CN101663997A - Regeneration and cultivation method of high frequency somatic embryos for overcoming shamrock variety genotypic disorder - Google Patents
Regeneration and cultivation method of high frequency somatic embryos for overcoming shamrock variety genotypic disorder Download PDFInfo
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Abstract
The invention discloses a regeneration and cultivation method of high frequency somatic embryos for overcoming shamrock variety genotypic disorder, which comprises the following steps: 1. material selection: selecting three explants, i.e. an epicotyl, a hypocotyls and a regeneration plant leaf of an aseptic seedling after a mature seed of the shamrock is flushed, sterilized, disinfected and dried;2. inoculating the selected explants onto an embryogenic callus inducing culture medium for cultivation; 3. inoculating the explants cultivated through the embryogenic callus inducement onto an embryo successive formation culture medium for cultivation; 4. inoculating the explants cultivated through the embryo successive formation cultivation onto an embryo maturation germination seedling culturemedium for cultivation to form seedlings; 5. carrying out rooting cultivation on strong seedlings; and 6. transplanting a regeneration plant for survival. The invention can effectively overcome the problems of different varieties and genotypic disorder in the regeneration and cultivation of the high frequency somatic embryos of the shamrock, greatly improve the cell embryogenesis frequency and the regeneration plant frequency, and improve the breeding efficiency.
Description
Technical field
The present invention relates to the high-frequency somatic embryo regeneration culture method of a kind of leguminous forage, relate to a kind of clover variety genotype barrier high-frequency somatic embryo regeneration culture method 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 orchard forest, 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, the kind of countries in the world application at present is synthetic variety more than 80%, genotypic difference is very big in the kind, some important breeding objectives of clover are difficult to reach by the conventional breeding method, as the clover drought resistance and salt tolerance, disease-resistant pest-resistant, improve seed and careless output, nitrogen fixing capacities etc. all do not reach desirable effect, therefore in recent years, people attempt by the modern genetic engineering technology, promptly obtain resistant plant and cultivate the clover new varieties by transgenosis, improve the adaptability of clover to various ecotopes, but the precondition as this technology energy successful Application is that clover has the high-frequency somatic embryo regeneration ability in its tissue culture procedures, and 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, more existing reports show, 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 by 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 by 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 by 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 is not subjected to the restriction of kind and genotype is the important prerequisite that obtains each veriety clover transgenosis success.
Summary of the invention
In order to overcome the deficiencies in the 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 culture method.
The present invention is achieved through the following technical solutions:
A kind ofly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, may further comprise the steps:
(1) selection: the clover mature seed is cultivated aseptic seedling on growth of seedling domestication medium after flushing, disinfection, drying, chooses epicotyl, hypocotyl and three kinds of explants of regeneration plant blade of the aseptic seedling after the sprouting;
(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 on the growth of seedling domestication medium carries out the strengthening seedling and rooting cultivation, form the regeneration plant of taking root;
The regeneration plant of (6) taking root is transplanted, is survived;
Described growth of seedling domestication medium 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 and its corresponding concentration are as follows:
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 and its corresponding concentration are as follows:
Manganese sulfate monohydrate 10mg/L;
Zinc sulphate 1.0mg/L;
Boric acid 5.0mg/L;
Potassium iodide 1.0mg/L;
Sodium molybdate 0.1mg/L;
Copper sulphate 0.2mg/L;
Cobalt chloride 0.1mg/L.
The component of organic reagent and its corresponding concentration are as follows:
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 and its corresponding concentration are as follows:
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 and its corresponding concentration are as follows:
Four water manganese sulphate 22.3mg/L;
Zinc sulphate 8.6mg/L;
Boric acid 6.2mg/L;
Potassium iodide 0.83mg/L;
Sodium molybdate 0.25mg/L;
Copper sulphate 0.025mg/L;
Cobalt chloride 0.025mg/L.
The component of organic reagent and its corresponding concentration are as follows:
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 by 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.Setting up clover high-frequency somatic embryo regeneration technical system is the precondition that guarantees clover somatic induction variation, efficient stable genetic transformation, obtains can supply in a large number the target plant of screening, simplify the generation of clover somatic embryo and plant regeneration medium and technical system by 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
Below in conjunction with the drawings and specific embodiments, describe the specific embodiment of the present invention in detail:
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.
