CN104871974A - Method and special culture medium for inducing seedless grape young embryos to generate somatic embryos - Google Patents
Method and special culture medium for inducing seedless grape young embryos to generate somatic embryos Download PDFInfo
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Abstract
The invention discloses a special culture medium for inducing seedless grape young embryos to generate somatic embryos. The culture medium is characterized in that in the process of ovule cultivation and somatic embryo generation induction, the same basic formula is adopted and different supplementary elements are added in different cultivation stages to achieve the expected effect. The invention further discloses a method for inducing the seedless grape young embryos to generate the somatic embryos. The method comprises the following steps: 6 weeks after blooming of a grape tree, collecting young fruits in the field, disinfecting, sequentially conducting ovule endodermal cultivation, inducing cultivation on embryonic callus and differentiation cultivation on the somatic embryos to obtain a somatic embryo in-vitro regenerated plant, and then conducting hardening seedling and transplanting to obtain a robust grape plant. The somatic embryos subjected to induced differentiation is more in number, ordered in development, and robust in growth after germinating to be a seedling, and has high possibility of surviving when transplanted.
Description
Technical field
The invention belongs to technical field of plant culture, be specifically related to a kind of special culture media of inducing currant rataria shedder blast, the present invention also provides a kind of method of inducing currant rataria shedder blast.
Background technology
Grape is one of the most ancient in the world fruit tree species, the cultivation history of existing more than 5000 year so far, be loved by the people in China because it has high, the nutritious and plurality of health care functions of strong adaptability, output, grape production is the important component part of agricultural production always, in recent years, China's grape industry obtains fast development, also more and more higher to the requirement of Table Grape Varieties, especially currant, for eating, processing or make the dry favor being all subject to domestic and international consumer raw.Compared with other plant, the investigation and application progress of grape transgenic technology is relatively slow.This is because, the development of transgenic technology is strictly subject to the restriction of regenerating system.It is generally acknowledged, the regenerating system of grape mainly contains organ generation and two kinds of approach occur embryoid, because the latter has unicellular origin, not easily produces the advantages such as chimera, has larger value in transgenosis.Existing research shows, many positions of grape are as flower pesticide, ovary, column cap etc., and tendril etc. all have the ability of shedder blast, but regeneration efficiency is general lower.
Up to now, produce the research of embryoid using prematurity flower pesticide as explant induction and application in transgenic technology the most common.Generally include following step: (I) gathers and spend front immature grape inflorescence, after the sterilization of tap water, 70% alcohol disinfecting and 0.1% mercuric chloride, aseptic water washing is clean; (II) strips flower pesticide and is seeded on callus inducing medium, and light culture is until form callus; In (III) 3-4 month, embryo callus is forwarded on embryo differentiate medium, light culture 3-4 week; The somatic embryo that induction obtains is seeded on embryo germination, root media by (IV) successively, illumination cultivation 2-3 month; Good regeneration plant of taking root is carried out hardening and transplanting by (V).But the shortcoming doing the generation of explant induction body embryo with flower pesticide is (1) length consuming time, is inoculated into formation embryo callus from flower pesticide, generally need 3-4 month; (2) induced efficiency is low, and frequency of embryonic callus induction is usually below 60%; (3) there is strict genotype-independent, only seldom counting in kind and can occur by successful somatic embryos at present, and the flower pesticide of most of kind shedder blast after induction is very difficult, this is the bottleneck limiting the development of grape transgenic technology at present.
Zygotic embryo is grown after being combined with egg cell by grape sperm and is formed, and systematic growth has similar growth course to somatic embryo.The research of forefathers is thought, explant is done with the zygotic embryo of grape, also shedder blast can be induced, the research of this respect has been reported having on core grape, but on most currant, due to midway abortion in the zygotic embryo growth course of after fertilization, ovule can not form normal seed and only leave the seed vestige varied in size.This feature of currant embryo development makes its zygotic embryo do explant to draw materials difficulty, therefore with currant zygotic embryo in explant induction embryo callus then differentiation-inducing somatic embryo, still lack so far and comparatively systematically study.
Summary of the invention
The object of this invention is to provide a kind of special culture media of inducing currant rataria shedder blast, solve the currant induction shedder blast difficulty and the inefficient problem of regeneration plant that exist in prior art.
