CN103636505A - Complex breeding method of high-chlorophyll multi-tiller barley - Google Patents
Complex breeding method of high-chlorophyll multi-tiller barley Download PDFInfo
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Abstract
The invention provides a complex breeding method of barley with high chlorophyll content and/or large tiller number, a method for preparing barley green products and a method for improving the chlorophyll content and/or the tiller number of the barley. The method provided by the invention comprises the following steps: selecting spikelets with barley microspores in development single-nuclear early stage and/or middle stage, and performing treatment at the temperature of 2-10 DEG C for 10-30 days; harvesting the microspores, culturing the microspores and regenerating to obtain a homozygous doubled haploid; culturing the homozygous doubled haploid; and further screening to obtain the barley with the high chlorophyll content and/or the large tiller number, and preparing the barley into the required barley green products. According to the method, a plurality of excellent traits are polymerized through a hybridization technology, a microspore culture technology is utilized to perform fast homozygous stabilization on target traits, multi-index screening is performed under an artificial controllable environment, and a barley germplasm material with high chlorophyll content and multiple tillers is finally obtained. The material can be used as the excellent germplasm material for producing the barley green and other nutritional and health care products in a field planting or water culture way.
Description
Technical field
The invention belongs to agricultural technology field, be specifically related to the screening breeding method of a kind of high chlorophyll content and many stems tiller barley material.
Background technology
The seedling of wheat crops is a kind of Chinese traditional herbs, the record of China in < < Compendium of Materia Medica > > and < < typhoid fever summary > > the medical value of wheat seeding.1969, Japanese scholars reed original meaning show doctor has delivered < < gramineous plants greenery composition and as stable medicine, food research > > mono-literary composition, and is devoted to commercialized development.1991, he utilized the technology of " ultralow temperature fast spraying seasoning ", and barley seedling is squeezed the juice and is condensed into powder, is called " Barley Greeg " (Barely Green), and promotes to international market.Barley Greeg product is in the market that to take the tiller tender leaf (the high 20-30cm of wheat) of Sheng phase of the wheat crops such as barley or wheat be primary raw material, is aided with that the batchings such as dextrin process.Barley Greeg is rich in protein, chlorophyll, a large amount of organized enzyme (as SOD enzyme, catalase, amylase, protease etc.), beta carotene, multivitamin, and various trace elements (Guo Jianhua, 2008).
Modern biologic test and clinical research confirmation, Barley Greeg has following 9 aspect physiological functions: (1) promotes the restitution of damage dna; (2) anti-inflammatory analgesic; (3) strengthen immunity and anti-cancer, anticancer, radiation resistance; (4) suppress AIDS virus effect; (5) hypotensive; (6) antiulcer; (7) remove unnecessary free radical in body, anti-ageing and control cardiovascular and cerebrovascular disease; (8) prevent and treat diabetes; (9) accelerating wound healing, promotes hematopoiesis, eliminates halitosis (Qiao Wenjing, 2009).U.S. FDA approved yong barley leaves juice is as food supplement.In Japan, yong barley leaves juice product has obtained the sign (Niu Guangcai, 2011) of the healthy food of Japanese health association identification.Wheat leaf series of products have been developed at present abroad, as Semen Tritici aestivi fiber food, the short health food of barley leaf, wheat straw leaf beverage, Barley Greeg cola, blue or green wheat enzyme nutriment etc., Japan has also released the tonic food that adds dextrin, yeast, carrot meal, Korean ginseng powder in yong barley leaves juice powder.
Barley Greeg product has good alimentary health-care function, there are wide market prospects, the major production base of Barley Greeg is in the U.S., Canada, Australia etc., due to its pure natural property and health-care effect, catered to the pursuit of people " back to natures ", thereby fashionable North America, Europe, Australia, Southeast Asia, every annual sales amount has reached 23～2,700,000,000 dollars (yellow prime minister, 2003).The production of barley functional product will increase the added value of barley cultivation industry, makes increasing peasant income, it is reported, can produce fresh leaf 500kg for every mu, the output value can reach more than 1000 yuan, and farmers' income exceeds one times above (Wu Hongxia, 2002) than the conventional barley of plantation and other winter crops.
But the special-purpose barley variety of producing at present Barley Greeg lacks, and has seriously limited the development and utilization of Barley Greeg product.The seed selection of function barley at present does not also cause enough attention in China, and traditional breeding objective can not meet the requirement of function barley seed selection.Traditional breeding objective is mainly to consider that the economical character of kind is as Grain yield and quality, disease resistance etc., and takes from vegetative growth phase to produce the barley variety raw material of Barley Greeg special use, therefore, need to rethink its breeding goal.To Barley Varieties Applied for Barley Green seed selection, require the nutrient contents such as seedling growth is fast, and during jointing, vine yield is high, chlorophyll high.
