CN106561451A - Picea koraiensis nakai somatic embryo induced plant regeneration method and application - Google Patents
Picea koraiensis nakai somatic embryo induced plant regeneration method and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention discloses a picea koraiensis nakai somatic embryo induced plant regeneration method and application, and relates to a picea koraiensis nakai plant regeneration method and application thereof. The problems that by means of existing methods, the reproductive rate of picea koraiensis nakai is low, the reproductive cycle is long, and the extraction rate of flavonoid compounds of the picea koraiensis nakai is low are solved. According to the picea koraiensis nakai somatic embryo induced plant regeneration method and application, the germination rate of somatic embryos is increased by conducting drying treatment before picea koraiensis nakai somatic cell induction, the reproductive cycle is shortened by improving culture mediums and the culture condition, and low-cost raw materials are adopted so that the method and application can be suitable for industrial production; the PAI activity is improved by changing the temperature, light and inductive agents, accordingly, the synthetic amount of the flavonoid compounds of the picea koraiensis nakai is increased, and a foundation is laid for increasing the extraction rate of the flavonoid compounds subsequently; and the flavonoid compounds in the picea koraiensis nakai is extracted through a homogenate method combined with a microwave mode, the extraction rate of the flavonoid compounds is increased, and impurities are decreased.
Description
Technical field
The present invention relates to a kind of Picea koraiensis Nakai plant regeneration method and its application.
Background technology
Picea koraiensis Nakai (Picea koraiensis Nakai) also known as Folium Styracis Suberifoliae are smelly, are Pinaceae Picea aiphylliums, tree crown
Steeple shape, bough tiltedly stretches or open and flat, there is obvious fixing fillet shape pulvinus on sprig；Bud conico-acuminate, leaf taper, tip point, many spokes
Stretching, extension is penetrated, cross section rhombus has on four sides spiracle line.Cone ovate is cylindrical or cylindric square is circular, it is ripe after it is greenish-yellow brown or brown
Color；There is the long wing of film quality fruit scale veneer matter, triangle obovate, seed upper end.It is distributed in the northeast Xiaoxinanlin Mountains, Jilin mountain area height above sea level
1400--1800 meter bands, Korea and Ussuri Region are also produced.More resistance to shade, shallow root.Adaptability is stronger, and natural pond is removed in areal area
Outside the tailo of poolization area and drying, ridge, can grow under different condition area.The seeds attitude is graceful, and tool views and admires special
Property and gardens setting.
Somatic embryo is one of important channel of plant regeneration in cell engineering, can be used as the receptor of genetic transformation
System, while being also the ideal test system built up to study totipotent cell differentiation and its form.In application, somatic cell
Embryo is a kind of effectively extensive asexual reproduction method.In the plant of success inductor embryogenesis at present, draft
Plant account for great majority, and existing more than 40 kinds of xylophyta obtains somatic embryo, is especially difficult with conventional vegetative propagation technique
Research and achieve the progress for attracting people's attention in the acerose body embryo taken root, the different acerose explant success of kind more than 20 there is
Induce somatic embryo.
One complete plant somatocyte embryo generation technique includes the induction of embryo callus, embryo callus
The four-stages such as propagation, the differentiation of somatic embryo and the sprouting of mature somatic embryo；Wherein, the sprouting stage of mature somatic embryo
As the final step of whole technical system, the height and sprouting quality of somatic embryo germination rate, final body embryo is directly decide
The success or failure that the harvest yield of Seedling and whole body embryo technical system build.
But, existing Picea koraiensis Nakai renovation process breeds that efficiency is low, and germination rate is low, and it is long to sprout the cycle, is unsuitable for industrialization
Production problem, is unfavorable for breeding for Picea koraiensis Nakai.
And, for improving Picea koraiensis Nakai somatic cell germination rate, existing method does not provide good solution, greatly
Part means are improved in terms of culture medium and condition of culture, can so increase cost, and effect is not also very aobvious
Write.
In addition, the existing Flavonoid substances for Picea koraiensis Nakai are extracted, not good method can improve its extraction
Rate.
