BO 3 3.5mg，KI 3.0mg，FeSO 4 ·7H 2 O 27.8mg，Na 2 EDTA·2H 2 O37.3 mg, inositol 100mg, vitamin B 1 0.5mg, vitamin B 6 2.0mg, 2.0mg of nicotinic acid, 1.0mg of glycine, 50mg of adenosylmethionine, 0.1mg of auxin, 3.2mg of cytokinin, 30g of cane sugar and 7g of agar, and adding water to 1L and pH value is 5.8, wherein the induction culture medium is abbreviated as S7, and the cytokinin is two of adenine, 6-benzyladenine, kinetin, zeatin and thidiazuron.
The formula of the differentiation medium is as follows: 41.74g of MS minimal medium containing sucrose and agar, 0.06mg of auxin and 0.2mg of cytokinin, and adding water to 1L, pH5.8, wherein the differentiation medium is abbreviated as D9.
The steps for performing unfertilized ovary culture are as follows: placing in an incubator at 30-33 deg.C, and culturing in dark for 7-10 days; culturing at 28-32 deg.C under light of 14 hr/dark of 10 hr and light intensity of 600-.
The female-line cucumber double haploid regeneration plant prepared by the preparation method.
The female-line cucumber double haploid regeneration plant is applied to cucumber breeding as a breeding pure line.
The invention has the advantages that:
1. the donor material for cucumber unfertilized ovary culture takes a variety with high frequency embryo occurrence rate as a medium, realizes the reintegration of high frequency genes after hybridization combination preparation, and is beneficial to the improvement of the embryo occurrence rate and the high-efficiency regeneration of double haploid plants. 2. The composition, concentration and culture method of the unfertilized ovary culture induction culture medium and the differentiation culture medium are different from the existing research, the composition of the culture medium does not need to be adjusted according to the difference of donor materials, the embryogenesis rate and the plant regeneration rate are high, the embryogenesis rate is 50-80%, the highest plant regeneration rate is 12%, and the method is particularly suitable for female-line smooth fruit cucumbers; 3. the application of the technical system of the invention to the culture of the female smooth fruit cucumber unfertilized ovary has the advantages that the proportion of double haploid regeneration plants is high and is 60-80 percent, the regeneration plants do not need chromosome doubling treatment, the seedling death in the doubling process of the haploid plants is avoided, and the selfing and seed reservation can not be carried out. 4. The method can efficiently obtain the cucumber double haploid regeneration plant, and the excellent pure line obtained by screening can be directly used for cucumber breeding.
Drawings
FIG. 1 shows the culture of embryoid bodies and embryogenic callus from the non-fertilized ovaries of female-line smooth fruit cucumber.
FIG. 2 is a regenerated plantlet obtained from the culture of the unfertilized ovary of female line smooth fruit cucumber.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
The formulation of the cucumber unfertilized ovary culture screening medium (S5) is as follows: KNO 3 750mg，NH 4 NO 3 300mg，MgSO 4 ·7H 2 O 75mg，CaCl 2 .2H 2 O 165mg，KH 2 PO 4 420mg，(NH 4 ) 2 SO 4 150mg，Mn SO 4 15mg，Zn SO 4 10mg，H 3 BO 3 7.2mg，KI 1.5mg，Fe SO 4 .7H 2 O 27.8mg，Na 2 EDTA·2H 2 O37.3 mg, inositol 150mg, vitamin B 1 1.0mg, vitamin B 6 2.0mg, 5.0mg nicotinic acid, 5.0mg glycine, 2.5mg 6-benzyladenine (cytokinin), 0.1mg vitamin H, 10mg hydrolyzed milk protein, 30g sucrose, 7.0g agar, water to 1L, pH5.8, and making into culture medium by conventional method.
