CN101861831B - High-frequency somatic embryo regeneration culture medium capable of overcoming clover variety genotype barrier - Google Patents
High-frequency somatic embryo regeneration culture medium capable of overcoming clover variety genotype barrier Download PDFInfo
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Abstract
The invention discloses a high-frequency somatic embryo regeneration culture medium capable of overcoming a clover variety genotype barrier. The culture medium comprises a seedling growing domestication culture medium, an embryogeny callus induction culture medium, an embryo subculture forming culture medium and an embryo ripening and germinating culture medium, wherein the seedling growing domestication culture medium comprises 1/2 of improved MS, 20 g/L of white sugar and 0.7% of agar; the embryogeny callus induction culture medium comprises improved SH, 8-12 mg/L of 2,4-D, 0.2-0.5 mg/L of 6-BA, 30 g/L of cane sugar and 0,3% of phytagel; the embryo subculture forming culture medium comprises improved SH, 2-8 mg/L of 2,4-D, 0.2-0.5 mg/L of 6-BA, 50 g/L of cane sugar and 0,35% of phytagel; and the embryo ripening and germinating culture medium comprises improved SH, 30 g/L of white sugar and 0.7% of agar. The invention can effectively overcome the problems of different varities and genetype barriers during the regeneration culture of clover high-frequency somatic embryos, and greatly improve the embryogeny ratio of the somatic embryos, the plantlet regeneration frequency and the breeding efficiency.
Description
Technical field
The present invention relates to the high-frequency somatic embryo regeneration medium of a kind of leguminous forage, relate to a kind of clover variety genotype barrier high-frequency somatic embryo regeneration medium that overcomes more specifically, belong to the botany field.
Background technology
Clover (Clover) is a kind of good leguminous forage of worldwide distribution and cultivation; It is the first-selected grass seeds of area, temperate zone sown pasture planting; Also be the key component in all kinds of ornamental lawns and greenery patches, it in the greening in cities and towns, beautify, the fixed nitrogen culture fertility of garden and forest land, the aspects such as water and soil conservation of road dykes and dams all play irreplaceable important function.In view of trefoil important economic worth, many developed countries utilize biological technique method to study improvement clover one after another, solve the problem that exists in the clover production.
Clover is a heterogeneous heterozygote; Selfing is not affine, and the kind of countries in the world application at present is synthetic variety more than 80%, and genotypic difference is very big in the kind; Some important breeding objectives of clover are difficult to reach through the conventional breeding method; As the clover drought resistance and salt tolerance, disease-resistant pest-resistant, improve seed and careless output, nitrogen fixing capacity etc. and all do not reach desirable effect, therefore in recent years, people attempt through the modern genetic engineering technology; Promptly obtain resistant plant and cultivate the clover new varieties through transgenosis; Improve the adaptability of clover to various ecotopes, but be that clover has the high-frequency somatic embryo regeneration ability in its tissue culture procedures as the precondition that this technology can successful Application, this is a problem that is difficult to overcome always.
The research of Trifolium Plant Tissue Breeding regeneration techniques starts from the seventies in 20th century, and more existing reports show that most of trefoil tissue culture regeneration rate is all extremely low; Have only 1% even littler; And regenerative process is longer, generally needs about 4～6 months, and reviviscence and regeneration frequency are subject to the genotype influence; This has become the restriction different genotype, especially the genetically modified bottleneck of improved seeds.Nancy and Jerzy 1989 induce through red clover petiole callus, have obtained 70～81% shoot regeneration frequency; Quesenberry and Smith1993 have reported the research of red clover regeneration techniques, and nearly 4% shoot regeneration frequency is than higher, if further screening and regeneration again in the high plant of this 4% regeneration rate, the regeneration power of plant can bring up to 70%.Mostly the foundation of clover regenerating system is that through adventitious organogenesis the report of setting up from somatic embryo generation approach is seldom arranged, and does not more see the report that the high-frequency somatic embryo regenerating tissues culture technique system that is suitable for the clover genetic transformation is arranged.Because somatic embryo is clone's origin, body embryogenesis path more can guarantee the genetic identity (not forming chimera) of genetically modified plants, makes foreign gene pass to the offspring through pollen and gamete.So the somatic embryo regeneration technology is the most attractive method in the Plant Tissue Breeding, be widely used in the screening of plant genetic conversion, somatic variation system etc.
