CN1746294A - Method for setting up matured embryo regenerating system of zhonghuajielu grass - Google Patents
Method for setting up matured embryo regenerating system of zhonghuajielu grass Download PDFInfo
- Publication number
- CN1746294A CN1746294A CN 200510087733 CN200510087733A CN1746294A CN 1746294 A CN1746294 A CN 1746294A CN 200510087733 CN200510087733 CN 200510087733 CN 200510087733 A CN200510087733 A CN 200510087733A CN 1746294 A CN1746294 A CN 1746294A
- Authority
- CN
- China
- Prior art keywords
- substratum
- callus
- stage
- acid
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A method for constructing mature embryo regenerative system of Chinese Korea lawn grass is carried out by using mature seed of Chinese Korea lawn grass as material, culturing on induced culture medium to obtain wound tissue, adding 2,4-D and 6-BA and high-concentration carbon source into induced wound tissue culture medium proportionally, sub-culturing, and high-frequency inducing soma and regenerative tree. It has higher induction rate of wound tissue and regeneration rate.
Description
Technical field:
The present invention relates to a kind of method of setting up Zoysia sinica mature embryo regeneration system.Belong to biological technical field.
Background technology:
Since the nineties, along with the development of China's urban construction and the raising of living standards of the people, the lawn is sodded in the many cities of northern China energetically, has obtained good landscape effect, as 3000 hectares in Beijing turf plant, all has every year nearly 300 hectares of lawns to come out; The Daliang City catches up from behind, and annual newly-increased more than 240 hectare (Wang Qingkui etc., grassland and meadow, 2002,97 (7): 22-24) are looked back and countermeasure in China's Lawn Industry development.But in the majority with cool-season grasses, though the green phase is long, color and luster is graceful, water loss is big, growth needs rapidly often to prune easy sick insect pest in summer, maintenance costs height.China is developing country, and economic strength is limited, and Freshwater resources lack, the Northwest more so, so no matter be urban afforestation or erosion and torrent control works, all should be the long lawn of exploitation drought resisting, disease-resistant, extensive management, green period and cover plant as emphasis.And jielu grass just in time can remedy the deficiency of cold-season turfgrass, and it has many excellent characteristic: 1) drought-enduring, thermotolerance is extremely strong; 2) salt tolerant alkalescence is strong; 3) wear-resisting, anti-trample, good springiness; 4) anti-pruning; 5) has disease and insect resistance preferably; 6) anti-soil depletion; 7) anti-low-level maintenance.With English grass relatively in, the water loss of jielu grass lacks 30% to 40% than English grass, and lawn elastic force is 30 to 40 times of English grass, planting and overhead charges are than the low 80% (Hu Shuliang of English grass, the discussion of development green grassland key issue, the China meadow, 1997 (2): 67-70).Therefore, jielu grass has become the first-selected grass seeds of playground, open greenery patches, greening side slope and soil conservation, is the turf grass species that is fit to China's national situation.
Zoysia sinica (Zoysia sinica Hance) is the good lawn plant of homemade preciousness, except that the good characteristic that the general jielu grass of tool is possessed, its blade quality is thinner, color and luster U.S., expansion is fast, and its suitable habitats pH in soil is saline alkali tolerant plant at 7.2-9.3, can be used as the good plant of saltings and coastal cities lawn planting, potentiality to be exploited is very big.Scholarly forecast is arranged, and jielu grass, particularly Zoysia sinica will become one of pillar of Future in China Lawn Industry.
But also there are some problems in magnificent in actual applications jielu grass, and is poor as winter resistance, and the withered and yellow phase is long etc.Zoysia sinica is very responsive to low temperature, when being lower than 10 ℃, envrionment temperature enters dormancy, the green phase in northern area every year on average has only about 6 months, as mid or late April turning green in the area, Dalian, mid or late October is withered and yellow, and green period is about 160 days, and annual bluegrass is 280 days (Liu Jianxiu etc., the research and the improvement of warm-season turf grass seeds resource, external Animal husbandry-grassland and herbage, 1996 (3): 12-21).This shortcoming has seriously limited its popularization at northern area.Thereby jielu grass carried out genetic improvement, and delaying leaf senile, the seed selection green period prolongs, and the fine quality new variety (being) in lawn, has important theory and practice significance.
