CN1322117C - Method for establishing perennial ryegrass inheritance and transformation receptor system through embryoid way - Google Patents
Method for establishing perennial ryegrass inheritance and transformation receptor system through embryoid way Download PDFInfo
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- CN1322117C CN1322117C CNB2005100900773A CN200510090077A CN1322117C CN 1322117 C CN1322117 C CN 1322117C CN B2005100900773 A CNB2005100900773 A CN B2005100900773A CN 200510090077 A CN200510090077 A CN 200510090077A CN 1322117 C CN1322117 C CN 1322117C
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Abstract
The present invention discloses a method for establishing perennial ryegrass genetic conversion receptor system through embryoid generation approach. The main steps of the present invention comprise that mature seeds of perennial ryegrass are used as materials and are induced in an inducing culture medium to obtain calluses, and embryoids and regenerated plantlets are induced in an embryonic callus inducing culture medium and a regenerating culture medium in a high-frequency mode after successive transfer culture. The inducing rate of the calluses can be effectively improved by adding 2, 4-D, 6-BA, NAA and casein hydrolysate of a certain mixture ratio in an inducing callus culture medium. In the process of subculture to regenerating culture, by changing the concentration mixture ratio of plant growth regulators, the inducing rate of the embryoids reaches above 70 percent, and the plantlet regenerating rate reaches above 85 percent.
Description
Technical field:
The present invention relates to a kind ofly set up the method for perennial ryegrass inheritance and transformation receptor system, belong to biotechnology and modern agriculture biological technical field by embryoid way.
Background technology:
English ryegrass belongs to Gramineae lolium per nnial herb.Originate in southern Europe, Northern Europe and southwest, Asia.It is the extensively important graminous pasture of cultivation of the many areas of temperate zone, world rainfall amount.Because of its fibrous root system prosperity, characteristic such as the blade quality is thin, soft, the leaf look green, and planting speed is fast, and tillering ability and wear resistance are strong, the pioneer plant in the Chang Zuowei soil conservation mixed seeding kind is a kind of important good turfgrass.
But the English ryegrass root system buries not dark, and winter resistance is poor, and regional overwintering survival rate is lower to the north of Beijing reaches, its thermotolerance and drought tolerance are also relatively poor, when temperature more than 35 ℃ the time, growth retardation, it is also undesirable to grow in the arid and semi-arid area, thereby has seriously limited the widespread use of this kind.Many investigators have begun it is carried out the research of character improvement at English ryegrass as the shortcoming that turfgrass exists.And English ryegrass is a cross-pollinatd plant, and it is big to utilize traditional breeding method to carry out the breed improvement difficulty, and the cycle is long.Utilize modern biotechnology, particularly transgenic technology is carried out genetic improvement to plant and is had characteristics such as efficient, quick, successful Application in many plant breeding.For utilizing the modern biotechnology breeding, English ryegrass provides important reference.Isolated culture and the transgenic technology of English ryegrass more and more come into one's own.
The genetic transformation system of setting up the English ryegrass efficient stable is to utilize transgenic technology that it is carried out the important prerequisite of genetic improvement, but English ryegrass tissue culture and the equal imperfection of plant regeneration system reported at present, regeneration rate is on the low side, can not satisfy genetically modified needs.Dale (1977) cultivates by the tip of a root and obtains the English ryegrass regeneration plant.Kran (1980,1981); White (1987,1988) utilizes mature embryo to obtain regeneration plant for explant carries out tissue culture.Creemers-Molenaar et al, (1988) Wang et al, (1993), Dalton (1988) has studied the influence that different training methods are set up the English ryegrass regeneration system, and has tentatively set up solid culture, fluid suspension culture and the protoplastis suspension culture regeneration system of English ryegrass.Creemers-Molenaar et al, (1988) are explant with prematurity children fringe, have studied exogenous hormone and culture environment to its plant regeneration influence, and have obtained regeneration plant.1994, Chen Wenpin was an explant with perennial young fringe, directly induces regenerated adventitious bud from young fringe.And form complete plant.