Example 2
The formulation of the induction medium (S7) was:
KNO 3 300mg，NH 4 NO 3 600mg，MgSO 4 ·7H 2 O 135mg，CaCl 2 ·2H 2 O 100mg，KH 2 PO 4 90mg，NH 4 SO 4 50mg，Mn SO 4 3mg，Zn SO 4 3.5mg，H 3 BO 3 3.5mg，KI 3.0mg，FeSO 4 ·7H 2 O 27.8mg，Na 2 EDTA·2H 2 o37.3 mg, inositol 100mg, vitamin B 1 0.5mg, vitamin B 6 2.0mg, 2.0mg of nicotinic acid, 1mg of glycine, 50mg of adenosylmethionine, 0.1mg of naphthylacetic acid (auxin), 3.0mg of adenine (cytokinin), 0.2mg of thidiazuron (cytokinin), 30g of sucrose, 7g of agar, adding water to 1L, and adjusting the pH to 5.8 to prepare a culture medium according to a conventional method.
Example 3
The formula of the differentiation medium (D9) was:
a culture medium was prepared by adding water to 1L, pH5.8, to 41.74g of a MS minimal medium containing sucrose and agar, 0.06mg of indoleacetic acid (auxin), 0.2mg of zeatin (cytokinin), and then preparing the medium by a conventional method.
Example 4
A preparation method of a female-line cucumber double haploid regeneration plant comprises the following steps:
(1) commercial species of female-line smooth fruit cucumber F1 were introduced: saana, Raziao, Israel Hazila; dudostar, Cond, 22-33 and 22-36 are worn by Holland Raysurg Schwann company, and the commercial species 22-33 with high frequency embryo generation rate is screened by culturing through cucumber unfertilized ovary culture screening culture medium (S5);
(2) respectively planting the commercial F1 varieties 22-33 with the high-frequency embryo generating rate screened in the step (1) and other female-line smooth-type fruit cucumber F1 commercial varieties (Duoxing, Kande, Saburg, Tony and the like worn by Holland Rayscraper company) in fields, and performing hybrid pollination in flowering phases, wherein hybrid offspring are cucumber donor genotype materials containing high-frequency embryo generating rate genes;
the donor genotype material obtained was: 22-33 star, 22-33 constantan, 22-33 Sabog; 22-33 × toni;
(3) by cucumber unfertilized ovary culture technique: planting the cucumber donor genotype material obtained in the step (2) in a greenhouse, collecting unfertilized ovaries 3 days before flowering in the flowering period, performing surface sterilization, crosscut or longitudinal cut, and inoculating the ovaries on an induction medium S7 for unfertilized ovary culture, wherein the specific steps are as follows: placing in an incubator at 32 deg.C, and culturing in dark for 7 days; placing the cucumber in a culture room at 28 ℃, illuminating for 14 hours/dark for 10 hours, culturing at the illumination intensity of 1200lux, generating embryoid (shown in figure 1), subculturing the embryoid into a differentiation culture medium D9, and continuously culturing to obtain a female-line cucumber regenerated plantlet (shown in figure 2). The unfertilized ovary was used for embryogenesis and plant regeneration (see Table 1).
Table 1: the prepared donor genotype material is used for the conditions of embryo generation and plant regeneration of the unfertilized ovary culture
| Donor genotype | Inoculation ovary number (number) | Rate of embryo formation (%) | Plant regeneration rate (%) | Proportion of doubled haploid plant (%) |
| 22-33 Sabo lattice | 500 | 70 | 10 | 70 |
| 22-33 recovery | 380 | 80 | 12 | 80 |
| 22-33 star-shaped | 400 | 60 | 11 | 64 |
| 22-33 star Tunni | 500 | 70 | 9 | 73 |
| Raziao star of Daidaxing | 400 | 50 | 9 | 61 |
| Raziao kang de la ziao | 400 | 70 | 12 | 71 |
| Raziao Sabo lattice | 500 | 65 | 11 | 71 |
| Raziao Tornib | 500 | 75 | 10 | 70 |
Ploidy identification of female-line cucumber regenerated plantlet
(1) Morphological identification: and (4) carrying out ploidy identification on the regenerated plantlets of the female-line cucumbers according to the field morphological indexes. The haploid plants grow slowly; the blades are small; the male flower petal is small and deep-cracked, and can not normally open, and the pollen is sterile; the ovary and the female corolla are both smaller. The shape of the double haploid plant is the same as that of a normal diploid plant, and the size of leaves is normal; the shape of the male and female flowers is normal, the flowers are opened normally, and the pollen is fertile.
