CN103583369B - Induction medium for culturing callus of barley microspore - Google Patents
Induction medium for culturing callus of barley microspore Download PDFInfo
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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 0-2500 mg/L, the (NH4) SO4 concentration of 0-400 mg/L, and is added with 400-2500 mg/L of casein hydrolyzate and/ or 400-2500 mg/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, it is related to the inducing culture improvement of cereal crops sporidiole, is obtained with this
The method taking a large amount of calluss and doubled haploid (dh) regeneration plant.
Background technology
Plant cell has totipotency, and that is, each cell comprises whole hereditary informations of this species, thus possess sending out
It is bred as the hereditary potency of whole plant.Under optimum conditions, any one cell can develop into a new individuality.Plant
Cellular omnipotency is the theoretical basiss of plant tissue culture.The sporidiole of plant is a kind of cell being in special developmental stage,
There is monoploid, single celled characteristic, thus microspores culture system can be used for the cell dedifferentiation research of differentiation mechanism, structure again
Build doubled haploid (dh) mapping population, screening mutant, monoploid Protoplast cuhnre and fusion and haploid turn base
Factor receptor.In cross-breeding, the selection to hybrid generation is an important ring, because hybrid generation's genetic constitution is highly miscellaneous
Close, character constantly separates, often will select just can obtain the stable recombinant type material of character through 4-5 field, expend a large amount of
Manpower and materials.100% haplobiont can be produced using the induction of microspores culture method, by chromosome doubling, at 1
Can obtain in from generation to generation can normal solid, homozygosis liploid plant, allele will not in the generation afterwards due to point
From and lose, the serious separation of hybrid generation being avoided, thus accelerating character regrouping process, shortening the breeding cycle, making various
Character is showed, and is greatly enhanced efficiency of selection.The Callus yield of microspores culture and quality determine regeneration
The quantity of green Seedling, so, the quality of inducing culture determines the effectiveness of microspores culture method to a great extent.To mesh
Before till, the effect of cereal crops microspores culture is poor, and wherein, callus induction rate is relatively low to be main restriction factor.
Therefore, the method that research and development improve microspores culture callus induction rate, for setting up efficient cereal crops microspores culture
System has vital effect.
Content of the invention
The present invention is directed to the low deficiency of the restriction of genotype and Callus yield in microspores culture, provides a kind of wound healing
Tissue inducing culture.
The present invention is with existing n6Based on culture medium.n6Culture medium is the conventional of cereal crop cell and tissue culture
Inducing culture, nitrogen is most important composition in culture medium.Conventional n6The composition of culture medium is substantially as shown in table 1 below, no
Concentration with the heterogeneity described in manufacturer or document perhaps has little difference, but its function is all roughly the same:
Table 1:n6The formula (unit: mg/l) of culture medium:
| n6A great number of elements: | |
| (nh4)2so4 | 463 |
| kno3 | 2830 |
| cacl2·2h2o | 166 |
| mgso4·7h2o | 185 |
| kh2po4 | 400 |
| n6Trace element: | |
| h3bo3 | 1.6 |
| ki | 0.83 |
| mnso4·4h2o | 4.4 |
| znso4·7h2o | 1.5 |
| n6Iron salt: | |
| na2edta | 37.3 |
| feso4·7h2o | 27.8 |
| n6Organic element: | |
| Thiamine hydrochloride | 1.0 |
| Pyridoxine Hydrochloride | 0.5 |
| Glycine | 2.0 |
| Nicotinic acid | 0.5 |
The present inventor passes through substantial amounts of research and finds, can greatly shadow by changing the consumption of nitrogen in inducing culture
The yield of sound Fructus Hordei Vulgaris sporidiole calluss or embryoid and quality, that is, affect subsequent green plant regeneration quantity.Specifically, will
n6Kno in culture medium3Content drop to 0-2500mg/l, (nh from 2830mg/l4)so4Content drop to from 463.0mg/l
0-400mg/l, and add hydrolysed casein (ch) and each 400-2500mg/l of L-Glutamine (glu), with the induction of this culture medium
Callus yield is former n61.3-9.0 times of culture medium, the quantity of Shoot regeneration is former n61.1-8.6 times of culture medium.