Above-mentioned research report all is the regeneration plants that obtain with organ germination approach, and regeneration rate is all lower.Embryoid is meant that the cell mass of dedifferentiation is that callus is under suitable culture condition, the part cell is further grown, occur in the cell polarising, and form the somatocyte that has similar characteristics with the seed that forms through sexual propagation through several etap such as torpedo embryo, globular embryo, heart-shape embryo.Condition is suitable, and therefore embryoid that can be to utilize transgenic technology that English ryegrass is carried out the optimal acceptor material of genetic transformation by the plastidogenetic embryoid of inductor as the seed rudiment.Mature seed is drawn materials and is not subject to seasonal restrictions, and is easy to preserve.Therefore, focusing mostly on its mature seed at present is explant, and the method and the technology of English ryegrass regeneration system further improved in research.Regrettably, up to now, it is still undesirable with the mature embryo to be that explant is set up the stable regeneration system of high frequency.
Summary of the invention:
The advantage of setting up the genetic transformation acceptor at the deficiency and the embryoid way of above-mentioned technology, the present invention is an explant with the English ryegrass mature seed, the high-frequency induction callus, the callus of acquisition changes the plant regeneration substratum over to and induces the acquisition high-frequency regeneration plant after succeeding transfer culture and embryoid induction cultivation.Thereby set up the genetic transformation system of English ryegrass efficient stable, established technology platform for further utilizing genetically engineered that it is carried out genetic improvement.
Explant described in the present invention is meant under the aseptic condition, and isolated culture is used to reach the starting materials of culturing purposes on artificial medium, and the explant that uses among the present invention is the English ryegrass mature seed.
Genetic transformation system described in the present invention is meant to making the carrier that contains goal gene can be fused to the host plant body smoothly and can making the plant materials after the conversion form the technical system that complete of exploring can make the stable regeneration induction plant of its stripped material high frequency under suitable culture condition.It is the regeneration system that explant is set up that acceptor among the present invention is with the English ryegrass.
Cultural method of the present invention comprises four cultivation stages: (1) explant is cultivated evoked callus (2) callus succeeding transfer culture (3) callus induction embryoid (4) embryoid regeneration plant, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～500 luxs, light application time is 12h/d, (4) be the light cultivation of 1500～3000 luxs, light application time is 12h/d, whole culturing process, culture temperature is 20 ℃～30 ℃, and every cultivation renewed bright substratum in 20～30 days.
The present invention induces callus by the cultivation of above-mentioned four-stage from the English ryegrass mature seed, and further induces embryoid from callus, has obtained regeneration plant by embryoid regeneration approach.
To be N to the minimum medium of four-stage among the present invention
6Substratum (subordinate list).The used hormone of four-stage is 2,4-dichlorphenoxyacetic acid 2, select in the combination of 4-D, naphthylacetic acid NAA, 6-benzylamino VITAMIN B4 6-BA and kinetin KT any, two or three, in (1) stage, also add caseinhydrolysate CH in the substratum, the N6 substratum that the minimum medium of (4) stage employing reduces by half for the macroelement consumption, sucrose is 30g/L.
Method of the present invention is selected and excellently is: used exogenous hormone and concentration thereof are in the substratum in (1) stage: 2,4-dichlorphenoxyacetic acid 2,4-D 4～15mg/L, 6-benzylamino VITAMIN B4 6-BA 0.1～2.0mg/L, naphthylacetic acid NAA 0.1～3.0mg/L has also added caseinhydrolysate CH 200～1000mg/L, used exogenous hormone and concentration thereof are in the substratum in (2) stage: 2,4-dichlorphenoxyacetic acid 2,4-D2.0～10.0mg/L, kinetin KT 0.5～2mg/L.Used exogenous hormone and concentration thereof are in the substratum in (3) stage: 2,4-dichlorphenoxyacetic acid 2,4-D 0.1～3.0mg/L, kinetin KT 0.1～2.0mg/L is at the substratum interpolation 6-benzylamino VITAMIN B4 6-BA 0.5～2mg/L in (4) stage and the N of kinetin KT 0.1～1mg/L
6Substratum, wherein macroelement reduces by half, and the sucrose consumption reduces to 30g/L.
