CN101263786A - Method for tissue culture technique to high-efficiency reproduce perennial wild soybean - Google Patents
Method for tissue culture technique to high-efficiency reproduce perennial wild soybean Download PDFInfo
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- CN101263786A CN101263786A CNA2008100370982A CN200810037098A CN101263786A CN 101263786 A CN101263786 A CN 101263786A CN A2008100370982 A CNA2008100370982 A CN A2008100370982A CN 200810037098 A CN200810037098 A CN 200810037098A CN 101263786 A CN101263786 A CN 101263786A
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Abstract
The invention relates to an efficient propagation method for perennial wild soybean utilizing tissue culture technology, belonging to the technical field of agriculture, comprising the following steps: at first, seeds are sterilized, seed coat is scratched, and aseptic seedlings are cultivated on a germination medium; secondly, when the length of the aseptic seedlings reaches 10cm, 3cm to 4cm of the top of the aseptic seedling is cut off and inserted in a rooting medium; thirdly, the seedlings with the top cut off is transplanted in sterilized substrate or soil, and when new shoots appear, the seedlings are transplanted in open air; after a week, the seedlings are transplanted to fields or pots; fourthly, the young stem obtained from the second step is cultivated in the rooting medium until root is developed. The efficient propagation method for perennial wild soybean utilizing tissue culture technology has the advantages that, by adopting the method, the survival rate of perennial wild soybean can reach 99% to 100%; the invention can be used for the propagation of the materials of 22 subgenus of perennial wild soybean Glycine; the invention also provides method for the aseptic seeding and propagation of other species of glycine, species within the glycine, or other intergeneric hybrids.
Description
Technical field
The present invention relates to a kind of method of breeding wild soybean of agricultural technology field, be specifically related to a kind of method of utilizing tissue culture technique to high-efficiency to breed perennial wild soybean.
Background technology
Perennial wild soybean is pulse family (Leguminosae), Papillionoideae (Papiliomatae), Glycine, Glycine subgenus.The Glycine subgenus has 22 perennial wild species.Mainly be distributed in Australia and island, the South Pacific Ocean, China Taiwan and Fujian, Guangdong Coastal island have G.tabacina Benth. (cigarette beans) and 2 kinds of G.tomentella Hayata (glycine tomentella) to distribute.Glycine also has the soya subgenus, and two kinds are arranged, and is respectively the cultivated soybean (G.max Merr.) and annual wild soybean (G.soja Sieb.﹠amp; Zucc.).Annual wild soybean is the ancestors of the cultivated soybean.Wild soybean spreads all over China, Korea, Japan and the Far-east Area of Russia in East Asia.The cultivated soybean originates from China.The diplontic genome 2n=40 of Glycine species.Monoploid genome analysis, RFLP labeled analysis show that soybean is an ancient tetraploid, and its genome passes through long-term evolution and diplodization.Because human intervention, the habitat of wild soybean resource constantly reduces, and these plant resourceses are as oil, and mineral products are the same, are non-renewable.Wild soybean is listed in Chinese Second Class Key Protected Plant.The wild species of Glycine subgenus are compared with the cultivated soybean, a lot of proterties have potential using value, as many graininesses of Sino-Japan property, heat-resisting, drought-enduring, cold-resistant, salt tolerant, Chinese People's Anti-Japanese Military and Political College's beans cyst roundworm, mosaic disease resisting viral disease, anti-root rot, inflorescence floriferousness, pod etc.But so far the external source proterties of wild perennial soybean is introduced the cultivated soybean not success as yet.Along with deepening continuously of sexual hybridization and somatic hybridization research, and the application of gene clone and gene transfer technique, the germ plasm resource that is difficult to utilize in the modified soybeans before being expected to make adds in the breeding plan of soybean.
Wild soybean all has thick kind skin, and perhaps planting the skin foreign minister has the mud film, can prevent that seed from crossing premature eruption because of meeting the water imbibition in the environment of unfavorable growth.This characteristic is highly beneficial for being grown in the former habitat raw soybean of marching off into political wilderness, but for the wild soybean resource that need cultivate and breed under cultivation condition, is disadvantageous for sowing and emerging.Especially for seed amount perennial wild soybean resource seldom, mainly there are two problems in its breeding: the one, and emergence rate is low.For the seed that makes perennial wild soybean is in time sprouted, conventional method is to destroy kind of a skin prior to seeding, is seeded in then in soil or the matrix.But when field planting, draw the influence that the seed that breaks in the seed coat often is subjected to microorganism in the soil too early, loss part seed.Behind the seed germination before become strong sprout, can run into causing harm of soil pests, also can reduce planting percent.Another problem is, seed of perennial wild soybean can only grow a seedling, for at the material in seedling stage, perhaps those roots can not send the material of shoot, a seed can only grow up to a plant, if before the results mature seed, run into adverse environment, cause plant death just can not stay the offspring.
