CN103583369A - Induction medium for culturing callus of barley microspore - Google Patents
Induction medium for culturing callus of barley microspore Download PDFInfo
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- CN103583369A CN103583369A CN201310596632.4A CN201310596632A CN103583369A CN 103583369 A CN103583369 A CN 103583369A CN 201310596632 A CN201310596632 A CN 201310596632A CN 103583369 A CN103583369 A CN 103583369A
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Abstract
The invention provides an induction medium for culturing a callus of a barley microspore. The induction medium is prepared on the basis of a formula of an N6 culture medium, but the callus induction medium has the KNO3 concentration of 0-2500mg/ L, the (NH4) SO4 concentration of 0-400mg/ L, and is added with 400-2500mg/ L of casein hydrolyzate and/ or 400-2500mg/ L of glutamine. The invention also provides a method for culturing the callus of the barley microspore and obtaining regenerated plants, and a kit containing the induction medium, an optional differential medium and a rooting and seedling-strengthening medium. The whole process is carried out under controlled conditions in a laboratory; results are real and reliable; after differentiation of the obtained callus, plenty of homozygous regenerated plants can be obtained.
Description
Technical field
The invention belongs to plant biotechnology field, relate to the inducing culture improvement of cereal microspore, with this, obtain the method for a large amount of callus and doubled haploid (DH) regeneration plant.
Background technology
Plant cell has totipotency, whole hereditary information that each cell is comprising these species, thus possess the hereditary potency that develops into whole plant.Under optimum conditions, any one cell can develop into a new individuality.Totipotency of plant cell is the theoretical foundation of Plant Tissue Breeding.The microspore of plant is a kind of cell in special developmental stage, there is monoploid, single celled characteristic, thereby microspores culture system can be used for cell dedifferentiation research, structure doubled haploid (DH) mapping population, screening mutant, the cultivation of monoploid protoplast and fusion and haploid transgene receptor of differentiation mechanism again.In crossbreeding, to hybrid generation's selection, be an important ring, due to hybrid generation's genetic constitution height heterozygosis, proterties is constantly separated, often will select just to obtain the recombinant type material that proterties is stable through 4-5 field, expends a large amount of manpower and materials.Utilize the induction of microspores culture method can produce 100% haplobiont, pass through chromosome doubling, in 1 generation, can obtain normally liploid plant solid, that isozygoty, allelomorph can not lost due to separated in the generation afterwards, can avoid hybrid generation's serious separation, thereby accelerate proterties regrouping process, shortening the breeding cycle, various proterties are showed, greatly improve efficiency of selection.The callus output of microspores culture and quality have determined the quantity of Shoot regeneration, so the quality of inducing culture has determined the validity of microspores culture method to a great extent.Up to the present, the effect of cereal microspores culture is poor, and wherein, callus induction rate is lower is main limiting factor.Therefore, research and development improve the method for microspores culture callus induction rate, for setting up efficient cereal microspores culture system, have vital effect.
Summary of the invention
The present invention is directed to the deficiency that in microspores culture, genotypic restriction and callus yield poorly, a kind of callus inducing medium is provided.