Directly inductor cytometaplasia, or the employing genetic engineering technique, degeneration-resistant functional gene there is purpose, imports the callus or the protoplastis of certain species targetedly, obtain the transgenic plant of improvement, improve the resistance of turfgrass, become the research forward position that the field is learned on the lawn.Tissue culture (protoplastis cultivation) is the indispensable integral part of modern biotechnology, no matter be providing of transgene receptor, still the screening and the regeneration of transformant all need tissue culture technique to support, thereby set up one and overlap efficiently that regeneration system is the necessary condition of transgenic technology.
The turfgrass regeneration system to set up difficulty bigger, many turfgrasss researchs only are to have obtained regeneration plant, can't satisfy the needs of genetic transformation.And warm season turf is than more difficult induced embryonic callus of cold-season turfgrass and regeneration plant.The Zoysia plant belongs to warm season turf, and its Study on tissue culture has had more than ten years.Some Japan and Korea S scholar research in this respect are comparatively deep, inquired in the jielu grass regenerative process 2, the effect of 4-D, NAA and 6-BA and proportioning (Yoo Y K etc., Effects of plant growthregulators on callus formation and organogenesis from the shoot cultures of fiveturf-grass species, Journal of the Korean Society for HorticulturalSci., 1991,32 (2): 237-246; Noh, HeeYoung, Choi, JoonSoo, Ahn, ByungJoon.Plantregeneration through somatic embryogenesis in zoysiagrasses, Journal of the KoreanSociety for Horticultural Science, 1995,36 (4): 582-587; Li Ruifen etc., jielu grass callus induction and plant regeneration, gardening journal, 2003,30 (3): 355-357; Deng).
The jielu grass callus to induce many be that (Al-Khayri waits .In vitro plant regeneration of zoysiagrass, Arkansas Farm Research, 1989,38 (2): 11 to explant with mature seed; Chai etc., Application of biotechnology in turfgrass genetic improvement, Crop Science, 1998,38:1320-1338; Rim etc., Callus induction and plant regeneration from seeds of Zoysia japonica Steud, Journal of the Korean Society of Grassland Science, 2001,21 (2): 49-52; Deng), adding 2 of different concns, the MS substratum of 4-D, NAA and BA (seeing attached list 1) or LS substratum (seeing attached list 2) are gone up evoked callus and regeneration plant.In existing research, the zoysia japonica seed callus induction rate only is 30-50%, and wherein the embryo callus rate is usually less than 3%.With young fringe and young fruit is explant, also induces callus and has obtained regeneration plant.(Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants regenerated from pro-toplasts such as Inokuma, Plant Cell Reports, 1998,17 (5): 334-338) start the precedent of finishing the living plastid cultivation in thread grassland.
More than each regeneration system to set up research be material with Japanese lawn grass (Z.japonica) all, and callus induction rate and regeneration rate are still lower, and the foundation of relevant Zoysia sinica (Zoysia sinica Hance) tissue culture regeneration system research does not appear in the newspapers as yet, and correlative study is all handled the exploratory stage.
Summary of the invention
At the deficiencies in the prior art, the present invention is a material with Zoysia sinica mature seed embryo, induce the mature embryo high frequency to produce callus, cut callus, put into subculture medium and plant regeneration substratum and obtain high, frequently stable frequency of embryonic callus induction and shoot regeneration frequency.For genetic transformation provides the good receptor material.
