CN1418950A - Method for obtaining haploid plant strain by culturing anther or pollen - Google Patents
Method for obtaining haploid plant strain by culturing anther or pollen Download PDFInfo
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- CN1418950A CN1418950A CN 02158575 CN02158575A CN1418950A CN 1418950 A CN1418950 A CN 1418950A CN 02158575 CN02158575 CN 02158575 CN 02158575 A CN02158575 A CN 02158575A CN 1418950 A CN1418950 A CN 1418950A
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Abstract
The method for obtaining haploid plant by utilizing anther or pollen culture includes the following steps: directly inoculating the liquid culture medium with anther to make anther floating culture, after the anther is opened naturally, making pollen (microspore) freely spill into liquid culture medium, after anther floating culture for proper days, under the aseptic condition removing anther and making free pollen (microspore) culture, at the same time improving components in the culture medium. Said invention not only can simplify culture process, but also can obviously raise the induction of pollen (microspore) embryoid and frequency of plant regeneration.
Description
Technical field:
The present invention relates to the plant cell engineering field, relate to a kind of method that obtains haplobiont by flower pesticide or pollen culture technique more specifically.
Background technology:
At present, in method, generally all use solid medium to carry out with anther culture eggplant haplobiont.In this anther culture process, somatocyte such as anther wall, filigree is very easy to the dedifferentiation starting, thereby influence the dedifferentiation starting of pollen (sporule), cause the two kinds of callus or the embryoid of diploid cell and haploid cell to mix, being difficult to distinguish any is needed monoploid material.Because mostly anther culture is to be differentiated to form embryoid again or indefinite bud develops into plant again through callus cell, the ploidy of plant is easy to variation, so can not guarantee it is haplobiont.Vaulx RD de etal is 1982, and Agronomie.2:10 reported once on the 983-988 page or leaf that 100 flower pesticide obtained 25-35 strain regeneration plant, and wherein 15-50% is diplontic.Miyoshi K. is 1996, and Plant Cell Reports 15:6 reported once also on the 391-395 page or leaf that 100 flower pesticide obtained 12 strains altogether, and it is haploid having only 1 strain, and 7 strains are diplontic, and 3 strains are triploid, and 1 strain is tetraploid.In addition, during solid culture, flower pesticide is only in a fixed position of substratum, and the nutritive ingredient in the substratum is not fully utilized.
Utilizing pollen (sporule) to cultivate in the method for haplobiont, use liquid nutrient medium to carry out suspension culture mostly.The problem of its existence is, the process of making microspore suspension is very loaded down with trivial details, need push flower pesticide, pollen (sporule) is extruded, for the flower pesticide residue is removed, also to filter through stainless (steel) wire, also must be centrifugal through three times on whizzer in order to remove little anther wall residue, just and then add that prepared culture medium can be finished this seeded process in advance.Because the operation steps complexity has also increased the chance of polluting.
In addition, no matter be that anther culture or pollen (sporule) are cultivated, the induction frequency of its embryoid or callus is all very low, and according to the available data report, during anther culture, its induction frequency is between 1-35%.For example: Deng Li equality, 1991 at biotechnology 1:(5) report that during the eggplant anther culture, the induction frequency of pollen callus is between 5.6-18% on the 30-34 page or leaf; Academy of agricultural sciences, Beijing vegetables institute 1977 reports on anther culture academic discussion collected works. the induction frequency of pollen embryoid is between 0.7-9.09%; Tokumo K etal. is 1994, Scientific Reports of the Faculty of Agriculture, report on the OkayamaUniversity No.84 13-16., the induction frequency of pollen callus is up to 28%, and the induction frequency of its embryoid has only 5.1%.Rotino G.L. etc. reported on Capsicum Newsletter No.689-90 page or leaf in 1987, produced 531 embryoids in 9050 flower pesticide, and best is that 910 flower pesticide have 306 embryoids to form, and its induction frequency is 33.6%.When pollen was cultivated, its induction frequency just still less had only 0.1-12%.For example: k.1996 Miyoshi at Plant Cell Reports15:6, reported once on the 391-395 page or leaf that 100 flower pesticide obtained 12 strain regeneration plants altogether.Low like this induction frequency is difficult to reach the requirement of breeding, and this is unfavorable for quickening the seed selection of crop breeding process and improved seeds.