The present invention is with existing N
6medium is basis.N
6medium is the conventional inducing culture of cereal crop cell and tissue culture, and nitrogen is most important composition in medium.Conventional N
6the composition of medium is substantially as shown in table 1 below, and the concentration of the heterogeneity that different vendor or document are recorded perhaps has a little difference, but its function is all roughly the same:
Table 1:N
6the formula of medium (unit: mg/L):
| N 6Macroelement: | ? |
| (NH 4) 2SO 4 | 463 |
| KNO 3 | 2830 |
| CaCl 2·2H 2O | 166 |
| MgSO 4·7H 2O | 185 |
| KH 2PO 4 | 400 |
| N 6Trace element: | ? |
| H 3BO 3 | 1.6 |
| KI | 0.83 |
| MnSO 4·4H 2O | 4.4 |
| ZnSO 4·7H 2O | 1.5 |
| N 6Molysite: | ? |
| Na 2EDTA | 37.3 |
| FeSO 4·7H 2O | 27.8 |
| N 6Organic element: | ? |
| Thiamine hydrochloride | 1.0 |
| Pyridoxine Hydrochloride | 0.5 |
| Glycine | 2.0 |
| Nicotinic acid | 0.5 |
The inventor finds by a large amount of research, can greatly affect output and the quality of barley microspore callus or embryoid by changing the consumption of nitrogen in inducing culture, i.e. impact green seedling quantity of differentiation subsequently.Particularly, by N
6kNO in medium
3content from 2830mg/L, drop to 0-2500mg/L, (NH
4) SO
4content from 463.0mg/L, drop to 0-400mg/L, and add hydrolysis casein (CH) and each 400-2500mg/L of glutamine (Glu), by the callus output that this medium is induced, be former N
6doubly, the quantity of Shoot regeneration is former N to the 1.3-9.0 of medium
6the 1.1-8.6 of medium doubly.
Therefore, the application provides a kind of N of transformation
6inducing culture, this inducing culture is with the N shown in table 1
6medium is basis, and difference is the KNO that contains 0-2500mg/L
3(NH with 0-400mg/L
4) SO
4, and be added with the hydrolysis casein of 400-2500mg/L and the glutamine of 400-2500mg/L.
In a specific embodiment, KNO
3concentration be 0-2200mg/L.
In a specific embodiment, KNO
3concentration be 700-2200mg/L.
In a specific embodiment, KNO
3concentration be 707.5mg/L or 1415mg/L or 2122.3mg/L,
In a specific embodiment, (NH
4) SO
4concentration be 0-350mg/L.
In a specific embodiment, (NH
4) SO
4concentration be 110-240mg/L, more preferably 110-160mg/L.
One concrete shown in, (NH
4) SO
4concentration be 115.8mg/L or 231.5mg/L.
In a specific embodiment, inducing culture of the present invention is also added with the hydrolysis casein of 500-2500mg/L and the glutamine of 500-2500mg/L.
In a specific embodiment, in inducing culture of the present invention, be added with the hydrolysis casein of 600-2000mg/L and the glutamine of 600-2000mg/L.
In a specific embodiment, the Na that inducing culture of the present invention is contained
2eDTA and FeSO
47H
2o separately concentration is known N
6the 1.5-2 of medium doubly.For example, Na
2the concentration of EDTA can reach 74.6mg/L, and FeSO
47H
2the concentration of O can reach 55.6mg/L.
In a specific embodiment, inducing culture of the present invention also can be at N
6on the basis of medium, add inositol, its concentration can be 100-200mg/L.
In a specific embodiment, inducing culture of the present invention also can be added with 2,4,5-T(2,4,5-trichlorophenoxyacetic acid) 0.4-1.2mg/L, KT0.3-0.8mg/L and maltose 60-120g/L.
In a specific embodiment, the concentration of 2,4,5-T is 0.8-1.0mg/L.
In a specific embodiment, the concentration of KT is 0.4-0.65mg/L, more preferably 0.4-0.5mg/L.
In a specific embodiment, the concentration of maltose is 80-100g/L, more preferably 80-90g/L.
In a specific embodiment, can use 2,4-D(2,4-dichlorophenoxyacetic acid) alternative 2,4,5-T.The concentration 0.4-1.2mg/L of 2,4-D, more preferably 0.8-1.0mg/L.Or can use the mixture of 2,4,5-T and 2,4-D, both total concentrations are controlled in the scope of 0.4-1.2mg/L, more preferably 0.8-1.0mg/L.
In a specific embodiment, the pH of inducing culture of the present invention is pH5.5-6.2, preferably 5.6-5.9, more preferably 5.7-5.8.