The present invention has set up regeneration system by isolated culture Zoysia sinica mature embryo, and explant is drawn materials easily and convenient the storage, and the regeneration plant proterties homogeneous that is obtained, stable is easy to genetic engineering procedure.Cultural method of the present invention comprises four cultivation stages: (1) is explant with the mature embryo, the direct evoked callus of isolated culture, (2) succeeding transfer culture of callus, (3) callus induction somatic embryo, (4) somatic embryo inducement regeneration plant, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～500 luxs, (4) be the light cultivation of 1500～3000 luxs, whole culturing process, culture temperature is 25 ± 3 ℃, and (1) stage was cultivated switching in 40 days, and (2) (3) (4) every cultivation renewed bright substratum in 20 days.
Be preferably (1), (4) minimum medium in stage is the MS substratum, (2), (3) minimum medium in stage is the HMS substratum, used hormone is in the substratum in (1) stage: 2.5～6mg/L 2,4-dichlorphenoxyacetic acid 2,4-D, 0.1～1.0 naphthylacetic acid NAA, 0.1～1.0mg/L 6-benzylamino VITAMIN B4 6-BA, riboflavin VB2 0.5～2.0mg/L and caseinhydrolysate 200～500mg/L have also been added, used hormone is in the substratum in (2) stage: 0.1～5mg/L 2,4-dichlorphenoxyacetic acid 2,4-D, 0.1～3mg/L 6-benzylamino VITAMIN B4 6-BA.Used hormone is in the substratum in (3) stage: 0.05～5mg/L 2,4-dichlorphenoxyacetic acid 2,4-D, 0.01～2mg/L 6-benzylamino VITAMIN B4 6-BA, in the substratum in (4) stage, contain kinetin KT0～0.3mg/L, 2,4 dichlorophenoxyacetic acid 2,4-D 1.0～3.5mg/L, 6-benzylamino VITAMIN B4 6-BA 0.1mg/L.
Wherein, the carbon source of adding in the substratum in (1) stage is sucrose 20～30g/L, glucose 15～25g/L, (2), (3), (4) the stage carbon source of adding in cultivating is sucrose 30～60g/L.The composition of HMS substratum is: 1/2MS macroelement, MS trace element, MS molysite, riboflavin (VB2) 1.0mg/L, hydrochloric tiamide (VB1) 1.0mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, inositol 0.1g/L.
The method that the present invention sets up the Zoysia sinica regeneration system comprises the steps: (1) seed earlier with the NaOH solution-treated of 30% (w/v) 40 minutes, breaking dormancy, and soaked in the tap water under room temperature 12～20 hours then (2).With 70% (v/v) alcohol sterilization 30 seconds, after sterile distilled water cleans 3 times, 0.1% mercuric chloride soaked 10 minutes, clean 8-10 time with sterile distilled water again, (3) will the go out explant of bacterium is inoculated in and contains 2.5～6.0 2,4-dichlorphenoxyacetic acid 2,4-D, 0.1～1.0 naphthylacetic acid NAA, 0.1～1.0mg/L 6-benzylamino VITAMIN B4 6-BA, 0.5～2.0mg/L riboflavin VB
2, 200～500mg/L caseinhydrolysate CH and 20～30g/L sucrose and 15～25g/L glucose substratum in evoked callus, temperature is 25 ± 3 ℃.The explant rate of depolluting reaches more than 90%.After 10 days, produce on the bastem portion of seed germination or sprout, or begin to occur callus at the embryo position of seed, callus reached the peak period in 15-25 days, and the investigation callus induction rate reached more than 70% in 40 days.2,4-D and 6-BA and high density carbon source have very important effect in the evoked callus process.(4) under the aseptic condition, callus changed over to contain 1.0～4.0mg/L2,4-dichlorphenoxyacetic acid 2,4-D, on the HMS substratum of 0.1～0.5mg/L 6-benzylamino VITAMIN B4 6-BA, culture condition is: lose light and cultivate 25 ± 3 ℃ of temperature.Behind the succeeding transfer culture 60 days, the callus volume obviously increases, and the allometry amount is 2～4 times, and the callus phenotype can be divided into three classes significantly: a class is that appearance white arrives flaxen callus, has soft, sticking tissue and solids tissue concurrently; Two classes are that growth is fast, and outward appearance is relaxed and comfortable, inner soft, hygrophanous callus; Three classes are that growth is slow, the callus of color depth, brownization.