Therefore, the application provides a kind of n of transformation6Inducing culture, this inducing culture is with the n shown in table 16Culture medium
Based on, except that the kno containing 0-2500mg/l3(nh with 0-400mg/l4)so4, and it is added with 400-2500mg/
The hydrolysed casein of l and the L-Glutamine of 400-2500mg/l.
In a specific embodiment, kno3Concentration be 0-2200mg/l.
In a specific embodiment, kno3Concentration be 700-2200mg/l.
In a specific embodiment, kno3Concentration be 707.5mg/l or 1415mg/l or 2122.3mg/l,
In a specific embodiment, (nh4)so4Concentration be 0-350mg/l.
In a specific embodiment, (nh4)so4Concentration be 110-240mg/l, more preferably 110-160mg/l.
At one specifically shown in, (nh4)so4Concentration be 115.8mg/l or 231.5mg/l.
In a specific embodiment, the inducing culture of the present invention is also added with the hydrolysed casein of 500-2500mg/l
L-Glutamine with 500-2500mg/l.
In a specific embodiment, be added with inducing culture of the present invention 600-2000mg/l hydrolysed casein and
The L-Glutamine of 600-2000mg/l.
In a specific embodiment, the na contained by the inducing culture of the present invention2Edta and feso4·7h2O is each dense
Spend for known n61.5-2 times of culture medium.For example, na2The concentration of edta is up to 74.6mg/l, and feso4·7h2The concentration of o can
Reach 55.6mg/l.
In a specific embodiment, the inducing culture of the present invention also can be in n6Add inositol on the basis of culture medium,
Its concentration can be 100-200mg/l.
In a specific embodiment, the inducing culture of the present invention may further be enriched with 2,4,5-t(2,4,5- trichloro-benzenes oxygen
Acetic acid) 0.4-1.2mg/l, kt0.3-0.8mg/l and maltose 60-120g/l.
In a specific embodiment, 2,4,5-t concentration 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, 2,4-d(2,4- dichlorophenoxyacetic acid can be used) substitute 2,4,5-t.2,4-d's
Concentration 0.4-1.2mg/l, more preferably 0.8-1.0mg/l.Or 2,4,5-t and 2,4-d mixture can be used, both is total
Concentration controls in the range 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
For 5.7-5.8.
In a specific embodiment, the kno that the inducing culture of the present invention contains3Concentration be 0-2200mg/l,
(nh4)so4Concentration be 0-350mg/l, the concentration of hydrolysed casein is 400-2000mg/l, and the concentration of L-Glutamine is 400-
2000mg/l.
In a specific embodiment, the kno that the inducing culture of the present invention contains3Concentration be 700-2200mg/l,
(nh4)so4Concentration be 110-350mg/l, the concentration of hydrolysed casein is 600-2000mg/l, and the concentration of L-Glutamine is
600-2000mg/l.
In a specific embodiment, the kno that the inducing culture of the present invention contains3Concentration be 700-1500mg/l,
(nh4)so4Concentration be 110-240mg/l, the concentration of hydrolysed casein is 600-2000mg/l, and the concentration of L-Glutamine is
600-2000mg/l.
In a specific embodiment, the kno that the inducing culture of the present invention contains3Concentration be 707.5mg/l, (nh4)
so4Concentration be 115.8mg/l, the concentration of hydrolysed casein is 800-2000mg/l, and the concentration of L-Glutamine is 800-
2000mg/l.
In a specific embodiment, the inducing culture of the present invention does not contain kno3(nh4)so4, hydrolysed casein dense
Spend for 800-2000mg/l, the concentration of L-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 includes using n6The inducing culture that culture medium is prepared, its composition such as table 1 institute
Show, but this inducing culture does not contain kno3, (the nh containing 400-463mg/l4)2so4, the hydrolysis cheese of 800-2000mg/l
Element and the L-Glutamine of 800-2000mg/l.