The particularly preferred method of the present invention may further comprise the steps: (1) mature seed is after sterilization, be inoculated in and add 2,4-dichlorphenoxyacetic acid 2, the substratum of 4-D 1～15mg/L, 6-benzylamino VITAMIN B4 6-BA 0.1～2.0mg/L, naphthylacetic acid NAA 0.1～3.0 mg/L and caseinhydrolysate CH 200～1000mg/L; (2) induce the callus that obtains to be inoculated in and add 2,4 dichlorophenoxyacetic acid 2,4-D 2.0～10.0mg/L, the substratum of kinetin KT 0.5～2mg/L carries out succeeding transfer culture twice, subculture cycle 20～30 days; (3) select embryo callus to be inoculated in and add 2,4-dichlorphenoxyacetic acid 2,4-D 0.1～3.0mg/L, the substratum inducing embryoid body of kinetin KT 0.1～2.0mg/L, culture condition is that the low light level is cultivated, and intensity of illumination is 200～500 luxs, and (5) induce the embryoid that obtains to be inoculated in the substratum that adds 6-benzylamino VITAMIN B4 6-BA 0.5～2mg/L and kinetin KT 0.1～1mg/L, see the light cultivation, intensity of illumination is 1500～3000 luxs.
The present invention soaks English ryegrass (Lolium perenne L.) mature seed 0.5 hour with tap water; Washing composition and water soaked 1 hour with 1: 50 mixed, and the mobile tap water washed after 12 hours, 70% alcohol-pickled 30s, aseptic water washing 5 times; Soak 15min with 0.1% mercuric chloride again, use aseptic water washing then 5～10 times.Be seeded in the substratum of inducing for (1) stage after with scalper under the aseptic condition the seed crosscut, dark culturing after 30 days the incision at seed form the transparent callus of white.Under the substratum in above-mentioned (1) stage and culture condition, cultivated 30 days, callus induction rate reaches more than 85%, change the substratum in (2) stage over to, per 25 days subcultures once, the increment volume increases 2.0-3.0 doubly approximately, the quality of callus significantly improves, and it is tight structure to occur, and faint yellow or milky callus and quality are crisp, faint yellow or oyster white, surface have the callus of papilla.Choose wherein fine and close white or the faint yellow callus of structure, change the culture medium culturing 50 days in (3) stage over to, embryoid (somatic embryo) inductivity reaches more than 75%, embryoid is changed over to the substratum in (4) stage, be to cultivate 30 days under the illumination condition of 1500～3000 luxs in light intensity, shoot regeneration frequency reaches more than 86%.Regeneration plant opened in temperature is 25 ℃, the glasshouse of humidity about 75% seal film hardening 3～4d.Take out regeneration plant afterwards, clean root adheres to substratum, is transplanted to matrix (perlite: the peat composed of rotten mosses: fertilizer=4: 2: 1), preserve moisture with the plastic film covering.Behind the 10d, open plastic film, surviving rate reaches 95%.
The present invention induces callus from the English ryegrass mature seed, and kept the good quality of callus, the embryoid that is induced (being somatic embryo) can produce the regeneration plant of high quantity, stable, homogeneous, so this system can be used for genetically engineered or cell engineering breeding.
Description of drawings:
The callus that Fig. 1 induces
Inducing of Fig. 2 embryo callus
Fig. 3 differentiation of calli
Fig. 4 regeneration plant is induced
Fig. 5 transplant survival seedling
Embodiment:
Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.
