CN104396722A - Method for cultivating triploid dendrobium huoshanense by crossbreeding method of tetraploid plants and diploid plants - Google Patents
Method for cultivating triploid dendrobium huoshanense by crossbreeding method of tetraploid plants and diploid plants Download PDFInfo
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Abstract
The invention discloses a method for cultivating triploid dendrobium huoshanense by a crossbreeding method of tetraploid plants and diploid plants. The method comprises the following steps: (1) dendrobium huoshanense species which are from different producing areas and have characteristics of good stress resistance, rapid growth, thick stalk, long stem, many tillers, low fiber content and high active ingredient content are preferably selected as polyploidy cultivated original plant parents; (2) seeds of the different species of original plants undergo aseptic sowing in a solid medium so as to obtain diploid protocorms; (3) protoplasts of the different species of diploid protocorms are prepared and cell fusion is carried out; (4) detected tetraploid plants undergo asexual tissue culture so as to obtain tetraploid plant seedlings; (5) the tetraploid plant seedlings undergo hardening-seedling acclimatization and cultivation to flowering; (6) the tetraploid plants and diploid plants undergo artificial pollination and crossbreeding, and fructification is completed to obtain triploid dendrobium huoshanense seeds; and (7) the triploid seeds undergo aseptic sowing and culture so as to obtain a lot of triploid seedlings.
Description
Technical field
The present invention relates to the orchid family Dendrobium medicinal plant biological breeding methods, particularly a kind of method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness.
Background technology
Dendrobidium huoshanness (formal name used at school: Dendrobium huoshanense C.Z.Tang et S.J.Cheng) has the wild Dendrobidium huoshanness of the title of " medicine circle giant panda " in the world, be called the stem of noble dendrobium suddenly in history, be commonly called as a meter dry measure used in former times, apart from the history of modern existing more than 200 year.Originating in the ground such as China's Dabie Mts, anhui, is the first protection kind in National modern Chinese medicine key special subjects more than 200 Rare Kinds in imminent danger.The superfine product in medicinal dendrobium, Ye Shi Huoshan County exclusive, the rare traditional Chinese medicine that has original producton location geographical sign, be described as " first of Chinese nine immortal grass ", " healthy soft gold " in history." legendary god of farming's book on Chinese herbal medicine ", Compendium of Material Medica are all on the books.Polysaccharide contained by Dendrobidium huoshanness, alkaloids substance have throat clearly, liver-nourishing and eyesight-improving, hypoglycemic, the effect of promoting longevity.This product can increase substantially SOD in human body (main matter delayed senility), in develop immunitypty, antifatigue, delays senility and promotes there is obvious effect in cancer cell-apoptosis etc., thus reaching effect of health care health care.
Polyploid breeding is the important means that new variety of plant is cultivated, and polyploid breeding originated from for 20 beginnings of the century, had been widely used in now in the cultivation of new variety of plant.Polyploid, due to the increase of genome ploidy, often shows the features such as modal huge property and resistance enhancing.Wherein triploid also has sterile feature, namely can be used as permanent hybrid, is also conducive to business protection.
By genome tripling, can obtain the triploid with polyploid and crossbreed double dominant, its Heterosis goes out to have huge property and irreplaceability, can improve Dendrobidium huoshanness root yield, quality and resistance.Triploid Dendrobidium huoshanness then has the double dominant on seed output and quality.
The task that country's Eleventh Five-Year Plan and " 12 " plan all propose " directive breeding of the excavation of germ plasm resource, preservation, innovation and new varieties ".For implementing, implementing this task, we advocate the method for application " genome polyploidization combines with fusion hybridization ", realize the object of the innovation of germ plasm resource and the directive breeding of new varieties.Only have the breeding method of triploid dendrobium officinale and spring dendrobium to disclose and authorized at present, up to now, triploid Dendrobidium huoshanness (karyotype index is 2n=3x=57) and breeding method yet there are no open report.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness, the triploid Dendrobidium huoshanness that the method is cultivated is compared with dliploid Dendrobidium huoshanness, its main morphological traits of tea plant is substantially identical, but its stem, leaf, flower show obvious increase, output and polyoses content significantly improve, and resistance strengthens.The triploid Dendrobidium huoshanness that the present invention produces has the double dominant of polyploid and crossbreed.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
Utilize tetraploid plant and liploid plant breeding by crossing to cultivate a method for triploid Dendrobidium huoshanness, comprise the following steps:
(1) the source plant parent that good stress resistance, growth are fast, thick, the high length of stem, the Different sources many, fiber content is low, content of drug effect components is high of tillering the Dendrobidium huoshanness kind of stem stalk is cultivated as polyploid is optimized;
(2) in solid culture medium, aseptic seeding is carried out to the former plant seed of different cultivars, obtain 2 times of body protocorms;
(3) prepare the protoplast of different cultivars 2 times of body protocorms, carry out cell protoplast fusion, and be trained callus;
(4) carry out differentiation-inducing to the tetraploid callus detected, obtain tetraploid plant seedling;
(5) conventional hardening domestication carried out to tetraploid plant seedling and cultivate to blooming;
(6) tetraploid plant and liploid plant artificial pollination are hybridized, solid, obtain triploid Dendrobidium huoshanness seed;
(7) to triploid aseptically sowing seeds, cultivation, a large amount of triploid seedling is obtained.