Another object of the present invention is to provide the method for induction currant rataria shedder blast.
First technical scheme of the present invention is, a kind of special culture media of inducing currant rataria shedder blast, its component and content as follows: nitrate of lime 250.0mg/L, potassium nitrate 600.0mg/L, potassium chloride 75.0mg/L, ammonium nitrate 300.0mg/L, magnesium sulfate 1200.0mg/L, potassium dihydrogen phosphate 300.0mg/L, manganese sulphate 3.0mg/L, potassium iodide 0.8mg/L, boric acid 0.5mg/L, zinc sulphate 0.5mg/L, sodium selenite 0.25mg/L, cobalt chloride 0.025mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.025mg/L, ironic citrate 10.0mg/L, thiamine hydrochloride 0.25mg/L, pyridoxine hydrochloride 0.25mg/L, D-VB5 calcium 0.25mg/L, nicotinic acid 0.25mg/L, asparagine 300mg/L, glycine 5.0mg/L, arginase 12 .0mg/L, inositol 50.0mg/L, caseinhydrolysate 500.0mg/L, Cys 121.16mg/L, sucrose 30000mg/L, agar 6000mg/L, all the other are distilled water.
Second technical scheme of the present invention is, a kind of method of inducing currant rataria shedder blast, specifically implements according to following steps:
Step 1, grape Post flowering 6 weeks fields gather young fruit, tap water 10min; With after alcohol-pickled 1 minute of 70% on superclean bench, then use the HgCl of 0.1%
2soak 8 minutes, rinsed with sterile water 3 times;
Fruit grain after step 2, sterilization is placed in sterilized culture dish, takes out ovule, be seeded on zygotic embryo Development culture base and carry out embryo culture in ovule under aseptic condition; Zygotic embryo Development culture base is the TL medium of solid-liquid double-phase, wherein additional saccharose 6.0g/L, active carbon 1.5g/L;
After step 3, ovule cultivate 6 weeks on zygotic embryo Development culture base, the rataria of growth is taken out under aseptic condition, be seeded on the embryonic callus induction medium of solid, the composition of embryonic callus induction medium is TL+0.5mg/L6-BA+1.0mg/L 2,4-D, wherein additional saccharose 30g/L, agar 6.0g/L;
After step 4, rataria cultivate 4 weeks on embryonic callus induction medium, the embryo callus of the yellow of acquisition, graininess, growth consolidation is seeded on the embryo differentiate medium of solid, the composition of embryo differentiate medium is TL+0.5mg/L 6-BA+2.0mg/L NAA, wherein, additional saccharose 30g/L, agar 6.0g/L;
After step 5, embryo callus cultivate 3-4 week on embryo differentiate medium, be seeded in by the somatic embryo of acquisition on TL+0.2mg/L IBA medium, wherein additional saccharose 30g/L, agar 6.0g/L, make its Germination And Seedling; Every 4 weeks subcultures once, obtain the somatic embryo Regeneration in Vitro plant with normal root, stem and true leaf in February;
Step 6, in vitro root system development good stand to be cleaned on it after greenhouse hardening the agar of attachment with clear water, be transplanted into and be equipped with in the nutritive cube of Nutrition Soil, the seedling after surviving carries out Routine Management, develops into healthy and strong grapevine seedling.
In technique scheme, component and the content of TL medium are as follows: nitrate of lime 250.0mg/L, potassium nitrate 600.0mg/L, potassium chloride 75.0mg/L, ammonium nitrate 300.0mg/L, magnesium sulfate 1200.0mg/L, potassium dihydrogen phosphate 300.0mg/L, manganese sulphate 3.0mg/L, potassium iodide 0.8mg/L, boric acid 0.5mg/L, zinc sulphate 0.5mg/L, sodium selenite 0.25mg/L, cobalt chloride 0.025mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.025mg/L, ironic citrate 10.0mg/L, thiamine hydrochloride 0.25mg/L, pyridoxine hydrochloride 0.25mg/L, D-VB5 calcium 0.25mg/L, nicotinic acid 0.25mg/L, asparagine 300mg/L, glycine 5.0mg/L, arginase 12 .0mg/L, inositol 50.0mg/L, caseinhydrolysate 500.0mg/L, Cys 121.16mg/L, sucrose 30000mg/L, agar 6000mg/L, all the other are distilled water.