Embodiment 1:
English ryegrass (Lolium perenne L.) mature seed soaked 0.5 hour with tap water; Washing composition and water soaked 1 hour with 1: 50 mixed, and the mobile tap water washed after 12 hours, 70% alcohol-pickled 30s, aseptic water washing 5 times; Soak 15min with 0.1% mercuric chloride again, with aseptic water washing 5～10 times.Be seeded to the substratum of evoked callus under the aseptic condition after with the seed crosscut with scalper, add 5.0mg/L 2,4-dichlorphenoxyacetic acid 2,4-D, 6-benzylamino VITAMIN B4 6-BA 0.5mg/L, the substratum of naphthylacetic acid NAA0.5 mg/L and caseinhydrolysate CH 500mg/L, dark culturing 30 days.Wherein, the preparation of substratum is that hormone is directly made an addition to N
6In other compositions of substratum, autoclaving makes then.
Used plant and instrument is equipment such as the known ultra-clean working machine of plant tissue culture, automatic high pressure Autoclave.Cultivate in the whole process at whole regeneration system, the medium pH value is 5.8, and temperature is 25 ± 1 ℃.
Add 2,4 dichlorophenoxyacetic acid 2,4-D 5.0mg/L, the N of kinetin KT 0.5mg/L with inducing the callus that obtains to be transferred to
6Substratum, dark culturing 50 days, wherein per 25 days replacing fresh cultures.
White that choice structure is fine and close or flaxen callus are inoculated in and add 2,4 dichlorophenoxyacetic acid 2, and 4-D 1.0mg/L, the substratum inducing embryoid body of kinetin KT 1.0mg/L, culture condition are that the low light level is cultivated, and intensity of illumination is 200～500 luxs
Inductive embryoid (somatic embryo) is changed over to the N that adds 6-benzylamino VITAMIN B4 6-BA 0.5mg/L and kinetin KT 0.5mg/L
6On the substratum, be inducing culture under the illumination condition of 1500～3000 luxs in light intensity, make it grow up to whole plant.Light application time is at 12h/d, and embryoid grows up to the complete plant of root, stem and leaf through 30 days cultivation, and regeneration plant is opened in temperature is 25 ℃, the glasshouse of humidity about 75% and sealed film hardening 3～4d.Take out regeneration plant afterwards, clean the residual substratum of root, be transplanted to matrix (perlite: the peat composed of rotten mosses: fertilizer=4: 2: 1), preserve moisture with the plastic film covering.Behind the 10d, open plastic film, can carry out the land for growing field crops transfer.
The regeneration system that aforesaid method is set up, callus induction rate reaches 85.47%, and the embryoid induction rate reaches 76.83%, and regeneration rate reaches 86.2%, and transplanting survival rate reaches 100%.
Embodiment 2:
The cultivation program is all undertaken by embodiment 1, institute's difference is to add 2 in the callus inducing medium, 4-dichlorphenoxyacetic acid 2,4-D is 8.0mg/L, 6-benzylamino VITAMIN B4 6-BA is 0.6mg/L, naphthylacetic acid NAA is 0.6mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the callus subculture medium, 4-D is 8.0mg/L, kinetin KT is 0.6mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the embryoid induction substratum, 4-D is 0.7mg/L, kinetin KT is 1.0mg/L, and adding 6-benzylamino VITAMIN B4 6-BA in embryoid regeneration plant substratum is 0.4mg/L and kinetin KT 0.4mg/L.
The regeneration system that aforesaid method is set up, callus induction rate reaches 85.25%, and the embryoid induction rate reaches 75.04%, and regeneration rate reaches 85.72%, and transplanting survival rate reaches 97.5%.
Embodiment 3:
The cultivation program is all undertaken by embodiment 1, institute's difference is to add 2 in the callus inducing medium, 4-dichlorphenoxyacetic acid 2,4-D is 6.0mg/L, 6-benzylamino VITAMIN B4 6-BA is 0.8mg/L, naphthylacetic acid NAA is 0.3mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the callus subculture medium, 4-D is 6.0mg/L, kinetin KT is 0.8mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the embryoid induction substratum, 4-D is 0.5mg/L, kinetin KT is 1.5mg/L, and adding 6-benzylamino VITAMIN B4 6-BA in embryoid regeneration plant substratum is 0.8mg/L and kinetin KT 0.4mg/L.
The regeneration system that aforesaid method is set up, callus induction rate reaches 86.06%, and the embryoid induction rate reaches 75.33%, and regeneration rate reaches 85.2%, and transplanting survival rate reaches 98%.