(2) And (3) genetic observation: after the double haploid plants are subjected to selfing and seed reservation, the stability and consistency of the characters of more than 90 percent of double haploid plants are found through multi-generation planting and repeated observation, and the double haploid plants can be used as breeding pure lines.
Example 5
A preparation method of a female-line cucumber double haploid regeneration plant comprises the following steps:
(1) the same as in (1) of example 4;
(2) respectively planting the commercial variety F1 with the high-frequency embryo generating rate screened in the step (1) and other female-line smooth-type fruit cucumber F1 commercial varieties (Duoxing, Kande, Saburg, Tony and the like worn by Holland Rayscraper company) in a field, and performing hybrid pollination in the flowering period, wherein hybrid offspring are cucumber donor genotype materials containing high-frequency embryo generating rate genes;
the obtained donor genotype material was: lazi ao davidian, lazi zaokang, lazi zaburg; lazio atkini;
(3) by cucumber unfertilized ovary culture technique: planting the cucumber donor genotype material obtained in the step (2) in a greenhouse, collecting unfertilized ovaries 2 days before flowering in the flowering period, sterilizing the surface, transversely or longitudinally cutting, and inoculating the ovaries on an induction culture medium for unfertilized ovary culture, wherein the method comprises the following specific steps: placing in an incubator at 30 deg.C, and dark culturing for 10 days; placing the cucumber in a culture room at 30 ℃, illuminating for 14 hours/dark for 10 hours, culturing, generating buds, subculturing the buds into a differentiation culture medium D9, and continuously culturing to obtain the female-line cucumber double haploid regeneration plant. The unfertilized ovary was used for embryogenesis and plant regeneration (see Table 1).
The application is as follows:
the technical method developed by the invention belongs to haploid breeding technology in breeding practice. The haploid breeding technology has important effects on innovating breeding materials and improving breeding efficiency, and becomes an important means of modern commercial breeding. The double haploid regeneration plant obtained by the method can be used as a breeding pure line after field screening, is directly applied to cucumber breeding, shortens the material creation period from conventional breeding for 3-4 years to obtain a homozygous breeding material in the same year, and greatly improves the breeding efficiency. Is favorable for obtaining important agronomic character specific breeding materials controlled by recessive genes, and is difficult to obtain by using a conventional breeding high-generation selfing method.
The female-line cucumber double haploid regeneration plant has consistent field character performance, the character homozygosity rate reaches more than 90 percent, and a female-line cucumber double haploid breeding pure line with excellent performance in the aspects of disease resistance, earliness, fruit shape, fruit color, peel brightness, fruit regularity, continuous melon bearing capacity, cold resistance and the like can be obtained through comprehensive evaluation of identification systems of disease resistance, stress resistance, commodity performance and the like. The dihaploid breeding pure line with excellent performance can be directly applied to breeding new cucumber varieties.