In other embodiments, the present invention also includes using n6The inducing culture that culture medium is prepared, its composition such as table 1 institute
Show, but this inducing culture does not contain (nh4)2so4, the kno containing 2500-2830mg/l3, 800-2000mg/l hydrolysis do
Casein and the L-Glutamine of 800-2000mg/l.
The inducing culture of the present invention can be used for cultivating sporidiole induction production calluss.
Therefore, another aspect of the present invention provides a kind of method for inducing and cultivating of Fructus Hordei Vulgaris sporidiole calluss, the method bag
Include the inducing culture inducing culture Fructus Hordei Vulgaris sporidiole with the present invention, thus obtaining calluss.
In a specific embodiment, the method for the present invention also includes: takes young fringe to process at low temperature 10-30 days, sterilizing
After isolate flower pesticide；With isolate sporidiole from flower pesticide.
Present invention additionally comprises the method obtaining Fructus Hordei Vulgaris regeneration plant, the method includes:
(1) use the inducing culture inducing culture Fructus Hordei Vulgaris sporidiole of the present invention, thus obtaining calluss；With
(2) calluss being obtained step (1) with division culture medium culture, thus obtain regeneration plant.
In a specific embodiment, the method further comprises the steps of: (3) and uses Rooting and hardening-off culture base incubation step (2) institute
The regeneration plant obtaining.
In a specific embodiment, described Rooting and hardening-off culture base is to be added with naa0.02-0.08mg/l, paclobutrazol
(met) the 1/2ms Rooting and hardening-off culture base of 2.0-10.0mg/l and sucrose 10-40g/l, ph5.6-6.0.
In a specific embodiment, at 25 ± 1 DEG C, under conditions of illumination in daily 10～12 hours, 25～30 days are carried out
Rooting and hardening-off culture.
Present invention additionally comprises produce the box set of Fructus Hordei Vulgaris sporidiole calluss or Fructus Hordei Vulgaris regeneration plant, this box set for induction
Inducing culture containing the present invention, and optional division culture medium and Rooting and hardening-off culture base.
Specific embodiment
Table 1 above shows the n of the basal medium being suitable as inducing culture of the present invention6The basis of culture medium is joined
Side.It should be understood that different documents, different commercially available n6The formula of culture medium may have a little difference, but can be used in implementing the present invention.
More specifically, suitable variation can be made to the concentration of each composition in table 1, for example have about 5%(such as 3% about,
2% about, 1% about, or lower mobility scale) or lower variation, thus prepare the culture medium obtaining and still there is institute
The biological function needing, remains to for implementing the present invention.Change illustrative example can in table 2 by " ± " in the way of list.
Additionally, n6Composition in culture medium also can be replaced using function and the same or analogous composition of property.
In practical operation, for convenience, typically with former n6Kno in culture medium3、(nh4)2so4Amount 3/4,1/2 or 1/
4 amount uses kno3、(nh4)2so4, as shown in the application specific embodiment, 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 707.5-
In the range of 2122.5mg/l and 115.8-347.3mg/l.Or, as it was noted above, in inducing culture of the present invention, kno3
Concentration range can be 0-2500mg/l, (nh4)2so4Concentration range can be 0-400mg/l.
The division culture medium being applied to the present invention with 2/3ms as minimal medium, 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 division culture medium of the present invention, 6-ba(6- benzyl aminoadenine) preferred concentration be 0.4-0.8mg/l, more excellent
Elect 0.5-0.6mg/l as；Kt(6- bran amidopurin) preferred concentration be 1.0-1.8mg/l, more preferably 1.2-1.5mg/l；
Naa(naphthalene acetic acid) 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 a detailed embodiment, the concentration of naa is 0.05mg/l.