In step 3, ovule condition of culture on zygotic embryo Development culture base is: under 25 DEG C of low light conditions, intensity of illumination 800Lux, light application time 16h/d.
In step 4, rataria condition of culture on embryonic callus induction medium is: 25 DEG C of dark conditions.
In step 5, embryo callus condition of culture on embryo differentiate medium is: 25 DEG C of dark conditions.
Somatic embryo condition of culture on TL+0.2mg/L IBA medium is: cultivate under 25 DEG C of illumination conditions, intensity of illumination 2000Lux, light application time 16h/d.
The invention has the beneficial effects as follows: the active factors of growing containing multiple promotion cells,primordial in the culture medium raw material that the present invention uses, nutrition arrangement is reasonable, effectively can promote that currant zygotic embryo forms rapidly embryo callus clade blast then in vitro, produce embryo callus from inoculation zygotic embryo to induction and only need 20-30 days time, embryo callus forms ratio can reach more than 80%, apparently higher than with normally used take flower pesticide as the abductive approach of explant; Application the present invention, we are obtained successfully by test on multiple Seedless Grape Species.And differentiation-inducing somatic embryo quantity is many, grows neat, robust growth after Germination And Seedling, transplant and be easy to survive.The present invention is mainly used in occurring with the currant zygotic embryo embryo callus that is explant and somatic embryo inducement, also can be used for having the somatic embryo inducement of core grape rataria and mature embryo to occur, also the long-term preservation and the Germination And Seedling that can be used for vine plant somatic embryo are cultivated, and the embryo callus that induction obtains, archiblast group and somatic embryo can be used for the research of the aspects such as genetic transformation, cell engineering and artificial seed structure.The feature of basal culture medium can adopt same basic recipe in Ovule development and inductor embryo generating process, and adding different supplementary element at different cultivation stage can produce a desired effect.
Accompanying drawing explanation
Fig. 1 is the rataria generation shedder blast of induction currant and the process of plant regeneration cultivation; Wherein, A, Ovule development; B, the rataria of growing in ovule; C, the rataria taken out in ovule; D, the embryo callus that rataria obtains after induction; E, a large amount of somatic embryos that embryo callus surface occurs; F, the somatic embryo before sprouting; G, the somatic embryo after sprouting; H, the plant that somatic embryo regeneration obtains; I, the somatic embryo regeneration plant survived after transplanting.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
The invention provides a kind of special culture media of inducing currant rataria shedder blast, called after TL medium, its component and content as follows: nitrate of lime 250.0mg/L, potassium nitrate 600.0mg/L, potassium chloride 75.0mg/L, ammonium nitrate 300.0mg/L, magnesium sulfate 1200.0mg/L, potassium dihydrogen phosphate 300.0mg/L, manganese sulphate 3.0mg/L, potassium iodide 0.8mg/L, boric acid 0.5mg/L, zinc sulphate 0.5mg/L, sodium selenite 0.25mg/L, cobalt chloride 0.025mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.025mg/L, ironic citrate 10.0mg/L, thiamine hydrochloride 0.25mg/L, pyridoxine hydrochloride 0.25mg/L, D-VB5 calcium 0.25mg/L, nicotinic acid 0.25mg/L, asparagine 300mg/L, glycine 5.0mg/L, arginase 12 .0mg/L, inositol 50.0mg/L, caseinhydrolysate 500.0mg/L, Cys 121.16mg/L, sucrose 30000mg/L, agar 6000mg/L, all the other are distilled water.
The TL culture medium prescription component that the present invention proposes is reasonable, effectively can promote the generation of currant somatic embryo, differentiation and growth, and its culture efficiency is obviously better than grapes tissue and cultivates normally used MS and the NN medium in field, and comparison result is in table 1.
The Contrast on effect of table 1 different culture media induction " seedless white " rataria generation embryo callus and somatic embryo
Induction rataria generation embryo callus stage different culture media adds hormone and is 0.5mg/L6-BA+1.0mg/L2,4-D; Somatic embryos stage of development different culture media adds hormone and is 0.5mg/L 6-BA+2.0mg/L NAA.
As can be seen from Table 1, the frequency of embryonic callus induction of medium of the present invention and somatic embryo generation rate are all higher than MS medium and NN medium.
The present invention also provides a kind of method of inducing currant rataria shedder blast, and incubation as shown in Figure 1, is specifically implemented according to following steps:
Step 1, grape Post flowering 6 weeks fields gather young fruit, tap water 10min; With after alcohol-pickled 1 minute of 70% on superclean bench, then use the HgCl of 0.1%
2soak 8 minutes, rinsed with sterile water 3 times;
Fruit grain after step 2, sterilization is placed in sterilized culture dish, takes out ovule, be seeded on zygotic embryo Development culture base and carry out embryo culture in ovule under aseptic condition; Zygotic embryo Development culture base is the TL medium of solid-liquid double-phase, wherein additional saccharose 6.0g/L, and active carbon 1.5g/L, TL medium is the special culture media of above-mentioned induction currant rataria shedder blast;
Step 3, ovule cultivate 6 weeks afterwards (under 25 DEG C of low light conditions on zygotic embryo Development culture base, intensity of illumination 800Lux, light application time 16h/d), the rataria of growth is taken out under aseptic condition, be seeded on the embryonic callus induction medium of solid, the composition of embryonic callus induction medium is TL+0.5mg/L6-BA+1.0mg/L 2,4-D, wherein additional saccharose 30g/L, agar 6.0g/L;
Step 4, rataria are cultivated after 4 weeks (under 25 DEG C of dark conditions) on embryonic callus induction medium, the embryo callus of the yellow of acquisition, graininess, growth consolidation is seeded on the embryo differentiate medium of solid, the composition of embryo differentiate medium is TL+0.5mg/L 6-BA+2.0mg/L NAA, wherein, additional saccharose 30g/L, agar 6.0g/L;
After step 5, embryo callus cultivate 3-4 week on embryo differentiate medium (under 25 DEG C of dark conditions), the somatic embryo of acquisition is seeded on TL+0.2mg/L IBA medium, wherein additional saccharose 30g/L, agar 6.0g/L, its Germination And Seedling is made (to cultivate under 25 DEG C of illumination conditions, intensity of illumination 2000Lux, light application time 16h/d); Every 4 weeks subcultures once, can obtain the somatic embryo Regeneration in Vitro plant with normal root, stem and true leaf in February;
Step 6, in vitro root system development good stand to be cleaned on it after greenhouse hardening the agar of attachment with clear water, be transplanted into and be equipped with in the nutritive cube of Nutrition Soil, the seedling after surviving carries out Routine Management, can develop into healthy and strong grapevine seedling.
In the present invention, the incubation time of ovule on zygotic embryo Development culture base is 6 weeks, is greater than or less than this scope frequency of embryonic callus induction and obviously reduces (see table 2).Adopt different culture media formula induction currant generation embryo callus different with the successful that somatic embryo occurs, the TL culture medium prescription that the present invention proposes, its culture efficiency is obviously better than grapes tissue and cultivates normally used MS and the NN medium in field.
The table 2 different Ovule development time is on the impact of " seedless white " embryo-derived callus induction rate and somatic embryo inducement rate
Claims (7)
1. induce the special culture media of currant rataria shedder blast for one kind, it is characterized in that, its component and content as follows: nitrate of lime 250.0mg/L, potassium nitrate 600.0mg/L, potassium chloride 75.0mg/L, ammonium nitrate 300.0mg/L, magnesium sulfate 1200.0mg/L, potassium dihydrogen phosphate 300.0mg/L, manganese sulphate 3.0mg/L, potassium iodide 0.8mg/L, boric acid 0.5mg/L, zinc sulphate 0.5mg/L, sodium selenite 0.25mg/L, cobalt chloride 0.025mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.025mg/L, ironic citrate 10.0mg/L, thiamine hydrochloride 0.25mg/L, pyridoxine hydrochloride 0.25mg/L, D-VB5 calcium 0.25mg/L, nicotinic acid 0.25mg/L, asparagine 300mg/L, glycine 5.0mg/L, arginase 12 .0mg/L, inositol 50.0mg/L, caseinhydrolysate 500.0mg/L, Cys 121.16mg/L, sucrose 30000mg/L, agar 6000mg/L, all the other are distilled water.
2. induce a method for currant rataria shedder blast, it is characterized in that, specifically implement according to following steps:
Step 1, grape Post flowering 6 weeks fields gather young fruit, tap water 10min; With after alcohol-pickled 1 minute of 70% on superclean bench, then use the HgCl of 0.1%
2soak 8 minutes, rinsed with sterile water 3 times;
Fruit grain after step 2, sterilization is placed in sterilized culture dish, takes out ovule, be seeded on zygotic embryo Development culture base and carry out embryo culture in ovule under aseptic condition; Zygotic embryo Development culture base is the TL medium of solid-liquid double-phase, wherein additional saccharose 6.0g/L, active carbon 1.5g/L;
After step 3, ovule cultivate 6 weeks on zygotic embryo Development culture base, the rataria of growth is taken out under aseptic condition, be seeded on the embryonic callus induction medium of solid, the composition of embryonic callus induction medium is TL+0.5mg/L6-BA+1.0mg/L 2,4-D, wherein additional saccharose 30g/L, agar 6.0g/L;
After step 4, rataria cultivate 4 weeks on embryonic callus induction medium, the embryo callus of the yellow of acquisition, graininess, growth consolidation is seeded on the embryo differentiate medium of solid, the composition of embryo differentiate medium is TL+0.5mg/L 6-BA+2.0mg/L NAA, wherein, additional saccharose 30g/L, agar 6.0g/L;
After step 5, embryo callus cultivate 3-4 week on embryo differentiate medium, be seeded in by the somatic embryo of acquisition on TL+0.2mg/L IBA medium, wherein additional saccharose 30g/L, agar 6.0g/L, make its Germination And Seedling; Every 4 weeks subcultures once, obtain the somatic embryo Regeneration in Vitro plant with normal root, stem and true leaf in February;
Step 6, in vitro root system development good stand to be cleaned on it after greenhouse hardening the agar of attachment with clear water, be transplanted into and be equipped with in the nutritive cube of Nutrition Soil, the seedling after surviving carries out Routine Management, develops into healthy and strong grapevine seedling.
3. the method for induction currant rataria shedder blast according to claim 2, it is characterized in that, component and the content of described TL medium are as follows: nitrate of lime 250.0mg/L, potassium nitrate 600.0mg/L, potassium chloride 75.0mg/L, ammonium nitrate 300.0mg/L, magnesium sulfate 1200.0mg/L, potassium dihydrogen phosphate 300.0mg/L, manganese sulphate 3.0mg/L, potassium iodide 0.8mg/L, boric acid 0.5mg/L, zinc sulphate 0.5mg/L, sodium selenite 0.25mg/L, cobalt chloride 0.025mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.025mg/L, ironic citrate 10.0mg/L, thiamine hydrochloride 0.25mg/L, pyridoxine hydrochloride 0.25mg/L, D-VB5 calcium 0.25mg/L, nicotinic acid 0.25mg/L, asparagine 300mg/L, glycine 5.0mg/L, arginase 12 .0mg/L, inositol 50.0mg/L, caseinhydrolysate 500.0mg/L, Cys 121.16mg/L, sucrose 30000mg/L, agar 6000mg/L, all the other are distilled water.
4. the method for induction currant rataria shedder blast according to claim 2, it is characterized in that, in described step 3, ovule condition of culture on zygotic embryo Development culture base is: under 25 DEG C of low light conditions, intensity of illumination 800Lux, light application time 16h/d.
5. the method for induction currant rataria shedder blast according to claim 2, it is characterized in that, in described step 4, rataria condition of culture on embryonic callus induction medium is: 25 DEG C of dark conditions.
6. the method for induction currant rataria shedder blast according to claim 2, it is characterized in that, in described step 5, embryo callus condition of culture on embryo differentiate medium is: 25 DEG C of dark conditions.
7. the method for induction currant rataria shedder blast according to claim 2, it is characterized in that, described somatic embryo condition of culture on TL+0.2mg/L IBA medium is: cultivate under 25 DEG C of illumination conditions, intensity of illumination 2000Lux, light application time 16h/d.
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Cited By (1)
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| CN106244517A (en) * | 2016-10-12 | 2016-12-21 | 中国农业科学院特产研究所 | A kind of Vitis Amurensis somatic embryo and cultural method, culture medium and application |
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