Summary of the invention:
The invention provides a kind of method, adopt this method can obviously improve the induction frequency of embryoid and plant by flower pesticide and pollen cultivation acquisition haplobiont.
Method of the present invention is:
With the flower pesticide direct inoculation in liquid nutrient medium, carry out ANTHER FLOATING CULTURE, make the flower pesticide natural cracking, pollen (the alleged pollen of the present invention also comprises sporule) freely becomes scattered about in the liquid nutrient medium, behind the suitable fate of ANTHER FLOATING CULTURE, remove flower pesticide under aseptic condition, the pollen that dissociates is cultivated.
In the method for the invention, described liquid nutrient medium can be to well known to a person skilled in the art that MS (can be referring to Murashige T.and F.Skoog, in 1962 at Physiol.Plant, 15:473-497) or other liquid nutrient medium, preferably, contain maltose in the liquid nutrient medium, its preferred concentration is 3-15%.
In the method for the invention, preferably contain 2 in the described liquid nutrient medium, 4-D and KT, wherein 2, the preferred concentration of 4-D is the 0.05-0.5 mg/litre, and the preferred concentration of KT is the 0.5-5 mg/litre, and is preferred, and both usage ratios are 1: 5-20.
In the method for the invention, preferably also contain vitamins C in the described liquid nutrient medium, its working concentration is preferably the 0.5-50 mg/litre.
In the method for the invention, took out flower pesticide after ANTHER FLOATING CULTURE 10-25 days, the pollen that dissociates is cultivated.
The method of the invention described above is cultivated applicable to flower pesticide, the pollen of the plant of the easy dedifferentiation of somatocyte such as eggplant, capsicum, tomato, cucumber starting.
In the present invention, owing to be that anther culture and pollen cultivation are combined, flower pesticide directly floated in the liquid nutrient medium cultivate, and in culturing process, remove anther wall in good time, somatic interference such as medicine wall, filigree when just having overcome existing anther culture, thus make ploidy relatively stable.Because of pollen is free in the liquid nutrient medium, can make full use of the nutrient in the substratum.On the other hand, method of the present invention has also overcome when pollen is cultivated to need to push, filtration, loaded down with trivial details operating process such as centrifugal, promptly reduces labor capacity and has reduced the chance of polluting again.And method of the present invention is adopted in the discovery that the applicant is also pleasantly surprised, has obviously improved the frequency with plant regeneration of inducing of eggplant pollen embryoid.For example in an embodiment of the present invention, 25 flower pesticide have just produced 70 embryoids, and its induction frequency is 280%, and existing 52 embryoids develop into plant, and survival rate of plant is 74.3%.
Provide below and adopt method cultivation eggplant flower pesticide of the present invention to obtain the example of haplobiont, but be not limited to present embodiment.
Embodiment: eggplant ANTHER FLOATING CULTURE
Fetching pollen development from the field healthy plant is the eggplant bud to middle and advanced stage in monokaryon mid-term period, remove calyx, with 70% alcohol disinfecting several seconds, again with 5-10% aqueous sodium hypochlorite solution sterilization 7-8 minute, in super clean bench, with aseptic water washing 3-4 time, water is fallen to do the back from bud, take out flower pesticide and be seeded in the EGA liquid nutrient medium, make flower pesticide float on fluid surface.Described liquid nutrient medium is: saltpetre 2150 mg/litre, ammonium nitrate 1240 mg/litre, sal epsom 410 mg/litre, calcium chloride 240 mg/litre, potassium primary phosphate 140 mg/litre, nitrocalcite 50 mg/litre, Sodium phosphate dibasic 40 mg/litre, ammonium sulfate 30 mg/litre, Repone K 10 mg/litre; Manganous sulfate 22 mg/litre, zinc sulfate 4.0 mg/litre, boric acid 3.0 mg/litre, potassiumiodide 0.7 mg/litre, Sodium orthomolybdate 0.2 mg/litre, copper sulfate 0.02 mg/litre, cobalt chloride 0.02 mg/litre; Molysite (forming) by ferrous sulfate 27.85 mg/litre and ethamine tetraacethyl disodium 37.25 mg/litre, inositol 50 mg/litre, vitamin B6 6 mg/litre, VITMAIN B1 0.5 mg/litre, nicotinic acid 0.5 mg/litre, glycine 0.1 mg/litre, 2,4-D0.2 mg/litre, KT2 mg/litre, maltose 5%, Catergen 0 mg/litre.Then, material that inoculation is good places in 25 ± 1 ℃ the culturing room and cultivates.Cultivate after 15 days, take out flower pesticide in super clean bench, still put back to and continue in the culturing room to cultivate, passed through 10-15 days again, visible pollen forms embryoid, and statistics shows that 25 flower pesticide have just produced 70 embryoids, and its induction frequency is 280%.Then, the embryoid immigration is made on its substratum that further develops into plant, 1-2 week just can obtain the monoploid whole plant of eggplant.At present, existing 52 embryoids develop into plant, and survival rate of plant is 74.3%.