The ph of division culture medium is preferably 5.7-5.8.
The formula of ms culture medium is as shown in table 3 below (unit: mg/l):
Table 3
| Ms a great number of elements: | |
| nh4no4 | 1650 |
| kno3 | 1900 |
| cacl2·2h2o | 440 |
| mgso4·7h2o | 370 |
| kh2po4 | 170 |
| Ms trace element: | |
| h3bo3 | 6.2 |
| ki | 0.83 |
| mnso4·4h2o | 22.3 |
| znso4·7h2o | 8.6 |
| na2moo4·2h2o | 0.25 |
| cuso4·5h2o | 0.025 |
| cocl2·6h2o | 0.025 |
| Ms iron salt: | |
| na2edta | 37.3 |
| feso4·7h2o | 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 voluntarily press above-mentioned formula and prepare, and also can buy finished product to chemical reagents corporations such as sigma.2/3ms culture medium
It is then that a great number of elements of ms culture medium is reduced 1/3, and trace element, iron salt and organic element are constant.Then according to this paper institute
State the 2/3ms inducing culture that formula prepares this paper.It will be understood by those skilled in the art that the concentration shown in above-mentioned table 3 can be made
Go out suitable variation, for example have about 5% or lower (such as 3% about, 2% about, 1% about, or lower variation model
Enclose) variation, thus prepare the culture medium obtaining and still there is required biological function, remain to for implementing the present invention.Example
As, taking inositol as a example, can the inositol containing about 95-105mg (5% amplitude of fluctuation) in every liter of ms culture medium.Other compositions
Concentration also so change.Additionally, composition in 2/3ms inducing culture also can using function same or analogous with property become
Divide and replaced.
Similarly, the inducing culture of the present invention also can voluntarily be pressed above-mentioned formula and prepare, or can be to chemistry examinations such as sigma
Agent company buys and obtains n6Culture medium finished product.Then the inducing culture of this paper is prepared according to formula described herein.
As it was previously stated, the calluss producing Fructus Hordei Vulgaris sporidiole can be induced using the inducing culture of the present invention, including
Cultivate Fructus Hordei Vulgaris sporidiole with this inducing culture.
Generally, when implementing the application, middle part little Hua can be chosen from land for growing field crops, its microspore development is in monokaryon early stage, mid-term
Tassel, put into refrigerator cold-storage 10-30 days, typically 15-25 days, such as 15 days about.The temperature of cold preservation can be in 0-8 DEG C of model
Between enclosing, for example, can be 3-8 DEG C.Generally can be in about 5 DEG C of cold preservations.
During inoculation, tassel is first sterilized, for example, use the Eusol sterilization 10-20min of saturation, aseptic water washing 2-4 time.
Access the flower pesticide of 10 tassels in each centrifuge tube, pour the manna alcohol extract of 15-20ml0.2-0.5m into, use high speed disperser
Hypervelocity (such as 3000-4000rpm) rotary-cut, 150 mesh sieve net filtrations, filtrate low speed (such as 800-1000rpm) is centrifuged, and repeats 2-
3 times, collect sporidiole.
Concentration for the manna alcohol extract of pretreatment flower pesticide can be about 0.3-0.4m, preferably from about 0.3m.At a high speed
The concentration of the manna alcohol extract being used during disperser rotary-cut flower pesticide can be 0.3-0.4m, preferably 0.3m.
Extract the Isolated microspore obtaining and be placed in the inducing culture of the present invention carrying out inducing culture.Can be by before culture
First with washing sporidiole 1 to 2 times with this inducing culture, then with culture medium by sporidiole Auto-regulating System of Density of Heavy Medium to 1.0～1.2 ×
105/ ml, takes about 1.5-3ml microspore suspension to be inoculated in culture dish (30 × 15mm), parafilm seals, (example under room temperature
Such as from about 25-28 DEG C) light culture 15-25 days.It is in 25 DEG C of light culture about 21 days about under normal circumstances.Wound healing thus can be obtained
Tissue.