(5) substratum that the tissue block classification is transferred to embryonic callus induction and plant regeneration carries out differentiation culture.The first kind i.e. white can be seen a small amount of bud original hase to callus faint yellow, that contain solids after differentiation culture 10-15 days, can see tangible budlet clump after 30-40 days, and regeneration rate can reach 30-65%.Plant regeneration sends palpus shape root at base portion based on the organ occurring mode when Miao Cong forms or subsequently.At room temperature hardening is after 3 days for regrowth, and transplanting survival rate is more than 95%.The promptly softening callus of second class, in the continued growth of differentiation culture initial stage, along with the prolongation of time, brownization is dead gradually.The 3rd class is the callus of color depth, brownization, and is most dead behind the differentiation culture, has only indivedual tissue block can form green callus, but regeneration plant seldom.Wherein (2) (3) are dark culturing, and (4) are that the low light level of 200～500 luxs is cultivated, and (5) are that the light of 1500～3000 luxs is cultivated, whole culturing process, and culture temperature is 25 ± 3 ℃, except that (2) stage, fresh culture was changed in every cultivation in 20 days one time.
Among the present invention, callus is to produce on the bastem portion of seed germination or sprout, or directly sends at the embryo position of seed, thereby can think that callus is to be produced by the mature embryo of seed.
Described callus is meant the cell mass of inducing the not organized somatoblast that expands of generation to constitute from the Zoysia sinica mature embryo.
Described embryo callus is meant that the somatoblast group of inorganizationization is after further cultivating, cell mass surface or internal portion cell content increase, and polarity appears, behind further growth experience globular embryo, torpedo embryo and the cotyledon shape embryo is the organizer somatic embryo, can directly be divided into whole plant under optimum conditions.The ovary development process of the similar after fertilization of its growth course.
The present invention is an explant with the Zoysia sinica mature embryo, the genetic transformation acceptor systems of setting up has overcome the low and low shortcoming of shoot regeneration frequency of the ubiquitous callus induction rate of jielu grass platymiscium, its characteristics are: (1) callus induction significantly increases, and is up to more than 70%; (2) regeneration plant inductivity height, and stable.(3) draw materials and be not subject to seasonal restrictions.
Description of drawings
Fig. 1 embryo callus
Fig. 2 begins to form embryo callus
Fig. 3 non-embryonic callus tissue
Fig. 4 embryo callus differentiation and bud formation point
Fig. 5 somatic embryo is sprouted
Fig. 6 is from the embryonic callus induction adventitious shoot regeneration
The ripe regeneration induction plant of Fig. 7 Zoysia sinica
Embodiment:
Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.
Embodiment 1: callus induction
Test materials: Zoysia sinica mature seed.
Test design and substratum: design according to L9 (4 * 3) orthogonal table.Used substratum is the HMS substratum, sucrose 30g/L, and agar 6.0g/L, pH 5.8.
Culture condition: culture temperature is 25 ± 1 ℃, unglazed photograph.
Testing sequence: seed is with the NaOH solution-treated 40min of 30% (w/v), but breaking dormancy, and percentage of germination reached 89.9% in 7 days.Under room temperature, soaked 20 hours in the tap water then.With 70% (v/v) alcohol sterilization 30 seconds, after sterile distilled water cleans 3 times, 0.1% mercuric chloride soaked 10 minutes, clean to be inoculated in then for 8 times with sterile distilled water again and contain 2,4-dichlorphenoxyacetic acid 2,4-D 2.5mg/L, naphthylacetic acid NAA 0.5mg/L, 6-benzylamino VITAMIN B4 6-BA 0.25mg/L, VB
11.0mg/L and in the MS substratum of sucrose 30g/L, glucose 20g/L, lose light and cultivate, temperature is 25 ± 1 ℃.After 10 days, produce on the bastem portion of seed germination or sprout, or begin to occur callus at the embryo position of seed, callus reached the peak period in the 21st day.The investigation callus induction rate reached more than 70% in 40 days.2,4 dichlorophenoxyacetic acid 2,4-D, 6-benzylamino VITAMIN B4 6-BA and carbon source play a part very important in the evoked callus process.