Blade is that plant carries out photosynthetic vitals, and being plant, chlorophyll carries out photosynthetic important pigment, its effect is exactly to carry out luminous energy to catch, the height of its content not only can affect leaf look, and can directly affect photosynthetic efficiency, there are some researches show that chlorophyllous content is to become positively related with photosynthetic efficiency within the specific limits.Green plants is all to rely on photosynthesis to carry out biosynthesis, completes its morphogenesis and biomass accumulation.The chlcrophyll biosynthesis of green plants is one and participates in synthetic complex process by a series of enzymes, wherein participates in synthetic enzyme and gene more clearly in higher plant, but the hereditary pattern of control chlorophyll content is not clear.To the screening of chlorophyll content, be all to concentrate on land for growing field crops screening at present, adopt chlorophyll meter or chlorophyll fluorescence instrument to measure on the spot.There are some problems in this screening mode, there are some researches show that chlorophyllous content is subject to the impact of environmental factor obvious, the content of different growth periods also can change, land for growing field crops circumstance complication, planting conditions is subject to various factors impact and restriction, and chlorophyll content performance is also inconsistent, and chlorophyll is subject to recessive gene control to a great extent in addition, screening need to be carried out in high generation, and the time that this need to be longer obtains the offspring of isozygotying.
Except screening high chlorophyll content, its overground part biological yield is also an important indicator.The cauline leaf of tillering is the biological yield component part of acrial part in seedling stage, improve its biological yield, needs to select to tiller many, the eugonic material of cauline leaf.The screening of Barley Breeding concentrates on the growth and maturity phase mostly, and the overground part biological yield in seedling stage and the screening of chlorophyll content be have not been reported.
The present invention will provide a kind of some problems that overcome the screening of current land for growing field crops, enforcement is effective, the method for the barley of many stems of quick breeding high chlorophyll content tiller, comprise: by hybridization technique by a plurality of excellent proterties polymerizations, utilize microspore culture to isozygoty fast stable to objective trait, under artificial controllable environment, carry out many index screenings, finally obtain the barley germplasm materials of many stems of high chlorophyll content tiller.This material can be used as the elite germplasm material of the nutrition and health care productions such as Barley Greeg by field planting or water planting mode.
Summary of the invention
The object of the invention is to barley efficient, that Quick supplies high chlorophyll content, many stems tiller number, and the described proterties energy genetic stability of described barley.The present invention realizes this object by the following method, and described method has stability and repeatability.
One aspect of the present invention provides the method for the barley of high chlorophyll content and/or many stems tiller number to comprise:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, low temperature treatment 2-3 week;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys;
(3) doubled haploid that isozygotys described in water planting;
Obtain with screening the barley that chlorophyll content is high and/or stem tiller number is many.
The present invention provides a kind of method of preparing Barley Greeg product on the other hand, and described method comprises:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, low temperature treatment 2-3 week;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys; With
(3) doubled haploid that isozygotys described in water planting, screening obtains the barley that chlorophyll content is high and/or stem tiller number is many; With
(4) described barley is prepared into Barley Greeg product.
Further aspect of the present invention provides a kind of method that improves barley chlorophyll content and/or stem tiller number, and described method comprises:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, process 10-30 days for 2-10 ℃;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys;
(3) doubled haploid that isozygotys described in water planting, obtains regeneration plant;
Wherein, described regeneration plant is the plant that chlorophyll content improves and/or stem tiller number increases.
Should be understood that described " chlorophyll content raisings " and " stem tiller number increases " are for initial barley material, with respect to the described barley microspore of step (1), without the inventive method, process and for the chlorophyll content and stem tiller number of the barley of acquisition.
In a specific embodiment, said method of the present invention also comprises before in step (1):
(a) choose that a collection of leaf look dark green, seedling growth is vigorous, the more barley material of tillering, after water planting, measure the chlorophyll content of 21 heart stages of leaf, to tillering stage, start to add up tiller number, plant height and/or basal part of stem girth, according to these statistical indicators, filter out that chlorophyll content is high, tiller number is many, the kind rapidly of growing; With
(b) cultivate described kind, obtain the described microspore of step (1); With
(c) if barley material only has, chlorophyll content is high, tiller number is many, an objective trait in growth rapidly, after pollen maturation, carry out pollination hybridization mutually, obtain hybridization F0 for seed, breed this F0 for seed, obtain F1 generation plant, from this F1 generation plant, obtain the described microspore of step (1).
In a specific embodiment, in said method of the present invention, the barley that microspore is high available from chlorophyll content and stem tiller number is many.
In a specific embodiment, in said method of the present invention, described step (2) comprising:
(2a) the described tassel of sterilizing, then cleans;
(2b) from the separated microspore of described tassel;
(2c) in room temperature, dark processing microspore 36-60 hour;
(2d) with the inducing culture microspore that suspends, and suspension is inoculated in inducing culture to callus induction; With
(2e) induction gained callus is proceeded to differential medium, cultivate to obtain regrowth, doubled haploid isozygotys described in.