After inducing culture, gained calluss can be transferred on division culture medium.
Generally at 25 ± 1 DEG C, differentiation coercing cultivation 25～30 days under conditions of illumination in daily 10～12 hours, until differentiation
Go out green regenerating plant.Differentiation coercing cultivation can be carried out using the division culture medium of the present invention.
The regeneration plant differentiating can proceed to and be added with naa0.02-0.08mg/l, paclobutrazol (met) 2.0-10.0mg/l
With sucrose 10-40g/l, the 1/2ms Rooting and hardening-off culture base of ph5.6-6.0.At 25 ± 1 DEG C, illumination in daily 10～12 hours
Under the conditions of 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 to halve a great number of elements of the ms culture medium shown in table 3, and adds the application institute
Prepare after the naa0.02-0.08mg/l, paclobutrazol (met) 2.0-10.0mg/l and sucrose 10-40g/l that state and obtain.
Compared with traditional breeding way, the inventive method simplicity is it is easy to grasping, implementing；Do not affected by external condition；Profit
With sporidiole as parent material, substantial amounts of calluss can be obtained, whole process is carried out all in culturing room, reduce simultaneously again
The use of labour force and farmland, and can repeat in the anniversary to test, both shortened breeding time, and substantial amounts of kind of material can be obtained
Material.
Hereafter will taking Fructus Hordei Vulgaris as a example present invention is described.It should be understood that in the feelings without departing from spirit and scope of the invention
Various modifications and changes can be made to the present invention under condition.And the method for the present invention may also be employed for other cereal crops, and
Identical effect can be produced.Following examples are only illustrative, and the scope of the present invention is by claims hereof in addition
Limit.If no special instructions, the percentage ratio used by the present invention is percent weight in volume.Furthermore, it is to be understood that above-mentioned each culture medium
In each composition preferred scope can combination in any, as long as it enables the goal of the invention of the present invention.
Embodiment
Embodiment 1: the acquisition of calluss
Choose, from land for growing field crops, the tassel that middle part little Hua microspore development is in monokaryon early stage, mid-term, put into refrigerator cold-storage 15 days.
During inoculation, the Eusol sterilization 15min of tassel saturation, aseptic water washing 3-4 time.Each test tube connects 10 tassels,
Enter 15ml Mannitol 60g/l, cacl2The extracting solution of 1.1g/l, mes0.976g/l, with the high speed disperser hypervelocity rotation of 3000rpm
Cut, 150 mesh sieve net filtrations, filtrate, with 800rpm low-speed centrifugal, is repeated 3 times, collect sporidiole.Sporidiole extracting solution is in 25
DEG C, dark pretreatment 2d.Inducing culture is with n shown in table 16Culture medium is minimal medium, but na2The concentration of edta is
74.6mg/l, feso4·7h2The concentration of o is to reach 55.6mg/l, wherein also added with 2,4,5-t1.0mg/l, kt0.5mg/l, Fructus Hordei Germinatus
Sugared 90g/l, inositol 200mg/l, L-Glutamine and each 2.0g/l of hydrolysed casein, ph5.8.By sporidiole culture medium before culture
Washing 1 time, then with culture medium by sporidiole Auto-regulating System of Density of Heavy Medium to 1.0～1.2 × 105/ ml, takes 1.0ml microspore suspension to connect
Plant in culture dish (30 × 15mm), parafilm seals, 25 DEG C of light culture 21d.
Embodiment 2: the acquisition of regeneration plant
After inducing culture 21d, calluss are transferred on division culture medium.Division culture medium is cultivated with 2/3ms for basic
Base, wherein added with 6-ba0.5mg/l, kt1.5mg/l, naa0.05mg/l and maltose 30g/l, ph is 5.8.At 25 ± 1 DEG C,
Differentiation culture 25～30 days under conditions of illumination in daily 10～12 hours, until differentiate green regenerating plant.