Claims (2)
1. A preparation method of a female-line cucumber double haploid regeneration plant is characterized by comprising the following steps:
(1) introducing a female-line smooth fruit cucumber F1 commercial variety, culturing by using a cucumber unfertilized ovary culture screening culture medium, and screening commercial varieties with high frequency embryo occurrence rate;
(2) respectively planting the high-frequency embryo incidence F1 commercial variety screened in the step (1) and other female-line smooth fruit cucumber F1 commercial products in the field, hybridizing and pollinating in the flowering period, and substituting a cucumber donor genotype material containing a high-frequency embryo incidence gene after hybridization;
(3) obtaining a female-line cucumber double haploid regeneration plant by a cucumber unfertilized ovary culture technology;
the formula of the cucumber unfertilized ovary culture screening medium in the step (1) is as follows: KNO 3 750mg，NH 4 NO 3 300mg，MgSO 4 ·7H 2 O
75mg，CaCl 2 ·2H 2 O 165mg，KH 2 PO 4 420mg，(NH 4 ) 2 SO 4 150mg，MnSO 4 15mg，ZnSO 4 10mg，H 3 BO 3 7.2mg，KI 1.5mg，FeSO 4 ·7H 2 O 27.8mg，Na 2 EDTA·2H 2 37.3mg of O, 150mg of inositol and vitamin B 1 1.0mg, vitamin B 6 2.0mg, 5.0mg nicotinic acid, 5.0mg glycine, 2.5mg 6-benzyladenine, 0.1mg vitamin H, 10mg hydrolyzed milk protein, 30g sucrose, 7.0g agar, adding water to 1L, pH 5.8;
the cucumber unfertilized ovary culture technology in the step (3) comprises the following steps: planting the cucumber donor genotype material obtained in the step (2) in a greenhouse or a greenhouse, collecting unfertilized ovaries 2-4 days before flowering in the flowering period, sterilizing the surface, performing cross cutting or longitudinal cutting, inoculating the materials on an induction culture medium to perform unfertilized ovary culture, and after embryoids or buds appear, subculturing the culture into a differentiation culture medium to continue culture to obtain a female-line cucumber double haploid regeneration plant;
the formula of the induction culture medium is as follows: KNO 3 300mg，NH 4 NO 3 600mg，MgSO 4 ·7H 2 O 135mg，CaCl 2 ·2H 2 O 100mg，KH 2 PO 4 90mg，NH 4 SO 4 50mg，MnSO 4 3mg，ZnSO 4 3.5mg，H 3 BO 3 3.5mg，KI 3.0mg，FeSO 4 ·7H 2 O 27.8mg，Na 2 EDTA·2H 2 O37.3 mg, inositol 100mg, vitamin B 1 0.5mg, vitamin B 6 2.0mg, nicotinic acid 2.0mg, glycine 1.0mg, adenosylmethionine 50mg, naphthylacetic acid 0.1mg, 6-benzyladenine 3.0mg, thidiazuron 0.2mg, sucrose 30g, agar 7g, water to 1L, pH 5.8;
the formula of the differentiation medium is as follows: 41.74g of MS minimal medium containing sucrose and agar, 0.06mg of indoleacetic acid, 0.2mg of zeatin, pH5.8, and water to 1L.
2. The preparation method as set forth in claim 1, wherein the cucumber unfertilized ovary culture of step (3) comprises the steps of: placing in an incubator at 30-33 deg.C, and dark culturing for 7-10 days; culturing at 28-32 deg.C under light of 14 hr/dark of 10 hr and light intensity of 600-.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911320469.2A CN110915656B (en) | 2019-12-19 | 2019-12-19 | Female-line cucumber double haploid regeneration plant and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911320469.2A CN110915656B (en) | 2019-12-19 | 2019-12-19 | Female-line cucumber double haploid regeneration plant and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110915656A CN110915656A (en) | 2020-03-27 |
| CN110915656B true CN110915656B (en) | 2022-09-02 |
Family
ID=69864358
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911320469.2A Active CN110915656B (en) | 2019-12-19 | 2019-12-19 | Female-line cucumber double haploid regeneration plant and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110915656B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111837948B (en) * | 2020-06-24 | 2022-09-23 | 中国农业科学院郑州果树研究所 | Method for obtaining double haploid plants by utilizing non-pollinated ovary culture of watermelons |