Embodiment 2: the callus succeeding transfer culture
Callus induction is with embodiment 1.Callus is inoculated into to contain 6-benzylamino VITAMIN B4 6-BA be 0.1mg/L, 2,4 dichlorophenoxyacetic acid 2,4-D is on the HMS substratum of 1.6mg/L, callus is white in color more, has a small amount of faint yellow callus in the tissue block that has, and brownization takes place callus hardly.Behind the succeeding transfer culture 60 days, callus allometry amount is 3.2 times, but does not have tangible bud original hase differentiation.When 6-BA concentration was 0.25mg/L, tissue block is hardening gradually, formed white to flaxen budlet, and brownization of tissue block rate is 10.3%.When 6-BA concentration is 0.5mg./L, brownization rate reaches 25%.Explanation 6-benzylamino VITAMIN B4 6-BA concentration in the succeeding transfer culture of callus is answered≤0.1mg/L.When 6-BA concentration is high, cause the organ differentiation easily, be unfavorable for callus growth.
In the subculture medium 2,4-dichlorphenoxyacetic acid 2,4-D concentration is at 2.5mg/L, when 6-benzylamino VITAMIN B4 6-BA concentration is 0.1mg/L, behind the callus succeeding transfer culture 60 days, callus allometry amount is 3.6 times, the allometry amount difference of callus all not significantly (P=0.05) between each 2,4 dichlorophenoxyacetic acid 2,4-D concentration are handled.Can be divided into two classes (seeing description of drawings) significantly according to the callus phenotype: a class is that appearance white arrives flaxen callus, has soft, sticking tissue and solids tissue concurrently; Two classes are that growth is fast, inner soft, hygrophanous callus; Wherein based on the first kind.2,4 dichlorophenoxyacetic acid 2 is described, 4-D concentration is the maintenance that 2.5mg/L utilizes the callus growth quality.
Embodiment 3: regeneration plant is induced
Test materials: 60 days callus of succeeding transfer culture, preparation method is with embodiment 1,2.
Substratum: used substratum is the MS minimum medium that does not add plant-growth regulator, sucrose 30g/L, and agar 6.0g/L, pH 6.0.
Culture condition: culture temperature is 25 ± 3 ℃, and intensity of illumination is 2000 luxs, and light application time is 12 hours/day.
Choose wherein fine and close white or the faint yellow callus of structure, be inoculated into and contain: 1.5mg/L 2,4-dichlorphenoxyacetic acid 2,4-D, 0.05mg/L differentiation culture can be seen a small amount of bud original hase in the substratum of 6-benzylamino VITAMIN B4 6-BA after 15 days, can see tangible budlet clump after 30 days, regeneration rate can reach 70%.Plant regeneration sends palpus shape root at base portion based on the organ occurring mode when Miao Cong forms or subsequently.At room temperature hardening is after 3 days for regrowth, and transplanting survival rate is more than 95%.