As Fig. 1-shown in Figure 10:
A kind ofly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, may further comprise the steps:
(1) 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), red clover (Ruide) and three leaves of mixing;
Wash 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, with aseptic water washing 5 times, sterilization filter paper suck dry moisture is inoculated on the growth of seedling domestication medium;
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 vertical master pulse cutting at distance 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 on the growth of seedling domestication medium carries out the strengthening seedling and rooting cultivation, 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 survives;
Above-mentioned growth of seedling domestication medium is: 1/2 improvement MS+20g/L white sugar+0.7% agar;
Above-mentioned 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;
Above-mentioned embryo subculture forms medium: improvement SH+2～8mg/L 2,4-D+0.2～0.5mg/L6-BA+50g/L sucrose+0.35%phytagel;
Above-mentioned 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 and its corresponding concentration are as follows:
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 and its corresponding concentration are as follows:
Manganese sulfate monohydrate 10mg/L;
Zinc sulphate 1.0mg/L;
Boric acid 5.0mg/L;
Potassium iodide 1.0mg/L;
Sodium molybdate 0.1mg/L;
Copper sulphate 0.2mg/L;
Cobalt chloride 0.1mg/L.
The component of organic reagent and its corresponding concentration are as follows:
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 and its corresponding concentration are as follows:
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 and its corresponding concentration are as follows:
Four water manganese sulphate 22.3mg/L;
Zinc sulphate 8.6mg/L;
Boric acid 6.2mg/L;
Potassium iodide 0.83mg/L;
Sodium molybdate 0.25mg/L;
Copper sulphate 0.025mg/L;
Cobalt chloride 0.025mg/L.
The component of organic reagent and its corresponding concentration are as follows:
Thiamine hydrochloride 1.0mg/L;
Nicotinic acid 1.0mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100mg/L.
In specific embodiment, the combination of plant growth regulator is respectively:
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%phytage l)+and C. embryo maturation germination becomes seedling medium (improvement MS+30g/L white sugar+0.7% agar)+D. growth of seedling domestication medium (1/2 improvement MS+20g/L white sugar+0.7% agar), and 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 domestication medium (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 domestication medium (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 domestication medium (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 domestication medium (1/2 improvement MS+20g/L white sugar+0.7% agar), pH is about 5.8 behind above-mentioned all medium autoclavings.
Embodiment 6
A. embryo generation callus inducing medium (is improved SH+12mg/L 2,4-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 domestication medium (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.
As shown in Figure 1: in above-mentioned embryo generation callus induction step, the callus surface of seeing explant by microexamination forms some projections, protrusion cell matter is dense, separate with peripheral cell, obvious boundary is arranged, illustrate 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 to take place on the medium of the auximone that contains suitable concn or suitable ratio auximone/cytokinin, too high auximone or inappropriate auximone/cytokinin ratio all can reduce explant embryo callus incidence, the most 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, epicotyl and hypocotyl embryo callus incidence on average improve more than 21% and (see Table 1), as seen have only auximone/cytokinin proportioning suitable, could promote the embryo callus to produce and formation.
Extremely shown in Figure 4 as Fig. 2: as to form incubation step at above-mentioned embryo subculture, this experiment is handled discovery by 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 2,4-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, the most 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, make, hypocotyl embryoid differentiation rate has on average improved more than 23% and (has seen Table 2), the embryo callus also no longer increases and shoals or brownization occur, 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, Bai Sanye embryo callus surface develops into harder bulk particle, it is globular embryo, these green little stones or bulk particle are exactly the somatic embryo of growing, and it can be cut apart cultivation and further develop into single plantlet when looking bigger.
As 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, cotyledon and true leaf form, and grow 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 results are shown in Table 3 and table 4:
Table 3 clover different parts embryo callus incidence relatively
Table 4 clover different parts embryoid differentiation rate relatively
Find out from table 1 and table 2: 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.Upper and lower plumular axis different cultivars and genotype are described The above results because the difference of genetic background, and reaction has certain difference to condition of culture, but all can obtain embryo callus and embryoid differentiation seedling under consistent relatively condition of culture.
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.
By 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 replaces or technical scheme that the equivalent transformation mode is obtained, all drops within protection scope of the present invention.