In a specific embodiment, the KNO that inducing culture of the present invention contains
3concentration be 0-2200mg/L, (NH
4) SO
4concentration be 0-350mg/L, hydrolysis casein concentration be 400-2000mg/L, the concentration of glutamine is 400-2000mg/L.
In a specific embodiment, the KNO that inducing culture of the present invention contains
3concentration be 700-2200mg/L, (NH
4) SO
4concentration be 110-350mg/L, hydrolysis casein concentration be 600-2000mg/L, the concentration of glutamine is 600-2000mg/L.
In a specific embodiment, the KNO that inducing culture of the present invention contains
3concentration be 700-1500mg/L, (NH
4) SO
4concentration be 110-240mg/L, hydrolysis casein concentration be 600-2000mg/L, the concentration of glutamine is 600-2000mg/L.
In a specific embodiment, the KNO that inducing culture of the present invention contains
3concentration be 707.5mg/L, (NH
4) SO
4concentration be 115.8mg/L, hydrolysis casein concentration be 800-2000mg/L, the concentration of glutamine is 800-2000mg/L.
In a specific embodiment, inducing culture of the present invention is not containing KNO
3(NH
4) SO
4, the concentration of hydrolysis casein is 800-2000mg/L, the concentration of glutamine is 800-2000mg/L.
Therefore, the formula of inducing culture of the present invention can be as shown in table 2 below:
Table 2: the formula (unit: mg/L) of inducing culture of the present invention
In other embodiments, the present invention also comprises and uses N
6the inducing culture of medium preparation, its composition is as shown in table 1, but this inducing culture does not contain KNO
3, (the NH that contains 400-463mg/L
4)
2sO
4, the hydrolysis casein of 800-2000mg/L and the glutamine of 800-2000mg/L.
In other embodiments, the present invention also comprises and uses N
6the inducing culture of medium preparation, its composition is as shown in table 1, but this inducing culture does not contain (NH
4)
2sO
4, the KNO that contains 2500-2830mg/L
3, the hydrolysis casein of 800-2000mg/L and the glutamine of 800-2000mg/L.
Inducing culture of the present invention can be used for cultivating microspore induction and produces callus.
Therefore, the present invention provides a kind of method for inducing and cultivating of barley microspore callus on the other hand, and the method comprises with inducing culture induction of the present invention cultivates barley microspore, thereby obtains callus.
In a specific embodiment, method of the present invention also comprises: get young fringe and process at low temperatures 10-30 days, isolate flower pesticide after sterilizing; With from flower pesticide, isolate microspore.
The present invention also comprises the method that obtains barley regeneration plant, and the method comprises:
(1) with inducing culture induction of the present invention, cultivate barley microspore, thereby obtain callus; With
(2) with differential medium, cultivate the callus that step (1) is obtained, thereby obtain regeneration plant.
In a specific embodiment, the method also comprises step: the regeneration plant that (3) obtain with Rooting and hardening-off culture base incubation step (2).
In a specific embodiment, described Rooting and hardening-off culture base is to be added with NAA0.02-0.08mg/L, paclobutrazol (MET) 2.0-10.0mg/L and sucrose 10-40g/L, the 1/2MS Rooting and hardening-off culture base of pH5.6-6.0.
In a specific embodiment, at 25 ± 1 ℃, under the condition of illumination in 10～12 hours every day, carry out the Rooting and hardening-off culture of 25～30 days.
The present invention also comprises the cover box that produces barley microspore callus or barley regeneration plant for inducing, and this cover box contains inducing culture of the present invention, and optional differential medium and Rooting and hardening-off culture base.
Embodiment
Above table 1 shows the N of the basal medium that is suitable as inducing culture of the present invention
6the basic components of medium.Should be understood that different documents, different commercially available N
6the formula of medium may have a little difference, but all can be used for implementing the present invention.More specifically, the concentration of each composition in can his-and-hers watches 1 is made suitable change, for example there are about 5%(for example 3% left and right, 2% left and right, 1% left and right, or lower mobility scale) or lower change, the medium that preparation obtains thus still has required biological function, still can be for implementing the present invention.The illustrative example of change can the mode with " ± " be listed in table 2.In addition N,
6composition in medium also can be used the same or analogous composition of function and character to be replaced.