In a specific embodiment, in said method of the present invention, during separated microspore, the CaCl that adds tassel and contain 4-8% mannitol, 1.0-1.5g/L in test tube
2with the extract of 2-(N-morpholine) ethyl sulfonic acid of 0.8-1.2g/L, with the high speed disperser rotary-cut that exceeds the speed limit, filter, centrifugal filtrate, thus obtain microspore.
In a specific embodiment, in said method of the present invention, inducing culture be take N6 medium as minimal medium, is wherein added with 6-Furfurylaminopurine 0.3-0.8mg/L, 2,4-dichlorphenoxyacetic acid 0.5-1.5mg/L and maltose 70-120g/L; Differential medium be take MS as minimal medium, is wherein added with 6-benzyl aminoadenine 0.3-0.8mg/L, KT 1.0-2.0mg/L, methyl α-naphthyl acetate 0.03-0.08mg/L and maltose 15-50g/L, with 3-8g/L agar powder, solidifies.
In a specific embodiment, in said method of the present invention, step (3) also comprises afterwards:
The regeneration plant that microspores culture is obtained is put into barley standard liquid nutrient and is cultivated, and again screens, and selects the barley that chlorophyll content is high and/or stem tiller number is many, and collects its seed.
In a specific embodiment, described screening is again to adopt water planting mode to screen under artificial controllable environment.
In a specific embodiment, described artificial controllable environment comprises: in the controlled environment chamber, adopt fluorescent tube illumination, the about 1500-2500lx of intensity of illumination, preferred about 1800-2000lx; Keep room temperature about 18-22 ℃ on daytime, preferably approximately 20 ℃, evening about 16-20 ℃, preferably 18 ℃, the about 1-2 ℃ of temperature fluctuation at daytime, night.
In a specific embodiment, described Barley Greeg product is selected from: barley fiber food, the short health food of barley leaf, wheat straw leaf beverage, Barley Greeg cola, blue or green wheat enzyme nutriment, and the tonic food that adds dextrin, yeast, carrot meal, Korean ginseng powder in yong barley leaves juice powder.
In a specific embodiment, in said method of the present invention, the microspore obtaining uses pretreatment fluid in room temperature, dark pretreatment 36-60 hour, wherein, and the CaCl that described pretreatment fluid contains 4-8% mannitol, 1.0-1.5g/L
22-(N-morpholine) ethyl sulfonic acid with 0.8-1.2g/L.
Preferably, the present invention obtains barley strain chlorophyll content and stem tiller number all improve.
The method of the invention provides has following characteristics:
1. to draw materials be generally in the 6-7 leaf phase to Barley Greeg products material, so the water planting in 6-8 week can complete incubation, reaches predetermined growth period, during can conveniently measure and draw materials.According to statistical indicator described herein, filter out respectively the kind of proterties excellence.What the present invention adopted is artificial controlled unified condition of culture, completes in the controlled environment chamber, not controlled by season, and screening has stability and repeatability.21 heart stages of leaf be highstrung screening period, interracial difference is obvious, unconspicuous kind also can compare in the METHOD FOR CONTINUOUS DETERMINATION in later stage.Select tillering number as an index of evaluating the upperground part biomass simultaneously.
2. screening target is carried out around Parameters of Barley Seedling chlorophyll content and two targets of acrial part biological yield, does not investigate after material the proterties such as grain yield breeding time.
Pair Preliminary screening contain better objective trait barley material, adopt microspore culture to obtain doubled haploid (DH) strain of isozygotying, all proterties of germplasm are isozygotied completely, recessive character can show the present age.
4. the barley material of pair single excellent objective trait adopts hybridization technique polymerization objective trait.
5. according to objective trait, select different indexs to screen in a large number, screening relates to just material selection, the DH plant of isozygotying, the separated offspring of hybridization polymerization proterties.
6. the common 2-3 consuming time of existing land for growing field crops triage techniques, even the longer time, just likely screens the barley material that can be used to prepare Barley Greeg product that obtains having above-mentioned objective trait, but screening deficient in stability and repeatability.The present invention, due to the specially treated of microspore can be shortened to this screening time greatly, can realize conventionally in 1 year.
Embodiment
The above-mentioned method of the present invention comprises:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, low temperature treatment 10-30 days;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys; With
(3) doubled haploid that isozygotys described in water planting.
Described " low temperature " is often referred to 2-10 ℃, for example 3-5 ℃.Low temperature treatment refers to place at low temperatures the described small ear of fresh harvesting.The time of low temperature treatment is 10-30 days normally, such as can be 14-21 days not etc.
Microspore is taken from the barley that chlorophyll content is high, tiller number is many, the parent material seed plantation rapidly of growing obtains conventionally.
Herein, " chlorophyll content is high " refers to regard to screened batch, and the plant that chlorophyll content is relatively high for example, refers to before screen batch Determination of Chlorophyll content comes 20% strain, preferably front 10%, more preferably front 5%.Certainly can be selected from the highest or relatively the highest several strains of chlorophyll content.
Herein, " tiller number is many " refer to that tiller number is heterogeneous to more plant with regard to screened batch, for example, refer to screen batch in tiller number come before 20% strain, preferably front 10%, more preferably front 5%.
Herein, " growth rapidly " with regard to screened batch, the comparatively faster plant of growth rate, for example, refer to screen batch in growth rate come before 20% strain, preferred front 10%, more preferably front 5%.
Conventionally, can be first according to objective trait, choose that a collection of leaf look dark green, seedling growth is vigorous, the more barley material of tillering, after water planting, measure the chlorophyll content of 21 heart stages of leaf, to tillering stage, start to add up tiller number, plant height and/or basal part of stem girth, according to these statistical indicators, filter out respectively the kind of proterties excellence, for example, filter out that chlorophyll content is high, tiller number is many, the kind rapidly of growing.
Can adopt the standard barley mill water culture nutrient solution (improvement Hoagland solution) of having reported to carry out water planting, this nutrient source formula is as follows:
NH
4nO
3(0.2mM), KNO
3(5mM), Ca NO
3.2 (2mM), MgSO
4(2mM), KH
2pO
4(0.1mM), Na
2siO
3(0.5mM), NaFe (III) – EDTA (0.05mM), H
3bO
3(0.01mM), MnCl
2(0.005mM), ZnSO
4(0.005mM), CuSO
4(0.0005mM), Na
2moO
3(0.0001mM), use front pH to be adjusted to 6.0 left and right with watery hydrochloric acid.
Water planting is carried out in can be in the controlled environment chamber.The illumination of employing fluorescent tube, the about 2000lx of intensity of illumination, keeps room temperature degree on daytimes 20, and spend evenings 18, temperature fluctuation 1-2 degree.
Then, plant the seed (being parent material seed) of this kind, for microspores culture, draw materials.Can plant large Tanaka, or, if plant screening material is few, also can plantation in the controlled environment chamber after screening.
For the material that only has one of them objective trait excellence, after pollen maturation, can carry out pollination hybridization (positive and negative hybridization) mutually, obtain hybridization F0 for seed.Land for growing field crops or phytotron plantation F0, for seed, obtain F1 generation plant, for microspores culture, draw materials.
The chlorophyll content that can plant from land for growing field crops or phytotron is high, tiller number is many, choose middle part little Hua microspore development small ear early stage in monokaryon, mid-term in the kind of growing rapidly carries out low temperature treatment 10-30 days, as mentioned before.
After low temperature treatment, can adopt technology known in the art to obtain microspore.Comprise sterilization, high speed centrifugation etc.
Herein, sterilization is conventionally with 75% alcohol-pickled 20-60 second, the saturated Eusol 10-20 minute that sterilizes.Conventionally with 75% alcohol-pickled 30 seconds, saturated Eusol was sterilized 15 minutes.Known other sterilization means of those skilled in the art.
After sterilization, available aseptic water washing several, for example 3-4 time.Then, each test tube connects 5-15 tassel, for example 10 tassels of every pipe.Pour 10-20ml, preferred 15ml extract into, with the high speed disperser rotary-cut that exceeds the speed limit, filter (for example, with 150 eye mesh screens, filtering), centrifugal filtrate (for example, with the centrifugal 5min of 100 * g, repeating 3 times), collection microspore.Extract preferably contains the CaCl of 4-8% mannitol, 1.0-1.5g/L
2mES(2-(N-morpholine) ethyl sulfonic acid with 0.8-1.2g/L).In a specific embodiment, extract is the CaCl of 6% mannitol, 1.1g/L preferably
2mES with 0.976g/L.
Optionally use pretreatment fluid for example, in room temperature (25 ℃), dark pretreatment 36-60 hour, conventionally process 2 days.The same extract of composition of pretreatment fluid.
Then, optionally by 21%(quality percent by volume) maltose purifies microspore, then with inducing culture washing 1 time, then with inducing culture by microspore Auto-regulating System of Density of Heavy Medium to 0.5-2.0 * 10
5/ ml, preferably 1.0 * 10
5/ ml, gets about 1ml microspore suspension and is inoculated in culture dish (for example, in the culture dish of 35 * 15mm), and after sealing, room temperature (25 ℃) is secretly cultivated, callus induction; Then proceed to differential medium and obtain regrowth.Obtaining plant is herein the doubled haploid that isozygotys (DH) that unicellular cultivation obtains.Detected monoploid can directly double to become doubled haploid with colchicine treatment.
Inducing culture is with N
6medium is minimal medium, is wherein added with 6-Furfurylaminopurine (KT) 0.3-0.8mg/L, 2, and 4-dichlorphenoxyacetic acid (2,4-D) 0.5-1.5mg/L and maltose 70-120g/L.In an embodiment, inducing culture is added with KT 0.4-0.6mg/L and 2,4-D0.8-1.2mg/L, and maltose 80-100g/L.In an embodiment, inducing culture is added with KT 0.5mg/L and 2,4-D 1.0mg/L, and maltose 90g/L.
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 |
Can press voluntarily above-mentioned formulated inducing culture of the present invention, 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.It will be appreciated by those skilled in the art that, can make suitable change to the concentration shown in above-mentioned table 2, 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,, with KH
2pO
4for example, every liter of N
6in medium, can contain the KH of about 380-420mg
2pO
4(5% amplitude of fluctuation).The concentration of other composition is so change also.In addition N,
6composition in medium also can be used the same or analogous composition of function and character to be replaced.
Differential medium be take MS as minimal medium, is wherein added with 6-benzyl aminoadenine (6-BA) 0.3-0.8mg/L, KT1.0-2.0mg/L, methyl α-naphthyl acetate (NAA) 0.03-0.08mg/L and maltose 15-50g/L, with 3-8g/L agar powder, solidifies.
In an embodiment, differential medium is added with 6-benzyl aminoadenine (6-BA) 0.4-0.6mg/L, KT 1.3-1.8mg/L, methyl α-naphthyl acetate (NAA) 0.03-0.06mg/L and maltose 20-40g/L, with 4-6g/L agar powder, solidifies.
In an embodiment, differential medium is added with 6-BA 0.5mg/L, KT 1.5mg/L, NAA 0.05mg/L and maltose 30g/L, with 6g/L agar powder, solidifies.
The formula of MS medium as shown in table 2 below (unit: mg/L):
Table 2
| 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.It will be appreciated by those skilled in the art that, can make suitable change to the concentration shown in above-mentioned table 2, 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.Composition in inducing culture also can be used the same or analogous composition of function and character to be replaced.
The regeneration plant that microspores culture is obtained (when Length of shoot and root surpasses 2cm) shifts out from medium, and clear water is cleaned root agar, with sponge strip parcel foundation portion, be placed on floating plate, put into barley standard liquid nutrient and cultivate, phytotron is controlled growing environment condition, again screens.The material with excellent proterties that this time screening obtains can genetic stability, and can screen in single part of material all more satisfactory target materials of two proterties.The plant screening can be transplanted the cultivation of basin alms bowl earth, and phytotron is planted to Grain Ripening, results single-strain seed, the same breeding conservation.
Optionally, can plant described seed, thus the material of results high chlorophyll content, many stems tiller.
The condition of water planting, comprises illumination, temperature, culture fluid etc. as mentioned before.
The regeneration plant obtaining by cultivating microspore, this material is the doubled haploid (DH) being isozygotied by a set of chromosome doubling, and parent's genetic character is isozygotied completely, and recessive character can be in contemporary plant performance.
Therefore, the present invention also comprises barley strain and the seed thereof that adopts the inventive method to prepare, and the Barley Greeg product that adopts this barley strain to prepare, include but not limited to that the short health food of barley fiber food, barley leaf, wheat straw leaf beverage, Barley Greeg are laughable, blue or green wheat enzyme nutriment, and the tonic food etc. that adds dextrin, yeast, carrot meal, Korean ginseng powder in yong barley leaves juice powder.
Below by the mode with specific embodiment, present invention is described.Should be understood that following examples are only illustrative, can make various modifications and changes to the present invention in the situation that not departing from spirit and scope of the invention.Scope of the present invention is limited by the application's claim.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: parental line selection
According to field planting barley material range estimation situation, choose 200 parts of the materials that leaf look dark green, seedling growth is vigorous, tiller number is many, minute individual plant results seed.Get 100, seed, surface adopts 75% ethanol to soak 30 seconds, 1% clorox sterilization 15 minutes, distilled water flushing is clean, is placed on moistening gauze and spends the night in the vernalization of room temperature dark place, after germination, choose robust growth, seedling 30 strains that radical bud length is greater than 1 centimetre, wrap up seed with sponge strip, and (floating plate can punch by 5 centimetres of spacing with cystosepiment to be placed in floating plate, 1.5 centimetres of diameters) in circular hole, be placed in nutrient solution and cultivate.The standard barley mill water culture nutrient solution that nutrient solution employing has been reported (improvement Hoagland solution, fill a prescription as follows:
NH
4nO
3(0.2mM); KNO
3(5mM); Ca NO
3.2 (2mM); MgSO
4(2mM); KH
2pO
4(0.1mM); Na
2siO
3(0.5mM); NaFe (III) – EDTA (0.05mM); H
3bO
3(0.01mM); MnCl
2(0.005mM); ZnSO
4(0.005mM); CuSO
4(0.0005mM); Na
2moO
3(0.0001mM). before using, pH is adjusted to 6.0 left and right with watery hydrochloric acid.
The illumination of phytotron employing fluorescent tube, the about 2000lx of intensity of illumination, the air-supply of large-size air conditioning temperature control, keeps room temperature degree on daytimes 20, and spend evenings 18, temperature fluctuation 1-2 degree.
When seedling grows to 2 leaves during 1 heart stage, with Japanese SPAD-502 hand-held chlorophyll meter, measure the 2nd leaf, each plant METHOD FOR CONTINUOUS DETERMINATION central region 10 times, reads mean value as chlorophyll content value, and this, storeroom difference was obvious in period.Each week is measured once 2 leaves as method later, and extremely whole cultivation finishes (while growing to 6-7 leaf).Tiller after beginning, add up weekly tiller number, measure plant height and lobe numbers simultaneously and evaluate its growth rate.
After cultivation finishes, all material determination data is carried out to statistical analysis, find out the material with significant difference.
Embodiment 2: screening parent plantation
Selection has the material of one of above-mentioned excellent proterties or several (growth rapidly for high chlorophyll, many stems tiller) as the parent material for hybridizing, then field seeding.Select size evenly, the planting seed of full seed is in land for growing field crops, spacing 3cm, line-spacing 30cm.In soil containing organic 34.0g/kg, full nitrogen 2.41g/kg, available nitrogen 37.30mg/kg, basal dressing 20kg/ mu before sowing, tillering stage 10kg/ mu urea, during can its leaf look of sampling Detection and overground part biological yield.
Embodiment 3: hybridization
Two parts of materials that carry different target proterties (high chlorophyll or many tillers) are carried out to positive and negative hybridization, concrete grammar is when pollen maturation, cuts off maternal plant tassel grain husk shell, gets paternal plant little Hua pollen and pollinates, carry out listing mark, the ripe rear hybrid seed of results separately simultaneously.
Embodiment 4:F1 is for plant breeding
Hybrid seed the same in sowing season kind in land for growing field crops, obtain F1 generation growth plant, as Isolated microspore culture materials.
Embodiment 5: microspores culture obtains the DH plant of isozygotying
From large Tanaka, choose middle part little Hua microspore development small ear early stage in monokaryon, mid-term and carried out for 5 degree low temperature treatment 2-3 weeks, during inoculation, tassel was with 75% alcohol-pickled 30 seconds, saturated Eusol sterilization 15 minutes, aseptic water washing 3-4 time, each test tube connects 10 tassels, pour 15ml extract into and (contain 6% mannitol, 1.1g/L CaCl
2with 0.976g/L MES), with the high speed disperser rotary-cut that exceeds the speed limit, with 150 eye mesh screens, to filter, filtrate, with the centrifugal 5min of 100 * g, is repeated 3 times, collection microspore.
Microspore uses pretreatment fluid in 25 ℃, dark pretreatment 2d.
Before cultivating, microspore is first purified with 21% maltose, then washs 1 time with inducing culture, then with inducing culture by microspore Auto-regulating System of Density of Heavy Medium to 1.0 * 10
5/ ml, gets 1ml microspore suspension and is inoculated in culture dish (35 * 15mm), the sealing of Parafilm film, 25 ℃ of dark cultivations, callus induction.
Inducing culture is with the N of improvement
6medium is minimal medium, is wherein added with KT 0.5mg/L and 2,4-D 1.0mg/L, maltose 90g/L.
Callus is proceeded to differential medium and obtain regrowth.Differential medium be take MS as minimal medium, is wherein added with 6-BA 0.5mg/L, KT 1.5mg/L, NAA 0.05mg/L and maltose 30g/L, with 6g/L agar powder, solidifies.
The plant that obtains microspore regeneration by cultivation, this material is the doubled haploid (DH) being isozygotied by a set of chromosome doubling, and parent's genetic character is isozygotied completely, and recessive character can be in contemporary plant performance.
Can the samely carry out water planting and again screen the barley material that obtains two objective trait polymerizations (high chlorophyll content and many tillers).
The screening of embodiment 6:DH plant
The regeneration plant that microspores culture is obtained (when Length of shoot and root surpasses 2cm) shifts out from medium, clear water is cleaned root agar, with sponge strip parcel foundation portion, be placed on floating plate, putting into barley standard liquid nutrient cultivates, phytotron is controlled growing environment condition, and the same step is screened again.The material with excellent proterties that this time screening obtains can genetic stability, and can screen in single part of material all more satisfactory target materials (two characteristic indexs see the following form) of two proterties.The plantlet of transplant basin alms bowl earth cultivation screening, phytotron is planted to Grain Ripening, results single-strain seed, the same breeding conservation.
Choose not through the original strain of hybridization and microspores culture in contrast be unscreened the seed that roguing is (strain), by preceding method, germinate, after seedling, adopt under identical mill water culture nutrient solution and artificial climate growth conditions, be measured in the same method chlorophyll and individual plant stem tiller and count index and compare.
Following table 3 has shown chlorophyll relative content (adopting the test of SPAD method) and the individual plant stem tiller number of the different strains that adopt the inventive method and contrast method acquisition.
Table 3
The result of table 3 shows, through barley material chlorophyll content and the stem tiller number of the inventive method seed selection, all exceeds the contrast barley material of processing without the inventive method far away.For example, with regard to chlorophyll content, the chlorophyll content of barley of the present invention is with respect to the chlorophyll content of contrast barley 20% the raising (relatively strain L3 and strain L10) of at least having an appointment, the highest about 85%(relatively strain L2 and the L8 of improving); Equally, the stem tiller number of barley of the present invention is with respect to the stem tiller number of contrast barley 17% the increase (relatively strain L3/L4 and strain L6, and the highest about 230%(comparison strain L1 and the strain L7 of increasing) of at least having an appointment.And, from the data of table 3, can find out, the chlorophyll content of the barley of processing through the inventive method and stem tiller number are general all higher than the chlorophyll content and the stem tiller number that contrast barley.
Claims (10)
1. a method of producing the barley of high chlorophyll content and/or many stems tiller number, described method comprises:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, process 10-30 days for 2-10 ℃;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys;
(3) doubled haploid that isozygotys described in water planting; With
(4) screening obtains the barley that chlorophyll content is high and/or stem tiller number is many.
2. a method of preparing Barley Greeg product, described method comprises:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, low temperature treatment 2-3 week;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys; With
(3) doubled haploid that isozygotys described in water planting, screening obtains the barley that chlorophyll content is high and/or stem tiller number is many; With
(4) described barley is prepared into Barley Greeg product.
3. a method that improves barley chlorophyll content and/or stem tiller number, described method comprises:
(1) get the barley microspore development small ear in and/or mid-term early stage in monokaryon, process 10-30 days for 2-10 ℃;
(2) results microspore, cultivates microspore, and regeneration obtains the doubled haploid that isozygotys;
(3) doubled haploid that isozygotys described in water planting, obtains regeneration plant;
Wherein, described regeneration plant is the plant that chlorophyll content improves and/or stem tiller number increases.
4. the method as described in any one in claim 1-3, is characterized in that, described method also comprises before in step (1):
(a) choose that a collection of leaf look dark green, seedling growth is vigorous, the more barley material of tillering, after water planting, measure the chlorophyll content of 21 heart stages of leaf, to tillering stage, start to add up tiller number, plant height and/or basal part of stem girth, according to these statistical indicators, filter out that chlorophyll content is high, tiller number is many, the kind rapidly of growing; With
(b) cultivate described kind, obtain the described microspore of step (1); With
(c) if barley material only has, chlorophyll content is high, tiller number is many, an objective trait in growth rapidly, after pollen maturation, carry out pollination hybridization mutually, obtain hybridization F0 for seed, breed this F0 for seed, obtain F1 generation plant, from this F1 generation plant, obtain the described microspore of step (1).
5. the method as described in any one in claim 1-4, is characterized in that, described step (2) comprising:
(2a) the described tassel of sterilizing, then cleans;
(2b) from the separated microspore of described tassel;
(2c) in room temperature, dark processing microspore 36-60 hour;
(2d) with the inducing culture microspore that suspends, and suspension is inoculated in inducing culture to callus induction; With
(2e) induction gained callus is proceeded to differential medium, cultivate to obtain regrowth, doubled haploid isozygotys described in.
6. method as claimed in claim 5, it is characterized in that, in step (2b), during separated microspore, in test tube, add tassel and extract, with the high speed disperser rotary-cut that exceeds the speed limit, filter, centrifugal filtrate, thus obtain microspore, wherein, the CaCl that described extract contains 4-8% mannitol, 1.0-1.5g/L
22-(N-morpholine) ethyl sulfonic acid with 0.8-1.2g/L.
7. the method as described in claim 5 or 6, is characterized in that,
Inducing culture is with N
6medium is minimal medium, is wherein added with 6-Furfurylaminopurine 0.3-0.8mg/L, 2,4-dichlorphenoxyacetic acid 0.5-1.5mg/L and maltose 70-120g/L;
Differential medium be take MS as minimal medium, is wherein added with 6-benzyl aminoadenine 0.3-0.8mg/L, KT1.0-2.0mg/L, methyl α-naphthyl acetate 0.03-0.08mg/L and maltose 15-50g/L, with 3-8g/L agar powder, solidifies.
8. the method as described in any one in claim 1-7, is characterized in that, step (3) also comprises afterwards:
The regeneration plant that microspores culture is obtained is put into barley standard liquid nutrient and is cultivated, and again screens, and selects the barley that chlorophyll content is high and/or stem tiller number is many, and collects its seed.
9. method as claimed in claim 2, it is characterized in that, described Barley Greeg product is selected from: barley fiber food, the short health food of barley leaf, wheat straw leaf beverage, Barley Greeg cola, blue or green wheat enzyme nutriment, and the tonic food that adds dextrin, yeast, carrot meal, Korean ginseng powder in yong barley leaves juice powder.
10. method as claimed in claim 6, is characterized in that, the microspore obtaining uses pretreatment fluid in room temperature, dark pretreatment 36-60 hour, wherein, and the CaCl that described pretreatment fluid contains 4-8% mannitol, 1.0-1.5g/L
22-(N-morpholine) ethyl sulfonic acid with 0.8-1.2g/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106106157A (en) * | 2016-06-29 | 2016-11-16 | 无锡南理工科技发展有限公司 | A kind of breeding method of crisp stem Fructus Hordei Vulgaris |
| CN109105258A (en) * | 2018-08-15 | 2019-01-01 | 上海市农业科学院 | A kind of cultural method of barley microspore |
| CN112273219A (en) * | 2020-10-10 | 2021-01-29 | 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 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1418950A (en) * | 2002-12-26 | 2003-05-21 | 北京市海淀区植物组织培养技术实验室 | Method for obtaining haploid plant strain by culturing anther or pollen |
| CN101558735A (en) * | 2009-06-03 | 2009-10-21 | 西北农林科技大学 | Novel method for cultivating wheat stripe rust resistant polygene polymerized flower |
| CN101904296A (en) * | 2010-07-14 | 2010-12-08 | 江苏沿海地区农业科学研究所 | Breeding method of salt-tolerant malting barley variety |
| CN101946629A (en) * | 2010-09-26 | 2011-01-19 | 甘肃省农业科学院生物技术研究所 | Method for rapidly obtaining pure line of hybrid wheat |
| CN102106257A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Method for screening anti-fusarium graminearum toxin of wheat crops |
| CN102106258A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Method for improving low-nitrogen tolerance character of wheat crops |
| CN103249836A (en) * | 2010-09-24 | 2013-08-14 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-elated traits and method for making the same |
| WO2013123051A1 (en) * | 2012-02-14 | 2013-08-22 | The Board Of Trustees Of The Leland Stanford Junior University | Method for modulating the number of archesporial cells in a developing anther |
-
2013
- 2013-12-20 CN CN201310713460.4A patent/CN103636505B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1418950A (en) * | 2002-12-26 | 2003-05-21 | 北京市海淀区植物组织培养技术实验室 | Method for obtaining haploid plant strain by culturing anther or pollen |
| CN101558735A (en) * | 2009-06-03 | 2009-10-21 | 西北农林科技大学 | Novel method for cultivating wheat stripe rust resistant polygene polymerized flower |
| CN102106257A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Method for screening anti-fusarium graminearum toxin of wheat crops |
| CN102106258A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Method for improving low-nitrogen tolerance character of wheat crops |
| CN101904296A (en) * | 2010-07-14 | 2010-12-08 | 江苏沿海地区农业科学研究所 | Breeding method of salt-tolerant malting barley variety |
| CN103249836A (en) * | 2010-09-24 | 2013-08-14 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-elated traits and method for making the same |
| CN101946629A (en) * | 2010-09-26 | 2011-01-19 | 甘肃省农业科学院生物技术研究所 | Method for rapidly obtaining pure line of hybrid wheat |
| WO2013123051A1 (en) * | 2012-02-14 | 2013-08-22 | The Board Of Trustees Of The Leland Stanford Junior University | Method for modulating the number of archesporial cells in a developing anther |
Non-Patent Citations (2)
| Title |
|---|
| GENEVIÈVE WOJNAROWIEZ，ET AL.: "Barley anther culture: assessment of carbohydrate effects on embryo yield, green plant production and differential plastid development in relation with albinism", 《J.PLANT PHYSIOL.》 * |
| 王仁杯等: "不同大麦品种的麦绿素专用价值初步研究", 《浙江农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN106106157A (en) * | 2016-06-29 | 2016-11-16 | 无锡南理工科技发展有限公司 | A kind of breeding method of crisp stem Fructus Hordei Vulgaris |
| CN109105258A (en) * | 2018-08-15 | 2019-01-01 | 上海市农业科学院 | A kind of cultural method of barley microspore |
| CN112273219A (en) * | 2020-10-10 | 2021-01-29 | 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 |
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|---|---|
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