The content of the invention
The present invention provides a kind of Picea koraiensis Nakai somatic embryo induction plant regeneration to solve above-mentioned problem
Method and application.
A kind of Picea koraiensis Nakai somatic embryo induction plant regeneration method of the present invention, it is followed the steps below：
First, in mid-June to mid-July, clone cone is plucked, after sterilization, takes bulb immature zygotic embryos, as
Somatic embryo inducement explant material；
2nd, drying and other treatment is carried out to the explant material of step one：
1) explant material is placed in the culture dish for being placed with multi-layer filter paper, is sealed on culture dish with sealed membrane, then will
Culture dish is placed in culture vessel, then is poured sterilized water into into culture vessel and cultivated；
2) condition of culture：In 10～60 μm of olm-2·s-1Illumination, light application time 12h, temperature be 22～25 DEG C of conditions
Lower culture 1～3 week, takes the explant material that plumular axis and red radicle are processed as lower step；
3rd, by the explant material after step 2 drying and other treatment aseptically, it is linked on inducing culture, in temperature
Spend and go out embryo callus for inducing culture under 22～26 DEG C, dark condition；
In described inducing culture the concentration of 6-BA be 0～2.0mg/L, concentration of NAA be 1.0～5.0mg/L, water
Solution casein concentration is 0.7～1.0g/L, glutamine concentration is 450～500mg/L, sucrose concentration is 20～25g/L, gel
Concentration is 2～4g/L, kinetins concentration is 1～3g/L of 0.5～2.0mg/L and activated carbon；PH is controlled 5.6～5.8；
4th, the culture of somatic embryo：
1) embryo callus for obtaining previous step, it is 1g to cut embryo callus by mass volume ratio：10～
The ratio of 20mL adds it to mix in fluid medium, then filters, and filters out fluid medium, and remaining embryo is healed
Injured tissue is placed on inducing culture, temperature be 20～25 DEG C, the photoperiod of 13～15h, intensity of illumination be 1200～
20～30d of culture is carried out under conditions of 1800LX, calluss are obtained；
Inducing culture is 3/4MS culture medium；Wherein, 6-BA concentration be 0.1～2.0mg/L, zeatin ZT concentration be 0.3
～1.5mg/L, sucrose concentration be 20～25g/L, caseinhydrolysate concentration be 0.7～1.0g/L, concentration of activated carbon be 1mg/L with
And gel strength is 2～4g/L；
2) calluss cultivated in upper one-step inducing culture medium are transferred to into induction to sprout in culture medium, are 18 in temperature
～22 DEG C, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of, cultivate 15d；
Induction sprouts culture medium for 1/2MS culture medium；Wherein, gibberellins concentration is 0.1～1.0mg/L, concentration of NAA
For 0.01～0.1mg/L, glutamine concentration be 500mg/L, sucrose concentration be 30～40g/L, concentration of activated carbon be 1mg/L with
And gel strength is 7～8g/L；
3) by the adventitious bud grown after upper step culture temperature be 18～22 DEG C, the photoperiod of 10～12h, intensity of illumination be
4～5d is cultivated under conditions of 1000～1500LX, the ethephon of 280～320mg/L is then sprayed, and with 3～5h of blue light illumination,
Continue be 18～22 DEG C in temperature, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of train 1d, so
After be transferred on root media, light application time be 15～20h, temperature be 22～25 DEG C under the conditions of cultivate 20～30d；It is described
The illumination condition of culture is on root media：It is 5～10 μM of m in 1～10d illumination-2·s-1, in 11～18d light
According to for 15～20 μM of m-2·s-1, it is 25～40 μM of m in 19～30d illumination-2·s-1；
Root media is 1/2MS culture medium；Wherein, heteroauxing concentration be 0.1～5.0mg/L, sucrose concentration be 10
～30g/L and gel strength are 2～8g/L；
5th, seedling exercising：1.0～1.5cm is grown in adventitious root, test tube bottleneck is opened, under natural lighting, 3～5d of seedling exercising,
After taking-up, plant in the substrate of ratio mixing of fertile soil+perlite+Vermiculitum+fowl and animal excrement in mass ratio 6: 4: 3: 1, i.e.,
Complete described Picea koraiensis Nakai somatic embryo induction plant regeneration.
A kind of application of Picea koraiensis Nakai of the present invention, to the plant of above-mentioned acquisition flavone compound is extracted.
The present invention includes following beneficial effect：
The present invention by before Picea koraiensis Nakai somatic induction culture, by carrying out drying and other treatment to it, at the same will induction,
After the culture medium such as take root is improved, significantly improves Picea koraiensis Nakai and breed efficiency, the sprouting cycle effectively shortens and (shortens 45 days left sides
It is right), and, the present invention is using the raw material of low cost as culture medium, it is adaptable to industrialized great production.
PAI is a kind of inducible enzyme, and it has direct relation with the content of flavone compound in PiceameyeriRehd. Et Wils., is in positive
Close；When plant is subject to environmental stimuli, plant is adjusted by its own growth promoter so that PAI activity is raised, so as to promote
The synthesis of flavone compound.Affect the factor of PAI activity a lot, temperature, illumination, derivant etc. can stimulate PAI so as to
Activity is raised, so as to improve the synthetic quantity of flavone compound；Present invention discover that sending out in PiceameyeriRehd. Et Wils. tissue culture stage and seed
In the bud stage, be that the duration of response for improving flavone compound synthetic quantity, especially seed are sprouted the stage, and the present invention is by by 280
The ethephon of the ethephon of～320mg/L, especially 300mg/L is sprayed onto in the seedling for sprouting, and by being brought rapidly up, drop
Temperature, using blue light illumination, can significantly improve the activity of PAI, so as to improve the synthetic quantity of flavone compound, the Huang of the present invention
The synthetic quantity of ketone compounds improves 30～70%.So as to lay the first stone for the follow-up flavone compound extraction ratio that improves.
The Picea koraiensis Nakai Cheng Shu cultivated after the method for the present invention optimizes carries out flavone compound extraction, flavonoid
Compound extraction ratio is 5.012%～6.504%.
Specific embodiment
Specific embodiment one：A kind of Picea koraiensis Nakai somatic embryo induction plant regeneration method of present embodiment, it is
Follow the steps below；
First, in mid-June to mid-July, clone cone is plucked, after sterilization, takes bulb immature zygotic embryos, as
Somatic embryo inducement explant material；
2nd, drying and other treatment is carried out to the explant material of step one：
1) explant material is placed in the culture dish for being placed with multi-layer filter paper, is sealed on culture dish with sealed membrane, then will
Culture dish is placed in culture vessel, then is poured sterilized water into into culture vessel and cultivated；
2) condition of culture：In 10～60 μm of olm-2·s-1Illumination, light application time 12h, temperature be 22～25 DEG C of conditions
Lower culture 1～3 week, takes the explant material that plumular axis and red radicle are processed as lower step；
3rd, by the explant material after step 2 drying and other treatment aseptically, it is linked on inducing culture, in temperature
Spend and go out embryo callus for inducing culture under 22～26 DEG C, dark condition；
In described inducing culture the concentration of 6-BA be 0～2.0mg/L, concentration of NAA be 1.0～5.0mg/L, water
Solution casein concentration is 0.7～1.0g/L, glutamine concentration is 450～500mg/L, sucrose concentration is 20～25g/L, gel
Concentration is 2～4g/L, kinetins concentration is 1～3g/L of 0.5～2.0mg/L and activated carbon；PH is controlled 5.6～5.8；
4th, the culture of somatic embryo：
1) embryo callus for obtaining previous step, it is 1g to cut embryo callus by mass volume ratio：10～
The ratio of 20mL adds it to mix in fluid medium, then filters, and filters out fluid medium, and remaining embryo is healed
Injured tissue is placed on inducing culture, temperature be 20～25 DEG C, the photoperiod of 13～15h, intensity of illumination be 1200～
20～30d of culture is carried out under conditions of 1800LX, calluss are obtained；
Inducing culture is 3/4MS culture medium；Wherein, 6-BA concentration be 0.1～2.0mg/L, zeatin ZT concentration be 0.3
～1.5mg/L, sucrose concentration be 20～25g/L, caseinhydrolysate concentration be 0.7～1.0g/L, concentration of activated carbon be 1mg/L with
And gel strength is 2～4g/L；
2) calluss cultivated in upper one-step inducing culture medium are transferred to into induction to sprout in culture medium, are 18 in temperature
～22 DEG C, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of, cultivate 15d；
Induction sprouts culture medium for 1/2MS culture medium；Wherein, gibberellins concentration is 0.1～1.0mg/L, concentration of NAA
For 0.01～0.1mg/L, glutamine concentration be 500mg/L, sucrose concentration be 30～40g/L, concentration of activated carbon be 1mg/L with
And gel strength is 7～8g/L；
4) by the adventitious bud grown after upper step culture temperature be 18～22 DEG C, the photoperiod of 10～12h, intensity of illumination be
4～5d is cultivated under conditions of 1000～1500LX, the ethephon of 280～320mg/L is then sprayed, and with 3～5h of blue light illumination,
Continue be 18～22 DEG C in temperature, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of train 1d, so
After be transferred on root media, light application time be 15～20h, temperature be 22～25 DEG C under the conditions of cultivate 20～30d；It is described
The illumination condition of culture is on root media：It is 5～10 μM of m in 1～10d illumination-2·s-1, in 11～18d light
According to for 15～20 μM of m-2·s-1, it is 25～40 μM of m in 19～30d illumination-2·s-1；
Root media is 1/2MS culture medium；Wherein, heteroauxing concentration be 0.1～5.0mg/L, sucrose concentration be 10
～30g/L and gel strength are 2～8g/L；
5th, seedling exercising：1.0～1.5cm is grown in adventitious root, test tube bottleneck is opened, under natural lighting, 3～5d of seedling exercising,
After taking-up, plant in the substrate of ratio mixing of fertile soil+perlite+Vermiculitum+fowl and animal excrement in mass ratio 6: 4: 3: 1, i.e.,
Complete described Picea koraiensis Nakai somatic embryo induction plant regeneration.
Specific embodiment two：A kind of application of Picea koraiensis Nakai of present embodiment, obtains to specific embodiment one
Plant extracts flavone compound.
Specific embodiment three：Present embodiment from unlike specific embodiment two：Described plant refers to：Will tool
After the Transplantation of Regenerated Plantlets that body embodiment one is obtained, harvest its seed and sprouted, then transplant, the trees after transplanting are extracted
Flavone compound.Other are identical with specific embodiment two.
Specific embodiment four：Present embodiment from unlike specific embodiment two：Described sprouting be according to
What lower step was carried out：
First, seed is harvested in 8～October, by seed with snow according to 1:3 weight deep, wide is 1m's than being placed in after mixing
In ditch, snow and earthing, and stalks mulching are covered above；Wherein, the accumulated snow of 10～15cm of ditch heelpiece；
2nd, prior to seeding 5d takes out in seed, takes seed with 40～50 DEG C of 1～2d of immersion, stirs 5～6 times during immersion；
Then 2～3d of natural drying at a temperature of 23～26 DEG C,
3rd, it is 3 according to weight ratio by wet sand and seed:1 ratio mixing, is positioned over shady spot, is 5～8 DEG C in temperature
Under cultivate to sprouting, on the same day of sprouting, first, spray the ethephon of 280～320mg/L, temperature is warming up to into 15 DEG C after sprinkling
Or after 20 DEG C, 5～8 DEG C are cooled to immediately, then using 3～4h of blue light illumination, continue temperature be 5～8 DEG C at culture 1～
2d, that is, complete described sprouting.
Other are identical with specific embodiment two.
Specific embodiment five：Present embodiment from unlike specific embodiment two：Extract flavone compound
Process is as follows：
First, by the seedling replanting after sprouting；
2nd, take step one and transplant Picea koraiensis Nakai blade crushing air-dried afterwards for many years, sieve for subsequent use；
3rd, it is 1g by material ratio:Volumetric concentration is added to be 45～60% in powder of the ratio of 8～12mL to after sieving
Ethanol, is 60～70 DEG C in temperature in being placed in refiner, is homogenized 2～6min, obtains homogenate；After ethanol in homogenate is removed,
Material ratio is pressed again for 1g:The ratio of 18～20mL adds the ethanol that volumetric concentration is 50～70%, soaks 0.5～1.5h, then
Microwave power be 250～300W, temperature be 60～80 DEG C under conditions of, extract 4～7min, obtain extracting solution；
4th, it is by volume 1:1 ratio, the extracting solution obtained to step 2 adds ether, and oscillation extraction 30～
35min, collects extraction phase；Mutually carry out adding ether to extract according to above-mentioned volume ratio again more than extracting ether；
5th, repeat step four is operated 2～3 times, merges extraction phase；
6th, concentrating under reduced pressure is carried out to the extraction phase of step 5, the concentrated solution after concentration is crossed into silica gel column chromatography and is separated,
Collect eluent；Concentrating under reduced pressure, that is, complete described flavone compound and extract.
Other are identical with specific embodiment two.
Specific embodiment six：Present embodiment from unlike specific embodiment two：Described flavone compound
For isoflavone, isoflavanone, flavonol, flavanone, flavanonol, chalcone derivative and dihydrochalcone.Other with it is concrete
Embodiment two is identical.
Present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several specific embodiments
Contract sample can also realize the purpose invented.
By having with effect for the following examples checking present invention：
Embodiment 1
A kind of Picea koraiensis Nakai somatic embryo induction plant regeneration method of the present embodiment, it is to follow the steps below
's：
First, in mid-June to mid-July, clone cone is plucked, after sterilization, takes bulb immature zygotic embryos, as
Somatic embryo inducement explant material；
2nd, drying and other treatment is carried out to the explant material of step one：
1) explant material is placed in the culture dish for being placed with multi-layer filter paper, is sealed on culture dish with sealed membrane, then will
Culture dish is placed in culture vessel, then is poured sterilized water into into culture vessel and cultivated；
2) condition of culture：In 10～60 μm of olm-2·s-1Illumination, light application time 12h, temperature be 22～25 DEG C of conditions
Lower culture 3 weeks, takes the explant material that plumular axis and red radicle are processed as lower step；
3rd, by the explant material after step 2 drying and other treatment aseptically, it is linked on inducing culture, in temperature
Spend and go out embryo callus for inducing culture under 22～26 DEG C, dark condition；
In described inducing culture the concentration of 6-BA be 1.0mg/L, concentration of NAA be 3.0mg/L, caseinhydrolysate
Concentration is 1.0g/L, glutamine concentration is 500mg/L, sucrose concentration is 20g/L, gel strength is 2g/L, kinetins concentration
For 1.0mg/L and activated carbon 2g/L；PH is controlled 5.6～5.8；
4th, the culture of somatic embryo：
1) embryo callus for obtaining previous step, it is 1g to cut embryo callus by mass volume ratio：12mL
Ratio add it in fluid medium, then filter, filter out fluid medium, remaining embryo callus are put
On inducing culture, temperature be 20～25 DEG C, the photoperiod of 13～15h, the condition that intensity of illumination is 1200～1800LX
Under carry out culture 23d；
Inducing culture is 3/4MS culture medium：6-BA concentration is 2.0mg/L, zeatin ZT concentration is 0.3mg/L, sucrose
Concentration is 20g/L, caseinhydrolysate concentration is 1.0g/L, concentration of activated carbon is 1.0mg/L and gel strength is 2g/L；
2) calluss cultivated on above-mentioned inducing culture are transferred to into induction to sprout in culture medium, are 18 in temperature
～22 DEG C, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of, cultivate 15d；
Induction sprouts culture medium for 1/2MS culture medium：Gibberellins concentration is 0.1mg/L, concentration of NAA is 0.01mg/L,
Glutamine concentration is 500mg/L, sucrose concentration is 35g/L, concentration of activated carbon is 1mg/L and gel strength is 7g/L；
5) by the adventitious bud grown after above-mentioned culture temperature be 18～22 DEG C, the photoperiod of 10～12h, intensity of illumination be
4～5d is cultivated under conditions of 1000～1500LX, the ethephon of 300mg/L is then sprayed, and with 3～5h of blue light illumination, is continued
Be 18～22 DEG C in temperature, the photoperiod of 10～12h, intensity of illumination be 1000-1500LX under conditions of train 1d, then shift
To root media, light application time be 15～20h, temperature be 22～25 DEG C under the conditions of cultivate 20～30d；It is described to be transferred to
The illumination condition of root media is：It is 5～10 μM of m in 1～10d illumination-2·s-1, 11～18d illumination be 15～
20μM·m-2·s-1, it is 25～40 μM of m in 19～30d illumination-2·s-1；
Root media is 1/2MS culture medium：Heteroauxing concentration is 2.0mg/L, sucrose concentration is 20g/L and gel
Concentration is 5g/L；
5th, seedling exercising：1.0～1.5cm is grown in adventitious root, test tube bottleneck is opened, under natural lighting, 3～5d of seedling exercising,
After taking-up, plant in the substrate of ratio mixing of fertile soil+perlite+Vermiculitum+fowl and animal excrement in mass ratio 6: 4: 3: 1, i.e.,
Complete described Picea koraiensis Nakai somatic embryo induction plant regeneration.
Somatic embryo germination rate after the present embodiment process is up to 80.5%.The present embodiment induction differentiation before,
Drying and other treatment is carried out to somatic embryo, the somatic cell after the process of this kind of method significantly improves its induction germination rate.Together
When, the present embodiment is processed explant in the root culture stage, by adjusting temperature, illumination, and sprinkling ethephon
Mode, improves Picea koraiensis Nakai flavone compound growing amount, is subsequently to improve flavone compound extracted amount to lay the first stone.
By the method Picea koraiensis Nakai terminal bud of the present embodiment or the culture in 12 days or so of stem section Jing with lateral bud just can be lured
Bud differentiation is led, is within 25 days or so 1 subculture multiplication cycle, Jing cultures in 10 days just can be able to induce Multiple Buds, Jing to train for 20 days or so
Supporting just can bear root hair, and the whole breeding cycle is most short to have reached 45 days or so.
Embodiment 2
The process that the present embodiment extracts flavone compound is as follows：
Before extraction, after the Transplantation of Regenerated Plantlets for first obtaining embodiment 1, harvest its seed and sprouted, then transplant, it is right
Trees after transplanting extract flavone compound.
Described sprouting is followed the steps below：
First, seed is harvested in 8～October, by seed with snow according to 1:3 weight deep, wide is 1m's than being placed in after mixing
In ditch, snow and earthing, and stalks mulching are covered above；Wherein, the accumulated snow of 10～15cm of ditch heelpiece；
2nd, prior to seeding 5d takes out in seed, takes seed with 40～50 DEG C of 1～2d of immersion, stirs 5～6 times during immersion；
Then 2～3d of natural drying at a temperature of 23～26 DEG C,
3rd, it is 3 according to weight ratio by wet sand and seed:1 ratio mixing, is positioned over shady spot, is 5～8 DEG C in temperature
Under cultivate to sprouting, on the same day of sprouting, first, spray the ethephon of 300mg/L, after temperature is warming up to into 15 DEG C after sprinkling, stand
5～8 DEG C are cooled to, then using blue light illumination 4h, it is to cultivate 1～2d at 5～8 DEG C to continue in temperature, that is, complete described
Sprout.
Extract flavone compound：
First, by the seedling replanting after sprouting；
2nd, take step one and transplant Picea koraiensis Nakai blade crushing air-dried afterwards for many years, sieve for subsequent use；
3rd, it is 1g by material ratio:The second that volumetric concentration is 45～60% is added in powder of the ratio of 10mL to after sieving
Alcohol, is 60～70 DEG C in temperature in being placed in refiner, is homogenized 2～6min, obtains homogenate；After ethanol in homogenate is removed, then
It is 1g by material ratio:The ratio of 20mL adds the ethanol that volumetric concentration is 50～70%, 0.5～1.5h is soaked, then in microwave
Power is 280W, temperature under conditions of 70 DEG C, 4～7min of extraction obtains extracting solution；
4th, it is by volume 1:1 ratio, the extracting solution obtained to step 2 adds ether, and oscillation extraction 30～
35min, collects extraction phase；Mutually carry out adding ether to extract according to above-mentioned volume ratio again more than extracting ether；
5th, repeat step four is operated 2～3 times, merges extraction phase；
6th, concentrating under reduced pressure is carried out to the extraction phase of step 5, the concentrated solution after concentration is crossed into silica gel column chromatography and is separated,
Collect eluent；Concentrating under reduced pressure, that is, complete described flavone compound and extract.
It is 6.504% by the method flavone compound extraction ratio of the present embodiment, flavone compound is extracted in Folium Styracis Suberifoliae
Content in PiceameyeriRehd. Et Wils. increases 70%.
Claims (6)
1. a kind of Picea koraiensis Nakai somatic embryo induction plant regeneration method, it is characterised in that it is followed the steps below：
First, in mid-June to mid-July, clone cone is plucked, after sterilization, takes bulb immature zygotic embryos, it is thin as body
Blastula induces explant material；
2nd, drying and other treatment is carried out to the explant material of step one：
1) explant material is placed in the culture dish for being placed with multi-layer filter paper, is sealed on culture dish with sealed membrane, then will culture
Ware is placed in culture vessel, then is poured sterilized water into into culture vessel and cultivated；
2) condition of culture：In 10～60 μm of olm-2·s-1Illumination, light application time be 12h, temperature be 22～25 DEG C under the conditions of
Culture 1～3 week, takes the explant material that plumular axis and red radicle are processed as lower step；
3rd, by the explant material after step 2 drying and other treatment aseptically, it is linked on inducing culture, is in temperature
22～26 DEG C, inducing culture goes out embryo callus under dark condition；
In described inducing culture the concentration of 6-BA be 0～2.0mg/L, concentration of NAA be 1.0～5.0mg/L, hydrolysis cheese
Protein concentration is 0.7～1.0g/L, glutamine concentration is 450～500mg/L, sucrose concentration is 20～25g/L, gel strength
It is 1～3g/L of 0.5～2.0mg/L and activated carbon for 2～4g/L, kinetins concentration；PH is controlled 5.6～5.8；
4th, the culture of somatic embryo：
1) embryo callus for obtaining previous step, it is 1g to cut embryo callus by mass volume ratio：10～20mL
Ratio add it in fluid medium mix, then filter, fluid medium is filtered out, by remaining embryo callus subculture group
Knit and be placed on inducing culture, temperature be 20～25 DEG C, the photoperiod of 13～15h, intensity of illumination be 1200～1800LX's
Under the conditions of carry out 20～30d of culture, obtain calluss；
Inducing culture is 3/4MS culture medium；Wherein, 6-BA concentration be 0.1～2.0mg/L, zeatin ZT concentration be 0.3～
1.5mg/L, sucrose concentration be 20～25g/L, caseinhydrolysate concentration be 0.7～1.0g/L, concentration of activated carbon be 1mg/L and
Gel strength is 2～4g/L；
2) calluss cultivated in upper one-step inducing culture medium are transferred to into induction to sprout in culture medium, are 18～22 in temperature
DEG C, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of, cultivate 15d；
Induction sprouts culture medium for 1/2MS culture medium；Wherein, gibberellins concentration is that 0.1～1.0mg/L, concentration of NAA are
0.01～0.1mg/L, glutamine concentration be 500mg/L, sucrose concentration be 30～40g/L, concentration of activated carbon be 1mg/L and
Gel strength is 7～8g/L；
3) by the adventitious bud grown after upper step culture temperature be 18～22 DEG C, the photoperiod of 10～12h, intensity of illumination be 1000
4～5d is cultivated under conditions of～1500LX, the ethephon of 280～320mg/L is then sprayed, and with 3～5h of blue light illumination, is continued
Be 18～22 DEG C in temperature, the photoperiod of 10～12h, intensity of illumination be 1000～1500LX under conditions of train 1d, Ran Houzhuan
Move on root media, light application time be 15～20h, temperature be 22～25 DEG C under the conditions of cultivate 20～30d；It is described to give birth to
The illumination condition of culture is in root culture medium：It is 5～10 μM of m in 1～10d illumination-2·s-1, it is in 11～18d illumination
15～20 μM of m-2·s-1, it is 25～40 μM of m in 19～30d illumination-2·s-1；
Root media is 1/2MS culture medium；Wherein, heteroauxing concentration be 0.1～5.0mg/L, sucrose concentration be 10～
30g/L and gel strength are 2～8g/L；
5th, seedling exercising：1.0～1.5cm is grown in adventitious root, test tube bottleneck is opened, under natural lighting, 3～5d of seedling exercising takes out
Afterwards, plant in the substrate of ratio mixing of fertile soil+perlite+Vermiculitum+fowl and animal excrement in mass ratio 6: 4: 3: 1, that is, complete
Described Picea koraiensis Nakai somatic embryo induction plant regeneration.
2. a kind of application of Picea koraiensis Nakai, it is characterised in that flavone compound is extracted to the plant that claim 1 is obtained.
3. the application of a kind of Picea koraiensis Nakai according to claim 2, it is characterised in that described plant refers to：Will by right
After seeking the Transplantation of Regenerated Plantlets of 1 acquisition, harvest its seed and sprouted, then transplant, flavonoid is extracted to the trees after transplanting
Compound.
4. the application of a kind of Picea koraiensis Nakai according to claim 3, it is characterised in that described sprouting is according to following step
Suddenly carry out：
First, seed is harvested in 8～October, by seed with snow according to 1:3 weight is than being placed in deep, the wide ditch for being 1m after mixing
It is interior, snow and earthing, and stalks mulching are covered above；Wherein, the accumulated snow of 10～15cm of ditch heelpiece；
2nd, prior to seeding 5d takes out in seed, takes seed with 40～50 DEG C of 1～2d of immersion, stirs 5～6 times during immersion；Then
2～3d of natural drying at a temperature of 23～26 DEG C,
3rd, it is 3 according to weight ratio by wet sand and seed:1 ratio mixing, is positioned over shady spot, trains in the case where temperature is for 5～8 DEG C
Support to sprouting, on the same day of sprouting, first, spray the ethephon of 280～320mg/L, temperature is warming up to into 15 DEG C or 20 after sprinkling
After DEG C, 5～8 DEG C are cooled to immediately, then using 3～4h of blue light illumination, continue to cultivate 1～2d in the case where temperature is for 5～8 DEG C, i.e.,
Complete described sprouting.
5. the application of a kind of Picea koraiensis Nakai according to claim 4, it is characterised in that extract the process of flavone compound
It is as follows：
First, by the seedling replanting after sprouting；
2nd, take step one and transplant Picea koraiensis Nakai blade crushing air-dried afterwards for many years, sieve for subsequent use；
3rd, it is 1g by material ratio:The second that volumetric concentration is 45～60% is added in powder of the ratio of 8～12mL to after sieving
Alcohol, is 60～70 DEG C in temperature in being placed in refiner, is homogenized 2～6min, obtains homogenate；After ethanol in homogenate is removed, then
It is 1g by material ratio:The ratio of 18～20mL adds the ethanol that volumetric concentration is 50～70%, soaks 0.5～1.5h, Ran Hou
Microwave power is 250～300W, temperature under conditions of 60～80 DEG C, 4～7min of extraction obtains extracting solution；
4th, it is by volume 1:1 ratio, the extracting solution obtained to step 2 adds ether, 30～35min of oscillation extraction to receive
Collection extraction phase；Mutually carry out adding ether to extract according to above-mentioned volume ratio again more than extracting ether；
5th, repeat step four is operated 2～3 times, merges extraction phase；
6th, concentrating under reduced pressure is carried out to the extraction phase of step 5, the concentrated solution after concentration is crossed into silica gel column chromatography and is separated, collected
Eluent；Concentrating under reduced pressure, that is, complete described flavone compound and extract.
6. the application of a kind of Picea koraiensis Nakai according to claim 5, it is characterised in that described flavone compound is different
Flavone, isoflavanone, flavonol, flavanone, flavanonol, chalcone derivative and dihydrochalcone.
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