It is material that previous researcher has only selected 1～3 kind of a kind mostly, because the kind quantity of research is few, under-represented, these researchs all are difficult to solve the problem of clover different cultivars and genotype barrier.Therefore, setting up the high-frequency somatic embryo regeneration cultivating system that clover do not receive the restriction of kind and genotype is to obtain the successful important prerequisite of each veriety clover transgenosis.
Summary of the invention
In order to overcome the deficiency of prior art, the object of the present invention is to provide a kind of can effectively improve somatic embryo occurrence frequency and regeneration frequencies overcome clover variety genotype barrier high-frequency somatic embryo regeneration medium.
The present invention realizes through following technical scheme:
A kind ofly overcome clover variety genotype barrier high-frequency somatic embryo regeneration medium, comprise that growth of seedling acclimation shaking culture base, embryo generation callus inducing medium, embryo subculture form medium and become the seedling medium with the embryo maturation germination, it is characterized in that:
Described growth of seedling acclimation shaking culture base is: 1/2 improvement MS+20g/L white sugar+0.7% agar;
Described embryo generation callus inducing medium is: improvement SH+8～12mg/L 2,4-D+0.2～0.5mg/L 6-BA+30g/L sucrose+0.3%phytagel;
Described embryo subculture forms medium: improvement SH+2～8mg/L 2,4-D+0.2～0.5mg/L6-BA+50g/L sucrose+0.35%phytagel;
Described embryo maturation germination becomes the seedling medium to be: improvement MS+30g/L white sugar+0.7% agar.
Above-mentioned improvement SH comprises grand nutrition element, micronutrient element and organic reagent.
Wherein the component of grand nutrition element is following with its corresponding concentration:
Ammonium sulfate 463mg/L;
Potassium nitrate 2830mg/L;
Calcium chloride dihydrate 166mg/L;
Epsom salt 185mg/L;
Anhydrous potassium dihydrogenphosphate 400mg/L;
Iron edta sodium salt 1.4mg/L.
The component of micronutrient element is following with its corresponding concentration:
Manganese sulfate monohydrate 10mg/L;
Zinc sulphate 1.0mg/L;
Boric acid 5.0mg/L;
KI 1.0mg/L;
Sodium molybdate 0.1mg/L;
Copper sulphate 0.2mg/L;
Cobalt chloride 0.1mg/L.
The component of organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 5.0mg/L;
Nicotinic acid 5.0mg/L;
Puridoxine hydrochloride 5.0mg/L.
Above-mentioned modified MS medium comprises grand nutrition element, micronutrient element and organic reagent.Wherein the component of grand nutrition element is following with its corresponding concentration:
Ammonium sulfate 1650mg/L;
Potassium nitrate 1900mg/L;
Epsom salt 370mg/L;
Anhydrous potassium dihydrogenphosphate 170mg/L;
Calcium chloride dihydrate 440mg/L;
Disodium ethylene diamine tetraacetate 37.3mg/L;
Ferrous sulfate heptahydrate 27.8mg/L.
The component of micronutrient element is following with its corresponding concentration:
Four water manganese sulphate 22.3mg/L;
Zinc sulphate 8.6mg/L;
Boric acid 6.2mg/L;
KI 0.83mg/L;
Sodium molybdate 0.25mg/L;
Copper sulphate 0.025mg/L;
Cobalt chloride 0.025mg/L.
The component of organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 1.0mg/L;
Nicotinic acid 1.0mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100mg/L.
The invention has the beneficial effects as follows: the present invention is through selecting supplementary measures such as proper explant, improvement minimal medium and composition, adjustment condition of culture; Overcome different cultivars and genotype barrier problem in the cultivation of clover high-frequency somatic embryo regeneration effectively, increased substantially somatic embryo occurrence frequency and regeneration frequencies.Set up clover high-frequency somatic embryo regeneration technical system and be the precondition of the target plant that guarantees clover somatic induction variation, efficient stable genetic transformation, obtains supply to screen in a large number; Simplify the generation of clover somatic embryo and plant regeneration medium and technical system through optimizing; Can effectively bring into play the effects of biotechnology in the clover modern breeding such as transgenosis, improve breeding efficiency.