Subordinate list 1
MS minimum medium (Murashige ﹠amp; Skoog, 1962) composition
| Component | Composition | Content (mg/L) |
| Macroelement | Ammonium nitrate | 1650 |
| Saltpetre | 1900 | |
| Potassium primary phosphate | 170 | |
| Bitter salt | 370 | |
| Two hydration calcium chloride | 440 | |
| Trace element | Four anhydrous manganeses | 22.3 |
| Zinc vitriol | 8.6 | |
| Boric acid | 6.2 | |
| Potassiumiodide | 0.83 | |
| Two molybdic acid hydrate sodium | 0.25 | |
| Cobalt chloride hexahydrate | 0.025 | |
| Salzburg vitriol | 0.025 | |
| Organic element | Inositol | 100 |
| Nicotinic acid | 0.5 | |
| Vitamin | 0.1 | |
| Pyridoxine hydrochloride | 0.5 | |
| Glycine | 2.0 | |
| Sucrose | 30,000 | |
| Molysite | Ferrous sulfate | 27.8 |
| Disodium ethylene diamine tetraacetate | 37.3 |
Subordinate list 1
MS minimum medium (Murashige ﹠amp; Skoog, 1962) composition
| Component | Composition | Content (mg/L) |
| Macroelement | Ammonium nitrate | 1650 |
| Saltpetre | 1900 | |
| Potassium primary phosphate | 170 | |
| Bitter salt | 370 | |
| Two hydration calcium chloride | 440 | |
| Trace element | Four anhydrous manganeses | 22.3 |
| Zinc vitriol | 8.6 | |
| Boric acid | 6.2 | |
| Potassiumiodide | 0.83 | |
| Two molybdic acid hydrate sodium | 0.25 | |
| Cobalt chloride hexahydrate | 0.025 | |
| Salzburg vitriol | 0.025 | |
| Organic element | Inositol | 100 |
| Nicotinic acid | 0.5 | |
| Vitamin | 0.1 | |
| Pyridoxine hydrochloride | 0.5 | |
| Glycine | 2.0 | |
| Sucrose | 30,000 | |
| Molysite | Ferrous sulfate | 27.8 |
| Disodium ethylene diamine tetraacetate | 37.3 |
Claims (9)
1. a method of setting up the Zoysia sinica regeneration system is characterized in that with the Zoysia sinica mature embryo be explant, carries out isolated culture.
2. according to the method for claim 1, it is characterized in that described method comprises four cultivation stages:
(1) with the mature embryo is explant, the direct evoked callus of isolated culture, the succeeding transfer culture of (2) callus, (3) callus induction somatic embryo, (4) somatic embryo inducement regeneration plant, wherein (1) (2) are dark culturing, and (3) are that the low light level of 200～500 luxs is cultivated, and (4) are that the light of 1500～3000 luxs is cultivated, whole culturing process, culture temperature is 25 ± 3 ℃, and (1) stage was cultivated switching in 40 days, and (2) (3) (4) every cultivation renewed bright substratum in 20 days.
3. in accordance with the method for claim 2, it is characterized in that, (1), the minimum medium in (4) stage is the MS substratum, (2), the minimum medium in (3) stage is the HMS substratum, used plant-growth regulator is from auxin substance 2,4-dichlorphenoxyacetic acid 2, select in the combination of 4-D, naphthylacetic acid NAA and cytokinin-like substance 6-benzylamino VITAMIN B4 6-BA, kinetin KT and zeatin ZT any, two or three, in (1) substratum, also added riboflavin VB
2, sucrose and glucose.(1) cultivation stage has added caseinhydrolysate CH or milk-protein LH.The used medium pH value of whole culturing process is 5.5～6.2.
4. in accordance with the method for claim 3, it is characterized in that the composition of HMS substratum is: 1/2MS macroelement, MS trace element, MS molysite, riboflavin VB
21.0mg/L, hydrochloric tiamide (VB1) 1.0mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, inositol 0.1g/L.
5. in accordance with the method for claim 3, the MS that relates to is a minimum medium, and its composition sees attached list 1.