| CN112493122B (en) * | 2020-11-30 | 2022-02-08 | 北京市农林科学院 | Method for doubling haploid of cucumber |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300306C (en) * | 2005-07-29 | 2007-02-14 | 天津科润农业科技股份有限公司 | Medium for inducing cucumber macrospore in vitro haploid embryogenesis and its uses |
| CN104620988B (en) * | 2015-02-13 | 2017-01-18 | 中国农业科学院蔬菜花卉研究所 | Method for obtaining cucumber regenerated plantlet by inducing gynogenesis by virtue of culture of unfertilized ovules |
-
2019
- 2019-12-19 CN CN201911320469.2A patent/CN110915656B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110915656A (en) | 2020-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wan et al. | Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus | |
| Li et al. | Thidiazuron and silver nitrate enhanced gynogenesis of unfertilized ovule cultures of Cucumis sativus | |
| Abdollahi et al. | Induction and development of anther-derived gametic embryos in cucumber (Cucumis sativus L.) by optimizing the macronutrient and agar concentrations in culture medium | |
| Moqbeli et al. | In vitro cucumber haploid line generation in several new cultivars | |
| CN110915656B (en) | Female-line cucumber double haploid regeneration plant and preparation method and application thereof | |
| CN103548672B (en) | The seed selection of cabbage heart cytoplasmic male sterile line, breeding and producing method for seed | |
| CN101263783B (en) | Method for using herbicide barban to fast breed aloe all-male plant strain | |
| Vagera | Pepper (Capsicum spp.): In vitro induction of haploids | |
| Bottino | Vegetable crops | |
| Das et al. | Micropropagation of sweet orange, Citrus sinensis Osbeck. for the development of nucellar seedlings | |
| Cao et al. | Application of anther culture and isolated microspore culture to vegetable crop improvement | |
| US20190200553A1 (en) | Method for Producing Rice Haploid by Rice X Maize Hybridization | |
| CN101536673B (en) | High-frequency plant regeneration method of rice cropping mature embryo | |
| Thaneshwari | Effect of plant growth regulators and sucrose concentration on callus induction and shoot differentiation from ovary culture of marigold (Tagetes spp) | |
| CN103461099A (en) | Method for creating novel white flesh loquat strain by taking red flesh loquat variety as material | |
| Sourour et al. | Effect of 2, 4-dichlorophenoxyacetic acid and nitrate silver on the efficiency of haploid production in durum wheat× maize crosses | |
| CN104303999B (en) | A kind of beautiful Cuculus polioephalus and the mating system of big word Cuculus polioephalus bybrid embryo | |
| CN106508671A (en) | Method for breeding earlier germinated eriobotrya japonica seeds | |
| CN114424749A (en) | Liriope spicata in-vitro rapid propagation method | |
| CN103329792A (en) | Breeding, propagating and seed-producing method of purple pakchoi cytoplasmic male sterile line | |
| Chlyah et al. | Tomato (Lycopersicon esculentum L.): Anther culture and Induction of androgenesis | |
| CN108243949B (en) | Breeding method of light and simple type temperature-sensitive genic male sterile line | |
| CN101889548A (en) | Cabbage haploid breeding method | |
| CN105359961A (en) | Method for obtaining Apple Blossom hybrid cultivar of Hippeastrum Herb through immature embryo in vitro rescue | |
| CN105325300B (en) | The methods and applications of Eriobotrya distant hybrid progeny material are quickly bred by embryo culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220809 Address after: 300457 Tianjin Binhai New Area Economic and Technological Development Zone Huanghai Road 276 Taida small and medium sized Enterprise Park 2 building 310 Applicant after: TIANJIN KERUN AGRICULTURAL SCIENCE & TECHNOLOGY Co.,Ltd. Applicant after: Tianjin Academy of Agricultural Sciences Address before: 300192, 295 Bai Causeway Road, Tianjin, Nankai District Applicant before: TIANJIN KERUN AGRICULTURAL SCIENCE & TECHNOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |