CN105165603A - Ophiopogon japonicus f. nanus gamma-ray homogeneous mutant library rapid creation method - Google Patents
Ophiopogon japonicus f. nanus gamma-ray homogeneous mutant library rapid creation method Download PDFInfo
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Abstract
The invention provides an ophiopogon japonicus f. nanus gamma-ray homogeneous mutant library rapid creation method. The method comprises the first step of high-frequency somatic embryo explant material preparation, the second step of culture of an early-stage somatic embryo explant, the third step of physical mutagen preparation, the fourth step of gamma-ray treatment of the early-stage somatic embryo explant, the fifth step of somatic embryo differentiation culture, the sixth step of rapid propagation and proliferation culture of a homogeneous mutant embryoid, the seventh step of screening and rapid propagation and proliferation culture of the homogeneous mutant embryoid, the eighth step of plant regeneration through a mature homogeneous mutant embryoid, the ninth step of acclimatization and transplant of a regenerated plant of a homogeneous mutant and the tenth step of establishment of a field mutant library. By means of the method for rapidly establishing the ophiopogon japonicus f. nanus gamma-ray saturated homogeneous mutant library, a foundation is laid for intensive study of functional genomics and genetics and breeding problems of ophiopogon japonicus, and meanwhile homogeneous mutant materials which are most direct and effective are provided for breeding a novel variety of the ophiopogon japonicus f. nanus with strong cold resistance; important theoretical significance and application value are achieved.
Description
Technical field
The invention belongs to field of plant breeding, relate to the method that a Plants homogeneous mutant storehouse builds, particularly relate to a kind of by physical mutagen (
60co-gamma-rays) quick method of formulating lowgrow ophiopogon japonicus saturated gene homogeneous mutant storehouse.
Background technology
Lowgrow ophiopogon japonicus (Ophiopogonjaponicusf.nanus) belongs to the lowgrow ophiopogon japonicus kind in Liliaceae Ophiopogon, there is strong happiness shade, the four seasons are evergreen, cold-resistant, heat-resisting, regeneration capacity strong, good, the resistance to trample of sight, manage the features such as easy, under being suitable for amenity forest, building shelter from the sun and the cladding greenbelt inferior special habitats grass carpet & cover plant of afforesting, beautifying.Lowgrow ophiopogon japonicus is as a kind of important asexually propagated plant, not only breed improvement is constantly paid attention in recent years, and its more distinctive biological characters find some typical material having the gene of important value to discover and use as genetic resourceses such as short raw gene, resistance to shady genes as nanism and strong shade tolerance etc. also make it become.
Interaction between the function of Analysis and Identification gene and gene, the impact that gene pairs grows is the primary study content of functional genomics.Although have developed at present the new technologies of multiple Analysis and Identification gene, most effective method builds saturated gene mutation body storehouse, by the function of Analysis of Mutants identified gene.So the structure of mutant library is the Research foundation of functional genomics.The crops such as current model plant arabidopsis, paddy rice have all constructed saturated mutant library and have carried out the research of correlation function genomics, obtain a collection of Innovation Germplasm resource having important value.
In numerous physical mutagens,
60co-gamma-rays can produce multiple point mutation because of it on a genome, and is evenly distributed, and particularly r ray mainly causes disappearance and the chromosomal rearrangement of DNA fragmentation, is specially adapted to the establishment of the mutant library of multiple family gene afunction.Especially its less mutagenized populations just can obtain the abundant saturated mutant library of variation and become one and be widely used, not only efficient but also stable physical mutagen.
The acquisition of homogeneous mutant: method is exactly obtained by the tissue culture regeneration of single mutant cell the most fast and effectively, the variation proterties obtained by homogeneous mutant is because mutant is without chimerism, so can genetic stability, it be the basis in composition homogeneous mutant storehouse.But majority of plant tissue culture regeneration is not the regeneration depended on cellular level for a long time, but be confined to the regeneration of individuality or organ-tissue level.The Progenies from Regenerated produced by the mutagenesis of multicellular tissue organ, because the otherness of its numerous cytogene sudden change often causes mutant chimerism very serious, Character instability, poor repeatability, regeneration screening time extend, due to very difficult acquisition homogeneous mutant, thus degradation series of malpractice under causing breeding efficiency.
How to make one unicellularly to develop into a whole plant, be a difficult problem for many plant tissue cultures regeneration always.It is unicellular that current research shows that most plant somatic embryo originates from, somatic embryo development ways has recurred the morphogenetic process of zygotic embryo, so somatic embryo Regeneration Ways is regeneration seed source, seedling detoxifying fast breeding, inducing somatic variation or the desirable receptor system of genetic transformation.
The regeneration of somatic mutagenesis, somatic embryo is the homogeneous mutant obtaining extensively variation fast, the Perfected process avoiding chimera to be formed, being easy to obtain at short notice the homogeneous mutant that a large amount of variations is abundant, is build the visible and basis of the mutant library of inheritance stability of phenotype.Therefore somatic cell physical mutagenesis combine with technique somatic embryo regeneration techniques is the key technology ensureing to obtain fast r ray homogeneous mutant storehouse.
Research shows: the screening of mutant is more simple, and having become the effective way that much important germ plasm resource obtains, is also the prerequisite that many most important theories problems are solved.But the screening technique of mutant somatic selection combination blast regeneration acquires a certain degree of difficulty, and report is also more rare at present, it is the key of directed homogeneous mutant screening success or failure.
Not yet find that the lowgrow ophiopogon japonicus somatic cell utilizing genetic background identical is by the process of r irradiation induction at present both at home and abroad, somatic embryo regeneration plant obtains the abundant homogeneous mutant of variation and by the directed homogeneous mutant of the mutant somatic selection combination blast fast numerous acquisition of regeneration, builds the report in the saturated homogeneous mutant storehouse of lowgrow ophiopogon japonicus r ray with this.Therefore the present invention breaches most plants in the past and comprises lowgrow ophiopogon japonicus: 1) though formulated mutant with new approaches of physical mutagenesis, and the genetic background building the parent material of mutant library is not identical; 2) mutant is with plant that the is individual or regeneration of organ-tissue development ways mostly, thus causes mutant chimerism very serious, also therefore causes screening time to extend or even insignificant screening; 3) because mutant chimerism is very serious, cause the proterties of generation very unstable, extend the screening time to Mutant progeny, and be difficult to obtain homogeneous mutant fast, also play saturated homogeneous mutant storehouse that is that the variation of inheritance stability is enriched and orthomutation with regard to being difficult to by r ray rapid build.Therefore the method in the saturated homogeneous mutant storehouse of a kind of rapid build lowgrow ophiopogon japonicus r ray that provides of this invention, not only for dwarf lilyturf functional genomics, genetic breeding provide more germ plasm resource, simultaneously also for seed selection lowgrow ophiopogon japonicus new varieties provide the most effective mutant material, there is important practical value.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the problem that the present invention mainly solves is to provide a kind of somatic cell r irradiation induction, directed screening and regenerates with somatic embryo and combines, a kind of optimization method in rapid build lowgrow ophiopogon japonicus r ray saturated homogeneous mutant storehouse.
Technical scheme: in order to solve the problems of the technologies described above, the invention provides the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse, the method comprises the steps:
1) high frequency somatic embryo explant material prepares: choose the outdoor healthy lowgrow ophiopogon japonicus plant without damage by disease and insect, get its young young stem and leaf, after Aseptic sterilisation, in aseptic culture medium, group trains in vitro cuttings, get its cauline leaf base portion 0.5-1.0cm size explant, by the cultivation of high-frequency somatic embryo regeneration system, somatic embryo and embryoid and intactly regeneration plant will be produced continuously, originate using regeneration plant as high frequency somatic embryo explant material;
2) cultivation of early stage somatic embryo explant: the explant of above-mentioned tool high frequency somatic embryo regeneration capacity is cut its leaf, basal part of stem 0.5cm size tissue block, be placed on 25-28 DEG C of desinfection chamber light culture 7-10d on body embryo generation inducing culture, now most of somatic cell is unicellular period, be called early stage somatic embryo, in this, as mutagen process material;
3) physical mutagen prepares: comprise
60the setting of the preparation of Co-gamma ray projector and radiation dose;
60co-gamma ray projector is provided by Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov.;
4) the gamma-rays process of early stage somatic embryo explant: early stage somatic embryo explant is divided into 8 groups, for the process of 8 different radiation dose;
5) embryo differentiate is cultivated: by the early stage somatic embryo explant through gamma-ray and mutagenesis process, place back in desinfection chamber light culture 50-60d at temperature 25-28 DEG C, middle subculture once, be conducive to embryo differentiate, the big and small embryoid that suddenlys change in early days that grows out tens on visible explant; Normal temperature light culture can ensure the homogeneity saturated mutant material of the inheritance stability obtaining the extensive genetic mutation of tool fast.
6) with the fast numerous Multiplying culture of cytoplasmic mutation embryoid: above-mentioned early stage sudden change embryoid is cut into 0.5cm size block, numbering is transferred in the fast numerous proliferated culture medium of somatic embryo respectively, cultivate 50-60d under being placed on 25-28 DEG C of light, middle subculture once, can obtain a large amount of ripe same cytoplasmic mutation embryoid;
7) with screening and fast numerous Multiplying culture of cytoplasmic mutation embryoid: above-mentioned all kinds of same cytoplasmic mutation embryoid numerous is soon taken out a part, be cut into 0.5cm size block, numbering continuation is transferred in the fast numerous proliferated culture medium of somatic embryo respectively, under putting into the 5-8 DEG C of cryogenic sterile incubator low light level, (500-800lx) cultivates 25-30d, under subculture proceeds to 25-28 DEG C of light more afterwards, (1500-2000lx) cultivates 25-30d, can obtain the stronger maturation of cold resistance with cytoplasmic mutation embryoid;
8) ripe with cytoplasmic mutation somatic embryogenesis plant: to be the tool of above-mentioned fast numerous propagation is enriched the maturation sudden change embryoid of variation and the strong maturation sudden change embryoid of cold resistance to number respectively and transfer in plant regeneration medium, 20-25d is cultivated, acquisition tool root, stem, the homogeneous mutant regeneration plant that leaf is complete under desinfection chamber temperature 25-28 DEG C light;
9) homogeneous mutant regeneration plant acclimatization and transplants: when root is long-living grow to 1-1.5cm time, each mutant strain is numbered respectively and is transplanted to flowerpot, select light ground mass, be placed in green house, automatic intermittent spraying device waters; Survival rate can reach 100%.Throw off the sealed membrane of blake bottle, at indoor hardening 1 ~ 2d, carefully seedling is taken out with tweezers, clean the medium that root adheres to, be transplanted in the flowerpot that light ground mass (peat: vermiculite=1:1) is housed, putting into greenhouse temperature is 24 DEG C ~ 26 DEG C, suitably shades, or under seedling is put into seedbed, automatic intermittent spraying device waters.
10) field mutant library is set up: select the warm and moist cloudy day that each mutant strain is transplanted to outdoor sylvan life or examination garden, field district after 60d, number respectively, record correlated traits, mutant qualification, screening is carried out in each stage that homogeneous mutant grows, the saturated homogeneous mutant storehouse of dwarf lilyturf gamma-rays is set up, for the research of the aspects such as follow-up dwarf lilyturf selection and use and gene function group with this.Because seedling growth is vigorous, also not high to the conditional request of transplanting land for growing field crops, very easily plant, the survival rate of seedling can reach 100%.
Wherein, described step 3) in radiation dose arrange: be set to 0.0,5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy8 different disposal;
Wherein, described step 3) in
60co-gamma Rays dosage is 20Gy.
Wherein, described aseptic culture medium is cultivated as 1/2MS+2.0mg/LBA+0.5mg/LNAA+20g/L sucrose+0.7% agar.
Wherein, described inducing culture is MS+0.2mg/LBA+0.5mg/LNAA+0.5mg/L2,4-dichlorphenoxyacetic acid+30g/L sucrose+3.5g/Lphytagel.
Wherein, the cultivation of described high-frequency somatic embryo regeneration system: refer to and cultivate 50-60d under nascent embryoid being proceeded to high frequency somatic embryo medium glazing, middle subculture once, constantly will produce secondary embryoids and somatic embryo in large quantities.
Wherein, described high frequency somatic embryo medium is MS+2mg/LBA+0.5mg/LNAA+40g/L sucrose+3.5g/Lphytagel.
Wherein, described step 6) in the fast numerous proliferated culture medium of somatic embryo be MS+2mg/LBA+0.2mg/LNAA+40g/L sucrose+3.5g/Lphytagel, intensity of illumination is 1500-2000lx, light application time is 10-14 hour every day, can ensure that Fast-propagation various sudden change embryoid can obtain again the same cytoplasmic mutation embryoid of corresponding maturation.
Wherein, described step 8) in plant regeneration medium be 1/2MS+20g/L white sugar+0.7% agar.
Beneficial effect: compared with prior art, the invention has the advantages that: select in numerous physical mutagens,
60this not only efficiently not only stable but also easy to use physical mutagen of Co-gamma-rays, utilize it can produce multiple point mutation be evenly distributed on a genome, main to cause disappearance and the chromosomal rearrangement of DNA fragmentation, be specially adapted to the establishment of the mutant library of multiple family gene afunction, especially its less mutagenized populations just can obtain the abundant saturated mutant Sink Characteristics of variation, and the mutant obtained does not relate to transgenic event, safety advantages of higher; Overcome in the past simultaneously
60not identical, the chimerism of mutant genetics background of Co-gamma-ray and mutagenesis method initiative is serious, Character instability, poor repeatability, regeneration screening time extend, and is difficult to obtain saturated and directed homogeneous mutant, the series of malpractice such as breeding efficiency is low.The lowgrow ophiopogon japonicus somatic cell that the present invention utilizes genetic background identical passes through
60co-gamma-ray and mutagenesis combination blast regenerates, and the complex mutation technology of somatic selection combination blast regeneration, obtain the saturated mutant library that variation dissimilar in breeding time, green phase, flowering stage, leaf look, plant type, resistance etc. is abundant, particularly also orientation obtains the strong genetic resources of cold resistance, namely enriched lowgrow ophiopogon japonicus gene pool, germ plasm resource also can be applicable among breed breeding.So the method in the saturated homogeneous mutant storehouse of a kind of rapid build lowgrow ophiopogon japonicus r ray that the present invention provides, not only for the further investigation of the problems such as dwarf lilyturf functional genomics, genetic breeding is laid a good foundation, simultaneously also for the lowgrow ophiopogon japonicus new varieties that seed selection cold resistance is strong provide the most effective homogeneous mutant material, there is important theory significance and using value.
Accompanying drawing explanation
Fig. 1 embryo is unicellular;
Fig. 2 globular embryo and torpedo embryo;
Fig. 3 cotyledonary embryos (left side) and fast numerous mutant regeneration plant (right: lowgrow ophiopogon japonicus);
The same cytoplasmic mutation embryoid that Fig. 4 cold resistance is stronger;
The whole plant of Fig. 5 homogeneous mutant;
Fig. 6 potted plant lowgrow ophiopogon japonicus homogeneous mutant storehouse (left side) and Parental Germplasms (right side).
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1 is that experiment material is described with lowgrow ophiopogon japonicus.Step is:
1) cultivation of the preparation of high frequency somatic embryo explant material and early stage somatic embryo explant: choose the outdoor healthy lowgrow ophiopogon japonicus plant without damage by disease and insect, peel off Lao Ye, get its young tender stem foot and be with 3-4 sheet spire, (1/2MS+2.0mg/LBA+0.5mg/LNAA+ sucrose 20g/L+0.7% agar) 50-60d is cultivated under putting into aseptic culture medium light after Aseptic sterilisation, middle subculture once, group trains into test-tube plantlet, get its test-tube plantlet leaf, basal part of stem 0.5-1.0cm size explant, be placed on the upper light culture 50-60d of body embryo generation inducing culture (MS+BA (0.2mg/L)+NAA (0.5mg/L)+2.4D (0.5mg/L)+sucrose 30g/L+3.5g/Lphytagel), middle subculture once, 50-60d is cultivated under the nascent embryoid produced is proceeded to high frequency somatic embryo medium (MS+BA2mg/L+NAA0.5mg/L+ sucrose 40g/L+3.5g/Lphytagel) glazing, middle subculture once, somatic embryo (the globular embryo of each developmental stage will be produced continuously in a large number, torpedo embryo and cotyledonary embryos), the seedling medium (MS+ white sugar 30g/L+0.7% agar) proceeded to by ripe embryoid (cotyledonary embryos) without hormone is cultivated, complete regenerated plant is formed after 20-25 days, using it as high frequency somatic embryo material source.Cut its leaf, basal part of stem 0.5cm size tissue block as high frequency somatic embryo explant material, be placed on body embryo generation inducing culture and cultivate 7-10d, now most of somatic cell is unicellular period, be called early stage somatic embryo (Fig. 1: embryo is unicellular), body embryo occur early stage as mutagen process material.
2) physical mutagen prepares:
60co-gamma ray projector is provided by Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov.; Radiation dose is set to 0.0,5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy8 different disposal;
3) the gamma-rays process of early stage somatic embryo explant: early stage somatic embryo explant is divided into 8 groups, for the process of 8 different radiation dose; After process terminates, place back in desinfection chamber and carry out embryo differentiate cultivation;
4) embryo differentiate is cultivated: the early stage somatic embryo explant through gamma-ray and mutagenesis process being transferred to temperature is that 25-28 DEG C of desinfection chamber carries out light culture, on the visible explant of about 50-60d big and small grow out tens early stage with cytoplasmic mutation embryoid (Fig. 2: globular embryo, torpedo embryo).Normal temperature light culture can ensure the homogeneity saturated mutant material of the inheritance stability obtaining the extensive genetic mutation of tool fast.
5) with the fast numerous Multiplying culture of cytoplasmic mutation embryoid: be cut into 0.5cm size block with cytoplasmic mutation embryoid in early days by above-mentioned, numbering is transferred in the fast numerous proliferated culture medium of somatic embryo (MS+BA2mg/L+NAA0.2mg/L+ sucrose 40g/L+3.5g/Lphytagel) respectively, 50-60d is cultivated under being placed on 25-28 DEG C of light, middle subculture once, intensity of illumination is 1500-2000lx, light application time is 10-14 hour every day, can ensure that Fast-propagation various sudden change embryoid can obtain again the same cytoplasmic mutation embryoid of corresponding maturation.(Fig. 3: cotyledonary embryos and fast numerous mutant regeneration plant).
6) with screening and fast numerous Multiplying culture of cytoplasmic mutation embryoid: above-mentioned all kinds of same cytoplasmic mutation embryoid numerous soon is respectively taken out half, be cut into 0.5cm size block, numbering continuation is transferred in the fast numerous proliferated culture medium of somatic embryo respectively, under putting into the 5-8 DEG C of cryogenic sterile incubator low light level, (500-800lx) cultivates 25-30d, under subculture proceeds to 25-28 DEG C of light more afterwards, (1500-2000lx) cultivates 25-30d, can obtain the stronger maturation of cold resistance with cytoplasmic mutation embryoid (Fig. 4: the same cytoplasmic mutation embryoid that cold resistance is stronger).
7) ripe with cytoplasmic mutation somatic embryogenesis plant: the tool of above-mentioned fast numerous propagation to be enriched the maturation sudden change embryoid of variation and the strong maturation sudden change embryoid of cold resistance and number respectively and transfer in plant regeneration medium, 20-25d is cultivated, acquisition tool root, stem, the homogeneous mutant regeneration plant (Fig. 5: the whole plant of homogeneous mutant) that leaf is complete under desinfection chamber temperature 25-28 DEG C light.
8) homogeneous mutant regeneration plant acclimatization and transplants: when root is long-living grow to 1-1.5cm time, each mutant strain is numbered respectively and is transplanted to flowerpot, select light ground mass, be placed in green house, automatic intermittent spraying device waters.Survival rate can reach 100%.Throw off the sealed membrane of blake bottle, at indoor hardening 1 ~ 2d, carefully seedling is taken out with tweezers, clean the medium that root adheres to, be transplanted in the flowerpot that light ground mass (peat: vermiculite=1:1) is housed, putting into greenhouse temperature is 24 DEG C ~ 26 DEG C, suitably shades, or under seedling is put into seedbed, automatic intermittent spraying device waters.
9) homogeneous mutant storehouse in field is set up: select the warm and moist cloudy day that each mutant strain is transplanted to outdoor sylvan life or examination garden, field district after 60d, because seedling growth is vigorous, also not high to the conditional request of transplanting land for growing field crops, very easily plant, the survival rate of seedling can reach 100%.Later stage, according to recording mutant correlated traits breeding time, carries out mutant qualification, screening in each stage that mutant grows.Dwarf lilyturf gamma-rays saturated homogeneous mutant storehouse (Fig. 6: potted plant lowgrow ophiopogon japonicus homogeneous mutant storehouse and Parental Germplasms) is set up, for the research of the aspects such as follow-up dwarf lilyturf selection and use and gene function group with this.
Claims (9)
1. the quick method for creating in lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse, it is characterized in that, the method comprises the steps:
1) high frequency somatic embryo explant material prepares: choose the outdoor healthy lowgrow ophiopogon japonicus plant without damage by disease and insect, get its young young stem and leaf, after Aseptic sterilisation, in aseptic culture medium, group trains in vitro cuttings, get its cauline leaf base portion 0.5-1.0cm size explant, by the cultivation of high-frequency somatic embryo regeneration system, somatic embryo and embryoid and intactly regeneration plant will be produced continuously, originate using regeneration plant as high frequency somatic embryo explant material;
2) cultivation of early stage somatic embryo explant: the explant of above-mentioned tool high frequency somatic embryo regeneration capacity is cut its leaf, basal part of stem 0.5cm size tissue block, be placed on 25-28 DEG C of desinfection chamber light culture 7-10d on body embryo generation inducing culture, now most of somatic cell is unicellular period, be called early stage somatic embryo, in this, as mutagen process material;
3) physical mutagen prepares: comprise
60the setting of the preparation of Co-gamma ray projector and radiation dose;
4) the gamma-rays process of early stage somatic embryo explant: early stage somatic embryo explant is divided into 8 groups, for the process of 8 different radiation dose;
5) embryo differentiate is cultivated: by the early stage somatic embryo explant through gamma-ray and mutagenesis process, place back in desinfection chamber light culture 50-60d at temperature 25-28 DEG C, middle subculture once, be conducive to embryo differentiate, the big and small embryoid that suddenlys change in early days that grows out tens on visible explant;
6) with the fast numerous Multiplying culture of cytoplasmic mutation embryoid: above-mentioned early stage sudden change embryoid is cut into 0.5cm size block, numbering is transferred in the fast numerous proliferated culture medium of somatic embryo respectively, cultivate 50-60d under being placed on 25-28 DEG C of light, middle subculture once, can obtain a large amount of ripe same cytoplasmic mutation embryoid;
7) with screening and fast numerous Multiplying culture of cytoplasmic mutation embryoid: above-mentioned all kinds of same cytoplasmic mutation embryoid numerous is soon taken out a part, be cut into 0.5cm size block, numbering continuation is transferred in the fast numerous proliferated culture medium of somatic embryo respectively, under putting into the 5-8 DEG C of cryogenic sterile incubator low light level, (500-800lx) cultivates 25-30d, under subculture proceeds to 25-28 DEG C of light more afterwards, (1500-2000lx) cultivates 25-30d, can obtain the stronger maturation of cold resistance with cytoplasmic mutation embryoid;
8) ripe with cytoplasmic mutation somatic embryogenesis plant: the tool of above-mentioned fast numerous propagation to be enriched the maturation sudden change embryoid of variation and the strong maturation sudden change embryoid of cold resistance and number respectively and transfer in plant regeneration medium, 20-25d is cultivated, acquisition tool root, stem, the homogeneous mutant regeneration plant that leaf is complete under desinfection chamber temperature 25-28 DEG C light;
9) homogeneous mutant regeneration plant acclimatization and transplants: when root is long-living grow to 1-1.5cm time, each mutant strain is numbered respectively and is transplanted to flowerpot, select light ground mass, be placed in green house, automatic intermittent spraying device waters;
10) field mutant library is set up: select the warm and moist cloudy day that each mutant strain is transplanted to outdoor sylvan life or examination garden, field district after 60d, number respectively, record correlated traits, carry out mutant qualification, screening in each stage that homogeneous mutant grows, set up the saturated homogeneous mutant storehouse of dwarf lilyturf gamma-rays with this.
2. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, it is characterized in that, in described step 3), radiation dose is arranged: be set to 0.0,5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy8 different disposal.
3. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, is characterized in that, in described step 3)
60co-gamma Rays dosage is 20Gy.
4. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, is characterized in that, described aseptic culture medium is cultivated as 1/2MS+2.0mg/LBA+0.5mg/LNAA+20g/L sucrose+0.7% agar.
5. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, it is characterized in that, described inducing culture is MS+0.2mg/LBA+0.5mg/LNAA+0.5mg/L2,4-dichlorphenoxyacetic acid+30g/L sucrose+3.5g/Lphytagel.
6. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, it is characterized in that, the cultivation of described high-frequency somatic embryo regeneration system: refer to and cultivate 50-60d under nascent embryoid being proceeded to high frequency somatic embryo medium glazing, middle subculture once, constantly will produce secondary embryoids and somatic embryo in large quantities.
7. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 6, is characterized in that, described high frequency somatic embryo medium is MS+2mg/LBA+0.5mg/LNAA+40g/L sucrose+3.5g/Lphytagel.
8. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, it is characterized in that, in described step 6), the fast numerous proliferated culture medium of somatic embryo is MS+2mg/LBA+0.2mg/LNAA+40g/L sucrose+3.5g/Lphytagel, intensity of illumination is 1500-2000lx, and light application time is 10-14 hour every day.
9. the quick method for creating in a kind of lowgrow ophiopogon japonicus r ray homogeneous mutant storehouse according to claim 1, is characterized in that, in described step 8), plant regeneration medium is 1/2MS+20g/L white sugar+0.7% agar.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105123530A (en) * | 2015-09-24 | 2015-12-09 | 江苏农林职业技术学院 | Optimization method for efficiently and quickly building ophiopogon japonicus saturation high resistance homogeneous mutant library |
| CN113728919A (en) * | 2021-09-03 | 2021-12-03 | 湖南省园艺研究所 | Method for obtaining homogeneous mutant of kiwi fruit callus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101422136A (en) * | 2008-12-10 | 2009-05-06 | 江苏农林职业技术学院 | Lilyturf root embryoid induction and culture method |
| CN104186321A (en) * | 2014-08-22 | 2014-12-10 | 江苏省农业科学院 | Rooting culture method for pear test-tube plantlets and culture medium |
-
2015
- 2015-09-24 CN CN201510616695.0A patent/CN105165603B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101422136A (en) * | 2008-12-10 | 2009-05-06 | 江苏农林职业技术学院 | Lilyturf root embryoid induction and culture method |
| CN104186321A (en) * | 2014-08-22 | 2014-12-10 | 江苏省农业科学院 | Rooting culture method for pear test-tube plantlets and culture medium |
Non-Patent Citations (2)
| Title |
|---|
| 史清云等: "日本矮生沿阶草的快繁技术", 《江苏农业科学》 * |
| 李晶等: "日本矮生沿阶草愈伤组织的诱导及其分化", 《草业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105123530A (en) * | 2015-09-24 | 2015-12-09 | 江苏农林职业技术学院 | Optimization method for efficiently and quickly building ophiopogon japonicus saturation high resistance homogeneous mutant library |
| CN105123530B (en) * | 2015-09-24 | 2019-09-10 | 江苏农林职业技术学院 | A kind of optimization method in efficient rapid build dwarf lilyturf saturation highly resistance homogeneous mutant library |
| CN113728919A (en) * | 2021-09-03 | 2021-12-03 | 湖南省园艺研究所 | Method for obtaining homogeneous mutant of kiwi fruit callus |
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