Claims (10)
1, a kind of flower pesticide or pollen are cultivated the method for haplobiont, it is characterized in that the flower pesticide direct inoculation in liquid nutrient medium, treat the flower pesticide natural cracking, make pollen freely become scattered about in the liquid nutrient medium, behind the suitable fate of ANTHER FLOATING CULTURE, remove flower pesticide under aseptic condition, the pollen that dissociates (sporule) is cultivated.
2, the described method of claim 1 contains maltose in the liquid nutrient medium.
3, the described method of claim 2, the concentration of maltose is 3-15%.
4, the described method of claim 1 contains 2 in the liquid nutrient medium, 4-D and KT, and 2, the preferred concentration of 4-D is the 0.05-0.5 mg/litre, the preferred concentration of KT is the 0.5-5 mg/litre.
5, the described method of claim 4,2, the usage ratio of 4-D and KT is 1: 5-20.
6, the described method of claim 1 contains vitamins C in the liquid nutrient medium.
7, the described method of claim 6, ascorbic concentration is the 0.5-50 mg/litre.
8, the described method of claim 1 was taken out flower pesticide after ANTHER FLOATING CULTURE 10-25 days, the pollen that dissociates is cultivated.
9, the described method of claim 1-8 is characterized in that this method is applicable to flower pesticide or the pollen cultivation of the plant of the easy dedifferentiation starting of flower pesticide somatocyte.
10, the described method of claim 9, this method is suitable for the cultivation of flower pesticide such as eggplant, capsicum, tomato, cucumber.
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| CN 02158575 CN1226408C (en) | 2002-12-26 | 2002-12-26 | Method for obtaining haploid plant strain by culturing anther or pollen |
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| CN 02158575 CN1226408C (en) | 2002-12-26 | 2002-12-26 | Method for obtaining haploid plant strain by culturing anther or pollen |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100338998C (en) * | 2005-03-07 | 2007-09-26 | 四川农业大学 | Method for culturing embryoid of woody plant anther |
| CN100361569C (en) * | 2005-12-31 | 2008-01-16 | 四川农业大学 | Method for preparing plant haploid embryo and haploid plant |
| CN103004599A (en) * | 2012-12-20 | 2013-04-03 | 扬州大学 | Method for obtaining crowtoe regeneration plantlet by anther culture |
| CN103436482A (en) * | 2013-08-01 | 2013-12-11 | 南京年吉冷冻食品有限公司 | Preparation method of anther culture solution |
| CN103436483A (en) * | 2013-08-01 | 2013-12-11 | 南京年吉冷冻食品有限公司 | Anther culture solution |
| CN103636505A (en) * | 2013-12-20 | 2014-03-19 | 上海市农业科学院 | Complex breeding method of high-chlorophyll multi-tiller barley |
| CN104041415A (en) * | 2014-06-18 | 2014-09-17 | 南京市蔬菜科学研究所 | Method for obtaining haploid regenerated plant by eggplant anther culturing |
| CN104542274A (en) * | 2014-07-03 | 2015-04-29 | 郑州市蔬菜研究所 | Culture medium of induced haplobiont for culturing eggplant anther and method of culture medium |
| CN104041414B (en) * | 2014-06-18 | 2016-04-27 | 南京市蔬菜科学研究所 | A kind of pepper anther culture obtains the method for monoploid regeneration plant |
| CN105638455A (en) * | 2014-11-14 | 2016-06-08 | 石河子大学 | Method for obtaining dry chilli haploid plant by anther culture and culture medium |
| CN108522270A (en) * | 2018-03-06 | 2018-09-14 | 山东寿光蔬菜种业集团有限公司 | A method of based on cucumber anther culture and improvement virus elimination rate and hardening survival rate |