Claims (9)
1, a kind ofly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, may further comprise the steps:
(1) selection: the clover mature seed is cultivated aseptic seedling on growth of seedling domestication medium after flushing, disinfection, drying, chooses epicotyl, hypocotyl and three kinds of explants of regeneration plant blade of the aseptic seedling after the sprouting;
(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 on the growth of seedling domestication medium carries out the strengthening seedling and rooting cultivation, form the regeneration plant of taking root;
The regeneration plant of (6) taking root is transplanted, is survived;
Described growth of seedling domestication medium 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.
2, according to claim 1ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that described improvement SH comprises grand nutrition element, micronutrient element and organic reagent.
3, according to claim 2ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that the component of described grand nutrition element and its corresponding concentration are as follows:
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.
4, according to claim 2ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that the component of described micronutrient element and its corresponding concentration are as follows:
Manganese sulfate monohydrate 10mg/L;
Zinc sulphate 1.0mg/L;
Boric acid 5.0mg/L;
Potassium iodide 1.0mg/L;
Sodium molybdate 0.1mg/L;
Copper sulphate 0.2mg/L;
Cobalt chloride 0.1mg/L.
5, according to claim 2ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that the component of described organic reagent and its corresponding concentration are as follows:
Thiamine hydrochloride 5.0mg/L;
Nicotinic acid 5.0mg/L;
Puridoxine hydrochloride 5.0mg/L.
6, according to claim 1ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that described modified MS medium comprises grand nutrition element, micronutrient element and organic reagent.
7, according to claim 6ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that the component of described grand nutrition element and its corresponding concentration are as follows:
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.
8, according to claim 2ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that the component of described micronutrient element and its corresponding concentration are as follows:
Four water manganese sulphate 22.3mg/L;
Zinc sulphate 8.6mg/L;
Boric acid 6.2mg/L;
Potassium iodide 0.83mg/L;
Sodium molybdate 0.25mg/L;
Copper sulphate 0.025mg/L;
Cobalt chloride 0.025mg/L.
9, according to claim 2ly overcome clover variety genotype barrier high-frequency somatic embryo regeneration culture method, it is characterized in that the component of described organic reagent and its corresponding concentration are as follows:
Thiamine hydrochloride 1.0mg/L;
Nicotinic acid 1.0mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100mg/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224802A (en) * | 2011-04-19 | 2011-10-26 | 浙江大学 | Proliferation medium for strawberry |
| CN103896678A (en) * | 2014-03-28 | 2014-07-02 | 黄景诚 | Disease and insect-resistant high-efficiency fertilizer for perennial grasses |
| CN104969775A (en) * | 2015-07-29 | 2015-10-14 | 句容美华园林景观发展有限公司 | Method for cultivating Indian bread |
| CN105557521A (en) * | 2015-12-16 | 2016-05-11 | 青岛百瑞吉生物工程有限公司 | Seedling strengthening and rooting culture medium of pear tree somatic embryos |
| CN108056023A (en) * | 2018-02-06 | 2018-05-22 | 江苏农林职业技术学院 | No germplasm genotype limits high-frequency somatic embryo regeneration culture method |
| CN108243960A (en) * | 2018-02-06 | 2018-07-06 | 江苏农林职业技术学院 | The high-frequency somatic embryo regeneration culture medium of no germplasm genotype limitation and its application |
-
2009
- 2009-09-16 CN CN 200910034976 patent/CN101663997B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224802A (en) * | 2011-04-19 | 2011-10-26 | 浙江大学 | Proliferation medium for strawberry |
| CN103896678A (en) * | 2014-03-28 | 2014-07-02 | 黄景诚 | Disease and insect-resistant high-efficiency fertilizer for perennial grasses |
| CN103896678B (en) * | 2014-03-28 | 2015-12-09 | 黄景诚 | Perennial grass disease and insect resistance concentrated fertilizer |
| CN104969775A (en) * | 2015-07-29 | 2015-10-14 | 句容美华园林景观发展有限公司 | Method for cultivating Indian bread |
| CN105557521A (en) * | 2015-12-16 | 2016-05-11 | 青岛百瑞吉生物工程有限公司 | Seedling strengthening and rooting culture medium of pear tree somatic embryos |
| CN108056023A (en) * | 2018-02-06 | 2018-05-22 | 江苏农林职业技术学院 | No germplasm genotype limits high-frequency somatic embryo regeneration culture method |
| CN108243960A (en) * | 2018-02-06 | 2018-07-06 | 江苏农林职业技术学院 | The high-frequency somatic embryo regeneration culture medium of no germplasm genotype limitation and its application |
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| CN101663997B (en) | 2011-11-09 |
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