Embodiment 4:
The cultivation program is all undertaken by embodiment 1, institute's difference is to add 2 in the callus inducing medium, 4-dichlorphenoxyacetic acid 2,4-D is 10.0mg/L, 6-benzylamino VITAMIN B4 6-BA is 1.0mg/L, naphthylacetic acid NAA is 0.4mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the callus subculture medium, 4-D is 10.0mg/L, kinetin KT is 0.7mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the embryoid induction substratum, 4-D is 0.8mg/L, kinetin KT is 1.0mg/L, and adding 6-benzylamino VITAMIN B4 6-BA in embryoid regeneration plant substratum is 0.6mg/L and kinetin KT 0.6mg/L.
The regeneration system that aforesaid method is set up, callus induction rate reaches 86.06%, and the embryoid induction rate reaches 77.23%, and regeneration rate reaches 87.12%, and transplanting survival rate reaches 96%.
Embodiment 5:
The cultivation program is all undertaken by embodiment 1, institute's difference is to add 2 in the callus inducing medium, 4-dichlorphenoxyacetic acid 2,4-D is 9.0mg/L, 6-benzylamino VITAMIN B4 6-BA is 0.8mg/L, naphthylacetic acid NAA is 0.4mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the callus subculture medium, 4-D is 5.0mg/L, kinetin KT is 0.8mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the embryoid induction substratum, 4-D is 0.5mg/L, kinetin KT is 1.5mg/L, and adding 6-benzylamino VITAMIN B4 6-BA in embryoid regeneration plant substratum is 0.6mg/L and kinetin KT 0.6mg/L.
The regeneration system that aforesaid method is set up, callus induction rate reaches 86.06%, and the embryoid induction rate reaches 78.67%, and regeneration rate reaches 88.32%, and transplanting survival rate reaches 98%.
Embodiment 6:
The cultivation program is all undertaken by embodiment 1, institute's difference is to add 2 in the callus inducing medium, 4-dichlorphenoxyacetic acid 2,4-D is 7.0mg/L, 6-benzylamino VITAMIN B4 6-BA is 0.4mg/L, naphthylacetic acid NAA is 0.6mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the callus subculture medium, 4-D is 4.0mg/L, kinetin KT is 0.8mg/L, add 2,4 dichlorophenoxyacetic acid 2 in the embryoid induction substratum, 4-D is 1.2mg/L, kinetin KT is 1.0mg/L, and adding 6-benzylamino VITAMIN B4 6-BA in embryoid regeneration plant substratum is 0.4mg/L and kinetin KT 0.5mg/L.
The regeneration system that aforesaid method is set up, callus induction rate reaches 86.76%, and the embryoid induction rate reaches 77.12%, and regeneration rate reaches 85.06%, and transplanting survival rate reaches 100%.
Subordinate list:
N
6Minimal medium (Zhu Zhiqing, 1974)
| Component | Composition | Content (mg/L) |
| A great number of elements | Ammonium sulfate | 460 |
| Potassium nitrate | 2830 | |
| Potassium dihydrogen phosphate | 400 | |
| Bitter salt | 185 | |
| CALCIUM CHLORIDE DIHYDRATE | 166 | |
| Trace element | Four hydrated manganese sulfates | 10 |
| Zinc vitriol | 1.5 | |
| Boric acid | 1.6 | |
| KI | 0.8 | |
| Organic element | Glycine | 2.0 |
| Nicotinic acid | 0.5 | |
| Thiamine hydrochloride | 1 | |
| Puridoxine hydrochloride | 0.5 | |
| Molysite | Green vitriol | 27.8 |
| Disodium ethylene diamine tetraacetate | 37.3 | |
| Other | Sucrose | 50000 |
Claims (3)
1. set up the method for perennial ryegrass inheritance and transformation receptor system by embryoid way, it is characterized in that with the English ryegrass mature seed being that explant is cultivated, described method comprises four cultivation stages: (1) explant is cultivated evoked callus, (2) callus succeeding transfer culture, (3) callus induction embryoid, (4) embryoid regeneration plant, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～500 luxs, light application time is 12h/d, (4) be the light cultivation of 1500～3000 luxs, light application time is 12h/d, whole culturing process, culture temperature are 20 ℃～30 ℃, and every cultivation renewed bright substratum in 20～30 days.