In practical operation, for convenience, normally with former N
6kNO in medium
3, (NH
4)
2sO
43/4,1/2 or 1/4 amount of amount use KNO
3, (NH
4)
2sO
4, as shown in the application's specific embodiment, be specifically respectively 2122.5mg/L, 1415mg/L or 707.5mg/L, and 347.3mg/L, 231.5mg/L or 115.8mg/L.Or both concentration also can be in the scope of 707.5-2122.5mg/L and 115.8-347.3mg/L.Or, as mentioned before, in inducing culture of the present invention, KNO
3concentration range can be 0-2500mg/L, (NH
4)
2sO
4concentration range can be 0-400mg/L.
Be applicable to differential medium of the present invention and take 2/3MS as minimal medium, be wherein added with 6-BA0.3-1.2mg/L, KT0.5-2.0mg/L, NAA0.01-0.1mg/L, maltose 10-50g/L and inositol 95-105mg/L, pH is 5.5-6.2.
In differential medium of the present invention, 6-BA(6-benzyl aminoadenine) preferred concentration is 0.4-0.8mg/L, more preferably 0.5-0.6mg/L; KT(6-chaff aminopurine) preferred concentration is 1.0-1.8mg/L, more preferably 1.2-1.5mg/L; NAA(methyl α-naphthyl acetate) preferred concentration 0.02-0.08mg/L, more preferably 0.03-0.06mg/L; The preferred concentration of maltose is 20-40g/L, more preferably 20-30g/L.
In an embodiment, the concentration of NAA is 0.05mg/L.
The pH of differential medium is preferably 5.7-5.8.
The formula of MS medium as shown in table 3 below (unit: mg/L):
Table 3
| MS macroelement: | ? |
| NH 4NO 4 | 1650 |
| KNO 3 | 1900 |
| CaCl 2·2H 2O | 440 |
| MgSO 4·7H 2O | 370 |
| KH 2PO 4 | 170 |
| MS trace element: | ? |
| H 3BO 3 | 6.2 |
| KI | 0.83 |
| MnSO 4·4H 2O | 22.3 |
| ZnSO 4·7H 2O | 8.6 |
| Na 2MoO 4·2H 2O | 0.25 |
| CuSO 4·5H 2O | 0.025 |
| CoCl 2·6H 2O | 0.025 |
| MS molysite: | ? |
| Na 2EDTA | 37.3 |
| FeSO 4·7H 2O | 27.8 |
| MS organic element: | ? |
| Thiamine hydrochloride | 0.1 |
| Pyridoxine Hydrochloride | 0.5 |
| Nicotinic acid | 0.5 |
| Glycine | 2.0 |
| Inositol | 100.0 |
MS can press above-mentioned formulated voluntarily, also can buy finished product to chemical reagents corporations such as Sigma.2/3MS medium is that the macroelement of MS medium is reduced to 1/3, and trace element, molysite and organic element are constant.Then according to formulated described herein 2/3MS inducing culture herein.It will be appreciated by those skilled in the art that, can make suitable change to the concentration shown in above-mentioned table 3, for example have about 5% or lower (for example 3% left and right, 2% left and right, 1% left and right, or lower mobility scale) change, the medium that preparation obtains thus still has required biological function, still can be for implementing the present invention.For example, take inositol as example, in every liter of MS medium, can contain the inositol (5% amplitude of fluctuation) of about 95-105mg.The concentration of other composition is so change also.In addition, the composition in 2/3MS inducing culture also can be used the same or analogous composition of function and character to be replaced.
Similarly, inducing culture of the present invention also can be pressed above-mentioned formulated voluntarily, or can buy and obtain N to chemical reagents corporations such as Sigma
6medium finished product.Then according to formulated described herein inducing culture herein.
As previously mentioned, can induce the callus that produces barley microspore with inducing culture of the present invention, comprise with this inducing culture and cultivate barley microspore.
Conventionally, while implementing the application, can choose from land for growing field crops middle part little Hua, its microspore development is early stage in monokaryon, the tassel in mid-term, puts into refrigerator cold-storage 10-30 days, and 15-25 days normally, for example, about 15 days.The temperature of refrigeration can between 0-8 ℃ of scope, for example, can be 3-8 ℃.Conventionally can be approximately 5 ℃ of refrigerations.
During inoculation, tassel is first sterilized, for example, with saturated Eusol sterilization 10-20min, aseptic water washing 2-4 time.In each centrifuge tube, access the flower pesticide of 10 tassels, pour the mannitol extract of 15-20ml0.2-0.5M into, for example, with (3000-4000rpm) rotary-cut of high speed disperser hypervelocity, 150 eye mesh screens filter, (for example 800-1000rpm) is centrifugal for filtrate low speed, repeats 2-3 time, collects microspore.
The concentration that is used for the mannitol extract of pretreatment flower pesticide can be about 0.3-0.4M, is preferably about 0.3M.The concentration of the mannitol extract using during high speed disperser rotary-cut flower pesticide can be 0.3-0.4M, is preferably 0.3M.
The Isolated microspore that extracts acquisition can be placed in inducing culture of the present invention and induce cultivation.Can will be first before cultivation with washing microspore 1 to 2 time with this inducing culture, then with medium by microspore Auto-regulating System of Density of Heavy Medium to 1.0～1.2 * 10
5/ ml, gets about 1.5-3ml microspore suspension and is inoculated in culture dish (30 * 15mm), Parafilm sealing, and under room temperature, (for example about 25-28 ℃) secretly cultivates 15-25 days.Generally 25 ℃ of dark cultivations about about 21 days.Can obtain callus thus.
Induction can be transferred to gained callus on differential medium after cultivating.
Conventionally at 25 ± 1 ℃, under the condition of illumination in 10～12 hours every day, differentiation is coerced and is cultivated 25～30 days, until differentiate green regenerating plant.Differentiation is coerced cultivation and can be used differential medium of the present invention to carry out.
The regeneration plant differentiating can proceed to and be added with NAA0.02-0.08mg/L, paclobutrazol (MET) 2.0-10.0mg/L and sucrose 10-40g/L, the 1/2MS Rooting and hardening-off culture base of pH5.6-6.0.At 25 ± 1 ℃, under the condition of illumination in 10～12 hours every day, carry out the Rooting and hardening-off culture of 25～30 days.
In Rooting and hardening-off culture base, the preferred concentration range of NAA is 0.03-0.06mg/L, more preferably 0.04-0.05mg/L; The preferred concentration range of MET is 3.0-8.0mg/L, more preferably 4.0-6.0mg/L.In one embodiment, the concentration of MET is 5.0mg/L; The preferred concentration range of sucrose is 20-30g/L.In one embodiment, the concentration of sucrose is 20g/L.
The pH of Rooting and hardening-off culture base is preferably 5.8-5.9.
1/2MS Rooting and hardening-off culture base is that the macroelement of the MS medium shown in table 3 is reduced by half, and adds after NAA0.02-0.08mg/L, paclobutrazol (MET) 2.0-10.0mg/L described in the application and sucrose 10-40g/L and prepare and obtain.
Compare with traditional breeding way, the inventive method is easy, is easy to grasp, implement; Be not subject to the impact of external condition; Utilize microspore as starting material, can obtain a large amount of callus, in whole process Dou culturing room, carry out, reduced again the use in labour and farmland simultaneously, and can anniversary repeated test, both shortened breeding time, can obtain a large amount of germplasm materials again.
Present invention is described as example below will to take barley.Should be understood that in the situation that not departing from spirit and scope of the invention and can make various modifications and changes to the present invention.And also can adopt method of the present invention for other cereal crop, and can produce identical effect.Following examples are only illustrative, and scope of the present invention is limited by the application's claim.If no special instructions, the present invention's percentage used is percent weight in volume.In addition it, should be understood that the preferable range of each composition in above-mentioned each medium can be combined, as long as can realize goal of the invention of the present invention.
Embodiment
Embodiment 1: the obtaining of callus
From land for growing field crops, choose that middle part little Hua microspore development is early stage in monokaryon, the tassel in mid-term, put into refrigerator cold-storage 15 days.During inoculation, saturated Eusol sterilization 15min for tassel, aseptic water washing 3-4 time.Each test tube connects 10 tassels, pours 15ml mannitol 60g/L, CaCl into
2the extract of 1.1g/L, MES0.976g/L, with the high speed disperser hypervelocity rotary-cut of 3000rpm, 150 eye mesh screens filter, and filtrate, with 800rpm low-speed centrifugal, is repeated 3 times, collects microspore.Microspore uses extract in 25 ℃, dark pretreatment 2d.Inducing culture is with N shown in table 1
6medium is minimal medium, but Na
2the concentration of EDTA is 74.6mg/L, FeSO
47H
2the concentration of O, for reaching 55.6mg/L, is wherein also added with 2,4,5-T1.0mg/L, KT0.5mg/L, maltose 90g/L, inositol 200mg/L, glutamine and each 2.0g/L of hydrolysis casein, pH5.8.Before cultivating by medium washing for microspore 1 time, then with medium by microspore Auto-regulating System of Density of Heavy Medium to 1.0～1.2 * 10
5/ ml, gets 1.0ml microspore suspension and is inoculated in culture dish (30 * 15mm), Parafilm sealing, 25 ℃ of dark 21d that cultivate.
Embodiment 2: the obtaining of regeneration plant
Induction is transferred to callus on differential medium after cultivating 21d.Differential medium be take 2/3MS as minimal medium, is wherein added with 6-BA0.5mg/L, KT1.5mg/L, NAA0.05mg/L and maltose 30g/L, and PH is 5.8.At 25 ± 1 ℃, under the condition of illumination in 10～12 hours every day, differentiation is cultivated 25～30 days, until differentiate green regenerating plant.
Embodiment 3:
Preparation different K NO
3(NH
4)
2sO
4the N of modification of concentration
6inducing culture is (except KNO
3, (NH
4)
2sO
4, Glu and CH as follows outside, all the other constituent concentrations are with embodiment 1), by embodiment 1, collect the microspore evoked callus of 4 parts of barley materials, weigh after blotting medium while cultivating 28d, the results are shown in following table.
The callus output (mg/ ware) of inducing on different CH and Glu concentration medium
Inorganic nitrogen concentration in inducing culture
N
6-full nitrogen: KNO
3(NH
4)
2sO
4concentration be respectively 2830mg/L and 463mg/L, each 800mg/L of Glu and CH.
N
6-3/4 nitrogen: KNO
3(NH
4)
2sO
4concentration be full nitrogen 3/4, be respectively 2122.5mg/L and 347.3mg/L, each 600mg/L of Glu and CH.
N
6-1/2 nitrogen: KNO
3(NH
4)
2sO
4concentration be full nitrogen 1/2, be respectively 1415mg/L and 231.5mg/L, each 400mg/L of Glu and CH.
N
6-1/4 nitrogen: KNO
3(NH
4)
2sO
4concentration be full nitrogen 1/4, be respectively 707.5mg/L and 115.8mg/L, each 200mg/L of Glu and CH.
N
6-without nitrogen: in medium containing KNO
3(NH
4)
2sO
4, also not containing Glu and CH.
From table, result can be found out, KNO in inducing culture
3(NH
4)
2sO
4concentration be down to 3/4 o'clock, 4 parts are risen to some extent for the callus output of 3 parts in examination materials, 1 part maintains an equal level; Be down to 1/2 o'clock, 4 parts reach peak for the callus output of 2 parts in examination materials, only have the callus output of 1 part a little less than full nitrogen.
Embodiment 4
Prepare the N of the modification of different CH and Glu concentration
6inducing culture is (except KNO
3, (NH
4)
2sO
4, outside Glu and CH, all the other constituent concentrations are with embodiment 1), by embodiment 1, collect the microspores culture evoked callus of a collection of barley material, while cultivating 21d, blot medium, take culture dish as unit, the callus obtaining is weighed, the results are shown in following table.
The callus output (mg/ ware) of inducing on different CH and Glu concentration medium
N
6-full nitrogen: KNO
3(NH
4)
2sO
4concentration be respectively 2830mg/L and 463mg/L, each 800mg/L of Glu and CH.
N
6-800:KNO
3(NH
4)
2sO
4concentration be respectively 707.5mg/L and 115.8mg/L, each 800mg/L of Glu and CH.
N
6-1200:KNO
3(NH
4)
2sO
4concentration be respectively 707.5mg/L and 115.8mg/L, each 1200mg/L of Glu and CH.
N
6-1600:KNO
3(NH
4)
2sO
4concentration be respectively 707.5mg/L and 115.8mg/L, each 1600mg/L of Glu and CH.
N
6-2000:KNO
3(NH
4)
2sO
4concentration be respectively 707.5mg/L and 115.8mg/L, each 2000mg/L of Glu and CH.
To add the N of 1/4 inorganic nitrogen consumption
6medium, as basic inducing culture, adds Glu and the CH of variable concentrations, relatively the impact of different organic nitrogen usage amounts on microspores culture callus output in medium.In table, result shows, all callus output for examination material improves along with the raising of Glu in inducing culture and CH concentration, at the N that contains Glu and CH2000mg/L modification
6on medium, callus output/ware is 1.3-9.0 times of full nitrogen medium.
Embodiment 5
The callus that embodiment 4 obtains is transferred on differential medium and breaks up, and adds up the green regenerating plant quantity that every ware obtains, and the results are shown in following table.
The green seedling quantity (strain/ware) of the Calli Differentiation on different CH and Glu concentration medium
Medium " N
6-full nitrogen ", " N
6-800 ", " N
6-1200 ", " N
6-1600 " and " N
6-2000 " implication is with embodiment 4.
In table, result shows, nearly all green seedling output/ware for examination material increases along with the raising of callus output, at the N that contains Glu and CH2000mg/L modification
6on medium, the 1.1-8.6 that green seedling output/ware is full nitrogen medium doubly.
Embodiment 6
Preparation different K NO
3, (NH
4)
2sO
4, Glu and CH concentration the N of modification
6inducing culture is (except KNO
3, (NH
4)
2sO
4, outside Glu and CH, all the other constituent concentrations are with embodiment 1), by embodiment 1, collect the microspore evoked callus of 4 parts of barley materials, weigh after blotting medium while cultivating 28d, the results are shown in following table.
In medium, different nitrogen sources is on the impact of callus output (mg/ ware)
N
6-A:(NH
4)
2sO
4concentration be 463mg/L, without KNO
3, Glu and CH.
N
6-N:KNO
3concentration is 2830mg/L, without (NH
4)
2sO
4, Glu and CH.
N
6-AN:KNO
3(NH
4)
2sO
4concentration be respectively 2830mg/L and 463mg/L, without Glu and CH.
N
6each 800mg/L of-O:Glu and CH, without KNO
3(NH
4)
2sO
4.
N
6-full nitrogen: KNO
3(NH
4)
2sO
4concentration be respectively 2830mg/L and 463mg/L, each 800mg/L of Glu and CH.
N
6-AO:(NH
4)
2sO
4concentration be 463mg/L, each 800mg/L of Glu and CH, without KNO
3.
N
6-NO:KNO
3concentration is 2830mg/L, and each 800mg/L of Glu and CH, without (NH
4)
2sO
4.