| CN109089885A (en) * | 2018-09-03 | 2018-12-28 | 江苏高航农业科技有限公司 | A kind of induction cherry and tomato anther obtains the cultural method of regeneration plant |
| CN110317772A (en) * | 2019-07-24 | 2019-10-11 | 上海市农业科学院 | A kind of method of drawing material of barley microspore |
| CN111004765A (en) * | 2019-11-29 | 2020-04-14 | 北京市农林科学院 | Method for promoting cucumber microspore division and culture method of cucumber microspore |
| CN112970577A (en) * | 2019-12-13 | 2021-06-18 | 赵鑫 | Breeding method of white jade green cucumber |
-
2002
- 2002-12-26 CN CN 02158575 patent/CN1226408C/en not_active Expired - Lifetime
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100338998C (en) * | 2005-03-07 | 2007-09-26 | 四川农业大学 | Method for culturing embryoid of woody plant anther |
| CN100361569C (en) * | 2005-12-31 | 2008-01-16 | 四川农业大学 | Method for preparing plant haploid embryo and haploid plant |
| CN103004599A (en) * | 2012-12-20 | 2013-04-03 | 扬州大学 | Method for obtaining crowtoe regeneration plantlet by anther culture |
| CN103436482A (en) * | 2013-08-01 | 2013-12-11 | 南京年吉冷冻食品有限公司 | Preparation method of anther culture solution |
| CN103436483A (en) * | 2013-08-01 | 2013-12-11 | 南京年吉冷冻食品有限公司 | Anther culture solution |
| CN103636505A (en) * | 2013-12-20 | 2014-03-19 | 上海市农业科学院 | Complex breeding method of high-chlorophyll multi-tiller barley |
| CN103636505B (en) * | 2013-12-20 | 2016-06-29 | 上海市农业科学院 | A kind of compound selection of high chlorophyll many tillers Fructus Hordei Vulgaris |
| CN104041415B (en) * | 2014-06-18 | 2015-09-23 | 南京市蔬菜科学研究所 | A kind of eggplant anther culture obtains the method for monoploid regeneration plant |
| CN104041414B (en) * | 2014-06-18 | 2016-04-27 | 南京市蔬菜科学研究所 | A kind of pepper anther culture obtains the method for monoploid regeneration plant |
| CN104041415A (en) * | 2014-06-18 | 2014-09-17 | 南京市蔬菜科学研究所 | Method for obtaining haploid regenerated plant by eggplant anther culturing |
| CN104542274A (en) * | 2014-07-03 | 2015-04-29 | 郑州市蔬菜研究所 | Culture medium of induced haplobiont for culturing eggplant anther and method of culture medium |
| CN105638455A (en) * | 2014-11-14 | 2016-06-08 | 石河子大学 | Method for obtaining dry chilli haploid plant by anther culture and culture medium |
| CN105638455B (en) * | 2014-11-14 | 2018-03-20 | 石河子大学 | A kind of method and culture medium that Drying pepper haplobiont is obtained by Anther Culture |
| CN108522270A (en) * | 2018-03-06 | 2018-09-14 | 山东寿光蔬菜种业集团有限公司 | A method of based on cucumber anther culture and improvement virus elimination rate and hardening survival rate |
| CN109089885A (en) * | 2018-09-03 | 2018-12-28 | 江苏高航农业科技有限公司 | A kind of induction cherry and tomato anther obtains the cultural method of regeneration plant |
| CN110317772A (en) * | 2019-07-24 | 2019-10-11 | 上海市农业科学院 | A kind of method of drawing material of barley microspore |
| CN110317772B (en) * | 2019-07-24 | 2021-04-13 | 上海市农业科学院 | Method for taking barley microspores |
| CN111004765A (en) * | 2019-11-29 | 2020-04-14 | 北京市农林科学院 | Method for promoting cucumber microspore division and culture method of cucumber microspore |
| CN112970577A (en) * | 2019-12-13 | 2021-06-18 | 赵鑫 | Breeding method of white jade green cucumber |
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| CN1226408C (en) | 2005-11-09 |
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Owner name: BEIJING HAIHUA BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: PLANT TISSUE CULTURE TECHNOLOGY LAB, HAIDIAN DISTRICT, BEIJING Effective date: 20101216 |
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