Embodiment 3:
Prepare different kno3(nh4)2so4The modification of concentration n6Inducing culture is (except kno3、(nh4)2so4, glu and
Ch is as follows outer, and remaining constituent concentration is with embodiment 1), the sporidiole callus induction of 4 parts of barley materials is collected by embodiment 1
Tissue, weighs after Aspirate medium during culture 28d, result see table.
The Callus yield (mg/ ware) of induction on different ch and glu concentration cultures
Inorganic nitrogen concentration in inducing culture
n6- full nitrogen: kno3(nh4)2so4Concentration be respectively 2830mg/l and 463mg/l, each 800mg/l of glu and ch.
n6- 3/4 nitrogen: kno3(nh4)2so4Concentration be the 3/4 of full nitrogen, i.e. respectively 2122.5mg/l and 347.3mg/
The each 600mg/l of l, glu and ch.
n6- 1/2 nitrogen: kno3(nh4)2so4Concentration be the 1/2 of full nitrogen, i.e. respectively 1415mg/l and 231.5mg/l,
The each 400mg/l of glu and ch.
n6- 1/4 nitrogen: kno3(nh4)2so4Concentration be the 1/4 of full nitrogen, i.e. respectively 707.5mg/l and 115.8mg/l,
The each 200mg/l of glu and ch.
n6- nitrogen-free: do not contain kno in culture medium3(nh4)2so4, do not contain glu and ch yet.
From table, result can be seen that kno in inducing culture3(nh4)2so4Concentration when being down to 3/4,4 parts for examination
In material, 3 parts of Callus yield has risen, and 1 part maintains an equal level；When being down to 1/2,2 parts of calluss in 4 parts of materials to be tested
Yield reaches peak, and only 1 part of Callus yield is slightly below full nitrogen.
Embodiment 4
Prepare the n of the modification of different ch and glu concentration6Inducing culture is (except kno3、(nh4)2so4, outside glu and ch, remaining
Constituent concentration is with embodiment 1), collect the microspores culture callus induction of a collection of barley material by embodiment 1, cultivate 21d
When Aspirate medium, in units of culture dish, the calluss obtaining are weighed, result see table.
The Callus yield (mg/ ware) of induction on different ch and glu concentration cultures
n6- full nitrogen: kno3(nh4)2so4Concentration be respectively 2830mg/l and 463mg/l, each 800mg/l of glu and ch.
n6- 800:kno3(nh4)2so4Concentration be respectively 707.5mg/l and 115.8mg/l, each 800mg/ of glu and ch
l.
n6- 1200:kno3(nh4)2so4Concentration be respectively 707.5mg/l and 115.8mg/l, glu and ch be each
1200mg/l.
n6- 1600:kno3(nh4)2so4Concentration be respectively 707.5mg/l and 115.8mg/l, glu and ch be each
1600mg/l.
n6- 2000:kno3(nh4)2so4Concentration be respectively 707.5mg/l and 115.8mg/l, glu and ch be each
2000mg/l.
To add the n of 1/4 inorganic nitrogen consumption6Culture medium as basic inducing culture, add variable concentrations glu and
Ch, compares the different impacts to microspores culture wound healing yield for the organic nitrogen usage amount in culture medium.In table, result shows, all confessions
The Callus yield of examination material improves with the raising of glu and ch concentration in inducing culture, containing glu and
The n of ch2000mg/l modification6In culture medium, Callus yield/ware is 1.3-9.0 times of full nitrogen culture medium.
Embodiment 5
The calluss that embodiment 4 obtains are transferred to differentiation on division culture medium, count the green regenerating plant that every ware obtains
Strain number amount, result see table.
The green Seedling quantity (strain/ware) of the calluss differentiation on different ch and glu concentration cultures
Culture medium " n6- full nitrogen ", " n6-800”、“n6-1200”、“n6- 1600 " and " n6- 2000 " implication is with embodiment 4.
In table, result shows, the green plantlet yield/ware of nearly all material to be tested increases with the raising of Callus yield
Plus, in the n changing containing glu and ch2000mg/l6In culture medium, green plantlet yield/ware is 1.1-8.6 times of full nitrogen culture medium.
Embodiment 6
Prepare different kno3、(nh4)2so4, glu and ch concentration modification n6Inducing culture is (except kno3、(nh4)2so4、
Outside glu and ch, remaining constituent concentration is with embodiment 1), the sporidiole callus induction group of 4 parts of barley materials is collected by embodiment 1
Knit, weigh after Aspirate medium during culture 28d, result see table.
The impact (mg/ ware) to wound healing yield for the different nitrogen sources in culture medium
n6- a:(nh4)2so4Concentration be 463mg/l, no kno3, glu and ch.
n6- n:kno3Concentration is 2830mg/l, no (nh4)2so4, glu and ch.
n6- an:kno3(nh4)2so4Concentration be respectively 2830mg/l and 463mg/l, no glu and ch.
n6The each 800mg/l of-o:glu and ch, no kno3(nh4)2so4.
n6- full nitrogen: kno3(nh4)2so4Concentration be respectively 2830mg/l and 463mg/l, each 800mg/l of glu and ch.
n6- ao:(nh4)2so4Concentration be 463mg/l, each 800mg/l of glu and ch, no kno3.
n6- no:kno3Concentration is 2830mg/l, each 800mg/l of glu and ch, no (nh4)2so4.
As can be seen from the above table, lacking kno3Or (nh4)2so4In the case of, with n6- full nitrogen group is compared, add glu and
Ch also can significantly improve wound healing yield (n6- ao and n6- no group).Additionally, inducing culture does not contain kno3(nh4)2so4Situation
Under (n6- o group), with n6- full nitrogen group is compared, and adds glu and ch and also can significantly improve wound healing yield.
Claims (12)
1. a kind of Fructus Hordei Vulgaris sporidiole callus inducing medium for inducing Fructus Hordei Vulgaris Microsporogenesis calluss, described lures
Lead culture medium according to n6Culture medium prescription is formulated it is characterised in that kno in described inducing culture3Concentration is 650-
2200mg/l, (nh4)2so4Concentration be 100-350mg/l, and described inducing culture is also added with the water of 400-2500mg/l
Solution casein and the L-Glutamine of 400-2500mg/l；With described inducing culture be also added with inositol 100-200mg/l, 2,4,
5-t and/or 2,4-d0.4-1.2mg/l, kt 0.3-0.8mg/l and maltose 60-120g/l；Wherein, when using 2,4,5-t
During with the mixture of 2,4-d, total concentration controls in 0.4-1.2mg/l.
2. inducing culture as claimed in claim 1 is it is characterised in that kno in described inducing culture3Concentration be 700-
1500mg/l, (nh4)2so4Concentration be 110-240mg/l.
3. inducing culture as claimed in claim 1 or 2 is it is characterised in that described inducing culture contains 37.3-
74.6mg/l na2The feso of edta and 27.8-55.6mg/l4·7h2o.
4. inducing culture as claimed in claim 1 or 2 is it is characterised in that in described inducing culture, the concentration of kt is
0.4-0.65mg/l, the concentration of maltose is 80-100g/l.
5. inducing culture as claimed in claim 1 is it is characterised in that the formula of described culture medium is as follows:
.
6. inducing culture as claimed in claim 5 is it is characterised in that kno3Concentration be 700-800mg/l, (nh4)2so4
Concentration be 110-160mg/l, the concentration of L-Glutamine is 1200-2000mg/l, and the concentration of hydrolysed casein is 1200-
The concentration of 2000mg/l, kt is 0.4-0.65mg/l, and/or the concentration of maltose is 80-100g/l.
7. inducing culture as claimed in claim 6 is it is characterised in that the concentration of kt is 0.4-0.5mg/l, and/or Fructus Hordei Germinatus
The concentration of sugar is 80-90g/l.
8. a kind of box set, this box set contains inducing culture and optional differentiation culture any one of claim 1-7
Base and Rooting and hardening-off culture base.
9. box set as claimed in claim 8 it is characterised in that described division culture medium be with the addition of 6-ba0.3-1.2mg/l,
The 2/3ms division culture medium of kt 0.5-2.0mg/l, naa 0.01-0.1mg/l and maltose 10-50g/l, ph is 5.5-6.2；
Described Rooting and hardening-off culture base is to be added with naa 0.02-0.08mg/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.
10. a kind of method for inducing and cultivating of Fructus Hordei Vulgaris sporidiole calluss is it is characterised in that the method includes using claim
Inducing culture inducing culture Fructus Hordei Vulgaris sporidiole described in any one of 1-7, thus obtain calluss.
A kind of 11. methods preparing Fructus Hordei Vulgaris regeneration plant are it is characterised in that the method includes:
(1) use the inducing culture inducing culture Fructus Hordei Vulgaris sporidiole any one of claim 1-7, thus obtaining wound healing
Tissue；With
(2) calluss being obtained with division culture medium incubation step (1), thus obtain regeneration plant.
12. methods as claimed in claim 11 are it is characterised in that methods described also includes:
(3) regeneration plant being obtained with Rooting and hardening-off culture base incubation step (2).
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| CN103947550B (en) * | 2014-04-20 | 2016-04-13 | 浙江省农业科学院 | The direct seedling tissue culture method of barley immature embryos and used medium |
| CN105684895B (en) * | 2014-11-28 | 2020-01-17 | 上海市农业科学院 | Construction method of haploid population of cereal crop |
| 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 |
| CN106489726A (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 differentiation culture based formulas |
| CN105340755B (en) * | 2015-12-04 | 2018-09-07 | 上海市农业科学院 | Microspore continuously cultivates high-frequency regeneration plant method in cereal crop single plant source |
| 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 |
| CN106538381B (en) * | 2016-09-30 | 2018-01-23 | 海南波莲水稻基因科技有限公司 | A kind of plant establishment strong seedling culture base and preparation method and application containing paclobutrazol |
| CN108085334B (en) * | 2016-11-17 | 2021-04-30 | 上海市农业科学院 | Improved method for transforming barley microspore by agrobacterium |
| CN108401903A (en) * | 2018-03-16 | 2018-08-17 | 上海市农业科学院 | A method of improving barley microspores culture callus yield and green seedling |
| CN109105258A (en) * | 2018-08-15 | 2019-01-01 | 上海市农业科学院 | A kind of cultural method of barley microspore |
| CN112273219B (en) * | 2020-10-10 | 2022-05-17 | 上海市农业科学院 | A method for binding F by hybridization1Method for rapidly obtaining stable homozygous nitrogen high-efficiency material by culturing microspore |
| CN113881698B (en) * | 2021-10-29 | 2023-04-25 | 上海市农业科学院 | Method for transforming barley microspore callus by using agrobacterium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106259A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Method for improving salt endurance of cereal crops |
-
2013
- 2013-11-22 CN CN201310596632.4A patent/CN103583369B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106259A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Method for improving salt endurance of cereal crops |
Non-Patent Citations (3)
| Title |
|---|
| Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare. The effects of ammonium nitrate, glutamine and asparagine as nitrogen sources;Finn Lok Olsen;《Carlsberg Research Communications》;19871231;第52卷(第6期);第393-404页 * |
| 大麦单倍体细胞水平与植株水平耐低氮性的关系;陆瑞菊等;《麦类作物学报》;20111231;第31卷(第2期);第292-296页 * |
| 大麦游离小孢子培养技术的优化及单倍体耐盐、耐低氮胁迫筛选体系的建立;陆瑞菊;《南京农业大学博士学位论文》;20121001;摘要，第26，31页 * |
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