Description of drawings
The embryonic type cell that Fig. 1 cultivates on embryo generation callus inducing medium for the clover explant;
Fig. 2 forms the somatic embryo of growing on the medium for assorted three leaves at embryo subculture;
Fig. 3 forms the somatic embryo of growing on the medium for red clover at embryo subculture;
Fig. 4 forms the somatic embryo of growing on the medium for Bai Sanye at embryo subculture;
Fig. 5 becomes the torpedo embryo that develops on the seedling medium for somatic embryo in the embryo maturation germination;
Fig. 6 becomes the cotyledonary embryos that develops on the seedling medium for somatic embryo in the embryo maturation germination;
Fig. 7 cultivates the differentiation seedling of sprouting for the torpedo embryo subculture;
The differentiation seedling that Fig. 8 sprouts for the cotyledonary embryos successive transfer culture;
Fig. 9 is the seedling that the differentiation seedling of torpedo embryo develops into;
Figure 10 is the seedling that the differentiation seedling of cotyledonary embryos develops into.
Embodiment
To combine accompanying drawing and specific embodiment below, specify embodiment of the present invention:
The embryonic type cell that Fig. 1 cultivates on embryo generation callus inducing medium for the clover explant; Fig. 2 forms the somatic embryo of growing on the medium for assorted three leaves at embryo subculture; Fig. 3 forms the somatic embryo of growing on the medium for red clover at embryo subculture; Fig. 4 forms the somatic embryo of growing on the medium for Bai Sanye at embryo subculture; Fig. 5 becomes the torpedo embryo that develops on the seedling medium for somatic embryo in the embryo maturation germination; Fig. 6 becomes the cotyledonary embryos that develops on the seedling medium for somatic embryo in the embryo maturation germination; Fig. 7 cultivates the differentiation seedling of sprouting for the torpedo embryo subculture; The differentiation seedling that Fig. 8 sprouts for the cotyledonary embryos successive transfer culture; Fig. 9 is the seedling that the differentiation seedling of torpedo embryo develops into; Figure 10 is the seedling that the differentiation seedling of cotyledonary embryos develops into.
Like Fig. 1-shown in Figure 10:
A kind ofly overcome clover variety genotype barrier high-frequency somatic embryo regeneration medium, comprise that growth of seedling acclimation shaking culture base, embryo generation callus inducing medium, embryo subculture form medium and become the seedling medium with the embryo maturation germination,
Wherein growth of seedling acclimation shaking culture base is: 1/2 improvement MS+20g/L white sugar+0.7% agar; Embryo generation callus inducing medium is: improvement SH+8～12mg/L 2,4-D+0.2～0.5mg/L 6-BA+30g/L sucrose+0.3%phytagel; Embryo subculture forms medium: improvement SH+2～8mg/L 2,4-D+0.2～0.5mg/L 6-BA+50g/L sucrose+0.35%phytagel; The embryo maturation germination becomes the seedling medium to be: improvement MS+30g/L white sugar+0.7% agar.
Above-mentioned improvement SH comprises grand nutrition element, micronutrient element and organic reagent.
Wherein the component of grand nutrition element is following with its corresponding concentration:
Ammonium sulfate 463mg/L;
Potassium nitrate 2830mg/L;
Calcium chloride dihydrate 166mg/L;
Epsom salt 185mg/L;
Anhydrous potassium dihydrogenphosphate 400mg/L;
Iron edta sodium salt 1.4mg/L.
The component of micronutrient element is following with its corresponding concentration:
Manganese sulfate monohydrate 10mg/L;
Zinc sulphate 1.0mg/L;
Boric acid 5.0mg/L;
KI 1.0mg/L;
Sodium molybdate 0.1mg/L;
Copper sulphate 0.2mg/L;
Cobalt chloride 0.1mg/L.