2. in accordance with the method for claim 1, it is characterized in that in the substratum in (1) stage used exogenous hormone and concentration thereof are: 2,4-dichlorphenoxyacetic acid 2,4-D 1～15mg/L, 6-benzylamino VITAMIN B4 6-BA0.1～2.0mg/L and naphthylacetic acid NAA0.1～3.0mg/L, also added caseinhydrolysate CH200～1000mg/L, used exogenous hormone and concentration thereof are in the substratum in (2) stage: 2,4-dichlorphenoxyacetic acid 2,4-D2.0～10.0mg/L and kinetin KT0.5～2mg/L, used exogenous hormone and concentration thereof are in the substratum in (3) stage: 2,4-dichlorphenoxyacetic acid 2,4-D 0.1～3.0mg/L, kinetin KT0.1～2.0mg/L is at the substratum interpolation 6-benzylamino VITAMIN B4 6-BA 0.5～2mg/L in (4) stage and the N of kinetin KT0.1～1mg/L
6Substratum, wherein macroelement reduces by half, and the sucrose consumption reduces to 30g/L.
3. in accordance with the method for claim 1, it is characterized in that this method may further comprise the steps (1) English ryegrass mature seed after sterilization, be inoculated in and add 2,4-dichlorphenoxyacetic acid 2,4-D 1～15mg/L, 6-benzylamino VITAMIN B4 6-BA 0.1～2.0mg/L, the substratum of naphthylacetic acid NAA0.1～3.0mg/L and caseinhydrolysate CH200～1000mg/L; (2) induce the callus that obtains to be inoculated in and add 2,4 dichlorophenoxyacetic acid 2,4-D2.0～10.0mg/L, the substratum of kinetin KT0.5～2mg/L carries out succeeding transfer culture twice, subculture cycle 20～30 days; (3) select embryo callus to be inoculated in and add 2,4-dichlorphenoxyacetic acid 2,4-D0.1～3.0mg/L, the substratum inducing embryoid body of kinetin KT0.1～2.0mg/L, culture condition is that the low light level is cultivated, intensity of illumination is 200～500 luxs, (4) induce the embryoid that obtains to be inoculated in the substratum of interpolation 6-benzylamino VITAMIN B4 6-BA0.5～2 mg/L and kinetin KT0.1～1mg/L, see the light cultivation, intensity of illumination is 1500～3000 luxs, is N at used minimum medium of (1)～(3) stage
6Substratum.
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| CN1597933A (en) * | 2004-08-13 | 2005-03-23 | 山东大学 | Process for construvting transgene teceptor system of rye grass and its application |
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Non-Patent Citations (5)
| Title |
|---|
| 多年生黑麦草愈伤组织诱导和植株再生 冯霞等，草业科学，第21卷第10期 2004 * |
| 多年生黑麦草愈伤组织诱导和植株再生 冯霞等，草业科学，第21卷第10期 2004;多年生黑麦草转基因育种研究进展 陈季琴等，草业学报，第3卷第5期 2004;黑麦草幼穗离体培养及植株再生 杨爱芳等，草业科学，第12卷第5期 2004;种坯处理、不同激素浓度在暗培养条件下对多年生黑麦草愈伤组织诱导的影响 徐定炎等，浙江林业科技，第23卷第2期 2003 * |
| 多年生黑麦草转基因育种研究进展 陈季琴等，草业学报，第3卷第5期 2004 * |
| 种坯处理、不同激素浓度在暗培养条件下对多年生黑麦草愈伤组织诱导的影响 徐定炎等，浙江林业科技，第23卷第2期 2003 * |
| 黑麦草幼穗离体培养及植株再生 杨爱芳等，草业科学，第12卷第5期 2004 * |
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