6. in accordance with the method for claim 3, it is characterized in that used hormone is in the substratum in (1) stage: 2.5～6mg/L 2,4-dichlorphenoxyacetic acid 2,4-D, 0.1～1.0 naphthylacetic acid NAA, 0.1～1.0mg/L 6-benzylamino VITAMIN B4 6-BA has also added riboflavin VB
20.5～2.0mg/L and enzymic hydrolysis casein 200～500mg/L, used hormone is in the substratum in (2) stage: 0.1～5mg/L 2,4 dichlorophenoxyacetic acid 2,4-D, 0.1～3mg/L 6-benzylamino VITAMIN B4 6-BA.Used hormone is in the substratum in (3) stage: 0.05～5mg/L2,4-dichlorphenoxyacetic acid 2,4-D, 0.01～2mg/L 6-benzylamino VITAMIN B4 6-BA, in the substratum in (4) stage, contain kinetin KT 0.1～0.3mg/L, 2,4 dichlorophenoxyacetic acid 2,4-D1.0～3.5mg/L, 6-benzylamino VITAMIN B4 6-BA 0.1mg/L.
7. in accordance with the method for claim 3, it is characterized in that the carbon source of adding is sucrose 20～30g/L in the substratum in (1) stage, glucose 15～25g/L.The carbon source of additive is sucrose 30～60g/L in the substratum in (2) (3) (4) stages.
8. in accordance with the method for claim 2, it is characterized in that, be used for the callus succeeding transfer culture and keep the substratum of its growth quality to add in (2) stage: 2,4 dichlorophenoxyacetic acid 2,4-D 1.0～4.0mg/L, 6-benzylamino VITAMIN B4 6-BA 0.1～0.5mg/L.
9. in accordance with the method for claim 2, it is characterized in that this method may further comprise the steps to be inoculated in after (1) explant sterilization contains 2.5～6mg/L 2,4 dichlorophenoxyacetic acid 2,4-D, 0.1～1.0 naphthylacetic acid NAA, 0.1～1.0mg/L 6-benzylamino VITAMIN B4 6-BA, riboflavin VB
20.5 cultivated 40 days in the substratum of～2.0mg/L, enzymic hydrolysis casein 200～500mg/L and sucrose 20～30g/L and 15～25g/L glucose; (2) induce the callus of acquisition to be inoculated in interpolation 0.1～5mg/L 2,4 dichlorophenoxyacetic acid 2,4-D is in the substratum of 0.1～3mg/L6-benzylamino VITAMIN B4 6-BA; (3) callus of subculture is containing 1.0～4.0mg/L 2,4-two phenoxy acetic acids 2, and 4-D cultivates in the substratum of 0.1～0.5mg/L 6-benzylamino VITAMIN B4 6-BA; (4) callus crossed of succeeding transfer culture is inoculated in and contains 0.1～0.3mg/L kinetin KT, 1.0～2.5mg/L 2,4-dichlorphenoxyacetic acid 2 is cultivated induced embryonic callus and somatic embryo in the substratum of 4-D, 0.1mg/L 6-benzylamino VITAMIN B4 6-BA and 30～60g/L sucrose; (5) induce the somatic embryo that obtains to be inoculated in and contain kinetin KT0～0.3mg/L, 2,4-dichlorphenoxyacetic acid 2,4-D1.0～2.0mg/L, the substratum regeneration induction plant of 6-benzylamino VITAMIN B4 6-BA0.1mg/L, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～500 luxs, (5) be the light cultivation of 1500～3000 luxs, whole culturing process, culture temperature are 25 ± 3 ℃, and (1) stage was cultivated 40 days switching callus, in (2) (3) (4) stage, fresh culture was changed in every cultivation in 20 days one time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510087733 CN1746294A (en) | 2005-08-08 | 2005-08-08 | Method for setting up matured embryo regenerating system of zhonghuajielu grass |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510087733 CN1746294A (en) | 2005-08-08 | 2005-08-08 | Method for setting up matured embryo regenerating system of zhonghuajielu grass |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1746294A true CN1746294A (en) | 2006-03-15 |
Family
ID=36166007
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510087733 Pending CN1746294A (en) | 2005-08-08 | 2005-08-08 | Method for setting up matured embryo regenerating system of zhonghuajielu grass |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1746294A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102630565A (en) * | 2012-04-17 | 2012-08-15 | 浙江大学 | Method for inducing differentiation seedling of manilagrass callus |