The component of organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 5.0mg/L;
Nicotinic acid 5.0mg/L;
Puridoxine hydrochloride 5.0mg/L.
Above-mentioned modified MS medium comprises grand nutrition element, micronutrient element and organic reagent.
Wherein the component of grand nutrition element is following with its corresponding concentration:
Ammonium sulfate 1650mg/L;
Potassium nitrate 1900mg/L;
Epsom salt 370mg/L;
Anhydrous potassium dihydrogenphosphate 170mg/L;
Calcium chloride dihydrate 440mg/L;
Disodium ethylene diamine tetraacetate 37.3mg/L;
Ferrous sulfate heptahydrate 27.8mg/L.
The component of micronutrient element is following with its corresponding concentration:
Four water manganese sulphate 22.3mg/L;
Zinc sulphate 8.6mg/L;
Boric acid 6.2mg/L;
KI 0.83mg/L;
Sodium molybdate 0.25mg/L;
Copper sulphate 0.025mg/L;
Cobalt chloride 0.025mg/L.
The component of organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 1.0mg/L;
Nicotinic acid 1.0mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100mg/L.
Embodiment 1
A. embryo generation callus inducing medium (is improved SH+8mg/L 2; 4-D+0.2mg/L 6-BA+30g/L sucrose+0.3%phytagel)+B. embryo subculture formation medium (improvement SH+8mg/L 2; 4-D+0.2mg/L 6-BA+50g/L sucrose+0.35%phytagel)+C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling acclimation shaking culture base (1/2 improvement MS+20g/L white sugar+0.7% agar), pH is about 5.8 behind above-mentioned all medium autoclavings.
Embodiment 2
A. embryo generation callus inducing medium (is improved SH+8mg/L 2; 4-D+0.5mg/L 6-BA+30g/L sucrose+0.3%phytagel)+B. embryo subculture formation medium (improvement SH+8mg/L 2; 4-D+0.5mg/L 6-BA+50g/L sucrose+0.35%phytagel)+C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling acclimation shaking culture base (1/2 improvement MS+20g/L white sugar+0.7% agar), pH is about 5.8 behind above-mentioned all medium autoclavings.
Embodiment 3
A. embryo generation callus inducing medium (is improved SH+10mg/L 2; 4-D+0.2mg/L 6-BA+30g/L sucrose+0.3%phytagel)+B. embryo subculture formation medium (improvement SH+5mg/L 2; 4-D+0.2mg/L 6-BA+50g/L sucrose+0.35%phytagel)+C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling acclimation shaking culture base (1/2 improvement MS+20g/L white sugar+0.7% agar), pH is about 5.8 behind above-mentioned all medium autoclavings.
Embodiment 4
A. embryo generation callus inducing medium (is improved SH+10mg/L 2; 4-D+0.5mg/L 6-BA+30g/L sucrose+0.3%phytagel)+B. embryo subculture formation medium (improvement SH+5mg/L 2; 4-D+0.5mg/L 6-BA+50g/L sucrose+0.35%phytagel)+C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling acclimation shaking culture base (1/2 improvement MS+20g/L white sugar+0.7% agar), pH is about 5.8 behind above-mentioned all medium autoclavings.
Embodiment 5
A. embryo generation callus inducing medium (is improved SH+12mg/L 2; 4-D+0.2mg/L 6-BA+30g/L sucrose+0.3%phytagel)+B. embryo subculture formation medium (improvement SH+2mg/L 2; 4-D+ 0.2mg/L 6-BA+50g/L sucrose+0.35%phytagel)+C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling acclimation shaking culture base (1/2 improvement MS+20g/L white sugar+0.7% agar), and pH is about 5.8 behind above-mentioned all medium autoclavings.
Embodiment 6
A. embryo generation callus inducing medium (is improved SH+12mg/L2; 4-D+0.5mg/L 6-BA+30g/L sucrose+0.3%phytagel)+B. embryo subculture formation medium (improvement SH+2mg/L 2; 4-D+0.5mg/L 6-BA+50g/L sucrose+0.35%phytagel)+C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling acclimation shaking culture base (1/2 improvement MS+20g/L white sugar+0.7% agar), pH is about 5.8 behind above-mentioned all medium autoclavings.
Above-mentioned 360 explants are carried out embryo generation callus induction, embryo subculture formation cultivation, strengthening seedling and rooting cultivation, regeneration plant transplanting respectively in 6 described plant growth regulator combinations of embodiment.
At first be selection: select 11 kinds, that is: Bai Sanye (comprise that floor file, koala, Latin promise, Flying Carpet, hundred despots, sea are sent out, Ai Lisi, Hu Yia, 9 kinds of Rui Wende), the red clover (Ruide) and three leaves of mixing are washed the about 1～2h of above-mentioned seed with flowing water; At desinfection chamber with 75% alcohol disinfecting 20～30 seconds; Aseptic washing 3～5 times, 0.1% mercuric chloride sterilization 6～8 minutes is with aseptic water washing 5 times; Sterilization filter paper suck dry moisture is inoculated on the growth of seedling acclimation shaking culture base; The upper and lower plumular axis of getting above-mentioned sprouting 5-6 days of aseptic seedling is cut into the long segment of 3～4mm; The regeneration blade is at the vertical master pulse cutting apart from leaf base 2/3 place, band petiole part, vertical again master pulse crosscut 2 cuttves; Cause wound; Stem comprises the stem handle, and these materials are inoculated in embryo generation callus inducing medium on as explant (360) totally;
(2) embryo generation callus induction: the above-mentioned explant of choosing is inoculated on the embryo generation callus inducing medium, by day 27 ± 2 ℃ of room temperatures, night 20 ± 1 ℃ of room temperatures condition under dark the cultivation 28～30 days;
(3) embryo subculture forms and cultivates: above-mentioned explant after embryo generation callus induction is cultivated is inoculated in embryo subculture forms on the medium; 4～6 ℃ of low temperature climatic cabinates that at first carried out 5 days are secretly cultivated, then by day 27 ± 2 ℃ of room temperatures, night 20 ± 1 ℃ of room temperatures condition under dark the cultivation 30-35 days;
(4) the embryo maturation germination becomes seedling to cultivate: above-mentioned explant after embryo subculture forms cultivation is inoculated in the embryo maturation germination becomes on the seedling medium; 27 ± 2 ℃ of room temperatures, 20 ± 1 ℃ of nights, intensity of illumination are that the light that carries out 14h/d under the condition of 1000～1500lx was cultivated 20～25 days by day, form seedling;
(5) strengthening seedling and rooting is cultivated: above-mentioned seedling after the embryo maturation germination becomes the seedling cultivation is transferred to carries out the strengthening seedling and rooting cultivation on the growth of seedling acclimation shaking culture base, form the regeneration plant of taking root;
(6) regeneration plant that will take root is transplanted in the greenhouse small flower, the component of the soil body in the flowerpot and percentage by weight: peat soil: garden mould=7: 3, and transplant the back and water 1～2 time every day, treat to reduce the number of times that waters gradually after seedling becomes to live;
As shown in Figure 1: in above-mentioned embryo generation callus induction step; The callus surface of seeing explant through microexamination forms some projections; Protrusion cell matter is dense, separates with peripheral cell, and obvious boundary is arranged; Explain and these divide rapidly cell is a cells,primordial; And in the embodiment of the embryo generation callus induction base of above-mentioned 6 kinds of combinations, can both obtain embryo generation callus, but the highest inductivity of embryo callus all is on the medium of the auximone that contains suitable concn or suitable ratio auximone/cytokinin, to take place, too high auximone or inappropriate auximone/cytokinin ratio all can reduce explant embryo callus incidence; The effective combination of embryo generation callus is 2; 4-D 10mg/L and 6-BA0.2mg/L, this combination is easy to most of genotype and induces the more embryo generation callus of generation, and epicotyl and hypocotyl embryo callus incidence on average improve more than 21% (sees table 1); It is thus clear that have only auximone/cytokinin proportioning suitable, could promote the embryo callus to produce and formation.
Extremely shown in Figure 4 like Fig. 2: as to form incubation step at above-mentioned embryo subculture; This experiment is handled discovery through low temperature-normal temperature is set; Form the low temperature treatment of cultivating process certain hour in early stage at embryo subculture and help the embryo subculture maturation; Embryo subculture forms the characteristics of medium for reducing by 2, and 4-D concentration improves sucrose and phytagel concentration.6 embodiment results show: suitably reduce by 2,4-D concentration helps somatic embryo and forms, and reduces secondary somatic embryo and produces.Among 6 embodiment, effective combination is 2,4-D 5mg/L and 6-BA 0.2mg/L; Can improve the somatic embryo maturing rate by a relatively large margin; Upper and lower plumular axis embryoid differentiation rate has on average been improved more than 23% (see table 2), the embryo callus also no longer increases and shoals or brownization occur, and softer light yellow callus appears in assorted three leaf embryo callus surface; Inlaying the little stone of many greens inside and outside the red clover embryo callus; These green little stones develop into single plantlet later on, and Bai Sanye embryo callus surface develops into harder bulk particle, i.e. globular embryo; These green little stones or bulk particle are exactly the somatic embryo of growing, and when looking bigger, can it be cut apart cultivation and further develop into single plantlet.
Like Fig. 5-shown in Figure 10: become the seedling incubation step in the embryo maturation germination; After the medium dehydration processing of above-mentioned somatic embryo through 50g/L sucrose and 0.35%phytagel; Can promote that 80% globular embryo develops into torpedo embryo and cotyledonary embryos, be transferred on improvement MS+30g/L white sugar+0.7% agar medium and cultivate, can observe a large amount of differentiation seedlings; Each differentiation seedling is to be sprouted by somatic embryo to form; The sprouting of somatic embryo also comprises radicle, plumular axis elongation, and cotyledon and true leaf form, and grows at last to be free growing seedling.
In the somatic embryo development process, again with globular embryo for the highest, cotyledon idiophase embryo, mature embryo, banana-shaped embryo reduce successively, and are minimum with the pyriform embryo.
The embryo callus incidence of table 1 explant in embodiment 5 relatively
Table 2 explant embryoid differentiation rate on improvement MS compares
Compare 11 kind different genotype different parts embryo callus incidences and embryoid differentiation rate.
The result sees table 3 and table 4:
Table 3 clover different parts embryo callus incidence relatively
Table 4 clover different parts embryoid differentiation rate relatively
From table 1 and table 2, find out: the assorted upper and lower plumular axis embryo of three leaves callus incidence and embryoid differentiation rate are all the highest; Flying Carpet and Latin promise are taken second place; The red clover Ruide is poorer slightly than them, and 11 kind epicotyl embryo callus incidences are followed successively by assorted three leaves＞Flying Carpet＞Latin promise＞Ruide＞floor file＞koala＞sea and send out＞Hu Yia＞Ai Lisi＞hundred despot＞Rui Wende; Hypocotyl embryo callus incidence is followed successively by assorted three leaves＞Latin promise＞Ruide＞Flying Carpet＞koala＞floor file＞sea and sends out＞Hu Yia＞hundred despot＞Ai Lisi＞Rui Wende; Epicotyl embryoid differentiation rate is followed successively by assorted three leaves＞Flying Carpet＞Latin promise＞Ruide＞floor file＞koala＞Hu Yia＞sea and sends out＞Ai Lisi＞hundred despot＞Rui Wende; Hypocotyl embryoid differentiation rate is followed successively by assorted three leaves＞Latin promise＞Flying Carpet＞Ruide＞floor file＞koala＞sea and sends out＞Hu Yia＞hundred despot＞Ai Lisi＞Rui Wende; Regeneration leaf different cultivars embryo callus incidence and embryoid differentiation rate are all very high, and all more than 90%, the different cultivars differences is very little.The above results is described by upper and lower plumular axis different cultivars and genotype because the difference of genetic background, and reaction has certain difference to condition of culture, breaks up seedling but under consistent relatively condition of culture, all can obtain embryo callus and embryoid.
In the test of each 360 seed of 11 kinds of three kinds of clover, epicotyl embryo callus incidence on average reaches 39.7%; Hypocotyl on average reaches 35.3%; Regeneration blade embryo callus incidence on average reaches 92.8%; Epicotyl embryoid differentiation rate on average reaches 34.9%, and hypocotyl on average reaches 31.7%, and regeneration blade embryoid differentiation rate on average reaches 92.8%.All kinds of participating in the experiment all can obtain regeneration plant.
Through selecting supplementary measures such as proper explant, improvement minimal medium and composition, adjustment condition of culture; Overcome different cultivars and genotype barrier problem in the cultivation of clover high-frequency somatic embryo regeneration effectively, increased substantially somatic embryo occurrence frequency and regeneration frequencies.
Below disclose the present invention with preferred embodiment, so it is not in order to restriction the present invention, and all employings are equal to replacement or the technical scheme that obtained of equivalent transformation mode, all drop within protection scope of the present invention.
Claims (1)
1. one kind overcomes clover variety genotype barrier high-frequency somatic embryo regeneration medium, comprising: growth of seedling acclimation shaking culture base, embryo generation callus inducing medium, embryo subculture form medium and become the seedling medium with the embryo maturation germination, it is characterized in that:
Described growth of seedling acclimation shaking culture base is: 1/2 improvement MS+20g/L white sugar+0.7% agar;
Described embryo generation callus inducing medium is: improvement SH+8～12mg/L 2,4-D+0.2～0.5mg/L 6-BA+30g/L sucrose+0.3% phytagel;
Described embryo subculture forms medium: improvement SH+2～8mg/L 2,4-D+0.2～0.5mg/L6-BA+50g/L sucrose+0.35% phytagel;
Described embryo maturation germination becomes the seedling medium to be: improvement MS+30g/L white sugar+0.7% agar,
Described improvement SH and modified MS medium all comprise grand nutrition element, micronutrient element and organic reagent,
Wherein, the component of the grand nutrition element that comprises of improvement SH is following with its corresponding concentration:
Ammonium sulfate 463mg/L;
Potassium nitrate 2830mg/L;
Calcium chloride dihydrate 166mg/L;
Epsom salt 185mg/L;
Anhydrous potassium dihydrogenphosphate 400mg/L;
Iron edta sodium salt 1.4mg/L;
The component of the micronutrient element that improvement SH comprises is following with its corresponding concentration:
Manganese sulfate monohydrate 10mg/L;
Zinc sulphate 1.0mg/L;
Boric acid 5.0mg/L;
KI 1.0mg/L;
Sodium molybdate 0.1mg/L;
Copper sulphate 0.2mg/L;
Cobalt chloride 0.1mg/L;
The component of the organic reagent that improvement SH comprises is following with its corresponding concentration:
Thiamine hydrochloride 5.0mg/L;
Nicotinic acid 5.0mg/L;
Puridoxine hydrochloride 5.0mg/L;
And the component of the grand nutrition element that described modified MS medium comprises is following with its corresponding concentration:
Ammonium sulfate 1650mg/L;
Potassium nitrate 1900mg/L;
Epsom salt 370mg/L;
Anhydrous potassium dihydrogenphosphate 170mg/L;
Calcium chloride dihydrate 440mg/L;
Disodium ethylene diamine tetraacetate 37.3mg/L;
Ferrous sulfate heptahydrate 27.8mg/L;
The component of the micronutrient element that modified MS medium comprises is following with its corresponding concentration:
Four water manganese sulphate 22.3mg/L;
Zinc sulphate 8.6mg/L;
Boric acid 6.2mg/L;
KI 0.83mg/L;
Sodium molybdate 0.25mg/L;
Copper sulphate 0.025mg/L;
Cobalt chloride 0.025mg/L;
The component of the organic reagent that modified MS medium comprises is following with its corresponding concentration:
Thiamine hydrochloride 1.0mg/L;
Nicotinic acid 1.0mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100mg/L.
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| 杨丽莉等.影响白三叶草高频率植株再生因素的研究.《华北农学报》.2003,第18卷(第4期),第66-68页. * |
| 杨珍等.高加索三叶草和白三叶草再生体系的建立.《内蒙古农业大学学报》.2008,第29卷(第4期),第68-73页. * |
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