Find through literature search prior art, Chinese patent application numbers 200410050905.6, denomination of invention is: the aseptic seeding of vanda and tissue culture technique, this patent proposes to relate to a kind of aseptic seeding and tissue culture technique of vanda, adopt the artificial alimentation base to utilize the method for aseptic seeding can obtain a large amount of test-tube plantlets, and utilize the grow thickly propagation of bud and can form whole plant of the stem apex of aseptic seedling or stipes by culture of rootage.This patent relates to the aseptic seeding of vanda and the method and the employed medium of tissue culture propagating.Be to utilize aseptic seeding, utilize the grow thickly propagation of bud of the stem apex of aseptic seedling or stipes.This method utilizes aseptic seedling to obtain stem apex often, internode waits and produces the bud of growing thickly, breed in a large number, and no longer utilize for seedling itself, like this for some rare materials, or seed material seldom, directly adopt the tissue culture mode to breed in a large number and have some risks, must guarantee to have a quantity of seeds, just can carry out the method propagation of tissue culture.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of method of utilizing tissue culture technique to high-efficiency to breed perennial wild soybean is proposed, it is few both to have solved seed amount, carry out method for tissue culture propagation and can lose the risk of seed, it is low also to have solved present perennial wild soybean breeding emergence rate, and before the results mature seed, running into adverse environment or factor, plant is put in order strain death, can not stay offspring's problem.
The present invention is achieved by the following technical solutions, according to the difference of propagating materials character and seed number, can adopt following two kinds of schemes, promptly few at seed amount, and the material of homogeneity has only the 1-2 grain kind period of the day from 11 p.m. to 1 a.m, employing method one in every part of heredity; When the material that will breed has seed more than 2, adopt method two.
Described method one may further comprise the steps:
The first step, the acquisition of seed sterilization and perennial wild soybean aseptic seedling: get ripe perennial wild soybean seed, carry out the seed sterilization, will plant skin with scalpel and scratch with chlorine or supersaturation calcium hypochlorite tablet solution.Be inoculated on the germination medium drawing the perennial wild soybean seed that breaks in the seed coat, obtain aseptic seedling, for " second step " provides vegetable material through cultivating.If only directly breed with seed, do not need tissue culture propagation, this step does not need the seed sterilization.
Described germination medium is existing MS medium, B
5The inorganic constituents of medium or MS medium adds B
5The organic principle of medium, cultivation temperature are 24 ℃-26 ℃.
In second step, breed and take root: treat that seedling grows to 10cm, downward 3cm-4cm downcuts the stem end from the aseptic seedling top, the botany lower end of aseptic seedling stem sections is inserted in the root media take root, as the 4th material that goes on foot; Truncated wild soybean seedling is as the material in the 3rd step.
Described root media is the MS solid culture medium, and cultivation temperature is 24 ℃-26 ℃.The continuation propagation that is the potted plant of the 3rd step and the 4th step is prepared material.
In the 3rd step, potted plant: the bottle that truncated seedling will be housed is opened, and growth is 3 days in culturing room, is transplanted in the basin of the matrix (or soil) that sterilization is housed.Under 24 ℃ of-26 ℃ of conditions, cultivate, after waiting to send forth branches, basin is transferred to the open air.Potted plant wild soybean is grown out of doors and is transplanted to the field after a week.If this material can not be in the locality outdoor surviving the winter, then adopt potted plant mode.
In the 4th step, the seedling top of having taken root children's stem that second step obtained through the cultivation of two weeks, grows the root of a lot of health in root media.Continue breeding if desired, can repeat the step in second step under the stem end-grain cutting of having extended.
Described method two may further comprise the steps:
The first step, seed germination: get ripe perennial wild soybean seed, will plant skin with scalpel and scratch.Be inoculated on the germination medium drawing the perennial wild soybean seed that breaks in the seed coat, make seed germination, for " second step " provides vegetable material.
Described germination medium is existing MS medium, B
5The inorganic constituents of medium or MS medium adds B
5The organic principle of medium, cultivation temperature are 24 ℃-26 ℃.
Second step, potted plant: as to treat that seed grew young root, takes off kind of a skin seed germination 3-4 days, seedling is implanted through in the soil or matrix of sterilizing, after one month, change pot transplanting to the field.If this material can not be in the locality outdoor surviving the winter, then adopt potted plant mode.
Though the present invention also is to use aseptic seeding and tissue culture technique, it is long after enough big in aseptic seedling that different is, on downcutting, be transplanted in soil or the matrix lower end that will have normal root system, makes its Cheng Miao.Be used for sowing the seed that obtains aseptic seedling like this and also grow up to a plant, the seed that obtains for the material or the hybridization of seed rareness can adopt this mode to breed, adopt this method can guarantee that each seed can both obtain healthy plant, utilize the mode of tissue culture to breed the plant that homogeneity is gone up in heredity in a large number simultaneously.The root system of seedling is strong in addition, and the seedling in the medium is transplanted the plant that obtains, and is strongr than the plant that obtains through the blastogenesis root of growing thickly, well developed root system, and growth is bloomed solid morning, the survival rate height of transplanting rapidly.For the breeding of perennial wild soybean, still do not use the aseptic seeding tissue culture propagating in the prior art in addition.
Adopt above-mentioned two kinds of methods, from three steps of the first step to the of method one, in the first step to the second step of method two, if there is not the human operational error, the planting percent of seed can reach 99%-100%.Method one is through the 4th step, and a perennial wild soybean seed can obtain two strains or the above more plant of homogeneity in heredity of two strains.This method also can be used for crossbreed F
0The breeding in generation is with less F
0Seed obtains the F of more homogeneity
1Plant, and obtain more F
1Seed.For hybridization difficulty or long species of solid cycle, as long as once hybridize successfully, a hybrid seed just can be bred a plurality of F like this
1Plant.Therefore this method not only can be used for breeding, the preservation of resource, can also be used for crossbreeding and scientific research, can maximally utilise seed and germ plasm resource propagating materials.
The present invention can be used for the breeding of the material of 22 kinds of perennial wild soybean Glycine subgenus, and can be other kind of Glycine, belong to interior or provide method (thinner for kind of skin with other aseptic seeding of bigenering and breeding, the seed that seed can absorb water and sprout can save the step of scratching middle skin with cutter).
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
Step 1:
Seed sterilization: soaked the wild soybean seed 15 minutes with the calcium hypochlorite tablet supersaturated solution, rinsed with sterile water 3 times or with disinfection by chlorine 2-10 hour;
Cut and break in the seed coat: with scalpel wild soybean kind skin is scratched under the aseptic condition;
The sowing and germination medium: will draw the seed that breaks in the seed coat and be inoculated on the germination medium, germination medium be MS, B5 or MS inorganic+B5 organic solid medium;
Seed germination is cultivated: the seed on the medium is sprouted under 24 ℃ of-26 ℃ of conditions.
Step 2
Seed is cultivated in germination medium: treat that aseptic seedling grows to 10cm when high, downcuts top stem end;
Step 3
Take root: downward 3-4cm downcuts the stem section from the aseptic seedling top, and root media is inserted in the botany lower end of stem section, and root media is the MS solid culture medium, and cultivation temperature is 24 ℃-26 ℃;
Transplant: truncated seedling is transplanted in the basin of matrix (or soil) of sterilization.Cultivate under 24 ℃ of-26 ℃ of conditions, after waiting to send forth branches, basin transferred to the open air, if this material can not be in the locality outdoor surviving the winter, then adopt potted plant mode;
Step 4
Propagation: the seedling stem end that obtains in the step 2 grows healthy root (15 days) in root media.Continue breeding as need, under the stem end-grain cutting of having extended, the operation of repeating step 2, root media is the MS solid culture medium, cultivation temperature: 24 ℃-26 ℃;
The present embodiment effect: the planting percent from step the 1 step to the 3rd step reaches 99%-100%, the propagation by the 4th step again, perennial wild soybean seed can obtain two strains or two strains above more in heredity the plant of homogeneity.
Present embodiment is used: this method can be used for the crossbreed F0 breeding in generation, obtains the F1 plant of more homogeneity with less F0 seed, obtains more F1 seed.Be particularly suited for hybridizing difficulty or the long species of growth cycle.This method can be used for breeding, the preservation of resource, and crossbreeding and correlative study, can maximally utilise the seminal propagation material.
Embodiment 2
Step 1
Seed sterilization, cut break in the seed coat, germination medium is with embodiment 1.
Seed germination: the seed on the medium is sprouted under 24 ℃ of-26 ℃ of conditions.
Step 2
Seed is cultivated in germination medium and is transplanted: grow young root after 3 days, seedling is transferred in the soil or matrix of sterilization.Be transplanted to outdoor (potted plant in the time of can not surviving the winter on land) after 30 days.
The present embodiment effect: as long as seed can normally be sprouted, planting percent can reach 99.5%-100%.
Present embodiment is used: this method is applicable to the breeding of (comprising annual wild soybean and perennial wild soybean) of the less wild soybean material of seed.
Embodiment 3
Step 1
Seed sterilization, cut break in the seed coat, germination medium is with embodiment 1.
Seed germination: the seed on the medium is sprouted (18 ℃-28 ℃) in room temperature.。
Step 2
Seed is cultivated in germination medium and is transplanted: after 3-4 days seedling replanting to the sterilization soil or matrix in (18-23 ℃, 4d, 23-28 ℃, 3d) all the other are with embodiment 2.
The present embodiment effect: as long as seed can normally be sprouted, planting percent can reach 99.5%-100%.
Present embodiment is used: this method is applicable to the breeding of (comprising annual wild soybean and perennial wild soybean) of the less wild soybean material of seed.
Embodiment 4
Step 1
Cut and break in the seed coat: scalpel scratches wild soybean kind skin.
Germination medium: with MS, the B of seed kind in improvement
5Or MS+B
5Solid culture medium.
Seed germination: the seed on the medium is sprouted (18 ℃-28 ℃) in room temperature.。
Step 2 is with embodiment 3
The present embodiment effect: compare with embodiment 2, do not need sterile working, the planting percent height can reach 99%-100%.
Present embodiment is used: the sprouting that is suitable for the less wild soybean material of seed, prematurity soya seeds with becomes seedling, be fit to produce and R﹠D institution's usefulness.
The medium that uses among the above embodiment is prior art, specifies as follows:
Annotate: minimal medium prescription (mg/L)
MS solid culture based formulas:
Inorganic constituents: potassium nitrate KNO3 1900, ammonium nitrate NH4NO3 1650, potassium dihydrogen phosphate KH2PO4170, magnesium sulfate MgSO4.7H2O 370, calcium chloride CaCl2.2H2O 440, potassium iodide KI 0.83, boric acid H3BO3 6.2, manganese sulphate MnSO4.4H2O 22.3, zinc sulphate ZnSO4.7 H2O 8.6, sodium molybdate Na2MoO4.2 H2O 0.25, copper sulphate CuSO4.5 H2O 0.025, cobalt chloride CoCl2.620.025, disodium ethylene diamine tetraacetate Na2.EDTA 37.3, ferrous sulfate FeSO24.7H2O 27.8.
Organic principle: inositol 100, glycine 2, thiamine hydrochloride VB1 0.1, puridoxine hydrochloride VB60.5, nicotinic acid VB5 or VPP 0.5, sucrose 30000, agar 10000.
Ph value: 5.8.
B5 solid culture based formulas (mg/L)
Inorganic constituents: NaH2PO4H2O (sodium dihydrogen phosphate) 150, KNO3 (potassium nitrate) 2500, (NH4) 2SO4 (ammonium sulfate) 134, MgSO47H2O (magnesium sulfate) 250, CaCl22H2O (calcium chloride) 150, Na-Fe-EDTA (Ferric Sodium Edetate) 28, MnSO4H2O (manganese sulphate) 10, H3BO3 (boric acid) 3.0, ZnSO47H2O (zinc sulphate) 2.0, NaMoO42H2O (sodium molybdate) 0.25, CuSO45H2O (copper sulphate) 0.025, CoCl26H2O (cobalt chloride) 0.025, KI (potassium iodide) 0.75.
Organic principle: thiamine hydrochloride 10, pyridoxine hydrochloride 1, nicotinic acid 1, myo-inositol 100, sucrose 20000.Agar 10000.
PH value: 5.5.
The MS solid-based basal culture medium of improvement, the B of improvement
5The MS of solid-based basal culture medium and improvement is inorganic+B
5The organic solid medium: other composition is respectively with MS solid-based basal culture medium, B
5Solid-based basal culture medium and MS be inorganic+B
5The organic solid medium is not just with sucrose.
Claims (5)
1, a kind of method of utilizing tissue culture technique to high-efficiency to breed perennial wild soybean is characterized in that, may further comprise the steps:
The first step, the acquisition of seed sterilization and perennial wild soybean aseptic seedling: get ripe perennial wild soybean seed, carry out the seed sterilization with chlorine or supersaturation calcium hypochlorite tablet solution, to plant skin with scalpel scratches, be inoculated on the germination medium drawing the perennial wild soybean seed that breaks in the seed coat, obtain aseptic seedling through cultivating, for second step provided vegetable material;
Described germination medium is existing MS medium, B
5Medium, or the inorganic constituents of MS medium adds B
5The organic principle of medium;
Second step, propagation and taking root: treat that seedling grows to 10cm, downward 3cm-4cm downcuts the stem end from the aseptic seedling top, takes root in the botany lower end insertion root media with aseptic seedling stem sections, as the material in the 4th step, truncated wild soybean seedling is as the material in the 3rd step;
Described root media is the MS solid culture medium;
In the 3rd step, potted plant: the bottle that truncated seedling will be housed is opened, and growth is 3 days in culturing room, be transplanted to again in the basin of matrix that sterilization is housed or soil, after waiting to send forth branches, basin is transferred to the open air, potted plant wild soybean is grown out of doors and is transplanted to the field after a week;
In the 4th step, the seedling top of having taken root children's stem that second step obtained through the cultivation of two weeks, grows the root of a lot of health in root media.
2, the method for utilizing tissue culture technique to high-efficiency to breed perennial wild soybean according to claim 1 is characterized in that, the first step to the is in three steps, and the temperature of described cultivation is 24 ℃-26 ℃.
3, the method for utilizing tissue culture technique to high-efficiency to breed perennial wild soybean according to claim 1 is characterized in that, in the 4th step, continues breeding if desired, under the stem end-grain cutting of having extended, repeats the step in second step.
4, a kind of method of utilizing tissue culture technique to high-efficiency to breed perennial wild soybean is characterized in that, may further comprise the steps:
The first step, seed germination: get ripe perennial wild soybean seed, will plant skin with scalpel and scratch.Be inoculated on the germination medium drawing the perennial wild soybean seed that breaks in the seed coat, make seed germination, for second step provided vegetable material;
Described germination medium is existing MS medium, B
5Medium, or the inorganic constituents of MS medium adds B
5The organic principle of medium;
Second step, potted plant: as to treat that seed grew young root, takes off kind of a skin seed germination 3-4 days, seedling is implanted through in the soil or matrix of sterilizing, after one month, change pot transplanting to the field.
5, the method for utilizing tissue culture technique to high-efficiency to breed perennial wild soybean according to claim 4 is characterized in that, in the first step, cultivates in the described germination medium, and its cultivation temperature is 24 ℃-26 ℃.
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| CNA2008100370982A CN101263786A (en) | 2008-05-08 | 2008-05-08 | Method for tissue culture technique to high-efficiency reproduce perennial wild soybean |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103168684A (en) * | 2012-12-18 | 2013-06-26 | 新疆农业大学 | Tissue culture intermediate propagation method of chickpeas |
| CN104126507A (en) * | 2014-07-22 | 2014-11-05 | 青岛农业大学 | Rooting method of soybean tissue culture differentiated seedlings |
| CN115039621A (en) * | 2022-06-09 | 2022-09-13 | 深圳市仙湖植物园(深圳市园林研究中心) | Propagation method of Chinese Saraca indica seedlings |
-
2008
- 2008-05-08 CN CNA2008100370982A patent/CN101263786A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103168684A (en) * | 2012-12-18 | 2013-06-26 | 新疆农业大学 | Tissue culture intermediate propagation method of chickpeas |
| CN103168684B (en) * | 2012-12-18 | 2014-07-09 | 新疆农业大学 | Tissue culture intermediate propagation method of chickpeas |
| CN104126507A (en) * | 2014-07-22 | 2014-11-05 | 青岛农业大学 | Rooting method of soybean tissue culture differentiated seedlings |
| CN104126507B (en) * | 2014-07-22 | 2016-03-30 | 青岛农业大学 | The rooting method of differentiation seedling is cultivated by a kind of soyabean tissue |
| CN115039621A (en) * | 2022-06-09 | 2022-09-13 | 深圳市仙湖植物园(深圳市园林研究中心) | Propagation method of Chinese Saraca indica seedlings |
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