As can be seen from the above table, lacking KNO
3or (NH
4)
2sO
4situation under, with N
6-full nitrogen group is compared, and adds Glu and CH and also can obviously improve callus output (N
6-AO and N
6-NO group).In addition, inducing culture is not containing KNO
3(NH
4)
2sO
4situation under (N
6-O group), with N
6-full nitrogen group is compared, and adds Glu and CH and also can obviously improve callus output.
Claims (10)
1. an inducing culture, described inducing culture is according to N
6culture medium prescription is formulated, it is characterized in that, KNO in described medium
3concentration is 0-2500mg/L, (NH
4) SO
4concentration be 0-400mg/L, and described inducing culture is also added with the hydrolysis casein of 400-2500mg/L and the glutamine of 400-2500mg/L.
2. inducing culture as claimed in claim 1, is characterized in that, KNO in described medium
3concentration be 650-2200mg/L, (NH
4) SO
4concentration be 100-350mg/L.
3. inducing culture as claimed in claim 1 or 2, is characterized in that, the Na that described inducing culture contains 37.3-74.6mg/L
2the FeSO of EDTA and 27.8-55.6mg/L
47H
2o.
4. the inducing culture as described in any one in claim 1-3, is characterized in that, described inducing culture is also added with inositol 100-200mg/L, 2,4,5-T and/or 2,4-D0.4-1.2mg/L, KT0.3-0.8mg/L and maltose 60-120g/L.
5. inducing culture as claimed in claim 1, is characterized in that, the formula of described medium is as follows:
6. overlap a box, this cover box contains the inducing culture described in any one and optional differential medium and Rooting and hardening-off culture base in claim 1-5.
7. cover box as claimed in claim 6, is characterized in that, described differential medium is for having added 6-BA0.3-1.2mg/L, KT0.5-2.0mg/L, NAA0.01-0.1mg/L and maltose 10-50g/L2/3MS differential medium, and pH is 5.5-6.2; Described Rooting and hardening-off culture base is for being added with NAA0.02-0.08mg/L, paclobutrazol 2.0-10.0mg/L and sucrose 10-40g/L, the 1/2MS Rooting and hardening-off culture base of pH5.6-6.0.
8. a method for inducing and cultivating for barley microspore callus, is characterized in that, the method comprises with the inducing culture induction described in claim 1-5 any one cultivates barley microspore, thereby obtains callus.
9. a method of preparing barley regeneration plant, is characterized in that, the method comprises:
(1) with the inducing culture induction described in any one in claim 1-5, cultivate barley microspore, thereby obtain callus; With
(2) with differential medium, cultivate the callus that step (1) is obtained, thereby obtain regeneration plant.
10. method as claimed in claim 9, is characterized in that, described method also comprises:
(3) regeneration plant obtaining with Rooting and hardening-off culture base incubation step (2).
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| CN103947550A (en) * | 2014-04-20 | 2014-07-30 | 浙江省农业科学院 | Tissue culture method for directly growing seedling from barley embryo and culture medium used in method |
| CN105340755A (en) * | 2015-12-04 | 2016-02-24 | 上海市农业科学院 | Method for continuously culturing high frequency regeneration plants through cereal crop single plant source microspores |
| CN105684895A (en) * | 2014-11-28 | 2016-06-22 | 上海市农业科学院 | A method for constructing a haploid population of a cereal crop |
| CN106069768A (en) * | 2016-06-29 | 2016-11-09 | 无锡南理工科技发展有限公司 | A kind of Anthers of Hordeum Vulgare method for inducing and cultivating |
| CN106489728A (en) * | 2015-09-08 | 2017-03-15 | 无锡南理工科技发展有限公司 | A kind of Anthers of Hordeum Vulgare inducing culture based formulas |
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| CN108085334A (en) * | 2016-11-17 | 2018-05-29 | 上海市农业科学院 | A kind of Agrobacterium-mediated Transformation barley microspore method of improvement |
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