Dendrobidium huoshanness described in step (1) is the kind that the Different sources being distributed in Dabie Mountain mountain system has merit, wherein the kind of high latitude, high altitude localities has strong stress resistance, stem stalk is thick, content of drug effect components is high feature, and the kind of low latitudes, low altitude area has that fast growth, stem height are long, many, that fiber content is low feature of tillering.
Cultivate described in step (2) as solid culture medium is cultivated, be applicable to the medium that dedifferentiation forms callus.The 1/2MS medium that preferred macroelement reduces by half, adjustment sucrose concentration is 5%-8%, preferably adds the coconut palm breast of 10-15%, 2,4-D 0.2-2.0 mg/L, NAA 0.2-2.0 mg/L; Cultivation temperature is within the scope of the day and night temperature of 25-18 DEG C, and light application time 10-12 hour, intensity of illumination 2000-3000Lux, colour temperature is at 4500-5500k.
Preparation method's preferred enzyme solution preparation of protoplast described in step (3), cytomixis way preferably shocks by electricity fusion method.
Tetraploid described in step (4) is allodidiploid or homology amphidiploid; Wherein allodidiploid has heterosis, hybrid vigor, because this be by the individual cells of different cultivars between fusion form, this is different from the tetraploid utilizing colchicine and functional analogue thereof to be prepared by the mitotic method of T suppression cell, and tetraploid prepared by this way is only homology amphidiploid; Described detection chromosome G banding is normal dyeing microscopy way, chooses the seedling of chromosome number 2n=4x=76, is to cultivate successful tetraploid.
Tetraploid plant and liploid plant artificial pollination described in step (6) are hybridized, and tetraploid plant and liploid plant can male parent and female parents each other.
The present invention includes the triploid Dendrobidium huoshanness that any one breeding method above-mentioned obtains.
Beneficial effect of the present invention:
This method adopts the way of cytomixis the Different Individual of the Dendrobidium huoshanness kind with different merit to be constructed out tetraploid (allodidiploid or homology amphidiploid) plant, then tetraploid plant and liploid plant are hybridized obtains triploid Dendrobidium huoshanness plant.Breeding process is that chromogene group doubles process, is also the process merging hybridization.The triploid Dendrobidium huoshanness selected is compared with the dliploid Dendrobidium huoshanness stem of noble dendrobium, and its main morphological traits of tea plant is substantially identical, but its stem, leaf, flower show obvious increase, output and polyoses content significantly improve, resistance enhancing.The triploid Dendrobidium huoshanness that the present invention produces has the double dominant of polyploid and crossbreed.
Embodiment
With embodiment, the invention will be further described below, but the present invention is not limited to these embodiments, the equivalent replacement that the technical characteristic of those skilled in the art to content of the present invention is done, or improve accordingly, still belongs within protection scope of the present invention.
For convenience of describing, represent high latitude, Dendrobidium huoshanness kind that the karyotype index of high altitude localities is 2n=38 in following examples with " kind A ", this kind has strong stress resistance, stem stalk is thick, content of drug effect components is high feature; Represent low latitudes with " kind B ", Dendrobidium huoshanness kind that the karyotype index of low altitude area is 2n=38, this kind has that fast growth, cane are long, many, that fiber content is low feature of tillering.
EXAMPLE l
(1) by the plant of kind A and kind B growing and cultivation according to a conventional method;
(2) in solid culture medium, aseptic seeding is carried out to the former plant seed of different cultivars, obtain 2 times of body protocorms of kind A and kind B plant, solid culture medium is the 1/2MS medium that macroelement reduces by half, and adds 0.2g/L 2,4-D and 0.2g/L NAA, adjustment sucrose concentration is 5%, add 10% coconut palm breast, cultivation temperature within the scope of the day and night temperature of 25-18 DEG C, light application time 10-12 h, intensity of illumination 2000-3000 Lux, colour temperature is at 4500-5500 K;
(3) protoplast preparing kind A and kind B plant 2 times of bodies carries out cytomixis, and the preparation method of protoplast is enzymatic isolation method preparation, and cytomixis way adopts electric shocking method;
(4) continue to be cultured to callus to fusion cell, get callus cell dyeing compressing tablet, microscopy checking chromosome G banding, carries out differentiation-inducing cultivation to the tetraploid detected (allodidiploid or homology amphidiploid) fusion cell, obtains tetraploid plant seedling.Cultivation temperature in the temperature range of 25-18 DEG C, light application time 10-12 hour, intensity of illumination 2000-3000lux, colour temperature is at 4500-5500k;
(5) conventional hardening domestication and cultivation management are carried out to blooming to tetraploid plant seedling;
(6) with tetraploid plant be maternal with liploid plant for male parent artificial pollination is hybridized, solid, obtain triploid Dendrobidium huoshanness seed;
(7) to triploid aseptically sowing seeds, cultivation, a large amount of triploid seedling is obtained;
(8) triploid Dendrobidium huoshanness seedling carries out conventional hardening domestication and cultivates by management to strain.Strain has that strong stress resistance, stem stalk are thick, the high length of stem, many, that fast growth, fiber content are low, content of drug effect components is high feature of tillering, and wherein total starches content reaches 23%.
Embodiment 2
(1) by the plant of kind A and kind B growing and cultivation according to a conventional method;
(2) in solid culture medium, aseptic seeding is carried out to the former plant seed of different cultivars, obtain 2 times of body protocorms of kind A and kind B plant, solid culture medium is the 1/2MS medium that macroelement reduces by half, and adds 0.2g/L 2,4-D and 0.2g/L NAA, adjustment sucrose concentration is 6%, add 15% murphy juice, cultivation temperature within the scope of the day and night temperature of 25-18 DEG C, light application time 10-12 h, intensity of illumination 2000-3000 Lux, colour temperature is at 4500-5500 K;
(3) prepare the protoplast of kind A and kind B plant 2 times of bodies, carry out cytomixis, the preparation method of protoplast is enzymatic isolation method preparation, and cytomixis way adopts electric shocking method;
(4) continue to be cultured to callus to fusion cell, get callus cell dyeing compressing tablet, microscopy checking chromosome G banding, carries out differentiation-inducing cultivation to the tetraploid detected (allodidiploid or homology amphidiploid) fusion cell, obtains tetraploid plant seedling.Cultivation temperature in the temperature range of 25-18 DEG C, light application time 10-12 hour, intensity of illumination 2000-3000lux, colour temperature is at 4500-5500k;
(5) conventional hardening domestication and cultivation management are carried out to blooming to tetraploid plant seedling;
(6) take tetraploid plant as male parent and liploid plant for maternal artificial pollination is hybridized, solid, obtain triploid Dendrobidium huoshanness seed;
(7) to triploid aseptically sowing seeds, cultivation, a large amount of triploid seedling is obtained;
(8) triploid Dendrobidium huoshanness seedling carries out conventional hardening domestication and cultivates by management to strain.Strain has that strong stress resistance, stem stalk are thick, the high length of stem, many, that fast growth, fiber content are low, content of drug effect components is high feature of tillering, and wherein total starches content reaches 22%.
Claims (7)
1. utilize tetraploid plant and liploid plant breeding by crossing to cultivate a method for triploid Dendrobidium huoshanness, it is characterized in that comprising the following steps:
(1) the source plant parent that good stress resistance, growth are fast, thick, the high length of stem, the Different sources many, fiber content is low, content of drug effect components is high of tillering the Dendrobidium huoshanness kind of stem stalk is cultivated as polyploid is optimized;
(2) in solid culture medium, aseptic seeding is carried out to different cultivars source plant seed, obtain 2 times of body protocorms;
(3) prepare the protoplast of different cultivars 2 times of body protocorms, carry out cell protoplast fusion, and be trained callus;
(4) carry out differentiation-inducing to the tetraploid callus detected, obtain tetraploid plant seedling;
(5) conventional hardening domestication carried out to tetraploid plant seedling and cultivate to blooming;
(6) tetraploid plant and liploid plant artificial pollination are hybridized, solid, obtain triploid Dendrobidium huoshanness seed; (7) to triploid aseptically sowing seeds, cultivation, a large amount of triploid seedling is obtained.
2. the method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness according to claim 1, it is characterized in that: Dendrobidium huoshanness described in step (1) is the kind that the Different sources being distributed in Dabie Mountain mountain system has merit, wherein the kind of high latitude, high altitude localities has strong stress resistance, stem stalk is thick, content of drug effect components is high feature, and the kind of low latitudes, low altitude area has that fast growth, stem height are long, many, that fiber content is low feature of tillering.
3. the method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness according to claim 1, it is characterized in that: cultivate as to cultivate at solid culture medium described in step (2), be applicable to the medium of evoked callus, the 1/2MS medium that preferred macroelement reduces by half, adjustment sucrose concentration is 5%-8%, preferably adds the coconut palm breast of 10-15%, 2,4-D 0.2-2.0 mg/L, NAA 0.2-2.0 mg/L; Cultivation temperature is within the scope of the day and night temperature of 25-18 DEG C, and light application time 10-12 hour, intensity of illumination 2000-3000Lux, colour temperature is at 4500-5500k.
4. the method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness according to claim 1, it is characterized in that: the preparation of preparation method's preferred enzyme solution of protoplast described in step (3), protoplast fusion way preferably shocks by electricity fusion method.
5. the method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness according to claim 1, is characterized in that: tetraploid described in step (4) is allodidiploid or homology amphidiploid; Described detection chromosome G banding is normal dyeing microscopy way, chooses the seedling of chromosome number 2n=4x=76, is to cultivate successful tetraploid.
6. the method utilizing tetraploid plant and liploid plant breeding by crossing to cultivate triploid Dendrobidium huoshanness according to claim 1, it is characterized in that: described in step (6), tetraploid plant and liploid plant are hybridized in florescence artificial pollination, tetraploid plant and liploid plant can male parent and female parents each other.
7. the triploid Dendrobidium huoshanness that in claim 1-6, breeding method described in any one obtains.
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| CN105684896A (en) * | 2016-01-08 | 2016-06-22 | 玉林师范学院 | Dendrobium germplasm breeding method based on asymmetry protoplast hybridization technique |
| CN105906641A (en) * | 2016-05-20 | 2016-08-31 | 皖西学院 | Method for producing Dendrobium huoshanense alkaloids by cell suspension culture |
| CN107801633A (en) * | 2017-11-07 | 2018-03-16 | 安徽同济生生物科技有限公司 | A kind of stem of noble dendrobium polyploid seedling abductive approach |
| CN107896972A (en) * | 2017-11-09 | 2018-04-13 | 四川农业大学 | A kind of method by the perennial feeding Job's tears of distant hybridization selection and breeding |
| CN108739402A (en) * | 2018-06-24 | 2018-11-06 | 合肥扬扬农业科技有限公司 | A method of improving corn hybrid seed production efficiency |
| CN109430061A (en) * | 2018-12-28 | 2019-03-08 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| CN109601382A (en) * | 2018-12-28 | 2019-04-12 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| CN109618931A (en) * | 2018-12-28 | 2019-04-16 | 武汉博立达农业科技发展有限公司 | The method for cultivating triploid giantreed using somatic hybridization |
| CN109618929A (en) * | 2018-12-28 | 2019-04-16 | 武汉博立达农业科技发展有限公司 | The breeding method of triploid giantreed |
| CN109662030A (en) * | 2018-12-28 | 2019-04-23 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| WO2020135712A1 (en) * | 2018-12-28 | 2020-07-02 | 武汉兰多生物科技有限公司 | Method for growing arundo donax plant |
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| CN105684896A (en) * | 2016-01-08 | 2016-06-22 | 玉林师范学院 | Dendrobium germplasm breeding method based on asymmetry protoplast hybridization technique |
| CN105906641A (en) * | 2016-05-20 | 2016-08-31 | 皖西学院 | Method for producing Dendrobium huoshanense alkaloids by cell suspension culture |
| CN107801633A (en) * | 2017-11-07 | 2018-03-16 | 安徽同济生生物科技有限公司 | A kind of stem of noble dendrobium polyploid seedling abductive approach |
| CN107896972A (en) * | 2017-11-09 | 2018-04-13 | 四川农业大学 | A kind of method by the perennial feeding Job's tears of distant hybridization selection and breeding |
| CN107896972B (en) * | 2017-11-09 | 2019-12-31 | 四川农业大学 | Method for breeding perennial feeding coix seeds by distant hybridization |
| CN108739402A (en) * | 2018-06-24 | 2018-11-06 | 合肥扬扬农业科技有限公司 | A method of improving corn hybrid seed production efficiency |
| CN109430061A (en) * | 2018-12-28 | 2019-03-08 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| CN109601382A (en) * | 2018-12-28 | 2019-04-12 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| CN109618931A (en) * | 2018-12-28 | 2019-04-16 | 武汉博立达农业科技发展有限公司 | The method for cultivating triploid giantreed using somatic hybridization |
| CN109618929A (en) * | 2018-12-28 | 2019-04-16 | 武汉博立达农业科技发展有限公司 | The breeding method of triploid giantreed |
| CN109662030A (en) * | 2018-12-28 | 2019-04-23 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| WO2020135712A1 (en) * | 2018-12-28 | 2020-07-02 | 武汉兰多生物科技有限公司 | Method for growing arundo donax plant |
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