| CN102907322A (en) * | 2012-10-31 | 2013-02-06 | 北京农业生物技术研究中心 | Method for creating high-frequency regeneration system of zoysia japonica |
| CN106234217A (en) * | 2016-07-28 | 2016-12-21 | 江苏农林职业技术学院 | A kind of method utilizing Result Fuse phyllopodium callus induction |
| 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 |
-
2005
- 2005-08-08 CN CN 200510087733 patent/CN1746294A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102630565A (en) * | 2012-04-17 | 2012-08-15 | 浙江大学 | Method for inducing differentiation seedling of manilagrass callus |
| CN102907322A (en) * | 2012-10-31 | 2013-02-06 | 北京农业生物技术研究中心 | Method for creating high-frequency regeneration system of zoysia japonica |
| CN106234217A (en) * | 2016-07-28 | 2016-12-21 | 江苏农林职业技术学院 | A kind of method utilizing Result Fuse phyllopodium callus induction |
| 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 |
| WO2019153690A1 (en) * | 2018-02-06 | 2019-08-15 | 江苏农林职业技术学院 | High-frequency somatic embryo regeneration growth medium without germplasm genotype restriction and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100334946C (en) | Tissue culturing, rapid propagating and transplanting method of Rhododendron mucronulatum Turcz. | |
| CN102499090B (en) | Method for isolated culture of Haworthia succulent plants | |
| CN102301952B (en) | Method for breeding chamomile | |
| CN101720670B (en) | Rapid breeding method for pinellia tuber tissue culture | |
| CN1746294A (en) | Method for setting up matured embryo regenerating system of zhonghuajielu grass | |
| CN103155862B (en) | Sinocalamus latiflorus flower pesticide inductor embryo the method obtaining regeneration plant | |
| CN104365479B (en) | A kind of tissue culture and rapid propagation method of tail alpine ash DH32-28 kind | |
| CN101663997B (en) | Regeneration and cultivation method of high frequency somatic embryos for overcoming shamrock variety genotypic disorder | |
| CN1596600A (en) | Long tube lycoris fast breeding method | |
| CN104285815B (en) | A kind of tissue culture and rapid propagation method of tail alpine ash DH32-13 kind | |
| CN104094848B (en) | The method of the induction of tung oil tree hypocotyledonery axis callus and highly efficient regeneration plant | |
| CN1600082A (en) | Tissue culture technique for peony | |
| CN112400696B (en) | Tissue culture method of evergreen common selfheal fruit-spike bamboo | |
| CN101861831B (en) | High-frequency somatic embryo regeneration culture medium capable of overcoming clover variety genotype barrier | |
| CN1232167C (en) | Quick reproduction of xiangxuelan | |
| CN104054579B (en) | A kind of method of tung oil tree petiole directly regenerated plant | |
| CN102119664B (en) | Method for culturing muscari botryoides mill with plant tissues | |
| CN101828528B (en) | Tissue culture and quick propagation method suitable for pteridophyte scarecrow | |
| CN1320110C (en) | Method for establishing buffalo grass regeneration system through somatic embryogenesis way | |
| CN1748477A (en) | Method for generating Japan lawn grass plant by mature embryo calls induction and culture medium | |
| CN1883258A (en) | Method for building regenerative system of kostelezkya virginica | |
| CN1860860A (en) | High frequency plant stem regeneration tech. by cell-culture of Xiangcao grass | |
| CN1322117C (en) | Method for establishing perennial ryegrass inheritance and transformation receptor system through embryoid way | |
| CN104686333B (en) | A kind of ripple leaf Stranvaesia Davidiana Dcne tissue cultivation rapid breeding method | |
| CN112400695B (en) | Culture medium for culturing evergreen common summer pink |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |