CN102771393B - Method for picea balfouriana somatic embryo generation and plant regeneration - Google Patents
Method for picea balfouriana somatic embryo generation and plant regeneration Download PDFInfo
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Abstract
The invention relates to a method for picea balfouriana somatic embryo generation and plant regeneration, which comprises steps of collecting picea balfouriana open pollination clonal cones, and separating immaturate zygotic embryos out for somatic embryogenetic culture, wherein the somatic embryogenetic culture comprises four stages, i.e. an embryogenetic callus inducing stage, an embryogenetic callus multiplicating stage, a somatic embryo maturity stage and a somatic embryo germination stage. Compared with the prior art, the method has the advantages that the seedling stage is greatly shortened, and natural maturing rate, seed maturation rate, differentiation rate and germination rate are all improved. The seedling culture method having short period, high multiplying rate, closely linked cultivation steps and low cost is provided for popularizing and planting picea balfouriana in a large scale for forestry production, is suitable for a rapid propagation technical system of picea balfouriana cell engineering and satisfies the requirements of markets on picea balfouriana.
Description
Technical field
The present invention relates to forestry cell engineering sapling multiplication field, particularly, relate to a kind of balfour spruce somatic embryo Somatic Embryogenesis and plant regeneration method.
Background technology
Picea (Picea dietr) the plant whole world approximately has 40 kinds, mainly be distributed in cool temperate zone or the tundra zone of 50 ° ~ 60 ° of north latitude, minority is distributed on temperate zone or the semi-tropical clammy high mountain, namely most the kind is the distribution of circumpolar formula, and near China and the U.S. can be distributed to 30 ° of north latitude, be that area low in calories important built group seeds.
Picea seeds unit are year increment is high, and wood quality is good, and purposes is wide, becomes West Europe, Northern Europe, Baltic Sea coastwise contries, Russia and Canadian essential industry commerical tree species.China's Picea has 17 kinds and 9 mutation, is the area of this genus most species in the world, mainly is distributed in the alpine belts such as northeast, North China, northwest, southwest, is important reproducting tree species and the best pulpwood seeds of China alpine region.
Balfour spruce (Picea balfouriana) has another name called Xikang dragon spruce, Xikang-Tibet dragon spruce, Bollinger body dragon spruce.The balfour spruce woods is the main forest types in Sichuan, is the peculiar forest vegetation of In The Eastern Region of Xizang Plateau, and it not only distributes vast, and aboundresources, is important timber forest seeds.Simultaneously, it or Chuan Xi highlands form the company edge forest of sylvosteppe zone of transition, to preventing that south, grassland from moving, forest line decline and water conservation, conserving water and soil and the protection in the Sources of the Yangtze River area has very important effect.
The somatic embryo Somatic Embryogenesis is one of important channel of plant regeneration in the cell engineering, can be used as the receptor system of genetic transformation, also is simultaneously the ideal test system of building up for studying totipotent cell differentiation and form thereof.On using, the somatic embryo Somatic Embryogenesis is a kind of effectively extensive asexual reproduction method.
Balfour spruce natural forest growth is slower, and the seed maturity rate is very low, causes the very large difficulty of breeding and breed breeding, and seedling stage and Juvenile Stage growth are slowly, having a strong impact on the genetic improvement process, particularly the genetic evaluation process.And can greatly shorten seedling stage by the somatic embryo Somatic Embryogenesis, so can the balfour spruce the Development of Somatic Embryogenesis can become and accelerate one of key of breeding.If can develop a kind of balfour spruce somatic embryo Somatic Embryogenesis and plant regeneration method, overcome the deficiency that prior art exists, be necessary.
Summary of the invention
The purpose of this invention is to provide a kind of balfour spruce somatic embryo Somatic Embryogenesis and plant regeneration method.
A kind of balfour spruce somatic embryo Somatic Embryogenesis provided by the invention and plant regeneration method comprise: gather balfour spruce open pollination clone cone, isolate immature zygotic embryos and carry out the body embryo and cultivate; Described body embryo occurs to cultivate and comprises four-stage: embryonic callus induction stage, embryo callus multiplicative stage, the somatic embryo stage of ripeness and somatic embryo are sprouted the stage.
Wherein, isolating immature zygotic embryos carries out the body embryo and occur to cultivate and to carry out under aseptic condition.Immature zygotic embryos refers to that globular stage is to the immature zygotic embryos of cotyledon period.
Wherein, described embryonic callus induction stage and embryo callus multiplicative stage: all adopt the 1/2LM minimal medium, its additional hormone is respectively: 2 of 0.55 ~ 2.2mg/L, (2,4-dichlorophenoxyacetic acid is called for short 2 to 4-Benzene Chloride fluoroacetic acid, 4-D), the 6-glycosyl aminopurine (6-Furfurylamino purine is called for short KT) of the 6-benzylaminopurine of 0 ~ 1.1mg/L (benzylaminmopurine is called for short 6-BA) and 0 ~ 0.44mg/L.
Further, described 2,4-D is preferred 2.2mg/L all.Described 6-BA is at the preferred 0.55 ~ 1.1mg/L of induction period, more preferably 1.1mg/L; 6-BA is at preferred 0.8 ~ 1.1mg/L of multiplicative stage, more preferably 0.8mg/L.Described KT is at the preferred 0 ~ 22mg/L of induction period, more preferably 0.22mg/L; KT is at preferred 0 ~ 0.44mg/L of multiplicative stage, more preferably 0mg/L.
Wherein, described embryonic callus induction stage and embryo callus multiplicative stage: in the additional hormone, the consumption of 2,4-D is more than 2 times of 6-BA, preferred 2 times in the 1/2LM minimal medium.
Wherein, in the medium of described embryonic callus induction stage and multiplicative stage, all also comprise: concentration is 1% sucrose, the glutamine of 500mg/L, the casein hydrolysis of 1g/L and 0.2% plant gel.
The method of the invention, also comprises after the multiplicative stage (somatic embryo is before the stage of ripeness) at embryo callus: the state adjusting stage of embryo callus, for adopting the 1/2LM minimal medium, do not add any hormone and cultivated 7 days.Its purpose and effect are: embryo callus is changed to differentiation state by vegetative state.
Wherein, the described somatic embryo stage of ripeness: adopt the 1/2LM minimal medium, it adds the dormin (abscisic acid, abscisic acid are called for short ABA) of 12 ~ 20mg/L and 0 ~ 1% active carbon.Described ABA is preferably 16 ~ 20mg/L, more preferably 16mg/L.Active carbon is preferably 0.4 ~ 0.8%, and more preferably 0.4%.
Wherein, in the medium in the described somatic embryo stage of ripeness, also comprise: 0.6% plant gel, 6% sucrose, the glutamine of 500mg/L and the caseinhydrolysate of 1g/L.
Wherein, the condition of culture in described embryonic callus induction stage, embryo callus multiplicative stage and the somatic embryo stage of ripeness is: the pH value of medium is transferred to 5.8,121 ℃, pressure 101kp, high temperature, autoclaving 20 minutes, cultivation temperature is 24 ± 1 ℃, and dark condition is cultivated.
The method of the invention, embryonic callus induction stage and multiplicative stage, and the hormone concentration of the sprouting stage of somatic embryo use, can play good synergy, farthest bring into play its effect, to the inducing and breed and play more excellent effect of balfour spruce embryo callus, be conducive to keep the embryo sexuality of embryo callus.
Wherein, described somatic embryo is sprouted the stage: adopt the 1/4LM minimal medium, it is additional: 0 ~ 0.25% active carbon.Active carbon is preferred 0.125 ~ 0.25%, and more preferably 0.25%.
Wherein, described somatic embryo is sprouted in the medium in stage, also comprises: glutamine 500mg/L, 2% sucrose, 1g/L caseinhydrolysate and 0.4% plant gel.
Wherein, the condition of culture in the sprouting stage of described somatic embryo is: at 24 ± 1 ℃, and 80 μ Em
-2s
-1Lower illumination cultivation 16h.
Method of the present invention also comprises: balfour spruce open pollination clone cone is carried out disinfection, then take out immature zygotic embryos under aseptic environment before the body embryo cultivates immature zygotic embryos is carried out.
Wherein, described disinfection way is: utilize 75 ~ 95% ethanol to balfour spruce open pollination clone cone sterilization 15 ~ 20min, use aseptic water washing 3 ~ 4 times again.Preferably: utilize 95% ethanol disinfection to balfour spruce open pollination clone cone 15min, use again aseptic water washing 3 times.
Further, before sterilization, can also under 4 ℃ of conditions, refrigerate 0 ~ 30 day to balfour spruce open pollination clone cone.Refrigeration is 7 days under preferred 4 ℃ of conditions.
Through the cone that refrigerates and disinfect, meeting is so that inductivity increases, and pollution rate is lower than 0.1%.
The percent concentration that occurs among the present invention " % " need in quality-volumetric concentration g/mL(100mL solution all to refer to the grams of adding).
The present invention adopts immature zygotic embryos, especially utilizes globular stage to the immature zygotic embryos of cotyledon period to carry out the body embryo and cultivates, and can successfully induce the balfour spruce embryo callus, and advantage is that the tender immature zygotic embryos of children can improve inductivity effectively.
Method provided by the invention, low for the natural ripening rate of balfour spruce specially, the seed maturity rate is very low, seedling stage and Juvenile Stage growth are slow, the sapling multiplication technology can't satisfy the present situation of large tracts of land afforestation needs, seeks a kind of somatic embryo Somatic Embryogenesis of balfour spruce choiceness and the technology of plant regeneration.Be compared with existing technology, the method has shortened seedling stage greatly, and natural ripening rate, seed maturity rate and differentiation rate and germination rate are improved.For the plantation of large-scale promotion in production of forestry balfour spruce provides a kind of cycle short, reproduction rate is high, production stage is connected seedling-cultivating method tight, with low cost, be applicable to balfour spruce cell engineering quick propagating technology system, satisfying the market is to the needs of balfour spruce.
Description of drawings
Fig. 1 is the immature zygotic embryos of balfour spruce.
Fig. 2 is the embryo callus of balfour spruce.
Fig. 3 is the mature somatic embryo of balfour spruce.
Fig. 4 is the plant that the somatic embryo of balfour spruce is sprouted.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The present invention used plant gel Gelrite is available from U.S. Sigma company.It is a kind of epoxy resin, and is identical with the effect of agar, but effect is well a lot, solidifying fast of medium and be transparent.Used other raw materials and reagent are conventional raw material and the reagent of buying from the market.
The composition of LM liquid nutrient medium is: 1.65g/L NH
4NO
3, 1.9g/L KNO
3, 1.85g/L MgSO47H
20,0.34g/L KH
2PO
4, 0.2g/L Myo-Inositol(inositol), 2g/L N-Z-Amine A(casein hydrolysis), 0.022g/L CaCl
22H
20,0.028g/L, FeSO
47H
20,0.038g/L EDTA2H
2The 0(ethylenediamine tetra-acetic acid), 0.0304g/LH
3BO
3, 0.0210g/L MnSO
4H
20,0.043g/L ZnSO
47H
20,0.001g/LNiacinamide(niacinamide), 0.0002g/L Pyroxidone-HCl(Vitamin B6), 0.0002g/L Thiamine-HCl(Vitamin B1) and, 0.0042g/L KI, 0.00126g/LNa
2MoO
427H
20,0.0005g/L CuSO
45H
20,0.00013g/L CoCl
26H
20, add water to 1L.
The used minimal medium of the present invention is exactly the semisolid culturemedium that adds the required plant gel of each corresponding cultivation stage on the basis of the liquid nutrient medium of mentioned component.
Embodiment 1
Isolate immature zygotic embryos: gather the open pollination in spring, the balfour spruce open pollination clone cone that obtains by the end of August is with the deepfreeze 7 days under 4 ℃ of conditions of the cone under adopting.Ethanol Treatment cone 15min with 95%, aseptic water washing 3 times.Under the germ-free condition, take out seed and peel off kind of a skin, immature zygotic embryos (as shown in Figure 1) is carried out the embryonic callus induction of next stage and cultivate.
Embodiment 2
The embryonic callus induction stage: with the immature zygotic embryos that embodiment 1 obtains, carry out embryonic callus induction and cultivate.Adopt the 1/2LM minimal medium, additional hormone concentration is respectively 2,4-D 2.2mg/L, 6-BA1.1mg/L and KT0.22mg/L.And, in medium, add the natural complex casein hydrolysis of 1g/L, 1% sucrose, the glutamine of 500mg/L (filtration sterilization) and 0.2% plant gel (Gelrite, Sigma).Before the sterilization pH was transferred to 5.7,121 ℃, pressure 101kp high temperature, autoclaving 20 minutes, 24 ± 1 ℃ of cultivation temperature, dark condition is cultivated, to inducing embryo callus.Inductivity is: 61.55%.
Embodiment 3
The embryonic callus induction stage: with the immature zygotic embryos that embodiment 1 obtains, carry out embryonic callus induction and cultivate.Adopt the 1/2LM minimal medium, additional hormone concentration is respectively 2,4-D 0.55mg/L, 6-BA 0.8mg/L, and, the natural complex casein hydrolysis of interpolation 1g/L in medium, 1% sucrose, the glutamine of 500mg/L (filtration sterilization) and 0.2% plant gel (Gelrite, Sigma).Before the sterilization pH was transferred to 5.7,121 ℃, pressure 101kp high temperature, autoclaving 20 minutes, 24 ± 1 ℃ of cultivation temperature, dark condition is cultivated, to inducing embryo callus.Inductivity is 60.56%.
Embodiment 4
The embryonic callus induction stage: with the immature zygotic embryos that embodiment 1 obtains, carry out embryonic callus induction and cultivate.Adopt the 1/2LM minimal medium, additional hormone concentration is respectively 2,4-D 2.2mg/L and 6-BA 1.1mg/L(does not add KT).And, in medium, add the natural complex casein hydrolysis of 1g/L, 1% sucrose, the glutamine of 500mg/L (filtration sterilization) and 0.2% plant gel (Gelrite, Sigma).Before the sterilization pH is transferred to 5.7,121 ℃, pressure 101kp, high temperature, autoclaving 20 minutes, 24 ± 1 ℃ of cultivation temperature, dark condition is cultivated, to inducing embryo callus.Inductivity is: 59.67%.
Immature zygotic embryo approximately began to start after accessing above-mentioned inducing culture in 2 ~ 3 days, and the callus that grows is yellowish, translucent.About 25 days, the hard callus of the green warty of some formation, subculture (conditionally complete in subculture mode and this stage is consistent) is brownization afterwards, and what have then grows translucent, loose embryo callus.This callus is examined under a microscope, and mostly is the cell that cell nucleus is large, cytoplasm is dense.
Embodiment 5
The multiplicative stage of embryo callus: the embryo callus that embodiment 2 ~ 4 induces is proceeded the propagation cultivation.Adopt the 1/2LM minimal medium, additional hormone concentration is respectively 2,4-D 2.2mg/L and 6-BA 1.1mg/L(does not add KT).And, in medium, add the natural complex casein hydrolysis of 1g/L, 1% sucrose, the glutamine of 500mg/L (filtration sterilization) and 0.2% plant gel (Gelrite, Sigma).And condition of culture is all identical with induction period, and the embryo callus that obtains as shown in Figure 2.The amount of growth of the embryo callus that obtains according to the present embodiment is 2.5 times of initial inoculum concentration.
The state adjustment of embryo callus, medium still adopts the 1/2LM minimal medium, do not add hormone and cultivate a week, other composition and condition of culture in the medium are all constant, purpose is that embryo callus is changed to differentiation by vegetative state, can shorten cultivation cycle on the basis that uses manpower and material resources sparingly.
Embodiment 6
The multiplicative stage of embryo callus: other conditions are with embodiment 5.Difference is: additional hormone concentration is respectively 2,4-D 2.2mg/L, 6-BA 1.1mg/L and KT 0.22mg/L, and the amount of growth of the embryo callus that obtains according to the present embodiment is 2 times of initial inoculum concentration.
Embodiment 7
The multiplicative stage of embryo callus: other conditions are with embodiment 5.Difference is: additional hormone concentration is respectively 2,4-D 2.2mg/L, and the amount of growth of the embryo callus that 6-BA0.55mg/L and KT 0.44mg/L obtain according to the present embodiment is 1.5 times of initial inoculum concentration.
Embodiment 8
The stage of ripeness of somatic embryo: with the embryo callus that embodiment 5 ~ 7 obtains, carry out the ripe cultivation of somatic embryo.Adopt the 1/2LM minimal medium, additional hormone and active carbon are respectively the ABA of 16mg/L, 0.4% active carbon.Sucrose concentration in the medium is 6% at this moment, and plant gel (Gelrite, Sigma) is 0.6%, caseinhydrolysate substitutes the casein hydrolysis of inducing with the multiplicative stage, be 1g/L, the amount of glutamine is 500mg/L, and wherein ABA and glutamine are filtration sterilization.And condition of culture is all identical with induction period and multiplicative stage.Average every 100mg callus can form the cotyledonary embryos of 143 maturations on this differential medium.The mature somatic embryo that obtains as shown in Figure 3.
Embodiment 9
The stage of ripeness of somatic embryo: with the embryo callus that embodiment 5 ~ 7 obtains, carry out the ripe cultivation of somatic embryo.Adopt the 1/2LM minimal medium, additional hormone and active carbon are respectively the ABA of 16mg/L, 0.6% active carbon.Sucrose concentration in the medium is 6% at this moment, and plant gel (Gelrite, Sigma) is 0.6%, caseinhydrolysate substitutes the casein hydrolysis of inducing with the multiplicative stage, be 1g/L, the amount of glutamine is 500mg/L, and wherein ABA and glutamine are filtration sterilization.And condition of culture is all identical with induction period and multiplicative stage.Average every 100mg callus can form the cotyledonary embryos of 129 maturations on this differential medium.
Embodiment 10
The stage of ripeness of somatic embryo: with the embryo callus that embodiment 5 ~ 7 obtains, carry out the ripe cultivation of somatic embryo.Adopt the 1/2LM minimal medium, additional hormone and active carbon are respectively the ABA of 20mg/L, 0.8% active carbon.Sucrose concentration in the medium is 6% at this moment, and plant gel (Gelrite, Sigma) is 0.6%, caseinhydrolysate substitutes the casein hydrolysis of inducing with the multiplicative stage, be 1g/L, the amount of glutamine is 500mg/L, and wherein ABA and glutamine are filtration sterilization.And condition of culture is all identical with induction period and multiplicative stage.Average every 100mg callus can form the cotyledonary embryos of 106 maturations on this differential medium.
Embryo callus begins differentiation after changing above-mentioned stage of ripeness medium over to, can pass through successively globular embryo, heart-shape embryo, torpedo embryo and cotyledonary embryos through bimestrial growth.On improved culture medium of the present invention, successful adjustment the upgrowth situation of cells,primordial, so that being effectively controlled of the most difficult key technical problem-Embryos and asynchronization in the somatic embryo Somatic Embryogenesis, and differentiation rate is high, and average every 100mg callus can form the cotyledonary embryos of 106 ~ 143 maturations.
The somatic embryo that in adding the stage of ripeness medium of active carbon, forms on form more near its zygotic embryo (because the somatic embryo Somatic Embryogenesis has dipolar characteristics, so, ripe cotyledonary embryos can form cotyledon and root simultaneously, become complete seedling, then well solved the difficult problem of taking root in the somatic embryos of coniferous trees Somatic Embryogenesis) and divergaence time to shorten half, only need 40 ~ 50 days.
Embodiment 11
The sprouting stage of somatic embryo: the somatic embryo that embodiment 8 ~ 10 obtains is proceeded to sprout.Use the 1/4LM minimal medium, add active carbon 0.25%, glutamine 500mg/L, 2% sucrose, 1g/L caseinhydrolysate and 0.4% plant gel (Gelrite, Sigma).Temperature is 24 ± 1 ℃, 80 μ Em
-2s
-1Lower illumination cultivation 16h.
Because the somatic embryo Somatic Embryogenesis has dipolar characteristics, so ripe cotyledonary embryos can form cotyledon and root simultaneously, becomes complete seedling, then well solved the difficult problem of taking root in the somatic embryos of coniferous trees Somatic Embryogenesis.The germination rate that obtains plant (as shown in Figure 4) according to the present embodiment is 22.52%.
Embodiment 12
The sprouting stage of somatic embryo: other conditions are with embodiment 11, and concentration of activated carbon is respectively: 0,0.1%, 0.125%, 0.2 and 0.25(with embodiment 11), the germination rate of somatic embryo is as shown in the table:
| Active carbon (%) | 0 | 0.1 | 0.125 | 0.2 | 0.25 |
| Germination rate (%) | 12.95% | 18.38% | 20.49% | 21.31% | 22.52% |
As can be seen from the above table: add the sprouting that active carbon can improve somatic embryo significantly in sprouting the medium in stage, the germination rate that wherein adds 0.25% active carbon is the highest, and namely 22.52%.
Among the present invention, for the stages of somatic embryo Somatic Embryogenesis clear and definite medium scheme, hormone are arranged, and the combination of other nutriments.The balfour spruce callus induction rate is up to 59.67% ~ 61.55%, and the number that every 100mg callus can differentiate mature somatic embryo is 106 ~ 143.
Adopt technology provided by the invention, can provide the method that a kind of cycle is shorter, reproductive efficiency is high, coefficient is large for the extensive asexual multiplication seedling of balfour spruce.Broken through the natural ripening rate of balfour spruce low, the cottage propagation difficulty, the sapling multiplication technology can't satisfy the restriction of clonal planting needs.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (11)
1. a balfour spruce somatic embryo Somatic Embryogenesis and plant regeneration method is characterized in that, comprising: gather balfour spruce open pollination clone cone, isolate immature zygotic embryos and carry out the body embryo and cultivate; Described body embryo occurs to cultivate and comprises four-stage: embryonic callus induction stage, embryo callus multiplicative stage, the somatic embryo stage of ripeness and somatic embryo are sprouted the stage;
The described embryonic callus induction stage, adopt the 1/2LM minimal medium, its additional hormone is: 2 of 0.55～2.2mg/L, 4-Benzene Chloride fluoroacetic acid, the 6-glycosyl aminopurine of the 6-benzylaminopurine of 0～1.1mg/L and 0～0.44mg/L;
The described embryo callus multiplicative stage, adopt the 1/2LM minimal medium, its additional hormone is: 2 of 0.55～2.2mg/L, 4-Benzene Chloride fluoroacetic acid, the 6-glycosyl aminopurine of the 6-benzylaminopurine of 0～1.1mg/L and 0～0.44mg/L;
The described somatic embryo stage of ripeness: adopt the 1/2LM minimal medium, the dormin of its additional 12～20mg/L and 0～1% active carbon;
Described somatic embryo is sprouted the stage, adopts the 1/4LM minimal medium, and it is additional: 0～0.25% active carbon.
2. method according to claim 1 is characterized in that, in the described embryonic callus induction stage, 2,4-Benzene Chloride fluoroacetic acid is 2.2mg/L, and the 6-benzylaminopurine is 0.55～1.1mg/L, and 6-glycosyl aminopurine is 0～0.22mg/L.
3. method according to claim 1 is characterized in that, the described embryo callus multiplicative stage, 2,4-Benzene Chloride fluoroacetic acid is 2.2mg/L, and the 6-benzylaminopurine is 0.8～1.1mg/L.
4. according to claim 2 or 3 described methods, it is characterized in that, in the medium of described embryonic callus induction stage and multiplicative stage, all also comprise: 1% sucrose, the glutamine of 500mg/L, the casein hydrolysis of 1g/L and 0.2% plant gel.
5. the described method of any one is characterized in that according to claim 1～3,, also comprises after the multiplicative stage at embryo callus: the state adjusting stage of embryo callus, for adopting the 1/2LM minimal medium, do not add any hormone and cultivated 7 days.
6. method according to claim 1 is characterized in that, in the described somatic embryo stage of ripeness, dormin is 16～20mg/L, and active carbon is 0.4～0.8%.
7. method according to claim 6 is characterized in that, in the medium in the described somatic embryo stage of ripeness, also comprises: 0.6% plant gel, 6% sucrose, the glutamine of 500mg/L and the caseinhydrolysate of 1g/L.
8. method according to claim 1 is characterized in that, described somatic embryo is sprouted the stage, and active carbon is 0.125～0.25%.
9. the described method of any one according to claim 8 is characterized in that, described somatic embryo is sprouted in the medium in stage, also comprises: glutamine 500mg/L, 2% sucrose, 1g/L caseinhydrolysate and 0.4% plant gel.
10. according to claim 1～3 or the described method of 6～9 any one, it is characterized in that, the method also comprises: cone is carried out disinfection, then take out immature zygotic embryos under aseptic environment before the body embryo cultivates immature zygotic embryos is carried out; Described disinfection way is: utilize 75～95% ethanol to cone sterilization 15～20min, use aseptic water washing 3～4 times again.
11. method according to claim 10 is characterized in that, described disinfection way is: utilize 95% ethanol to the cone 15min that sterilizes, use aseptic water washing 3 times again.
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| CN103461119B (en) * | 2013-08-20 | 2015-10-28 | 中国林业科学研究院林业研究所 | PIECA ASPERATA somatic embryo occurs and plant regeneration method |
| CN103497928B (en) * | 2013-09-30 | 2016-02-03 | 中国林业科学研究院林业研究所 | Drying method for treating before the sprouting of PIECA ASPERATA somatic embryo |
| CN104304034B (en) * | 2014-11-11 | 2017-01-11 | 新疆林科院造林治沙研究所 | Induction culture method and special induction culture medium for somatic embryo calluses of picea schrenkiana |
| CN105372368A (en) * | 2015-11-12 | 2016-03-02 | 中国林业科学研究院林业研究所 | Method for evaluating 6-BA-caused influence on picea balfouriana callus development by GC-MS combined technology |
| CN108200864A (en) * | 2018-01-24 | 2018-06-26 | 广西心壶城人家企业管理有限公司 | A kind of dragon spruce seed seedling-raising method |
| CN113826550B (en) * | 2021-09-03 | 2022-05-27 | 江西省科学院生物资源研究所 | Somatic embryogenesis and tissue culture method for camphor trees |
| CN115299343B (en) * | 2022-08-17 | 2023-05-09 | 中国林业科学研究院 | Method for establishing somatic embryogenesis system of huperzia serrata and application of somatic embryogenesis system |
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| 云杉属树种的体细胞胚胎发生;杨金玲;《植物学通报》;19920228;第16卷(第1期);第60页右栏第2段，第61页倒数第3段 * |
| 北美蓝云杉体细胞胚发生技术研究;孙敬爽;《北京林业大学学报》;20100131;第32卷(第1期);摘要，1.1试验材料及处理 * |
| 孙敬爽.北美蓝云杉体细胞胚发生技术研究.《北京林业大学学报》.2010,第32卷(第1期), |
| 杨金玲.云杉属树种的体细胞胚胎发生.《植物学通报》.1992,第16卷(第1期),第60-61页. |
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